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LWT 40 (2007) 1246–1252

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Antioxidative effect of compounds isolated from Globularia alypum L.structure–activity relationship

Nour-Eddine Es-Safia,b,�, Albert Kollmanna, Samira Khlific, Paul-Henri Ducrota

aUnite de Phytopharmacie et Mediateurs Chimiques, INRA, Route de Saint-Cyr., 78026 Versailles Cedex, FrancebLaboratoire de Chimie Organique et d’Etudes Physico-Chimiques, Pole de Competences Pharmacochimie, Ecole Normale Superieure,

B.P. 5118 Takaddoum Rabat, MoroccocLaboratoire de Biochimie Appliquee et Biotechnologie, Faculte des Sciences, B.P. 20 El Jadida, Morocco

Received 3 January 2006; received in revised form 24 August 2006; accepted 31 August 2006

Abstract

The antioxidant activity of the Globularia alypum phytochemicals were evaluated for their capacity to scavenge the 1,1-diphenyl-2-

dipicrylhydrazyl (DPPH1) free radical and some structure–activity relationships were obtained. Assay guided fractionation led to the

isolation of syringin, four phenylethanoids, four flavonoids and six iridoids as the main constituents of the extract and their antioxidant

activity was determined. The obtained results showed that the activity towards the DPPH1 free radical was mainly due to the flavonoid

and phenyl ethanoid constituents which were most active free radical scavengers than iridoids. Among the tested flavonoids,

6-hydroxyluteolin glycosides showed the strongest activity, suggesting that the presence of the 6-hydroxyl group was a favourable

structural feature of flavonoids with regard to DPPH1 scavenging effect. The isolated phenylethanoid glycosides all showed potent

antioxidant activity and their capacity to scavenge free DPPH1 radical was greater than BHT. Their high antioxidant activity could be

attributed to the caffeoyl moieties contained in them, while iridoids showed moderate free radical scavenging activity. The obtained

results demonstrated that some of the isolated compounds play an important role for the antioxidant activity of G. alypum and give a

scientific basis to the use of this plant in traditional medicine. The hydromethanolic extract of G. alypum could thus be considered as a

source of potential antioxidants and will promote the reasonable usage of this plant in food technology and processing as well as for

medical use.

r 2006 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved.

Keywords: Antioxidant activity; Radical scavenging; Flavonoids; Phenylethanoids; Iridoids; DPPH1; Globularia alypum; Globulariaceae

1. Introduction

Free radicals are charged and highly reactive specieswhich are made of unstable molecules or atoms due to theirsingle and unbalanced electrons. The common free radicalsare oxygen-reactive species (ROS), which are formednaturally, both internally by metabolism and externallyby chemicals. ROS are unstable and through chain reaction

0 r 2006 Swiss Society of Food Science and Technology. Pu

t.2006.08.019

ing author. Laboratoire de Chimie Organique et d’Etudes

ues, Pole de Competences Pharmacochimie, Ecole

ieure, B.P. 5118 Takaddoum Rabat, Morocco.

5 22 61; fax: +21237 75 00 47.

ess: nouressafi@yahoo.fr (N.-E. Es-Safi).

can attack vital biomolecules (DNA, lipids, proteins) incells and body fluids (Diplock et al., 1998). ROS can alsoaffect the quality of foods which are rich in polyunsatu-rated fatty acids. The latter are inherently unstable tooxidation, reducing thus the nutritional content of foodsand promoting the development of food rancidity and off-flavours (Ladikos & Lougovois, 1990). In particular, lipidperoxidation is a toxic reaction that commonly occursin food via organoleptic deterioration during processing,distribution, and later storage stages. Therefore, theprevention of such adverse reactions is obvious withpotential financial and nutritional gains to be attained forthe food chemistry. All of these considerations indicate apossible useful role for antioxidants which are a class of

blished by Elsevier Ltd. All rights reserved.

