Études des anti-oxydants-antimicrobiens provenant de fruits et ...

Études des anti-oxydants-antimicrobiens

provenant de fruits et légumes

Thèse

Cuong Ho

Doctorat en sciences et technologie des aliments

Philosophiae Doctor (Ph. D.)

Québec, Canada

© Cuong Ho, 2017

Études des anti-oxydants-antimicrobiens provenant de fruits et légumes

Thèse

Cuong Ho

Sous la direction de :

Joseph Arul, directeur de recherche

Paul Angers, codirecteur de recherche

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Résumé

Plusieurs études ont rapporté l’activité antimicrobienne et anti-oxydante d'extraits de sous-

produits végétaux. Le recours à l’utilisation de ces extraits dans les produits alimentaires

pourrait constituer une stratégie innovante et prometteuse. Les résidus de fruits et légumes

peuvent être une source potentielle de composés bioactifs. L’enjeu de cette étude était

d’initier des recherches qui pourront aboutir au développement d’agents anti-oxydants-

antimicrobiens à partir des sous-produits végétaux destinés à la conservation des aliments,

comme une alternative aux produits chimiques synthétiques. L'étude initiale a été

d'identifier des extraits de fruits et légumes qui possèdent à la fois de grandes propriétés

antimicrobiennes et anti-oxydantes. Enfin, des moyens simples pour augmenter l'activité

antimicrobienne de ces extraits ont été étudiés.

Environ 160 extraits aqueux de sous-produits de fruits et légumes ont été criblés pour

évaluer leur potentiel antimicrobien et anti-oxydant. L’activité antimicrobienne a été

déterminée par l’inhibition de la croissance d’Escherichi coli et de Bacillus subtilis.

L’activité anti-oxydante a été mise évidence par le test au DPPH (2,2-diphenyl -1-

picrylhydrazyl). L'étude a conduit à l'identification des extraits présentant à la fois un

potentiel antimicrobien et des propriétés anti-oxydantes. Les propriétés bioactives des

extraits sont influencées par le pH de l'extrait, le type de tissu (fruits, feuilles et racines) et

le type physiologique des fruits (climactérique). Les résultats ont montré l’existence d’une

relation entre les propriétés anti-oxydantes et antimicrobiennes des extraits de plantes. De

ce fait, l'indice anti-oxydant-antimicrobien développé pourrait être utile dans le choix des

sources végétales bioactives. Les extraits de plantes aux propriétés antimicrobiennes et anti-

oxydantes présentent un potentiel comme agent de conservation sécuritaire, bénéfique pour

la santé et économique.

La considération de la vitesse de piégeage des radicaux (facteur du temps) pour définir

l'efficacité réelle (capacité) du système anti-oxydant a été considérée comme la meilleure

manière d’exprimer avec plus de fiabilité le pouvoir anti-oxydant réel. La nouvelle

expression, le pouvoir antiradicalaire - ARP, produite à partir du test DPPH, peut être plus

utile pour identifier l'activité anti-oxydante des échantillons biologiques. Certains

échantillons présentaient une valeur ARP élevée tels que les extraits de rambutan, feuille de

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canneberge, feuille de bleuet, feuille de vigne (sauvage), feuille de framboise, feuille de

bétel, avocat, grenade et cherimoya. Les extraits de feuilles possèdent, en général, une

valeur d’ARP supérieure à celle des fruits tandis que, les extraits de racines présentent les

plus faibles ARP. De plus, cette étude montre également que le nombre moyen d’oxydation

du carbone de mélanges complexes tels que des extraits de plantes peut prédire leur pouvoir

anti-oxydant, en dépit de la disparité entre les tissus de feuilles et de fruits.

Le spectre d'activité anti-radicalaire d'extraits sélectionnés a été exploré. L'activité anti-

oxydante a été évaluée par différentes méthodes incluant, capacité anti-oxydante

équivalente de Trolox (TEAC), essai de radicaux libres (DPPH), test du pouvoir réducteur

d’ion ferrique (FRAP), mesure du potentiel d'oxydoréduction, réduction du peroxyde

d'hydrogène, du radical hydroxyle, d’anion superoxyde, d’oxyde nitrique et de l’activité de

chélation du fer. Les teneurs totales en composés phénoliques (TPC) et en flavonoïdes

(TFC) des extraits ont également été déterminées. Les extraits de feuille de bétel, fruit de

bleuet, feuille de cassis, feuille de canneberge ont montré une bonne activité de piégeage

des radicaux (essai TEAC); ceux de pomme, oseille, vigne rouge et racine de pissenlit

étaient efficaces contre SOA; ceux de feuilles de canneberge, feuille de bleuet, cassis et

romarin contre le radical hydroxyle; ceux de bette à carde, panais, brocoli et orange contre

H2O2; et ceux de pomme de terre, banane, oseille, feuille d'argousier contre l'oxyde

nitrique. Les extraits de feuille et fruit de bleuet, grenade, cassis et feuille de bétel ont

montré un bon pouvoir réducteur d’ion ferrique. Les extraits possédant la capacité élevée de

chélation du fer étaient: feuille d’argousier, radis, panais, feuille de bétel et mangoustan.

L'extrait de feuille de bétel présentait des activités élevées, y compris de fixation du fer et

de piégeage de divers radicaux, à l'exception de l'oxyde nitrique, où l'activité était

néanmoins modérée. Les essais de TEAC, de DPPH et de FRAP fournissent

essentiellement la même réponse concernant l'activité anti-oxydante des extraits de plantes,

ce qui suggère que l'un de ces essais serait suffisant pour évaluer leur capacité anti-

radicalaire. Cette étude suggère également que l'activité anti-oxydante d'une substance,

déterminée par un ou plusieurs essais, ne donne pas une image complète de son efficacité

contre les diverses espèces de radicaux oxygénés; et elle montre que la détermination du

spectre de l'activité anti-radicalaire serait nécessaire.

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L’activité antimicrobienne des extraits de plantes sélectionnés a été évaluée en détail par

turbidimétrie en milieu liquide et par diffusion en milieu solide (gel). L’activité

antimicrobienne a été exprimée dans la première méthode par l’inhibition de la croissance

(GI), par la concentration minimale inhibitrice (MIC) et par une nouvelle expression,

dénommée index antimicrobien (AMI). Dans la seconde méthode, l’activité a été exprimée

en zone d’inhibition. L'AMI prend en compte les trois phases de croissance des bactéries.

Bien que les méthodes turbidimétriques soient fiables dans l’évaluation de l’activité

antimicrobienne des substances, la nouvelle expression (AMI) semble être plus

significative car, elle comprend l’information sur l’interaction entre la substance et le

micro-organisme et sa vitesse de croissance en présence de cette substance. De plus, l'AMI

démarque l’activité des échantillons, même ceux qui sont très actifs. Le spectre d’activité

antimicrobienne des extraits de plantes sélectionnés a été également étudié. Seulement

quelques extraits ont montré un spectre d’activité antimicrobienne assez large. En effet,

seul l’extrait de feuille de bétel a un large spectre d’activité contre les bactéries, les levures

et les champignons, suivis des extraits de grenade et de fruits d’argousier qui semblent être

efficaces contre les bactéries et les levures. L’extrait de cassis est effectivement une

substance antibactérienne.

Finalement, différentes stratégies pour améliorer l’activité antimicrobienne d'extraits anti-

oxydants-antimicrobiens sélectionnés ont été explorées. Trois approches, incluant

l’extraction par solvants sélectifs, le mélange binaire des extraits et l'addition de composés

végétaux, ont été étudiées. L’activité anti-oxydante-antimicrobienne et le profil

phytochimique ont été analysés. Le résultat montre que l’eau chaude peut être utilisée

comme solvant pour l’extraction d’agents anti-oxydants-antimicrobiens d’origine végétale,

bien qu’un solvant polaire extrait en préférence les substances phénoliques et modestement

les substances non polaires comme les terpénoïdes. Toutefois, compte tenu du rendement et

de l’activité des extraits, l’eau semble être appropriée, et elle peut être considérée comme

un solvant efficace pour les fruits qui sont riches en substances phénoliques. Néanmoins,

d'autres solvants sélectifs doivent être considérés pour l’extraction des substances actives

non-polaires issues de matière première végétale comme les feuilles. Certaines

augmentations de l’activité antimicrobienne sont possibles par le mélange de deux extraits

de plantes ou par l’addition de composés végétaux. On a aussi observé que la composition

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des mélanges peut être importante, car les interactions synergiques ou antagonistes se

retrouvent dans certaines proportions. Le mélange des extraits de grenade et cassis d’une

part et le mélange des extraits de thé vert et pomme de cajou d’autre part ont montré une

amélioration d’activité. L’ajout de glyoxal de méthyle et mono-caprine a également produit

une augmentation de l’activité des extraits de plantes. L'addition de glyoxal de méthyle à 25

% (p/p) a amélioré l’activité des extraits de grenade et de cassis.

Dans l'ensemble, ce travail a exploré le potentiel des extraits des sous-produits de fruits et

de légumes comme agent anti-oxydant-antimicrobien. Les extraits de plantes montrant des

potentiels anti-oxydants et antimicrobiens regroupent: olive, canneberge, noni, feuille de

bétel, cassis, grenade, citronnelle, épinard, raisin vert (vin), cassis (résidu), aubergine,

ramboutan, prune indienne, feuille de canneberge, feuille de romarin, feuille de vigne

(sauvage), thé vert, mangoustan et feuille de framboise. Cependant, de cette liste, seuls

quelques extraits (feuille de bétel, grenade, résidus de cassis) présentent un large spectre

d’activité anti-oxydante et antimicrobienne. En outre, cette étude a introduit de nouvelles

expressions pour l’activité anti-oxydante (ARP) et antimicrobienne (AMI) des mélanges

complexes tels que les extraits de plantes. Ces expressions peuvent être utiles pour le

criblage de matériels d’origine végétale dans la recherche de ces activités. Un autre

enseignement de cette étude est que l'amélioration de l'activité antimicrobienne des extraits

de plantes ne peut pas être possible simplement par le mélange d'extraits, en raison des

interactions potentielles entre les composants des extraits. La connaissance de la

composition phytochimique est essentielle pour comprendre de telles interactions, dans la

sélection des mélanges et pour déterminer les approches possibles pour améliorer l'activité

antimicrobienne. Les sous-produits de végétaux peuvent être une source potentielle

d'antioxydant-antimicrobiens pour une application dans la conservation des aliments,

comme alternative aux agents synthétiques, mais beaucoup de travaux sont encore

nécessaires pour atteindre ce but, ce qui nécessiterait une approche systémique.

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Abstract

There is a growing interest in the development of strategies to use agricultural and

industrial residues as a source of high value-added, including bioactive products. The

residues of fruits and vegetables may be a potential source of bioactive compounds. The

major objective of this work is to conduct studies to pave the way for the development of

antioxidant-antimicrobials from plant by-products for use in food preservation, as

alternative to synthetic chemicals. The starting point was the identification of extracts of

fruit and vegetable extracts exhibiting high antimicrobial and antioxidant properties. The

selected extracts were then characterized for their spectrum of antioxidant and

antimicrobial activities. Finally, simple ways of enhancing the antimicrobial activity of the

selected extracts were examined.

Aqueous extracts of about 160 fruit and vegetable by-products were evaluated for their

potential as antimicrobial and antioxidant agents. The growth inhibiting activity of all the

160 extracts were tested against Escherichia coli, and Bacillus Subtilis; and the antioxidant

activity was determined by DPPH radical scavenging assay. The pH of the extract, type of

tissue (fruit, leaf and root) and physiological type of fruits (climacteric) had impact on the

bioactive properties. There was some relationship between antioxidant and antimicrobial

properties of the plant extracts. The proposed antioxidant-antimicrobial index may be

useful in the selection of plant sources of bio-actives.

Consideration of rate of radical quenching (time factor) to define actual efficacy (capacity)

of antioxidant system was found to be a better and reliable way of expressing the real

antioxidant power. The new expression, antiradical power - ARP, generated from DPPH

assay may be more useful in identifying the antioxidant activity of biological samples.

Some samples exhibited high ARP value such as rambutan, cranberry leaf, blueberry leaf,

grape leaf (wild), raspberry leaf, betel leaf, avocado, pomegranate and custard apple. Leaf

extracts possess, in general, higher ARP than fruits, and root extracts possess low ARP. In

addition, this study also suggests that average carbon oxidation number of complex

mixtures such as plant extracts may portend their antioxidant power, in spite of the

disparity between leaf and fruit materials.

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In chapter 4, plant extracts selected from the original 160 were investigated for their

spectrum of anti-radical activity. The antioxidant activity was evaluated by Trolox

equivalent antioxidant capacity (TEAC), DPPH free radical assay (DPPH), ferric reducing

power assay (FRAP), redox potential measurement, hydrogen peroxide, hydroxyl radical,

superoxide anion, nitric oxide and iron chelating activities. Their total phenolic content

(TPC) and total flavonoid content (TFC) were also determined. Betel leaf, blueberry fruit

and black currant and cranberry leaf showed high radical scavenging activity (TEAC

assay); apple, sorrel, red grape and dandelion root were effective against SOA; cranberry

leaf, blueberry leaf, black currant and Rosemary against hydroxyl radical; rainbow chard,

parsnip, broccoli and orange against H2O2; and potato, banana, sorrel, sea buckthorn leaf

against nitric oxide. Blueberry leaf and fruit, pomegranate, black currant and betel leaf

showed high ferric ion reducing power. The extracts showing high iron binding capacity

were: sea buckthorn leaf, radish, parsnip, betel leaf and mangosteen. Betel leaf extract

exhibited high activities, including iron binding and scavenging of various radicals except

nitric oxide, where the activity was moderate. TEAC, DPPH and FRAP assays provide

essentially the same response with respect to the antioxidant activity of plant extracts,

suggesting that any one of them would be adequate to evaluate their anti-radical capacity.

This study also suggests that antioxidant activity of a substance, determined by one or

more related assays, does not give the complete picture of its effectiveness against various

species of oxygen radicals; and emphasizes that determination of the spectrum of the anti-

radical activity would be necessary.

In chapter 5, the antimicrobial activity of selected plant extracts was evaluated in detail by

turbidimetric methods in liquid medium, and by well-diffusion method in gel medium. The

antimicrobial activity was expressed in the former by growth inhibition, minimum

inhibitory concentration - MIC and by a new expression, the antimicrobial index – AMI;

and in the latter, expressed by zone of inhibition. AMI takes into account all the three

growth phases of the bacteria. Although the turbidimetric methods were in good agreement

in the assessment of the activity of the substances, the new expression, AMI, appears to be

more meaningful since it carries the information regarding interaction between the

substance and the microorganism and the growth rate in the presence of that substance. In

addition, AMI demarcates the activity of samples, even those found to be highly active.

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Furthermore, the spectrum of antimicrobial activity of selected plant extracts was also

examined. Only a few extracts showed some broad spectrum in their activities. In effect,

only betel leaf extract showed a broad spectrum of antimicrobial activity against bacteria,

yeasts and fungi; whereas pomegranate and sea buckthorn fruit extracts appear to be

effective against both bacteria and yeasts. Black currant extract is effectively an

antibacterial substance.

In chapter 6, various ways of enhancing the antimicrobial activity of selected antioxidant-

antimicrobial extracts were explored. Three approaches, including selective solvent

extraction, binary blending of extracts and addition of plant compounds were investigated.

Antioxidant, antimicrobial activity and the profile of phytochemical classes were analyzed.

The results showed that hot water could be used for solvent extraction of antioxidant-

antimicrobials from plant materials, albeit a polar solvent that extracts phenolic substances

preferably and only modestly non-polar substances such as terpenoids. However,

considering the yield of the extracts and the activity, water appeared to be an effective

solvent solvent for fruit sources that are rich in phenolic substances. Other selective

solvents must be considered for extraction of active non-polar substances for plant sources

such as leaves. Some enhancement in antimicrobial activity was possible by either mixing

plant extracts or by the addition of plant compounds. It was also observed that the

composition of blends or mixtures might be important, since synergistic or antagonistic

interactions occurred at certain proportions. Pomegranate and black currant extract blends

and green tea and cashew apple extract blends showed enhancement in activity. The

addition of methyl glyoxal and mono-caprin also showed enhancement in the activity of

plant extracts. Methyl glyoxal at 25 % (w/w) addition improved the activity of pomegranate

and black currant extracts.

Overall, this work explored the potential of extracts of fruit and vegetable by-products as

anti-oxidant-antimicrobials. Some plant extracts having potential as antioxidant-

antimicrobial agents. They include: olive, cranberry, noni, betel leaf, black currant,

pomegranate, lemon grass, spinach, green grape (wine), black currant (residue), egg plant,

rambutan, Indian plum, cranberry leaf, rosemary leaf, grape leaf (wild), green tea,

mangosteen and raspberry leaf. However, the list is reduced to a few (betel leaf,

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pomegranate, black currant residue), should broad antioxidant and antimicrobial activities

are taken into account.

In addition, this study introduces new expressions for antioxidant (ARP) and antimicrobial

(AMI) activities of complex mixtures such as plant extracts, and they can be useful in the

screening of plant materials for these activities. The knowledge of the phytochemical

composition is essential to understand such interactions, in the selection of mixtures and to

determine possible approaches to enhance the antimicrobial activity. Plant by-product can

be a potential source of antioxidant-antimicrobial for use in food preservation, as

alternative to synthetic agents, but much work is needed to realize this goal, and that would

require a systemic approach.

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Table des matières

Résumé .................................................................................................................................. iv

Abstract .............................................................................................................................. viii

Table des matières .............................................................................................................. xii

Liste des tableaux ............................................................................................................. xvii

Liste des figures ................................................................................................................. xix

Liste des abréviations et des sigles .................................................................................. xxii

GENERAL INTRODUCTION ........................................................................................... 1

Chapter 1 REVUE DE LITERATURE .............................................................................. 5

1.1 Plant by-products: eco-friendly source of novel bioactive compounds ................. 6 1.1.1 Why plant by-products? ......................................................................................... 7 1.1.2 Source, provider and type of by-products .............................................................. 8

1.1.3 Methods of extraction of bioactive compounds from plant ................................... 9 1.1.4 Trend and challenges in using plant by-products ................................................. 11

1.2 Plant secondary metabolites .................................................................................... 13 1.2.1 Primary and secondary metabolites ..................................................................... 13 1.2.2 Plant secondary metabolism ................................................................................. 14

1.2.3 Biosynthesis of secondary metabolites ................................................................ 16 1.2.3.1 Terpenes......................................................................................................... 18 1.2.3.2 Phenolic compounds ...................................................................................... 19

1.2.3.3 Nitrogen-containing compounds ................................................................... 23

1.2.3.4 Polyketides..................................................................................................... 23

1.3 Antioxidants .............................................................................................................. 25 1.3.1 Concepts ............................................................................................................... 25

1.3.2 Why to examine antioxidants from plants? .......................................................... 26 1.3.3 Production of free radical in biology ................................................................... 28

1.3.4 Natural sources of antioxidant compounds .......................................................... 29 1.3.5 Modulation of free radical by antioxidants .......................................................... 30

1.3.5.1 Mechanisms of antioxidant action ................................................................. 31 1.3.5.2 Defense system mechanism in vivo against oxidative damage ..................... 31 1.3.5.3 Plant antioxidant systems .............................................................................. 33

1.3.6 The structure–activity relationships of antioxidant .............................................. 35 1.3.7 Methods of in vitro antioxidant activity determination .................................... 37

1.3.7.1 In vitro antioxidant capacity assays ............................................................... 37 1.3.7.2 Mode of action of in vitro antioxidant activity assays ................................... 38

1.3.8 Trends and challenges in application of antioxidants from plant sources. .......... 46

1.4 Plants as a source of antimicrobials ........................................................................ 49 1.4.1 Why antimicrobials from plant sources? .............................................................. 49

1.4.2 Major groups of phytochemicals with antimicrobial properties .......................... 50 1.4.3 Modes of action of antimicrobials ........................................................................ 53 1.4.4 Bacterial resistance to antimicrobials and mechanisms ....................................... 56

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1.4.5 In vitro methods to evaluate plant extracts for antimicrobial activity.................. 57

1.4.5.1 Diffusion method ........................................................................................... 58

1.4.5.2 Dilution method ............................................................................................. 58 1.4.5.3 Broth dilution ................................................................................................. 58 1.4.5.4 Agar dilution .................................................................................................. 59 1.4.5.5 Time kill assay ............................................................................................... 59

1.4.6 Research trends and challenges of antimicrobials from plant products ............... 59

1.4.7 Recent literature on bioactivities of some specific fruits and vegetables - A

random walk .................................................................................................................. 61

1.5 Hypotheses ................................................................................................................. 66 1.6 Objectives .................................................................................................................. 67

1.6.1 General Objective ................................................................................................. 67

1.6.2 Specific Objectives ............................................................................................... 67

Chapter 2 NATURAL ANTIOXIDANT-ANTIMICROBIALS: SCREENING OF

FRUITS, VEGETABLES AND THEIR BY-PRODUCTS ............................................. 68

2.1 Abstract ...................................................................................................................... 69 2.2 Introduction ............................................................................................................... 70 2.3 Materials and Methods ............................................................................................. 72

2.3.1 Plant materials ...................................................................................................... 72 2.3.2 Chemicals and reagents ........................................................................................ 72 2.3.3 Preparation of extracts .......................................................................................... 72

2.3.4 Bacterial strains and inoculum preparation .......................................................... 72 2.3.5 Antimicrobial activity assay ................................................................................. 73

2.3.6 Antioxidant assay - DPPH radical-scavenging capacity ...................................... 73 2.3.7 Statistical analyses ............................................................................................... 74

2.4 Results ........................................................................................................................ 75 2.4.1 Antimicrobial activity .......................................................................................... 75

2.4.2 Antioxidant activity of fruit and vegetable extracts (DPPH assay) ..................... 84 2.4.3 Correlation between the antimicrobial and antioxidant activity of plant extracts 84

2.5 Discussion .................................................................................................................. 86 2.5.1 Antioxidant and antibacterial activities of plant by-products .............................. 86 2.5.2 E. coli and B. subtilis sensitivity. ......................................................................... 86

2.5.3 Effect of pH of extracts on AM and AO properties ............................................. 88 2.5.4 Effect of physiological type of fruit: climacteric vs. non-climacteric.................. 89 2.5.5 Effect of plant tissue type ..................................................................................... 90 2.5.6 Antioxidant activity of Brassica and Allium extracts .......................................... 90 2.5.7 Correlation between antioxidant and antimicrobial activities .............................. 92

2.5.8 Antioxidant-antimicrobials index (AO-AM Index) ............................................. 93

2.6 Conclusions ................................................................................................................ 95

Chapter 3 ANTI-RADICAL POWER (ARP) OF PLANT EXTRACTS: A NEW

MEASURE OF ANTIOXIDANT PROPERTY FROM DPPH ASSAY ....................... 96

3.1 Abstract ...................................................................................................................... 97 3.2 Introduction ............................................................................................................... 98 3.3 Materials and methods ........................................................................................... 100

3.3.1 Plant materials .................................................................................................... 100

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3.3.2 Chemicals and reagents ...................................................................................... 100

3.3.3 Plant extract preparation .................................................................................... 100

3.3.4 DPPH free radical scavenging assay .................................................................. 100 3.3.5 Elemental and metal analyses ............................................................................ 101 3.3.6 Reaction kinetics and calculation of reaction rate constants .............................. 102 3.3.7 Anti-Radical Power (ARP): The overall antioxidant activity ............................ 104 3.3.8 Average carbon oxidation number ..................................................................... 105

3.3.9 Data analysis ...................................................................................................... 105

3.4 Results and discussion ............................................................................................ 106 3.4.1 Estimation of antioxidant activity by DPPH• assay ........................................... 106

3.4.1.1 Antioxidant capacity .................................................................................... 107 3.4.1.2 Rate of radical scavenging ........................................................................... 108

3.4.2 Relation between scavenging rate and capacity of antioxidant activity ............ 113

3.4.3 Anti-Radical Power ............................................................................................ 116 3.4.4 Average carbon oxidation number (ACON) ...................................................... 119

3.5 Conclusions .............................................................................................................. 124

Chapter 4 ANTI-RADICAL ACTIVITY SPECTRUM OF SECLETED FRUIT AND

VEGETABLE EXTRACTS AGAINST SEVERAL REACTIVE OXYGEN SPECIES

............................................................................................................................................ 125

4.1 Abstract .................................................................................................................... 126

4.2 Introduction ............................................................................................................. 127 4.3 Materials and methods ........................................................................................... 129

4.3.1 Plant materials .................................................................................................... 129

4.3.2 Chemicals and reagents ...................................................................................... 130 4.3.3 Plant extract preparation .................................................................................... 130

4.3.4 Total phenolic content ........................................................................................ 130 4.3.5 Total flavonoid assay ......................................................................................... 131

4.3.6 Antioxidant Assays ............................................................................................ 131 4.3.6.1 TEAC (Trolox equivalent antioxidant capacity) ......................................... 131 4.3.6.2 DPPH free radical scavenging assay ........................................................... 132

4.3.6.3 Reducing power property ............................................................................ 132 4.3.6.4 Hydrogen peroxide scavenging activity ...................................................... 133

4.3.6.5 Hydroxyl radical scavenging activity .......................................................... 133 4.3.6.6 Superoxide anion scavenging capacity ........................................................ 133 4.3.6.7 Nitric oxide scavenging activity .................................................................. 134 4.3.6.8 Metal chelating assay (Spectrometric assay) ............................................... 134

4.3.7 Data analysis ...................................................................................................... 134

4.4. Results and Discussion ........................................................................................... 136 4.4.1 Total phenolic and flavonoid contents ............................................................... 136

4.4.2 Antioxidant activity ............................................................................................ 138 4.4.2.1 Test Radicals (ABTS tand DPPH)............................................................... 138 4.4.2.2 Relation between anti-radical activities (TEAC, DPPH) and total phenolic

content (TPC) and total flavonoid content (TFC) ................................................... 141 4.4.2.3 Reducing power property (FRAP) ............................................................... 143 4.4.2.4 Hydroxyl radical scavenging activity .......................................................... 146 4.4.2.5 Hydrogen peroxide scavenging activity ...................................................... 147

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4.4.2.6 Superoxide anion scavenging activity ......................................................... 147

4.4.2.6 Nitric oxide scavenging activity .................................................................. 147

4.4.2.7 Metal chelating activity ............................................................................... 148 4.4.3 Inter-relationships between anti-radical activities ............................................. 150 4.4.4 Anti-radical activity spectrum ............................................................................ 153

4.5 Conclusions .............................................................................................................. 155

Chapter 5 CHARACTERIZATION OF ANTIMICROBIAL ACTIVITY OF

SELECTED PLANT EXTRACTS: ANTIMICROBIAL ACTIVITY INDEX AND

SPECTRUM OF ACTIVITY .......................................................................................... 156

5.1 Abstract .................................................................................................................... 157 5.2 Introduction ............................................................................................................. 158

5.3 Materials and methods ........................................................................................... 161 5.3.1 Plant materials .................................................................................................... 161

5.3.2 Extraction and stock solution preparation .......................................................... 162

5.3.4 Bacterial strains and inoculum preparation ........................................................ 162 5.3.5 Antimicrobial activity assays ............................................................................. 162

5.3.5.1 Zone inhibition agar gel diffusion assay...................................................... 162 5.3.5.2 Micro-titer broth dilution (growth inhibition or inhibition capacity) and time

kill curve assay ........................................................................................................ 163 5.3.5.3 Broth micro-dilution (MIC) assay ............................................................... 163 5.3.5.4 Agar dilution (yeasts) .................................................................................. 164

5.3.5.5 Anti-fungal assay (agar dilution) ................................................................. 164 5.3.6 Empirical expression for antimicrobial activity ................................................. 164

5.3.7 Data analysis ...................................................................................................... 167

5.4 Results and discussion ............................................................................................ 168 5.4.1 Antimicrobial activity of plant extracts .............................................................. 168

5.4.1.1 Antimicrobial Index (AMI) ......................................................................... 168

5.4.1.2 Growth inhibition or antimicrobial capacity ............................................... 173 5.4.1.3 Minimum inhibitory concentration (MIC) .................................................. 173 5.4.1.4 Zone inhibition (ZI) ..................................................................................... 173

5.4.1.5 Comparison of methods in the characterization plant extracts .................... 174 5.4.2 Spectrum of Antimicrobial activity .................................................................... 182

5.4.2.1 Antimicrobial activity of plant extracts against bacteria ............................. 182 5.4.2.2 Antimicrobial activity of plant extract against yeasts ................................. 183 5.4.2.3 Antimicrobial activity of plant extract against fungi ................................... 184

5.5 Conclusions .............................................................................................................. 186

Chapter 6 ENHANCEMENT OF THE ANTIMICROBIAL ACTIVITY OF

SELECTED PLANT EXTRACTS BY FRACTIONATION AND MIXING ............. 187

6.1 Abstract .................................................................................................................... 188

6.2 Introduction ............................................................................................................. 189 6.3 Materials and methods ........................................................................................... 190

6.3.1 Plant materials .................................................................................................... 190 6.3.3 Plant extracts and stock solution preparation ..................................................... 190 6.3.4 Bacterial strains and inoculum preparation ........................................................ 191 6.3.5 Antimicrobial activity assays ............................................................................. 191

xvi

6.3.5.1 Growth Inhibition using micro-titer broth dilution assay (GI) .................... 191

6.3.5.2 MIC - Broth microdilution assay ................................................................. 192

6.3.6 Antioxidant assay - DPPH radical-scavenging capacity .................................... 192 6.3.7 Phyto-chemical analysis ..................................................................................... 193

6.3.7.1 Total phenolics ............................................................................................ 193 6.3.7.2 Total flavonoid assay ................................................................................... 193 6.3.7.3 Estimation of total tannin content ................................................................ 194

6.3.7.4 Determination of proanthocyanidin content ................................................ 194 6.3.7.5 Determination of alkaloid content ............................................................... 194 6.3.7.6 Terpenoids ................................................................................................... 195

6.3.8 Enhancement of antimicrobial activity of plant extracts .................................... 196 6.3.8.1 Selective solvent extraction ......................................................................... 196

6.3.8.2 Binary blends of selected extracts ............................................................... 197

6.3.8.3 Addition of plant compounds ...................................................................... 197 6.3.9 Statistical analysis .............................................................................................. 197

6.4 Results and discussion ............................................................................................ 198 6.4.1 Solvent fractionation of extracts ........................................................................ 198

6.4.1.1 Extract yields ............................................................................................... 198

6.4.1.2 Composition of solvent fractions and their antioxidant-antimicrobial

activities ................................................................................................................... 199 6.4.2 Binary blends of selected extracts ...................................................................... 203

6.4.3 Addition of plant compounds ............................................................................. 206

6.5 Conclusions .............................................................................................................. 212

GENERAL CONCLUSIONS AND PERSPECTIVES ................................................. 213

BIBLIOGRAPHIE ........................................................................................................... 218

ANNEXES ......................................................................................................................... 233

Annexe 1. Yields of extracts from plant materials (Chapter 1) ................................ 234

Annexe 2. Numerical data for figures in Chapter 6 ................................................... 238

xvii

Liste des tableaux

Table 1.1 Solvents used for active component extraction .................................................... 10

Table 1.2 Natural plant sources of some antioxidants .......................................................... 29

Table 1.3 Natural sources of polyphenol .............................................................................. 30

Table 1.4 In vitro antioxidant capacity assays ...................................................................... 38

Table 1.5 Major classes of antimicrobial compounds from plants ....................................... 52

Table 1.6 Phyto-chemical composition and bioactivities of fruit and vegetable

extracts .................................................................................................................................. 63

Table 2.1 Antimicrobial and Antioxidant activity of Climacteric fruits .............................. 78

Table 2.2 Antimicrobial and Antioxidant activity of Non-climacteric fruits ....................... 79

Table 2.3 Antimicrobial and Antioxidant activity of leaf extracts ....................................... 81

Table 2.4 Antimicrobial and antioxidant activity of root peel extracts ................................ 83

Table 3.1 Antioxidant activity of fruits and vegetables by products extracts ................... 109

Table 3.2 Elemental composition, average carbon oxidation number value of fruits

and vegetables by-products extracts ................................................................................... 120

Table 3.3 Comparison of ACON, Capacity, rate and ARP values of plant by product

(fruit and leaf) extracts (1.0 mg/mL) .................................................................................. 120

Table 4.1 Total phenolic and flavonoid contents of aqueous extracts of 36 selected

plant materials: Total phenolic content was expressed as gallic acid (GAE) and

ascorbic acid equivalent (AAE) and total flavonoid content was expressed as rutin

equivalent (RE). (±) SD ...................................................................................................... 137

Table 4.2 Free radical scavenging activity and reducing property of the aqueous

extracts obtained from selected plant materials. (±) denotes standard deviation ............... 140

Table 4.3 Free radical scavenging activity and reducing property of the aqueous

extracts obtained from selected plant materials. (±) denotes standard deviation: Ferric

reducing power assay (FRAP) and Nitric Oxide Radical Scavenging (NO) ...................... 144

Table 4.4 Ferrous ion chelating activity of aqueous extract obtained from selected

plant material. This activity was expressed as Na2EDTA equivalent and (±) means the

standard deviation ............................................................................................................... 149

Table 5.1 Antimicrobial growth kinetic parameters against E.coli for plant extracts at

the concentration of 10 mg/mL .......................................................................................... 170

Table 5.2 Comparison of antimicrobial activity (AMI) against E.coli of plant extracts

(10 mg/mL) determined by agar gel diffusion assay (Zone of inhibition, ZI), Micro-

titer broth dilution (antimicrobial capacity), Broth microdilution assay (MIC) ................. 176

Table 5.3 Growth inhibition of bacteria by plant extracts at 10.0 mg/mL (Growth

inhibition ± SD) .................................................................................................................. 182

xviii

Table 5.4 Zone of inhibition (mm) of bacterial growth by well-diffusion assay at 5.0

mg/mL ................................................................................................................................ 183

Table 5.5 Inhibitory activity of selected plant extracts on the growth inhibition of

yeasts at 10.0 mg/mL .......................................................................................................... 184

Table 5.6 Inhibitory activity of selected plant extracts on the growth inhibition of

fungi (%) at 10.0 mg/mL .................................................................................................... 184

Table 6.1 Solubility parameter of the solvent systems ....................................................... 196

Table 6.2 The MIC against E.coli of 12 selected plant extracts and 10 plant products ..... 206

Table 6.3 The GI (%) against E.coli of mixture of 12 plant extracts with added plant

products at total concentration of 5.0 mg/mL .................................................................... 208

Table 6.4 The GI (%) against E. coli of mixtures of plant extracts and 5 plant

compounds at the proportion of plant extract 97.5%, (v/v). ............................................... 209

xix

Liste des figures

Figure 1.1 Overview of plant primary and secondary metabolism ...................................... 15

Figure 1.2 Biosynthetic relationships of major groups of secondary metabolites ............... 17

Figure 1.3 Biosynthetic of terpenes: Mevalonic acid pathway ............................................ 18

Figure 1.4 Biosynthetic phenolic compounds: Shikimate + Malonate pathway .................. 19

Figure 1.5 Balance between antioxidant (AO) and reactive oxygen species (ROS) in

plants ..................................................................................................................................... 34

Figure 1.6 ROS and antioxidant defense mechanisms ......................................................... 34

Figure 1.7 (a) catechol moiety of the B-ring, (b) 2,3-double bond in conjugation with

a 4-oxofunction of a carbonyl group in the C-ring and (c) presence of hydroxyl

groups at the 3 and 5 positions ............................................................................................. 35

Figure 1.8 Intra-molecular hydrogen bonding of ortho substituted phenols ........................ 36

Figure 1.9 Radical trapping mechanism of phenolic antioxidants ....................................... 36

Figure 1.10 Radical trapping mechanism of carotenoids ..................................................... 36

Figure 1.11 Reduction of ABTS+ radical by Trolox (H-donor)........................................... 39

Figure 1.12 Protonation of DPPH (violet) to most stable DPPH-H form (light yellow

color) ..................................................................................................................................... 40

Figure 1.13 Mechanism of Ferric Reduction ........................................................................ 40

Figure 1.14 Reduction of Titanium compound by H2O2 ...................................................... 41

Figure 1.15 Superoxide anion radical generation by PMS-NADH system .......................... 42

Figure 1.16 Formation of azo dye ........................................................................................ 43

Figure 1.17 Mechanism of metal chelation by ferrozine ...................................................... 45

Figure 1.18 Mechanisms of action of antimicrobial agents. PABA, paraminobenzoic

acid; DHFA, dihydrofolic acid; THFA, tetrahydrofolic acid ............................................... 54

Figure 1.19 Mechanisms of bacterial resistance to antimicrobials ....................................... 57

Figure 2.1 A comparison of sensitivity of E.coli and B.subtilis against 164 plant

extracts .................................................................................................................................. 75

Figure 2.2 Effect of physiological type of fruits on the growth inhibition of B.subtilis ..... 77

Figure 2.3 Effect of physiological type of fruits on the growth inhibition of E.coli ........... 77

Figure 2.4 Effect of the plant tissue type on the growth inhibition of E. coli ...................... 77

Figure 2.5 Effect of the plant tissue type on the growth inhibition of B. subtilis ................ 77

Figure 2.6 Effect of ripening process of fruit on DPPH radical scavenging capacity .......... 85

Figure 2.7 Effect of plan part origin of sample on DPPH radical scavenging capacity ....... 85

file:///C:/Users/Cuong%20Ho/Desktop/Cuong%20Ho%20PhD/final%20versions%2019%20jan%202017/New%20folder/910141839.doc%23_Toc476735839






xx

Figure 2.8 Correlation between, the pH, growth inhibitory activity (GI) against E.coli

and B.subtilis and DPPH radical scavenging capacity of the extracts. ................................ 85

Figure 3.1 Time course of DPPH• scavenging by selected extracts ................................... 107

Figure 3.2 Relation between rate of radical scavenging and capacity of the radical

scavenging capacity of plant extracts ................................................................................. 115

Figure 3.3 Relation between ARP and radical scavenging capacity of plant extracts ....... 118

Figure 3.4 Correlations between pH, rate, capacity and ARP ............................................ 119

Figure 3.5 Relationship between ACON and capacity of selected plant extracts .............. 121

Figure 3.6 Relationship between ACON and rate of selected plant extracts ..................... 121

Figure 3.7 Relationship between ARP and ACON of selected plant extracts .................... 121

Figure 4.1 Correlation between Trolox equivalent antioxidant capacity and DPPH

free radical scavenging activity .......................................................................................... 141

Figure 4.2 Correlation between Trolox equivalent antioxidant capacity (TEAC),

DPPH and total phenolic content (TPC)............................................................................. 142

Figure 4.3 Relationship between redox (potentiometric) and FRAP analysis ................... 145

Figure 4.4 Relationship between TEAC and FRAP values of plant extracts ..................... 146

Figure 4.5 Correlation between Total polyphenol content (TPC), Total flavonoid

content (TFC), DPPH, TEAC, Hydroxyl radical (HR), Superoxide anion (SOA),

Hydrogen peroxide (HP) and Nitric oxide (NO) ................................................................ 151

Figure 4.6 Multi correlation between Total polyphenol content (TPC), Total

flavonoid content (TFC), DPPH, TEAC, Hydroxyl radical (HR), Superoxide anion

(SOA), Hydrogen peroxide (HP) and Nitric oxide (NO) ................................................... 152

Figure 5.1 Growth curve of E. coli in Nutrient broth at 37oC temperature during 24 h

period .................................................................................................................................. 168

Figure 5.2 Linear representation of growth curves of E. coli according to equation (9).

A: Control; B: Nutrient broth containing pomegranate extract at 10 mg/mL

concentration ...................................................................................................................... 169

Figure 5.3 Relationship between interaction constants log Ka and log K of plant

extracts ................................................................................................................................ 171

Figure 5.4 Antimicrobial activity of plant extracts: correlation loading of

antimicrobial evaluation methods by PCA ......................................................................... 179

Figure 5.5 Antimicrobial activity of plant extracts: scatter plot of antimicrobial

activity of plant extracts by PCA. The number corresponds to the product as listed in

Table 5.1 and 5.2. ............................................................................................................... 179

Figure 5.6 Multi-correlation between different methods for assay of antimicrobial

activity ................................................................................................................................ 181

Figure 6.1 Yield of solvent extracts of pomegranate, black currant residue and betel

leaf ...................................................................................................................................... 198









xxi

Figure 6.2 Composition of solvent fractions of pomegranate and their antimicrobial

activity against E. coli and anti-radical activity by DPPH assay........................................ 201

Figure 6.3 Composition of solvent fractions of black currant residue and their

antimicrobial activity against E. coli and anti-radical capacity by DPPH assay ................ 201

Figure 6.4 Composition of solvent fractions of betel leaf and their antimicrobial

activity against E. coli and anti-radical activity by DPPH assay........................................ 202

Figure 6.5 Antibacterial activity of blends of plant extracts in varying proportions.......... 203

Figure 6.6 Antibacterial activity of blends of plant extracts in varying proportions

against B.subtilis at total concentration of 5.0 mg/mL ...................................................... 205

Figure 6.7 Antibacterial activity (MIC) against E.coli at varying compositions of

plant extract – plant compound mixtures ........................................................................... 210

xxii

Liste des abréviations et des sigles

AAE: Ascorbic Acid Equivalent

ABTS: 2,2-azinobis 3-ethylbenzothiazoline-6-sulphonic acid

ACAC: acetone: acetonitrile (solvent)

ACON: Average Carbon Oxidation Number

ACS: acridone synthase

AM: antimicrobial

AMI: Antimicrobial Index

ANOVA: Analysis of Variance

AOA: Antioxidant Activity

APX: ascorbate peroxidase

ARP: anti-radical power

ASH: ascorbic acid

ATCC: American Type Culture Collection

ATP: adenosine triphosphate

BHA: butylated hydroxyanisole

BHT: butylated hydroxytoluene

BPS: benzophenone synthase

BS: Bacillus subtilis

CAT: catalase

CE: catechol equivalent

CFU: colony-forming unit

CHS chalcone synthase

CLSI: Clinical and Laboratory Standards Institute

CTAS: coumaroyl triacetic acid synthase

DHAR: dehydroascorbate reductase

DHFA: dihydrofolic acid

DMAPP: dimethylallyl pyrophosphate

DNA: deoxyribonucleic acid

DPPH: 2,2-diphenyl-1-picrylhydrazyl

DR: Dragendorff’s Reagent

EC: Escherichia coli

EC50: effective concentration of a drug that gives half-maximal response

EO: Essential oils

ET: electron transfer

EUCAST: European Committee on Antimicrobial Susceptibility Testing

FCR: Folin Ciocalteu reagent

FRAP: Ferric ion Reducing Power

FW: Fresh weight

GAE: Gallic Acid Equivalent

GI: Growth Inhibitory Activity

GOPX: guaicol peroxidase

GPX: glutathione peroxidase

GR: glutathione reductase

GSH: glutathione

GSH: glutathione peroxidase

xxiii

GSSG: glutathione disulfide

GST: glutathione-S- transferase

H2O: water

HAT: hydrogen atom transfer

HEEA: hexane: ethyl acetate (solvent)

HEX: Hexane (solvent)

HFA: tetrahydrofolic acid

HH2O hot water

HIV: human immunodeficiency virus

HMG-CoA: 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase

HPLC: High Performance Liquid Chromatography

HSD: honest significant difference

iNOS: inducible nitric oxide synthase

IOU: inhibits oxygen uptake

IPP: isopentenyl diphosphate

LDL: Low-density lipoprotein

MBC: minimum bactericidal concentration

MDHAR: monodehydroascorbate reductase

MDR: multidrug-resistance

MEET: methanol: ethanol (solvent)

MEH2O: methanol 80 (v): water 20 (v) (solvent)

MEP: 2-Methyl-D-erythritol-4-phosphat pathway

MHB: Mueller-Hinton Broth

MIC: Minimum inhibitory concentration

MPO myeloperoxidase

Na2EDTA: sodium ethylene diamine tetra acetate

NADH: nicotinamide adenine dinucleotide

NADPH: Nicotinamide adenine dinucleotide phosphate

NAO: natural antioxidant

NBT: nitroblue tetrazolium

NBT: nitro-blue tetrazolium

NCCLS: National Committee for Clinical Laboratory Standards

ND: not detected

NO: nitric oxide

OD: optical density

OH: hydroxyl

ONOO−: peroxynitrite

ORAC: oxygen radical absorbance capacity

PA: proanthocyanidin

PABA: paraminobenzoic acid

PAL: phenylalanine ammonia lyase

PBS: phosphate buffered saline

PCA: Principal Component Analysis

PG: propyl gallate

PKS: Polyketide synthases

PMS: phenazine methosulfate

RE: rutin equivalent

xxiv

RNS: reactive nitrogen species

ROS: Reactive Oxygen Species

SD: standard deviation

SOA: superoxide anion

SOD: superoxide dismutase

STS: stilbene synthase

TBHQ: tert-butyl hydroquinone

TBHQ: tertiary-butyl-hydroquinone

TEAC: Trolox equivalent antioxidant capacity

TFC: total flavonoid contents

TPC: total phenolic content

TRAP: total radical trapping antioxidant parameter

UV: Ultraviolet

ZI: Zone of Inhibition

xxv

Dévouement

Je tiens à exprimer mes plus profonds regrets au décès du Professeur Khaled Belkacemi. Il

a été une des victimes de l’attentat du 29 janvier 2017 au Centre culturel islamique de

Québec, une semaine après avoir siégé au comité de jury de la soutenance de cette thèse.

Nous nous souviendrons toujours de Dr. Belkacemi comme d’un grand professeur très

passionné, cultivé, dévoué, compétent et aimé de ses collègues et de ses étudiants.

xxvi

Remerciements

Je tiens tout d’abord à remercier mon directeur de recherche professeur Joseph Arul qui

m’a accueilli au sein de son laboratoire et m’a offert un projet si intéressant ainsi qu’un

inestimable appui scientifique et moral. La première leçon que tu m’as donnée est

inoubliable, "Attitude et Aptitude font Altitude". Cela m’a guidé tout au long de mes

études. Merci Joseph pour ta confiance en moi, tes encouragements, et tes conseils à toutes

les rencontres, merci aussi pour ton aide financière. Je voudrais remercie aussi professeur

Paul Angers, mon co-directeur de recherche, de m’avoir accepté de travailler dans son

laboratoire et utiliser ses équipements, merci pour sa gentillesse et ses encouragements. Je

remercie également professeur Khaled Belkacemi, du département des sols et de génie

agroalimentaire, d’avoir fait la pré-lecture de cette thèse. Je tiens à merci aussi professeur Jean-

Christophe Vuillemard, Dr. Wilhelmina Kalt (cherceure, Agricuture et agroalimentaire Canada,

Kentville, Nouvelle-Écosse) d’avoir accepté d’être membre de commité de jury. Vos

commentaires ont vraiment contribué à la qualité de cette thèse. Un remerciement particulier

est également adressé au PCBF-ACDI de m'avoir accordé une bourse d'études de 5 ans.

J'aimerais également remercier Ronan Corcuff, pour son support technique dont j’ai pu

bénéficier tout au long de mes études. Merci pour sa grande disponibilité et

particulièrement pour sa rigueur au labo qui ont contribué aussi à la qualité de tous mes

résultats. Un gros merci aussi à Diane Gagnon, Pascal Dubé, Anne-Françoise Allain,

Catherine Viel, Marie Michèlle Gagnon, Benoit Fernandez, pour leur supporte technique,

leur gentillesse, leurs conseils.

J’ai eu la chance de travailler dans une excellente équipe. Merci pour mes collègues Arturo,

Fayaz, Navina, Likun, Maria, Mialy, Tom Richard, Marine Dubois, et mention spéciale

pour Mayank Pathak, Deepak Kumar Jha et Denis Dallie pour leurs participations dans mes

travaux de thèse et pour leurs connaissances et encouragements. Un merci particulier à

Thang et Vinh pour leur amitié.

Un remerciement spécial à mes parents, ma sœur Huyen, ma femme Khuong et mes enfants

Han et Phong, pour leurs soutiens lors des moments difficiles et leur amour, leur

compréhension de même que pour n'avoir jamais cessé de croire en moi.

1

GENERAL INTRODUCTION

2

Agricultural industry produces billions of tons of residues in non-edible portions derived

from the cultivation and processing of a particular crop. Waste and by-products cause

pollution, management and economic problems worldwide (Santana-Méridas et al., 2012).

These residues of fruits and vegetables may be an abundant source of bioactive

compounds. There is a growing realization and interest in the development of different

strategies to use agricultural and industrial residues as a source of high value-added

products (Santana-Méridas et al., 2012). Vegetables and some fruits yield between 25%

and 75% of non-edible by-products as wastes (e.g., apple, 30 %; pineapple, 45 %; and

citrus fruits, 70 %) (Van Dyk et al., 2013), after processing of fruits and vegetables in the

food processing industry; and they contain a large amount of bioactive compounds such as

polyphenols. The most abundant by-products of minimally processed fresh fruit and

vegetable products are fruit peels and seeds which have been reported to contain high

amounts of phenolic compounds with antioxidant and antimicrobial properties

(Balasundram et al., 2006). The antimicrobial activities of a variety of naturally occurring

phenolic compounds from different plant sources have been studied in detail (Cueva et al.,

2010b; Daglia, 2012b; Quideau et al., 2011). Phenolic compounds play an important role in

the protection of crops against pathogenic agents by damaging microbial cell membrane

and causing lysis of the cells. Phenolic compounds from spices such as gingerone,

zingerone, and capsaicin have been found to inhibit the germination of bacterial spores

(Burt, 2004). Polyphenols from green tea have also been explored for their broad spectrum

activity against pathogens (Taylor et al., 2005). In addition, flavonoids, when used in

conjunction, have been reported to enhance the antibacterial, antiviral, and anticancer

activities of compounds such as naringenin, acycloguanosine, and tamoxifen (Ayala-

Zavala et al., 2010).

Antioxidants play a significant role in protecting the body from damage caused by free

radical- induced oxidative stress (Ozsoy et al., 2008). Natural anti-oxidants, like

polyphenols, found in medicinal and edible plants, are very effective in preventing

oxidative damages (Silva et al., 2005). It is desirable to establish and standardize

antioxidant assay methods that can measure the real antioxidant power directly for all

classes of anti-oxidants, irrespective of their origin, either plant extracts or biological

fluids. Free radicals play a very important role in the oxidative damage of biological

3

systems (McCord, 2000; Scalbert et al., 2005). Plant extracts are complex mixture of

compounds, where phytochemicals may function in synergism or antagonism with each

other, and they may exhibit ‘Broad spectrum antioxidant activity’ against various radicals

of importance in biology.

Plants have been a focus of attention for a longtime as source of antimicrobial compounds

and it has been reported that two-thirds of the world’s plant species have medicinal value

(Craig, 1999; Krishnaiah et al., 2011). In recent years, there has been an increasing interest

in antimicrobial properties of medicinal plants, and enormous attention is being paid in the

discovery of new antimicrobial from natural sources. (Brown et al., 2014; Khanam et al.,

2015), as multi-drug resistances of microbes are becoming a concern that can have

considerable impact on public health with potential treatment failures (Balouiri et al.,

2016). However, the research on antimicrobial compounds from plant sources has yet to see

a systematic approach with respect to screening for possible candidates active against

microbial pathogens, yeasts and fungi (Aqil and Ahmad, 2003). Furthermore, the screening

processes, as of now, do not necessarily take into account of the morphological (Gram-

positive or negative) or growth requirement (aerobe or anaerobe) characteristics of the

bacteria. It is expected that testing of selected plant extracts against wide variety of test

microorganisms will be helpful in developing plant extract formulations exhibiting broad

spectrum antimicrobial activity as well as new antimicrobial substances.

Several bioassays are used to determine the activity of antimicrobial substances, such as the

well-known and commonly used disk diffusion or well diffusion and broth or agar dilution

(Balouiri et al., 2016). These methods can be qualitative and quantitative in nature. A

qualitative test such as the Kirby-Bauer disk diffusion method helps screening candidate

antimicrobial compounds, whereas quantitative methods (such as MIC, growth inhibition)s,

also known as end point methods, help quantify the potency of the compound. Absorbance

readings can help monitor lag phase, growth rate, and maximum population in real time and

ascertain the effectiveness of an antimicrobial agent. Although, only the maximum growth

is typically noted to determine the effect of environmental conditions from these assays,

other parameters such as the lag time and growth rate reflecting bacterial growth through its

life cycle can help to understand bacteria-environment interactions and build bacterial

4

growth prediction models for use in food and fermentation microbiology. Mathematical

modeling can be important to incorporate a few parameters, or very useful to help advance

the understanding a wide variety of intrinsic and extrinsic factors governing microbial

growth.

Many studies have shown that naturally derived compounds and natural products may have

applications in controlling pathogens in foods (Lucera et al., 2012; Negi, 2012). However,

the use of natural antimicrobials is limited due to the fact that they are of low potency, and

it requires high concentrations, which can affect the organoleptic properties of foods

(Davidson et al., 2013; Lucera et al., 2012). However, enhancing the antimicrobial activity

of plant extracts to increase their potency could enable their use as antimicrobial agents in

foods at low levels without affecting the sensory properties of foods. Three approachs for

enhancement used in this study were extraction using selective solvents (Miyasaki et al.,

2013), binary blending of extracts and addition of plant products that may potentiate the

activity of the extracts (Worthington and Melander, 2013).

The objective of this work was to conduct studies to evaluate the potential of extracts

obtained from various fruit and vegetable by-products and to develop antioxidant-

antimicrobials for use as food preservation agents, as alternatives to synthetic agents. The

thesis is organized in six chapters starting with a review on plant by-products, plant

secondary metabolites, and antioxidants and antimicrobials from plant sources (chapter 1).

The identification of promising antioxidant-antimicrobial extracts from fruits and vegetable

by-products, generated during harvesting or processing is described in chapter 2. The

chapter 3 introduces a new measure to characterize the antioxidant property of plant

extracts from DPPH assay, the anti-radical power (ARP). The spectrum of anti-radical

activity of selected fruit and vegetable extracts is reported in chapter 4. The chapter 5

depicts the characterization of antimicrobial activity of selected plant extracts by a new

measure, the antimicrobial activity index (AMI) and the evaluation of the spectrum of their

antimicrobial activity. Finally, chapter 6 describes the work that explored ways to enhance

the antimicrobial activity of selected antioxidant-antimicrobial extracts.

5

Chapter 1 REVUE DE LITERATURE

Cette partie de revue bibliographique porte sur des sous-produits de plantes, les méthodes

d’extraction des composés bioactifs, les métabolites secondaires, les antioxydants et les

antimicrobiens. Ce chapitre se termine par la présentation de la problématique, de

l’hypothèse de recherche, du but et des objectifs spécifiques de cette étude.

6

1.1 Plant by-products: eco-friendly source of novel bioactive compounds

Avant propos

The following is a description of some of the terminologies often used in both business and

scientific literature to describe product sources. They include: by-products, co-products,

secondary products, intermediate products and sub-products We will use the definitions of

the Waste Framework Directive (EuropeanUnion, 2006) as follows:

Waste: A material that the holder discards, and indended or is required to discard.

Production residue: A material that is not deliberately produced in a production process

that may or may not be a waste.

By-product: A production residue that can be used directly without any additional

processing, other than normal industrial practice.

There are two main categories of agricultural residues: crop residues (generated in the

farm) and agro-industrial residues (generated during post-harvest process).

Crop residues (primary biomass residues) are non-edible plant parts that are left in the field

or orchard after the main crop part has been harvested. These residues, which mainly

include straw, stove, stubble, stalks, sticks, leaves, haulms, roots, branches, twigs, brushes,

trimmings and pruning, are produced from sources including seeds, fruits, nuts, vegetables

and energy crops.

Agro-industrial residues (secondary biomass residues) are those materials from the

processing of the crop into a main resource product, including residues from wood and food

processing industries in the form of husks, hulls, peels, dust, straws, bagasse, sawdust,

corncobs, pomace, etc. In addition, the remaining residues after the use of processed

materials may be considered as a tertiary biomass (UNIDO, 2007).

7

1.1.1 Why plant by-products?

The generation of food waste occurs all through the food life cycle; from agriculture,

industrial food processing and manufacturing, retail and up to household consumption. In

developed countries, 42% of food waste is produced by households, while 39% losses

occur in the food processing industry, 14% in food service sector, and the remaining 5% at

the retail and distribution level (Mirabella et al., 2014).


from cultivation and processing of a particular crop. Waste and by-products can cause

pollution, management and economic problems worldwide (Santana-Méridas et al., 2012).

These residues of fruits and vegetables may be an abundant source of bioactive compounds.

This is the reason for the development of different strategies to use agricultural and

industrial residues as a source of high value-added products (Santana-Méridas et al., 2012).

Because the consumers are more and more discouraged by the presence synthetic additives

in foods they consume, food industry is increasingly paying attention to obtain functional

ingredients from natural sources. This is particularly true for phenolic compounds, which in

contrast to certain ingredients such as carotenoids and vitamins, are not chemically

synthesized and need to be extracted from plant materials. The exploitation of by-products

of fruit and vegetable processing as a source of functional compounds and their application

in food is a promising field which requires interdisciplinary research of food technologists,

food chemists, nutritionists and toxicologists (A. Schieber, 2001).

Amongst the fruits, vegetables and herbals, agricultural and industrial residues are also

sources of natural antioxidants (Moure et al., 2001). Special attention is being paid to their

extraction from inexpensive or residual sources from agricultural industries. By-products,

remaining after processing of fruits and vegetables in the food processing industry, still

contain a considerable quantity of phenolic compounds (Cowan, 1999). Some studies have

already been done on by-products, which could be potential sources of antioxidants.

Recycling of the by-products has been supported by the fact that polyphenols have been

located specifically in the peels (Moure et al., 2001). One of the richest sources of

8

polyphenols are grape berry skins, which during wine and juice making remain as residue

are usually converted into compost (Lapornik et al., 2005). The olive mill wastes are also a

major potential source of phenolic compounds. The phenolic content of the olive mill waste

water is reported to fluctuate between 1.0 % and 1.8 % (Visioli and Galli, 2001), depending

on varietal factors and processing effects. Besides olive mill waste water, olive leaves are

another by-product of the olive industry that has been explored as a source of phenolics

(Lee and Lee, 2009). Besides having a strong antioxidant activity, polyphenols often also

exhibit antimicrobial activity (Agourram et al., 2013).

1.1.2 Source, provider and type of by-products

The food processing industry would be the ideal source of such wastes. Residue from juice

production, waste from canning factory, harvest, minor crops and other agricultural and

processed food by-products would be ideal candidates (Peschel et al., 2006).

The number of studies devoted to exploit residual sources for bioactive compounds has

increased considerably, which is driven not only to add value to the by-products, but also to

improve recycling of the wastes for sustainable development of the agro- and food industry.

Such studies have contributed to the knowledge base regarding specific locations of active

compounds and their modifications during processing. Although numerous studies have

been carried out on this subject, only a few by-product derived antioxidants have been

developed successfully from the vast quantities of plant residues produced by the food

processing industry. Various examples of such successes include the work done in Europe,

primarily on grape seed and olive waste extracts (Alonso et al., 2002; Amro et al., 2002).

Potential crop candidates with a high annual production and already confirmed high

antioxidant potential include apple (DuPont et al., 2002), tomato (Lavelli et al., 2000), and

artichoke (Jimenez-Escrig et al., 2003).

The peels of several other fruits also contain higher amounts of phenolic compounds than

the edible fleshy parts. Apple peels contain up 3.3 % in comparison to apple pomace, which

yields 11.8 % of phenolic compounds (Schieber et al., 2003). The peels and seeds of

tomatoes have also been reported to be richer sources of phenolic compounds in

comparison to their fleshy pulp (George et al., 2004). Louli et al (2004) investigated the

9

effect of various process parameters such as solvent type and feed pre-treatment (crushing,

removal of stems) on the efficiency of the extraction of phenolic antioxidants from grape

residue; whereas Negro et al (2003) investigated the content of total polyphenols and

antioxidant activity of grape residue extracts (Louli et al., 2004; Negro et al., 2003).

The citrus industry produces large quantities of peel and seed residues, which may account

for up to 50% of the total fruit weight (Bocco et al., 1998). Citrus industry by-products, if

utilized optimally, could be a major source of terpenes and phenolic compounds,

particularly flavonoids, as the peels are known to contain higher amounts of total phenolics

compared to the edible portions (Balasundram et al., 2006). Likewise, by-products obtained

after the processing of artichoke, cauliflower, carrot, celery and onion have also been

reported (Larrosa et al., 2002).

Although the antioxidant potential of less small volume crops such as strawberry

(Kahkonen et al., 2001), pear (Imeh and Khokhar, 2002), red beet root (Kujala et al., 2001),

or broccoli (Kurilich et al., 2002) is known, little information is available in the literature

with regards to practical means of obtaining bio-active substances from by-products as

well as their utilization in foods. This might be caused by three limiting factors often

overlooked in scientific studies: the effectiveness of recovery and extraction, the

marketability of resulting extracts and the practical suitability for the food, cosmetic or

pharmaceutical products.

1.1.3 Methods of extraction of bioactive compounds from plant

Extraction of bioactive compound from plant is the separation of active portions of plant

tissues using selective solvents. During extraction, solvents diffuse into the solid plant

material and solubilize compounds with similar polarity. The products so obtained from

plants are relatively complex mixtures of metabolites such as alkaloids, glycosides,

terpenoids, flavonoids and lignans (Joana Gil-Chávez et al., 2013).

The commonly used methods of extraction of plant are the conventional liquid–liquid or

solid–liquid extraction and the advanced include pressurized-liquid extraction, subcritical

and supercritical extractions, and microwave- and ultrasound-assisted extractions. In

10

addition, these extraction techniques have been improved with preparative treatment steps

(enzyme-and instant controlled pressure drop-assisted extractions), which help to release

the compounds from the matrix. These technologies could provide in the next few years an

innovative approach to increase the production of specific compounds, particularly

flavonoids, for use as nutraceuticals as ingredients in the design of functional foods (Joana

Gil-Chávez et al., 2013).

Table 1.1 Solvents used for active component extraction

Water Ethanol Methanol Ether Acetone Chloroform

Anthocyanins Tannins Anthocyanins Alkaloids Phenol Terpenoids

Starches Polyphenols Terpenoids Terpenoids Flavonols Flavonoids

Tannins Polyacetylenes Saponins Coumarins

Saponins Flavonol Tannins Fatty acids

Terpenoids Terpenoids Xanthoxyllines

Polypeptides Sterols Totarol

Lectins Alkaloids Quassinoids

Lactones

Flavones

Phenones

Polyphenols

(Cowan, 1999)

Table 1.1 summarizes the use of different solvents for extraction of various bioactive

compounds. Solvent is selected for its capacity for the species being separated into it, i.e.,

the solubility of the targeted compounds in that solvent. The solvent should also be

selective, extracting primarily one or more of the related class of compounds from the solid

matrix. Thus, polar solvents are more appropriate for polar components, and non-polar

solvents for non-polar components. Some times, a solvent mixture may be used to obtain

properties that cannot be achieved with a single solvent. For laboratory preparations, hot

water, hexane, ethyl acetate, chloroform, dichloromethane, ether, acetone and alcohols

(ethanol and methanol) are frequently used solvent mixtures offering a range of polarity.

Hot water is a universal solvent, and it is used frequently to extract phyto-compounds.

Acetone dissolves many hydrophilic and lipophilic components, and is volatile and

miscible with water, and has a low toxicity for use in the bioassay of extracts. Acetone is

used for extraction phenolic compounds from plant extracts. Alcohols are effective in the

11

degradation of cell walls, and seed coats that have non-polar character, and facilitate the

release of substances from the cells. The frequently used alcohols are ethanol and methanol.

Ethanol can permeate through the cellular membrane and facilitates the extraction of intra-

cellular components from plant materials. Higher recoveries of bioactive flavonoids can be

achieved with aqueous ethanol (70%, v/v) due to the higher polarity of the mixture than

pure ethanol (Khoddami et al., 2013). While methanol is more polar than ethanol, it is

often a suitable solvent for extraction of components of higher polarity such as

polyphenols. Since most of the antimicrobial compounds isolated from plant sources

possess relatively more non-polar character (aromatic or unsaturated organic compounds),

they are initially extracted using either ethanol or methanol and purified subsequently.

Although tannins can be modestly extracted by aqueous medium, they are efficiently

extracted by less polar solvents. Ether is commonly used selectively for the extraction of

coumarins and non-polar lipids. Terpenoids are generally extracted with more non-polar

solvents such as dichloromethane and ethyl acetate, when the former is more selective

towards terpenoids. Terpenoid lactones have been obtained by successive extractions of

dried barks with hexane, followed by solvent mixture (chloroform-methanol) with higher

bioactivity concentrating in chloroform-methanol fraction (Cowan, 1999).

1.1.4 Trend and challenges in using plant by-products

Recently, polyphenolic content was also examined in some plant by-products, which are

available in large quantities and at low cost, but are currently used only as animal feed or

fertilizer. Their use as food additives could help industries to solve the environmental

problems related to the disposal of these materials, and provide new sources of natural

antioxidants.

The future of food processing in a sustainable way should warrant not only minimize

wastes from processing, but also would require value-addition to the waste streams. An

integrated system should also incorporate nutrional, health-functional and food safety

characteristics of the foods and by-products. The food processes need optimization to

minimize the amounts of waste generation, and focus on complete utilization of by-

products resulting from large-scale processing at affordable costs. There is a need for

specific analytical methods for the characterization and quantification of organic

12

micronutrients and other functional compounds. The bioactivity, bioavailability and of

phytochemicals need to be carefully assessed by in vitro and in vivo studies. In addition,

there is also a need to eliminate natural and anthropogenic toxins such as solanin, patulin,

ochratoxin, dioxins and polycyclic aromatic hydrocarbons need by efficient quality control

systems.

The ‘activity’ of many phytochemicals has only been tested in in vitro models, and this may

bear no relationship to the situation in vivo. Undoubtedly, functional foods represent an

important, innovative and rapidly growing part of the overall food market. However, their

design, i.e. their complex matrix and their composition of bioactive principles, requires

careful assessment of potential risks which might arise from isolated compounds recovered

from by-products. Furthermore, investigations on stability and interactions of

phytochemicals with other food ingredients during processing and storage need to be

initiated. Since functional foods are on the boundary between foods and drugs, their

regulation still proves to be difficult. In any case, consumer protection must take priority

over economic interests, and health claims need to be substantiated by standardized,

scientifically sound and reliable studies (A. Schieber, 2001).

13

1.2 Plant secondary metabolites

1.2.1 Primary and secondary metabolites

Given that plants are sessile and cannot flee in inimical situations, they must have ways to

adapt themselves to both favourable and unfavourable changes in their environment. In

order to defend themselves, plants have evolved many strategies for survival, including the

capability to produce over 200,000 highly varied and diverse assortment of specialized low

molecular weight organic compounds that are collectively termed as secondary metabolites,

secondary products, or natural products. The synthesis and storage of secondary

metabolites can be regarded as a strategy of plants for defense and communication (Dixon,

2001; Wink, 2013). Even though secondary metabolites do not play a direct role in growth

and development of plants, they play a role in plant protection and signalling, and they

exhibit a wide range of medicinal, pharmacological properties in humans that make them

valuable. Numerous epidemiological studies indicate that an increase in the consumption of

fruits and vegetables is associated with a decrease in the incidence of cancer, cardiovascular

disease, osteoporosis and diabetes (Kris-Etherton et al., 2002; Temple and Kaiser Gladwin,

2003). Interestingly, some of the secondary metabolites such as flavonoids, polyacetylenes,

stilbenes, glucosinolates and others exhibit both antimicrobial and health-promoting

properties.

Phenolic and polyphenolic compounds, characterized by an aromatic or phenolic ring

structure, include flavonoids, phenolic acids, and lignans. Phenolic compounds are feeding

deterrents for many insects and other animals; high concentrations of phenolic compounds

are often associated with increased resistance to fungal plant pathogens (Nicholson and

Hammerschmidt, 1992). Some phenolic compounds determine the color and smell of

plants, attracting pollinators. Phenolic compounds also play a role in cold acclimation and

protection against UV radiation. In plant cells, most phenolic compounds are coupled to

sugars, thereby reducing the concentration of free phenolic compounds, and likely reducing

their endogenous toxicity.

Plant secondary metabolites are not mere randomly produced compounds, but ones that

have been shaped and optimized during evolution. It is not surprising that many of them are

14

used as pharmaceuticals, spices, fragrances, pesticides, poisons, hallucinogens, stimulants,

or colors. Many of the compounds also are used as medicinal, pharmaceutical and industrial

chemical precursors, pesticides, flavorings, perfumery ingredients, plant hormones, and

drugs of abuse. Some still play exotic roles in some cultures as arrow poisons, pesticides,

and hallucinogens (Seigler, 1998).

1.2.2 Plant secondary metabolism

Plants synthesize a vast range of organic compounds that are traditionally classified as

primary and secondary metabolites, although the precise boundaries between the two

groups can, in some instances, be somewhat blurred (Figure 1.1). Primary metabolites are

compounds that have essential roles associated with photosynthesis, respiration, growth and

development. They include phytosterols, acyl lipids, nucleotides, amino acids and organic

acids. Other phytochemicals, the secondary metabolites, many of which accumulate in

surprisingly high concentrations in some species, and they can be species or strain specific,

with genetic and environmental factors playing influence in their production (Sato, 2003;

Seigler, 1998).

Phytochemical investigations have revealed a high structural diversity of plant secondary

metabolites, comprising more than alkaloids, non-protein amino acids, cyanogenic

glucosides and glucosinolates, terpenoids, polyphenols, polyacetylenes and fatty acids,

polyketides, and carbohydrates (Harborne, 1993; Kretzschmann, 1981). The synthesis and

storage of secondary metabolites can be regarded as a strategy of plants for defense and

communication. Plants being devoid of motility and immune system, have elaborated

alternative defense strategies, involving the huge variety of secondary metabolites as tools

to overcome stress constraints, adapt to the changing environment and survive. The large

complexity of chemical types and and their interactions underly various protective

functions they perform: structure stabilizing compounds, produced by polymerization and

condensation of phenols and quinones or by electrostatic interactions of polyamines with

negatively charged loci in cell components; photo-protective compounds, related to

absorbance of visible light and UV radiation characterized by the presence of conjugated

15

(Seigler, 1998)

Figure 1.1 Overview of plant primary and secondary metabolism

double bonds; antioxidant and antiradical compounds, governed by the availability of –OH,

–NH2 , and –SH groupings, as well as aromatic nuclei and unsaturated aliphatic chains; and

signal transducing molecules (Edreva et al., 2008).

High concentrations of secondary metabolites might result in more resistant plant but such

continuous production is thought to be costly in terms of resource and energy usage causing

a burden on the plant that reduces its growth and reproduction (Siemens et al., 2002).

Therefore, plant defense metabolites can be divided into constitutive substances, also called

prohibitins or phytoanticipins and induced metabolites formed in response to an infection

16

involving de novo enzyme synthesis, known as phytoalexins (Grayer, 1994).

Phytoanticipins involve high energy and carbon consumption and exhibit fitness cost under

natural conditions (Mauricio, 1998), but recognized as the first line of chemical defense

that potential pathogens have to overcome. In contrast, phytoalexin production may take

two or three days, since the enzyme system needs to be synthesized first (Grayer and

Harborne, 1994). There are various other modes of defense include the construction of

polymeric barriers to pathogen penetration and the synthesis of enzymes that degrade

pathogen cell wall (Hammond-Kosack). In addition, plants employ specific recognition and

signaling systems enabling the rapid detection of pathogen invasion and initiation of

vigorous defensive responses (Schaller and Ryan, 1996). Some plants are primed upon

infection and develop defense responses, secondary compounds and proteins, quickly to

ward off subsequent microbial attacks (Putnam, 1983).

1.2.3 Biosynthesis of secondary metabolites

Secondary metabolites are synthesized from intermediate metabolites of primary

metabolism and mainly derived from major metabolic pathways of glycolysis, Krebs cycle

and Calvin cycle result in production of major primary metabolites such as acetyl-CoA,

shikimic acid and mevalonic acid, from which secondary metabolites are ultimately

synthesized. The majority of these compounds belong to one of a number of families, each

of which have particular structural characteristics arising from the way in which they are

built-up in nature, i.e., from their biosynthesis. These secondary metabolites include the

following (Hartmann, 2007; Mazid et al., 2011): a) Terpenoids derived from acetate-

mevalonate; b) Aromatic amino acids, aromatic glucosinolates and polyphenols derived

from shikimic acid; c) Alkaloids, aliphatic glucosinolates and peptide antibiotics derived

from amino acids; and d) Polyketides and fatty acids derived from acetate and malonate.

17

There are three secondary principal pathways (shown in Figure 1.2) that lead to the diverse

array of chemical compounds found in plants (Hartmann, 2007; Mazid et al., 2011). These

include:

- Shikimic acid pathway that leads to the formation of lignin, courmarins, tannins,

phenols, and various aromatics.

- Acetate-malonate pathway that forms the precursors of fatty acids, phospholipids,

glycerides, waxes, glycolipids, and polyketides.

- Acetate-mevalonate pathway that results in various terpenoids (gibberellins,

carotenoids, abscisic acid) and sterols.

Figure 1.2 Biosynthetic relationships of major groups of secondary metabolites

18

1.2.3.1 Terpenes

Figure 1.3 Biosynthetic of terpenes: Mevalonic acid pathway

Terpenes or terpenoids constitute the large class of secondary metabolites and are united by

their common biosynthetic origin from acetyl-coA or glycolytic intermediates (Taiz and

Zeiger, 2002). Terpenes are grouped by number of the five-carbon isoprene structural units

that they are composed of. Ten-carbon terpenes, which contain two C5 units, are called

monoterpenes; 15-carbon terpenes (three C5 units) are sesquiterpenes; and 20-carbon

terpenes (four C5 units) are diterpenes. Larger terpenes include triterpenes (30 carbons),

tetraterpenes (40 carbons), and polyterpenoids ([C5]n carbons, where n> 8). In the well-

studied HMG-CoA reductase pathway, isopentenyl diphosphate (IPP) and dimethylallyl

pyrophosphate (DMAPP), are formed by joining three units of acetyl-CoA via a mevalonic

acid intermediate (Chen et al., 2011). An alternative, totally unrelated biosynthesis pathway

of IPP is known in some bacterial groups and the plastids of plants, the MEP (2-Methyl-D-

erythritol-4-phosphate) pathway, which is initiated from C5 sugars. In both pathways, IPP is

isomerized to DMAPP by the enzyme isopentenyl pyrophosphate isomerase. Certain

19

terpenes have well characterized function in plant metabolism, and therefore, are

considered primary metabolites. For example, gibberellins and cytokinins are phyto-

hormones, and carotenoids play a role in the protection of the photosynthetic apparatus,

besides their ecological role as pigments. Other terpenes that are produced by plants as

secondary metabolites are presumed to be involved in defense as toxins and feeding

deterrents to a large number of plant feeding insects and mammals such as anti-herbivore

compounds limonoids and insecticidal monoterpenes pyrethroids.

1.2.3.2 Phenolic compounds

(Flamini et al., 2013)

Figure 1.4 Biosynthetic phenolic compounds: Shikimate + Malonate pathway

20

Plant phenolics are a chemically heterogeneous group of nearly 10,000 individual

compounds. Phenolic compounds are a diverse group of phytochemicals classified into

phenolic acids, flavonoids, proanthocyanidins (non-hydrolysable tannins) and hydrolysable

tannins, chalcones, coumarins, lignans, lignins, phenols, phenylpropanoids, quinines,

stilbenoids, and xanthones (Dai and Mumper, 2010). Many serve as defense compounds

against herbivores and pathogens. Others function in mechanical support, in attracting

pollinators and fruit dispersers, in absorbing harmful ultraviolet radiation, or in reducing the

growth of nearby competing plants (Imperato, 2006). Two basic biosynthetic pathways are

involved: the shikimic acid pathway and the malonic acid pathway. The shikimic acid

pathway participates in the biosynthesis of most plant phenolics whereas malonic acid

pathway is of less significance in higher plants. The shikimic acid pathway converts simple

carbohydrate precursors derived from glycolysis and the pentose phosphate pathway to the

aromatic amino acids (Herrmann and Weaver, 1999). The most abundant classes of

secondary phenolic compounds in plants are derived from phenylalanine via the elimination

of an ammonia molecule to form cinnamic acid. This reaction is catalyzed by phenylalanine

ammonia lyase (PAL), which is at a branch point between primary and secondary

metabolism, so it is the regulatory step in the formation of many phenolic compounds. The

activity of PAL is increased by environmental factors, such as low nutrient levels, light and

fungal infection (K Hahlbrock and Scheel, 1989).

Phenolic acids come from phenyl propanoid pathway are further sub-classed as hydroxyl

cinnamic acids, hydroxyl benzoic acids, hydroxyl phenylacetic acids, and hydroxyl

phenylpropanoic acids, and they are all synthesized from cinnamate precursor. Two classes

of phenolic acids can be distinguished as derivatives of cinnamic acid and derivatives of

benzoic acid (Manach et al., 2004). The hydroxy cinnamates include 4-coumaric acid,

caffeic acid, ferulic acid, sinapic acid, 3,4,5- Trihydroxy cinnamic acid, 2-hydroxy

coumaric acid and 2,5-Dihdroxy cinnamic acid. Ferulic acid is the most abundant phenolic

acid found in cereal grains. The corresponding hydroxyl benzoates include 4-hydroxy

benzoic acid, procatechuic acid, vanillic acid, syringic acid, gallic acid, salicylic acid and

gentisic acid. Tea is an important source of gallic acid. Ellagic acid is built from oxidative

coupling of two molecules of gallic acid. Chlorogenic acids are esters of hydroxyl cinnamic

21

acids, and coffee bean is rich containing about 30 chlorogenic acids (Gordon and Wareham,

2010; Henning et al., 2004).

Coumarins are classed by their benzopyran-2-on nucleus, and they originate from

cinnamate intermediate. Some well-known coumarins from vegetables include coumarin

(cinnamic acid), umbelliferone (4-coumaric acid) and scopoletin (ferulic acid) (Yang et al.,

2015). They exhibit a wide range of anti-microbial activity against both fungi and bacteria

(Brooker et al., 2008). It is believed that these cyclic compounds play pesticidal defense

role for plants (Seigler, 1998). Furano-coumarins are coumarins with furan ring either

linear or angular configuration. The most well studied furanocoumarins are psoralens for

their phyto-toxicity. They are abundant in members of the family Apiaceae including

celery, parsnip and parsley. These compounds turn toxic when exposed to UV-A light,

some become activated to a high energy electronic state, which can insert themselves into

the double helix of DNA and bind to the pyramidine bases, and thus, blocking transcription

and repair, and eventually leading to cell death (Rice, 1984).

Lignans are essentially phenyl propanoid dimers linked by C-C between the units. The

monomeric precursors (monolignols, e.g., coniferyl or 4-coumaryl alcohol) are the same for

lignin and lignin formation. Flaxseed is the richest source of lignans (resinols), but they are

also found in Brassica vegetables. Lignins are defense polymeric barriers to pathogen

penetration by a highly branched polymer of phenyl propanoid compounds. Lignification

blocks the growth of pathogens and are a frequent response to infection or wounding

(Vanholme et al., 2010).

Tannins are also plant phenolic polymers with molecular masses between 600 and 3000,

and are divided into two major groups: condensed tannins (or proanthocyanidins, PAs),

which are of flavonoid origin; and berries and plums are good sources of PAs. The

hydrolysable tannins, which are esters of gallic acid with a sugar moiety, mainly -D-

glucose, are grouped into gallotannins and ellagitannins. Grape seed and persimmon seeds

are rich sources of hydrolysable tannins. Berries such as strawberry, raspberry and

blackberry, and pomegranate are good sources of ellagitannins, and mango for gallotannins.

22

They are general toxins that significantly reduce the growth and survival of many

herbivores and also act as feeding repellents to a great diversity of animals.

Flavonoids. They make up the largest classes of phenolic compounds with flavane ring

system, having a basic carbon skeleton of 15 carbons arranged in two aromatic rings

connected by a three-carbon bridge. They are synthesized on a separate pathway from

cinnamoyl CoA esters with addition of three units of malonyl CoA (Figure 1.4). Flavonoids

are classified into different groups: the flavonones (e.g., hesperetin and naringenin in citrus

fruits); flavones (e.g., apigenin in celery and parsley; luteolin in pepper and red grape,

mainly as glycosides); flavonols (quercetin, kaempferol, myricetin and iso-hamnetin in

berries, pears, onion, kale and leek); favon-3-ols or catechins (tea, berries, peaches, red

grape and bannanas); procyanidins or flavan-3,4-diols; anthocyanidins such as cyanidin,

delphinidin, malvidin pelargonidin and others in berries, red grape, cherry and other

pigmented produce; and proanthocyanidins. Isoflavonoids are derived from a flavonone

intermediate, naringenin, ubiquitously present in plants and play a critical role in plant

developmental and defense response. Isoflavones such as genistein, daidzein and glycitein

occur mainly in leguminous plants such as soy (Manach et al., 2004)

Different types of flavonoids perform very different functions in plants, including

pigmentation and defense. Flavones and flavonols function to protect cells from UV-B

radiation because they accumulate in epidermal layers of leaves and stems and absorb light

strongly in the UV-B region (Lake et al., 2009). Moreover, it seems that synthesis of these

flavonoids is an effective strategy against reactive oxygen species (Posmyk et al., 2009).

Stilbenes (resveratrol and viniferins) are present in grape as constitutive compounds of the

woody organs (roots, canes, stems) and as induced substances (in leaves and fruit) acting as

phytoalexins in the mechanisms of grape resistance against certain pathogens (Bavaresco et

al., 1999).

23

1.2.3.3 Nitrogen-containing compounds

Nitrogenous secondary metabolites include well-known anti-herbivore defenses such as

alkaloids and cyanogenic glycosides, which are of considerable interest because of their

potential toxicity to humans and their medicinal properties. Most are biosynthesized from

common amino acids (Taiz and Zeiger, 2002). Alkaloids are toxic to some degree and

appear to serve primarily in defense against microbial infection and herbivore attack. They

are usually synthesized from one of the few common amino acids, in particular, aspartic

acid, lysine, tyrosine and tryptophan. Within plants, pyrrolizidine alkaloids occur naturally

as nontoxic N-oxides. In herbivore digestive tracts, however, they are quickly reduced to

uncharged, hydrophobic tertiary alkaloids, which can easily migrate through cell

membranes and they can be toxic.

Cyanogenic glycosides and glucosinolates also constitute a group of N-containing

protective compounds that are not inherently toxic but are readily broken down to give off

volatile poisons such as HCN and H2S when the plant is crushed; and their presence deters

feeding by insects and other herbivores such as snails and slugs. Found principally in the

Brassicaceae and related plant families, glucosinolates give off the compounds responsible

for the smell and taste of vegetables such as cabbage, broccoli, and radishes. The release of

these mustard-smelling volatiles from glucosinolates is catalyzed by a hydrolytic enzyme,

thioglucosidase or myrosinase, which cleaves glucose from its bond with the sulfur atom.

The resulting aglycone, the nonsugar portion of the molecule, rearranges with loss of the

sulfate to give pungent and chemically reactive products, including isothiocyanates,

thiocyanates and nitriles, depending on the conditions of hydrolysis. These products

function in defense as herbivore toxins and feeding repellents.

1.2.3.4 Polyketides

Polyketides are one of the most pharmacologically relevant families of natural products

found in microorganisms and plants. Extremely diverse in terms of structure and function,

polyketides of clinical importance include antibiotics, antitumor agents and cholesterol-

lowering agents; and they are biosynthesized from acyl CoA precursors. Polyketide

synthases (PKS) are responsible for the biosynthesis of polyketides and use a common

24

chemical strategy: chain elongation by a decarboxylase condensation reaction, followed by

cyclization to generate the final polyketide product. They are classified according to their

architectural configurations as type I, II and III (Staunton and Weissman, 2001). Several

type III PKSs, also known as chalcone synthase-PKSs, have been found in plants. They are

home-dimeric or condensing enzymes that essentially act iteratively. They utilize a variety

of different starter substrates substrates ranging from aliphatic-CoA to aromatic-CoA

substrates, from small (acetyl-CoA) to bulky (p-coumaroyl-CoA) substrates or from polar

(malonyl-CoA) to nonpolar (isovaleroyl-CoA) substrates, giving the plants an extraordinary

synthetic and functional diversity (Flores-Sanchez and Verpoorte, 2009).

25

1.3 Antioxidants

1.3.1 Concepts

Oxidation: In the past, oxidation was considered simply as a reaction, where an oxygen

molecule is added to a molecule or a hydrogen atom is taken away from a molecule. In the

larger sense, oxidation is a reaction that turns the oxidation number of a molecule more

positive or less negative. Therefore, the molecule that loses the electrons (received by an

oxidant) is the reductant. Redox reaction implies a transfer an electron of a reductant to an

oxidant. Oxidation is the loss of electrons or an increase in the oxidation state of a

molecule, atom, or ion.

Reactive oxygen species (ROS): The O2 molecule is a di-radical, as it has two impaired

electrons that have the same spin quantum number. This spin restriction makes O2 to accept

its electrons one at a time, leading to the generation of the so-called ROS, which can

damage the cells. ROS are also produced continuously as by-products of various metabolic

pathways.

Antioxidant is any substance that, when present at low concentrations compared to those

of an oxidisable substrate, significantly delays or prevents its oxidation (Halliwell et al.,

1995). Antioxidant may act by decreasing oxygen concentration, intercepting singlet

oxygen, preventing first-chain initiation by scavenging initial radicals such as hydroxyl

radicals, binding metal ion catalysts, decomposing primary products to non-radical

compounds, and chain breaking to prevent continued hydrogen abstraction from the

substrates. The extent to which oxidation of lipids occurs also depends on the chemical

structure of the fatty acids involved as well as other factors related to the storage of foods

and reaction conditions (Shahidi, 1997).

Food manufacturers have used food grade antioxidants to prevent quality deterioration of

products and to maintain their nutritional value (Shahidi and Zhong, 2007). Antioxidants

have also been of interest to biochemists and health professionals, because they may help

the body protect itself against damage caused by ROS and consequently, against the

incidence of degenerative diseases (Shahidi and Zhong, 2007).

26

Pro-oxidants are chemicals that induce oxidative stress, either by generating reactive

oxygen species or by inhibiting antioxidant systems (Puglia and Powell, 1984). For

example, as well as many of so-called antioxidants, also flavonoids can act, under certain

circumstances, as pro-oxidants and, hence, promote the oxidation of other compounds

(Halliwell, 2009). Human diets contain not only antioxidants but also many pro-oxidants:

hydrogen peroxide; lipid peroxides; redox–cycling toxins; heme and heme compounds; iron

and copper ions; ascorbate (that generates hydroxyl radicals by Fenton reaction); aldehyde

from lipid peroxides decomposition; and nitrite (nitrite at low pH in the stomach produces

reactive oxides of nitrogen).

1.3.2 Why to examine antioxidants from plants?

Fresh fruits and vegetables are normally consumed raw or minimally processed and are

considered to be an important part of a healthy diet as they provide needed nutrients, fiber

and antioxidants. Many plants and plant by-products have been studied as sources of

potentially safe natural antioxidants for the food industry; various bioactive compounds

have been isolated, many of them being polyphenols (Kennedy and Wightman, 2011;

Nicholson and Hammerschmidt, 1992)

Plants expose themselves to ROS, and so, they are rich in antioxidant defenses and repair

systems against oxidative damage to help them deal with O2 toxicity. Plants supply a range

of antioxidants to humans some known to be important in vivo such as ascorbate, α-

tocopherol, tocotrienols, flavonoids and carotenoids. Diets rich in plants are associated with

lower risk of developing many age-related diseases (e.g., some cancers, diabetes,

atherosclerosis, and dementia…) and most people in “advanced” countries would gain

better health if they consume diets containing more fruits and vegetables (Pandey and

Rizvi, 2009).

The growing interest in the substitution of synthetic food antioxidants by natural ones has

fostered research on vegetable sources and the screening of raw materials for identifying

new antioxidants. Oxidation reaction is not an exclusive concern for food industry, and

antioxidants are widely used to prevent deterioration of oxidation susceptible products,

such as cosmetics, pharmaceuticals. In addition, other biological properties such as anti-

https://en.wikipedia.org/wiki/Oxidative_stress

https://en.wikipedia.org/wiki/Reactive_oxygen_species

https://en.wikipedia.org/wiki/Reactive_oxygen_species

https://en.wikipedia.org/wiki/Antioxidant

27

carcinogenicity, anti-mutagenicity, anti-allergenicity and anti-aging activity have been

reported for natural and synthetic antioxidants.

Recently, special attention is being paid on their extraction from a variety of inexpensive or

residual sources from agricultural industries (Moure et al., 2001), since many studies have

reported that plants contain a wide variety of compounds with beneficial health effects.

Particularly, many herbs and spices used to aromatize foods have been screened as sources

of natural antioxidants and antimicrobial compounds. (Ramirez et al., 2007).

The importance of the antioxidants contained in foods is well appreciated for both

preserving the foods themselves and supplying essential antioxidants in vivo. With

increasing experimental, clinical and epidemiological data that show the beneficial effects

of antioxidants against oxidative stress-induced degenerative and age-related diseases,

cancer and aging, the importance and role of antioxidants have received renewed attention

(Yanishlieva et al., 2001).

Humans are protected from oxidative stress by various anti-oxidants that have different

functions; some are enzymes and proteins and others are small molecule antioxidants.

Foods are important as an essential source of such antioxidants, components and trace

elements. In addition, numerous synthetic antioxidants have been developed and some of

them have been used in practice, for example, as food additives, supplements and drugs.

The phenolic compounds such as vitamin E and flavonoids are typical antioxidants.

As endogenous antioxidants synthesised by aerobes (e.g. SOD, catalase, GSH) do not

completely prevent damage by reactive species in vivo (Halliwell, 1999); efficient repair

systems are needed to reduce the damage and humans must also obtain antioxidants from

the diet. There is currently a considerable amount of interest in dietary antioxidants as

bioactive components of food. The physiological role of some of these, such as vitamin E

and vitamin C, is well established. The interest in flavonoids has increased in recent years,

because of their ubiquitous presence as antioxidants in food (Yanishlieva et al., 2001).

28

1.3.3 Production of free radical in biology

The generation of ROS begins with rapid uptake of oxygen, activation of NADPH oxidase,

and the production of the superoxide anion radical (O2˙−) (Nimse and Pal, 2015)

(1)

The O2˙− is then rapidly converted to H2O2 by SOD:

(2)

These ROS can act by either of the two oxygen dependent mechanisms resulting in the

destruction of microorganism or other foreign matter. The reactive species can also be

generated by the myeloperoxidase–halide–H2O2 system. The enzyme myeloperoxidase

(MPO) is present in the neutrophil cytoplasmic granules. In the presence of chloride ion,

which is ubiquitous, H2O2 is converted to hypochlorous acid (HOCl), a potent oxidant and

antimicrobial agent (Babior, 1999).

(3)

ROS are also generated from O2˙−and H2O2 via ‘respiratory burst’ by Fenton (4) and/or

Haber–Weiss (5) reactions

H2O2 + Fe2+ → ˙OH + OH− + Fe3+ (4)

O2˙− + H2O2 → ˙OH + OH− + O2 (5)

The enzyme nitric oxide synthase produces reactive nitrogen species (RNS), such as nitric

oxide (NO˙) from arginine

L-Arg + O2 + NADPH → NO˙ + citrulline (6)

An inducible nitric oxide synthase (iNOS) is capable of continuously producing large

amount of NO˙, which act as a O2˙−quencher. The NO˙ and O2˙

− react together to produce

peroxynitrite (ONOO−), a very strong oxidant, hence, each can modulate the effects of the

other. Although neither NO˙ nor O2˙− is a strong oxidant, peroxynitrite is a potent and

versatile oxidant that can attack a wide range of biological targets (Zhu et al., 1992).

NO˙ + O2˙− → ONOO− (7)

Peroxynitrite reacts with the aromatic amino acid residues in the enzyme resulting in the

nitration of the aromatic amino acids. Such a change in the amino acid residue can result in

29

enzyme inactivation. However, nitric oxide is an important cytotoxic effector molecule in

the defense against tumor cells, various protozoa, fungi, helminths, and mycobacteria

(Nathan and Hibbs, 1991). The other sources of free radical reactions are cyclooxygenation,

lipooxygenation, lipid peroxidation, metabolism of xenobiotics, and ultraviolet radiations

(Shahidi and Zhong, 2010).

1.3.4 Natural sources of antioxidant compounds

Numerous plants have been studied as sources of potentially safe natural antioxidants for

the food industry; various compounds have been isolated, many of them being polyphenols

(Ferrazzano et al., 2011; Quideau et al., 2011). Many of the antioxidants occur as dietary

constituents, these include tocopherols, vitamin C, carotenoids, and phenolic compounds

(Table 1.2).

Table 1.2 Natural plant sources of some antioxidants

Compounds Example of source

Vitamin E (Tocolpherols

and Tocotrienols)

Oilseeds, palm oil, nuts, eggs, dairy products, whole grains,

vegetables, cereals, margarine, etc.

Vitamin C (Ascorbic acid) Fruits and vegetable, berries, citrus fruits, sprouts, green peppers,

potatoes, etc.

Carotenoids Dark leafy vegetables, carrots, sweet potatoes, yams, tomatoes,

cantaloupes, apricots, citrus fruits, kale, turnip greens, palm oil, etc.

Flavonoids/Isoflavones Fruits and vegetables, oilseeds, berries, eggplant, peppers, citrus

fruits, cruciferous vegetables, yams, tomatoes, onions, etc.

Phenolic acids/Derivatives

of Catechins

Oilseeds and certain oils, cereals, grains, etc.

Green tea, berries, certain oilseeds, etc.

(Shahidi, 1997)

30

Table 1.3 Natural sources of polyphenol

Poly phenols Source (serving size) mg/kg fresh wt (or mg/L) mg/serving

Hydroxybenzoic acids Blackberry (100 g) 80–270 8–27 Protocatechuic acid Raspberry (100 g) 60–100 6–10 Gallic acid Black currant (100 g) 40–130 4–13 p-Hydroxybenzoic acid Strawberry (200 g) 20–90 4–18

Hydroxycinnamic acids Blueberry (100 g) 2000–2200 200–220 Caffeic acid Kiwi (100 g) 600–1000 60–100 Chlorogenic acid Cherry (200 g) 180–1150 36–230 Coumaric acid Plum (200 g) 140–1150 28–230 Ferulic acid Aubergine (200 g) 600–660 120–132 Sinapic acid Apple (200 g) 50–600 10–120

Pear (200 g) 15–600 3–120 Chicory (200 g) 200–500 40–100 Artichoke (100 g) 450 45

Potato (200 g) 100–190 20–38 Corn flour (75 g) 310 23

Flour: wheat, rice, oat (75 g) 70–90 5–7 Cider (200 mL) 10–500 2–100 Coffee (200 mL) 350–1750 70–350

Anthocyanins Aubergine (200 g) 7500 1500 Cyanidin Blackberry (100 g) 1000–4000 100–400 Pelargonidin Black currant (100 g) 1300–4000 130–400 Peonidin Blueberry (100 g) 250–5000 25–500 Delphinidin Black grape (200 g) 300–7500 60–1500 Malvidin Cherry (200 g) 350–4500 70–900

Rhubarb (100 g) 2000 200 Strawberry (200 g) 150–750 30–150

Red wine (100 mL) 200–350 20–35 Plum (200 g) 20–250 4–50 Red cabbage (200 g) 250 50

Flavonols Yellow onion (100 g) 350–1200 35–120 Quercetin Curly kale (200 g) 300–600 60–120 Kaempferol Leek (200 g) 30–225 6–45 Myricetin Cherry tomato (200 g) 15–200 3–40

Broccoli (200 g) 40–100 8–20 Blueberry (100 g) 30–160 3–16 Black currant (100 g) 30–70 3–7 Apricot (200 g) 25–50 5–10 Apple (200 g) 20–40 4–8 Beans, green or white (200

g) 10–50 2–10

Black grape (200 g) 15–40 3–8 Tomato (200 g) 2–15 0.4–3.0 Black tea infusion (200 mL) 30–45 6–9 Green tea infusion (200 mL) 20–35 4–7 Red wine (100 mL) 2–30 0.2–3

Flavones Parsley (5 g) 240–1850 1.2–9.2 Apigenin Celery (200 g) 20–140 4–28 Luteolin Capsicum pepper (100 g) 5–10 0.5–1

Flavanones Orange juice (200 mL) 215–685 40–140 Hesperetin Grapefruit juice (200 mL) 100–650 20–130 Naringenin Lemon juice (200 mL) 50–300 10–60 Eriodictyol

Isoflavones Soy flour (75 g) 800–1800 60–135 Daidzein Soybeans, boiled (200 g) 200–900 40–180 Genistein Miso (100 g) 250–900 25–90 Glycitein Tofu (100 g) 80–700 8–70

Tempeh (100 g) 430–530 43–53 Soy milk (200 mL) 30–175 6–35

Monomeric flavanols Chocolate (50 g) 460–610 23–30 Catechin Beans (200 g) 350–550 70–110 Epicatechin Apricot (200 g) 100–250 20–50

Cherry (200 g) 50–220 10–44 Grape (200 g) 30–175 6–35 Peach (200 g) 50–140 10–28 Blackberry (100 g) 130 13

Apple (200 g) 20–120 4–24 Green tea (200 mL) 100–800 20–160 Black tea (200 mL) 60–500 12–100 Red wine (100 mL) 80–300 8–30

(Manach et al., 2004)

31

1.3.5 Modulation of free radical by antioxidants

1.3.5.1 Mechanisms of antioxidant action

Antioxidants act in three ways, namely, free radical chain breaking mechanism, metal

chelating mechanism and singlet oxygen quenching mechanism (Aruoma and Cuppett,

1997).

Free radical chain breaking mechanism: Commonly known as the (hydrogen donor

mechanism). This method is currently classified under two primary classifications: chain

breaking donor mechanism and chain breaking acceptor mechanism.

Metal chelating mechanism: This method can be described as preventive in nature, since

there is no interaction with the radical species. Instead the compound complexes with the

metal ions and retards its ability to catalyze hydro-peroxide decomposition and free radical

formation.

Singlet oxygen quenching: The third mechanism involves the quenching of 1O2 this

process occurs through an energy transfer between the 1O2 and the antioxidant, e.g.,

carotenoids quenching 1O2 and resulting in 3O2 formation. The antioxidant can also react

with 1O2 to form stable products.

However, none of these antioxidant mechanisms work in isolation, usually it is a

synergistic effect of combined action of one or more mechanisms.

1.3.5.2 Defense system mechanism in vivo against oxidative damage

There are several in vivo lines of defense exhibited by antioxidants. The first defense line is

to prevent the formation of ROS and free radicals by sequestering metal ions, reducing

hydro-peroxides and hydrogen peroxide and to quench superoxide and singlet oxygen.

(a) Non-radical decomposition of hydro-peroxides and hydrogen peroxide

-Decomposition of hydrogen peroxide by catalase:

2H2O2 → 2H2O + O2

-Decomposition of hydrogen peroxide and free fatty acid hydro-peroxides by

glutathione peroxidase (cellular):

H2O2 + 2GSH → 2 H2O + GSSG

32

LOOH + 2GSH → LOH + H2O+GSSG

-Decomposition of hydrogen peroxide and phospholipid hydro-peroxides by

glutathione peroxidase (plasma):

PLOOH + 2GSH → PLOH + H2O + GSSG

-Decomposition of phospholipid hydro-peroxides by glutathione peroxidase:

-Decomposition of hydrogen peroxide and lipid hydro-peroxides by peroxidase:

LOOH + AH2 → LOH + H2O + A

H2O2 + AH2 → 2H2O + A

-Decomposition of lipid hydro-peroxides by glutathione-S-transferase:

(b) Sequestration of metal by chelation:

- Transferrin, lactoferrin: sequestration of iron

- Haptoglobin: sequestration of haemoglobin

- Haemopexin: stabilisation of haem

- Ceruloplasmin, albumin: sequestration of copper

(c) Quenching of active oxygen species:

- Superoxide dismutase (SOD): disproportionation of superoxide

2O2•-+ 2H+ → H2O2 + O2

- Carotenoids, vitamin E: quenching of singlet oxygen

The second line defense is the radical-scavenging antioxidants. Vitamin E and vitamin C

are major lipophilic and hydrophilic radical-scavenging antioxidants. Phenolic compounds

may also work as important radical-scavenging antioxidants. They scavenge radicals and

inhibit chain initiation or break chain propagation. They include the hydrophilic agents

such as vitamin C, uric acid, bilirubin and albumin; and lipophilic agents such as vitamin E,

ubiquinol, carotenoids and flavonoids.

The third-line of defense is the repair and de novo enzymes responsible for defenses such as

repair of the damages and reconstitution of membranes: lipases, proteases, DNA repair

enzymes, and transferases (Gill and Tuteja, 2010).

33

1.3.5.3 Plant antioxidant systems

Plant systems have developed extensive protective mechanisms to combat harmful effects

of free radicals, including a variety of small molecules and enzymes. The equilibrium

between production and scavenging of ROS may be perturbed by various biotic and abiotic

stress factors such as salinity, UV radiation, drought, heavy metals, temperature extremes,

nutrient deficiency, air pollution, herbicides and pathogen attacks. These disturbances lead

to sudden increase in intracellular levels of ROS, which can cause significant damage to

cell structures (Figure 1.5). Through a variety of reactions, O2●- leads to the formation of

H2O2, OH● and other ROS. The ROS are highly reactive and toxic at high levels and causes

damage to proteins, lipids, carbohydrates, DNA which ultimately results in cell death. ROS

affect many cellular functions by damaging nucleic acids, oxidizing proteins, and causing

lipid peroxidation. Stress-induced ROS accumulation is counteracted by enzymatic

antioxidant systems that include a variety of scavengers, such as superoxide dismutase

(SOD), ascorbate peroxidase (APX), catalase (CAT), glutathione reductase (GR),

monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR),

glutathione peroxidase (GPX), guaicol peroxidase (GOPX) and glutathione-S- transferase

(GST) and non-enzymatic low molecular metabolites, such as ascorbic acid (ASH),

glutathione (GSH), α-tocopherol, carotenoids and flavonoids (Gill and Tuteja, 2010).

Exposure of plants to unfavorable environmental conditions such as temperature extremes,

heavy metals, drought, water availability, air pollutants, nutrient deficiency, or salt stress

can increase the production of ROS. Figure 1.6 summarizes the antioxidant defense systems

of plant cellular antioxidant machinery to protect themselves against abiotic stresses. The

components of antioxidant defense system are enzymatic and non-enzymatic antioxidants.

Enzymatic antioxidants include SOD, CAT, APX, MDHAR, DHAR and GR and non-

enzymatic antioxidants are GSH, ASH (both water soluble), carotenoids and tocopherols

(lipid soluble) (Yanishlieva et al., 2001).

34

Figure 1.5 Balance between antioxidant (AO) and reactive oxygen species (ROS) in plants

(Yanishlieva et al., 2001).

Figure 1.6 ROS and antioxidant defense mechanisms

35

1.3.6 The structure–activity relationships of antioxidant

The effectiveness of the natural antioxidants (NAOs) is dependent on the participation of

the phenolic hydrogen in radical reactions, the stability of the NAO radical formed during

radical reactions, and chemical substitutions present on the structure. The substitutions on

the structure are probably the most significant contributors to the ability of an NAO to

participate in the control of radical reactions, and the formation of resonance stabilized

NAO radicals (Yanishlieva et al., 2001).

The antioxidant activity of flavonoids depends on their chemical structure. Generally, there

are three structure groups in the determination, the free radical scavenging and/or

antioxidative potential of flavonoids.

(Yanishlieva et al., 2001)

Figure 1.7 (a) catechol moiety of the B-ring, (b) 2,3-double bond in conjugation with a 4-oxofunction of

a carbonyl group in the C-ring and (c) presence of hydroxyl groups at the 3 and 5 positions

The electron-donating capability of methyl, ethyl, and tertiary butyl alkyls at positions

ortho and para to the hydroxyl groups greatly enhances the antioxidant activity (AOA) of

phenol. In addition, hydroxyl substitutions at these positions would further enhance the

AOA. Ortho substituted phenols (e.g., 1,2-dihydroxybenzene), tend to form intra-molecular

hydrogen bonds during radical reactions (Figure 1.7), which could enhance the stability of

the phenoxy radical. The presence of a methoxy (OCH3) substitution at ortho position to

the hydroxyl group is unlikely to undergo hydrogen bonding resulting in a weaker AOA.

36

Figure 1.8 Intra-molecular hydrogen bonding of ortho substituted phenols

NAO would be expected to participate in radical trapping and singlet oxygen quenching

mechanisms. Radical trapping mechanisms can occur via interactions between radical

species, such as an antioxidant radical and lipid peroxyl radical (Figure 1.9).

Figure 1.9 Radical trapping mechanism of phenolic antioxidants

Alternatively, lipid peroxy radicals can interact with electron dense regions of a molecule.

For example, the conjugated polyene system of carotenoids has been found to interact with

peroxy radicals (Figure 1.10).

Figure 1.10 Radical trapping mechanism of carotenoids

37

1.3.7 Methods of in vitro antioxidant activity determination

1.3.7.1 In vitro antioxidant capacity assays

There are numerous methods for measuring antioxidant activity, involving various redox

reactions. The assays can roughly be classified into two types: assays based on hydrogen

atom transfer (HAT) reactions and assays based on electron transfer (ET) (Table 1.4). The

majority of HAT-based assays apply a competitive reaction scheme and substrates compete

for thermally generated peroxyl radicals through decomposition of azo compounds. These

assays include inhibition of induced low-density lipoprotein autoxidation, oxygen radical

absorbance capacity (ORAC), total radical trapping antioxidant parameter (TRAP), and

crocin bleaching assays. ET-based assays measure the capacity of an antioxidant in the

reduction of an oxidant, which changes color when reduced. ET-based assays include the

Trolox equivalence antioxidant capacity (TEAC), ferric ion reducing antioxidant power

(FRAP), “total antioxidant potential” assay using a Cu (II) complex as an oxidant, and 2,2-

diphenyl-1-picrylhydrazyl (DPPH). In addition, other assays have been designed to

measure the capacity of a sample to scavenge biologically relevant oxidants such as singlet

oxygen, superoxide anion, hydroxyl and peroxynitrite radicals, and they are summarized in

Table 1.4. Based on such analyses, it has been suggested that the total phenols assay by

Folin Ciocalteu reagent (FCR) could be useful for quantification of the reducing capacity of

the antioxidants in the sample, and the ORAC assay to quantify peroxyl radical scavenging

capacity. (Huang et al., 2005).

Each method has its own merits and drawbacks, it has been found that the most common

and reliable methods are the ABTS and DPPH methods; and they have been modified and

improved in recent years (Krishnaiah et al., 2011).

The oxidative damage can be attenuated not only by scavenging radicals, but also by

sequestering metal ions, decomposing hydrogen peroxide and/or hydro-peroxides,

quenching active pro-oxidants. It should be appreciated that the antioxidant can exert its

effect by different mechanisms and it is essential in evaluating antioxidant capacity to

clarify which function is being measured by the method employed (Aruoma, 2003). Thus,

as often pointed out, there is no single method that correctly evaluates the total antioxidant

activity.

38

Table 1.4 In vitro antioxidant capacity assays

Assays involving hydrogen atom

transfer reactions (HAT)

ROO● + AH → ROOH + A●

ROO● + LH → ROOH + L●

ORAC (oxygen radical absorbance capacity)

TRAP (total radical trapping antioxidant

parameter)

Crocin bleaching assay

IOU (inhibits oxygen uptake),

Inhibition of linoleic acid oxidation,

Inhibition of LDL oxidation

Assays by electron-transfer reaction

(ET)

M(n) + e (from AH) → AH●+ + M(n-

1)

TEAC (Trolox equivalent antioxidant capacity)

FRAP (ferric ion reducing antioxidant

parameter)

DPPH (diphenyl-1-picrylhydrazyl)

Copper (II) reduction capacity

Total phenols assay by Folin-Ciocalteu reagent

(Huang et al., 2005)

1.3.7.2 Mode of action of in vitro antioxidant activity assays

Trolox equivalent antioxidant capacity (TEAC)

The TEAC assay is based on the ability of antioxidants to scavenge the ABTS●+ radical

and can measure antioxidant capacities of hydrophilic and lipophilic compounds in the

same sample (Pellegrini et al, 1999). The mechanism of single hydrogen transfer takes

place in two steps:

39

Step 1:

N

S S

O

OOH

N

S

N

S

O

OOH

N

CH3

CH3

ABTS

-e- ↕ +e- K2S2O8 (Potassium persulfate)

N

S S

O

OO

-

N

S

N+

S

O

OO

-

N

CH3

CH3

Step 2: Reduction of ABTS°+ and oxidation of Trolox

O

OH

CH3

CH3CH3

O

OH

CH3

Trolox

+

CH3 O

CH3

CH3

O

OH

CH3

O-

ABTS+ (reduced) Oxidized Trolox

Figure 1.11 Reduction of ABTS+ radical by Trolox (H-donor)

* ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)),

* Trolox ((6-hydroxy-2,5,7,8-tetramethylchroman-2- carboxylic acid) (Pellegrini et al, 1999)

CH3

NH+

S

O

O

O-

S

N

N

N

S S

O

O

O-

CH3

40

DPPH free radical scavenging assay

The stable pre-formed DPPH radical has been used widely for the determination of primary

antioxidant activity. It is a free radical that can accept H+/e- to become a stable molecule

(Groupy et al. 2003). The antioxidant process of this reaction is thought to be expressed as

(Figure 1.18):

2,2-diphenyl-1-picrylhydrazyl (DPPHo) (Groupy et al. 2003)

Figure 1.12 Protonation of DPPH (violet) to most stable DPPH-H form (light yellow color)

Reducing power property (FRAP)

The ability of biological samples to reduce ferric ions is determined using FRAP method

previously described by Oyaizu (1986), and by redox potential (potentiometric assay). An

antioxidant with ablity to donate a single electron to the ferric-complex would cause the

reduction of this complex into the blue ferrous complex, which absorbs strongly at 700 nm.

The reducing power of antioxidant compounds can be easily predicted from equations (1),

(2) and (3) (Figure 1.19).

K3[Fe(CN)6] + e-(Extract) → K4[Fe(CN)6] (1)

Fe3+ + e- → Fe2+ (2)

Fe2+(aq) + K3Fe(CN)6(aq) → KFe[Fe(CN)6](s) + 2K+ (3)

Deep Blue Colour

Figure 1.13 Mechanism of Ferric Reduction

41

Hydrogen peroxide scavenging activity

Hydrogen peroxide is a weak oxidizing agent and can inactivate antioxidant enzymes such

as, Superoxide dismutase, Catalase, Glutathione-S-Transferase, Glutathione Peroxidase,

and Glutathione etc., directly, usually by oxidation of essential thiol (-SH) groups. It can

cross cell membranes rapidly, once inside the cell, hydrogen peroxide can probably

undergo Fenton’s reaction by interacting with metal ions such as, Fe2+, Cu2+ in the

cytoplasm (as shown in Figure 1.20, and forms reactive hydroxyl radical and this may be

the origin of many of its toxic effects.

Hydrogen peroxide scavenging activity was analysed by the reduction of titanium

compound by hydrogen peroxide (Figure 1.20).

Titanium compound + χH2O2 ↔ Titanium compound χH2O2 + γH2O2 [Red Solution]

I ↕ II

Titanium compound (χ+γ) H2O2 [White Precipitate]

The antioxidant process of this reaction is thought to be divided into the

following stages:

TiCl4 + H2O2 = TiCl3-O-OH + HCl Step 1

TiCl3-O-OH+2H+ = TiO2+3HCl Step 2

TiCl4 + 4NH3 + 4H2O = Ti (OH)4 + 4NH4Cl Step 3

2Ti(OH)4 + 4H2SO4 = Ti2(SO4)4 + 8H2O Step 4

Figure 1.14 Reduction of Titanium compound by H2O2

42

Superoxide anion scavenging activity

Superoxide anion is also another harmful reactive oxygen species as it damages cellular

component in biological systems. It can act directly or indirectly by forming H2O2, OH●,

peroxy nitrite, or singlet oxygen (Sharma and Singh, 2012). The ability of the plant extract

to scavenge superoxide radical from reaction mixture is reflected in the decrease of the

absorbance at 560 nm. The mechanism of single electron transfer takes place in two steps

(Figure 1.21):

Tetrazolium Salt Formazan + O2

.-

N

N+

O CH3

CH3

NH

N

CH3

O CH3

NAD(P)H

CH3SO4

_

1-Methoxy PMS 1-Methoxy PMS Reduced form

e-

In the next step O2.- is neutralized by the antioxidant compounds.

Figure 1.15 Superoxide anion radical generation by PMS-NADH system

43

Nitric oxide scavenging activity

Nitric oxide radicals display important roles in various types of inflammatory. It is

potentially toxic with a free radical character, and therefore, it is responsible for many

physiologic and pathologic events such as juveniles’ diabetes, multiple sclerosis, and

ulcerative colitis (Huie and Padmaje, 1993). Additionally, increasing evidence shows that

NO modulates neurotoxin induced cell damage and is involved in neuronal cell death in

Parkinson’s disease and other neurodegenerative disorders including Alzheimer disease

(Zhan et al; 2006, Aliev et al. 2009 ). Griess assay is generally used to assess nitric oxide

inhibition activity of the plant extracts. The antioxidant process of this reaction is thought

to be divided into the following stages as shown in Figure 1.22.

Figure 1.16 Formation of azo dye

44

Metal chelating activity

Bivalent transition metal ions play an important role as catalysts of oxidative processes,

leading to the formation of hydroxyl radicals and hydrogen peroxide decomposition

reaction via Fenton reaction (Aruoma et al., 1997). Therefore, minimizing their

concentration by chelating them would reduce radical oxygen species (ROS) generation

associated with Fenton’s reaction, and thereby, offering protection against oxidative

damage. The assay involves chelation of the metal ion using ferrozine that forms a

complex with ferrous ion. The chelation reaction is thought to be occurring in the

following stages (Figure 1.23). In the presence of other chelating agents, the ferrozine

complex formation is inhibited. Thus measurement of the reduction of colour of the

reaction media, allows the estimation of the chelating activity of the co-existing chelating

agent.

45

N

OOH

OH

O

N

OH

O

OH

O

+

FeCl2

O

OH

Fe2+

O-

OH

O

OH OH

OH

OH

O-

OH

OH

OH

OH

+Fe

2+ Residual Residual

EDTA

+ Fe2+

Ferrozine

Fe2+

N

N

N

N

SO

OO

-

S

O

O

O-

NN

N N

S

O

O

O-

SO

O

O-

Blue Colour Formation

Figure 1.17 Mechanism of metal chelation by ferrozine

NN

N N

S

O

O

OH

SO

O

O-

46

1.3.8 Trends and challenges in application of antioxidants from plant sources.

The application of antioxidants from natural sources in food can have dual benefits of food

preservation and health promotion. In effect, with increasing consumer consciousness with

regard to food additive safety, the replacement of synthetic antioxidants by natural ones can

have the added dimension of health function, in addition to physico-chemical functionality

such as solubility in both oil and aqueous phases such as emulsion systems in foods.

However, naturally occurring antioxidant substances may also need safety evaluation for

use as food additives (Rangan and Barceloux, 2009).

Antioxidants have been used in the food industry to preserve flavor and color and to avoid

vitamin destruction. Antioxidants such as ascorbic acid (vitamin C) and -tocopherol

(vitamin E) are frequently used as antioxidants for food. Because of high manufacturing

costs and lower efficiency of natural antioxidants, the most used are the synthetic

antioxidants. Among the synthetic types, the most frequently used are butylated

hydroxyanisole (BHA), butylated hydroxytoluene (BHT), propyl gallate (PG) and tert-

butyl hydroquinone (TBHQ) (Moure et al., 2001). However, there are some reports

suggesting that BHA and BHT could be toxic, causing concerns among the consumers with

regard to the safety of such synthetic antioxidants, leading to increased attention of the

industry in finding natural and safer alternatives for antioxidants (Wanasundara and

Shahidi, 1998).

Spices and herbs such as oregano, thyme, dittany, marjoram, lavender, and rosemary have

limited applications in spite of their high antioxidant activity, as they impart a characteristic

herbal flavor to the food, and deodorization steps are required (Moure et al., 2001).

Polyphenolic compounds that contribute to the sensory and organoleptic properties of fruits

and vegetables (color, taste, astringency), can affect the functional and nutritional values of

proteins by binding to them, reducing the overall nutritional values of foodstuffs. Besides

the formation of strong complexes with dietary proteins, polyphenols have other

undesirable effects in the digestion of foods such as binding to salivary proteins and

digestive enzymes. Polyphenol polymerization, due to autoxidation, is responsible for color

loss in processed vegetables. Therefore, the potential of tannins to diminish nutrient

47

availability should be considered when using them as biological antioxidants (Moure et al.,

2001).

Although the current understanding of the role of natural antioxidants is derived from in

vitro studies, their biological effects have not been studied rigorously in animal models and

humans. The results obtained from in vitro investigations show many contradictions,

because AOA depends strongly on many factors, including the presence of oxygen and

metals, pH and polarity of the medium, and temperature. Thus there is a need to standardize

AOA assays (Aruoma, 2003). Direct observation of antioxidant reactions in living

systems, generally, is not possible because of the low concentrations of the reactants

involved, and because of the rapid rates of oxidant-antioxidant reactions (Halliwell, 2009).

Despite decades of research, it is currently impossible to state what are the contributions of

fruits and vegetables to the health-promoting effects through antioxidant protection. In

particular, it is unclear whether flavonoids and other plant polyphenols have any direct

antioxidant effect in vivo. One challenge is to measure the low levels of oxidized products

in the presence of a vast excess of unpertubed biomolecules in human tissues and body

fluids (Halliwell, 2009).

Phenolic compounds could be both antioxidants and pro-oxidants in the gastrointestinal

tract. In practice, there may be little difference in the results of these various actions.

However, increased cellular antioxidant protection can be achieved either by supplying

more antioxidants or by a mild degree of pro-oxidant stress, leading to upregulation of

endogenous antioxidant defense systems (Halliwell, 2009).

While phenolic compounds are putative antioxidants, the radicals emenating from their

radical scavenging activity may exhibit pro-oxidant activity as well. In practice, there may

be little difference in the results of these various actions. However, increased cellular

antioxidant protection can be achieved either by supplying more antioxidants or by a mild

degree of pro-oxidant stress, leading to upregulation of endogenous antioxidant defense

systems (Halliwell, 2009).

48

The failure of antioxidant in demonstrated benefits in humans is debatable. First, in some

diseases, reactive oxygen species may not actually be important. Second, intervention trials

often tested antioxidants on subjects who already had extensive pathology, where

preventative actions may be too late. Third, the doses used may be wrong. For cancer and

several neurodegenerative diseases, the evidence supporting a role for oxidative damage in

their progression is compelling (Halliwell, 2009), but the evidence is mixed for

atherosclerosis and several other diseases. Recently it is very interesting to note the

interplay of factors causing neuronal cell death in Alzheimer's and Parkinson's diseases,

including mitochondrial dysfunction, oxidative damage, and defects in the turnover of

abnormal proteins (especially affecting the proteasome system). This line of thinking

ensued the discovery that inhibition of proteasome function can lead to cellular oxidative

stress. For chronic inflammatory diseases, the overall role of reactive oxygen species, both

in their origin and progression is less clear, nonetheless the reactive oxygen species may

help explain why such diseases also increase cancer risk. Ironically, these species can

sometimes be anti-inflammatory, e.g., by modulating lymphocyte function. Hence the use

of antioxidants to treat chronic inflammatory diseases may not be as simple as it originally

sounded (Halliwell, 2009).

49

1.4 Plants as a source of antimicrobials

1.4.1 Why antimicrobials from plant sources?

Microbial and plant products occupy the major part of the antimicrobial compounds

discovered until now (Balouiri et al., 2016). The folk medicines and alternative medicines

which have been prevalent for hundreds of years prove a point that it is possible that nature

might have enough for us to offer so that we might not need to turn to synthetic

antimicrobials at all (Dorai, 2012). In fact, plants synthesize over 100,000 small-molecular

compounds, many of which have antimicrobial activity (Lewis and Ausubel, 2006).

Isolation of phytochemicals with potential antimicrobial activities are being explored as

potential therapeutic agents to fight infections. Naturally occurring phytochemicals,

predominantly secondary metabolites, are the source of the many of drugs in clinical use

today (Taylor, 2013).

After the revolution in the “golden era”, when almost all groups of important antibiotics

(tetracyclines, cephalosporins, aminoglycosides and macrolides) were discovered and the

main problems of chemotherapy were solved in the 1960s. As history repeats itself these

days and these exciting compounds are in danger of losing their efficacy because of

increased incidences of microbial resistance (Brown and Wright, 2016). Currently, its

impact is considerable with treatment failures associated with multidrug-resistant bacteria,

and it has become a global concern to public health (Balouiri et al., 2016). Phytochemicals

have already demonstrated their potential as antibacterials when used alone, and as

synergists/potentiators of less effective antibacterial products. Moreover, studies have

demonstrated that phytochemicals can be used, where bacterial resistance mechanisms

make conventional treatments ineffective and also in the control of biofilm (Simoes et al.,

2009). Phytochemicals can act through different mechanisms than conventional antibiotics,

and therefore, could be of use in the treatment of resistant bacteria (Abreu et al., 2012).

Nowadays, consumers are increasingly aware of diet-related health problems, therefore

demanding natural ingredients which are expected to be safe and health-promoting. They

want organically grown and manufactured foods containing minimal synthetic

preservatives (Odonovan, 2002; Yeung, 2001). Because of greater consumer awareness and

50

concern regarding synthetic chemical additives, foods preserved with natural additives are

becoming popular. This has led researchers and food processors to look for natural food

additives with a broad spectrum of antimicrobial activity (Bhalodia and Shukla, 2011;

Marino, 2001).


from the cultivation and processing of a particular crop. Waste and by-products can cause

disposal problems, pollution, management and economic problems worldwide (Santana-

Méridas et al., 2012). Food grade agro-industrial waste is often utilized as animal feed or as

fertilizer. Fruit and vegetable wastes and by-products can range from pomace (leftovers

after pressing), to vegetable trimmings and dicarded whole fruits and vegetables

(Wijngaard et al., 2009), and their utilization would add profit value to the by-products. It

could also help save energy and money that go into research and development costs for

synthetic food additives (Kabuki, 2000; Mirabella et al., 2014).

1.4.2 Major groups of phytochemicals with antimicrobial properties

Nature antimicrobial compounds are present in abundance in fruits and vegetables, and they

are present in all parts of the plant such as bark, stalks, leaves, fruits, roots, flowers,

pods, seeds, stems, latex and hull (Amarowicz, 2009; Amarowicz et al., 2005; Ramos,

2006; Ramos et al., 2005). The antimicrobial compounds in plant materials are commonly

found in leaves (rosemary, sage, basil, oregano, thyme, and marjoram), flowers or buds

(clove), bulbs (garlic and onion), seeds (caraway, fennel, nutmeg, and parsley), rhizomes

(asafetida), fruits (pepper and cardamom), or other parts of plants (Tiwari et al., 2009).

Plants have an almost limitless ability to synthesize aromatic substances, most of which are

phenols or their oxygen-substituted derivatives (Cowan, 1999; Naidu and Davidson, 2000).

Most are secondary metabolites, of which at least 12,000 have been isolated, a number

estimated to be less than 10 % of the total (Cowan, 1999). In many cases, these substances

serve as plant defense mechanisms against predation by microorganisms, insects, and

herbivores. Some, such as terpenoids impart to plants their odors; while others such as

anthocyanins, betalains, carotenoids and tannins) are responsible for plant pigmentation.

Many compounds are responsible for plant flavour (e.g., the terpenoid capsaicin from chilli

pepper), and some of the same herbs and spices used by humans to season food yield useful

51

medicinal compounds. Useful antimicrobial phytochemicals can be divided into several

categories, described and summarized by Cowan (1999) in Table 1.5.

Secondary metabolites produced by plants constitute a major source of bioactive

substances. The scientific interest in these metabolites has increased today with the search

of new therapeutic agents from plant sources. Major components with antimicrobial activity

found in plants, herbs, and spices are phenolic compounds, terpenes, aliphatic alcohols,

aldehydes, ketones, acids, and isoflavonoids (Davidson et al., 2005). Nonphenolic

constituents such as allyl isothiocyanate and garlic oil are also effective against gram-

negative bacteria and fungi (Tiwari et al., 2009).

Plants, herbs, and spices as well as their derived essential oils, and isolated compounds

from them such as eugenol, carvacrol, thymol are known to inhibit various metabolic

activities of bacteria, yeast, and molds (Deba et al., 2008; Djenane et al., 2012; Ichrak et al.,

2011; Jia et al., 2010; Joshi et al., 2010). Antimicrobial activity of essential oils depends on

the chemical structure of their components and on their concentration. Katayama and Nagai

(1960) recognized eugenol, carvacrol, thymol, and vanillin as active antimicrobial

compounds in essential oils. Oils with high levels of eugenol (allspice, clove bud and leaf,

bay, and cinnamon leaf), cinnamamic aldehyde (cinnamon bark and cassia oil), and citral

(lemon myrtle, Litsea cubeba, and lime) are typically strong antimicrobials. In mustard and

horse radish, precursor glucosinolates are converted by enzyme myrosinase to yield a

variety of isothiocynates including the allyl form, which are strong antimicrobial agents

(Katayama and Nagai, 1960).

Simple phenols and phenolic acids. Some of the simplest bioactive phytochemicals

consist of a single substituted phenolic ring. Cinnamic and caffeic acids are common

representatives of a wide group of phenylpropane-derived compounds. The mechanisms

thought to be responsible for phenolic toxicity to microorganisms include enzyme

inhibition by the oxidized compounds, possibly through reaction with sulfhydryl groups or

through more non-specific interactions with the proteins (Cowan, 1999).

52

Table 1.5 Major classes of antimicrobial compounds from plants

Class Subclass Mechanism

Phenolics

Simple phenols Substrate deprivation, membrane disruption

Phenolic acids Substrate deprivation, membrane disruption

Quinones Bind to adhesins, complex with cell wall, inactivate

enzymes

Flavonoids Bind to adhesins

Flavones Complex with the cell wall, inactivate enzyme, inhibit

HIV reverse transcriptase

Flavonols Complex with the cell wall, inactivate enzyme, inhibit

HIV reverse transcriptase

Tannins Bind to proteins, Bind to adhesions, Enzyme inhibition,

substrate deprivation, complex with cell wall, membrane

disruption, metal ion complexation

Coumarins Interaction with eukaryotic DNA (antiviral activity)

Terpenoids,

essential oils

Membrane disruption

Alkaloids Intercalate into cell wall and/or DNA

Lectins and

polypeptides

Block viral fusion or adsorption, form disulfide bridges

(Cowan, 1999)

Quinones. A quinone is a class of organic compounds that are formally "derived from

aromatic compounds [such as benzene or naphthalene] by conversion of an even number of

–CH= groups into –C(=O)– groups with any necessary rearrangement of double bonds",

resulting in "a fully conjugated cyclic dione structure (IUPAC, 2009). Quinones are

aromatic rings with two ketone or carbonyl groups. They are ubiquitous in nature and are

highly reactive. These compounds, being colored, are responsible for the browning

reaction in cut or injured fruits and vegetables, and are intermediates in the melanin

synthesis pathway. Quinones are known to complex irreversibly with nucleophilic amino

acids in proteins (Stern, 1996), often leading to inactivation of proteins and loss of their

function.

Flavonoids: Flavones, flavonoids, and flavonols. Flavones are a class of flavonoids

containing one carbonyl group (as opposed to the two carbonyls in quinones). The addition

of a 3-hydroxyl group yields a flavonol . Since they are known to be synthesized by plants

in response to microbial infection, it should not be surprising that they have been found to

be effective antimicrobial substances in vitro against a wide array of microorganisms. Their

https://en.wikipedia.org/wiki/Organic_compound

https://en.wikipedia.org/wiki/Aromatic

https://en.wikipedia.org/wiki/Benzene

https://en.wikipedia.org/wiki/Naphthalene

https://en.wikipedia.org/wiki/Double_bond

https://en.wikipedia.org/wiki/Conjugated_system

https://en.wikipedia.org/wiki/Diketone

53

activity is probably due to their ability to complex with extracellular and soluble proteins

and to complex with bacterial cell walls similar to quinones. More lipophilic flavonoids

may also disrupt microbial membranes (Cowan, 1999).

Tannins.“Tannin” is a general descriptive name for a group of polymeric phenolic

substances capable of tanning leather or precipitating gelatin from solution, a property

known as astringency. Many human physiological activities, such as stimulation of

phagocytic cells, host-mediated tumor activity, and a wide range of anti-infective actions,

have been assigned to tannins (SantiestebanLópez, 2007). One of their molecular actions is

to complex with proteins through so-called secondary bonding such as hydrogen bonding

and hydrophobic effects. Thus, their mode of antimicrobial action, may be related to their

ability to inactivate microbial adhesins, enzymes, cell envelope transport proteins, etc. They

can also complex with polysaccharides (Cowan, 1999; SantiestebanLópez, 2007).

1.4.3 Modes of action of antimicrobials

Antimicrobials can fatally affect a microbial cell in various ways by specifically and

selectively killing or damaging cellular structures and their functions. Much of knowledge

regarding the mechanisms of action of antimicrobials is derived from studies on antibiotics.

Various mechanisms have been proposed by which antibiotics act with different

specificities with regards to target sites on or in microbial cells (Kohanski et al., 2010).

Figure 1.17 summarizes the general mechanisms of antibiotics against bacterial cell as well

as the major cellular targets (Neu H. C., 1996). Antibiotic acts by interferring with: (1) cell

wall synthesis, (2) plasma membrane integrity, (3) nucleic acid synthesis, (4) ribosomal

function, and (5) folate synthesis. Cell wall synthesis is inhibited by ß-lactams, such as

penicillins and cephalosporins, which inhibit peptidoglycan polymerization, and by

vancomycin, which combines with cell wall substrates. Polymyxins disrupt the plasma

membrane, causing leakage. The plasma membrane sterols of fungi are attacked by

polyenes (amphotericin) and imidazoles. Quinolones bind to a bacterial complex of DNA

and DNA gyrase, blocking DNA replication. Nitroimidazoles damage DNA. Rifampin

blocks RNA synthesis by binding to DNA directed RNA polymerase. Aminoglycosides,

tetracycline, chloramphenicol, erythromycin, and clindamycin interfere with ribosome

54

function. Sulfonamides and trimethoprim block the synthesis of the folate needed for DNA

replication.

(Neu H. C., 1996).

Figure 1.18 Mechanisms of action of antimicrobial agents. PABA, paraminobenzoic acid; DHFA,

dihydrofolic acid; THFA, tetrahydrofolic acid

Although there are dozens of plant secondary metabolites that show activity in the micro to

sub-micro molar range, at least against Gram-positive species, surprisingly little is known

about their mechanisms of action (Lewis and Ausubel, 2006). Antimicrobials from plant

sources can affect the structure and function of some of the biomolecules. Although plant

antimicrobials can be expected to affect microbial cell organelles such as such as cell

membrane, interference with DNA/RNA function and cell metabolism, and modification of

cell environment, the available information suggests that cell membranes may be the

primary target with disruption of their integrity, since the effectiveness of antimicrobial

activity of these compounds generally increases with their lipophilicity (Daglia, 2012b;

Hendrich, 2006; Kanjee and Houry, 2013; Kohanski et al., 2010). One complication that

arises in establishing antimicrobial mechanisms of plant extracts is that they are a mixture

of compounds, and methodologies commonly used to elucidate the mode of action of a pure

compound are less useful. The addition of phytochemical compounds to a medium can

alter the conditions of the milieu such as pH, depending on their solubility in water

(hydrophilicity), their concentration, and temperature condition of the antimicrobial assay.

55

However, determination of the physico-chemical properties of phytochemicals related to

antimicrobial activity may shed some light on their antimicrobial potential. They may

include: capacity to acidify or alkalinize microbial cells (acid dissociation constant), lipid

solubility (anesthetics), water solubility, surface activity and ability to form complexes with

proteins, metal binding capacity, and redox potential (e.g., oxygen scavenging ability or

ability to generate reactive oxygen groups) (Kanjee and Houry, 2013; Kohanski et al.,

2010). The polycationic nature of some of the antimicrobials can also contribute to

antimicrobial activity by binding to cellular membrane and causing membrane dysfunction

(Sikkema, 1995).

Some examples of mechanisms of action of natural compounds are shown in Table 1.5.

Various mechanisms have been elucidated in detail. For example, tea tree oil, which is

essentially a terpenic hydrocarbon. It is known that hydrocarbons can partition

preferentially into biological membranes and disrupt their vital functions. Tea tree oil has

been shown to be effective against a plethora of micro-organisms (Carson, 2006). The

major antimicrobial constituent of garlic and onion is allicin, along with several other

sulfur-containing compounds (Barone and Tansey, 1977).

The possible mechanism of action for phenolic compounds is not very clear. The effect of

phenolic compounds is generally concentration-dependent. At low concentrations, phenols

affect enzyme activity, particularly those associated with energy production; while at high

concentrations, they cause protein denaturation (Bajpai et al., 2008). The antimicrobial

effect of phenolic compounds may be, in part, due to their ability to alter microbial cell

permeability, thereby accentuating the loss of biomolecules and ions from the interior

(Dorman and Deans, 2000). They could also interfere with membrane function such as

electron transport, nutrient uptake, protein, nucleic acid synthesis, and enzyme activity

(Bajpai et al., 2008), and interact with membrane proteins, perturbing membrane structure

and functionality (Dorman and Deans, 2000).

The antimicrobial activity of isothiocynates derived from broccoli, onion and garlic is

related to the inactivation of extracellular enzymes through oxidative cleavage of disulfide

bonds and the formation of the reactive thiocyanate radical was proposed to mediate the

antimicrobial activity (Delaquis and Mazza, 1995). Carvacrol, (β)-i-carvone, thymol, and

56

trans-cinnamaldehyde are reported to decrease the intracellular ATP (adenosine

triphosphate) content of E. coli O157:H7 cells, while simultaneously increasing

extracellular ATP, indicating that the disruptive action of these compounds occurs on the

plasma membrane (Helander et al., 1998a).

The accumulation of lipophilic compounds in the (cytoplasmic) membrane of

microorganisms has also considerable effect on the structural and functional properties of

the membrane. The numerous observations of toxic effects of terpenes, and phenols can be

explained largely by the interactions of these compounds with the membrane and with

membrane constituents. As a result of accumulation of lipophilic molecules, the membrane

loses its integrity, causing increased permeability to protons and ions. In addition, it has

been observed that proteins embedded in the membrane are also affected (Carson, 2006;

Cox, 2001; Sikkema, 1995).

1.4.4 Bacterial resistance to antimicrobials and mechanisms

Infectious diseases are still one of the most important causes of human mortality. The quest

for novel effective antimicrobial agents from natural products has gained much momentum

particularly in the health care sector, where microbial resistance is increasing at an alarming

rate and offering new challenges (Aumeeruddy-Elalfi et al., 2016). The overuse, underuse

and general misuse of antibiotics are major factors in the emergence and dissemination of

resistance.

Bacteria compete against each other for food resources, by manufacturing lethal

compounds that they direct against each other. Other bacteria, to protect themselves, evolve

defense against that chemical attack. By this way, penicillin-the first antibiotic discovered

in 1928 by Alexander Fleming, and the bacterial resistance to penicillin occurred by 1945,

just 4 years after its mass production (Brown and Wright, 2016).

The major mechanisms of bacterial resistance to antimicrobials are depicted in Figure 1.18

(Abreu et al., 2012) and include: (1) active drug efflux systems from the cell via a

collection of membrane-associated that effectively remove toxic compounds from cells; (2)

mutations resulting in altered cell permeability; (3) enzymatic degradation of antimicrobials

57

by the synthesis of enzymes that either modify or inactivate selectively, targeting and

destroying antimicrobial compounds; and (4) alteration/modification of the target site, e.g.,

through mutation of key binding elements, such as ribosomal RNA.

By understanding the mechanism of resistance to antimicrobial, there seems to be

considerable potential to develop at least three different types of antibacterials from plants:

traditional antibiotics, multidrug-resistance (MDR) inhibitors and compounds that target

bacterial virulence (Lewis and Ausubel, 2006).

(Abreu et al., 2012)

Figure 1.19 Mechanisms of bacterial resistance to antimicrobials

1.4.5 In vitro methods to evaluate plant extracts for antimicrobial activity

A variety of methods are currently in use for evaluation and/or screening for in-vitro

antimicrobial activity of plant extracts. These methods have been traditionally categorized

as qualitative and quantitative assays. They can also be classified as kinetic and end-point

methods, depending upon whether the antimicrobial compound is being evaluated for its

efficiency or potential. The most commonly known methods are the disk or well diffusion

and broth or agar dilution methods. For a more detailed investigation time kill assays,

Minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC)

estimation are also carried out (Balouiri et al., 2016).

58

1.4.5.1 Diffusion method

Since its inception the Kirby Bauer diffusion method has been extensively used. Clinical

and Laboratory Standards Institute (CLSI) regularly publishes standards of testing for

bacteria, yeasts and fungi (Jorgensen and Ferraro, 2009). In this method, agar plates are

inoculated with a standardized inoculum of chosen test strain. Then depending upon if it’s a

disc diffusion or well diffusion method, either paper discs (4-6mm diameter) saturated with

the extract are placed on the agar surface or wells are bored on the surface which are filled

with the extract (20-100µL). The antimicrobial agent radially diffuses into the agar surface

inhibiting the bacteria, and thereby forming a clear “zone of inhibition”. The zone of

inhibition is measured in mm excluding the well or disc diameter. It is a simple, low cost

and relatively fast method, which can handle a large number of samples easily. However, it

is important to point out the qualitative nature of this method, which limits its application

as a screening method.

1.4.5.2 Dilution method

Dilution methods are appropriate once the antimicrobial compounds have been screened

and their MIC, MBC values need to be estimated. Either agar dilution or broth dilution can

be used to estimate the quantitative effect of the antimicrobial compound on bacteria and

fungi. Similar to diffusion methods, CLSI and EUCAST regularly update the standards and

guidelines for dilution methods as well (NCCLS, 1991; Wiegand et al., 2008).

1.4.5.3 Broth dilution

A micro or macro dilution is the most commonly used antimicrobial susceptibility test

(NCCLS, 1991). The assay involves testing a serially diluted antimicrobial compound (e.g.,

2, 4, 8, 16 µg/mL) in liquid medium dispensed in tubes containing a minimum volume of 2

mL (macro) or with smaller volume using a 96 wells microtiter plate (micro). Thereafter,

each tube or well is inoculated with a standardized inoculum of a microbial test strain,

which had previously been adjusted to 0.5 McFarland scale. After inoculation, the setup

(tube/plate) is incubated as per growth condition of the test strain. The resulting MIC value

is the lowest concentration of the antimicrobial compounds, which completely inhibit

microbial growth. The tubes/wells for this purpose can be observed visually or through a

turbidity meter (Wiegand et al., 2008).

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1.4.5.4 Agar dilution

The method is similar to broth dilution with the difference being that a known, serially

diluted antimicrobial compound is incorporated into molten agar before being poured in the

petri. Thereafter, the agar surface is inoculated with a standardized and known

concentration of microbial inoculum. The MIC endpoint is the lowest concentration of

antimicrobial agent that completely inhibits growth of the test strain. This technique is

useful for both bacteria and fungi, and especially, when a large number of target organism

need to be tested against (NCCLS, 1991; Wiegand et al., 2008).

1.4.5.5 Time kill assay

Time kill assay is a dynamic antimicrobial assay, and it helps to understand the direct

interaction between the antimicrobial compound and the microorganism in a time

dependent or concentration dependent manner. The CLSI-M26-A document provides easy

to follow assay guidelines for time kill assay (Jorgensen and Ferraro, 2009). The

antimicrobial compound is tested at 0.25xMIC and 1xMIC concentration in a growth

medium against a bacterial suspension of minimum log 5 CFU/mL. The incubation is

carried out for progressively increasing durations (4, 6, 8, 12, 16, 18, 24 hours). The tubes

containing the bacterial suspension and the antimicrobial compound are then plated to

enumerate the bacteria. The resulting bacterial population is compared against a control to

calculate the minimum bactericidal concentration MBC. This methods is reliable and

reproducible and correlates well with other antimicrobial susceptibility assays (Li et al.,

1993).

1.4.6 Research trends and challenges of antimicrobials from plant products

The need for natural antimicrobials is very pertinent with the growing awareness of the

consumers about synthetic additives in foods and with the rise of antibiotic resistance in the

health sector. Understandably, there is increasing number of studies and reports on

antimicrobials from microbial sources (bacteriophages, bacteria, algae and fungi)

(Challinor and Bode, 2015), and plant products (essential oils, phytochemicals and

bioactive compounds from fruit and vegetable by-products) (Guil-Guerrero et al., 2016;

Lewis and Ausubel, 2006; Munuswamy et al., 2013; Taylor, 2013).

60

Essential oils (EO) are complex natural mixtures of biologically active substances can have

therapeutic use in the treatment of a panoply of human diseases (Aumeeruddy-Elalfi et al.,

2016). EO of a large number of plants possess useful biological and therapeutic activities,

and are extensively used in the preparation of pharmacologic drugs (Djenane et al., 2012;

Joshi and Mathela, 2012; Prakash et al., 2010; Taga et al., 2012). Many studies have

showed that antimicrobial activity of essential oils (EO), phytochemicals from plants,

herbs, and spices depends not only on the extraction method but also on geographic zone of

cultivation (Rather et al., 2012; Shi et al., 2010).

Unfortunately, many of the published reports on the application of plant extracts as

antimicrobials have been accomplished in model and laboratory systems, and there are few

studies that have been carried out in real foods (Tiwari et al., 2009). The essential oils of

spices and plant extracts as well as their major components are more effective in

microbiological media than when evaluated in real foods (Davidson et al., 2005). In most

cases, the concentrations of of those natural substances required to inhibit a microorganism

in food systems are significantly higher than those found to be inhibitory in model systems;

and hence, it is necessary to use higher concentrations for their applications in real foods,

but it often compromises the sensory quality of the foods. Consequently, few of the

applications of phenolic antioxidants as antimicrobials have been successful (Maqsood et

al., 2013). This reduction in the effectiveness observed in vivo represents an important

limitation to the use of EO and phenolic antioxidants as antimicrobial agents in foods

(Davidson et al., 2005).

Moreover, the interactions among phenolic compounds and proteins and lipids could

explain also the reduction of the antimicrobial effect of the plant extracts, where the major

constituents are typically phenols. The chemical complexity of plant extracts, often

undocumented toxicity, poor water solubility and the lack of standardization may be

responsible for the apparent lack of industrial interest in phytochemicals. Difficulties in

access and supply, the inherent slowness of working with natural products and the costs of

collection, extraction and isolation are additional limitations (Abreu et al., 2012).

Plant extracts compounds from medicinal herbs and dietary plants possess a range of

bioactivities like - antibacterial, antifungal, antiviral, antimutagenic and anti-inflammatory

61

activities. However, limiting the use of natural antimicrobials is the fact that they are of low

potency and require to be used in high quantity, which can detrimental for the organoleptic

properties of food (Davidson et al., 2013; Lucera et al., 2012). For example, the

concentrations of extracts from Cirsium spp required to control and/or to inhibit

successfully Staphylococcus aureus, Bacillus subtilis and Pseudomonas aeruginosa was

found at 1.56–50.0mg/mL. Concentrations of 1.56–25.0 mg/mL for Gram- positive bacteria

and that for Gram-negative (12.5–50.0 mg/mL) were found to be too high to be used in

foods (Borawska et al., 2010).

Several studies have proposed that natural compounds in combination with antibiotics and

other antimicrobials are a new strategy for developing therapies for infections caused by

bacterial species, and that natural plant products can potentiate their activity (Borawska et

al., 2010; Holloway et al., 2011; Jayaraman et al., 2010; Worthington and Melander, 2013).

The use of combinations of EOs and their isolated components are proposed as new

approaches to increase the efficacy of EOs in foods, taking advantage of their synergistic or

additive effects (Bassole and Juliani, 2012). The use of bacterial resistance modifiers such

as efflux pump inhibitors, derived from natural sources mainly from plants can suppress the

emergence of MDR strains (Stavri et al., 2007). Plants extract have also been studied as

sources of new co-therapeutics and resistance-modifying agents (Abreu et al., 2012).

1.4.7 Recent literature on bioactivities of some specific fruits and vegetables - A

random walk

There is growing interest in exploring bioactivities of edible plant materials and their by-

products, herbs, spices and medicinal plants as seen by increasing number of scientific

reports appearing in the literature recently. While some studies have focused mainly on the

bioactivity of plant extracts, some have determined the phytochemical composition of the

extracts and attempted to relate it to their bioactivity. Again, most of studies on fruit and

vegetable extracts have examined antioxidant and antimicrobial activities, while some have

evaluated a range of activities including anti-cholesterolemic, anti-inflammatory, anti-

cancer, analgesic, anti-aging and antiviral activities. Some selected studies reporting on

phytochemical composition and biological activities of fruit and vegetable extracts are

summarized in Table 1.6. Red onion and green pepper exhibited a range of bioactivities;

62

bluberry fruit, rose mary and cranberry appear to be promising sources of antioxidants,

while mangosteen, sea buckthorn and pomgrenate were shown to be good sources of

antimicrobials. These studies consistently support the idea that fruit and vegetables are

potentially a vast reservoir of antioxidant and antimicrobial compounds. Additionally, the

principle compounds responsible for the AO-AM activity in the plant-based or herbal

extractions also contribute to variety of health benefits (Gao et al., 2013; Johanningsmeier

and Harris, 2011; Slavin and Lloyd, 2012; Wang et al., 2011). Although plant-sourced

compounds show bioactivity, a high concentration of the extracts is required in most cases

(Seydim and Sarikus, 2006; Vegara et al.; Yai). For example, the MIC of plant extracts

required to successfully inhibit bacteria in foods ranged (1.56–25.0 mg/mL for gram

positive), and, (12.5–50.0 mg/mL for Gram negative bacteria) (Borawska et al., 2010).

Furthermore, it is evident that factors such as geographical zone of habitation, time of

collection of plants, maturity of the plants affect the levels of phytochemical constituents,

and therefore, their bioactivity (Rathee et al., 2006). In addition, the solvent used for

extraction appears to have a significant impact, since a particular class of phytochemicals is

extracted depending on the nature of solvents. In view of the aforementioned factors and a

lack of standardized testing and screening method for plant-sourced compounds, a

reasonable comparison of results becomes difficult (Halliwell, 2009).

63

Table 1.6 Phyto-chemical composition and bioactivities of fruit and vegetable extracts

Product Extraction, analytical and assay methods

Phyto-compounds present Potential activity Reference

Anise - Hot water and ethanol extraction - Total phenolic compounds -Reducing power assay, superoxide anion scavenging activity, free radical scavenging activity, scavenging of hydrogen peroxide - Zone inhibition method

Anethole, eugenol pseudoisoeugenol methylchavicol, coumarins, scopoletin, umbelliferon, terpene hydrocarbons, polyenes and polyacetylenes

Antioxidant, antimicrobial

(Faculty, 2003)

Asparagus (by-product)

-Ethanol extraction -HPLC -DPPH

Saponins, flavonoids, and hydroxycinnamates,

-sitosterol

Antioxidant

(Fuentes-alventosa et al., 2009)

Broccoli -Solvent extraction (methylene chloride, methanol) -GC-MS -DPPH, reducing power assay, superoxide anion radical and hydroxyl radical assays

Flavones, ascorbic acid, quecetin, larciresinol, pinoresinol,

secoisolariciresinol, -carotene, -

carotene, -tocopherol,

campestanol, -sitosterol, -sitostanol, glucoiberin, glucorapinin, glucoalyssin

Antioxidant

(Yuan et al., 2010)

Black berry (leaves)

-Solvent extraction (methanol) - HPLC - TEAC, ABTS - MBC

Ellagic acid, quercetin, rutin, ferulic acid, gallic acid, caffeic acid, kaemferol

Anti-inflammatory, antiviral and antimicrobial, antioxidant

(Martini et al., 2009)

Blue berry fruit

- Acetone extraction - Total phenolic content -Oxygen radical scavenging activity, hydroxyl radical scavenging activity, DPPH, H2O2 assay, , pH differential method

Phenolics, anthocyanins, flavanols, hydroxycinnamic acid, ascorbic acid

Antioxidant and antimicrobial (Wang et al., 2010)

Cabbage - Methanol extraction - Free radical scavenging activity-DPPH and ABTS radicals

Flavanols, sterols, glucosinolates Antioxidant

(Bartoszek et al., 2008)

Carrot - Solvent extraction (water and alcohol) - HPLC-DAD -MIC

Alkaloids, naphtho- and anthraquinones, terpenoids, saponins, flavones, flavonols, phenolic acids derivatives, lactones, isocourmarin(6-methoxymellein), polyacetylenes (falcarionl and falcarindiol)

Antimicrobial

(Vegara et al., 2011)

Cherry -Solvent extraction: (acetone/water/acetic acid (70:29:1, v/v/v) -Total phenolic content, HPLC MS/MS, -DPPH radical scavenging activity

Myricetin, quercetin, catechins, flavonols, and proanthocyanidins

Antioxidant

(Bonat et al., 2011)

Coriander - Hydrodistillation extraction - Total phenolic content, GC-MS

- DPPH, reducing power assay, -carotene/ linoleic acid bleaching assay

Linalool, pinene, phellandrene, eucalyptol, linalool, borneol, β-caryophyllene, citronellol, geraniol, thymol, linalyl acetate, geranyl acetate, caryophyllene oxide, elemol and methyl heptenol

Antioxidant

(Neffati et al., 2011)

Cranberry -Water extraction and ethanol extraction -Total soluble phenolic assay, HPLC, -DPPH, -Plate count

Proanthocyanidins, flavanols, quercitin, ellagic acid, rosmarnic acid, resveratrol

Antioxidant, antimicrobial (Vattem et al., 2004)

http://en.wikipedia.org/wiki/Linalool

http://en.wikipedia.org/wiki/Pinene

64

Table 1.6 Phyto-chemical composition and bioactivities of fruit and vegetable extracts (Continued)

Product Extraction, analytical and assay methods Phyto-compounds

present Potential activity

Reference

Fenugreek -Solvents and water extraction -Lipid peroxidation inhibitory activity, DPPH scavenging activity, FRAP - MIC, disc diffusion method

Polyphenols, flavonoids Antioxidant, antimicrobial (Premanath,

2011)

Grape (seeds)

- Hot water (80-90OC) extraction

--carotene, linoleic acid peroxidation assay, FRAP, DPPH - MIC

Stilbenes, catechins, epicatechins, and gallic acid, and the polymeric and oligomeric procyanidins

Antioxidant, antimicrobial (Adamez et al.,

2012)

Green pepper

- Methanol extraction -GC-MS, total polyphenols estimation, - DPPH, ABTS radical scavenging, ß-carotene bleaching assay

Carotenoids, terpenoids, alkaloids, flavonoids, lignans, simple phenols and phenolic acids,3 4 dihydroxyphenyl ethanol glucoside,3 4-dihydroxy 6 (N-ethylamino) benzamide

Antioxidant, anti- inflammatory, anti-cancer, anti-aging, anti-bacterial

(Chatterjee et al., 2007)

Kale -Soxhlet extraction (n-hexane and petroleum ether) - LC-MS/MS, total phenolic content -DPPH -Zone inhibition assay

Ferulic and caffeic acids, glucosinolates, flavonoids, phenolics

Antioxidant, antimicrobial (Ulrichova and

Strnad, 2008)

Kiwi - Water extraction -Total phenolic contents assay, total flavonoid content by calorimetric assay, LC-MS/MS, - DPPH, ABTS, Fe3+ reducing activity, cupric ion (Cu2+) reducing-CUPRAC assay, ferrous ion (Fe2+) chelating activity, superoxide anion radical scavenging activity

α-tocopherol), ascorbate acid, phenolics, carotenoids

Antioxidant

(Bursal and Galain, 2011)

Lettuce - Methanol extraction and water extraction - Total phenolic content, - DPPH, reducing power assay - MIC, cyto-pathic effect assay, cell toxicity assay

Flavonoid, tannins, polyphhenols

Antioxidant, antiviral, antibacterial, analgesic, anti-inflammatory

(Edziri et al., 2011)

Lychee seeds

- Ethanol extraction - Total phenolic content assay, fatty acid profile by gas chromatography DPPH

Flavonoids, citric acid, ascorbic acid, tocopherols, carotenoids

Antioxidant, serum fatty acid profile

(Maria et al., 2011)

Mango - Solvent extraction (methanol) - Total phenolic content - DPPH, FRAP

Anacardic acid, phenolic compounds, carotenoids, ascorbic acid

Antioxidant

(Palafox-carlos et al., 2012)

Mangosteen - Ethanol extraction -Total phenolic and flavonoid content assay - DPPH - MIC, MBC,

Prenylated and oxygenated xanthones, essential oils, tannins, flavonoids

Antioxidant, antimicrobial (Pothitirat and

Traidej, 2009) (Yai, 2010)

Orange (peel and juice)

- Hydro-distillation and methanol extraction -DPPH, reducing power and inhibition of lipid

peroxidation using -carotene–linoleate model system in liposomes, TBARS assay in brain homogenates

Flavanone glycosides, hydroxycinnamic acids,vitamin C, and carotenoids

Antioxidant

(Guimaraes et al., 2010)

Pineapple - Ethyl acetate extraction - Total phenolics, total flavonoids - Lipid peroxidation β-carotene-linoleate lipid model system, DPPH

Flavonoids, isoflavones, flavones, anthocyanins, catechins and other phenolics

Antioxidant

(Hossain and Rahman, 2011)

http://www.scialert.net/asci/result.php?searchin=Keywords&cat=&ascicat=ALL&Submit=Search&keyword=lipid+peroxidation

65

Table 1.6 Phyto-chemical composition and bioactivities of fruit and vegetable extracts (Continued)

Products Extraction, analytical and assay methods

Phyto-compounds present Potential activity

Reference

Pomegranate - Petroleum ether extraction - Total phenolic content assay, HPLC - DPPH, TEAC, - agar-well diffusion method

Polyphenols, ascorbic acid, tannins, flavonoids

Antioxidant, antimicrobial (Ferrara et al.,

2009)

Raspberry - Solvent extraction (acetone, methanol) - Total phenolic content, HPLC, - ORAC, DPPH, pH differential method

Flavonoids, phenolic acids, stilbenes, and procyanidins, cyanidins, anthocyanins, ellagitannins, phenolic acids, conjugates of ellagic acid and quercetin

Anti-inflammation, and anti-proliferation, antioxidant

(Zhang et al., 2010)

Red onion - Methanol extraction -HPLC, MS/MS,

--carotene and linoleic acid coupled reaction method, FRAP, DPPH, hydroxyl ions scavenging

Polyphenols, anthocyanins, flavonoids, quercetin, kaempferol, glycosides, tocopherol, carotenoids, angiotensin, melatonin, alliins (sulphur compounds), enzymes (superoxide dismutase, glutathione peroxidase and catalase)

Antimicrobial, antispasmodic, anticholesterolemic, hypotensive, hypo-glycemic, anti-asthmatic, and anti-cancer activities, antioxidant

(Dini et al., 2008), (Prakash et al., 2007)

Rhubarb - Solvent extraction (chloroform and methanol) - Total phenolic content, total flavonoids content

- DPPH, -carotene-bleaching assay, Free radical scavenging activity, Cupric reducing antioxidant capacity (CUPRAC), reducing power, metal chelating activity

Flavonoids, stilbenes and anthraquinones, carotenoids and vitamins C and E

Antioxidant

(Aydog and Duru, 2007)

Rosemary - Steam-distillation - DPPH - Zone inhibition assay, MIC

Carvacrol, eugenol, thymol, tocopherols, flavonoids, carnosic acid (CA), carnosol (COH) and rosmarinic acid (RA)

Antimicrobial, antioxidant (Seydim and

Sarikus, 2006)

Sea buckthorn -Liquid–liquid extraction (Hexane, ethyl acetate and water) -Total phenolic content, condensed tannin content, HP-TLC -FRAP, DPPH

Ascorbic acid, flavonoids, carotenoids, proanthocyanidins

Antioxidant, Antimicrobial (Michel et al.,

2012b)

Strawberry - Solvent extraction (ethanol) - Total phenolic content assay - Reducing power assay, DPPH

Ascorbic acid, -tocopherol, glutathione, carotenoids and flavonoids, total phenolics, proanthocyanidines

Antioxidant

(Oliveira et al., 2011)

Thai gac -Solvent extraction (hexane, acetone, ethanol and methanol) -Total flavonoid content, HPLC-diode array, DPPH, FRAP

-carotene, vitamin A, lycopene, lutein Antioxidant

(Kubola and Siriamornpun, 2011)

Tomato - Total phenolic content, total flavonoids content, HPLC- lycopene and ascorbic acid,

- FRAP, ORAC, DPPH, -carotene linoleic acid assay (lipid peroxidation)

Phenolics, carotenoids, ascorbic acid,

-tocopherol, tomatine (alkaloid)

Anti oxidant

(Liu et al., 2011)

66

1.5 Hypotheses

A variety of phytochemical compounds can be extracted from fruit and vegetable wastes,

and some of which may exhibit both antioxidant and antimicrobial properties. There are

certain common physico-chemical features that may confer to them both antioxidant and

antimicrobial properties, including lipophilicity-hydrophilicity balance, acidity-alkalinity

balance, redox potential and metal complexing ability. Identification of plant extracts

exhibiting both antioxidant and antimicrobial activityies could lead to the development of

effective antioxidant-antimicrobials for use in the preservation of foods, potentially

replacing synthetic preservatives.

67

1.6 Objectives

1.6.1 General Objective

The objective of this work is to conduct studies to evaluate the potential of extracts

obtained from various fruit and vegetable by-products towards the development of

antioxidant-antimicrobials for use as food preservation agents, as alternative to synthetic

agents.

1.6.2 Specific Objectives

Specifically, the research involves:

(1) The identification of promising antioxidant-antimicrobial extracts from fruits and

vegetable by-products, generated in the field or processing;

(2) Antioxidant characterization of plant extracts for their capacity and efficacy;

(3) Evaluation of selected edible plant extracts for their spectrum of anti-radical activity

of importance in biology;

(4) Comprehensive evaluation of selected edible plant extracts for their antimicrobial

activity; and

(5) Exploration of ways to enhance the antimicrobial activity of selected antioxidant-

antimicrobial extracts, primarily by blending of extracts in various proportions and

by the addition of various phyto-compounds that may be inherently antimicrobial or

not.

A positive result marked by an appreciable inhibitory activity against microbial growth and

viability would qualify an extract for evaluation at the next level, where the extract would

be tested with defined paradigms such as, colony count tests, MIC, MBC and kill kinetics.

The most promising extracts would be evaluated for spectrum spectrum of antimicrobial

action against selected aerobic Gram-positive and Gram-negative bacteria, yeasts and fungi.

68

Chapter 2 NATURAL ANTIOXIDANT-ANTIMICROBIALS: SCREENING OF

FRUITS, VEGETABLES AND THEIR BY-PRODUCTS

Environ 160 extraits aqueux de sous-produits de fruits et légumes ont été criblés pour évaluer leur potentiel

antimicrobien et anti-oxydant. L’activité antimicrobienne a été déterminée par l’inhibition de la croissance

d’Escherichi coli et de Bacillus subtilis. L’activité anti-oxydante a été mise évidence par le test au DPPH (2,2-

diphenyl -1-picrylhydrazyl). L'étude a conduit à l'identification des extraits présentant à la fois un potentiel

antimicrobien et une propriété anti-oxydante. Les propriétés bioactives des extraits sont influencées par le pH

de l'extrait, le type de tissu (fruits, feuilles et racines) et le type physiologique des fruits (climactérique). Les

résultats ont montré l’existence d’une relation entre les propriétés anti-oxydantes et antimicrobiennes des

extraits de plantes. De ce fait, l'indice anti-oxydant-antimicrobien développé pourrait être utile dans le choix

des sources végétales bioactives. Les extraits de plantes aux propriétés antimicrobiennes et anti-oxydantes

présentent un potentiel comme agent de conservation sécuritaire, bénéfique pour la santé et économique. Ce

résultat permet aussi de sélectionner 36 échantillons pour des études suivantes.

69

2.1 Abstract

Exploration of naturally occurring food additives for food preservation has received

enormous attention with increasing consumer concern for the synthetic chemical additives

in food. The antimicrobial and antioxidant properties of aqueous extracts of 164 fruit and

vegetable by-products were assayed. The growth inhibiting activity (GI) of the extracts

were tested against Escherichia coli (ATCC 25922), and Bacillus subtilis (ATCC 6633);

and the antioxidant activity (AO) was determined by DPPH radical scavenging assay. The

results showed that the samples exhibited varying degrees of growth inhibitory activity

against both test bacteria. About 53% of the samples showed growth inhibition of ≥70%.

Pomegranate fruit (98 %), betel leaf (97 %) and black currant fruit residue (95 %) exhibited

the highest antibacterial activity against E.coli, while black currant fruit (97 %), strawberry

leaf (96 %) and egg plant (96 %) showed the best antibacterial property against B.subtilis.

About 41 % of the samples displayed DPPH radical scavenging capacity of higher than 100

µg of ascorbic acid equivalent/mg-dry weight. Samples that showed the best anti-radial

capacity were: betel leaf (143.59 µg AAE/mg), green grape fruit (141.38 µg AAE/mg) and

pomegranate (141.34 µg AAE/mg). Non-climacteric fruits, in general, had a higher growth

inhibiting capacity against E.coli and B.subtilis compared with the climacteric fruits. The

samples from the Brassicaceae and Amaryllidaceae family displayed a low antioxidant

activity by DPPH assay. It was found that there was a positive correlation between the

antimicrobial and antioxidant activity of the plant extracts (r=0.65 for E.coli and r=0.57 for

B.subtilis). The pH and the type of plant tissue had an effect on the GI and AO activity.

Furthermore, fruit peels were observed to possess a higher activity compared to leaf

samples followed by root peels. E.coli 25922 (a Gram-negative bacteria) was found to be

significantly more sensitive to the plant extracts than B.subtilis 6633 (a Gram-positive

bacteria). A certain number of non-climacteric fruit and leaf extracts were found to have

potential as antioxidant-antimicrobials.

Key words: Bioactive compounds, antibacterials, anti-radical, solvent extraction,

climacteric, plant parts, Brassicaceae, Amaryllidaceae

70

2.2 Introduction

Vegetables and fruits yield between 25 % and 75 % of non-edible by-products as wastes

(for example, in 2010, apple 30%, pineapple 45%, citrus 70%) (Van Dyk et al., 2013), after

processing of fruit and vegetable in the food processing industry; and they contain a large

amount of bioactive compounds. The by-products of fruits and vegetables are made up of

skins, and seeds of different shapes and sizes that normally have no further usage, and are

usually discarded. These raw by-products may be good sources of AO and AM for use in

food applications.

Fruit and vegetable peels are first barrier-rich substances in plants which protect against

attack from the pests such as insects and other plant-feeding animals, and environmental

stresses such as UV radiation, heat, cold, heavy metals, and chemicals. Therefore, the fruit

and vegetable peel residues could be an excellent, inexpensive and readily available

resource for bioactive compounds for use in the food and pharmaceutical industries;

thereby, adding value to the processing wastes and also contributing to the economic

benefit of food processors.

The most abundant by-products of minimally processed fresh fruit and vegetable products

are fruit peels and seeds which have been reported to contain high amounts of phenolic

compounds with antioxidant and antimicrobial properties (Balasundram et al., 2006). The

antimicrobial activities of a variety of naturally occurring phenolic compounds from

different plant sources have been studied in detail (Cueva et al., 2010b; Daglia, 2012b;

Quideau et al., 2011). These compounds play an important role in the protection of crops

against pathogenic agents, damaging the cell membrane of microorganisms, and causing

lysis of the cells. Phenolic compounds from spices such as gingeron, zingerone, and

capsaicin have been found to inhibit the germination of bacterial spores (Burt, 2004).

Polyphenols from green tea have also been explored for broad spectrum activity against

pathogens (Taylor et al., 2005). In addition, flavonoids when used in conjunction have been

reported to enhance the antibacterial, antiviral, and anticancer activities of compounds such

as naringenin, acycloguanosine, and tamoxifen (Ayala-Zavala et al., 2010).

Extensive research also points to the potential of bioactive compounds from plant by-

71

products as a source of antioxidants (Agourram et al., 2013; Moure et al., 2001; Santana-

Méridas et al., 2012; Van Dyk et al., 2013). Of late, the use of synthetic antioxidants has

come under the scanner and their use is being strictly regulated (Lobo et al., 2010). Similar

to the changing perception towards synthetic antioxidants, increasing reports of resistance

of pathogens against synthetic antimicrobials has led to limitations on their use (Davidson

and Branen, 2005). It was found that total phenolic and flavonoid content was higher in

peels in comparison to other parts (Gözlekçi et al., 2011). The radical scavenging activity

was found to correlate well with the total phenolic content and flavonoids, where samples

with the lowest total phenolic content and flavonoids displayed the lowest radical

scavenging activity (Aksoy et al., 2013; Sen et al., 2013).

The objective of this study was to identify plant extracts exhibiting both antimicrobial and

antioxidant properties, and examine possible relationships between them. We performed

exhaustive screening of various edible plant by-products and wastes for potential

antimicrobial and antioxidant properties. Furthermore, the antimicrobial and antioxidant

properties of the extracts were also examined in relation to their sources such as plant tissue

type and the physiological type of fruit (climacteric and non-climacteric).

72

2.3 Materials and Methods

2.3.1 Plant materials

All fruit, vegetable and plant by-products were collected from farms, farmer’s markets and

fruit and vegetable processors in and around Quebec City. All collected samples were

transported the same day and stored at 4°C until further processing. Subsequently within 48

hours, they were lyophilized, ground to powder, vacuum-sealed and stored at -30°C.

2.3.2 Chemicals and reagents

Chemicals, diphenyl-2-picrylhydrazyl, ascorbic acid, gallic acid and Murielle Hilton Broth

were procured from Sigma Aldrich USA. All other reagents were of analytical grade.

2.3.3 Preparation of extracts

About 50 gram of the lyophilized powder of raw sample was extracted with 500 mL of

distilled water at 85-90°C for 60 minutes with constant stirring. The slurry was then

vacuum filtered (Whatman No. 1 filter paper), and the filtrate was concentrated under

vacuum using a rotary evaporator at 65°C to about 100 mL and then lyophilized. The

lyophilized extracts were vacuum-packed and stored at -30oC until further use. For the

antimicrobial and antioxidant assays the samples were dispersed in Mueller-Hinton broth

and methanol, to obtain a stock solution of 100 mg/mL and 1 mg/mL, respectively. All the

stock solutions were purged with argon, stored at 4oC and used within 24 hours.

2.3.4 Bacterial strains and inoculum preparation

The antimicrobial activity of the extracts was determined against Escherichia coli ATCC

25922 (a Gram-negative) and Bacillus subtilis ATCC 6633 (a Gram-positive) bacteria.

Bacterial cultures were revived from stocks kept at -80°C by three successive growth cycles

at 37 °C for 24 h in Mueller Hilton Broth (Sigma Aldrich). The standard inoculums were

prepared following NCCLS guidelines (Jorgensen and Ferraro, 2009) with organisms in

their log growth phase (4h after incubation), and then adjusted to match 0.5 McFarland

standard (corresponds to approximately to 1.5 X 108 CFU/mL). The standardized

suspensions were used within 15 minutes.

73

2.3.5 Antimicrobial activity assay

The antimicrobial activity of plant extracts was determined using a microtitre broth dilution

assay (NCCLS, 1991; Wiegand et al., 2008). Briefly, 190 µl of plant extract in MH broth

(10 mg/mL) was pipetted in the 96-well plate. Subsequently, the wells were inoculated with

the test strain (10µl) to achieve a final concentration of 104 to 105 CFU/mL in each well.

The blank and assay control was the plant extract (190 µl at 10 mg/mL) and 10 µl of MHB

broth without the bacterial strain, respectively; where the latter also served as the sterility

control. Chloramphenicol (1 mg/mL) was used as the positive control, while the inoculated

MHB broth as the negative control. The prepared plates were incubated at 37°C in a micro-

plate reader (Biotek Power XS2), and absorbance readings (OD600nm) were recorded hourly

for 24 hours. All assays were performed in triplicate. The percentage of growth inhibition

was calculated by the difference between the negative control (inoculum without plant

extract) and that contains the sample.

2.3.6 Antioxidant assay - DPPH radical-scavenging capacity

The DPPH radical scavenging was analysed through a standard spectrophotometric method

(Sharma and Bhat, 2009) with modifications. A stock solution of DPPH (6.1x 10-5 M) was

freshly prepared in methanol and was used immediately. The DPPH stock solution was

diluted with methanol and adjusted to OD of 0.7 (± 0.02) at 515 nm. Subsequently, 190 µL

of the DPPH solution was pipetted into a 96 well micro-plate (Becton Dickinson Falcon

353072), followed by addition of 10 µL of plant extracts (1.0 mg/mL). Thereafter, the plate

was incubated in a (Biotek Power XS2 Logicel Gen 5) spectrophotometer at 25°C for a

period of 1 hour, with absorbance readings (515 nm) recorded every 1 min. Ascorbic acid

(1 mg/mL) was used as a standard and the results were expressed as ascorbic acid

equivalent (µg AAE/mg extract). The assays were carried out in triplicate.

Radical scavenging activity = [(A0 − A1)/A0 ]× 100 (%)

Where A0 was the absorbance of the control sample (without plant extract) and A1 was the

absorbance in the presence of the test sample.

74

2.3.7 Statistical analyses

The results of the growth inhibition assay were expressed as mean of the percentage of

inhibition ± standard deviation (SD) of three replicates. The result of the DPPH radical

scavenging assay was presented as µg AAE/mg extract ± standard deviation (SD) of three

replicates. Antimicrobial and antioxidant activities were analyzed as a function of plant

tissue types (fruit peel, leaf, root peel); physiological type of the fruit (climacteric fruit vs.

non-climacteric fruit); and test strains used (E. coli (Gram -) vs B. subtilis (Gram +)). The

data were subjected to means comparison using Kruscal-Wallis test (Wilcoxon test

followed by Kruscal-Wallis post hoc analysis) using R software at a significance level of

5% (p=0.05).

75

2.4 Results

2.4.1 Antimicrobial activity

The antimicrobial activity of 164 samples (80 fruit peels, 67 leaves, and 17 root peels)

against the two test strains (E.coli and B.subtilis) is presented in Table 2.1. The inhibition

activity (GI) is presented as percentage of growth inhibition. The results showed that all the

samples exhibited antimicrobial activity, with 53 % of the samples showing >70 % GI at

the tested concentration of 10 mg/mL; about 6 % of the samples exhibiting GI of > 90 %.

Among them, a small number of extracts exhibited GI of > 90 %: black currant, betel leaf,

cranberry, egg plant, pomegranate, rambutan, raspberry, grape leaf and sea buckthorn leaf.

Among the materials evaluated, three samples exhibiting the highest antibacterial activity

against E.coli were pomegranate fruit (98 %), betel leaf (97 %) and black currant fruit

residue (95 %); whereas the three best extracts active against B. subtilis were black currant

fruit (97 %), strawberry leaf (96.07%) and egg plant peel (96 %). The extracts in general

displayed antimicrobial activity towards both bacteria. The mean growth inhibition activity

against E.coli was 65 %, and was significantly higher (p<0.05) than the mean of GI against

B.subtilis 6633 (59 %) as seen in Figure 2.1.

Figure 2.1 A comparison of sensitivity of E.coli and B.subtilis against 164 plant extracts

E. coli B. subtilis

20

40

60

80

10

0

Gro

wth

in

hib

itio

n, %

76

The inhibition activity of 80 fruit peel samples comprising of 30 climacteric fruits and 50

non-climacteric fruits is presented in Figure 2.2. The antimicrobial activity of non-

climacteric fruit peels (E.coli, 74 % and B.subtilis, 68 %) was significantly higher (p<0.05)

than those of climacteric fruit peels (E.coli 60.89% and B.subtilis, 54 % (Figures 2.2, 2.3).

The samples were divided according to their tissue types (fruit peels, leaf and root peels),

and their GI was compared. The type of plant tissue (fruit, leaf and root) was also corelated

with the inhibitory activity of the extracts. The fruit peel extracts exhibited a higher

antibacterial activity than leaf extracts (p<0.05), followed by root peel extracts (Figures 2.4

and 2.5).

It was observed that the pH of the extract was a factor influencing the GI of extracts. The

pH of the extract negatively correlated with the growth inhibition of both the test strains, as

seen in Figure 2.7, which shows coefficient of correlation, r of -0.39 for E.coli and r of -

0.37 for B.subtilis.

77

Climacteric Non-climacteric

30

40

50

60

70

80

90

Gro

wth

inh

ibiti

on

, % )

Figure 2.3 Effect of physiological type of fruits on the

growth inhibition of E.coli


20

40

60

80

10

0

Gro

wth

in

hib

itio

n, %

)

Figure 2.2 Effect of physiological type of fruits on the

growth inhibition of B.subtilis

Fruit Leaf Root

30

40

50

60

70

80

90

Gro

wth

in

hib

itio

n, %

)

Figure 2.4 Effect of the plant tissue type on the

growth inhibition of E. coli

Fruit Leaf Root

20

40

60

80

10

0

Gro

wth

in

hib

itio

n, %

)

Figure 2.5 Effect of the plant tissue type on the

growth inhibition of B. subtilis

78

Table 2.1 Antimicrobial and Antioxidant activity of Climacteric fruits

No. Sample name Botanical name pH E. coli *

(% GI ±SD)

EC

Index

B. subtilis *

(% GI ±SD)

BS

index

DPPH ±SD **

(µg AAE/mg

extract)

DPPH

index

AO-

AM

index

1 Apple Malus domestica 4.72 31.46± 6.31 0.32 15.86± 5.86 0.16 32.19± 1.41 0.19 0.22

2 Artichoke (base) Cynara cardunculus 5.67 39.36± 6.05 0.33 42.1± 1.27 0.41 21.6± 2.49 0.08 0.28

3 Artichoke (petal) Cynara cardunculus 5.93 33.24± 2.09 0.39 40.68± 0.96 0.43 14.1± 1.2 0.13 0.32

4 Avocado Persea americana 5.64 77.52± 6.61 0.78 76.95± 1.48 0.78 138.71± 4.69 0.82 0.79

5 Banana Musa acuminata 5.42 26.44± 8.43 0.26 19.86± 7.27 0.20 16.67± 3.66 0.10 0.19

6 Black fig Ficus carica 5.25 79.28± 2.05 0.79 83.26± 0.36 0.85 127.89± 4.41 0.76 0.80

7 Cantaloupe Cucumis melo var.

cantalupensis 5.12 51.3± 3.61

0.51 66.87± 9.71

0.68 28.64± 1.33

0.17 0.45

8 Cape

Gooseberry

Cucumis melo var.

cantalupensis 4.98 62.88± 0.87

0.63 63.47± 3.58

0.65 43.98± 8.91

0.26 0.51

9 Coffee beans

(Roasted) Coffea arabica 4.93 78.33± 1.53

0.78 51± 4.24

0.52 120.16± 1.96

0.71 0.67

10 Coing fruit Cydonia oblonga 4.18 66.67± 6.03 0.67 49.5± 6.36 0.50 134.67± 4.1 0.80 0.66

11 Custard apple Annona cherimola 5.31 79.95± 8.97 0.80 75.54± 0.66 0.77 136.41± 2.69 0.81 0.79

12 Date (Raw) Phoenix dactylifera 4.98 67.07± 4.64 0.67 66.19± 4.24 0.66 50.66± 1.19 0.31 0.55

13 Date (Red) Phoenix dactylifera 4.73 67.33± 5.77 0.75 67.5± 3.54 0.26 108.46± 6.99 0.51 0.51

14 Date molasses Phoenix dactylifera 4.98 57.69± 2.52 0.30 70.24± 1.07 0.19 39.57± 1.29 0.18 0.22

15 Dates (Fresh) Phoenix dactylifera 5.67 67.03± 2.4 0.68 64.99± 4.26 0.59 51.78± 2.3 0.74 0.67

16 Gac fruit Momordica

cochinchinensis 7.11 75.08± 2.89

0.69 25.87± 9.98

0.76 85.9± 1.02

0.83 0.76

17 Granadilla Passiflora ligularis 5.26 67.67± 5.51 0.68 58± 5.66 0.52 124.06± 1.92 0.25 0.48

18 Green almond Prunus amygdalus 5.18 30.35± 5.01 0.37 18.39± 4.32 0.25 29.93± 3.45 0.75 0.46

19 Jujuba Simmondsia chinensis 4.96 68.67± 8.14 0.62 74.5± 2.12 0.57 139.77± 3.25 0.39 0.53

20 Kaki Diospyros kaki 5.17 67.67± 5.77 0.67 51.5± 3.54 0.68 42.03± 3.54 0.80 0.72

21 Kiwi Actinidia deliciosa 4.2 62.37± 7.53 0.42 56.22± 5.35 0.45 66.41± 5.01 0.40 0.42

22 Langsat Lansium domesticum 4.83 67.22± 4.36 0.58 66.5± 0.71 0.71 134.58± 3.34 0.24 0.51

23 Leafy Kale Acephala Group 6.14 36.51± 1.44 0.44 24.5± 0.71 0.30 126.96± 1.37 0.19 0.31

24 Mango Caesia Mangifera

(cultivar Ataulfo) 3.87 41.58± 1.51

0.77 44.35± 0.92

0.65 66.86± 3.78

0.83 0.75

25 Papaya (Raw) Carica papaya 5.54 63.44± 5.76 0.85 38.4± 9.33 0.70 68.8± 7.65 0.76 0.77

26 Passion Fruit Passiflora edulis 5.01 43.67± 7.09 0.67 29.11± 2.83 0.67 32.67± 1.64 0.30 0.55

27 Pistachio Pistacia vera 4.42 77.03± 8.89 0.67 64.12± 2.83 0.69 139.1± 2.76 0.64 0.67

28 Plum Prunus domestica 3.45 85.1± 10.15 0.84 69.32± 2.83 0.89 128.57± 3.26 0.57 0.77

29 Raw mango Caesia Mangifera 4.3 84.04±

10.45

0.64 87.53± 0.74

0.39 95.76± 8.6

0.41 0.48

30 Tomato Solanum lycopersicum 4.76 71.07± 2.92 0.71 53.5± 2.13 0.54 74.89± 3.25 0.44 0.57

*GI (%) of chloramphenicol at 1 mg/mL for E.coli is 99.81%

*GI (%) of chloramphenicol at 1 mg/mL for B.subtilis is 98.38%

** AO activity of acid ascorbic at 1mg/mL is 168.33 µg AAE/mL

79

Table 2.2 Antimicrobial and Antioxidant activity of Non-climacteric fruits


(% GI ±SD)

EC

Index

B. subtilis *

(% GI ±SD)

BS

index

DPPH±SD **

(µg AAE/mg

extract)

DPPH

index

AO-

AM

index

31 Areca nut Areca catechu 5.8 80.35± 4.12 0.81 75.96± 2.3 0.77 138.52± 2.17 0.82 0.80

32 Bangladesh lemon

(Satkara) Citrus Macroptera 4.74 61.32± 5.29

0.61 58.5± 2.12

0.59 30.45± 2.41

0.18 0.46

33 Bitter Gourd Momordica charantia 4.99 79.04± 10.24 0.79 88.16± 3.05 0.90 103.59± 7.26 0.62 0.77

34 Black currant Ribes nigrum 3.27 91.23± 1.69 0.91 97.38± 3.36 0.99 133.12± 1.2 0.79 0.90

35 Black currant

(Fermented residue) Ribes nigrum 3.2 69.32± 5.23

0.69 79.56± 1.41

0.81 127.5± 2.47

0.76 0.75

36 Black currant (residue) Ribes nigrum 3.55 94.73± 3.52 0.95 80.5± 0.71 0.82 135.12± 6.98 0.80 0.86

37 Blueberry Vaccinium corymbosum 3.85 79.45± 8.1 0.80 65.82± 5.92 0.67 137.87± 4.54 0.82 0.76

38 Cactus Opuntia ficus-indica 5.56 63.81± 1.09 0.64 47.7± 3.82 0.48 53.95± 7.55 0.32 0.48

39 Canadian cucumber Cucumis sativus 6.23 78.64± 7.19 0.79 69± 4.25 0.70 11.34± 1.69 0.07 0.52

40 Canadian Yew taxus canadensis 4.78 77.39± 6.5 0.78 79.75± 1.77 0.81 34.24± 2.92 0.20 0.60

41 Carambola Averrhoa carambola 4.13 72.53± 6.08 0.73 67.5± 3.54 0.69 118.4± 2.26 0.70 0.71

42 Carob molasses Ceratonia siliqua 4.56 61.05± 5.29 0.61 73.96± 1.36 0.75 28.43± 1.1 0.17 0.51

43 Cashew apple Anacardium occidentale 5.23 78.86±5.61 0.79 72.65±4.32 0.74 96.34±3.97 0.57 0.70

44 Cherry Prunus avium 4.5 50.05± 7.07 0.50 73.18± 0.25 0.74 42.86± 2.92 0.25 0.50

45 Cranberry Vaccinium macrocarpon 2.69 94.33± 8.08 0.95 95± 2.83 0.97 138.52± 5.51 0.82 0.91

46 Drum Sticks Moringa oleifera 5.02 63.82± 2.87 0.64 27.25± 3.89 0.28 16.55± 7.04 0.10 0.34

47 Egg plant Solanum melongena 5.09 87.4± 2.1 0.88 95.61± 1.3 0.97 125.39± 4.9 0.74 0.86

48 English cucumber Cucumis sativus 6.12 34.03± 3.55 0.34 19.04± 8.44 0.19 12.7± 3.43 0.08 0.20

49 Grape (Black) Vitis vinifera 3.8 66.21± 4.11 0.66 48.41± 4.82 0.49 77.17± 2.32 0.46 0.54

50 Grape Green (wine)

(Residue) Vitis vinifera 3.55 78.33± 6.26

0.78 76± 1.41

0.77 138.07± 4.37

0.82 0.79

51 Grape Red Vitis vinifera 3.92 81.67± 6.17 0.82 72.5± 3.54 0.74 140.5± 2.75 0.83 0.80

52 Grape Red (wine) Vitis vinifera 4.17 70.2± 5.2 0.70 72.5± 0.71 0.74 130.86± 5.07 0.78 0.74

53 Grape (Red) seed

(Fermented residue) Vitis vinifera 4.31 88.33± 2.08

0.88 82.5± 2.12

0.84 132.64± 1.41

0.79 0.84

54 Grape Red (wine)

(Fermented residue) Vitis vinifera 3.49 82.67± 2.08

0.83 75± 1.41

0.76 132.69± 2.84

0.79 0.79

55 Green Chilly Cultivar Capsicum 5.62 82.13± 7.68 0.82 68.15± 4.04 0.69 29.66± 7.79 0.18 0.56

56 Green grape (Table) Vitis vinifera 4.12 78.46± 7.43 0.79 74.43± 5.05 0.76 134.47± 2.19 0.80 0.78

57 Green grape (wine) Vitis vinifera 4.26 87.67± 1.15 0.88 90.5± 0.71 0.92 141.38± 4.45 0.84 0.88

58 Green Italian pepper Capsicum annuum 6.07 73.65± 6.84 0.74 93.77± 5.32 0.95 113.43± 5.06 0.67 0.79

59 Indian Plum Ziziphus mauritiana 4.89 88.33± 1.15 0.88 84.5± 6.36 0.86 136.11± 1.19 0.81 0.85

60 Ivy Gourd Coccinia grandis 5.18 41.03± 5.24 0.41 29.34± 3.76 0.30 14.96± 5.84 0.09 0.27

61 Kumquat Citrus japonica 4.4 76.83± 7.19 0.77 66± 8.49 0.67 90.53± 4.18 0.54 0.66

62 Lemon albido Citrus lemon 4.95 71.3± 2.07 0.71 71.47± 0.76 0.73 117.64± 5.01 0.70 0.71

63 Lemon flavido Citrus lemon 5.02 70.46± 4.01 0.71 74.49± 7.76 0.76 84.68± 1.64 0.50 0.66

64 Lychee Litchi chinensis 4.11 78.96± 3.18 0.79 71.91± 4.11 0.73 130.96± 1.37 0.78 0.77

65 Mangosteen Garcinia mangostana 4.58 80.44± 7.88 0.81 82.78± 3.93 0.84 136.93± 1.84 0.81 0.82

66 Noni Morinda citrifolia 5.82 94.39± 7.8 0.95 92.08± 2.83 0.94 140.86± 2.83 0.84 0.91

67 Olive (Jolpai) Olea europaea 3.51 93.33± 5.13 0.94 80.5± 7.78 0.82 113.02± 6.71 0.67 0.81

68 Olive (dried mill

effluent) Olea europaea 4.12 97.55±8.14

0.98 98.23±3.22

1.00 136.75±4.45

0.81 0.93

69 Orange albido Citrus sinensis 4.56 50.8± 1.93 0.51 23.09± 2.7 0.23 111.55± 3.26 0.66 0.47

70 Orange flavido Citrus sinensis 5.01 42.81± 4.84 0.43 19.21± 1.12 0.20 53.28± 9.78 0.32 0.31

71 Pineapple Ananas comosus 3.89 66.65± 3.39 0.67 66.32± 0.45 0.67 25.29± 0.69 0.15 0.50

72 Pomegranate Punica granatum 3.55 97.81± 2.05 0.98 87.32± 8.03 0.89 141.34± 3.54 0.84 0.90

73 Pomegranate seeds Punica granatum 5.08 91.67± 1.15 0.92 90.5± 0.71 0.92 87.56± 3.01 0.52 0.79

74 Pomelo Citrus maxima 4.98 72.84± 8.87 0.73 27.16± 4.02 0.28 70.28± 6.48 0.42 0.47

80

Table 2.2 Antimicrobial and Antioxidant activity of Non-climacteric fruit (Continued)


(% GI ±SD)

EC

Index

B. subtilis *

(% GI ±SD)

BS

index

DPPH±SD **

(µg AAE/mg

extract)

DPPH

index

AO-

AM

index

75 Pumpkin Cucurbita pepo 6.44 71.48± 3.06 0.72 64.6± 0.57 0.66 32.41± 8.41 0.19 0.52

76 Rambutan Nephelium lappaceum 3.76 83.94± 4.33 0.84 90.79± 1.12 0.92 136.3± 3.68 0.81 0.86

77 Raspberry Rubus idaeus 3.27 93.83± 6.01 0.94 95.43± 2.02 0.97 133.63± 0.74 0.79 0.90

78 Sea buckthorn Hippophae (cultivar Indian

Summer) 3.11 87.29± 5.8

0.87 71.5± 4.96

0.73 125.69± 2.07

0.75 0.78

79 Spiky Gourd (Kantola) Momordica dioica 5.17 53.66± 3.86 0.54 39.18± 1.17 0.40 21± 7.68 0.12 0.35

80 Strawberry Fragaria × ananassa 3.41 58.37± 7.26 0.58 63.02± 2.83 0.64 135.5± 3.43 0.80 0.68

81 Tamarind Red Tamarindus indica 3.87 72.73± 5.36 0.73 70.5± 0.71 0.72 109.83± 6.78 0.65 0.70

82 Water-melon Citrullus lanatus 4.79 43.04± 10.8 0.43 15.3± 6.65 0.16 39.87± 6.78 0.24 0.27




81

Table 2.3 Antimicrobial and Antioxidant activity of leaf extracts


(% GI ±SD)

EC

Index

B. subtilis *

(% GI ±SD)

BS

index

DPPH±SD **

(µg AAE/mg

extract)

DPPH

index

AO-

AM

index

83 Alpha-alpha Medicago sativa 6.56 38.07± 2.55 0.38 42.15± 4.2 0.43 14.61± 4.53 0.09 0.30

84 Anise Pimpinella anisum 5.63 41.08± 1.22 0.41 32.47± 2.13 0.33 76.44± 3.34 0.45 0.40

85 Asparagus Asparagus officinalis 5.1 40.78± 4.13 0.39 59.99± 5.64 0.45 22.44± 5.17 0.28 0.37

86 Baby arugula Eruca sativa 5.01 38.47± 1.1 0.41 43.87± 1.6 0.61 47.71± 4.43 0.13 0.38

87 Basil Ocimum basilicum 5.7 76.67± 3.51 0.77 62.02± 1.41 0.63 95.14± 2.93 0.57 0.65

88 Betel Piper betle 5.92 97.02± 2.09 0.97 90.12± 1.41 0.92 143.59± 1.77 0.85 0.91

89 Black currant Ribes nigrum 5.44 43.12± 4.12 0.43 57.48± 3.5 0.58 60.83± 7.02 0.36 0.46

90 Blueberry Vaccinium corymbosum 3.52 77.29± 3.74 0.77 48.87± 1.6 0.50 136.47± 3.88 0.81 0.69

91 Bok Choy Brassica rapa chinensis, 5.92 36.28± 5.15 0.36 50.81± 5.38 0.52 45.26± 2.34 0.27 0.38

92 Broccoli Brassica oleracea group

Italica 5.44 64.73± 6.79

0.73 63.57± 2.02

0.81 39.79± 2.92

0.16 0.57

93 Broccoli

inflorescence

Brassica oleracea group

Italica 6.24 72.43± 7.41

0.65 79.88± 4.24

0.65 27.17± 1.65

0.24 0.51

94 Brussels sprouts Brassica oleracea group

Gemmifera 5.93 71.16± 10.22

0.71 62.59± 3.41

0.64 14.71± 6.1

0.09 0.48

95 Burdock Arctium lappa 5.95 56.98± 5.43 0.57 60.18± 1.36 0.61 132.92± 3.21 0.79 0.66

96 Cabbage Brassica oleracea group

Capitata 5.88 41.51± 6.83

0.42 31.4± 5.09

0.32 17.82± 8.07

0.11 0.28

97 Carrot Daucus carota 6.06 31.83± 3.81 0.32 17.85± 3.04 0.18 40.58± 3.6 0.24 0.25

98 Celery Apium graveolens var. dulce 4.35 41.6± 4.02 0.42 16.55± 4.96 0.17 26.06± 5.32 0.15 0.25

99 Celery rave Apium graveolens 5.02 66.71± 3.85 0.67 62.5± 3.54 0.64 67.37± 2.09 0.40 0.57

100 Celery oriental Chinese 5.92 41.41± 5.28 0.44 10.45± 6.44 0.47 74.96± 4.86 0.75 0.55

101 Chicory lettuce Cichorium intybus 5.93 43.53± 2.14 0.62 46.69± 2.39 0.83 126.06± 5.34 0.41 0.62

102 Coriander

(Fresh) Coriandrum sativum 6.01 61.73± 3.59

0.41 81.66± 2.34

0.11 68.65± 3.56

0.45 0.32

103 Cranberry Vaccinium macrocarpon 4.6 87.87± 6.18 0.88 88.43± 0.6 0.90 131.12± 1.3 0.78 0.85

104 Cress Lepidium sativum 5.85 36.95± 4.86 0.37 15.5± 0.71 0.16 15.19± 3.6 0.09 0.21

105 Curry Murraya koenigii 5.76 38.16± 2.98 0.38 37.5± 2.12 0.38 27.48± 10.3 0.16 0.31

106 Dandelion salad Taraxacum officinale 5.73 68.46± 2.9 0.56 28.73± 1.8 0.49 44.42± 3.52 0.22 0.42

107 Dandelion (wild) Taraxacum officinale 5.51 55.58± 9.42 0.69 48.27± 2.45 0.29 37.79± 1.86 0.26 0.41

108 Dill (Fresh) Anethum graveolens 6.19 40.61± 2.26 0.41 49.58± 0.81 0.50 64.25± 1.89 0.38 0.43

109 Echalot Allium cepa var.

aggregatum 4.59 63.67± 8.5

0.64 64.5± 0.71

0.66 10.41± 3.43

0.06 0.45

110 Endive Cichorium endivia 5.84 45.67± 2.95 0.46 57.7± 3.82 0.59 10.89± 6.49 0.06 0.37

111 Fenugreek Trigonella foenum-graecum 4.59 39.36± 9.72 0.39 41.05± 2.76 0.42 106.33± 1.89 0.63 0.48

112 Frisse Cichorium endivia

var crispum 6.73 59.33± 3.56

0.71 70.34± 1.89

0.66 88.31± 8.69

0.26 0.54

113 Grape (Wild) Vitis vinifera 3.33 92.27± 4.05 0.59 71.39± 5.11 0.71 140.78± 2.17 0.52 0.61

114 Grape (Wild) Vitis vinifera 3.49 92.16± 3.76 0.92 91.34± 1.89 0.73 137.14± 5.32 0.84 0.83

115 Green tea Camellia sinensis 4.48 87.08± 5.25 0.87 76.5± 2.12 0.78 139.55± 3.42 0.83 0.83

116 Hops (pellets) Humulus lupulus 5.73 67.53± 2.53 0.68 57.36± 17.88 0.58 130.66± 1.32 0.78 0.68

117 Horse radish pulp Armoracia rusticana 6.22 77.58± 4.61 0.78 66.55± 4.88 0.68 61.44± 8.02 0.36 0.61

118 Indian Round

Gouard (Tinda) Praecitrullus fistulosus 4.32 74.11± 4.62

0.52 44.39± 0.87

0.45 120.91± 3.26

0.64 0.54

119 Jewish Mallow Corchorus olitorius 5.23 52.25± 5.43 0.58 44.1± 1.56 0.52 107.01± 5.49 0.58 0.56

120 Kangkong Ipomoea aquatica 5.71 57.67± 7.51 0.38 51.5± 0.71 0.52 98.13± 3.39 0.48 0.46

121 Kohlrabi Brassica oleracea group

Gongylodes 5.54 37.55± 9.34

0.39 51.46± 3.47

0.59 80.7± 3.04

0.09 0.36

122 Leek Allium ampeloprasum group 5.31 39.06± 9.9 0.51 57.58± 2.02 0.63 14.96± 1.72 0.61 0.58

123 Leek term Allium ampeloprasum group 5.46 81.04± 3.98 0.81 52± 1.12 0.53 28.02± 2.2 0.17 0.50

124 Lemon Citrus lemon 5.43 51.13± 1.41 0.70 61.5± 0.71 0.67 103.05± 9.67 0.25 0.54

82

Table 2.3 Antimicrobial and Antioxidant activity of leaf extracts (Continued)


(% GI ±SD)

EC

Index

B. subtilis *

(% GI ±SD)

BS

index

DPPH±SD

**

(µg

AAE/mg

extract)

DPPH

index

AO-AM

index

125 Lemon grass Cymbopogon citratus 4.68 69.67± 8.62 0.92 65.5± 7.78 0.93 42.8± 2.96 0.81 0.89

126 Mint (Fresh) Mentha spicata 3.45 70.45± 3.22 0.74 65± 2.01 0.71 43.53± 2.4 0.63 0.69

127 Nicosse N.A 5.15 73.91± 4.96 0.43 69.64± 0.9 0.29 105.49± 2.96 0.50 0.40

128 Oleander Nerium oleander 5.65 87.11± 6.04 0.87 66.02± 1.56 0.67 115.49± 5.21 0.69 0.74

129 Oregano (Fresh) Origanum vulgare 6.02 42.51± 4.97 0.82 28.09± 9.78 0.63 84.51± 5.49 0.50 0.65

130 Parsley (Fresh) Petroselinum crispum 6.38 41.55± 1.12 0.42 67.69± 3.8 0.69 31.8± 1.24 0.19 0.43

131 Parsnip Pastinaca sativa 5.51 81.4± 10.44 0.88 62.25± 8.03 0.87 84.72± 8.29 0.70 0.81

132 Piper lolot Piper lolot 5.67 87.6± 10.18 0.49 85.13± 2.64 0.90 117.38± 7.08 0.69 0.69

133 Potato Solanum tuberosum 3.73 49.14± 8.09 0.30 88.67± 2.35 0.44 115.43± 6.66 0.19 0.31

134 Purselane Portulaca oleracea 5.01 30.43± 2.03 0.43 43.51± 2.12 0.61 31.52± 3.1 0.24 0.43

135 Radish Raphanus sativus 5.12 42.83±6.52 0.78 59.92± 2.95 0.39 40.58± 3.26 0.39 0.52

136 Rainbow chard B. vulgaris subsp. cicla 7.33 77.89± 2.03 0.30 38.62± 9.03 0.42 65.87± 0.21 0.11 0.28

137 Rapini Brassica rapa ruvo 5.77 30.2± 1.4 0.84 41.22± 0.3 0.78 18.36± 4.51 0.80 0.81

138 Raspberry Rubus idaeus 5.4 83.77± 7.34 0.85 76.65± 2.33 0.82 134.56± 2.1 0.78 0.82

139 Red onion Allium cepa L. 5.08 49.56± 4.39 0.50 53.91± 0.13 0.55 16.38± 9.57 0.10 0.38

140 Rhubarb Rheum rhabarbarum 3.19 85.26± 6.7 0.43 80.55± 3.47 0.18 130.73± 4.8 0.16 0.26

141 Roquette Eruca sativa 5.72 43.07± 10.36 0.76 17.5± 3.54 0.60 27.42± 3.26 0.78 0.71

142 Rosemary Rosmarinus officinalis 5.94 75.75± 10.83 0.90 58.58± 6.16 0.85 131.41± 6.01 0.82 0.85

143 Sea buckthorn Hippophae (cultivar Indian

Summer) 5.22 90.02± 10.74

0.78 83.15± 1.41

0.63 137.48± 0.76

0.16 0.52

144 Sorrel Rumex acetosa 3.21 77.65± 3.83 0.66 61.58± 4.84 0.59 26.56± 0.26 0.57 0.61

145 Spinach Spinacia oleracea 6.31 65.7± 4.59 0.89 58.5± 3.54 0.98 96.75± 4.68 0.79 0.89

146 Strawberry Fragaria × ananassa 5.22 89.1± 4.81 0.33 96.07± 4.34 0.70 133.53± 3.72 0.14 0.39

147 Swiss Chard Beta vulgaris subsp. cicla 6.54 33.16± 10.56 0.56 68.49± 3.52 0.46 23.48± 5.49 0.53 0.52

148 Taro Colocasia esculenta

esculenta 5.93 56.09± 3.33

0.74 45.08± 3.45

0.45 89.56± 4.29

0.72 0.64

149 Tomato Solanum lycopersicum 4.78 51.33± 3.67 0.51 36.78± 4.56 0.37 2.8± 4.71 0.02 0.30




83

Table 2.4 Antimicrobial and antioxidant activity of root peel extracts


(% GI ±SD)

EC

Index

B. subtilis *

(% GI ±SD)

BS

index

DPPH±SD **

(µg AAE/mg

extract)

DPPH

index

AO-AM

index

150 Beetroot Beta vulgaris 7.01 46.74± 7.96 0.47 31.45± 5.03 0.32 33.85± 1.54 0.20 0.33

151 Carrot Daucus carota 5.67 29.45± 2.76 0.30 5.58± 6.25 0.06 69.4± 1.12 0.41 0.25

152 Celery rave Apium graveolens 4.92 31.21± 8.57 0.31 23± 3.57 0.23 14.93± 1.4 0.09 0.21

153 Dandelion (Wild) Taraxacum officinale 7.55 45.52± 2.25 0.46 30± 4.14 0.30 12.67± 4.36 0.08 0.28

154 Galangal Alpinia galanga 4.9 65.08± 2.31 0.65 54± 1.78 0.55 23.93± 1.34 0.14 0.45

155 Ginger Zingiber officinale 6.23 85.33± 3.21 0.85 82± 2.83 0.83 73.38± 1.01 0.44 0.71

156 Horse radish Armoracia rusticana 5.01 73.87± 1.87 0.74 58.5± 2.12 0.59 59.78± 1.99 0.36 0.56

157 Parsnip Pastinaca sativa 5.4 72.4± 5.05 0.73 59.07± 2.83 0.60 112.7± 3.6 0.67 0.67

158 Potato Solanum tuberosum

(Cultivar Idoha) 5.95 71.3± 1.41

0.71 72.5± 0.71

0.74 20.83± 7.28

0.12 0.53

159 Potato Baby Solanum tuberosum 5.7 47.25± 14.9 0.46 27.5± 3.54 0.52 30.47± 2.3 0.28 0.42

160 Potato Mature Solanum tuberosum 5.92 46.09± 5.26 0.47 51.03± 2.79 0.28 47.94± 1.54 0.18 0.31

161 Radish Raphanus sativus 5.61 83.25± 3.03 0.83 85.82± 0.25 0.87 128.49± 7.91 0.76 0.82

162 Radish (Black) Raphanus sativus 6.03 58.57± 2.7 0.59 38.29± 9.49 0.39 58.27± 2.57 0.35 0.44

163 Radish Purple Raphanus sativus (Purple) 5.7 72.16± 4.46 0.72 67.44± 10.69 0.69 19.82± 6.21 0.12 0.51

164 Rutabaga Brassica napus var.

napobrassica 5.23 51.17± 4.47

0.51 69.68± 0.96

0.71 37.06± 3.49

0.22 0.48

165 Tapioca Manihot esculenta 6.57 75.12± 1.17 0.75 42.11± 4.09 0.43 24.29± 1.41 0.14 0.44

166 Turmeric Yellow Curcuma longa 5.19 72.32± 7.55 0.72 70.5± 0.71 0.72 72.67± 1.56 0.43 0.62

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2.4.2 Antioxidant activity of fruit and vegetable extracts (DPPH assay)

The results of radical scavenging activity of 164 aqueous plant extracts are given in Table

2.1. A total of 67 samples out of the 164 displayed excellent radical scavenging activity of

the DPPH radical at >100 µg of AAE (ascorbic acid equivalent/mg of dry weight); where

the radical scavenging activity of ascorbic acid was 168.33 µgAAE/mg at the concentration

1mg/mL. The three best samples which exhibited exceptional anti-radial capacity were:

betel leaf (143.59 µg AAE/mg), green grape fruit (141.38 µg AAE/mg) and pomegranate

peel (141.34 µg AAE/mg). The other materials that exhibited strong antioxidant activity

(126-140 µg AAE/mg) were: avocado, custard apple, blueberry, black currant, cranberry,

jujuba, mangosteen, rambutan, raspberry, chicory lettuce, grape leaf, green tea, rosemary,

raspberry leaf and sea buckthorn leaf. Non-climacteric fruit had a significantly higher anti-

radical activity than the climacteric fruit (Figure 2.6)

The plant tissue type showed an effect on the DPPH radical scavenging capacity of the

extracts. Fruit peel extracts exhibited significantly higher DPPH radical scavenging

capacity in comparison to leaf tissue and root peel extracts (p<0.05) (Figure 2.7). The effect

of pH was also assessed, and there was a negative correlation between the pH of the

extracts and their DPPH value (r= -0.42) (Figure 2.8).

Surprisingly, the plants belonging to the family Brassicaceae (mustard family) genus

Brassica (broccoli, cabbage and brussels sprout) and family Amaryllidaceae, genus Allium

(endive, shallot, leek and onion) displayed a low antioxidant activity.

2.4.3 Correlation between the antimicrobial and antioxidant activity of plant extracts

The antimicrobial activity of the aqueous plant extracts could be positively correlated with

the antioxidant activity. As seen in Figure 2.8, the antioxidant activity, assayed by DPPH

radical scavenging capacity exhibited a good correlation with the antimicrobial activity

(growth inhibition) against E.coli (r=0.65) and B.subtilis (r=0.57)

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pH

30 50 70

-0.39 -0.37

20 60

35

7

-0.42

30

50

70 E.Coli

0.76 0.65

B.Subtilis

20

50

80

0.57

3 5 7

20

60

20 50 80

DPPH

Figure 2.8 Correlation between, the pH, growth inhibitory activity (GI) against

E.coli and B.subtilis and DPPH radical scavenging capacity of the extracts.


20

40

60

80

12

0

Gro

wth

inh

ibiti

on

, %

)

Figure 2.6 Effect of ripening process of fruit on

DPPH radical scavenging capacity

DP

PH

(µ

g A

AE

/mg

extr

act)

Fruit Leaf Root

02

04

06

08

01

20

Gro

wth

inh

ibiti

on

, %

)

Figure 2.7 Effect of plan part origin of sample

on DPPH radical scavenging capacity

DP

PH

(µ

g A

AE

/mg

extr

act)

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2.5 Discussion

2.5.1 Antioxidant and antibacterial activities of plant by-products

This study shows that plants can be a good source of antioxidant-antimicrobials; 67

samples out of 164 displayed radical scavenging activity of >100 µg of AAE and, 53% of

the samples showed >70% GI.

The anti-radical activity could relate to the phenolic compounds, carotenoids, ascorbic acid

and tocopherol content (El-Massry et al., 2009; Erden et al., 2013). The data suggests that

aqueous plant extracts are free radical scavengers and act as primary antioxidants that react

with DPPH radical, attributable to their H-donating or electron transferring ability (Huang

et al., 2005).

The results show that plant by-products may also be a potential source of antimicrobials.

Phenolic compounds (phenolic acid, quinones, flavonoid, flavones, flavonols, tannins,

courmarins), terpenoids, alkaloids, lectins, polypeptides and polyacetylenes are the major

classes of antimicrobial compounds from plants (Cowan, 1999). Antimicrobials from plant

sources can fatally affect various aspects of microbial cell function, metabolism and life

cycle by different mechanisms (Kohanski et al., 2010). They include capacity to acidify or

alkalinize cytoplasm, negatively impact cell membranes (anesthetics) and surface enzyme

activity. Furthermore, they can complex with proteins and metals, and also alter the redox

potential of the medium. Polycationic nature of molecules such as chitosan also contributes

to antimicrobial activity (El Ghaouth et al., 1992).

2.5.2 E. coli and B. subtilis sensitivity.

The hot water extracts of plants have a significant growth inhibiting potential against both

the test strains, although E.coli was found to be significantly more sensitive than B. subtilis.

Gram-negative bacteria are generally observed to be more resistant to antimicrobial

compounds due to its cell membrane structure (Cueva et al., 2010a; Perry et al., 2009).

However, other authors have reported that Gram-negative bacteria more susceptible to plant

extracts than Gram-positive bacteria (Gülçın et al., 2003; Pereira et al., 2007a). Consistent

with our observation, in particular, is the report of (Puupponen‐ Pimiä et al., 2001), where

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phenolic extracts using acetone (70 %) according to the method of (Kähkönen et al., 1999)

from berry fruits including blueberry, raspberry, lingonberry, black currant, cloudberry, sea

buckthorn berry, strawberry, were found to inhibit the growth of Gram-negative, but not

Gram-positive bacteria. They explained that the difference in cell-wall structures between

Gram-negative and Gram-positive bacteria might be the likely basis for the difference in

their sensitivity to phenolic compounds.

The relatively higher sensitivity of the Gram-negative strain to the plant extracts can be

attributed to a variety of reasons. First, the composition of the plant extracts in their active

constituents and their corresponding function may be responsible for the plant extracts

being more active against the Gram-negative strain. The plant extracts that displayed

antimicrobial activity have also high concentrations of different organic acids. The weak

acids in the non-dissociated form, can more easily migrate through the lipid-containing cell

wall and cell membrane of Gram-negative bacteria (Dorman and Deans, 2000), and result

in acidification of the cytoplasm, thereby adversely affecting the microbial metabolism.

Second, hot water solvent that enhances the extract yields, is also efficient in extracting

phenolic acids, flavonoids, anthocyanins, tannins, saponins, terpenoids, polypeptides and

lectins (Cowan, 1999; Xu et al., 2008). These polyphenolic compounds exert antimicrobial

activity in a concentration-dependent manner (Dorman and Deans, 2000; Medina et al.,

2006b; Tajkarimi et al., 2010), where their activity generally depends on compromising the

integrity of cell membrane and/or disruption of the proton motive force (membrane

potential) (Helander et al., 1998b; Heller et al., 1999; Matsuzaki, 1998; Nowakowska,

2007; Rauha, 2000; SantiestebanLópez, 2007; Scalbert, 1991). Phenolic acids can pass

through the lipid membrane and in the process disrupt the cell surface enzyme rendering

them inactive (Cueva et al., 2010a). Hydroxy-cinnamic acids, due to their propanoid side

chain, are less polar than the corresponding hydroxy-benzoic acids, and this property might

facilitate the transport of these molecules across the cell membrane (Campos et al., 2003).

Presence of permeabilizers, amphipathic molecules, and polyphenolic compounds capable

of specifically interacting with the phospholipid membrane, may in combination

compromise the integrity of the outer lipid; thereby, rendering the Gram-negative bacteria

comparatively more sensitive and vulnerable compared to Gram positive bacteria.

However, to validate the result further, the antimicrobial activity needs to be tested against

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a wider spectrum of bacteria, also, the nature of the compounds in the extract need to be

determined to come to a firm conclusion.

2.5.3 Effect of pH of extracts on AM and AO properties

As expected, the pH of the extract was a factor influencing the GI, which was inversely

related to the pH of the extract against both bacteria (Figure 2.8), as the organic acids

present in the extracts in the non-dissociated form can enhance the antimicrobial activity of

weak acids. This is in accordance with similar studies done on plant derived antimicrobials

(Gutierrez et al., 2008; Hsieh et al., 2001). The inhibitory effect of plant extracts is

generally greater at acidic pH values (Campo and Amiot, 2000). The susceptibility of

bacteria to polyphenolic antimicrobials also appears to increase with lower pH values since

the hydrophobicity of compounds, such as terpenoids, flavonoids increases at low pH,

consequently enabling easier dissolution in lipid rich cell membrane of target bacteria

(Juven et al., 1994). The mode of action of phenolic acids is based on the ability of their

non-dissociated form to pass through the negatively charged outer membrane. Thus, the

membrane acts as a barrier for the conjugated base forms of acids. In their non-dissociated

state (pH below the pKa value of the acid), the acids may alter the membrane permeability

and interfere with enzymatic processes in the cell (Davidson and Branen, 2005). The pKa

of most of the organic acids lies between pH 3 and 5. Since the cytoplasmic pH is generally

higher than that of the growth medium, the weak acid dissociates, releasing the protons and

leading to acidification of the cytoplasm (Cotter and Hill, 2003). Plant phytochemicals can

be found in different chemical forms, which depend on their inherent pH. In the acidic pH,

the antioxidant activity of polyphenols would be significantly higher and would decrease

proportionately with increases in the pH, due to the deprotonation of hydroxyl groups.

Furthermore, resonance and delocalisation of electrons depending on the type of attached

electronegative group also plays a vital role.

As most of the processed foods are in the slightly acidic pH range, the increased AM

activity at acidic pH would be an advantage; although, these results need to be supported

with investigation of the effect of changing pH on the antioxidant and antibacterial activity

of plant extracts.

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2.5.4 Effect of physiological type of fruit: climacteric vs. non-climacteric

The antioxidant and antimicrobial activity of fruit peel was also analyzed according to the

classification based on their ripening characteristic. Fruits have been classified as

climacteric or non-climacteric, depending on whether or not a fruit exhibits a peak in

ethylene production and respiration during ripening. Environmental factors such as

temperature and sunlight affect plant secondary metabolism. Variation in the climate has

also been seen to affect the accumulation of phytochemical compounds in plants

(Schreiner, 2005). The difference in the distribution of phytochemical compounds is also

seen to vary between the cultivar types and geographical region. In the case of lettuce

cultivars, some are very poor in flavonoids and other phenolic compounds, whereas other

types contain larger amounts of flavonols and anthocyanins (DuPont et al., 2000). This

variation may explain the different antimicrobial activity observed, as the antimicrobial

potential of a plant extract is ultimately linked to their phytochemical composition.

Non-climacteric fruits are generally grown in temperate climates where the average

temperature lies between 0 and 20° C, with the nights being colder (McKnight and Hess,

2000). This diurnal variation in the minimum temperature means that the plants are

subjected to an abiotic stress due to the cold temperature, in comparison to climacteric

fruits growing in the tropical regions; where typically, the minimum temperature is at least

18° C (McKnight and Hess, 2000). The plants growing in cold regions respond to cold

stress by increasing secondary metabolism and in turn phytochemical concentration (Gould,

2004; Oh et al., 2009). For example, it was observed that daily mean temperatures below

16.5°C were beneficial for the β-carotene synthesis in broccoli (Shewfelt and Brückner,

2000), whereas the best temperature was 18°C for carrots (Rosenfeld et al., 1998).

Beneficial temperatures for lycopene formation in tomato were found in the range from

16°C to 21°C (Gross, 1991). The increased concentration of various phytochemicals such

as phenolic acids, flavonoids may explain the comparative higher antimicrobial and

antioxidant activity of non-climacteric fruits. In addition, non-climacteric fruits, generally,

tend to contain higher level of ascorbic acid and tocopherols than the climacteric fruit

(Hernández et al., 2006; Rodriguez-Burruezo et al., 2010). Furthermore, the climacteric

fruits undergo respiratory surge before ripening, subjecting them to oxidative stress, which

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can possibly deplete the stored antioxidant compounds to some extent. Moreover, with

advances in senescence, their ability to synthesize bioactive compounds may be

significantly compromised. The climacteric behaviour, regardless of the climatic habitat,

appears to be a more plausible factor contributing to the differences in the activity of fruits.

For example, non-climacteric and tropical fruits such as pomegranate or rambutan exhibit

higher activities (Table 2.2) than apple, a climacteric and temperate fruit (Table 2.1).

2.5.5 Effect of plant tissue type

The type of plant tissue (fruit, leaf and root) also had an effect on the inhibitory activity of

the extracts. The presence of polyphenols is ubiquitous in the plant kingdom, but their

presence may greatly vary, because of differences in exposure to light and other

environmental stresses. Consequently, these compounds are usually found at higher

concentrations in leaves and outer parts of plants, compared to the subterranean organs,

where they are present in trace amounts (Herrmann, 1988). Furthermore, plants produce a

diverse array of constitutive and/or induced secondary metabolites (phenylpropanoids,

flavonoids, terpenoids, glucosinolates and alkaloids), varying in concentration and

composition in different plant organs, which can impact antimicrobial and antioxidant

activities or other bioactivities of the extracts derived from them (Cowan, 1999). Berries

and fruits contain a wide range of phenolic acids, which makes them less vulnerable to

bacterial spoilage (Hamad, 2012). Fruit residues were found to possess strongest

antioxidant properties (Deng et al., 2012). Leaves and fruit peels have also been shown to

exhibit significant antioxidant activity (Lombardo et al., 2010; Siddhuraju et al., 2002).

Similar to our finding, leaves, fruit peels, pomace, fruit residue, grape and olive residue,

plant-food processing by-products have been shown to have a higher antimicrobial activity;

and in general, a higher bioactive potential than other parts of the plant such as flowers,

seeds, roots or stems (Riihinen et al., 2008; Romani et al., 2003; Siddhuraju et al., 2002;

Silva et al., 2014; Wijngaard, 2009).

2.5.6 Antioxidant activity of Brassica and Allium extracts

In our screening, extracts from broccoli, cabbage, Brussel sprout, endive, shallot, leek and

onion displayed a low antioxidant (DPPH) assay value. This was in contradiction to the

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prevailing knowledge about these plants. Allium and Brassica vegetables have long been

recognized for antioxidant activity. The principal bioactive compounds of Allium and

Brassica are commonly referred to as thiosulfinates and glucosinolates (unique to

Brassica). Specific examples include allicin (S-allyl-L-propenethiosulfinate) and methyl

methanethiosulfonate, present in Allium and Brassica, respectively.

An antioxidant is a molecule that inhibits the oxidation of other molecules while being

oxidized itself; meaning that antioxidants are often reducing agents (Sies, 1997). During the

extraction process, the maceration of the cruciferous plant tissue the enzyme myrosinase

cleaves off the glucose moiety in the glucosinolate from a isothiocyanates, thiocyanates and

nitrites depending on the pH of the medium (Burow et al., 2007). For the plants of the

Allium family the enzyme alliinase would act on the S-alkyl-L-cysteine S-oxides to yield

alkyl sulfinates. Further, once subjected to hot-water extraction, the thiosulfinates would

yield simpler thiol derivatives owing to thermal degradation (Oerlemans et al., 2006).

Direct thermal degradation of glucosinolates is also known to produce a variety of thiol

derivatives (Jin et al., 1999). These authors found that increasing the temperature of the

aqueous solutions increased the rate of formation of degradation products. Compounds

identified were dimethyl disulfide, S-methyl methyl-thio-sulfinate, S-methyl methyl-thio-

sulfonate and methyl (methyl-thio) methyl-disulfide. The major volatile compounds

generated by thermal degradation of alliin (S-allyl-L-cysteine sulfoxide) are di-allyl

sulfides (mono-, di-, tri-, and tetra-sulfide) and allyl alcohol, with the types of compounds

formed from alliin depends mainly on temperature of heating, water activity of the medium

and time of heating (Kubec et al., 1997). All of these organosulphur compounds and thiol

derivatives, thus formed have an important effect on the antioxidant properties of the

extracts from Allium and Brassica families. Thiols are organosulphur compounds that

contain a carbon-bonded sulfhydryl group (–C–SH or R–SH). They are one of the few

compounds, which are able to remove O2∙− , H2O2, although the rate constants for these

reactions are low, generally <103M-1*s-1 (Halliwell, 1995). Therefore, only very high thiol

concentrations would have any significant antioxidant effect. Furthermore, the reactive

oxygen species formed from H2O2 such as (HO•) hydroxyl radical, can react with thiols to

produce thiyl radicals (RS•) which can also combine with oxygen to give reactive

oxysulphur radicals such as RSO• and RSO2• (thiyl peroxyl) (Halliwell, 1995; Manoj et al.,

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2011). These oxysulphur radicals formed can further react with other polyphenols and

antioxidant compounds present in the extract, which will render them unavailable to react

with the DPPH radical, resulting in a reduced radical scavenging activity, thereby

decreasing the absorbance. The lower rate constant of reaction of thiols with ROS and the

formation of oxysulphur radicals may also be a possible reason behind why thiol containing

plant extracts did not exhibit a significant radical scavenging activity.

It is possible that hot water may not be a suitable medium for extraction for these plant

species rich in sulfur compounds that are prone to thermal degradation. On the other hand,

the DPPH assay may not be suitable assay as it is also possible that the antioxidant thiols

may be converted to thiyl radicals in the presence of reactive oxygen species such H2O2,

formed during extraction, can react with antioxidant compounds in the extract, rendering

the latter unavailable for scavenging the DPPH radical.

2.5.7 Correlation between antioxidant and antimicrobial activities

The result showed there was good correlation between the antioxidant and antimicrobial

activity against E.coli (r=0.65) and B.subtilis (r=0.57), suggesting that plant-derived

bioactive compounds have the potential to act both as antimicrobial and antioxidant

compounds. This dual nature of bioactivity can be presumably attributed to oxygen

scavenging ability of certain constituents present in the extracts, a characteristic desirable

for inhibition of aerobic microbial growth as well as antioxidant activity.

Among the phytochemicals, polyphenols are a group of secondary metabolites are

associated with antioxidant activity. The relation between phenolic content and antioxidant

activity has been fairly well documented (Medina et al., 2006a; Nehete et al., 2010). They

also play an important role in plants, providing protection against pathogens and predators

(Bravo, 1998). In addition, several studies have demonstrated antimicrobial activity of

phenols and/or phenolic extracts (Pereira et al., 2007b; Pereira et al., 2006; Proestos et al.,

2005; Rauha et al., 2000). A number of studies have reported that the antioxidants also have

antimicrobial activity (Daglia, 2012a; Djenane et al., 2012; Jia et al., 2010; Katalinić et al.,

2010; Ortega-Ramirez et al., 2014). In particular, the fungi are found to be very sensitive to

antioxidants, presumably, due to depletion of oxygen in the system by antioxidants, which

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is required for metabolism of aerobic bacteria and fungi. The common phenolic

antioxidants used in food such as BHA (butylated hydroxy anisole), BHT (butylated

hydroxy toluene) and TBHQ (tertiary-butyl-hydroquinone), are also known to exhibit anti-

fungal properties (Passone et al., 2008).

2.5.8 Antioxidant-antimicrobials index (AO-AM Index)

Knowing the plant extracts have both antioxidant and antimicrobial properties to varying

extents, an empirical evaluation was performed in order to identify potent antioxidant-

antimicrobial plant extracts by using the empirical index, giving a higher weight to

antimicrobial activity:

Antioxidant-antimicrobial index = (EC index+BS index+DPPH index)/3

where, EC index is the inhibitory activity (GI) index of extracts against E. coli (the ratio of

the GI of the test samples at 10 mg/mL to that of chloramphenicol at 1 mg/mL (inhibition

of 99.81%); BS index is the inhibitory activity index of extracts against B. subtilis; and

DPPH index is the antioxidant activity of the test samples (the ratio of antiradical activity

of the samples at 1.0 mg/mL to that of ascorbic acid at 1 mg/mL (168.88 µg AAE/mg).

The AO-AM indices are presented in Tables 2.1- 2.4. On this classification, none of the

root or climacteric fruit extracts displayed AO-AM index values of > 0.85. The leaf extracts

possessing high values were: betel, rosemary, lemon grass, cranberry, sea buckthorn and

spinach. The non-climacteric fruit extracts were: black currant, green grape (wine), olive oil

mill effluent, pomegranate, rambutan, raspberry, egg plant and Indian plum.

A small number of extracts from were identified as antioxidant-antimicrobials; and

interestingly, they are mostly non-climacteric fruit sources, while some are leaf materials. It

would seem that these materials are concentrated in few classes of phyto-compounds, in

contrast to climacteric fruit sources that they may contain a variety of phytochemical

classes but at low concentrations. Although the antioxidant activity of the selected extracts

is reasonably comparable to ascorbic acid, their antimicrobial activity is not comparable to

synthetic food preservatives such as benzoate or sorbate, and that their application as

antimicrobial agents would require higher concentrations. Because they inherently possess

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antioxidant activity, their incorporation at high concentrations would impart to foods added

advantages of health benefits as well as improved protection of foods against oxidation.

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2.6 Conclusions

The screening of various fruit and vegetable sources including by-products and wastes

permitted the identification of potential antimicrobial extracts exhibiting antioxidant

property. The pH of the extract, type of tissue, and the difference in physiology of fruit also

significantly affect the bioactive properties investigated. The results show that the

antioxidant and antimicrobial properties of the plant extracts are related. The AO-AM index

of the study could be used in the evaluating and selection of bio-actives from plant sources.

This index can be potentially used as the basis of investigating the mode of antimicrobial

action and the physicochemical properties of the molecules responsible for the bioactive

properties. Furthermore, the structure-function relationship of the bioactive compounds

responsible for the antioxidant-antimicrobial activity need to be investigated, which would

help in getting a better understanding of the bioactive properties of plant-derived

compounds. The plant extracts could be a safe, health-beneficial and economical food

preservation agent with antimicrobial and antioxidant properties.

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Chapter 3 ANTI-RADICAL POWER (ARP) OF PLANT EXTRACTS: A NEW

MEASURE OF ANTIOXIDANT PROPERTY FROM DPPH ASSAY

La capacité antioxydante ne décrit que la valeur quantitative du test antioxydant, tandis que la qualité

d’antioxidante a été toujours inaperçue dans l'étude de réaction d'antioxydant. Dans cette étude, la vitesse

de piégeage des radicaux (facteur du temps) pour définir l'efficacité réelle (capacité) du système anti-

oxydant a été considérée comme la meilleure manière d’exprimer avec plus de fiabilité le pouvoir anti-

oxydant réel. La nouvelle expression, le pouvoir antiradicalaire - ARP, produite à partir du test DPPH, peut

être plus utile pour identifier l'activité anti-oxydante des échantillons biologiques. Certains échantillons

présentaient une valeur ARP élevée tels que les extraits de rambutan, feuille de canneberge, feuille de bleuet,

feuille de vigne (sauvage), feuille de framboise, feuille de bétel, avocat, grenade et cherimoya. Les extraits de

feuilles possèdent, en général, une valeur d’ARP supérieure à celle des fruits tandis que, les extraits de

racines présentent les plus faibles ARP. De plus, cette étude montre également que le nombre moyen

d’oxydation du carbone de mélanges complexes tels que des extraits de plantes peut prédire leur pouvoir

anti-oxydant, en dépit de la disparité entre les tissus de feuilles et de fruits.

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3.1 Abstract

DPPH radical assay is a reliable method to determine the antioxidant capacity of biological

substrates. The DPPH radical scavenging activity is generally quantified in terms of the

capacity of antioxidants to quench the pre-formed free radicals, and is a typically employed

parameter to express the antioxidant activity and to compare the activity of different

substrates. But antioxidant capacity only describes the quantitative value of antioxidant

assay, while quality of antioxidant efficacy has always been unaccounted for. In this study,

the rate of radical quenching was measured along with capacity in the DPPH assay. The

anti-radical power (ARP) that takes into consideration of both the rate (time factor) and the

actual efficacy (capacity) of antioxidant systems was found to be a better measure of

antioxidant activity. Among all tested extracts, rambutan, cranberry leaf, blueberry leaf,

grape leaf (wild), raspberry leaf, betel leaf, avocado, pomegranate, custard apple, grape

red, grape leaf, grape red (wine), mangosteen, indian plum were found to have ARP value

more than 200 mg-1.min-1. Leaf extracts possess, in general, higher ARP than fruits, and

root extracts possess low ARP. Average carbon oxidation number (ACON) of complex

mixtures such as plant extracts may portend their antioxidant power, in spite of apparent

disparity between leaf and fruit materials.

Keywords: DPPH• radical, capacity, rate, oxidation number

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3.2 Introduction

Oxidation occurs in many living organisms, and it is an essential process for the production

of energy and fuel the biological processes smoothly. The continuous generation in vivo of

oxygen-centered free radicals as well as other reactive oxygen species (ROS) during

metabolic processes result in cell death and tissue damage. The role of oxygen radicals has

been implicated in several diseases, including cancer, diabetes and cardiovascular diseases,

aging, etc. (Halliwell and Gutteridge, 1999). Human body has multiple enzymatic and non-

enzymatic anti-oxidant systems that protect the cellular molecules against ROS induced

damages. However, severe or continued oxidative stress makes the innate defenses

insufficient for total protection. Hence, in order to balance the ROS in human body certain

amounts of exogenous anti-oxidants are constantly required to maintain adequate levels of

anti-oxidants.

Anti-oxidants play a significant role in protecting the body from damage caused by free

radical- induced oxidative stress (Ozsoy et al., 2008). Natural anti-oxidants, like

polyphenols, found in medicinal and edible plants, are very effective in preventing

oxidative damages (Silva et al., 2005). Such activity is due to the structure of the

polyphenols, which helps to eradicate free radical, and in vitro they have been shown to be

more effective anti-oxidants than tocopherols and ascorbate. Ability of the polyphenol-

derived radicals to stabilize and delocalize the unpaired electron throughout the structure,

and to chelate transition metal ions makes polyphenols ideal antioxidants (Rice-Evans et

al., 1997). Synthetic antioxidants such as butylated hydroxyl anisole (BHA) and butylated

hydroxyl toluene (BHT) are used at regulation limits to protect the quality of food by

retarding oxidation (Taghvaei and Jafari, 2015). However, there are some serious problems

concerning the safety and toxicity of such synthetic antioxidants due to their metabolites

and their possible absorption and accumulation in the body organs and tissues (Chen and

Tappel, 1996).

Antioxidants exert their action through simple or complex mechanisms including

prevention of chain initiation, binding with transition metal ion catalysts, decomposition of

peroxides, prevention of continued hydrogen abstraction, and radical scavenging (Sharma

and Singh, 2012). Till date, many in vitro tests are available from a simple chemical assay

performed in an homogeneous solutions to more complex methods using exogenous

99

sensitive probes to detect oxidation, like oxygen radical absorbance capacity (ORAC),

conjugated autoxidizable triene assay or use of fluorescent probes to detect oxidation

(Grajeda-Iglesias et al., 2016). Although many literature methods do not distinguish

between “antioxidant capacity” and “antioxidant activity” of non-enzymatic antioxidants,

end-point assays measuring the efficiency of antioxidant action (i.e., reactive species

inactivation) are not often differentiated from kinetic-based assays measuring reaction rate.

Antioxidant capacity, i.e., the capacity of antioxidants at a specific concentration to

neutralize radicals present in the reaction mixture, is reported often as their antioxidant

activity (Huang et al., 2005; Kurilich et al., 2002; Niki and Noguchi, 2000; Prior et al.,

2005). More recently however, some reports express the antioxidant activity based on the

kinetics of the redox reaction, and the time required for reduction of 50 % of the radicals

present in the reaction mixture (Philip, 2004; Sharma and Bhat, 2009). The complexities

and physiological applications of antioxidants, either separately or in mixtures, require

rigorous consideration and analysis of all aspects of the chemistry, reaction mechanisms,

and reaction/ radical/target specificity in various test systems, as well as careful and

accurate measure of all reactants and products involved. The DPPH radical assay for

estimating antioxidant activity has been used commonly since the last 20 years and

considered as the most important artificial radical to enlighten the scientific knowledge on

free radicals.

Thus, it is desirable to establish and standardize antioxidant assay methods that can

measure the real antioxidant power directly for all classes of anti-oxidants, irrespective of

their origin, either plant-based extracts or biological fluids. In this context, this study

proposes to determine the antioxidant activity by “Anti-Radical power” that takes into

account both antioxidant capacity, i.e., at at a fixed concentration and time point to

scavenge radicals and the rate at which the radicals are scavenged.

100

3.3 Materials and methods


Hundred and forty plant materials were obtained fresh from fields in and around Quebec

City. The plants materials that were investigated are presented in Table 1. Upon arrival to

the laboratory, samples were washed with water and bleaching agents (NaOCl-300 ppm) to

remove dirt and debris. Subsequently within 48 hours, they were deep frozen, lyophilized,

ground to powder, vacuum-sealed and stored at -30°C until ready for extraction.


Ascorbic acid and 1,1 diphenyl-2-picrylhydrazine (DPPH) were purchased from sigma-

Aldrich (Canada). All reagents used in this investigation were of analytical grade.

3.3.3 Plant extract preparation

About 50 gram of the lyophilized powder of raw sample was extracted in 500 mL of




lyophilized extracts were vacuum-packed and stored at -30oC until further use. The stock

solution of plant extract at 1.0 mg/mL was prepared in distilled water before the test

performed.

3.3.4 DPPH free radical scavenging assay

Free radical scavenging capacity, the main mechanism by which antioxidants protect foods

against oxydation, was determined by DPPH assay (Faleiro et al., 2005). The DPPH radical

scavenging was analysed through a standard spectrophotometric method (Sharma and Bhat,

2009) with modifications. A stock solution of DPPH (6.1x 10-5 M) was freshly prepared in

methanol and was used immediately. The DPPH stock solution was diluted with methanol

and adjusted to O.D of 0.7 (± 0.02) at 517 nm. Subsequently, 190 µL of the DPPH solution

was pipetted out into 96 wells microplate (Becton Dickinson Falcon 353072), followed by

addition of 10 µL of the test plant extract (1.0 mg.mL-1). Thereafter, the plate was

101

incubated in a spectrophotometer (Biotek Power XS2) at 25°C for a period of 1 hour, with

absorbance readings (517 nm) recorded every 1 min. The assays were carried out in

triplicate.

Radical scavenging capacity (%) = [(Ao − A1)/Ao ] × 100

where, Ao was the absorbance of the control sample (without plant extract) and A1 was the


The rate of radical scavenging was determined from the slope of the initial linear part of the

quenching curve (Figure 3.1), typically within 10 min of the quenching reaction.

where, rate is given in min-1; y is given in O.D. unit; and t is time of the reaction (min).

3.3.5 Elemental and metal analyses

The element analysis was carried out by Material Characterization Center, Sherbrooke

University, Canada. The equipment SC632 (LECO cooperation) was used to analyze

carbon and sulfur, Truspec for Nitrogen and Hydrogen and micro Truspec for Oxygen.

Briefly, for the determination of carbon, hydrogen, nitrogen and sulfur, the sample falls in

a furnace at 1100°C filled with oxygen for a very fast and complete combustion. The

detection and quantification of the elements (carbon, hydrogen and sulfur) were performed

by infra-red cells, and nitrogen detected by thermal conductivity. Oxygen was analyzed

independently, where the material was pyrolized in an oven at a temperature of 1300°C

equipped with Truspec for detection and quantification by infrared cells.

The metals (Cu, Fe, Mn) were analyzed by Department of Wood Sciences, Laval

University, Quebec, Canada. The samples were dissolved according to Microwave assisted

acid digestion. The sample solutions were then analyzed on an ICP-OES Optima4300DV

(Perkin-Elmer) (Barnes, 1997).

102

3.3.6 Reaction kinetics and calculation of reaction rate constants

A number of competing reactions may take place during ROS/radical processing, either in

series or in parallel in a complex biomaterial as a plant extract. The complete removal of

radicals may be the results of many interacting and complex reactions rather than a single e-

transfer step. An effective first-step kinetic modeling is to study simple e-/H+ transfer

systems rather than real radical scavenging (Huang et al., 2005). Chemical reaction kinetics

can be applied to quantify individual attribute of an ideal antioxidant system in form of the

general rate law (Van Boekel, 2008):

nkPdt

dP (1)

where, k is the rate constant (min-1), t the reaction time (min), and n is the order of the

reaction. In general, P represents a quantitative value for units of e-/H+ donating molecules

in the extracts. The core of kinetic studies on radical scavenging is to quantify an

antioxidant attribute as a function of time. Under specified conditions, using oxidation-

dependent reaction rate constants after the order of that reaction is determined. The order of

reaction is determined based on the goodness of fit of the observations to a pre-selected

reaction order model. Kinetics of antioxidant potential is an unclear phenomenon, and has

been speculated as zero-, first-, or second-order reactions as follows:

P = P0–kt for zero−order reactions (n=0) (2)

P = P0e-kt for first−order reactions (n=1) (3)

1/P = kt + 1/P0 for second−order reactions (n=2) (4)

where, P0 is the initial value of the antioxidant activity at t=0.

Calculation of reaction rate constant:

DPPH• scavenging reaction kinetics was followed as described earlier in methanol medium

(Mishra et al., 2012; Suja et al., 2004). Scavenging reaction between [DPPH•] and the

antioxidant [AH] is represented by Eq. (5-7), assuming secondary reactions may be of

limited occurrence.

[DPPH•] + [AH] ↔ [DPPH-H] + [A•] (5)

[DPPH•] + [e-/H+] ↔ [DPPH2] (6)

[A•] + [A•] ↔ [A-A] (7)

103

The order of reaction of [DPPH•] and [AH] depends on the relative concentration of the

reactants, and hence, it can be expected to follow second order kinetics. Since the

concentration of DPPH• is fixed at 0.1mM and with increasing concentration of antioxidant

such that ([DPPH•]<< [AH]), the reaction may follow a pseudo-first order kinetics as

shown in Eq. (8)

r = k [DPPH•][AH] (8)

where, r is the overall reaction rate.

Since [DPPH•] << [AH]

r = k [DPPH•], (9)

Equation (9) can be also written as:

DPPHkdt

DPPHd (10)

Integrating equation (6):

ln[DPPH•] = ln[DPPH•]o- kobst (11)

Or [DPPH•], = [DPPH•]o. e- kt (12)

Thus, the time required to obtain saturation, will be dependent on concentration and the

type of antioxidant.

104

3.3.7 Anti-Radical Power (ARP): The overall antioxidant activity

Calculation of antioxidant activity

The overall anti-oxidant activity = Antioxidant capacity x Conversion rate (or radical

scavenging rate), where, capacity signifies the maximum steady state equilibrium and

Conversion rate signifies the velocity of the reaction.

The reaction is:

DPPH•+ AH k

DPPH2 + A (13)

where k = reaction rate constant, and AH = Antioxidant

With complete conversion, [DPPH2] = [DPPHo]

AH

A

AHDPPH

ADPPHKeq

2

Velocity of reaction,

S

Sk

eff

where, Seff = Effective concentration of antioxidant in the sample

So = Concentration of the sample

dt

ODdk

)(

and the decrease in OD is the measure of DPPH• scavenging by antioxidant.

So, the overall antioxidant activity or anti-radical power (ARP) is defined as:

S

Sk

AH

AARP

KARP

eff

eq

Placing [AH] = [Seff]

AH

AkARP

where, DPPHA

and thus,

105

S

OD

dt

ODdARP

S

DPPHkARP

max)(

where, ΔODmax is the measure of maximum capacity of antioxidant to scavenge DPPH• at

steady state.

Expressing [So] as 1 unity of the sample,

max).()(

_OD

dt

ODd

sampleunit

ARP

With unit sample in mg and time in min, the unit of ARP is mg-1.min-1

3.3.8 Average carbon oxidation number

ACON calculation

The average carbon oxidation number (ACON) was calculated according to the method of

(Kroll et al., 2011) from the elemental composition of the extracts with some modification

(with corrections in the elemental composition for sugar content).

ACON = 2 (O/C) – (H/C) + 2 (N/C) + 2 (S/C)

Where (O/C) is the molar ratio of oxygen (OS = -2) to carbon; (H/C) is the molar ratio of

hydrogen (OS = +1) to carbon; (N/C) is the molar ratio of nitrogen (OS = -2) to carbon; and

(S/C) is the molar ratio of sulphur (OS = -2) to carbon. OS stands for oxidation state. The

OS of nitrogen in organic compounds can be either -2 or -3. The OS of sugars is ~ 0.0.

3.3.9 Data analysis

All experiments were done in triplicate and were set as a complete randomized design, and

the data were analyzed by one-way analysis of variance (one-way ANOVA) using a

significant level of 0.05. Tukey HSD difference test at the same significant level was done

when the analysis of variance found significant differences.

106

3.4 Results and discussion

Oxidation can be attenuated by antioxidants by different mechanisms, including scavenging

radicals, but also by sequestering metal ions, decomposing hydrogen peroxide and/or

hydro-peroxides, quenching active pro-oxidants, and repairing protein damage (Rahman,

2007). Some are potent radical-scavenging antioxidants, but are poor antioxidants against

lipid peroxidation by lipoxygenase (e.g., -tocopherol) (Niki and Noguchi, 2000).

Carotenoids are weak radical-scavenging antioxidants but can be potent inhibitors of the

oxidation induced by singlet oxygen (Niki and Noguchi, 2000). The activity of the

antioxidants in vivo is determined not only by the reactivity toward radical, but also by

several other factors such as concentration, distribution, localization, fate of antioxidant-

derived radical, interaction with other antioxidants, and metabolism. Plant extracts contain

a mixture of compounds with potential antioxidant activity at varying concentrations. They

may also perform this function by different mechanisms. Although DPPH radical assay

monitors essentially the quenching of the radical, other reactions that take place may

interfere this process. Consequently, the overall antioxidant activity of the extracts is

revealed.

3.4.1 Estimation of antioxidant activity by DPPH• assay

The stable radical, DPPH•, has been used widely for the determination of the free radical

scavenging activities of pure antioxidant compounds such as plant and fruit extracts and

food materials. The assay is based on the reduction of DPPH• in methanol, which causes a

decrease in absorbance at 517 nm as DPPH• is reduced. The absorbance decreases, noted by

color change from purple to yellow, as the radical is scavenged by antioxidants present in

the extract by donation of hydrogen to give the reduced form of DPPH–H (equation (5)). In

this study, decrease in the absorbance at 517 nm due to DPPH reduction was measured in

the presence of the plant extracts until the antioxidants in the extract were depleted under

pseudo-first order assay conditions. The pseudo-first order rate constant, k is linearly

dependent on the concentration of initial radical scavenger (AH).

.

107

3.4.1.1 Antioxidant capacity

The fall in absorbance is an indicator of the capacity of radical scavenging activity of

extract, and is expressed as percentage of decrease in the absorbance at steady state. The

time course of the scavenging of DPPH• radical by a few plant extracts and ascorbic acid is

shown in Figure 3.1 Some extracts exhibited the extent of radical quenching capacity (or

capacity) as ascorbic acid (e.g., betel leaf and grape leaf extracts). While betel leaf extract

neutralized the radical at a faster rate similar to ascorbic acid, grape leaf extract quenched

the radical at a slower rate. Strawberry leaf extract on the other hand, quenched the radical

to a smaller extent than ascorbic acid, and also at somewhat smaller rate than ascorbic acid,

but faster than grape leaf extract. Although, in general, extracts having higher radical

scavenging capacity scavenge the DPPH• at a higher rate, and those with lower capacity

took longer to reduce the amount of DPPH• present in the reaction mixture; it was not as

straight forward, as seen for grape leaf extract (Figure 3.1).

Figure 3.1 Time course of DPPH• scavenging by selected extracts

108

The antioxidant capacity as determined by DPPH assay of 140 plant extracts is shown in

Table 3.1. The antioxidant capacity of all the extracts ranged from 1.2 to 74.76 %. It was

found that there wasn’t significant different between capacity of fruit extracts and that of

leaf extracts (p=0.05). However, when taking account only ten top performances, leaf

extracts (73.42 %) showed higher capacity than climacteric fruit extracts (69.81 %). The

antioxidant capacity of the extracts ultimately depends on the composition of the extracts,

nature and concentration of phytochemical compounds in the extract mixture that exhibit

may possess with varying scavenging capacity, and in this case, the scavenging of DPPH•

radical.

3.4.1.2 Rate of radical scavenging

The reaction of DPPH• with antioxidant is basically a kinetic driven process. Thus in this

study, an attempt was made to assess the nature of kinetic behavior of disappearance of

DPPH• with ascorbic acid (1.0 mg/mL) and various plant extracts. Time course of the

reaction was monitored for individual extracts till steady state under conditions of substrate

depletion, i.e., maximum decrease in DPPH• radical was reached (Figure 3.1). It was clear

that DPPH• scavenging by extracts occurred at different rates. In a similar kinetic scan of

plant extracts, the time to reach the steady state was found to be between 20-30 min

depending upon concentration. However, a time duration of 20-30 min is usually

considered by most authors in fixed reaction time mode for estimation of antioxidant

capacity (Foti, 2015), although our measurements showed that the time to attain steady

state may vary, depending on the nature of antioxidants. The reaction of ascorbic acid with

DPPH• is very fast and attains completion within few minutes (Brand-Williams et al.,

1995), 8 minutes for this study (Figure 3.1). The time required to achieve saturation is

highest for standards like, ascorbic acid, BHT and gallic acid. Plant extracts found to show

a wide range of variability to attain saturation. Regardless of the capacity, antioxidants can

be categorized as slow, medium and fast on the basis of their rate of reaction. The kinetics

of scavenging was found to be concentration-dependent, where scavenging rate increases

with increase in concentration of the extracts (Jha et al., 2014).

109

Table 3.1 Antioxidant activity of fruits and vegetables by products extracts

Sample name Scientific name pH

Rate

(min-1)

Capacity

(%)

ARP

(mg-1.min-1)

Acid ascorbic (1.0 mg/ml) ND 0.531 76.69 321.55

1 Alpha alpha Medicago sativa 6.56 0.060 4.61 13.0

2 Anise Pimpinellaanisum 5.63 0.108 60.68 56.2

3 Areca nut Areca catechu 5.8 0.290 74.03 171.4

4 Artichoke (Base) Cynaracardunculus 5.67 0.084 35.44 32.1

5 Artichoke (Petal) Cynaracardunculus 5.93 0.045 28.40 15.5

6 Asparagus Asparagus officinalis 5.1 0.010 12.14 2.5

7 Avocado Perseaamericana 5.64 0.435 72.57 253.7

8 Bangladesh lemon (Satkara) Citrus Macroptera 4.74 0.042 16.75 11.8

9 Basil Ocimumbasilicum 5.7 0.308 73.06 180.6

10 Betel leaf Piper betle 5.92 0.452 71.60 261.1

11 Bitter Gourd Momordicacharantia 4.99 0.008 11.17 1.9

12 Black currant Ribesnigrum 3.27 0.208 70.15 118.8

13 Black currant Ribesnigrum 5.44 0.234 32.28 85.4

14

Black currant (Fermented

residue) Ribesnigrum

3.2 0.301 70.63 172.2

15 Black currant (residue) Ribesnigrum 3.55 0.183 74.76 109.0

16 Black Fig Ficuscarica 5.25 0.089 38.83 35.8

17 Blueberry fruit Vacciniumcorymbosum 3.85 0.259 69.66 146.8

18 Blueberry leaf Vacciniumcorymbosum 3.52 0.470 72.09 272.9

19 Bokchoy Brassica rapachinensis, 5.92 0.063 34.71 23.7

20 Broccoli Brassica oleracea group Italica 5.44 0.076 54.85 36.8

21 Broccoli inflorescence Brassica oleracea group Italica 6.24 0.094 31.55 33.9

22 Brussels sprouts Brassica oleracea group Gemmifera 5.93 0.121 45.63 53.1

23 Burdock Arctiumlappa 5.95 0.231 73.30 135.7

24 Cabbage Brassica oleracea group Capitata 5.88 0.072 25.24 23.5

25 Cactus Opuntiaficus-indica 5.56 0.098 34.22 36.7

26 Canadian cucumber Cucumissativus 6.23 0.020 6.55 4.5

27 Canadian Yew Taxus canadensis 4.78 0.003 20.39 0.9

28 Cantharanthus Cantharanthus ND 0.298 73.06 174.8

29 Cape Gooseberry Cucumismelo var. cantalupensis 4.98 0.283 36.65 110.2

30 Carambolla Averrhoa carambola 4.13 0.089 55.58 43.9

31 Carrot leaf Daucuscarota 6.06 0.027 24.27 8.9

32 Carrot Daucuscarota 5.67 0.024 19.17 7.0

33 Cashew Anacardiumoccidentale ND 0.135 71.36 77.9

34 Celery Apiumgraveolens var. dulce 4.35 0.030 33.01 11.0

35 Celery rave leaf Apiumgraveolens 5.02 0.023 8.98 5.5

36 Celery rave Apiumgraveolens 4.92 0.006 1.20 0.8

37 Celery oriental Chinese 5.92 0.043 48.79 19.4

38 Cherry Prunusavium 4.5 0.064 25.00 20.9

39 Chicory lettuce Cichoriumintybus 5.93 0.061 67.72 34.0

40 Coing Cydonia oblonga 4.18 0.109 73.30 64.1

41 Coriander (Fresh) Coriandrumsativum 6.01 0.028 36.17 10.8

42 Cranberry leaf Vacciniummacrocarpon 2.69 0.466 73.79 274.5

110

Table 3.1 Antioxidant activity of fruits and vegetables by products extracts (Continued)


Rate

(min-1)

Capacity

(%)

ARP

(mg-1.min-1)

43 Cranberry fruit Vacciniummacrocarpon 4.6 0.327 73.79 193.1

44 Cress Lepidiumsativum 5.85 0.034 10.68 8.4

45 Curry Murrayakoenigii 5.76 0.068 41.99 28.4

46 Custard apple Annona cherimola 5.31 0.422 64.56 227.8

47 Dandelion salad Taraxacumofficinale 5.73 0.008 22.09 2.4

48 Dandelion Taraxacumofficinale 5.51 0.025 38.83 9.8

49 Dandelion Taraxacumofficinale 7.55 0.003 21.12 1.0

50 Date (Raw) Phoenix dactylifera 4.98 0.001 17.96 0.2

51 Date (Red) Phoenix dactylifera 4.73 0.128 60.92 66.7

52 Dates (Fresh) Phoenix dactylifera 5.67 0.021 10.44 5.1

53 Drum Sticks Moringaoleifera 5.02 0.048 13.83 12.8

54 Echalot Allium cepa var. aggregatum 4.59 0.010 6.99 1.0

55 Eggplant Solanum melongena 5.09 0.083 56.55 41.3

56 Endive Cichoriumendivia 5.84 0.001 2.43 0.2

57 English cucumber Cucumissativus 6.12 0.028 9.22 6.8

58 Fenugreek Trigonellafoenum-graecum 4.59 0.127 55.58 62.6

59 Frisse Cichoriumendiviavar crispum 6.73 0.014 68.20 7.7

60 Galangal Alpiniagalanga 4.9 0.047 38.83 19.0

61 Ginger Zingiberofficinale 6.23 0.073 55.34 35.9

62 Granadilla Passifloraligularis 5.26 0.066 64.56 35.4

63 Grape leaf Vitisvinifera 3.33 0.379 74.03 224.1

64 Grape leaf (wild) Vitisvinifera 3.49 0.446 76.46 269.2

65 Grape Green (wine) (Residue)

Vitisvinifera 3.55 0.122 75.49 73.1

66 Grape Red Vitisvinifera 3.92 0.406 68.20 227.2

67 Grape Red (wine) Vitisvinifera 4.17 0.377 69.42 213.4

68 Green almond Prunusamygdalus 5.18 0.066 5.10 14.5

69 Green grape (wine) Vitisvinifera 4.26 0.297 74.27 175.8

70 Hops (pellets) Humuluslupulus 5.73 0.137 70.87 78.8

71 Horse radish Armoracia rusticana 5.01 0.044 20.15 13.3

72 Indian Plum Ziziphusmauritiana 4.89 0.360 71.84 208.2

73 Indian Round Gouard (Tinda) Praecitrullusfistulosus 4.32 0.100 64.08 53.7

74 Ivy Gourd Cocciniagrandis 5.18 0.002 3.88 0.3

75 Jewish Mallow Corchorusolitorius 5.23 0.025 53.40 12.1

76 Jujuba Simmondsiachinensis 4.96 0.195 73.79 115.2

77 Kaki Diospyros kaki 5.17 0.205 67.23 113.8

78 Kangkong Ipomoea aquatica 5.71 0.104 41.02 42.8

79 Kiwi Actinidiadeliciosa 4.2 0.099 52.91 47.3

80 Kohlrabi Brassica oleracea group Gongylodes 5.54 0.037 52.43 17.4

81 Kumquat Citrus japonica 4.4 0.007 2.43 1.3

82 Langsat Lansiumdomesticum 4.83 0.121 67.48 67.4

83 Leafy Kale Acephala Group 6.14 0.156 66.99 86.5

84 Leek Allium ampeloprasum group 5.31 0.030 9.22 7.2

85 Leek term Allium ampeloprasum group 5.46 0.014 6.80 3.1

111



Rate

(min-1)

Capacity

(%)

ARP

(mg-1.min-1)

86 Lemon Citrus lemon 5.43 0.096 58.25 48.6

87 Lemon grass Cymbopogoncitratus 4.68 0.024 36.89 9.2

88 Lychee Litchi chinensis 4.11 0.236 72.33 137.2

89 Mango CaesiaMangifera (cultivar Ataulfo) 3.87 0.302 64.08 162.4

90 Mangosteen Garcinia mangostana 4.58 0.361 71.60 208.6

91 Mint Menthaspicata 3.45 0.021 21.60 6.4

92 noni fruit Morindacitrifolia

0.063 52.67 30.1

93 Noni juice Morindacitrifolia 5.82 0.079 59.47 40.3

94 Oleander Nerium oleander 5.65 0.037 72.57 21.8

95 Orange (albedo) Citrus sinensis 4.56 0.115 35.44 43.8

96 Papaya (Raw) Carica papaya 5.54 0.073 32.77 26.7

97 Parsley (Fresh) Petroselinum crispum 6.38 0.060 24.76 19.6

98 Parsnip root Pastinaca sativa 5.51 0.056 2.10 13.4

99 Parsnip leaf Pastinaca sativa 5.4 0.043 19.66 12.7

100 Passiflora foetida Passiflorafoetida ND 0.044 49.76 20.0

101 Passion Fruit Passiflora edulis 5.01 0.066 5.58 14.5

102 Phyllanthus phyllanthus ND 0.302 68.20 168.7

103 Pineapple Ananascomosus 3.89 0.042 23.06 13.3

104 Piper lolot Piper lolot 5.67 0.105 62.38 55.6

105 Pistachio Pistaciavera 4.42 0.221 73.06 129.6

106 Plum Prunusdomestica 3.45 0.024 67.72 13.6

107 Pomegranate Punicagranatum 3.55 0.432 72.09 250.7

108 Pomelo Citrus maxima 4.98 0.129 49.27 59.0

109 Potato Solanum tuberosum 3.73 0.123 38.59 49.1

110 Potato Solanum tuberosum 5.95 0.089 25.97 29.6

111 Potato Baby Solanum tuberosum 5.7 0.204 64.81 110.3

112 Potato Mature Solanum tuberosum 5.92 0.116 66.99 64.1

113 Pumpkin Cucurbita pepo 6.44 0.114 23.30 36.1

114 Purselane Portulacaoleracea 5.01 0.048 7.28 11.1

115 Radish Raphanussativus 5.12 0.019 19.42 5.6

116 Radish Raphanussativus 5.61 0.165 59.47 84.5

117 Radish (Black) Raphanussativus 6.03 0.085 29.37 29.9

118 Radish Purple Raphanussativus 5.7 0.100 26.21 33.3

119 Rainbow chard B. vulgaris subsp. cicla 7.33 0.083 59.71 42.7

120 Rambutan Nepheliumlappaceum 3.76 0.466 74.76 276.9

121 Rapini Brassica raparuvo 5.77 0.032 9.47 7.8

122 Raspberry leaf Rubusidaeus 3.27 0.451 72.82 263.6

123 Raspberry fruit Rubusidaeus 5.4 0.221 71.12 126.9

124 Raw mango CaesiaMangifera 4.3 0.297 50.73 138.1

125 Red onion Allium cepa L. 5.08 0.031 15.53 8.4

126 Rhubarb Rheum rhabarbarum 3.19 0.166 74.27 98.5

127 Roquette Eruca sativa 5.72 0.018 12.38 4.7

128 Rosemary Rosmarinus officinalis 5.94 0.205 55.10 100.3

112



Rate

(min-1)

Capacity

(%)

ARP

(mg-1.min-1)

129 Sea buckthorn fruit Hippophae (cultivar Indian Summer) 3.11 0.145 52.18 68.6

130 Sea buckthorn leaf Hippophae (cultivar Indian Summer) 5.22 0.334 52.91 159.1

131 Sorrel Rumexacetosa 3.21 0.169 70.63 97.0

132 Spinach Spinaciaoleracea 6.31 0.059 24.76 19.1

133 Strawberry fruit Fragaria × ananassa 3.41 0.133 69.66 75.3

134 Strawberry leaf Fragaria × ananassa 5.22 0.314 48.79 142.6

135 Swiss Chard Beta vulgaris subsp. cicla 6.54 0.049 18.69 14.3

136 Tamarind Red Tamarindusindica 3.87 0.050 31.07 18.1

137 Taro Colocasiaesculentaesculenta 5.93 0.103 50.97 48.1

138 Tomato leaf Solanum lycopersicum 4.76 0.147 36.41 56.8

139 Tomato fruit Solanum lycopersicum 4.78 0.008 3.12 1.4

140 Tumeric (Yellow) Curcuma longa 5.19 0.031 42.72 13.2

On the basis of rate of radical scavenging at a specific concentration (1.0 mg/mL), extracts

can be empirically categorized in groups as follows:

Rate greater than 0.3

Blueberry, cranberry, rambutan, betel, raspberry, grape (wild), avocado, pomegranate,

custard apple, grape red, grape, grape red (wine), mangosteen, indian plum, sea buckthorn,

cranberry, strawberry, basil, mango, phyllanthus, black currant (fermented residue)

Rate between 0.1 and 0.3

Cantharanthus, green grape (wine), raw mango, arecanut, cape gooseberry, blueberry,

lychee, black currant, burdock, pistachio, raspberry, black currant, kaki, rosemary, potato

baby, jujuba, black currant (residue), sorrel, rhubarb, radish, leafy kale, tomato, sea

buckthorn, hops (pellets), cashew, strawberry, pomelo, date (red), fenugreek, potato, grape

green (wine), (residue), langsat, brussels sprouts, potato mature, orange albido, pumpkin,

coing, anis, piper, lolot, kangkong, taro, indian round gouard (tinda), radish purple

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Rate below 0.1

Kiwi, cactus, lemon, brocoli inflorescence, carambolla, black fig, potato, radish (black),

artichoke (base), eggplant, rainbow chard, noni juice, broccoli, ginger, papaya (raw),

cabbage, curry, green almond, granadilla, passion fruit, cherry, noni fruit, bocchoy, chicory

lettuce, alpha alpha, parsley (fresh), spinash, parsnip, tamarind red, swiss chard, drum

sticks, purselane, galangal, artichoke (petal), horse radish, passiflora foetida, cerlery

oriental, parsnip, bangladesh lemon (satkara), pinapple, oleander, kohlrabi, cress, rapini,

tumeric yellow, red onion, celery, leek, english cucumber, corriander, carrot, jewish

mallow, dandelion (wild), plum, lemon grass, carrot, celery rave, mint, dates , canadian

cucumber, radish, roquette, frisse, leek term, asparagus, echalot, tomato, bitter gourd,

dadelion salad, kumquat, celery rave, dandelion (wild), canadian yew, ivy gouard, endive,

date (raw)

3.4.2 Relation between scavenging rate and capacity of antioxidant activity

An important issue in studying the reactions of antioxidants and radicals is to distinguish

the rate and capacity, that is, quality and quantity. For equivalent capacity, some

antioxidants may be quite reactive and scavenge radicals very rapidly, while other

antioxidants may react with radicals slowly, as shown in Figure 3.1. In our study, the

reaction of the faster antioxidant extract (E1) is completed after about 12 min, the extracts

with intermediate antioxidant (E2) efficacy takes about 30 min to completion, while the

reaction of least antioxidant extract (E3) still continues after 60 min. Hence, if the percent

discoloration is determined at 30 min, the three antioxidants appear to have identical

performance, despite the substantial difference in reactivity. Instead, measurement at 10

min would give the correct rate of reactivity (E1> E2> E3). It should be noted that a

measurement taken at 60 min would yield a completely different rate of antioxidant

performance, merely reflecting the actual rate of the reaction.

In the present study, relation between rate and capacity was found to be hyperbolic (Figure

3.2). The relation between the rate and capacity was linear up to the scavenging rate of

0.15 min-1. Beyond that rate, the capacity reached maximum, i.e., rate had no effect on

capacity. In other words, beyond the rate of 0.15 min-1, the samples exhibiting higher rates

114

of radical scavenging and reaching the maximum capacity could not be differentiated.

Furthermore, some extracts fall into a category of extracts that show an increase in

scavenging capacity in proportion to or linearly with the rate of scavenging (Figure 3.2).

This group includes purselane (number 114), piper lolot (104), black currant (13), Cape

gooseberry (29), raw mango (124), sea buckthorn leaf (130), strawberry leaf (134), grape

red (66), custard apple (46), rambutan (120), raspberry leaf (122) and others. For this group

of extracts, either the capacity or the rate could characterize their antioxidant activity.

Moreover, some samples may not reach the maximum capacity, but may arrive at a

capacity at a faster rate (Figure 3.2). For example, some extracts such as betel leaf extract

reacts with radicals more rapidly than grape leaf extract, while scavenging almost same

amount of radicals. The slow scavenging of radicals by grape leaf extract is likely due to

regeneration of phyto-compound radicals produced by the rapid scavenging of DPPH

radicals; and such reaction occurs with betel leaf or pure ascorbic acid (Figure 3.1).

Evidently, characterizing a sample either by capacity or by rate alone cannot be straight-

forward; they cannot be taken as the sole criterion of antioxidant activity determination.

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Rate (min-1

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2456 3

Figure 3.2 Relation between rate of radical scavenging and capacity of the radical scavenging capacity

of plant extracts

* The number corresponds to the product as listed in Table 3.1.

Antioxidant activity can be more fully expressed by taking both the capacity and rate taken

into consideration. Capacity of an extract towards the radical scavenging is sum total of

amount of radical neutralized by the antioxidant components present in the extract. While,

the rate of radical scavenging depends on the nature of phytochemicals, reaction condition

(i.e., pH, temperature and solvents), and the transient antioxidant-derived radical formation

during scavenging of the active radicals. When an antioxidant scavenges active radicals, the

radical is transformed to a non-radical and stable product, while the antioxidant yields one

antioxidant-derived radical. For example, hydro-quinones and polyphenols may play

important role as antioxidants in vivo; and as they scavenge radicals, semi-quinone radicals

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are formed, which in turn, may scavenge another radical to produce a quinone, or

disproportionate with another semi-quinone radical to give the parent compound and a

quinone (Flora, 2009). They may also react with oxygen, yielding a quinone and a

hydroperoxyl radical. Therefore, it is not always certain to state that high rate of radical

quenching would have high antioxidant activity or capacity, or vice versa (as seen in Figure

3.2).

Plant extracts can differ quantitatively and qualitatively not only in the composition of

antioxidants but also of natural acids. In consequence, their pH can be different. For most

of the examined extracts the pH was found in between 4 and 5. Between the pH range of 4

and 5 (rate up to 0.15 min-1), capacity was found directly proportional to the rate. But the

extracts, having pH value below 4, showed similar antioxidant capacity with various rates

(plateau region in Figure 3.2). The influence of pH on the antioxidant rate and capacity

was found to be higher at lower pH, which might be due to the nature of phytochemicals at

lower pH and interference of pH on radical stability. De-protonation of hydroxyl groups at

lower pH enhances the antioxidant activity of polyphenols and it decreases monotonously

with increasing pH (Han et al., 2009).

3.4.3 Anti-Radical Power

The DPPH radical assay was developed by (Brand-Williams et al., 1995) and a vast

majority of antioxidant research community have adopted it. Briefly, different antioxidant

concentrations are used in such a way to determine the concentration of antioxidants that

quench the initial DPPH• radical concentration in a specific time interval. The higher the

DPPH• radical quenching by an antioxidant system, the higher would be its antiradical

ability. Most of the time, efficacy of antioxidant system is presented in terms of EC50

(Philip, 2004; Prior et al., 2005; Sharma and Bhat, 2009). However, there are many issues

associated with the EC50 parameter and its interpretation as well. For example, the foremost

is the time interval for the reaction. And EC50 is time-dependent, and the effect of time can

vary from compound to compound. Usually, increasing the reaction time may improve the

evaluation of a given compound (lower EC50), and hence, the test can be customized so that

a poor anti-radical/antioxidant could appear to be an excellent antiradical/antioxidant. It is

117

clear that the interpretation of this test is quite arbitrary, and any comparison between

results is not possible, if the adopted reaction time is different.

Thus the consideration of both rate (time factor) and the capacity of antioxidant system

would be a better and reliable way to define the true efficacy of the antioxidant activity, the

real antioxidant power or antiradical power (ARP). Defined ARP will have a kinetic

parameter (not just a concentration), and as such it will express the antioxidant or

antiradical ability of a compound. Among the tested plant extracts, rambutan, cranberry

leaf, blueberry leaf, grape leaf (wild), raspberry leaf, betel leaf, avocado, pomegranate,

custard apple, grape red, grape leaf, grape red (wine), mangosteen, indian plum, cranberry

fruit, basil, green grape (wine), cantharanthus, black currant (fermented residue), areca nut,

phyllanthus, mango, sea buckthorn leaf, blueberry fruit, strawberry leaf, raw mango,

lychee, burdock, pistachio, raspberry fruit, black currant, jujuba, kaki, potato baby, cape

gooseberry, black currant (residue), rosemary were found to have ARP value of > 200 mg-

1.min-1. The complete ARP values of all tested extracts are presented in Table 3.1.

The ARP value of plant extracts ranged from 0.37 mg-1.min-1 (endive) to 553.88 mg-1.min-1

(rambutan). Statistical analysis showed that the average ARP value of extracts follows the

order: leaf extracts (409.2) ~ non-climacteric fruit extracts (407.6) > climacteric fruit

extracts (283.0) > root crop extracts (86.6 mg-1.min-1) (p<0.05). The average ARP value of

leaf extracts was found to be higher than that of the climacteric fruit extracts.

The relation between ARP of the plant extracts and their antioxidant capacity is shown in

Figure 3.3. The relationship was hyperbolic as was seen for the rate and capacity, if in a

more coherent pattern, indicating that the contribution of the scavenging rate to ARP is

significant. Typically, the scavenging capacity increases with ARP and it reaches maximum

value above the ARP value of 150 mg-1.min-1 (Figure 3.3) and remains steady thereafter.

Thus, ARP can differentiate samples for their antioxidant activity more effectively than the

scavenging capacity. It is evident that extracts even though having high values of capacity,

have lesser value of ARP as their rate of radical quenching is lower, and vice versa. For

example, lychee, pomegranate and orleander have the same antioxidant capacity of 72.33,

72.09 and 72.57 % respectively, but they exhibited the ARP value of 137.2, 250.7 and 21.8

mg-1.min-1 respectively (Table 3.1).

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Figure 3.3 Relation between ARP and radical scavenging capacity of plant extracts

* The number corresponds to the product as listed in Table 3.1

By the virtue of taking into account of both scavenging capacity and rate, i.e., the extent

and action time, ARP effectively expresses the power of the antioxidant substances. As

shown in Figure 3.4, ARP is nearly co-linear with the scavenging rate (R=0.99), and

correlates well with the scavenging capacity (R=0.72). Most of studies evaluating the

antioxidant activity of natural products determine the scavenging capacity by DPPH assay,

differentiation of most active samples by this expression constrained because of its

limitation, and it is evident that ARP would be marker to estimate the activity of and to

discriminate natural products for their efficacy. Furthermore, pH of the extracts has some

effect on their radical scavenging activity, but it is not significant.

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Figure 3.4 Correlations between pH, rate, capacity and ARP

3.4.4 Average carbon oxidation number (ACON)

Phytochemicals present in plants are organic in nature and they are crucial because of the

protective roles they play in the life of plants. However, their make up at any specific

condition describes the state of their life. But understanding the organic pool is much

difficult and hindered due to the immense chemical complexity of cellular metabolite

mixtures.

Oxidation number or state indicates the total number of electrons, which have been

removed from an element (a positive oxidation state) or added to an element (a negative

oxidation state) to reach its state (Jensen, 2007). Since the primary element in organic

compounds is carbon, its oxidation number is determined by its bonding to other elements

(H, O, N, S) depending on their electronegativity. Higher and positive oxidation number of

carbon would signify the presence of more electronegative elements O, N and S; and

consequently, presence of more reducing functional groups can be expected. Since plant

extracts are mixtures of many phyto-compounds, and information on their composion and

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structure of thosition often lacking; it is conceivable to obtain a view of their reducing

activity by knowing the average carbon oxidation number (ACON) from the elemental

composition of the extract.

Elemental analysis was performed on a few selected extract samples for their composition

in C, H, O, N and S, and the ACON was calculated from the mass composition of the

elements of those extracts. The ACON values were corrected for sugar contents and oxalic

acid (rhubarb) for which ACON is zero. The abundance of various elements in the plant

extracts is presented in Table 3.2.

Table 3.2 Elemental composition, average carbon oxidation number value of fruits and vegetables by-

products extracts

Sample Cu

(ppm) Fe

(ppm) Mn

(ppm) C

(%) H

(%) N

(%) S

(%) O

(%) ACON

1.Cranberry leaf 1.9 1822.6 317.26 49.18 5.41 0.202 0.342 42.31 -0.0072

2.Black currant residue 5.3 68.0 15.73 41.42 5.80 0.358 0.100 51.34 0.207*

3.Sea buckthorn leaf 2.4 17.8 134.31 46.41 5.47 1.241 0.095 44.86 -0.092

4.Betel leaf 8.3 42.9 149.05 32.88 4.00 3.133 0.226 39.27 0.508

5.Pomegranate 3.4 24.4 0.30 45.01 5.15 0.280 0.075 48.04 0.364

6.Rhubarb 139.2 46.2 10.64 35.79 4.66 2.345 0.113 51.1 0.15**

7.Blueberry leaf 2.4 17.9 397.11 47.70 5.80 0.254 0.105 44.95 -0.027

8.Grape leaf 11.7 65.9 15.40 41.07 4.92 0.576 0.098 47.84 0.346

9.Cashew 7.3 52.4 7.38 40.75 6.54 0.440 0.056 54.06 0.107*

10.Sea buckthorn fruit 5.5 38.8 9.62 43.70 5.74 0.911 0.136 51.96 0.241*

* Corrected for sugar

**Corrected for oxalic acid

Table 3.3 Comparison of ACON, Capacity, rate and ARP values of plant by product (fruit and leaf)

extracts (1.0 mg/mL)

Sample's name ACON Capacity (%) Rate (min-1) ARP (mg-1.min-1)

Acid ascorbic (1.0 mg/mL) 0.67 76.5 0.531 321.6

Cranberry leaf -0.0072 73.8 0.466 274.5

Black currant residue (F) 0.207 74.8 0.183 109.0

Sea buckthorn leaf -0.092 52.9 0.334 159.1

Betel leaf 0.508 71.6 0.452 261.1

Pomegranate (F) 0.364 72.1 0.432 250.7

Rhubarb 0.15 74.3 0.166 98.5

Blueberry leaf -0.027 72.1 0.47 172.9

Grape leaf 0.346 74.0 0.446 269.2

Cashew (F) 0.107 71.4 0.135 77.9

Sea buckthorn fruit 0.241 52.2 0.145 68.6

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Figure 3.5 Relationship between ACON and capacity of selected plant extracts

Figure 3.6 Relationship between ACON and rate of selected plant extracts

Figure 3.7 Relationship between ARP and ACON of selected plant extracts

R=0.76

R=0.67

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The relationships between ACON and antioxidant capacity, rate of radical quenching and

ARP are shown in Table 3.3 and Figures 3.5, 3.6 and 3.7. One would expect linear or other

relationships between them, if the hypothesis were to be correct. There was no apparent

relationship between ACON and antioxidant capacity (Figure 3.5), and there was no

differentiation between leaf and fruit samples. ACON and radical scavenging rate showed

some linear relationship separately for fruit and leaf sources (Figure 3.6). Yet ACON

related to ARP in a linear fashion separately to leaf (R=0.67) and fruit (R=0.76) sources

(Figure 3.7), suggesting that ARP that contains information on both capacity and rate may

be more meaningful, albeit the sample population is small. It is interesting to note that

rhubarb extract falls in the fruit line, although it is a vegetable.

One striking observation is that ACON-rate and ACON-ARP relationships clearly

distinguish the sources of the extracts, and helps us to visualize the compositional diversity

of metabolites in relation to redox chemistry in sub-cellular compartments of these sources.

Leaf extracts exhibited a higher antioxidant activity than fruit extracts, indicating that the

former group possesses more potentially active compounds and/or in higher concentrations.

In fact, metabolites in different sub-cellular locations of plant have a wide compositional

variation. The separate trend between ACON and ARP of extracts may be an indication of

links between redox status and phytochemical richness of extracts. Compositional

divergences among extracts are also apparent, showing that more reduced metabolites are

found in leaf in comparison to fruits, and hence leaf having higher ARP than fruits. In fact,

leaf contains both specific (NADPH) and non-specific (chlorophyll, phytophytine,

carotenoids, plastaquinone and phenolic compounds) antioxidant components, which can

confer to it a greater potential radical scavenger than fruit, which has mostly non-specific

antiradical components such as low pH, vitamin C, and abundant phenolic compounds to

combat oxidative stress. Presence of polyphenols is ubiquitous in the plant kingdom, but

their presence may greatly vary, because of differences in environmental conditions.

Consequently, the polyphenols are usually found in higher concentrations in leaves and

outer parts of plants, compared to the subterranean organs, where they are present in trace

amounts (Herrmann, 1988).

This study has identified some promising candidate-extracts using ARP metric from 140

samples. They include rambutan, cranberry leaf, blueberry leaf, grape leaf (wild), raspberry

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leaf, betel leaf, avocado, pomegranate, custard apple, grape red, grape leaf, grape red

(wine), mangosteen, indian plum Although only DPPH radical scavenging activity was the

focus in this study, many reactive oxygen and nitrogen species are encountered in biology.

It is imperative, therefore, to examine antioxidant activity of natural products against an

array of radicals in order to determine their potency as antioxidants. In addition, it may be

useful to identify antioxidant substances possessing high antioxidant capacity but acting at

different rates.

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3.5 Conclusions

DPPH assay is a widely used for preliminary evaluation of the antioxidant capacity of

natural products, as it is reliable and reproducible. However, the value of the method is

somewhat compromised, when the reaction is not allowed to reach completion (fixed time

measurement) or only either the anti-radical capacity or the scavenging rate is noted. This

work shows that the true antioxidant activity may lie on both the capacity as well as the rate

of radical scavenging. The new expression, ARP, generated from DPPH assay may be more

useful in identifying the antioxidant activity of biological samples. Some samples exhibited

high ARP value such as rambutan, cranberry leaf, blueberry leaf, grape leaf (wild),

raspberry leaf, betel leaf, avocado, pomegranate and custard apple. Leaf extracts possess, in

general, higher ARP than fruits, and root extracts possess low ARP. In addition, this study

also suggests that average carbon oxidation number (ACON) of complex mixtures such as

plant extracts may portend their antioxidant power, in spite of the disparity between leaf

and fruit materials.

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Chapter 4 ANTI-RADICAL ACTIVITY SPECTRUM OF SECLETED FRUIT AND

VEGETABLE EXTRACTS AGAINST SEVERAL REACTIVE OXYGEN SPECIES

Le spectre d'activité anti-radicalaire des extraits sélectionnés a été exploré. L'activité anti-oxydante a été évaluée par différentes méthodes incluant, la capacité anti-oxydante équivalente de Trolox (TEAC), la détermination des radicaux libres (DPPH), le pouvoir réducteur d’ion ferrique (FRAP), la mesure du potentiel d'oxydoréduction, la réduction du peroxyde d'hydrogène, du radical hydroxyle, de l’anion superoxyde, de l’oxyde nitrique et de l’activité de chélation du fer. La teneur totale en composés phénoliques (TPC) et en flavonoïdes (TFC) des extraits ont également été déterminées. Feuille de bétel, Fruit de bleuet et de cassis, feuille de canneberge ont montré une bonne activité de piégeage des radicaux (essai TEAC); pomme, oseille, vigne rouge et racine de pissenlit étaient efficace contre SOA; feuilles de canneberge, feuille de bleuet, cassis et rosemary contre le radical hydroxyle; rainbow chard, panais, brocoli et orange contre H2O2; et pomme de terre, banane, oseille, feuille d'argousier contre oxyde nitrique. Feuille et fruit de bleuet, grenade, cassis et feuille de bétel ont montré un bon pouvoir réducteur d’ion ferrique. Les extraits possédant la capacité élevée de chélation du fer étaient: feuille d’argousier, radis, panais, feuille de bétel et mangoustan. Extrait de feuille de bétel présentait des activités élevées, y compris de fixation du fer et de piégeage de divers radicaux, à l'exception de l'oxyde nitrique, où l'activité était néanmoins modérée. Les essais de TEAC, de DPPH et de FRAP fournissent essentiellement la même réponse concernant l'activité anti-oxydante des extraits de plantes, ce qui suggère que l'un de ces essais serait suffisant pour évaluer leur capacité anti-radicalaire. Cette étude suggère également que l'activité anti-oxydante d'une substance, déterminée par une ou plusieurs essais, ne donne pas une image complète de son efficacité contre les diverses espèces de radicaux oxygénés; et montre que la détermination du spectre de l'activité anti-radicalaire serait nécessaire.

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4.1 Abstract

Trolox equivalent antioxidant capacity (TEAC), DPPH radical assay (DPPH) and ferric

reducing power (FRAP) assays are used to assess their antioxidant activity. However, the

activity is rarely evaluated against various reactive oxygen and nitrogen species of

importance in biology. Thus the objective of this study was to examine the antioxidant

properties of selected plant extracts by evaluating their spectrum of anti-radical activity.

Thirty-six aqueous plant extracts from fruit peel and the leaf materials were analysed for

their antioxidant activity by TEAC, DPPH, and FRAP assays, redox potential (reducing

power), quenching capacity against hydrogen peroxide, hydroxyl radical, superoxide anion,

nitric oxide, and iron (pro-oxidant metal ion) chelating activity. Their total phenolic

content (TPC) and total flavonoid contents (TFC) were also determined. It was found that

avocado, betel leaf, blueberry fruit, blueberry leaf, black currant residue, cranberry leaf,

custard apple, grape leaf, sea buckthorn leaf, mangosteen, pomegranate, raspberry and

rosemary exhibited significant antioxidant capacities as determined by the related assays,

TEAC, DPPH and FRAP, among those tested, betel leaf, blueberry fruit and black currant

and cranberry leaf showing high activity (TEAC assay). Apple, sorrel, red grape and

dandelion root were against SOA; cranberry leaf, blueberry leaf, black currant and

rosemary against hydroxyl radical; rainbow chard, parsnip, broccoli and orange against

H2O2; and potato, banana, sorrel, sea buckthorn leaf against nitric oxide. Blueberry leaf and

fruit, pomegranate, black currant and betel leaf showed high ferric ion reducing power. The

extracts showing high iron binding capacity were: sea buckthorn leaf, radish, parsnip, betel

leaf and mangosteen. Betel leaf extract exhibited a broader spectrum of activities,

including iron binding and scavenging of various radicals except nitric oxide, where the

activity was moderate nonetheless. A satisfactory correlation between TEAC & DPPH and

TEAC & FRAP values was found suggesting that any one of them would be adequate to

evaluate their anti-radical capacity. And a strong correlation of TPC with TEAC (R2=

0.954) and DPPH (R2= 0.950) was obtained indicating that the TPC of the extracts were

the main contributors of the antioxidant activity. In addition, TPC and TFC did not

correlate with metal chelating activity, or superoxide anion, hydrogen peroxide, and nitric

oxide, suggesting that a range of phytochemicals must be present to confer a broad

spectrum activity to the extracts, not merely phenolic compounds.

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4.2 Introduction

Free radicals play a very important role in the oxidative damage of biological systems

(McCord, 2000; Scalbert et al., 2005). Oxidation is an essential process for the production

of energy and run the biological processes smoothly in living organisms. The continuous in

vivo generation of free radicals and other reactive oxygen species (ROS) during metabolic

processes result in cell death and tissue damage. The role of oxygen radicals has been

reported in several diseases, including cancer, diabetes and cardiovascular diseases, ageing,

etc. (Halliwell et al., 1999). The most common free radical derivatives of oxygen include

hydroxyl, superoxide anion, peroxyl radicals, superoxide, singlet oxygen and hydrogen

peroxide; and the reactive nitrogen species include nitric oxide and peroxynitrite anion.

Among them, hydroxyl radicals are highly reactive and can not be eliminated by

endogenous enzymes, SOD (superoxide dismutase) or glutathione cycle; and can damage

all bio-molecules. Singlet oxygen is linked to the oxidation of LDL-cholesterol.

Superoxide anion (SOA), a precursor of other ROS, contributes to lipid and DNA damage.

The scavenging of this radical may also help reduce the formation of hydrogen peroxide

and peroxy radicals (Nimse and Pal, 2015). There are also RNS (Reactive nitrogen species)

that can be harmful to proteins (Patel et al., 1999).

Anti-oxidants have a significant role in protecting the body from damage caused by free

radical induced oxidative stress (Ozsoy et al., 2008). Natural anti-oxidants, such as

polyphenols, found in medicinal and dietary plant products are very effective in preventing

oxidative damages (Silva et al., 2005). Their radical scavenging activity of polyphenols is

due to their structure, which helps to eradicate free radical, and in vitro they have been

shown to be more effective anti-oxidants than tocopherols and ascorbic acid. Ability of the

polyphenol derived radical to stabilize and delocalize the unpaired electron throughout the

structure, and ability to chelate transition metal ions make polyphenols to be used as an

ideal antioxidant (Evans et al., 1997). Human body has multiple enzymatic and non-

enzymatic antioxidant systems that protect the cellular molecules against ROS induced

damages. However, severe or continued oxidative stress makes the innate defense

insufficient for total protection. Hence, in order to balance the ROS in human body certain

amounts of exogenous anti-oxidants are constantly required to maintain an adequate level

of anti-oxidants.

128

Research efforts are also made in screening fruit and vegetable sources for identifying

novel antioxidants. This is particularly true for plant materials that might act as alternative

antioxidant sources (Kalpana et al. 2011). There is a plethora of plants that has been found

to possess strong antioxidant activity (Badami et al. 2003). The natural antioxidant-

compounds are present in all parts of the plant including barks, stalks, leaves, fruits, roots,

flowers, pods, seeds, stems, latex and hull (Ramarathnam et al. 1995; Rajaei et al. 2009;

Gholivand et al. 2010). Antioxidants exert their action through simple or complex

mechanisms including prevention of chain initiation, binding with transition metal ion

catalysts, decomposition of peroxides, prevention of continued hydrogen abstraction, and

radical scavenging (Diplock, 1999). Many studies showed that antioxidant such as

flavonoids and other phenolic compounds present in plant are associated in reducing the

risk of chronic diseases (Singh and Marimuthu, 2005; Cieslik et al. 2006).

The ability of the pure compounds or extracts to trap free radicals is measured in order to

estimate their antioxidant properties. Various methods has been developed to measure the

radical-scavenging capacity of plant derived compounds against free radicals like the 2,2-

diphenyl-1-picrylhydrazyl radical (DPPH•), 2,2-azinobis(3-ethylbenzothiazoline-6-

sulphonic acid) cation radical (ABTS•+); the superoxide anion radical or the hydroxyl

radical (Williams et al., 1995; Pelligrini et al., 1999; Huang et al., 2005 and Siddhuraju et

al., 2007), ferric reducing antioxidant power (FRAP) (Guo et al., 2003; Jiménez-Escrig et

al., 2001). During the reactions, either hydrogen atom is donated from an antioxidant or

electron is transferred.

Plant extracts are complex mixture of compounds, where phytochemicals may function in

synergism or antagonism with each other, and they may exhibit ‘Broad spectrum

antioxidant activity’ against various radicals in biology. The objective of this study was to

investigate the in vitro activity of selected plant extracts against free radicals, hydroxyl and

superoxide anion radicals, hydrogen peroxide, and nitric oxide; reducing activity, and iron

chelating activity. Moreover, the total phenolic compounds and total flavonoid contents of

the extracts was also assessed. Inter-relationships between activities against specific

radicals and their relationship with the titers of total phenolic compounds and total

flavonoids were also examined.

129



Thirty-six plant materials vegetable and plant by-products were collected from farms,

farmer’s markets and fruit and vegetable processors in and around Quebec, QC, Canada.

The plants material that were investigated include apple (malus communis), avocado

(Persea americana), banana (Musa acuminate), betel leaf (Piper betle leaf), blueberry fruit

(Vaccinium corymbosum fruit), blueberry leaf (Vaccinium corymbosum leaf), broccoli

(Brassica oleracea), black currant residue (Ribes nigrum residue), black currant fruit (Ribes

nigrum fruit), celery oriental (Apium graveolens), chinese litchi (Litchi chinensis),

cranberry leaf (Vaccinium macrocarpon leaf), custard apple (Annona squamosa. Lin),

dandelion root (Taraxacum officinalis root), dandelion leaf (Taraxacum Officinalis), grape

leaf (Vitis vinifera leaf), green pepper (green Capsicum annuum), hops (Humulus lupulus),

sea buckthorn fruit (Rudbeckia hirta fruit), sea buckthorn leaf (Rudbeckia hirta leaf),

mango peel (mangifera indica peel), mangosteen (Garcinia mangostana), orange peel

(Citrus sinensis), parsnip (Pastinaca sativa), pomegranate (Punica granatum), potato

mature (Solanum tuberosum mature), radish peel (Raphanus sativus peel), rainbow chard

(Beta vulgaris), raspberry leaf (Rubus idaeus leaf), red grape (Vitis vinifera), rhubarb

(Rheum rhabarbarum), rosemary (Rosmarinus officinalis), sorrel (Hibiscus sabdariffa),

kiwi peel (Actinidia chinensis ), canadian yew (Taxus Canadensis) and tomato (Solanum

lycopersicum). Upon arrival to the laboratory, samples were first washed with distilled

water and bleaching agents (Sodium Hypochlorite-300 ppm) to remove dirt and debris.

Thereafter, within 48 hours, the samples were lyophilized, ground to powder, vacuum-

sealed and stored at -30°C until ready for extraction.

http://en.wikipedia.org/wiki/Solanum

130


Ascorbic acid, aluminum chloride, ammonium hydroxide, 2,2′azino-bis(3-ethyl

benzothiazolin e-6-sulfonic acid) diammonium salt (ABTS), 1,1 diphenyl-2-

picrylhydrazine (DPPH), ferrous sulfate, ferrous chloride, Folin Ciolcalteu’s reagent,

hydrogen peroxide, 1,10-phenanthroline, naphthylethylenediamine dihydrochloride,

nicotinamide adenine dinucleotide (NADH), nitroblue tetrazolium (NBT), phenazine

methosulfate (PMS), rutin, phosphoric acid, potassium persulfate, potassium

hexacyanoferrate (II), potassium hexacyanoferrate (III), sodium carbonate, sodium

ethylene diamine tetra acetate (Na2EDTA) sodium nitrite, sodium nitroprusside, sodium

phosphate monobasic, sodium phosphate dibasic, sulfanilic acid, sulfuric acid, 3-(2-

Pyridyl)-5,6-diphenyl-1,2,4-triazine-4′,4′′-disulfonic acid sodium salt and trichloroacetic

acid were purchased from Sigma-Aldrich (Canada). All the reagents were of analytical

grade.

4.3.3 Plant extract preparation

About 50 gram of the lyophilized powder of raw sample was extracted with 500 mL of




lyophilized extracts were vacuum-packed and stored at -30oC until further use. The stock

solution of plant extract at 1.0 mg/mL was prepared in distilled water before the assay.

4.3.4 Total phenolic content

Total phenolic content of plant extracts was determined by the Folin-Ciocaulteu assay

according to the method described by Singleton et al. (1999) with some modifications.

From the stock solution of plant extract at 1.0 mg/mL, an aliquot of 0.5 mL was mixed

with 0.5 mL of Folin-Ciocalteau reagent. After 3 min, 0.5 mL of saturated sodium

carbonate solution was added, and the solution was diluted to 5 mL with deionized water.

The reaction mixture was kept in the dark for 2 hr and its absorbance was measured at 760

nm against water in UV-Vis spectrophotometer (Biotek, Power XS2, Logiciel Gen 5).

131

Gallic acid and ascorbic acid were used as standards. The results were expressed in terms

of gallic acid equivalent (GAE) and ascorbic acid equivalent (AAE). All experiments were

performed in triplicate.

4.3.5 Total flavonoid assay

Total flavonoid content was determined with aluminium chloride colorimetric assay

(Chang et al., 2002). Plant extract (1.0 mg/mL) was added to 10 mL volumetric flask

containing 4 mL of water. To the above mixture, 0.3 mL of 5% NaNO2 was added. After 5

minutes, 0. 3 mL of 10 % AlCl3 was added. After 6 min, 2.0 mL of 1M NaOH was added,

and the total volume was made up to 10 mL with distilled water. The solution was mixed

and the absorbance was measured against a blank at 510 nm. Rutin was used as a standard,

and the results were expressed as rutin equivalent (RE).

4.3.6 Antioxidant Assays

Antioxidant capacity of each extract from plant was performed using Trolox equivalent

antioxidant capacity (TEAC), DPPH free radical assay, reducing power assay (FRAP),

hydrogen peroxide, hydroxyl radical, superoxide anion and nitric oxide scavenging assays

and metal chelation capacity assay.

4.3.6.1 TEAC (Trolox equivalent antioxidant capacity)

Trolox equivalent antioxidant capacity (TEAC) was carried out according the method

reported by Zulueta et al. (2009) with some modifications. ABTS and potassium persulfate

were dissolved in deionized water to make up to a concentration of 7 mM and 140 mM,

respectively. This reaction mixture was left at ambient temperature for 12 to 16 hours in

the dark to obtain the blue-green coloured radical cation of ABTS+. On the day of analysis,

the ABTS+ solution was diluted with phosphate buffered saline (PBS) (10 mM, pH 7.4) to

obtain an absorbance of 0.700 ± 0.001 at 734 nm. For the spectrophotometric assay 190 µL

of the ABTS+ solution 10 µL of plant extract (1.0 mg/mL) were mixed and the absorbance

was determined at 734 nm during 2 hours with a microplate spectrophotometer (Biotek,

Power XS2, Logiciel Gen 5). Trolox was used as a standard and the results were expressed

as Trolox equivalent. The assay was carried out in triplicate.

132

4.3.6.2 DPPH free radical scavenging assay

The free-radical scavenging activity of plant extract was assessed by the decrease in

absorbance of methanolic solution of DPPH (López-Galilea et al., 2006). A stock solution

of DPPH (6.1 x10-5 M) was prepared immediately before use. The DPPH solution was

diluted with methanol to obtain an absorbance of 0.7 (± 0.02) at 515 nm. Aliquots of 10 µL

of each plant extract (1.0 mg/mL) and 190 µL of DPPH solution were transferred to a

microplate. A microplate spectrophotometer (Biotek, Power XS2, Logiciel Gen 5) was

used for incubation and reading of microplates (96 wells). Incubation was performed at a

temperature of 25°C for a period of 1 hour. Absorbance was measured at 515 nm at

interval of 1 min. Ascorbic acid was used as a standard and the results were expressed as

ascorbic acid equivalent. The assay was carried out in triplicate.

4.3.6.3 Reducing power property

a) Ferric reducing power assay (FRAP)

The reducing power of the extracts was performed according to the method of Oyaizu

(1986), based on the reduction reaction from Fe (III) to Fe (II) with some modifications.

1.0 mg/mL of plant extract (2.5 mL) was mixed with 2.5 mL of 0.2 M sodium phosphate

buffer (pH 6.6) and 2.5 mL of 1% potassium ferricyanide. The mixture was incubated at

50°C for 20 min. After 2.5 mL of 10% trichloroacetic acid (w/v) was added, and the

mixture was centrifuged at 3000 rpm for 10 min. The upper layer (2.5 mL) was mixed with

2 mL of de-ionised water and 0.5 mL of 0.1% of ferric chloride and the absorbance was

measured spectrophotometrically at 700 nm. Ascorbic acid was used as standard and the

results were expressed as ascorbic acid equivalent. The assay was carried out in triplicate.

b) Potentiometry

The reducing property of the plant extract was assessed according to the method of

Brainina et al. (2007) with some modifications. Chemical interaction of the plant extract

with mediator system K3[Fe(CN6)]/K4[Fe(CN6)] leading to its redox potential shift was

used. Briefly, the redox potential measurements were made with a platinum electrode

connected to a voltmeter (YSI Pro 1020 ORP meter). Calibration was performed against

420 mV redox standard solutions (VWR ORP standard) at room temperature. Solution

containing 0.5 mM of ferricyanide and ferrocyanide were prepared by mixing 1 mM of tri-

133

potassium hexacyanoferrate (III) and 1 mM of potassium hexacyanoferrate (II) trihydrate

solution. Both solutions were freshly prepared by dissolving solids with 10 mM phosphate

buffer (pH 7.0). An electrode (Symphony platinum electrode, Ag/AgCl) was placed in a 50

mL plastic tube containing 28 mL of ferricyanide/ferrocyanide solution (0.5 mM) and 2

mL of plant extract (1.0 mg/mL). Prior to analysis, oxygen was removed from the system

by continuous flushing with argon for a period of 30 min. The potential was read

approximately 10 min after dipping the electrodes into the solution. A stable redox

potential was arbitrarily defined as a change of less than 1mV in a 3-min period. Ascorbic

acid was used as standard and the results were expressed as ascorbic acid equivalent. The

assay was carried out in triplicate.

4.3.6.4 Hydrogen peroxide scavenging activity

Hydrogen peroxide scavenging activity was carried out following procedure previously

described by Patterson et al. (1984). 135 µL of 20% TiCl4 in concentrated HCl was added

to 100 µL of plant extract (1.0 mg/mL), 815 µL of phosphate buffer (0.17 M, pH 7.4), 200

µL of NH4OH (17.0M), and 100 µL of H2O2 to give a precipitate which was dissolved in 3

mL of 1M H2SO4. Absorbance of the solution was measured spectrophotometrically at 410

nm.

4.3.6.5 Hydroxyl radical scavenging activity

The scavenging activity for hydroxyl radical was measured with Fenton reaction

(Sachindra et al., 2007). One mL of 1.865 mM 1.10 phenanthroline was mixed with 1.0 mL

of 1.865 mM FeSO4, 2.0 mL of 0.2 M phosphate buffer (7.4), 1.0 ml of 1.0 mg/mL plant

extract and 1.0 mL of 0.03 % H2O2. The mixture was incubated at 37°C for 1 hr. After

incubation, absorbance of the mixture was measured at 510 nm with a spectrophotometer.

Ascorbic acid (1.0 mg/mL) was used as reference. All the tests were carried out in

triplicate.

4.3.6.6 Superoxide anion scavenging capacity

Superoxide radical scavenging capacity is based on the anion radical which is associated

with PMS-NADH system (Liu et al., 1997). They are generated through PMS-NADH

system by the oxidation of NADH and are assayed by the reduction of nitro-blue

tetrazolium (NBT). 1.0 mL of NBT solution (156 µM NBT in 100 mM phosphate buffer,

pH 7.4), 1 mL of NADH solution (468 µM in 100 mM phosphate buffer, pH 7.4) and 0.1

134

mL of extract (1.0 mg/mL) were mixed and the reaction started by adding 100 µL of

phenazine methosulphate (PMS) solution (60 µM PMS in 100 mM phosphate buffer, pH

7.4). The reaction mixture was incubated at 25°C for 5 min, and the absorbance was

measured at 560 nm. Decrease in the absorbance of the reaction mixture indicates

increased superoxide anion scavenging capacity. Ascorbic acid was used as standard

solution and the results were expressed as acid ascorbic equivalent. All the tests were

performed in triplicate.

4.3.6.7 Nitric oxide scavenging activity

Sodium nitroprusside in aqueous solution at physiological pH spontaneously generates

nitric oxide, which interacts with oxygen to produce nitrite ion which can be determined by

using Griess reaction (Marcocci et al. 1994). 1.0 mL of 10 mM sodium nitroprusside

prepared in phosphate buffer (50 mM, pH 7.4) was mixed with 0.5 mL of the extract (1.0

mg/mL) and the mixture was incubated at 25°C for 150 min. From the incubation mixture,

1.0 mL was taken out and added to 1.0 mL of Griess reagent (1% sulphanilamide, 2%

orthophosphoric acid, and 0.1% napthlethyline diamine hydrochloride). The absorbance of

the chromophore formed was read at 546 nm. A decrease in absorbance indicates the

increase of nitric oxide scavenging capacity. Ascorbic acid (1.0 mg/mL) was used as

standard antioxidant. All the tests were performed in triplicate.

4.3.6.8 Metal chelating assay (Spectrometric assay)

The chelating abilities on ferrous ion by plant extract were estimated by the method as

described by Gülçin (2006) with some modifications. To 0.5 mL of extract (1.0 mg/mL), 1.

6 mL of deionized water and 0.05 mL of FeCl2 (2 mM) were added. Reaction mixture was

left at room temperature for 10 min with continuous stirring. Finally, the reaction was

completed by the addition of 5 mM of ferrozine (0.1 mL). Then, the mixture was shaken

vigorously and left at room temperature for 10 min. Absorbance of the solution was

measured at 562 nm. Na2EDTA (0.1mg/mL) was used as standard and the results were

expressed as Na2EDTA equivalent. The assay was carried out in triplicate.

4.3.7 Data analysis



135


when the analysis of variance found significant differences. Principal component analysis

(PCA) was also performed using R with the statistical package Rcmdr.

136

4.4. Results and Discussion

4.4.1 Total phenolic and flavonoid contents

The TPC (total phenolic compounds) and TFC (total flavonoid content) values of the

extracts were ranged from 168.16 to 7.57μg of gallic acid equivalent/mg of dry extract (or

119.72 to 8.20 μg of Ascorbic acid equivalent/mg of dry extract) and 762.33 to 1.67 μg of

rutin equivalent/mg of dry extract, respectively (Table 4.1). Among the extracts, blueberry

leaf was found to contain the highest phenolic and flavonoid contents. The lowest value of

total phenolic and flavonoid contents was found in dandelion root extract. Among thirty-

six plant extracts studied, the extracts those exhibiting high values of both TPC and TFC

were betel leaf, black currant fruit, black currant residue, chinese litchi, cranberry leaf,

custard apple, sea buckthorn fruit and leaf, mangosteen, parsnip, pomegranate, raspberry,

avocado and rosemary.

Generally, extracts that contain high amount of polyphenols also exhibited high antioxidant

activity. Phenolic compounds are the most widespread secondary metabolites in plants.

This class of compounds have received much attention as potential natural antioxidants in

terms of their ability to act as both efficient radical scavengers and metal chelators (Sharma

and Singh, 2012). It has been reported that the antioxidant activity of phenolic compounds

is mainly due to their redox, hydrogen donating and singlet oxygen quenching properties

(Dutra et al., 2008). The TPC of plant extracts was determined using the Folin-Ciocalteu’s

method. The exact mechanism of the reaction is complex, but it is essentially a redox

reaction occurring between antioxidants with phosphotungstic and phosphomolybic acids.

Thus the assay would not be specific to just phenolic compounds, but also to other

substances that could be oxidized by Folin-reagent; numerous reports have dealt with the

poor specificity of this assay (Singleton et al. 1999; Escarpa and Gonzalez, 2001).

Flavonoids, including flavones, flavanols and condensed tannins, are major class of

secondary metabolites, the antioxidant activity of which depends on the presence of free

OH groups, especially 3-OH. Plant flavonoids have antioxidant activity in vitro and also

act as antioxidants in vivo (Geetha et al., 2003; Shimoi et al., 1996).

137

Table 4.1 Total phenolic and flavonoid contents of aqueous extracts of 36 selected plant materials:

Total phenolic content was expressed as gallic acid (GAE) and ascorbic acid equivalent (AAE) and total

flavonoid content was expressed as rutin equivalent (RE). (±) SD

Extract Total Phenolic Content (TPC) Total Flavonoids Content

(TFC)

(1mg of dry extract)

(μg of Gallic acid

equivalent/mg of dry

extract)

(μg of Ascorbic acid

equivalent/mg of dry

extract)

(μg of Trolox equivalent/mg

of dry extract)

Betel leaf 154.03±0.40 109.91±0.28 24.33±2.52

Blueberry fruit 152.64±1.33 108.94±0.92 300.67±7.51

Brocoli 68.27±0.49 50.35±0.34 90±4.58

Black currant residue 156.80±0.42 111.83±0.29 432.67±9.61

Black currant Fruit 163.89±0.64 116.76±0.45 265.33±5.69

Celery oriental 47.39±1.07 35.85±0.75 39.00±3.61

Chinese leechi 136.64±1.83 97.83±1.27 19.67±2.08

Cranberry leaves 158.03±1.34 112.69±0.93 668.33±9.29

Custard apple 159.68±0.45 113.83±0.31 25.33±5.03

Grape leaf, wild 154.77±1.64 110.43±1.14 687.33±13.50

Green pepper 27.28±0.71 21.89±0.49 2.67±1.53

Hops 81.33±0.40 59.43±0.28 35.67±1.53

Sea buckthorn Fruit 137.12±2.07 98.17±1.44 101.33±3.51

Sea bruckthorn leaf 129.36±10.05 92.78±6.98 106.67±3.06

Mango peel 53.63±0.20 40.19±0.14 38.67±1.15

Mangosteen 159.44±3.15 113.67±2.19 41.67±0.58

Parsnip 139.17±0.53 99.59±0.37 251.67±2.08

Pomegranate 158.80±0.37 113.22±0.25 321.67±3.51

Radish peel 73.41±1.36 53.93±0.94 30.33±1.53

Rainbow chard 58.29±2.30 43.43±1.60 68.00±1.00

Raspberry leaf 154.03±0.69 109.91±0.48 270.33±7.64

Red grape 39.92±0.45 30.67±0.31 3.67±1.15

Rhubarb 95.33±1.19 69.15±0.82 7.33±1.15

Sorrel 23.89±4.14 19.54±2.87 31.33±3.06

Kiwi peel 17.81±0.18 15.31±0.13 4.00±1.00

Tomato 33.15±1.57 25.96±1.09 3.67±0.62

Avocado 161.95±2.13 115.41±1.48 83±3.46

Dandelion leaf 52.35±0.95 39.30±0.66 84.33±2.08

Dandelion root 7.57±0.33 8.20±0.23 1.67±0.58

Blueberry leaf 168.16±1.45 119.72±1.00 762.33±18.01

Rosemary 163.09±2.04 116.20±1.42 338.67±10.69

Orange 21.09±0.20 17.59±0.14 5.33±1.53

Canadian yew 17.15±0.32 14.85±0.22 2.33±1.15

Apple 24.32±0.70 19.83±0.48 2.33±0.58

Banana 24.85±0.56 20.20±0.39 3.67±1.15

Potato Mature 34.59±0.33 26.96±0.23 20.00±2.65

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4.4.2 Antioxidant activity

Plants, rich in secondary metabolites, including phenolic compounds, and flavonoids

mainly, have antioxidant activity due to their chemical structure and redox properties.

Generally, the aqueous extracts exhibited activities against various radicals investigated.

4.4.2.1 Test Radicals (ABTS tand DPPH)

Trolox equivalent antioxidant capacity

The TEAC (ABTS● scavenging activity) value is a quantification of the effective

antioxidant activity of the extracts. The higher TEAC value implies a greater antioxidant

activity. Table 4.2 shows TEAC activity of thirty-six samples. Betel leaf, blueberry fruit

and leaf, black currant residue, cranberry leaf, custard apple, grapes leaf, sea buckthorn

leaf, mangosteen, pomegranate and raspberry were found to possess high TEAC values,

suggesting that these extracts might function as efficient antioxidants. Moderate ABTS●

scavenging activity was recorded for Chinese litchi (216.30 μg of Trolox equivalent/mg of

dry extract), sea buckthorn fruit (191.79), parsnip (172.14) and rhubarb (120.63), while the

lowest activity was shown by green pepper (31.74).

DPPH free radical scavenging assay

The DPPH free radical scavenging capacity of the 36 aqueous plant extracts are shown in

Table 4.2. Orange showed the lowest DPPH free radical scavenging capacity, while betel

leaf, blueberry fruit, black currant residue, pomegranate, grape leaf, custard apple,

raspberry, avocado, blueberry leaf, and rosemary extracts exhibited promising activities,

suggesting that these extracts may contain some compounds which are efficient hydrogen-

donating antioxidants. Other than these extracts with highest activity, black currant fruit,

chinese litchi, cranberry leaf, sea buckthorn fruit and leaf, and parsnip also showed

relatively high DPPH free radical scavenging activities. DPPH free radical scavenging

activity of plant extracts is commonly reported (Parejo et al. 2003; Wong et al. 2005, Luo

et al. 2010, Beyhan et al. 2010, Sharma et al 2012). The DPPH assay is technically simple

and rapid that might explain its widespread use in antioxidant screening (Karadag et al

2009). However, there are some drawbacks, which may limit its application. It was

reported that reaction of DPPH with eugenol was reversible (Huang et al., 2005), which

139

could result in unexpected low reading of antioxidant capacity of sample containing

eugenol, and other compounds bearing a similar structure. This hypothesis may, in part,

explain the low DPPH free radical scavenging activity exhibited by some extracts in this

study. In addition, the low DPPH values registered by of some extracts could be related to

the presence of compounds not reactive towards DPPH.

140

Table 4.2 Free radical scavenging activity and reducing property of the aqueous extracts obtained from

selected plant materials. (±) denotes standard deviation

Extract TEAC assay DPPH assay Superoxide

Radical

Scavenging

Hydroxyl

Radical

Scavenging

Hydrogen

Peroxide

Radical

Scavenging

(1mg of dry

extract)

(μg of Trolox

equivalent/m

g of dry

extract)

(μg of

Ascorbic acid

equivalent/m

g of dry

extract)

(μg of

Ascorbic acid

equivalent/m

g of dry

extract)

(μg of

Ascorbic

Acid

equivalent/m

g of dry

extract)

(μg of

Ascorbic

Acid

equivalent/m

g of dry

extract)

Betel leaf 281.72±0.11 137.14±0.62 259.36±2.59 230.80±26.35 162.43±3.36

Blueberry fruit 280.69±0.11 134.08±2.36 32.40±5.87 98.57±13.91 124.22±4.24

Brocolli 56.18±4.22 39.27±6.15 80.01±1.61 32.29±6.25 382.70±8.94

Black currant

residue 281.39±0.11 135.88±0.83 37.73±3.86 398.91±9.64 155.83±6.97

Black currant

Fruit 271.50±1.73 124.34±9.00 179.29±13.95 27.68±3.35 27.68±2.60

Celery oriental 38.76±6.05 6.83±0.83 15.74±4.00 26.40±5.10 289.10±8.58

Chinese leechi 216.30±2.50 129.39±4.72 108.83±10.13 44.25±14.03 54.50±5.20

Cranberry leaves 279.67±0.22 136.60±1.65 28.98±4.36 446.36±5.11 84.88±3.28

Custard apple 280.24±0.22 130.83±1.43 23.64±2.67 185.50±14.14 166.62±5.44

Grape leaf, wild 253.18±5.65 131.37±3.24 38.37±9.11 29.20±5.43 46.88±4.27

Green pepper 31.74±1.46 7.91±1.90 39.65±14.05 18.69±3.80 60.07±8.44

Hops 99.00±0.48 59.82±3.84 75.31±10.01 14.09±3.78 117.35±20.44

Sea buckthorn

Fruit 191.79±3.05 122.54±7.34 106.91±15.08 39.10±10.80 87.05±1.41

Sea bruckthorn

leaf 229.76±5.42 134.98±2.25 119.72±5.45 44.95±9.87 175.75±5.02

Mango peel 75.01±4.13 61.08±1.43 189.33±11.33 58.98±13.75 128.22±5.30

Mangosteen 280.24±0.22 135.88±0.83 15.31±8.46 190.76±3.28 171.75±1.98

Parsnip 172.14±2.01 106.50±4.29 162.00±2.06 20.09±4.84 463.49±31.00

Pomegranate 280.37±0.38 134.62±0.94 51.40±8.98 55.23±10.00 267.38±21.21

Radish peel 102.13±1.83 34.40±4.72 172.03±4.00 15.20±4.42 11.83±2.47

Rainbow chard 54.26±4.82 50.26±1.95 44.35±7.96 16.60±4.28 760.97±14.37

Raspberry leaf 280.63±0.11 133.35±2.25 24.07±7.39 99.56±22.40 74.88±14.85

Red grape 39.71±3.65 28.99±2.19 251.46±2.79 23.27±8.40 122.13±17.50

Rhubarb 120.63±9.45 98.21±8.38 15.95±3.03 15.10±1.57 28.68±0.89

Sorrel 39.46±4.73 13.67±2.05 270.04±0.64 19.92±4.77 117.20±15.52

Kiwi peel 39.33±3.22 11.15±1.90 198.08±6.32 18.30±1.78 105.87±2.68

Tomato 57.01±6.26 9.35±2.05 209.19±12.86 18.31±2.46 123.55±5.44

Avocado 264.93±15.32 133.35±2.05 116.73±3.03 75.75±13.26 4.60±0.20

Dandelion leaf 61.67±5.53 28.99±2.77 241.64±11.82 16.88±4.00 37.38±3.46

Dandelion root 35.31±1.89 23.95±1.13 257.65±3.70 30.30±2.01 260.05±6.50

Blueberry leaf 281.07±0.22 135.16±0.54 194.88±11.21 368.47±19.51 130.72±16.16

Rosemary 279.80±0.38 130.29±1.08 14.03±4.63 288.25±12.18 115.22±14.50

Orange 37.67±1.73 22.15±2.16 36.88±1.69 22.02±10.95 306.55±14.01

Canadian yew 26.12±2.32 7.01±1.08 103.07±11.05 19.61±1.22 141.38±2.40

Apple 37.86±6.94 14.76±2.72 272.17±2.43 23.06±0.73 109.05±5.26

Banana 41.18±4.39 12.23±1.13 67.62±9.57 22.56±2.69 136.72±0.35

Potato Mature 63.58±0.58 10.97±2.25 229.04±12.86 19.28±6.31 54.55±16.26

141

4.4.2.2 Relation between anti-radical activities (TEAC, DPPH) and total phenolic

content (TPC) and total flavonoid content (TFC)

There was a strong correlation between TPC and DPPH activity (Figure 4.3). The

relationship between total antioxidant capacities obtained from TEAC and DPPH assays

was parabolic (R2 =0.954) (Figure 4.4), suggesting that antioxidant components in the

extract mixtures could scavenge free radicals by donating single electron and hydrogen

atom, and any one of them could be used for total antioxidant assay, barring certain

constraints with DPPH assay as pointed out before.

(TEAC value)0.5

g of Trolox equivalent/mg of dry extract

4 6 8 10 12 14 16 18

DP

PH

valu

e

g o

f A

scorb

ic a

cid

eq

uiv

ale

nt/

mg

of

dry

ext

ract

0

20

40

60

80

100

120

140

160

R2=0.951

Figure 4.1 Correlation between Trolox equivalent antioxidant capacity and DPPH free radical

scavenging activity

142

The linear relationship between TPC and total antioxidant capacity expressed by either

DPPH (R2 = 0.954) or TEAC (R2 = 0.950) value, as seen in Figure 4.4, suggests that

phenolic compounds could be major contributors of free radical scavenging activity of most

of the plant extracts. This result is consistent with many previous studies (e.g., Lamien-

Meda et al., 2008, Ikram et al., 2009). The small variations in both assays is possibly due to

the relatively higher contribution of hydrophobic components to the activity in the DPPH

assay, while the hydrophilic components mainly contribute to the activity in the ABTS

assay (Moniruzzaman et al., 2011; Müller et al., 2011).

Figure 4.2 Correlation between Trolox equivalent antioxidant capacity (TEAC), DPPH and total

phenolic content (TPC)

143

4.4.2.3 Reducing power property (FRAP)

The ferric ion reducing activities (FRAP) and redox potential (potentiometric assay) values

of the thirty-six plant extracts is shown in Table 4.3 Among the samples, betel leaves,

blueberry fruit, black currant residue, mangosteen, pomegranate and raspberry showed

high FRAP and reducing power values. However, the crude extract of blueberry leaf

displayed, in particular, a considerable reducing power, possibly due to the effect of its

phytochemical components as electron donors and thereby halting radical chain reaction by

converting free radicals to more stable products. Moderate high activity was recorded with

black currant fruit, cranberry leaf, custard apple, sea buckthorn leaf, and avocado; and

dandelion root showed the lowest ferric reducing power.

Naczk et al. (2003) indicated that antioxidant activity of a crude phenolic compound

extract from wild blueberry leaf was comparable to that exhibited by the synthetic

antioxidant butylated hydroxyanisole (BHA). However, Yasoubi et al. (2007) found that

pomegranate peel extract at a 0.050 % level, its antioxidant activity was noticeably higher

than synthetic antioxidants. Betel leaf had been reported to be effective antioxidant, as the

extract of the leaf retarded rancidity of butter cakes and extended their self life (Lean and

Mohamed, 1999).

144

Table 4.3 Free radical scavenging activity and reducing property of the aqueous extracts obtained from

selected plant materials. (±) denotes standard deviation: Ferric reducing power assay (FRAP) and

Nitric Oxide Radical Scavenging (NO)

Extract

(1.0 mg of dry

material)

FRAP

(Spectroscopic Study)

Nitric Oxide Radical

Scavenging


equivalent/mg of dry extract)


equivalent/mg of dry extract)

Betel leaf 295.57±6.44 94.21±1.76

Blueberry fruit 329.67±0.87 93.00±2.29

Brocoli 25.14±0.53 68.41±2.02

Black currant residue 315.52±4.16 75.43±0.89

Black currant Fruit 222.51±4.30 44.77±0.60

Celery oriental 18.61±0.45 94.20±6.26

Chinese leechi 95.33±6.35 100.52±4.63

Cranberry leaves 223.15±0.94 112.79±3.48

Custard apple 142.21±1.03 91.39±6.11

Grape leaf, wild 262.36±6.06 106.94±2.21

Green pepper 20.12±0.63 61.36±3.72

Hops 57.79±0.44 108.52±6.38

Sea buckthorn Fruit 94.54±0.57 109.29±0.69

Sea bruckthorn leaf 124.23±2.97 134.04±2.45

Mango peel 49.38±0.71 133.58±2.36

Mangosteen 273.23±8.24 116.66±2.29

Parsnip 82.35±0.28 128.62±3.96

Pomegranate 340.22±0.61 109.08±2.04

Radish peel 30.37±0.15 110.60±1.36

Rainbow chard 19.99±0.07 134.49±0.24

Raspberry leaf 294.23±8.89 131.47±0.51

Red grape 23.76±0.35 128.94±2.53

Rhubarb 71.97±0.64 100.38±1.72

Sorrel 27.98±0.18 97.36±4.32

Kiwi peel 11.45±0.39 134.89±0.83

Tomato 17.15±1.18 132.04±3.57

Avocado 124.98±1.48 92.85±3.95

Dandelion leaf 30.20±1.23 120.64±2.43

Dandelion root 3.29±0.13 65.80±3.56

Blueberry leaf 361.79±12.12 104.59±1.68

Rosemary 216.61±28.61 118.32±3.39

Orange 7.28±1.05 121.09±1.72

Canadian yew 7.33±0.36 109.37±2.27

Apple 11.57±0.07 124.38±5.56

Banana 16.85±1.59 138.20±1.37

Potato Mature 22.61±0.21 143.09±1.89

145

The FRAP and redox values showed a strong linear relationship (R2 =0.937) (Figure 4.6).

Thus, it seems that either one of the methods can be considered for evaluation of the

reducing power of biological samples.

Figure 4.3 Relationship between redox (potentiometric) and FRAP analysis

The DPPH free radical scavenging capacity and Trolox equivalent antioxidant capacity of

the extracts were significantly different from that of FRAP. The correlation between

Trolox equivalent antioxidant capacity and FRAP showed a strong linear correlation (R2

=0.991) (Figure 4.7), suggesting that the extracts contain a class of compounds,

presumably the total phenolic compounds, have not only the ability the ability to scavenge

organic radicals (ABTS, TEAC and DPPH) (Figure 4.4), but also reduce the oxidants such

as ferric ions. Among the plant extracts tested in this study, the leaf extracts (betel,

blueberry, cranberry, raspberry and rose mary), and fruit residue extracts (blueberry, black

currant, custard apple and mangosteen) exhibited high activity to the three related

antioxidant activities, i. e., TEAC, DPPH and FRAP.

146

(TEAC value)0.5

( g of Trolox equivalent/mg of dry extract)

4 6 8 10 12 14 16 18

FR

AP

va

lue

g o

f A

sco

rbic

acid

eq

uiv

ale

nt/m

g o

f d

ry e

xtra

ct)

0

50

100

150

200

250

300

R2=0.991

Figure 4.4 Relationship between TEAC and FRAP values of plant extracts

4.4.2.4 Hydroxyl radical scavenging activity

From the results given in Table 4.2, cranberry leaf exhibited the highest activity among the

extracts since it was able to scavenge 446.36 μg of AAE (ascorbic acid equivalent)/mg of

dry extract, followed in the decreasing order: cranberry leaf, blueberry leaf, black currant,

rose mary, betel leaf, mangosteen and custard apple. Blueberry fruit, avocado and

raspberry leaf exhibited a moderately high activity, and the lowest activity was observed

for hops (14.09 μg of ascorbic acid equivalent/mg of dry extract). Our results were

partially consistent with those of Wang et al (2000), who reported high hydroxyl radical

scavenging activity among different cranberry cultivars. It has been reported that hydroxyl

radical targets lipid peroxidation by subtracting out hydrogen atoms from the membrane

and leads to membrane damages (Lobo et al., 2010). Therefore, we hypothesized the

extract from cranberry leaves having strong activity would suppress lipid peroxidation on

biomembranes and quenched the hydroxyl radicals at the stage of initiation and termination

of peroxy radicals.

147

4.4.2.5 Hydrogen peroxide scavenging activity

The hydrogen peroxide scavenging activity of most plant extracts was generally low,

except for a few. Among of the 36 studied plant extracts (Table 4.2), only rainbow chard

exhibited relatively high hydrogen paroxide scavenging activity (760.97 μg of ascorbic

acid equivalent/mg of dry extract), followed by parsnip (463.49), broccoli (382.70), orange

(306.55), and celery oriental (289.10). Moderately high activity was observed for

pomrgranate, dandelion root, sea buckthorn leaf, mangosteen, custard apple and betel leaf.

Black currant fruit showed the lowest activity (27.68). Our result was in agreement with

those earlier reported by Hazra et al. (2010), who found negligible hydrogen peroxide

scavenging activity in some fruits (Terminalia chebula, Terminalia belerica and Emblica

officinalis) in comparison to that of the standard (ascorbic acid). In contrast, our results

were opposed to those obtained by Ogunlana and Ogunlana (2008) who reported high

hydrogen peroxide activity in papaya (Psidium guajava) and Wang et al. (2000) who

indicated strong hydrogen peroxide activity for different blackberry cultivars.

4.4.2.6 Superoxide anion scavenging activity

As shown in Table 4.2, it can be put forward that apple (272.17 μg of Ascorbic acid

equivalent/mg of dry extract), sorrel (270.04), betel leaves (259.36), dandelion root

(257.65), dandelion leaf (241.64), red grape (251.46), potato (229.04) and tomato (209.19)

were more potent scavengers of superoxide radical than the other plant extracts. However,

moderately high activities were exhibited by sorrel, kiwi peel, blueberry leaf, mango peel,

black currant fruit, radish peel, parsnip and sea buckthorn. Higher inhibitory effects of the

plant extracts on superoxide anion formation noted herein possibly render them as

promising antioxidants.

4.4.2.6 Nitric oxide scavenging activity

The assay involves generation of nitric oxide using sodium nitroprusside system, which

forms nitrite ion by auto-oxidation. The stable nitrite ion, is assayed spectrophotometrically

(546 nm) measured using Griess reagent system (Marcocci et al. 1994). From the result

given in Table 4.3, it appears that potato, banana, sea buckthorn leaf, mango peel, rainbow

chard, raspberry leaf, tomato, parsnip, apple and orange were potent scavengers of nitric

148

oxide radical. A number of studies reported the potentiality of plant extracts to scavenge

nitric oxide in vitro (Alisi and Onyeze, 2008, Balakrishnan et al. 2009, Ebrahimzadeh et al.

2010, Banerjee et al. 2011). The nitric oxide radical scavenging ability of some flavonoids

was has been reported, and anthocyanidins were found to be the most effective class (Apak

et al., 2016).

4.4.2.7 Metal chelating activity

All the extracts tested were able to chelate ferrous ion to varying extents (Table 4.4). Sea

buckthorn leaf, radish, parsnip, betel leaf, mangosteen, banana showed high iron chelating

activity. While sea buckthorn fruit and rainbow chard, hops, potato mature, tomato,

dandelion leaf and red grape exhibited relativity high activity suggesting that these extracts

contain chelatins and/or or polyacids which were able to chelate ferrous ion, causing the

loss of colour development in the assay.

149

Table 4.4 Ferrous ion chelating activity of aqueous extract obtained from selected plant material. This

activity was expressed as Na2EDTA equivalent and (±) means the standard deviation

Metal chelating activity (spectroscopic study)

(1mg of dry extract) (μg of EDTA equivalent/mg of dry extract)

Betel leaf 71.52±0.21

Blueberry fruit 35.29±1.91

Brocoli 5.79±0.28

Black currant residue 4.40±0.03

Black currant Fruit 0.57±0.11

Celery oriental 0.63±0.28

Chinese leechi 0.25±0.18

Cranberry leaves 39.57±0.18

Custard apple 2.24±1.13

Grape leaf, wild 22.39±0.89

Green pepper 30.91±3.10

Hops 57.11±0.35

Sea buckthorn Fruit 58.40±0.34

Sea bruckthorn leaf 75.77±0.08

Mango peel 3.91±1.14

Mangosteen 70.49±0.16

Parsnip 74.42±0.12

Pomegranate 24.65±0.00

Radish peel 75.69±0.08

Rainbow chard 57.51±0.37

Raspberry leaf 8.28±1.04

Red grape 51.67±0.44

Rhubarb 40±0.62

Sorrel 34.49±0.72

Kiwi peel 26.04±0.20

Tomato 52.96±0.06

Avocado 28.26±0.49

Dandelion leaf 52.36±0.04

Dandelion root 7.25±0.05

Blueberry leaf 13.86±0.07

Rosemary 27.70±0.17

Orange 20.58±0.12

Canadian yew 25.47±0.20

Apple 13.91±0.06

Banana 65.67±0.09

Potato Mature 56.50±0.00

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4.4.3 Inter-relationships between anti-radical activities

The correlation between various radical scavenging activities of the 36 plant extracts is

shown in Figure 4.12. The total antioxidant activity as determined by test radicals, the

TEAC and DPPH values and the reducing power, determined either by FRAP or the redox

potential are inter-related with high correlation coefficients (r > 0.91, p < 0.05) (Figures

4.3, 4.7 and 4.12). This suggests that same class of compounds in the extracts play

antioxidant role in these assays. Hydroxyl radical scavenging activity of the samples was

also related positively to TEAC, and FRAP values, but at lower correlation coefficient (r)

of 0.64 and 0.67, respectively (Figure 4.12), suggesting that only a sub-class of the

compounds in the samples are active scavengers of hydroxyl radicals.

The TEAC, DPPH and FRAP values of the plant extracts strongly related to their total

phenolic content (TPC) and to a lesser extent with total flavonoid content (TFC). A better

correlation was also observed between hydroxyl radical scavenging activity and TFC than

TPC (Figure 4.12), suggesting that flavonoids may be main components in the extract

contributing to this activity; but again certain flavonoids with potent antioxidant activity

such as quercetin - a flavonol or luteolin - a flavone (Arora et al. 1998) may be present at

higher levels in some extracts than in others. Phenolic compounds are largely hydrophilic

antioxidants, and these secondary metabolites that are most abundant in fruits (Macheix et

al., 1990). Gil et al. (2002) found high correlation (r=0.90) between antioxidant activities

as determined by DPPH or FRAP assay and TPC in nectarines, peaches and plums. Also,

high correlation between TPC and antioxidant activity as determined by FRAP were

reported in fruit juices (Gardner et al., 2000).

The superoxide anion (SOA) radical, H2O2 and NO scavenging activities did not show any

relationship to TEAC, DPPH and FRAP values of the extracts nor to TPC and TFC (Figure

4.12). It suggests that other classes of compounds in the extracts than the phenolic

compounds may be the active compounds playing a role in those scavenging activities.

Principal component analysis (PCA) was performed on the data and is illustrated as a

correlation circle in Figure 4.13, with principal dimensions accounting for 56 % variations

(Dimension 1) and 13 % of variations (Dimension 2), respectively. It confirms the above

conclusions that TEAC, DPPH and FRAP values are very closely and co-linearly related

151

with a high level of correlation. These values also correlate to the TPC of the extracts.

Furthermore, hydroxyl radical scavenging activity and TFC are also closely related to the

variables mentioned above, albeit correlated to a lesser level (Figure 4.12). The SOA

radical, H2O2 and NO scavenging activities are little related to TEAC, DPPH, FRAP, TPC,

FFC and hydroxyl radical scavenging activity, suggesting that the compounds present in

the extracts contributing to those activities are less likely to phenolic compounds. Again,

they are not closely related to each other, and that each of those activities originates from

different group of compounds. However, the H2O2 scavenging activity (positive in

dimension 2) may be exhibited only by a few of the extracts tested in this study, but they

possess strong activity.

TPC

0 600

0.63 0.98

0 200

0.86 0.59

50 250

-0.40 -0.14

60 140

20

100

-0.21

0600

TFC0.64 0.73 0.65 -0.26 -0.07 -0.11

TEAC0.91 0.64 -0.37 -0.20

50

250

-0.20

0250 FRAP

0.67 -0.32 -0.15 -0.20

HR-0.24 -0.10

0300

-0.13

50

250

SOA-0.16 0.11

HP

0600

0.12

20 100

60

140

50 250 0 300 0 600

NO

Figure 4.5 Correlation between Total polyphenol content (TPC), Total flavonoid content

(TFC), DPPH, TEAC, Hydroxyl radical (HR), Superoxide anion (SOA), Hydrogen

peroxide (HP) and Nitric oxide (NO)

152

-1.0 -0.5 0.0 0.5 1.0

-1.0

-0.5

0.0

0.5

1.0

Variables factor map (PCA)

Dim 1 (55.95%)

Dim

2 (

12

.95

%)

TPCTFC DPPH

TEACHR

SOA

HP

NO

FRAP

Figure 4.6 Multi correlation between Total polyphenol content (TPC), Total flavonoid content (TFC),

DPPH, TEAC, Hydroxyl radical (HR), Superoxide anion (SOA), Hydrogen peroxide (HP) and Nitric

oxide (NO)

153

4.4.4 Anti-radical activity spectrum

Present investigation confirms that the plant extracts are a rich potential source of natural

antioxidants, and they can be beneficial in preventing the progress of various oxidative

damages. Among the plant extracts, there was none exhibiting high activities against all:

organic radicals, SOA radical, hydroxyl radical and nitric oxide scavenging and iron

binding activities. This is understandable that a specific plant material may not possess an

array of compounds to exhibit all the activities evaluated in this study (Tables 4.2 – 4.4).

However, betel leaf extract exhibited high activities, including iron binding and scavenging

of various radicals except nitric oxide, where the activity was moderate nonetheless. Thus

this search, although not exhaustive, could identify one plant material exhibiting a wide-

spectrum antioxidant activity. Mangosteen that is less active against SOA radical, and, blue

berry leaf that less active against H2O2 scavenging and iron chelating activities could be

considered wide spectrum active materials to a lesser extent. Other moderate spectrum

active extracts were sea buckthorn leaf, pomegranate, rose mary cranberry leaf, custard

apple and black currant fruit.

Many materials that exhibited low TEAC, DPPH or FRAP values exhibited high NO

scavenging or SOA radical scavenging activities, including potato, dandelion leaf, mango,

tomato, kiwi, and rainbow chard. Interestingly, some materials such as rainbow chard,

broccoli, orange, oriental celery, parsnip exhibited very high H2O2 scavenging activity, and

some of them showed this activity.

Overall, some generalisation can be made: all extracts exhibiting high hydroxyl radical

scavenging extracts also exhibited high TEAC, DPPH and FRAP values. Likewise, all

SOA radical scavenging extracts and metal chelating extracts also exhibited high NO

scavenging activity. High H2O2 scavenging activity was exhibited by extracts that did not

exhibit any other activity, including oriental celery and broccoli and some such as rainbow

chard, orange and parsnip that showed good activity against NO. Dandelion root, sorrel and

hops showed high activity only against SOA radical.

This radical specificity could be linked to the presence of phytochemicals, which are

variably present in all plant genera, for example, antioxidant activity in fruits could be due

to abundance of flavonoids and also phenylpropanoid derivatives (Gordon, 1996; Rapta et

154

al., 1995; Yokozawa et al., 1997). Present study explains that screening of plant extracts

using the various radical quenching methods could be effective for the selection of

materials exhibiting either wide spectrum anti-radical activity or a specific radical

scavenging activity. In any event, more detailed studies on the chemical composition of

those extracts, as well as studies with other models, such as lipid peroxidation and in vivo

assays are essential to characterize them as biological antioxidants.

155

4.5 Conclusions

This work evaluated the spectrum of anti-radical activity of 36 plant extracts against

organic (ABTS and DPPH), superoxide anion (SOA), hydroxyl and nitric oxide radicals.

Ferric ion reducing activity and ion binding capacity of the extracts were also examined.

TEAC, DPPH and FRAP assays provide essentially the same response with respect to the

antioxidant activity of plant extracts, suggesting that any one of them would be adequate to

evaluate their anti-radical capacity. Betel leaf, blueberry fruit and black currant and

cranberry leaf showed high radical scavenging activity (TEAC assay); apple, sorrel, red

grape and dandelion root were against SOA; cranberry leaf, blueberry leaf, black currant

and Rosemary against hydroxyl radical; rainbow chard, parsnip, broccoli and orange

against H2O2; and potato, banana, sorrel, sea buckthorn leaf against nitric oxide. Blueberry

leaf and fruit, pomegranate, black currant and betel leaf showed high ferric ion reducing

power. The extracts showing high iron binding capacity were: sea buckthorn leaf, radish,

parsnip, betel leaf and mangosteen. Betel leaf extract exhibited high activities, including

iron binding and scavenging of various radicals except nitric oxide, where the activity was

moderate nonetheless. Thus, this search could identify one plant material exhibiting a

wide-spectrum antioxidant activity. Mangosteen that is less active against SOA radical,

and, blue berry leaf that less active against H2O2 scavenging and iron chelating activities

could be considered wide spectrum active materials to a lesser extent. This study also

shows that antioxidant activity of a substance, determined by one or more related assays,

does not give the complete picture of its effectiveness against various species of oxygen

radicals; and emphasizes that determination of the spectrum of the anti-radical activity

would be necessary.

156

Chapter 5 CHARACTERIZATION OF ANTIMICROBIAL ACTIVITY OF

SELECTED PLANT EXTRACTS: ANTIMICROBIAL ACTIVITY INDEX AND

SPECTRUM OF ACTIVITY

L’activité antimicrobienne des extraits de plantes sélectionnés a été évaluée en détail par turbidimétrie en

milieu liquide et par diffusion en milieu solide (gel). L’activité antimicrobienne a été exprimée dans la

première méthode par l’inhibition de la croissance (GI), par la concentration minimale inhibitrice (MIC) et par

une nouvelle expression, dénommée index antimicrobien (AMI). Dans la seconde méthode, l’activité a été

exprimée en zone d’inhibition. L'AMI prend en compte les trois phases de croissance des bactéries. Bien que

les méthodes turbidimétriques soient fiables dans l’évaluation de l’activité antimicrobienne des substances,

la nouvelle expression (AMI) semble être plus significative car, elle comprend l’information sur l’interaction

entre la substance et le micro-organisme et sa vitesse de croissance en présence de cette substance. De plus,

l'AMI démarque l’activité des échantillons, même ceux qui sont très actifs. Le spectre d’activité

antimicrobienne des extraits de plantes sélectionnés a été également étudié. Seulement quelques extraits ont

montré un spectre d’activité antimicrobienne assez large. En effet, seul l’extrait de feuille de bétel a un large

spectre d’activité contre les bactéries, les levures et les champignons, suivis des extraits de grenade et de

fruits d’argousier qui semblent être efficaces contre les bactéries et les levures. L’extrait de cassis est

effectivement une substance antibactérienne.

157

5.1 Abstract

There is much interest in natural antimicrobials from plant sources to replace synthetic

antimicrobial agents in foods. The antimicrobial activity of compounds or natural

preparations is generally evaluated by turbidimetric methods, zone of inhibition or by plate

count against selected test organisms. The responses from these methods may be

contradictory at times either because of the method itself or constituents of the test

substance such as plant extracts that are complex mixtures. The ability of natural substances

to act against a variety of microorganisms is also an important consideration in the

evaluation of their potential as antimicrobial agents. Thus the objective of this study was to

identify suitable assays for the evaluation of the antimicrobial activity of plant extracts, and

to evaluate the spectrum of activity of selected extracts against bacteria, yeasts and fungi.

Thirty-five aqueous plant extracts were characterized for antimicrobial activity against

Escherichia coli (ATCC 25922) by agar diffusion method and micro-titer broth dilution

method (growth inhibition capacity and MIC). We have developed an expression, the

antimicrobial index (AMI) that takes into account of the antimicrobial action during all the

three phases of bacterial growth from the broth dilution method. Although the turbidimetric

methods were in good agreement in the assessment of the antimicrobial activity of the

substances, the new expression AMI appears to be more meaningful since it carries the

information regarding interaction between the substance and the microorganism. In

addition, AMI demarcates the activity of samples, even those found to be highly active. It

was found that betel leaf, blackcurrant, pomegranate peel, sea buckthorn leaf and wild

grape leaf exhibited significant antimicrobial activity. Only betel leaf extract showed a

broad spectrum of antimicrobial activity against bacteria (Escherichia coli, Bacillus

subtili,s and Listeria monocytogenes); yeasts (Candida tropicalis and Zygo

saccharomyces)s); and molds (Botrytis cineria and Penicillium chrysogenum); whereas

pomegranate and sea buckthorn leaf extracts were effective against both bacteria and

yeasts. Black currant extract is an effective antibacterial substance. These extracts could be

potentially rich sources of natural antimicrobials for food applications.

Key words: agar diffusion, microdilution, turbidimetry, time kill curve, MIC, growth

inhibition, growth rate.

158

5.2 Introduction

Plants have been focus of attention for a longtime as source of antimicrobial compounds

and it has been reported that two-thirds of the world’s plant species have medicinal value

(Craig, 1999; Krishnaiah et al., 2011). In recent years, there has been an increasing interest

in antimicrobial properties of medicinal plants, and enormous attention is being paid in the

discovery of new antimicrobial from natural sources. (Brown et al., 2014; Khanam et al.,

2015), as multi-drug resistance are becoming a concern that can have considerable impact

on public health with treatment failures (Balouiri et al., 2016). However, the research on

plants for antimicrobial compounds has yet to see a systematic approach on screening for

possible candidates active against microbial pathogens, yeasts and fungi (Aqil and Ahmad,

2003). Furthermore, the screening processes, as of now, do not necessarily take into

account of the morphological (Gram-positive or negative) or growth requirement (aerobe or

anaerobe) characteristics of bacteria. It is expected that testing of selected plant extracts

against wide variety of test microorganisms will be helpful in obtaining a broad-spectrum

herbal formulation as well as new antimicrobial substances.

Growth parameters are important identifiers for determining the impact of an antimicrobial

compound on the viability of bacteria. The general growth curve of bacteria is characterized

by four phases: the lag phase, the exponential growth phase, the stationary phase and the

death phase. In the lag phase, there is little growth as the bacteria adapt to the new

environment and synthesize enzymes required for growth. The exponential phase describes

the multiplication of bacterial cells at a rate proportional to the number of cells present

initially, leading to an exponential increase in the number of cells at growth rate that is

characteristic of the bacteria, and dependent on various factors such as pH, temperature,

presence of antimicrobial constituents and others. As the nutrients are depleted in the

medium and inhibitory metabolites accumulate, or other environmental conditions

restricting the number of cells that can be sustained, bacterial growth ceases and the

bacteria moves into the stationary phase. Finally, cellular death and a declining population

occur when the surroundings cannot support the population (Davidson et al., 2005).

Several bioassays are used to determine the activity of antimicrobial substances, such as the

well known and commonly used disk diffusion or well diffusion and broth or agar dilution

(Balouiri et al., 2016). These methods can be qualitative and quantitative in nature. A

159

qualitative test such as the Kirby-Bauer disk diffusion method helps screening candidate

antimicrobial compounds, whereas quantitative methods (such as MIC, growth inhibition),

also known as end point methods, help quantify the potency of the compound.

Turbidimetry based techniques are routinely used to estimate the concentration of pure

cultures and have the advantage of being rapid and non-destructive (Li et al., 1993; Lima et

al., 2012). Tubidity techniques are based on light scattering by suspended particles in a

solution or dispersion, and it can be related to the population of microbial cells and is used

follow bacterial growth through various phases. The absorbance readings are often checked

and confirmed with the plate counts. Although it is based on Beer's Law (absorbance is

proportional to the concentration of the substance being measured). Absorbance readings

can help monitor lag phase, growth rate, and maximum population in real time and

ascertain the effectiveness of an antimicrobial agent. Although, only the maximum growth

is noted to describe the effect of environmental conditions from these measurements, other

criteria such as lag time and growth rate can also be derived from the growth curves that

may reflect bacterial growth through its life cycle under different physico-chemical

conditions, which can help understand the effects of antimicrobials and help build

prediction models for use in food microbiology. Mathematical modelling incorporating

growth kinetics can be useful in advancing the understanding a wide variety of intrinsic and

extrinsic factors governing microbial growth.

The aim of this study was two-fold. The first aim was to develop a primary three-phase

linear kinetic model to determine the impact of selected plant extracts on the growth curve

of E. coli, using absorbance measurement through its growth cycle, and compare it with

other standard methods of antimicrobial assay, including maximum growth inhibition or

inhibition capacity, MIC and diffusion assay. While all these assays would allow a detailed

characterization of the antimicrobial activity of extracts and would help identify the more

promising extracts for antimicrobial activity, there is an opportunity to determine the

kinetic parameters arising from the three-phase model to evaluate antimicrobial effect of

the extracts. Second, some of the promising fruit and vegetable extracts were evaluated for

their spectrum of antimicrobial activity, including against E. coli (Gram-negative) B.

subtilis (Gram-positive), Listeria monocytogenes (pshrotropic and Gram-positive

pathogen), and Geobacillus stearothermopilus (thermo-resistant); yeasts: Candida

160

tropicalis and Zygo saccharomyces; and molds (Botrytis cineria, Penicillium

chrysogenum).

161



Thirty-five vegetable and plant by-products were collected from farms, farmer’s markets

and fruit and vegetable processors in and around Quebec, QC, Canada. The selected plant

materials were from a second harvest. In a previous work, they had already been tested for

antimicrobial activity. The plants material that were investigated include Apple peel (Malus

communis), Avocado peel (Persea americana), Banana peel (Musa acuminate), Betel

leaves (Piper betle), Blueberry fruit peel (Vaccinium corymbosum), blueberry leaves

(Vaccinium corymbosum), Broccoli florets(Brassica oleracea), Black currant residue (Ribes

nigrum), Black currant peel (Ribes nigrum), oriental celery (Apium graveolens), Chinese

litchi (Litchi chinensis), Cranberry leaves (Vaccinium macrocarpon), Custard apple

(Annona squamosa), Dandelion root (Taraxacum officinalis), Dandelion leaves

(Taraxacum Officinalis), Grapes leaves (Vitis vinifera), Green pepper (Capsicum annuum),

Hops (Humulus lupulus), Indian summer fruit peel (Rudbeckia hirta fruit), Indian summer

leaves (Rudbeckia hirta leaves), Mango peel (mangifera indica peel), Mangosteenn peel

(Garcinia mangostana), Orange peel(Citrus sinensis), Parsnip peel (Pastinaca sativa),

Pomegranate peel (Punica granatum), Potato peel (Solanum tuberosum), Radish peel

(Raphanus sativus) Rainbow Chard (Beta vulgaris), Raspberry leaves (Rubus idaeus

leaves) red grape, rhubarb (Rheum rhabarbarum), Rosemary (Rosmarinus officinalis),

Sorrel (Hibiscus sabdariffa), Kiwi peel (Actinidia chinensis peel), Canadian yew (Taxus

Canadensis) and Tomato peel (Solanum lycopersicum). Upon arrival to the laboratory,

samples were first washed with distilled water and 300 ppm bleach solution to remove dirt

and debris. Thereafter, within 48 hours, the samples were lyophilized, ground to powder,

vacuum-sealed and stored at -30°C until ready for extraction.

http://en.wikipedia.org/wiki/Solanum

162

5.3.2 Extraction and stock solution preparation

About 50 gram of the lyophilized plant material powder was extracted with 500 mL of

distilled water at 80-90°C for 60 minutes in a water bath with continuous agitation. The

decoction was then vacuum filtered (Whatman No. 1 filter paper) and the filtrate was then

concentrated under vacuum using a rotary evaporator at 65°C until about the 100 mL of

volume was left, thereafter the liquid was lyophilized. The lyophilized samples were then

vacuum-packed and stored at -30oC until further use. For the antimicrobial assays the

samples were dissolved in Mueller-Hinton broth to obtain a stock solution of 80 mg/ml. All

the stock solutions were stored at 4oC and were used within 24 hours.


The antimicrobial activity of the extracts was determined against E. coli (ATCC 25922).

Bacterial culture was revived from stock at -80°C by three successive growth cycles at 37

°C for 24 h in Muller Hilton Broth (Sigma Aldrich). The standard inoculum was prepared

following NCCLS guidelines (Jorgensen and Ferraro, 2009) with bacteria in log phase

growth (4h after incubation) and then was standardized to match that of a 0.5 McFarland

standard (corresponds to approximately 1.5 x 108 CFU/ml). The adjusted bacterial culture

suspension was used within 15 minutes.

5.3.5 Antimicrobial activity assays

5.3.5.1 Zone inhibition agar gel diffusion assay

Plant extracts were tested according to NCCLS guidelines for antimicrobial susceptibility

tests with modifications (Jorgensen and Ferraro, 2009). The 100 mm MH agar (MHA)

plates were inoculated by confluent swabbing from the standard bacterial suspension. Four

6 mm wells were made in each MHA plate and then 80ul of plant extract at the

concentration of 10 mg/ml was added. Chloramphenicol (1.0 mg/ml) and sterile water were

used as positive and negative controls, respectively. The prepared plates were kept for 30

minutes at room temperature for diffusion and then incubated at 37oC. The zones of

inhibition were measured after 24h.

163

5.3.5.2 Micro-titer broth dilution (growth inhibition or inhibition capacity) and time

kill curve assay

The GI (%) value of plant extracts and the potential to reduce the bacterial population over

time was determined turbidimetrically using a micro-titer broth dilution assay (NCCLS,

1991; Wiegand et al., 2008). Briefly, 190 µl of plant extract in MH broth (10 mg/mL) was

pipetted in a 96-well plate. Subsequently, the wells were inoculated with the test strain

(10µl) to achieve a final concentration of 104- 105 CFU/mL in each well. The blank and

assay control was the plant extract (190 µl at 10 mg/mL) and 10 µl of MH broth without the

test strain, respectively; the latter also served as the sterility control. Chloramphenicol (1.0

mg/mL) was used as the positive control, while the inoculated MH medium as the negative

control. The microplates were incubated at 37°C on a micro-plate reader (Biotek Power

XS2). For time kill assay, hourly absorbance readings (OD600nm) were recorded for a period

of 24 hours, and the growth inhibition or inhibition capacity was expressed as a percentage.

The percentage of growth inhibition was calculated by the difference between the OD

reading of the sample and the negative control (sterile water). All assays were performed in

triplicate.

5.3.5.3 Broth micro-dilution (MIC) assay

Plant extracts were tested as per NCCLS guidelines for micro-dilution susceptibility tests

for aerobic bacteria (NCCLS, 1991) with modifications. The 96 wells micro-titer plates

were prepared with 12 plant extracts in the columns at 80 mg/mL in MH broth. Eight serial

dilutions were made in the rows with the lowest concentration tested being 0.6125 mg/mL.

Thereafter, 10µl of standard inoculum was dispensed in each well to give a final

concentration of 104- 105 CFU/mL. The MIC value after 24 hours of incubation at 37oC,

was the lowest concentration of plant extract at which no growth could be detected by

visual inspection. Chloramphenicol (1.0 mg/mL) was used as a positive control.

164

5.3.5.4 Agar dilution (yeasts)

Antimicrobial activity against yeasts was tested through an agar dilution method (NCCLS,

1991). This method involved incorporating the plant extract into the agar medium at the

concentration of 10 mg/mL. Thereafter, the prepared plates were inoculated with 100 µl of

yeast suspension (103 CFU/ml) onto the agar plate surface. The plates were then incubated

at 30oC and CFU was recorded at 48 hours and 96 hours. The result was expressed in

percentage of inhibition with reference with the control (without the incorporation of plant

extract)

5.3.5.5 Anti-fungal assay (agar dilution)

Anti-fungal activity was evaluated by radial growth method on agar plates amended with

the extracts (El Ghaouth et al., 1992). PDA plates containing 10 mg/ml of plant extracts

were seeded with 5 mm diameter agar plugs taken from the margin of 10-day old Botrytis

cineria cultures (3 day old Penicillium chrysogenum cultures). The test was performed in

triplicate and the plates were incubated in the dark at 24oC. Growth measurements were

determined visually by when the growth on the negative control (sterile water containing no

plant extract) reached the edge of the plate.

5.3.6 Empirical expression for antimicrobial activity

Antimicrobial index (AMI) derivation

The general growth curve of bacteria is characterized by three phases: the lag phase, the

exponential growth phase and the stationary phases, where specific growth rate is zero in

the lag and stationary phases and the logarithm of the bacterial population increases linearly

with time in the exponential phase. The presence of antimicrobial compounds in the

medium can significantly alter the growth kinetics of bacteria. The impact of antibacterial

can manifest by affecting the ability of the bacteria to use its growth medium to prepare for

exponential growth, thereby prolonging the lag phase; and lowering the growth rate during

the log phase as well as the maximum cell count.

The bacterial growth curve without a lag phase can be described by a hyperbolic function

(Michaelis-Menton equation in enzyme kinetic):

165

tK

tNN

max (1)

where, N is cell count or in O.D. units, Nmax is maximum cell count, t is time in hours, and

K is a constant characterizing the interaction between the microorganism and the nutrient

medium.

The overall growth curve is effectively sigmoidal with the lag phase, and can be described

from Hill equation by:

n

n

tK

tNN

max (2)

Where the exponent n is a measure of the sigmoidal character, and contains information on

growth rate.

Equation (2) can be rearranged in the linear form:

The values of K and n can be extracted by plotting equation (9) as intercept and slope of the

linear plot, respectively. The value of K, the interaction constant, contains information on

lag time, growth rate and maximum growth of a specific organism in the medium,

assuming that the interaction between the organism and the medium continues until the

KtnNN

N

tnKN

NN

tKN

NN

t

K

N

NN

t

tK

N

N

t

tK

N

N

tK

t

N

N

n

n

n

n

n

n

n

n

logloglog

logloglog

logloglog

11

max

max

max

max

max

max

max

(3)

(4)

(5)

(6)

(7)

(8)

(9)

166

nutrients in the medium are depleted. However, it can be expected that these kinetic values

would be modified when the nutrient medium contains antimicrobial substances in the

medium, as the antimicrobial substance in the medium interferes with the normal growth

process of the organism.

Thus, higher K values for the organism in the medium amended with potentially

antimicrobial substances relative to that in the growth medium alone would suggest

inhibitory action of those substances, encompassing all phases of growth. On the other

hand, smaller values of n compared to that in the growth medium would indicate growth

inhibition. Howerver, the K and n for a specific organism and a specific antimicrobial test

substance would depend on the concentration of the test substance in the medium,

temperature, pH, and redox status as the latter two factors might be altered by the presence

of the test substance. In addition, the interaction of the substance with the organism may

involve induction of inhibitory metabolites by the organism.

The values of K and n can be expressed relative to those of the growth medium alone as:

K1 = K/K0 and n1 = n/n0

where K and K0 are interaction constants; and n and n0 are growth rates in the medium

containing antimicrobial substance and in the medium alone, respectively; and K1 and n1

are inhibition constant and growth constant, respectively. When K1 is unity, no inhibition

occurs, and values >1.0 signify inhibitory activity. Likewise, a value of unity for n1

signifies no modification in the growth rate of the organism by the substance, and the

growth rate is reduced when the values of n1 are <1.0.

Combining these two kinetic constants, K1 and n1, we can obtain a measure of the

antimicrobial effect of the substance, the antimicrobial index (AMI):

AMI = K1/n1 (10)

167

5.3.7 Data analysis






168


5.4.1 Antimicrobial activity of plant extracts

5.4.1.1 Antimicrobial Index (AMI)

The growth curves of E.coli in nutrient broth (control) and nutrient broth containing 10.0

mg/mL of pomegranate, dandelion root, orange, lychee, taxus canadianxus, as examples,

are shown in Figure 5.1. The linearized growth curves (Figure 5.2) of the organism in

nutrient broth and that containing pomegranate at 10.0 mg/mL concentration, as examples,

show that the curves were not linear, as expected from equation (9); but they broken linear,

either for the control or for those containing plant extracts. Consequently, two intercepts

were evident, i.e., a true intercept (log K) and an apparent intercept (log Ka) obtained by

extrapolation; and the slope of the curves presents the value of bacterial growth rate (na)

(Figure 5.2). The values from the plots for the control and various plant extracts, log K, log

Ka, K and na, as well as the reduced values relative to the control, K1 and n1, are listed in

Table 5.1

Time kill curve against E.coli

Time (h)

0 5 10 15 20 25

OD

uni

t

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6 Control

Pomgranate

Dandelion root

Orange

Lychee

Taxus canadianxus

Nmax

Growth rate

Lag phase

Figure 5.1 Growth curve of E. coli in Nutrient broth at 37oC temperature during 24 h period

169

Figure 5.2 Linear representation of growth curves of E. coli according to equation (9). A: Control; B:

Nutrient broth containing pomegranate extract at 10 mg/mL concentration

log t (h)

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4

log

[N

/(N

ma

x-N

)]

-1

0

1

2

logKa=-1.35

logK=0.68

Pomegranate

na=0.84

B

A

log t (h)

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4

-4

-3

-2

-1

0

1

2

log [

N/(

Nm

ax-N

)]

logKa=3.04

logK=1.28

Control

170

Table 5.1 Antimicrobial growth kinetic parameters against E.coli for plant extracts at the concentration

of 10 mg/mL

Extract Log K Log Ka K na K1 na1

1 Control* -1.28 -3.04 5.25 3.43 1.00 1.00

2 Apple fruit peel -1.07 -1.81 8.51 1.40 1.62 0.41

3 Avocado fruit peel -0.46 -1.15 34.67 1.72 6.60 0.50

4 Banana fruit peel -1.05 -1.41 8.91 1.23 1.70 0.36

5 Betel leaf 0.53 0.16 338.84 0.76 64.54 0.22

6 Black currant 0.29 0.2 194.98 0.28 37.14 0.08

7 Black currant residue 0.75 0.42 562.34 0.75 107.11 0.22

8 Blueberry fruit peel -0.42 -1.44 38.02 1.79 7.24 0.52

9 Blueberry leaf -0.46 -1.5 34.67 2.41 6.60 0.70

10 Broccoli leaf -0.66 -1.55 21.88 2.07 4.17 0.60

11 Celery oriental -0.97 -1.72 10.72 1.85 2.04 0.54

12 Cranberry leaf 0.01 -0.49 102.33 1.05 19.49 0.31

13 Custard apple -0.38 -0.83 41.69 0.84 7.94 0.24

14 Dandelion leaf -0.83 -1.98 14.79 2.77 2.82 0.81

15 Dandelion root -0.86 -2.4 13.80 3.07 2.63 0.90

16 Grape leaf, wild 0.54 0.48 346.74 0.78 66.05 0.23

17 Green pepper -0.35 -0.27 44.67 0.71 8.51 0.21

18 Hops (pellets) -0.72 -1.34 19.05 2.40 3.63 0.70

19 Kiwi peel -0.61 -1.6 24.55 2.65 4.68 0.77

20 Lychee -0.46 -1.44 34.67 2.00 6.60 0.58

21 Mango (Ataulfo) -0.92 -1.64 12.02 1.65 2.29 0.48

22 Mangosteen -0.37 -0.82 42.66 0.82 8.13 0.24

23 Orange (albedo) -0.85 -1.88 14.13 2.02 2.69 0.59

24 Parsnip peel -0.53 -1.6 29.51 2.51 5.62 0.73

25 Pomegranate 0.68 -1.35 478.63 0.84 91.17 0.24

26 Potato mature -0.90 -1.61 12.59 2.01 2.40 0.59

27 Radish root peel -0.20 -0.71 63.10 1.15 12.02 0.34

28 Rainbow chard -0.51 -1.22 30.90 2.12 5.89 0.62

29 Raspberry leaf -0.20 -0.65 63.10 1.11 12.02 0.32

30 Red grape Fruit peel 0.11 -0.28 128.82 0.71 24.54 0.21

31 Rhubarb -0.17 -0.65 67.61 0.89 12.88 0.26

32 Sea buckthorn -0.09 -0.39 81.28 0.54 15.48 0.16

33 Sea buckthorn leaf 0.17 -0.57 147.91 1.63 28.17 0.48

34 Sorrel -0.42 -1.22 38.02 1.99 7.24 0.58

35 Taxus canadensis -0.65 -1.69 22.39 1.95 4.26 0.57

36 Tomato peel -0.63 -1.34 23.44 2.09 4.46 0.61

171

In spite of the deviation between the log K and log Ka; interestingly, they are related in a

linear fashion (Figure 5.3), allowing either one of the intercepts for interpretation of the

growth characteristic of bacteria, E. coli, in this instance. The true intercept would be more

appropriate, and thus, log K and na were used as kinetic parameters to characterize the

growth of E. coli.

log K

-1.5 -1.0 -0.5 0.0 0.5 1.0

Log K

a

-3.0

-2.5

-2.0

-1.5

-1.0

-0.5

0.0

0.5

1.0

R2=0.921

Figure 5.3 Relationship between interaction constants log Ka and log K of plant extracts

The inhibition or interaction constant (K1) ranges from 1.0 (control) to 107.1 (black currant

residue), and na1 values from 0.08 (black currant peel) to 1.0 (control) for the selected fruit

and vegetable extracts (Table 5.1). Among the 35 samples, only four samples (black currant

residue, pomegranate, betel leaf and wild grape leaf) were characterized by interaction

constant (K) of > 50.0; another eight samples with values between 10.0 and 50.0 and the

rest with values of < 10.0. This constant clearly demarcates those samples that exhibit high

inhibitory interaction with E. coli, which is also seen by the prolonged lag phase of E. coli

172

in the presence of these extracts. Plant extracts, being a mixture of phytochemicals, where

some of them may be inhibitory to the growth of the bacteria, while some may promote

their growth. Thus the interaction constant could be an indicator of the abundance of highly

inhibitory compounds in the extract, and hence, could be a powerful marker for inhibitory

activity of substances.

High growth rate (n1) values may suggest the possible presence of growth promoting

compounds in the extract mixture. Among the extracts, black currant peel and sea

buckthorn fruit peel were characterized by very low n1 values (< 0.20), reflecting that these

samples contain only small quantity of growth promoting substances. All the samples

possessing high interaction constants had n1 values between 0.20 and 0.25, indicating that

they contain modest quantities of growth promoting substances (Table 5.1).

The overall inhibitory activity, the antimicrobial index (AMI), should reveal the potential of

the extracts per se. The AMI value ranged from 1.0 (control) to 490.0 (black currant

residue) (Table 5.2). High values of AMI (> 290.0) were obtained for black currant peel

and residue, pomegranate and betel leaf, and wild grape leaf extracts, thus demarcating

them from all other samples (Table 5.2). However, they also contain modest levels of

growth promoters, as seen by the n1 values; and there is room to enhance their antimicrobial

activity by removing those substances. Such a purification step has a potential to improve

the activity of sea buckthorn leaf and cranberry leaf extracts (n1 value of 0.48 and 0.31,

respectively).

173

5.4.1.2 Growth inhibition or antimicrobial capacity

Growth inhibition capacity of various extracts was determined from the growth curves, as

depicted in Figure 5.1. This expression takes into account only maximum growth of E. coli

in the nutrient broth relative to the control. The inhibition activity is presented as

percentage of growth inhibition capacity in Table 5.2. The results show that almost all plant

extracts have antimicrobial activity to varying extents. The growth inhibition capacity of

plant extracts ranges from 26.4 % (banana fruit) to 94.39 % (pomegranate). More than 68%

of sample had 70% or more GI value with 17% having 90% of GI capacity. Pomegranate

showed a highest % of GI against E.coli (94.39%) following by black currant residue

(93.83%), grape leaf (92.27%), betel leaf (92.16%), sea buckthorn leaf (90.02%), cranberry

leaf (87.87%), sea buckthorn (87.29%) and rhubarb (85.26%).

5.4.1.3 Minimum inhibitory concentration (MIC)

The lowest value of minimum inhibitory concentration in broth micro-dilution assay was

found at 2.5 mg/mL of betel leaf and 5.0 mg/mL for pomegranate, grape leaf, sea buckthorn

and rhubarb.

5.4.1.4 Zone inhibition (ZI)

The ZI values indicated that black currant residue and pomegranate exhibited the highest

antibacterial against E.coli (28 mm), followed by dandelion leaf (27 mm) and avocado fruit

peel, custard apple, rhubarb (25 mm). About 70% of the samples showed a zone of

inhibition of 15 mm or more; of which 60% showing 20 mm or more. Generally, acidic

leaf or stalk extracts (pH < 4.0) such as dandelion leaf, sorrel, and oriental celery exhibited

high activity by this method, whereas their activities were only modest as determined by

other methods. However, leaf extracts having higher pH (pH> 5.0) that exhibited high or

moderate activities by the other methods such as betel, wild grape, sea buckthorn leaf,

cranberry leaf and raspberry leaf exhibited low ZI. In addition, only ZI method marked

avocado peel extract (pH, 5.64) as active (25 mm).

174

5.4.1.5 Comparison of methods in the characterization plant extracts

The antimicrobial activity of the plant extracts calculated through the different methods

(AMI, antimicrobial capacity, MIC and ZI) is presented in Table 5.2. Although there was a

trend in the identification of samples possessing antimicrobial activity by turbidimetric

methods (capacity, MIC and AMI) and zone inhibition method (ZI), the latter was not

consistent with the former for many samples. As mentioned above, some samples were

classed active only by ZI, while some samples found active by turbidimetric methods were

found poorly active by ZI. There was good agreement between the turbidimetric methods,

although the order of potency for the samples is different as determined by each of these

methods. The relatively high activity of the samples - pomegranate, black currant, sea

buckthorn and rhubarb - and moderate activity of the samples – radish root, mangosteen

and custard apple - was registered by all the four methods; whereas the turbidimetry

methods identified additional samples as highly active, including betel leaf, wild grape leaf,

sea buckthorn leaf, raspberry leaf and cranberry leaf.

High activity was registered by ZI for acidic leaf extracts such as dandelion, oriental celery

and sorrel, while these samples were registered consistently as a poorly active substance by

other methods (Table 5.2). On the contrary, betel, wild grape and other leaf extracts did not

exhibit high zone inhibition, but highly inconsistent with other methods that identified them

as highly active substances. Such striking differences can be inherent in the methods; the

zone inhibition involves diffusion of the active compounds in a solid matrix; whereas the

other methods involve liquid medium, where diffusion may not be a limitation. Acidic leaf

extracts can be expected to contain higher levels of organic acids that can effectively

diffuse out into the gel matrix, thereby contributing to the inhibition of the test

microorganism via pH effects.

The other leaf extracts with higher pH values can be expected to contain less amounts of

easily diffusible organic acids, and their inhibitory activity likely depends on the diffusion

of other active substances they contain. The disparity in the expression of activities between

the solid and liquid media may well be due to the nature of the active substances in those

extracts and their mobility in both the media. Unlike in liquid medium where high mobility

175

of the active compounds can be expected, their diffusion is likely hindered in the solid

medium due to possible interactions with components in the solid network. When the active

substances are soluble in the aqueous medium as it appears be so for fruit extracts such as

pomegranate and black currant that contain high levels of phenolic compounds (Chapter 6),

their migration appears to be little hindered and ZI registers high activity for these samples

(Table 5.2). On the other hand, if the extracts contain active lipophilic substances, as it

would seem to be with betel leaf extract that contain alkaloids and terpenoids, in addition to

phenolic compounds (Chapter 6), the lipophilic compounds can be adsorbed on to the solid

matrix and may not migrate far into the solid medium. Nonetheless, betel leaf other such

leaf extracts show some activity in the zone inhibition method, suggesting that this sample

contains some water-soluble (hydrophilic) active compounds such as phenolic compounds.

Zone inhibition method can provide useful insights into the activity of complex mixtures

such as plant extracts with respect to the nature of active compounds and their interactions,

although it cannot be a stand-alone method. This method should be coupled to a liquid

medium assay in the evaluation of the antimicrobial activity of substances. Of particular

interest is the differentiation of fruit and less acidic leaf extracts, otherwise deemed active

extracts by liquid medium assays, with respect to the nature of active compounds by ZI.

Detailed information on the composition of the extracts would be required, however, to

appreciate the differences underscored by ZI method.

An analysis of variance (ANOVA) was performed on antimicrobial activity data obtained

by those methods in order to get an idea of the most adept and useful expression for

antimicrobial activity that could demarcate the proficiency of the samples. On this count,

AMI demarcates more effectively the samples between the active and poorly active

samples, and also among the extracts that exhibit high activity (Table 5.2). AMI value

could also provide some indication whether the activity of a particular extract could be

improved subject to purification of the extract.

176

Table 5.2 Comparison of antimicrobial activity (AMI) against E.coli of plant extracts (10 mg/mL)

determined by agar gel diffusion assay (Zone of inhibition, ZI), Micro-titer broth dilution

(antimicrobial capacity), Broth microdilution assay (MIC)

* Positive control: Chloramphenicol (1.0 mg/ml)

** Means with the same letter are not significantly different.

No Extract AMI **

Antimicrobial

capacity (%) ** MIC (mg/mL) **

ZI

(mm) **

Positive control* - 100 0.25 0

1 Control (without extract) 1.00m 0.00s - 0g

2 Apple fruit peel 3.97klm 31.46r 80e 5g

3 Avocado fruit peel 13.17h-m 77.52hij 40d 25abc

4 Banana fruit peel 4.73klm 26.44r 80e 5g

5 Betel leaf 291.3d 92.16abc 2.5 a 19def

6 Black currant 455b 91.23abc 10b 23a-e

7 Black currant residue 489.9a 93.83ab 10b 28a

8 Blueberry fruit peel 13.88h-m 79.45gh 20c 24a-d

9 Blueberry leaf 9.4i-m 77.29hij 10b 18ef

10 Broccoli 6.91i-m 64.73lm 40d 5g

11 Celery oriental 3.79klm 41.41q 80e 23a-e

12 Cranberry leaf 63.67f 87.87a-e 20c 20cde

13 Custard apple 32.43g-l 79.95gh 10b 25abc

14 Dandelion leaf 3.49lm 55.58no 80e 27ab

15 Dandelion root 2.94lm 45.52pq 80e 5g

16 Grape leaf, wild 290.4d 92.27ab 5a 22.5b-e

17 Green pepper 41.1fgh 82.13e-h 40d 18ef

18 Hops 5.19klm 67.53klm 40d 22.5b-e

19 Kiwi peel 6.05j-m 62.37mn 40d 14f

20 Lychee 11.33h-m 78.96ghi 20c 21cde

21 Mango (Ataulfo) 4.76klm 41.58q 20c 5g

22 Mangosteen 33.99f-k 80.44fgh 10b 23a-e

23 Orange (Albedo) 4.57klm 50.8op 40d 21cde

24 Parsnip peel 7.68i-m 72.4ijk 20c 5g

25 Pomegranate 372.3c 94.39a 5 a 28a

26 Potato mature 4.09klm 46.09pq 40d 5g

27 Radish root peel 35.85f-j 83.25d-h 20c 23a-e

28 Rainbow chard 9.52i-m 77.89hij 40d 22b-e

29 Raspberry leaf 37.14f-i 83.77d-h 10b 20cde

30 Red grape Fruit peel 118.5e 81.67e-h 20c 5g

31 Rhubarb 49.63fg 85.26c-g 5 a 25abc

32 Sea buckthorn 98.34e 87.29b-f 5 a 24a-d

33 Sea buckthorn leaf 59.28fg 90.02a-d 10b 19def

34 Sorrel 12.48h-m 77.65hij 20c 24a-d

35 Taxus canadensis 7.5i-m 77.39hij 20c 22b-e

36 Tomato peel 7.33i-m 71.07jkl 80e 22b-e

177

The inter-relationship between antimicrobial capacity (Capacity), zone of inhibition (ZI),

MIC and AMI is visualized in principal component analysis (PCA) correlation circle shown

in Figure 5.4. The variables ZI, Capacity and AMI are related, but not closely; and MIC is

inversely (or negatively) related to the other expressions of antimicrobial activity.

However, it appears that the relationship between AMI and MIC is more direct and inverse

than either capacity or ZI, i.e., higher the AMI value, the smaller the MIC value. In

dimension 1 (accounting for 62.3 % of the variations), Capacity shows a very strong

correlation coefficient, but a poor coefficient in dimension 2 (accounting for 18.5 % of the

variations). ZI shows strong correlation coefficients on both dimensions; whereas AMI and

MIC show strong correlation coefficient on dimension 1 and moderate coefficients in

dimensions 2 and 3. This suggests that AMI and MIC are more closely related, albeit

inversely, i.e., higher the interaction of the substance with the microorganism, lower is the

concentration of that substance, required toinhibit the growth of the microorganism.

Although AMI points to the potential of a substance to inhibitory interaction with

microorganism and bacterial growth rate in its presence, and MIC is indicative of dose-

response relationship, they are inter-related, and may provide nearly the same information

regarding the antimicrobial activity of substances. Hence, either one of them may be useful

in practice for screening purposes, but AMI appears to have the capacity to clearly

distinguish even samples showing high activities than MIC. ZI appears to contain

information regarding antimicrobial activity as well as other information, presumably,

diffusivity of the substance, and therefore, constitutes a specific method to obtain additional

characteristic of the substance. The capacity determination remains a non-descript method.

The scatter plot of PCA for the samples is shown in Figure 5.5. Four distinct and

homogenous clusters were apparent. Cluster 1 includes: the control, broccoli, dandelion

root, apple fruit, banana fruit, mango, parsnip, potato and red grape fruit peels. This group

showed very low antimicrobial activity, and none of the methods showed them to be active.

The second cluster includes oriental celery, dandelion leaf and potato, tomato peel. This

group also exhibited low antimicrobial activity, but showed moderate activity by ZI, and

appear to possess additional attribute than group I. not relate to any used methods but with

different manner with group 1.

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About nineteen of the thirty-six samples clustered in the group 3. This group the center

cluster (Figure 5.5) showed intermediate antimicrobial property, and exhibited moderate

activities by all the methods (ZI, Capacity and AMI) except MIC. The most active and

promising is group 4 that includes betel leaf, black currant, black currant, wild grape leaf

and pomegranate. Interestingly, these samples were identified as the most promising

extracts by AMI alone. It adds credence to the potential of AMI in demarcating highly

active antimicrobial substances or complex mixtures such as the extracts.

179

Figure 5.4 Antimicrobial activity of plant extracts: correlation loading of antimicrobial evaluation

methods by PCA

Figure 5.5 Antimicrobial activity of plant extracts: scatter plot of antimicrobial activity of plant

extracts by PCA. The number corresponds to the product as listed in Table 5.1 and 5.2.

-1.0 -0.5 0.0 0.5 1.0

-1.0

-0.5

0.0

0.5

1.0

Variables factor map (PCA)

Dim 1 (62.28%)

Dim

2 (

18

.46

%)

Capacity

ZI

MIC

AMI

-4 -2 0 2 4

-2-1

01

2

Factor map

Dim 1 (62.28%)

Dim

2 (

18

.46

%)

1

42

15

26

11

21

10

14

36

19

23

24

18

30

17

28

3

9

2035

34 8

27

29 12

22

13

32

3133

5

16

6

25

7

cluster 1

cluster 2 cluster 3 cluster 4

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The antimicrobial activity expressed by different methods for the thirty-five samples were

correlated as shown in Figure 5.6. The analysis showed that the inhibition capacity

correlated with MIC the most (correlation coefficient of 0.79), and correlates well with

zone inhibition (0.64), but only moderately with AMI (0.51). The AMI correlates with MIC

only modestly (0.45), and poorly with zone inhibition (0.33). Likewise, zone inhibition and

MIC were related poorly (0.34). Given the interplay of activity and physico-chemical

nature of active compounds in the zone inhibition assay, it may be a useful assay to provide

additional information on the nature of the antimicrobial compound under study, and hence,

the activity expressed by this assay can be useful.

A comparison would be meaningful among the methods where the assay is performed in

liquid medium. Given the high correlation between inhibition capacity and MIC, it would

seem that any one of them could be adequate to identify the promising candidates for

antimicrobial activity. However, AMI relates to both capacity and MIC somewhat

similarly, albeit modestly, and knowing the ability of AMI to demarcate the most promising

candidates from a large pack, it becomes clear that AMI would be a more powerful marker

for characterizing potential antimicrobials. It takes into account of the entire growth cycle,

and provides insight into the potential interactions that occur with the organism as well as

the presence of compounds that could potentially promote growth.

181

Capacity

0.2 0.8

-0.62 0.53

0 200 500

0.51 0.64

0 40 80

040

80

-0.790.2

0.8 na1

-0.53 -0.60 -0.35 0.53

K1

0.92 0.37

040

100

-0.47

0200

500

AMI

0.33 -0.45

ZI

010

25

-0.34

0 40 80

040

80

0 40 100 0 10 25

MIC

Figure 5.6 Multi-correlation between different methods for assay of antimicrobial activity

182

5.4.2 Spectrum of Antimicrobial activity

5.4.2.1 Antimicrobial activity of plant extracts against bacteria

The micro-titer broth dilution assay results are presented in Table 5.3. All the samples

exhibited high or moderate inhibitory activities. Briefly, all extracts displayed the GI (%) of

77-97 % against E.coli, 71-90 % against B.subtilis and 80-100 % against L. monocytogenes.

The plant extracts showing promising activity against the bacteria included black currant

residue, betel leaf, and pomegranate peel. While pomegranate peel (97.8 %), betel leaf

(97%) had the highest antimicrobial activity against E. coli; betel leaf (90.1 %),

pomegranate peel (87.3 %) and cranberry leaf (88.43%) performed well against B. subtilis.

L. monocytogenes was found to be the most sensitive to plant extracts amongst the three

tested bacteria, with pomegranate peel showing 100 % of inhibition.

Table 5.3 Growth inhibition of bacteria by plant extracts at 10.0 mg/mL (Growth inhibition ± SD)

Sample Growth inhibition (%)

E. coli B. subtilis L.monocytogenes

Cranberry leaf 87.87 ± 6.18 88.43 ± 0.60 89.5 ± 0.1

Black currant (residue) 94.73 ± 3.52 80.50 ± 0.71 94.9 ± 0.4 Sea buckthorn leaf 90.02 ± 10.74 83.15 ± 1.41 88.1 ± 0.4 Betel leaf 97.02 ± 2.09 90.12 ± 1.41 98.7 ± 0.2 Pomegranate 97.81 ± 2.05 87.32 ± 8.03 99.99 Rhubarb 85.26 ± 6.70 80.55 ± 3.47 96.1 ±0.6 Blueberry leaf 77.29 ± 3.74 48.87 ± 1.60 92.3 ± 1.0 Grape leaf, wild 92.27 ± 4.05 71.39 ± 5.11 89.5 ± 1.4 Cashew apple 78.86 ± 5.61 72.65 ± 4.32 80.2 ± 0.9 Sea buckthorn (fruit) 87.29 ± 5.8 71.50 ± 4.96 85.6 ± 2.1

The antibacterial activity of plant extracts determined by well-diffusion assay is presented

in Table 5.4. Here, only pomegranate and sea buckthorn fruit were found to be effective,

presumably because of the diffusion limitation of certain active compounds in certain

extracts as discussed before (Section 5.4.1.4). Most of the antimicrobial compounds from

plant sources are either of intermediate polarity or are non-polar, this limits their ability to

diffuse sufficiently in the aqueous agar gel matrix, and hence, often results in a low zone of

inhibition (Balouiri et al., 2016; Khanam et al., 2015).

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Table 5.4 Zone of inhibition (mm) of bacterial growth by well-diffusion assay at 5.0 mg/mL

Sample E.coli B.subtilis L.monocytogenes G.stearothermophilus

(mm) (mm) (mm) (mm)

Cranberry leaf 5.0 5.0 11.5 8.0 Black currant 5.0 6.0 5.5 7.5 Sea buckthorn leaf 5.0 5.0 5.5 7.0 Betel leaf 15.0 5 .0 8.5 10.0 Pomegranate 18.5 22.0 26.5 33.5 Rhubarb 5.0 6.0 5.5 9.0 Blueberry leaf 6.0 5.0 5.0 8.5

Grape leaf, wild 5.0 8.0 5.5 5.5

Cashew apple 5.0 5.0 5.5 8.0 Sea buckthorn 17.0 15.0 23.5 23.0 Chloromphenicol (1 mg/ml) 27.0

25.5 39.0

41.5

5.4.2.2 Antimicrobial activity of plant extract against yeasts

The inhibitory activity of plant extracts (10 mg/mL) against C. tropicalis and Z.

saccharamyces was determined by agar dilution method is shown in Table 5.5.

Pomegranate, betel leaf, and sea buckthorn fruit consistently inhibitory activity against the

yeasts, most samples showed only static effect as the yeasts seem to resume their growth.

While black currant residue that showed high activity against bacteria, its activities against

yeasts (49.6 % against C. tropicalis and 45.1% against Z. saccharamyces) were low.

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Table 5.5 Inhibitory activity of selected plant extracts on the growth inhibition of yeasts at 10.0 mg/mL

Sample

C.tropicalis (%) Z.saccharamyces (%)

48h 96h 48h 96h

Cranberry leaf 34.8 21.5 11.2 11.1

Black currant 49.6 48.8 45.1 40.6

Sea buckthorn leaf 60.1 41.9 32.8 30.1

Betel leaf 91.4 90.1 99.99 99.99

Pomegranate 99.99 98.3 99.99 88.7

Rhubarb 36.7 23.8 45.6 17.5

Blueberry leaf 59.8 25.6 44.1 34.9

Grape leaf, wild 37.5 25.6 22.7 21.5

Sea buckthorn fruit 87.5 68.7 99.99 99.99

5.4.2.3 Antimicrobial activity of plant extract against fungi

The anti-fungal activity of plant extracts is summarized in Table 5.6. Here, only betel leaf

extract was effective in the inhibiting the growth of fungi (66.67% for B. cinerea and 100%

for P. chrysogenum).

Table 5.6 Inhibitory activity of selected plant extracts on the growth inhibition of fungi (%) at 10.0

mg/mL

Sample Inhibition of radial growth (%)

B. cinerea P. chrysogenum

Cranberry leaf 6.67 0.00

Black currant residue 0.00 8.33

Sea buckthorn leaf 13.33 0.00

Betel leaf 66.67 99.99

Pomegranate 33.33 16.67

Rhubarb 20.00 16.67

Blueberry leaf 13.33 0.00

Grape leaf, wild 6.67 0.00

Sea buckthorn fruit 20.00 0.00

Nystatin (0.5 mg/mL) 99.99 99.99

The minimum inhibitory concentration (MIC) against E.coli was determined for selected

extracts (Table 5.2). For most of the plant extracts the MIC value against E.coli were 10

mg/mL, except for pomegranate (5 mg/mL), sea buckthorn fruit (5 mg/mL) and betel leaf

(2.5 mg/mL) showing a lower value.

Since the result above showed 99.99 % of GI against P. chrysogenum of betel leaf. The

MIC against P. chrysogenum of betel leaf have been also carried out. Its MIC against P.

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chrysogenum was 2.5 mg/mL, compared to that of the positive control nystatin, which was

0.25 mg/mL. The exceptional activity of betel leaf extract against fungi is attributable to its

contents of alkaloids and terpenoids (Chapter 6). These non-polar compounds could perturb

the external coat of hydrophobins, which are moderately hydrophobic proteins secreted by

fungi creating an interfacial self-assembled amphipathic protein films (Wessels, 1996).

Considering the inhibitory activity against bacteria, yeasts and fungi, only betel leaf extract

appears to possess a broad spectrum of antimicrobial activity; whereas pomegranate and sea

buckthorn leaf extracts appear to be effective against both bacteria and yeasts, and black

currant extract is effectively an antibacterial substance.

The compounds that show activity in most of the samples may belong to the same class of

compounds, and may be present in abundance. As seen in betel leaf extract, the broad

spectrum activity may result from the presence of a variety of compounds with wide range

of polarities. The results confirm the statement of Tiwari et al. (2009) that there are

limitations to the application of natural antimicrobials from plant sources; and the search

must continue.

Although, phytochemicals that produce minimum inhibitory concentrations (MIC) in the

range 100–1000 g/mL in vitro susceptibility tests can be classified as antimicrobials

(Simoes et al., 2009), it is clear from these results that enhancement of the antimicrobial

potency of the extracts seems essential so that their application at lower levels in foods can

be envisaged. It is imperative to have information about the composition of the selected

extracts to determine their potential as broad spectrum antimicrobial.

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5.5 Conclusions

This study examined the antimicrobial activity of selected plant extracts by turbidimetric

methods, where antimicrobial activity was expressed by Capacity, MIC and AMI; and the

ZI-well-diffusion method, where the activity is expressed by zone of inhibition. Although

the turbidimetric methods were in good agreement in the assessment of the activity of the

substances, the new expression AMI appears to be more meaningful since it carries the

information regarding interaction between the substance and the microorganism. In

addition, AMI demarcates the activity of samples, even those found to be highly active. The

second part of the study examinded the spectrum of antimicrobial activity of selected plant

extracts. Effectively, only betel leaf extract showed a broad spectrum of antimicrobial

activity against bacteria, yeasts and fungi; whereas pomegranate and sea buckthorn leaf

extracts appear to be effective against both bacteria and yeasts, and black currant extract

was effectively an antibacterial substance.

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Chapter 6 ENHANCEMENT OF THE ANTIMICROBIAL ACTIVITY OF

SELECTED PLANT EXTRACTS BY FRACTIONATION AND MIXING

Différentes stratégies pour améliorer l’activité antimicrobienne d'extraits anti-oxydants-antimicrobiens

sélectionnés ont été explorées. Trois approches, incluant l’extraction par solvants sélectifs, le mélange binaire

des extraits et l'addition de composés végétaux, ont été étudiées. L’activité anti-oxydante-antimicrobienne et

le profil phytochimique ont été analysés. Le résultat montre que l’eau chaude peut être utilisée comme

solvant pour l’extraction d’agents anti-oxydants-antimicrobiens d’origine végétale, bien qu’un solvant polaire

extrait en préférence les substances phénoliques et modestement les substances non polaires comme les

terpénoïdes. Toutefois, compte tenu du rendement et de l’activité des extraits, l’eau semble être appropriée,

et elle peut être considérée comme un solvant efficace pour les fruits qui sont riches en substances

phénoliques. Néanmoins, d'autres solvants sélectifs doivent être considérés pour l’extraction des substances

actives non-polaires issues de matière première végétale comme les feuilles. Certaines augmentations de

l’activité antimicrobienne sont possibles par le mélange de deux extraits de plantes ou par l’addition de

composés végétaux. On a aussi observé que la composition des mélanges peut être importante, car les

interactions synergiques ou antagonistes se retrouvent dans certaines proportions. Le mélange des extraits

de grenade et cassis d’une part et le mélange des extraits de thé vert et pomme de cajou d’autre part ont

montré une amélioration d’activité. L’ajout de glyoxal de méthyle et mono-caprine a également produit une

augmentation de l’activité des extraits de plantes. L'addition de glyoxal de méthyle à 25 % (p/p) a amélioré

l’activité des extraits de grenade et de cassis.

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6.1 Abstract

Plant materials have been used over centuries as rich sources of antimicrobials. However,

the activity of the extracts from natural sources is rather low in comparison with chemical

antimicrobials, and thus, their application requires higher concentrations that may alter taste

and other properties of food. The aim of this study was to enhance the antimicrobial activity

of selected plant extracts. Three approaches were investigated, including selective solvent

extraction, binary blending of extracts and addition of plant compounds. Micro-titer broth

dilution and DPPH assay were used to measure the antimicrobial and antioxidant activity,

respectively. The profile of phytochemical classes was also analyzed. Based on the yield of

extracts and their activity, hot water could be a suitable solvent for fruit and vegetable

materials that are rich in phenolic substances. Nonetheless, other selective solvents must be

considered for extraction of active non-polar substances for rich plant sources such as

leaves. Some enhancement in antimicrobial activity was possible by mixing plant extracts

or by the addition of certain plant compounds. It was also observed that the composition of

blends or mixtures might be important, since synergistic or antagonistic interactions occur

at certain proportions. Pomegranate and black currant extract blends (both rich in phenolic

substances) and green tea and cashew apple extract blends showed enhancement in activity.

The addition of methyl glyoxal at 25 % (w/w) improved the activity of pomegranate and

black currant extracts. Although some improvement in the antimicrobial activity could be

achieved for some plant extracts, concentration of the extract required for effective control

of microorganisms in foods may not be a very promising by these approaches.

Key words: Phytochemicals, plant extracts, enhancement, antimicrobial, selective

extraction, blends

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6.2 Introduction

In recent years, there has been a growing interest in the exploration of new antimicrobial

agents from natural sources. Many studies have shown that naturally derived compounds

and natural products may have applications in controlling pathogens in foods (Lucera et al.,

2012; Negi, 2012). However, the use of natural antimicrobials is limited due to the fact that

they are of low potency, and it requires high concentrations, which can affect the

organoleptic properties of foods (Davidson et al., 2013; Lucera et al., 2012). Indeed, the

minimum concentration of plant extract required to successfully control and/or inhibit

(MIC) bacteria was found to be too high to be used in foods, where a concentration of

1.56–25.0 mg/mL for gram positive, and 12.5–50.0 mg/mL for Gram negative bacteria

(Borawska et al., 2010).

In the previous sections, we have shown that plant extracts have antimicrobial activity, and

the extracts of betel leaf and pomegranate exhibited a broad spectrum of activity against

bacteria, yeasts and molds, whereas balck currant residue extract was an effective

antibacterial substance. Other reports have indicated the good antibacterial property of

pomegranate peel extracts (Akhtar et al., 2015; Al-Zoreky, 2009; Ismail et al., 2012), betel

leaf (Nagori et al., 2011), black currant (Ikuta et al., 2012) and green tea (Reygaert, 2014).

Nevertheless, their activity was too low to use at concentrations comparable to chemical

antimicrobials. This is a major constraint in the application of naturally occuring

antibacterials. However, enhancement of antimicrobial activity of plant extracts may be a

possible option towards that goal and should be pursued. Pomegranate, betel leaf, black

currant and green tea extracts and others were selected for enhancement of antimicrobial

activity by blending. It was hypothesized that enhancing the antimicrobial activity of plant

extracts would be possible and would advance the development of antimicrobial agents

antimicrobial agents in foods. Three approachs for enhancement used in this study were

fractionation (Miyasaki et al., 2013), binary blending of extracts and addition of plant

products that may potentiate the activity of the extracts (Worthington and Melander, 2013).

190



Pomegranate (Punica granatum), black currant residue (Ribes nigrum), betel leaf (Piper

betle) and green tea (Camellia sinensis) were selected for this study. Samples were

collected from farms, farm markets and fruit and vegetable processors in and around

Quebec City. All collected samples were transported the same day and stored at 4°C until

further processing. Subsequently within 48 hours, they were lyophilized, ground to powder,

vacuum-sealed and stored at -30°C6.3.2 Chemicals and reagents.

Sulfuric acid, chloroform, linalool, sodium nitrite, sodium hydroxide, aluminum chloride,

Folin Ciolcalteau reagent, sodium carbonate, gallic acid, rutin, caffeine, +- catechin, tannic

acid, vanillin, potassium ferricyanide, ferric chloride, potassium iodide, bismuth nitrate

pentahydrate, thiourea, disodium sulphide are bought from Sigma Aldrich (Canada). All

reagents used in this investigation were of analytical grade.

6.3.3 Plant extracts and stock solution preparation

About 50 gram of the lyophilized powder of sample was extracted with 500 mL of distilled

water at 85-90°C for 60 minutes with constant stirring. The slurry was then vacuum filtered

(Whatman No. 1 filter paper), and the filtrate was concentrated under vacuum using a

rotary evaporator at 65°C to about 100 mL and then lyophilized. The lyophilized extracts

were vacuum-packed and stored at -30oC until further use. For the antimicrobial and

antioxidant assays the samples were dispersed in MHB (Mueller Hinton broth) and

methanol, respectively, to obtain a stock solution of 100 mg DW/mL for the antimicrobial

activity and 1.0 mg/mL for the antioxidant assay. All the stock solutions were purged with

argon, stored at 4oC and used within 24 hours.

191


The antimicrobial activity of the extracts was determined against E.coli ATCC 25922

(Gram-negative) and B. subtilis ATCC 6633 (Gram-positive) bacteria. Bacterial cultures

were revived from stocks at -80°C by three successive growth cycles at 37 °C for 24 h in

Muller Hilton Broth (Sigma Aldrich). The standard inoculums were prepared following

NCCLS guidelines (Jorgensen and Ferraro, 2009) with organisms in log phase growth (4h

after incubation) and then were standardized to match that of a 0.5 McFarland standard

(corresponds to approximately 1.5 x 108 CFU/mL). The adjusted bacterial suspensions were

used within 15 minutes.

6.3.5 Antimicrobial activity assays

6.3.5.1 Growth Inhibition using micro-titer broth dilution assay (GI)

The GI (%) value of plant extracts and the potential to reduce the bacterial population over

time was determined turbidimetrically using a micro-titer broth dilution assay (NCCLS,

1991; Wiegand et al., 2008). Briefly, 190 µl of the plant extract stock made in MHB broth

(10 mg/mL) was pipetted in a 96-well plate. Subsequently, the wells were inoculated with

the test strain (10µl) to achieve a final concentration of 104- 105 CFU/mL in each well. The

blank and assay control were the plant extract (190 µl at 10 mg/mL) and 200 µl of MHB

without the test strain, respectively; where the latter also served as the sterility control.

Chloramphenicol (1.0 mg/mL) was used as the positive control, while the inoculated MHB

medium as the negative control. The prepared plates were incubated at 37°C in a micro-

plate reader (Biotek Power XS2). For the time kill curve assay, hourly absorbance readings

(OD600nm) were recorded for 24 hours and for the time kill curve assay, the OD reading at

the end of 24 hours was recorded for calculating the %GI value. All assays were performed

in triplicate. The percentage of growth inhibition was calculated by the difference between

the final OD600 reading of the sample and the negative control.

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6.3.5.2 MIC - Broth microdilution assay

Plant extracts were tested as per NCCLS guidelines for microdilution susceptibility tests for

aerobic bacteria (NCCLS, 1991) with modifications. Micro-titer plates (96 wells) were

prepared with 12 plant extracts in the columns at the concentration 80 mg/mL in MH broth.

Eight serial dilutions were made in the rows. Thereafter, 10µl of standard inoculum was

dispensed in each well to give a final concentration of 104- 105 CFU/mL. The MIC value

after 24 hours of incubation at 37o C, was the lowest concentration of plant extract at which

no visible growth could be detected by visual inspection. Chloramphenicol (1.0 mg/mL)

was used as a positive control

6.3.6 Antioxidant assay - DPPH radical-scavenging capacity

Free radical scavenging capacity is the main mechanism by which antioxidants protect food

against oxidation and thus was determined through DPPH radical scavenging assay (Faleiro

et al., 2005). The DPPH radical scavenging was analysed through a standard

spectrophotometric method (Sharma and Bhat, 2009) with modifications. A DPPH stock

solution of 6.1x 10-5 M was freshly prepared in methanol and adjusted to O.D of 0.7 (±

0.02) at 515 nm. A volume 190 µL of DPPH solution was transferred into each microwell

compartement (96 well micro plate, Becton Dickinson Falcon 353072), followed by

addition of 10 µL of the test plant extract (1.0 mg/mL). Thereafter the plate was incubated

in a (Biotek Power XS2 Logicel Gen 5) spectrophotometer at 25°C for 1 hour, and the

absorbance at 515 nm was recorded each min. Ascorbic acid (1.0 mg/mL) was used as a

standard and the results were expressed as ascorbic acid equivalent (µg AAE/mg extract).

The assays were carried out in triplicate.

Radiacal scavenging capacity = [(A0 − A1)/A0 ]× 100 (%)

where A0 was the absorbance of the control sample (without plant extract) and A1 was the


193

6.3.7 Phyto-chemical analysis

6.3.7.1 Total phenolics

The method described by (Wolfe and Liu, 2003) with little modification by using Folin-

Ciocalteu reagent was used to determine total phenolic content in aqueous extract and

extracts of different solvent mixtures. A volume of 0.5 mL of the extract (1 mg/mL), was

mixed with 0.5 mL of 1N Folin-Ciocalteu reagent and 1mL of Na2CO3 (20% w/v) and the

final volume was made up to 5 mL The resulting mixture was vortexed for 15 sec and

incubated at room temp for 30 min for color development. The absorbance of total phenolic

was measured at 725 nm. Total phenolics content was expressed as mg/g gallic acid

equivalent (Michel et al., 2012a) (concentration 20-100µg/ml) from gallic acid calibration

curve. The experiment was conducted in triplicate and the results were expressed as mean

SD values.

6.3.7.2 Total flavonoid assay

The total flavonoid content was determined by using aluminium chloride colorimetric

method (Zhishen et al., 1999). Briefly, 250 µL of each extract (1.0 mg/mL) was mixed with

1.0 mL of distilled water and subsequently with 75 µL of sodium nitrite solution [5% (w/w)

NaNO2]. After 6 min of incubation, 75 µL of aluminium trichloride solution (10% AlCl3)

was added and then allowed to stand for 6 min, followed by addition of 1.0 mL 4% sodium

hydroxide solution to the mixture. Immediately, water was added to bring the final volume

to 2.5 mL, and then the mixture was thoroughly mixed and allowed to stand for another 15

min at room temperature. Absorbance of the pink color of the mixture was measured at 510

nm versus reagent blank containing water instead of the sample (Basma et al., 2011).

Catechol was used as a standard compound for the quantification of total flavonoid. Results

were expressed as mg of catechol equivalents per gram of dry weight of extracts (µg

CE/mg). Total content of flavonoid compounds in the plant extract was calculated using

this formula:

Total flavonoid content = CE.V/m

194

where, CE is the catechol equivalence (mg/mL) or concentration of catechin solution

established from the calibration curve; V is the volume of extract (mL) and m is the weight

(mg) of the pure plant extract.

6.3.7.3 Estimation of total tannin content

The quantitative tannin content in plant extracts was estimated by the method of Price and

Butler (Paaver et al., 2010; Price and Butler, 1977) with some modifications. Briefly, 0.5

ml aliquots of plant extracts having concentration of 1.0 mg/ml were transferred to vials, 1

ml 1% K3Fe(CN)6 and 1.0 ml 1% FeCl3 were added, and water was added to make 5 ml

volume. After five min, the solutions were measured spectrophotometrically at 760 nm.

The actual tannin concentration was calculated on the basis of the optical absorbance values

obtained for the standard solutions in range 20 - 100 µg/ml.

6.3.7.4 Determination of proanthocyanidin content

The proanthocyanidin content of the solvent fractions were determined by the vanillin- H2SO4

method (Lee et al., 2009; Sun et al., 1998). Briefly, a sample solution 200 μL was added to

500 μL of 1.2% vanillin solution and 500 μL of 20% H2SO4 solution. The reaction was

carried out in the dark at room temperature for 20 min, and then absorbance was measured

absorbance at 500 nm. Proanthocyanidin content was expressed as µg (+)-catechin equivalent

per ml of aquous extracts.

6.3.7.5 Determination of alkaloid content

Alkaloid content was determined by the method of (Sreevidya and Mehrotra, 2003) with

some modifications. The Dragendorff’s reagent (DR) was prepared by mixing a solution of

bismuth (0.8 g bismuth nitrate pentahydrate in 40 mL distilled water), 10 mL glacial acetic

acid, and a solution of potassium iodide (8.0 g potassium iodide in 20 mL distilled water).

The standard bismuth nitrate stock solution was made by dissolving 10 mg Bi(NO3)3·5H2O

in 5 mL of concentrated nitric acid, and diluting it to 100 mL with distilled water. The other

reagents were thiourea, 3% (w/v), and disodium sulfide, 1% (w/v). The stock solutions of

alkaloid was prepared as follows: 10 mg of pure alkaloid (caffeine “purine alkaloids”) was

dissolved in warm distilled water and then made up to 10 mL with warm distilled water.

Similarly, stock solutions of piperine in 95% alcohol were prepared.

195

The plant extract solution was prepared at the concentration 1.0 mg/mL. A 5 mL of the

extract solution was then taken and the pH was maintained at 2–2.5 with dilute HCl. A 2

mL amount of DR was added to it, and the precipitate formed was centrifuged. The

supernatant was checked for complete precipitation by adding 2 drops of DR. After

centrifugation, the centrifugate was completely and meticulously decanted. The residue was

then treated with 2 mL disodium sulfide (Na2S) solution. The brownish black precipitate

formed was then centrifuged. The completion of precipitation was checked qualitatively by

adding 2 drops of disodium sulfide. The residue was dissolved in 2 mL concentrated nitric

acid, with slight warming, as needed. This solution was diluted to 10 mL in a standard flask

with distilled water, an aliquot of 1.0 mL was then pipetted out, and to which 5 mL thiourea

solution was added. The absorbance was measured at 435 nm.

6.3.7.6 Terpenoids

Terpenoid content was determined by the method of (Ghorai et al., 2012). Briefly, 1.5 ml

chloroform was added in 2 ml microcentrifuge tube and then 200 µl plant sample was

added. Thereafter, the sample mixture was thoroughly vortexed and was keep aside 3 min.

Then 100µl concentrated H2SO4 was added to each microcentrifuge tube. Thereafter, the

assay tube was incubated at room temperature for 1.5h-2h in the dark. At the end of

incubation time a reddish brown precipitation was formed in each assay microcentrifuge

tube. Subsequently, all supernatant reaction mixture liquid was carefully decanted without

disturbing the precipitate. 1.5 ml of methanol 95% (v/v) was added and vortexed

thoroughly until all the precipitate completely dissolved in methanol. Then the solution was

read at the absorbance 538 nm. Methanol was used as blank. Total terpenoids concentration

of plant samples was expressed as linalool equivalents using the regression equation of

linalool standard curve.

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6.3.8 Enhancement of antimicrobial activity of plant extracts

Three approaches were chosen for improving the antimicrobial activity of the selected plant

extracts: Selective solvent extraction, binary blends of selected extracts and addition of

plant compounds.

6.3.8.1 Selective solvent extraction

Selective extraction was used to obtain fractions enriched in classes of phyto-compounds of

different polarity using solvents of varying polarity. The fractions enriched in different

classes of phytochemicals may exhibit a different antimicrobial activity compared with that

of the whole sample.

Solvent extraction of selected plant materials were carried out using sevent solvent systems

ranging in polarity (Table 6.1) solubility parameter was used to guide the polarity of

solvents, where higher the solubility parameter, higher its polarity. Hexane (HEX); hexane:

ethylacetate (HE-EA) = 1:1; was taken to extract the non-polar lipophilic compounds.

Acetone: acetonitrile (ACAC) = 1:1 was taken to obtain the less polar, oligomeric

components. Methanol: Ethanol (MEET) = 1:1, and methanol 80% (MEH2O), water (H2O)

and hot water (HH2O) were chosen to extract the hydrophilic compounds.

Table 6.1 Solubility parameter of the solvent systems

Solvents Solubility parameter

(Cal/cm3)0.5

Internal pressure

(Cal/cm3)0.5

H-bonding parameter

(Cal/cm3)0.5

HEX 7.5 7.5 0.0

HEA 8.3 7.7 2.3

ACAC 10.9 10.4 3.2

MEET 13.6 9.2 10.2

ME80 16.1 10.9 12.1

H2O 23.5 16.4 16.7

HH2O 22.6 - -

Lyophilized powder sample (1.0 g) was extracted with 10 mL of solvent with agitation at

room temperature for 6 h. Then the mixture was centrifuged at 5000 rpm for 10 min. The

supernatant was recovered and evaporated in Speed-Vac (Thermo scientific) with radiant

heat. The concentrates were further dried by manifold to ensure the complete removal of

197

solvent from the fractions. The extract in the solid form were used for antimicrobial test

against E.coli by micro-dilution assay at concentrations of 10,000, 5,000 and 2,500 ppm.

The yield of each solvent fraction was recorded, and its composition was analyzed for

various classes of phytocompounds (total phenols, hydrolysable tannins, proanthocyanidins

(condensed tanin), flavonoids, terpenoids and alkaloids), followed by antimicrobial and

antioxidant (DPPH) activity assays.

6.3.8.2 Binary blends of selected extracts

Binary blends of two selected extracts were prepared in different proportions (1.00:0.00,

0.75:0.25, 0.50:0.50, 0.25:0.75 and 0.00:1.00 by volume) and their antimicrobial activity

was evaluated against E. coli and B. subtilis by micro-dilution assay at total concentration

of 10,000, 5,000 and 2,500 ppm.

6.3.8.3 Addition of plant compounds

For this study, 12 selected plant extracts and 15 plant products (Sigma-Aldrich) were

initially assessed for their MIC (minimum inhibitory concentration). Subsequently, the

mixtures were evaluated for antimicrobial activity (12 plant extracts x15 plant products at a

total concentration of 5.0 mg/mL. The antimicrobial assay for plant products was the same

as for plant extracts. Based on the result, selected plant extracts and plant products were

mixed in difference proportions and the mixtures were evaluated for their activity (Growth

inhibition, GI (%); and MIC).

6.3.9 Statistical analysis

All experiments were done in triplicate and were set as a completely randomized design,

and the data were analyzed by one-way analysis of variance (one-way ANOVA) using a




198


6.4.1 Solvent fractionation of extracts

6.4.1.1 Extract yields

The yield of the fractions (% dried mass) by different solvent systems is presented in Figure

6.1. In general, the yield of the fractions increased with increase in the polarity of the

solvents for three plant samples. Higher yields of fractions were obtained with

methanol/ethanol, methanol/water and water solvents than in non-polar solvents (hexane,

hexane/ethyl acetate or acetone/acetonitrile). For example, the yield of fractions for

pomegranate (Figure 6.1, Table A6.1, Table A6.2, Table A6.3) were: 7.47, 7,08, 8.81,

35.51, 45.22, 29.81, 52.11 mg/g dry material, for Hex, HE-AE, AC-AC, ME-ET, ME-H2O,

H2O, HH2O repectively.

0

10

20

30

40

50

60

Hex HEA ACAC MEET ME80 H2O HH2O

Solvent fractions

Yie

ld,

%,

w/w

dry

sam

ple

PomegranateBlack currantBetel leaf

Figure 6.1 Yield of solvent extracts of pomegranate, black currant residue and betel leaf

199

The highest mass yields were obtained with hot water for all the three plant samples (52.1%

for pomegranate, 32.5% for black currant residue and 47% for betel leaf). This is so,

because hot water appears to be less selective in the extraction of compounds in the plant

materials, having solvent capacities to varying degrees for compounds of different polarity.

There is a much interest for new environmentally sustainable extraction techniques to

enhance the use of agricultural by-products, and hot water has been used in many studies to

extract bioactive compounds (Ko et al., 2014; Plaza et al., 2013; Vergara-Salinas et al.,

2013). The use of hot water at 85 °C was also found to be a suitable solvent to extract

flavonoid glycosides and amino acids (Bergeron et al., 2005).

6.4.1.2 Composition of solvent fractions and their antioxidant-antimicrobial activities

The phytochemical composition of the different solvent fractions of pomegranate, black

currant and betel leaf, and their corresponding antimicrobial activity against E. coli and

anti-oxidant activity are summarized in Tables 6.2 – 6.4 and Figures 6.2 - 6.4.

The polar solvent fractions were enriched in phenolic compounds (flavonoids,

proanthocyanidins and hydrolysable tannins) whereas non-polar solvents largely extracted

alkaloids and terpenoids, indicating their polar or non-polar nature. However, the non-polar

fractions were not highly enriched in alkaloids or terpenoids, presumably, high recovery of

these compounds may require high volumes of solvent. Nonetheless, hot water was able to

extract small amount of these non-polar compounds, in addition to enrichment in the

phenolic compounds.

The classes of phytochemicals, that were analyzed for, present in pomegranate and betel

leaf fractions, where the phenolic classes were enriched in polar solvents and the non-polar

classes were enriched in non-polar solvents. Although the same was true for black currant,

the non-polar alkaloids and terpenoids were nearly absent in its non-polar solvent fractions.

However, the non-polar fractions of black currant contained higher level of flavonoids

(44.52 and 28.88 mg/kg of dry mass for the fraction Hex and HE-AE, respectively) than

those of pomegranate (2.11 and 2.07 mg/kg dry mass), suggesting that the flavonoids in

black currant are more non-polar in nature than those of pomegranate.

200

The antioxidant and antimicrobial activities of the solvent fractions are shown in Table

A6.4, and in Figures 6.2 – 6.4 along with the phyto-compound composition of the fractions.

Higher concentrations of both polar phenolic compounds as well as the non-polar alkaloids

and terpenoids support antioxidant and antimicrobial activities of fractions of pomegranate

betel leaf and black currant. The poor activities of the non-polar fractions of black currant

can be attributed to the quasi-absence of alkaloids and terpenoids in those fractions. It is

also evident that alkaloids and terpenoids contribute significantly to the antimicrobial

activity of the non-polar fractions, even though they were present in smaller quantities

compared with the phenolic compounds. This suggests that isolation of these substances

may be worthwhile for more powerful antimicrobial agents (Nostro et al., 2000). In

contrast, in the polar solvent fraction, the phenolic compounds were responsible for the

antimicrobial activity, although, these compounds were present at higher concentrations,

indicating that their specific antimicrobial activity is smaller compared with alkaloids and

terpenoids, and consequently, higher concentrations would be required to achieve a similar

antimicrobial activity. Nevertheless, the antimicrobial activities of all fractions were still

comparable to that of hot water fraction, even though these fractions differed in their

composition of phyto-compounds. The results point to no significant enhancement in the

antimicrobial activity by fractionation alone compared with the extract with hot water.

201

0

100

200

300

400

500

600

700

800

900

1000


Solvent fractions

mg

/kg

d.m

.

0

10

20

30

40

50

60

70

80

90

100

Total polyphenol

Hydrolyzable tannins

Proanthocyanidins

Flavonoids

Antimicrobial activity

Antioxidant activity

Figure 6.2 Composition of solvent fractions of pomegranate and their antimicrobial activity against E.

coli and anti-radical activity by DPPH assay

0

100

200

300

400

500

600


Solvents fractions

mg

/kg

d.m

.

0

10

20

30

40

50

60

70

80

90Total polyphenol

Hydrolyzable tannins

Proanthocyanidins

Flavonoids

antimicrobial activity


Figure 6.3 Composition of solvent fractions of black currant residue and their antimicrobial activity

against E. coli and anti-radical capacity by DPPH assay

202

0

50

100

150

200

250

300


Solvent fractions

mg

/kg

d.m

.

0

10

20

30

40

50

60

70

80

90

100

Total polyphenol

alkaloids

Terpenoids

Antimicrobial activity


Figure 6.4 Composition of solvent fractions of betel leaf and their antimicrobial activity against E. coli

and anti-radical activity by DPPH assay

Furthermore, considering the productivity (yield and antimicrobial activity of the fractions),

it became evident that a simple fractionation as an approach to enhance the antimicrobial

activity of plant extracts might be less realistic to obtain antimicrobials for use in foods

with comparable effectiveness of chemical antimicrobials.

203

6.4.2 Binary blends of selected extracts

Two plant extracts were blended at a total concentration of 5.0 mg/mL. The proportions of

the blend is 0.0, 0.25, 0.50, 0.75, 1.0 (by mass). The blends were composed of extracts of

pomegranate, black currant, betel leaf, cashew and green tea. The antimicrobial activity is

shown in table A6.5 and Figure 6.5 (E.coli) and Table A6.6, Figure 6.6 (B.subtilis).

Blend composition - Extract (1), % (w/w)

0.0 25.0 50.0 75.0 100.0

GI,

%

0

20

40

60

80

100

120

Pomegranate (1)-Black currant (2)

Betel leaf (1)-Black currant (2)

Pomegranate (1) -Betel leaf (2)

Green tea (1)-Cashew (2)

Figure 6.5 Antibacterial activity of blends of plant extracts in varying proportions

The blend of pomegranate and betel leaf extracts that contain phenolic compounds or

alkaloids and terpenoids, respectively showed mild incompatibility or little interaction in all

proportions, although the antimicrobial activity was not affected significantly. But the

combination of betel leaf and black currant extracts showed synergy at 50.0 - 75.0 % of

betel leaf extract (100 % of inhibition against E. coli). This is surprising that black currant

extract, like pomegranate extract, is enriched in phenolic compounds should show synergy.

204

It is possible that the bridge class of compounds may be the flavonoids in black currant that

may be more lipophilic, as pointed earlier. Contrary to expectation, the blend of

pomegranate and black currant extracts, both being rich in phenolic compounds, did not

show any synergy, but significant antagonistic interaction at 25.0 % of pomegranate extract

(Figure 6.5). The blend of green tea and cashew apple extracts, that show moderate activity

alone, showed significant synergistic interaction in the antimicrobial activity at 50.0 – 75.0

% of green tea extract (82.39 and 86.34 % of inhibition against E. coli). It suggests that the

phyto-compounds present in these extracts may act in a similar mode against the bacteria.

While green tea is rich in flavon-3-ols (catechins) and hydrolysable tannins, gallo-catechins

and ellagi-catechins (Henning et al., 2004; Reygaert, 2014), cashew apple is known to

contain anacardic acid that is known to possess antimicrobial activity (Muroi et al., 1993).

The pattern of interaction between extracts appears to be similar for B. subtilis, as was

observed with E. coli (Table A6.6 and Figure 6.6), except that a higher synergy was evident

with the blend of green tea and cashew apple extracts (90.02% of inhibition). The similar

pattern of the blends against the bacteria of different morphology indicates that the nature

of interaction whether antagonistic, agonistic or neutral, between the constituents of the

blends may be the primary factor in the activity of the blends. The data on the composition

of phyto-compounds of the extracts are not adequate to understand such interactions, and

more detailed analysis of the extract constituents should be performed.

205

Blend composition -Extract (1), %, (w/w)

0.0 25.0 50.0 75.0 100.0

GI, %

30

40

50

60

70

80

90

100

Pomegranate (1)-Betel leaf (2)

Green tea (1)-Cashew (2)

Figure 6.6 Antibacterial activity of blends of plant extracts in varying proportions against B.subtilis at

total concentration of 5.0 mg/mL

The blends of betel leaf and black currant extracts in the proportion 50.0 % and 75.0 % of

betel leaf extract completely inhibited the growth of E. coli (Table A6.5). Thus there is

some scope to improve the antimicrobial activity of plant extracts. However, the

concentration of the blend used in this investigation may still be too high for consideration

as an antimicrobial additive in terms of efficacy. Although these extracts possess

antioxidant activity and are potentially health beneficial, their impact on the organoleptic

properties of foods containing these extracts at high concentrations may be adversely

affected. In short, it is difficult to qualify this approach as promising.

206

6.4.3 Addition of plant compounds

The new approach in enhancing the antimicrobial activity of plant extracts is mixing them

with salts of the following metals; Fe (II), Cu (II), Mn (II) or Zn (II), and vitamin C

(McCarrell et al., 2008), with sodium picolinate, sodium benzoate (Borawska et al., 2010),

or with antibiotics. For example, a synergistic effect of the essential oil of Citrus hystrix

were observed against Escherichia coli and Staphylococcus epidermidis when combined

with gentamicin (Aumeeruddy-Elalfi et al., 2016).

It was hypothesized that small molecular plant compounds, including plant hormones such

as gibberellins and signal molecules such as salicylic acid and jasmonic acid, that may or

may not possess antimicrobial activity, but could interact with plant extracts cooperatively

and thus could improve their activity. The antimicrobial activity of selected plant

compounds was evaluated by MIC as shown in Table 6.8, along with those for selected

plant extracts. All the plant compounds showed high activities with MIC values in the

range of 1.25 – 2.50 mg/mL, except mono-laurin at 5.0 mg/mL, while those ranged from

5.0 – 10.0 for the selected plant extracts, except for betel leaf extract with a MIC value of

2.50 mg/mL.

Table 6.2 The MIC against E.coli of 12 selected plant extracts and 10 plant products

Plant extract MIC (mg/mL) Plant compound MIC (mg/mL)

Cranberry leaf (CRL) 20 Mono-laurin 5.0

Black currant residue (BCR) 10 Mono-caprin 1.25

Sea buckthorn leaf (SBL) 10 Methyl glyoxal 1.25

Betel leaf (BL) 2.5 Azelaic acid 1.25

Pomegranate (PG) 5.0 Abietic acid 2.5

Rhubarb (RB) 10 Salicylic acid 1.25

Blueberry leaf (BBL) 10 Jasmonic acid 2.5

Grape leaf (GL) 10 Phenyl acetic acid 1.25

Cashew apple (CA) 10 Gibberellin 2.5

Sea buckthorn fruit (SBF) 5.0 Indole-3- butyric acid 1.25

Noni (N) 10

Green tea (GT) 10

207

The 10 plant compounds were added to the 12 selected plant extracts at a level of 10 %

(w/w) [9 volumes of plant extract at concentration of 5.0 mg/mL) + 1 volume of plant

compound at 5.0 mg/mL] at total concentration of 5.0 mg/mL, and the antimicrobial

activity was evaluated against E. coli, and the inhibitory activities of the mixtures are

summarized in Table 6.9. Generally, the addition of phyto-compounds to moderate active

extracts such as blueberry leaf, grape leaf and black currant extracts rendered them more

active, except sea buckthorn leaf and green tea that showed enhancement only with methyl

glyoxal among the plant compounds. Abeitic acid, mono-laurin, azelaic acid and jasmonic

acid did not impart any significant improvement in the antimicrobial activity of most of the

extracts (Table 6.9). For the most part, the addition of methyl glyoxal, mono-caprin,

salicylic acid, phenyl acetic acid, gibberellin and indole-3-butryric acid were generally

effective in enhancing the activity of most of the extracts, both high and moderate active

extracts, except sea buckthorn leaf and green tea extracts for which only methyl glyoxal

was effective in effectively enhancing their activity. For example, methyl glyoxal enhanced

antimicrobial activity of black currant residue from 76% to 99.99% of inhibition of E.coli,

of grape leaf and noni fruit from 78% to 99.99% of inhibition. And indole_3_butryric acid

exhibited an enhancement in antimicrobial activity of black currant residue (up to 99.5%),

grape leaf (up to 97.13%), and pomegranate (up to 99.36%) and cashew apple (up to

84.17%). The mixing of methyl glyoxal with black currant extract, or with pomegranate

extract or grape leaf extract exhibited complete inhibition of E. coli. Likewise, the mixing

of mono-caprin with pomegranate extract; salicylic acid with pomegranate extract; and

indole-3-butyric acid with betel leaf resulted in complete inhibition of the bacteria.

The nature of enhancement in the activity by plant compounds cannot be ascertained at this

time due to lack of data; however, it is reasonable to assume that these compounds may not

interact with the constituents of the plant extracts and may play a part in the

permeabilization of bacterial membrane, given their lipophilic character. When there is

interaction with the extract components, such an activity is compromised, as seen for sea

buckthorn leaf and green tea extracts (Table 6.9).

208

Table 6.3 The GI (%) against E.coli of mixture of 12 plant extracts with added plant products at total concentration of 5.0 mg/mL

Plant compound

Mono-laurin

Mono-caprin

Methyl glyoxal

Azelaic acid

Abietic acid

Salicylic acid

Jasmonic acid

Phenyl-acetic acid

Gibberellin

Indole-3-butyric acid

Plant extract Control, GI (%)

(79%)* (98%) (100%) (98%) (82%) (99%) (89%) (100%) (88%) (99%)

Cranberry leaf (73%)* 75.11 83.25 88.54 76.43 78.73 84.59 72.07 84.9 79.65 81.07

Black currant residue (76%) 82.15 99.66 99.99 88.64 80.26 98.13 79.76 87.96 84.56 99.5

Sea buckthorn leaf (74%) 70.2 76.42 97.05 78.06 76.72 78.62 75.48 81.2 78.93 80.01

Betel leaf (95%) 71.05 89.69 99.99 65.71 88.06 95.94 85.98 89.15 81.81 99.99

Pomegranate (87%) 76.87 99.99 99.99 81.52 88.08 99.99 89.1 97.67 77.49 99.63

Rhubarb (72%) 61.75 76.32 78.45 49.49 67.56 80.55 80.23 80.65 78.93 82.11

Blueberry leaf (68%) 69.83 78.5 92.16 78.77 74.34 79.54 80.04 76.51 81.81 86.53

Grape leaf (78%) 83.01 89.91 99.99 85.66 78.23 89.51 80.15 89.92 84.73 97.13

Cashew apple (64%) 66.69 81.12 85.03 57.92 65.23 78.9 78.14 76.3 76.09 84.17

Sea buckthorn fruit (83%) 73.11 84.27 95.55 86.01 75.56 81.17 71.65 77.64 86.32 83.33

Noni fruit (78%) 85.33 89.69 99.99 89.12 80.02 87.46 90.01 89.03 89.64 89.18

Green tea (73%) 53.77 68.09 93.97 57.24 60.11 78.65 75.66 79.31 65.64 79.89

* Value in brackets represent the GI (%) of plant extracts alone or added plant compounds alone at total concentration of 5.0 mg/mL

209

Five plant compounds and five plant extracts were selected based on the observations

(Table 6.9) for further examination. Mixtures were prepared in volumetric proportions of

97.5% plant extract at concentration of 2.5 mg/mL and 2.5% (2.5 mg/mL), in other words,

plant compound addition was at 2.5 %; and the antimicrobial activity against E. coli was

evaluated at total concentration of 2.5 mg/mL. Table 6.10 summarizes the inhibitory

activity of the mixtures. The addition of methyl glyoxal and mono-caprin generally

enhanced the antimicrobial activity of all extracts except betel leaf extract, where addition

of all the plant compounds had either negative or no impact. However, the addition of plant

compounds generally enhanced the activity of black currant, wild grape leaf and noni fruit

extracts. Among the plant compounds, methyl glyoxal and mono-caprin addition to either

pomegranate or black currant extract showed promising enhancement in their activity.

Table 6.4 The GI (%) against E. coli of mixtures of plant extracts and 5 plant compounds at the

proportion of plant extract 97.5%, (v/v).

Plant compound Methyl glyoxal (95%)*

Mono-caprin (90%)

Indole-3-Butyric acid (86%)

Salicylic acid (88%)

Plant extract

Pomegranate (78%)*

90.78 86.72 65.49 70.34

Betel leaf (89%)

89.42 78.32 70.05 74.44

Black currant (67%)

91.12 87.67 76.54 73.56

Grape leaf, wild (59%)

69.66 65.45 71.90 68.93

Noni (60%)

79.35 74.01 67.98 64.78

* Values in bracket represent the GI (%) of plant extracts alone or added compound alone

at total concentration of 2.5 mg/mL

210

Finally, the two plant compounds, methyl glyoxal and mono-caprin, were mixed with black

currant and pomegranate extracts in proportions of 100.0/0.0, 75/25, 50/50, 25/75 and

0.0/100.0 (w/w) The mixtures were assessed for MIC against E. coli by broth micro-

dilution assay, starting from total concentration of 10 mg/mL, with 2-fold series dilution.

Composition -Plant extract (w/w)

0.0 25.0 50.0 75.0 100.0

MIC

(m

g/m

L)

0.0

2.5

5.0

7.5

10.0

12.5Black currant-Methylglyoxal

Black currant-Monocaprin

Pomegranate-Methylglyoxal

Pomegranate-Monocaprin

Figure 6.7 Antibacterial activity (MIC) against E.coli at varying compositions of plant extract – plant

compound mixtures

211

The MIC values of different mixtures are shown in Table A6.7 and Figure 6.7. The addition

of mono-caprin to pomegranate extract did not alter the MIC of pomegranate extract at any

proportion. But methyl glyoxal at 50-75 % level of addition did show a reduction by 50 %

in MIC value of the extract. Besides, addition of such a high level of methyl glyoxal may

not be acceptable. It was quite similar with mono-caprin mixture with black currant.

However, methyl glyoxal mixed with black currant extract at 25 % level reduced the MIC

value against E. coli of black currant alone from 10.0 mg/mL to 2.5 mg/ml, a reduction of

75 %; presumably due to possible synergistic interactions. Yet, betel leaf extract alone

exhibited a MIC value of 2.5 mg/mL. Even though the addition level of 25 % may be

acceptable. In any event, this MIC value is still very high in comparison with synthetic

preservatives, and it would be to characterize them as antimicrobials. It has been suggested

that only those phytochemical products that produce minimum inhibitory concentrations

(MIC) in the range 100–1000 µg/mL in vitro susceptibility tests can be classified as

antimicrobials (Simoes et al., 2009).

212

6.5 Conclusions

This work shows that hot water can be used for solvent extraction of antioxidant-

antimicrobials from plant materials, albeit a polar solvent that extracts mainly phenolic

substances, but also non-polar substances such as terpenoids an alkaloids to some extent.

However, considering the yield of the extracts and the activity, water appears to be

appropriate as an effective solvent for fruit products that are rich in phenolic substances.

Thus, the extraction of multiple substances and the yield of extraction by hot water is still

disarable. Nonetheless, other selective solvents must be considered for extraction of active

non-polar substances for plant sources such as leaves. Some enhancement in antimicrobial

activity was possible by either mixing plant extracts or by the addition of plant compounds.

It was also observed that the composition of blends or mixtures might be important, since

synergistic or antagonistic interactions occur at certain proportions. Pomegranate and black

currant extract blends (both rich in phenolic substances) and green tea and cashew apple

extract blends showed enhancement in activity. The addition of methyl glyoxal and mono-

caprin also showed enhancement in the activity of plant extracts. Methyl glyoxal at 25 %

(w/w) addition improved the activity of pomegranate and black currant extracts. Although

some improvement in the antimicrobial activity could be achieved for the selected extracts,

concentration of the extract required for effective control of microorganisms may not be a

very promising by these approaches.

213

GENERAL CONCLUSIONS AND PERSPECTIVES

214

There is a growing realization and interest in the development of different strategies to use

agricultural and industrial residues as a source of high value-added products. By-products

of fruits and vegetables may be a potential source of bioactive compounds. Identification of

extracts exhibiting both antioxidant and antimicrobial activities could lead to the

development of natural products for possible use in the preservation of foods to replace

synthetic preservatives.

The major goal of this work was to conduct studies to pave the way for the development of

antioxidant-antimicrobials from plant by-products. Specifically, the study involved the

identification of promising antioxidant-antimicrobial extracts from fruits and vegetable by-

products; the characterization of plant extracts for their antioxidant capacity and efficacy;

the evaluation of selected edible plant extracts for their spectrum of anti-radical activity of

importance in biology; the evaluation of selected edible plant extracts for their

antimicrobial activity; and the exploration of ways to enhance the antimicrobial activity of

selected antioxidant-antimicrobial extracts.

The first objective centered on screening of various fruit and vegetable sources. About 160

samples (fruit and vegetable by-products) were evaluated for their potential as source of

antioxidant-antimicrobials for use in food preservation, as alternative to synthetic agents.

The study led to the identification of some potential extracts exhibiting both antimicrobial

and antioxidant properties. The pH of the extract, type of tissue (fruit, leaf and root) and

physiological type of fruits (climacteric) had impact on the bioactive properties. There was

also some relationship between antioxidant and antimicrobial properties of the plant

extracts. The proposed antioxidant-antimicrobial (AO-AM) index may be useful in the

selection of plant sources of such functional bio-actives. The plant extracts could be a safe,

health-beneficial and economical food preservation agent with antimicrobial and

antioxidant properties.

The second objective of this work focused on the antioxidant characterization of plant

extracts for their capacity and efficacy. The true antioxidant activity may lie on both the

capacity as well as the rate of radical scavenging. The new expression, anti-radical power -

ARP, generated from DPPH assay may be more useful in identifying the antioxidant

activity of biological samples. The samples that exhibited high ARP values were: rambutan,

215

cranberry leaf, blueberry leaf, grape leaf (wild), raspberry leaf, betel leaf, avocado,

pomegranate and custard apple. Leaf extracts possess, in general, higher ARP than fruits,

and root extracts possess low ARP. In addition, this study also suggests that average

carbon oxidation number (ACON) of complex mixtures such as plant extracts may portend

their antioxidant power, in spite of the disparity between leaf and fruit materials.

The third part of this study evaluated the spectrum of anti-radical activity of 36 selected

plant extracts against organic radicals (ABTS and DPPH), superoxide anion (SOA),

hydroxyl and nitric oxide radicals. Ferric ion reducing activity and iron binding capacity of

the extracts were also examined. TEAC, DPPH and FRAP assays provided essentially the

same response with respect to the antioxidant activity of plant extracts, suggesting that any

one of them would be adequate to evaluate their anti-radical capacity. Betel leaf, blueberry

fruit and black currant and cranberry leaf showed high radical scavenging activity (TEAC

assay); apple, sorrel, red grape and dandelion root were against SOA; cranberry leaf,

blueberry leaf, black currant and rosemary against hydroxyl radical; rainbow chard,

parsnip, broccoli and orange against H2O2; and potato, banana, sorrel, sea buckthorn leaf

against nitric oxide. Blue berry leaf and fruit, pomegranate, black currant and betel leaf

showed high ferric ion reducing power. The extracts showing high iron binding capacity

were: sea buckthorn leaf, radish, parsnip, betel leaf and mangosteen. Betel leaf extract

exhibited high activities with respect to scavenging of various radicals except nitric oxide,

and iron binding. Thus, this search could identify one plant material exhibiting a wide-

spectrum antioxidant activity. Mangosteen that is less active against SOA radical, and, blue

berry leaf that less active against H2O2 scavenging and iron chelating activities could also

be considered wide spectrum active materials to a lesser extent. This study also suggests

that antioxidant activity of a substance, determined by one or more related assays, does not

give the complete picture of its effectiveness against various species of oxygen radicals;

and emphasizes that determination of the spectrum of the anti-radical activity would be

necessary.

The fourth part of this work evaluated the antimicrobial activity of selected plant extracts in

detail by turbidimetric methods in liquid medium, and by well-diffusion method in gel

medium. The antimicrobial activity was expressed in the former by growth inhibition,

216

minimum inhibitory concentration - MIC and by a new expression, the antimicrobial index

– AMI; and in the latter, expressed by zone of inhibition. AMI takes into account all the

three growth phases of the bacteria. Although the turbidimetric methods were in good

agreement in the assessment of the activity of the substances, the new expression, AMI,

appears to be more meaningful since it carries the information regarding interaction

between the substance and the microorganism and the growth rate in the presence of that

substance. In addition, AMI demarcates the activity of samples, even those found to be

highly active. Furthermore, the spectrum of antimicrobial activity of selected plant extracts

was also examined. Only a few extracts showed some broad spectrum in their activities. In

effect, only betel leaf extract showed a broad spectrum of antimicrobial activity against

bacteria, yeasts and fungi; whereas pomegranate and sea buckthorn fruit extracts appear to

be effective against both bacteria and yeasts. Black currant extract is effectively an

antibacterial substance.

The final objective focused on the exploration of ways to enhance the antimicrobial activity

of selected antioxidant-antimicrobial extracts. The result shows that hot water can be used

for solvent extraction of antioxidant-antimicrobials from plant materials, albeit a polar

solvent that extracts phenolic substances preferably and only modestly non-polar

substances such as terpenoids. However, considering the yield of the extracts and the

activity, water appears to be appropriate, and it can be considered effective for fruit sources

that are rich in phenolic substances. Nonetheless, other selective solvents must be

considered for extraction of active non-polar substances for plant sources such as leaves.

Some enhancement in antimicrobial activity was possible by either mixing plant extracts or

by the addition of plant compounds. It was also observed that the composition of blends or

mixtures might be important, since synergistic or antagonistic interactions occur at certain

proportions. Pomegranate-black currant extract blends and green tea-cashew apple extract

blends showed enhancement in the activity. The addition of methyl glyoxal and mono-

caprin also showed enhancement in the activity of plant extracts. Methyl glyoxal at 25 %

(w/w) addition improved the activity of pomegranate and black currant extracts. Although

some improvement in the antimicrobial activity could be achieved for the selected extracts,

concentration of the extract required for effective control of microorganisms may not be a

very promising by these approaches.

217

Overall, this work explored the potential of extracts of fruit and vegetable by-products as

anti-oxidant-antimicrobials. Some plant extracts having potential as antioxidant-

antimicrobial agents. They include: olive, cranberry, noni, betel leaf, black currant,

pomegranate, lemon grass, spinach, green grape (wine), black currant (residue), egg plant,

rambutan, Indian plum, cranberry leaf, rosemary leaf, grape leaf (wild), green tea,

mangosteen and raspberry leaf. However, the list is reduced to a few (betel leaf,

pomegranate, black currant residue), should broad antioxidant and antimicrobial activities

are taken into account. In addition, this study introduces new expressions for antioxidant

(ARP) and antimicrobial (AMI) activities of complex mixtures such as plant extracts, and

they can be useful in the screening of plant materials for these activities. Another lesson

from this study is that the enhancement of antimicrobial activity of plant extracts may not

be possible merely by simple mixing of extracts, because of potential interactions between

the components of the extracts. The knowledge of the phytochemical composition is

essential to understand such interactions, in the selection of mixtures and to determine

possible approaches to enhance the antimicrobial activity.

Despite the huge number of studies concerning the antioxidant activity of fruits and

vegetables, their role in human health are objects of many studies in the past decades, some

important scientific issues are still rarely addressed. For example, current studies are

mainly focused on revealing the antioxidant potency of crude plant extracts. But, the

specificity of these phytochemicals towards specific radicals such as peroxy, superoxide,

singlet oxygen, hydroxyl radicals, and others is rarely explored. However, plant extract is a

complex system, where phytochemicals may individually function in synergism or

antagonism with each other. This study shows that antioxidant activity of a substance,

determined by one or more related assays, does not give the complete picture of its

effectiveness against various species of oxygen radicals in biology, and therefore,

determination of the spectrum of the anti-radical activity would be necessary.

218

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233

ANNEXES

234

Annexe 1. Yields of extracts from plant materials (Chapter 1)

No. Plant material Yield

(g dry matter/100g FW)

1 Alpha-alpha sprouts 2.1

2 Anise leaf 3.6

3 Apple 3.9

4 Artichoke base 3.4

5 Artichoke petal 2.5

6 Asparagus 2.8

7 Avocado 3.4

8 Banana peel 3.9

9 Beet root UV-C treated 6.5

10 Beetroot 6.2

11 Beetroot leaf 3.4

12 Betel leaf 1.7

13 Bitter gourd 2.8

14 Bitter melon peel 4.8

15 Black currant fruit 4.8

16 Black currant leaf 4.2

17 Black currant residue 6.1

18 Black figs 5.8

19 Black grape 37.1

20 Black radish 5.1

21 Black radish purple 12.1

22 Blueberry fruit 5.2

23 Blueberry leaf 10.2

24 Blueberry UV-C treated 6.8

25 Bok Choy 7.1

26 Broccoli leaf 6.3

27 Broccoli florets 5.3

28 Brussel sprouts 19.0

29 Cabbage 7.9

30 Cactus peel 5.6

31 Cantaloupe peel 6.2

32 Cape gooseberry 9.2

33 Carrot 7.0

34 Carrot leaf 4.8

35 Carrot peel 6.8

36 Carrot UV-C treated 7.0

235

Annexe 1. Yields of extracts from plant materials (Chapter 1) (continued)



37 Celery 24.7

38 Celery root (Rave) 4.7

39 Celery UV-C treated 24.5

40 Celery oriental 11.5

41 Cherry 12.9

42 Chicory lettuce 3.5

43 Chinese lychi 7.8

44 Coriander 3.7

45 Cranberry fruit 1.3

46 Cranberry leaf 13.1

47 Cress 17.6

48 Cucumber 4.2

49 Cucumber (Canada) 5.2

50 Curry leaf 6.4

51 Custard apple 17.1

52 Dandelion salad 6.8

53 Dandelion 7.8

54 Dandelion root 28.4

55 Dandelion root UV-C treated 25.8

56 Date 32.9

57 Dill 3.6

58 Drum sticks 4.3

59 Endive 6.2

60 Fenugreek leaf 2.9

61 Curly lectuce (Frisse) 2.1

62 Gac fruit peel 3.4

63 Gac fruit (purple) 6.7

64 Grape leaf 3.8

65 Green almond 4.2

66 Green grape 35.5

67 Green pepper 16.7

68 Hops 5.8

69 Horse radish 7.2

70 Indian summer fruit 2.5

71 Indian summer leaf 12.9

72 Italia green pepper 2.2

73 Ivy Gourd 5.6

74 Jewish Mallow 2.5

236




75 Kantula 2.1

76 Kiwi peel 5.3

77 Kohlrabi 6.5

78 Leafy Kale 3.2

79 Leek 13.1

80 Lemon 3.9

81 Lemon (albido) 5.1

82 Lemon (flavido) 4.8

83 Lime 18

84 Mango 4.2

85 Mangosteen 8.7

86 Molasses- Date 80.1

87 Molasses-Carob 77.7

88 Nicoise 1.1

89 Noni juice 5.4

90 Orange 7.2

91 Orange (albido) 21.9

92 Orange (flavido) 25

93 Oregano 5.6

94 Parsley leaf 4.1

95 Parsnip 2.1

96 Parsnip leaf 6.6

97 Parsnip peel 1.8

98 Pineapple peel 6.1

99 Pineapple leaf 6.7

100 Piper lolot 3.9

101 Pomegranate UV-C treated 10.3

102 Pomelo 5.4

103 Pomegranate 7.2

104 Potato leaf 6.5

105 Potato (mature) 4.6

106 Potato peel 6.0

107 Pumpkin 5.3

108 Radish leaf 2.1

109 Radish peal 3.2

110 Radish purle 12.3

111 Rainbow chard 2.8

112 Rambutan 7.3

237




113 Rapini 10.1

114 Raspberry leaf 11.1

115 Raw mango 1.1

116 Raw papaya 3.1

117 Red grape 20.2

118 Red grape UV-C treated 21.4

119 Red onion 10.3

120 Rhubarb 4.5

121 Rutabaga 6.4

122 Roquette leaf 3.4

123 Rosemary leaf 5.0

124 Sorrel 2.7

125 Spinach leaf 3.2

126 Strawberry leaf 4.2

127 Swiss chard 3.2

128 Taro leaf 2.7

129 Taxus Canadensis 3.3

130 Tinda 2.2

131 Tomato 4.6

132 Tomato leaf 5.3

133 Tapioca 4.0

134 Water melon 5.4

238

Annexe 2. Numerical data for figures in Chapter 6

Table A.6.1 Total phytochemical content antioxidant-antimicrobial activities of

pomegranate (for Figure 6.1)

Class of

compound (mg/g dry sample)

Fraction

Hex HE-AE AC-AC ME-ET ME-H2O H2O HH2O

Total polyphenolics 181.82 ± 21.77 216.59 ± 37.64 612.02 ± 87.84 649.18 ± 94.41 726.86 ± 49.29 660.02 ± 54.14 812.03 ± 83.76

Flavonoids 2.11±0.29 2.07±0.05 2.66±0.32 2.79±0.55 2.72±0.54 2.47±0.42 3.39±1.77

Tannins 74.81±2.15 54.02±3.12 136.49±5.14 32.71±2.47 26.38±1.25 24.49±0.98 24.01±1.58

Proanthocyanidins 17.52±7.87 7.88±0.99 46.21±8.94 15.47±2.21 12.95±2.81 10.1±2.79 12.16±3.98

Alkaloids 0.78 ± 0.05 0.55 ± 0.01 5.12 ± 0.13 0.22 ± 0.02 0.52 ± 0.03 0.99 ± 0.06 2.35 ± 0.09

Terpenoids 4.31 ± 0.12 11.05 ± 0.96 0.12 ± 0.00 1.34 ± 0.04 0.29 ± 0.00 0.76 ± 0.01 0.89 ± 0.04

Total yield (%) 7.47±0.13 7.08±0.08 8.81±0.1 35.51±0.64 45.22±1.97 29.81±2.21 52.11±2.83

Table A.6.2 Total phytochemical content antimicrobial antioxidant activity of black currant

residue

Class of compound

(mg/g dry sample)

Fraction



Flavonoids 44.52±4.18 28.88±3.49 96.2±8.35 128.6±8.57 260.36±12.27 170.16±12.6 175.04±12.53

Tannins 15.95±2 26.32±1.03 53.37±4.01 163.09±7.65 234.35±9.62 169.02±11.68 279.09±22.65


Alkaloids 1.23 0.78 0.11 0.23 0 0.13 0.58

Terpenoids 0.45 0.22 0.23 0 0 0 0.12

Total yield (%) 6.68±0.26 7.29±0.37 9.89±0.55 24.57±0.6 26.17±0.74 24.57±1.26 32.53±1.19

Table A.6.3 Total phytochemical content antimicrobial antioxidant activity of betel leaf

Class of compound

(mg/g dry sample)

Fraction



Flavonoids 10.71±4.98 10.21±3.18 14.29±8.44 16.52±3.94 18.6±0.69 7.79±1.06 11.36±3

Tannins 38.55±3.25 45.06±2.89 100.48±5.98 100.3±4.32 136.4±4.58 58.76±7.12 103.93±5.01


Alkaloids 36.56 ± 1.34 23.21 ± 0.98 29.45 ± 2.88 11.34 ± 0.56 10.99 ± 0.34 2.99 ± 0.11 3.23 ± 0.39

Terpenoids 83.45 ± 2.11 80.1 ± 4.32 35.66 ± 1.23 27.64 ± 2.01 17.61 ± 0.33 3.02 ± 0.12 5.64 ± 0.67

Total yield (%) 14.29±0.76 16.23±0.39 13.88±0.25 19.28±0.55 27.37±1.66 29.44±0.41 46.97±2.01

239

Table A.6.4 Antioxidant (AO)-antimicrobial (AM) activity of extracts (for Figures 6.2-6.4)

Sample

Activity

Fraction


Pomegranate

AO 67.1±2.21 64.54±4.02 74.05±1.56 73.04±2.14 72.89±1.98 74.05±3.7 71.02±4.15

AM 73.96±2.7 68.45±1.42 85.03±4.56 75.7±4.58 71.1±4.35 68.52±2.43 56.66±3.21

Black currant

residue

AO 23.73±1.18 22.35±2.54 58.76±4.73 72.12±1.93 74.42±5.67 76.73±3.4 74.65±3.28

AM 45.69±2.1 50.51±3.6 60.84±1.53 65.35±2.02 73.96±3.08 63.19±1.63 67.07±3.79

Betel leaf

AO 71.44±3.96 70.74±2.54 72.2±4.61 73.76±5.73 74.86±1.74 70.75±3.63 69.56±2.22

AM 79.96±2.7 82.94±2.9 73.33±4.67 67.83±1.39 77.92±5.15 66.64±4.33 69.6±5.48

Table A.6.5 Antibacterial activity against E.coli with varying proportions blends of plant

extracts at total concentration of 5.0 mg/Ml (for Figure 6.5)

Extract (1) Extract (2) Composition, extract (1), (w/w)

Inhibition (%)

Pomegranate Black currant

0.0 76.02

0.25 49.23

0.50 67.04

0.75 95.69

1.00 90.95

Betel leaf Black currant

0.0 79.5

0.25 88.81

0.50 100

0.75 100

1.00 86.82

Pomegranate Betel leaf

0.0 95.81

0.25 87.32

0.50 87.15

0.75 86.45

1.00 93.26

Green tea Cashew

0.0 47.67

0.25 55.78

0.50 82.39

0.75 86.34

1.00 56.24

240

Table A.6.6 Antibacterial activity against B. subtilis with varying proportions blends of

plant extracts at total concentration of 5.0 mg/mL (for Figure 6.6)

Extract (1) Extract (2) Composition, extract (1) % v/v

Inhibition (%)

Pomegrenate Betel leaf

0.0 86.22

0.25 84.73

0.50 87.58

0.75 81.34

1.00 88.43

Green tea Cashew

0.0 30.45

0.25 55.59

0.50 90.02

0.75 87.98

1.00 45.67

Table A.6.7 The MIC against E coli of the mixing of 2 plant extracts and 2 solutions (for

Figure 6.7)

Plant extract Plant compound Composition, Plant extract, % w/w

MIC

Black currant Methyl glyoxal

0.0 1.25

0.25 1.25

0.50 2.5

0.75 2.5

1.00 10

Black currant Mono-caprin

0.0 1.25

0.25 5

0.50 5

0.75 10

1.00 10

Pomegranate Methyl glyoxal

0.0 1.25

0.25 2.5

0.50 2.5

0.75 5

1.00 5

Pomegranate Mono-caprin

0.0 1.25

0.25 5

0.50 5

0.75 5

1.00 5

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