ARTICLE IN PRESSN.-E. Es-Safi et al. / LWT 40 (2007) 1246–1252 1247

bioactive nutrients that reduce the incidence of theseeffects. In the food industry, they have been widely usedas food additives to provide a number of benefits throughprotection against oxidative degradation of foods andcosmetics. Spices used in different types of food to improveflavours, since ancient time, are well known for theirantioxidant properties (Madsen & Bertelsen, 1995). Theiraddition to food is an effective way to prevent thedevelopment of various off-flavours and undesirablecompounds that result from lipid oxidation.

Both natural and synthetic antioxidants have beenshown to enhance product stability, quality, and shelf life.Many research works have mentioned the disadvantage ofsynthetic antioxidants, and their possible injurious proper-ties for human health in addition to their possible toxicityas well as general consumer rejection led to decreasing useof synthetic antioxidants such BHA or BHT which werereported to have some side effects (Branien, 1975;Ito, Fukushima, Hassegawa, Shibata, & Ogiso, 1983;Linderschmidt, Trylka, Goad, & Witschi.,1986; Namiki,1990; Tappel, 1995). Consequently, the development ofalternative antioxidants from natural origin has attractedconsiderable attention and many researchers have focusedon the discovery of new natural antioxidants aimed atquenching biologically harmful radicals (Aligiannis et al.,2003; Cuendet, Hostettmann, & Potterat, 1997; Lu & Foo,2001; Schwarz et al., 2001; Vagi et al., 2005). Therefore, weinitiated a systematic investigation of some Moroccanmedicinal plants for their potential antioxidant activity.

In the course of this research programme, we found thatthe hydro-methanolic extract of Globularia alypum L.(Globulariaceae) aerial parts showed significant antioxida-tive activity. The plant is a perennial shrub foundthroughout the Mediterranean area, and is known for itsuses in the indigenous system of medicine for a varietyof purposes such as hypoglycaemic agent, laxative,cholagogue, stomachic, purgative, sudorific and also inthe treatment of cardio-vascular and renal diseases(Bellakhdar, Claisse, Fleurentin, & Younos, 1991). Activ-ity-guided fractionation of the MeOH fraction ofG. alypum led to the isolation of flavonoids, phenyletha-noids and iridoids (Es-Safi et al., 2005, 2006). In the presentstudy, we investigate the antioxidative activity of thesecompounds against 2,2-diphenyl-1-picrylhydrazyl free ra-dical. Free radicals are known to be a major factor inbiological damages, and DPPH1 has been used to evaluatethe free radical-scavenging activity of natural antioxidants(Bondet, Brand-Williams, & Berset, 1997; Sanchez-Moreno, Larrauri, & Saura-Calixto, 1998). DPPH1 whichis a radical with a purple colour, changes into a stableyellow compound by reacting with an antioxidant, and theextent of the reaction depends on the hydrogen donatingability of the antioxidant (Bondet et al., 1997). Because ofthe ease and convenience of this reaction, it now haswidespread use in the free radical-scavenging activityassessment (Brand-Williams, Cuvelier, & Berset, 1995;Sanchez-Moreno et al., 1998).

2. Materials and methods

2.1. General experimental procedures

UV-visible spectra were recorded using a KontronUvikon 930 spectrophotometer fitted with a quartz cell.Column chromatography was performed on a SPE columnusing a mixture of MeOH/H2O (0/100–100/0) as eluent.Analytical HPLC was performed on a Kromasil reversed-phase C18 5 mm (250� 4.6mm) column using a Varianapparatus including a 9012 solvent delivery system, a 9100autosampler, and a 9065 polychrom diode array detector.Semi-preparative HPLC was performed on a Kromasil C18

10 mm column (250� 20mm) and using an apparatusincluding a Millipore Waters 600 Multisolvent Deliverysystem, a Waters U6K manual injector and a TSP-UV200Dual-Wavelength UV/visible programmable detector.

2.2. Extraction and isolation

G. alypum L. was collected from Taza region, Moroccoin April 2003. Taxonomic identification was performed byDr. R. Tellal, Department of Biology, University of ElJadida, Morocco. A voucher specimen (KS2) has beendeposited in the Herbarium of the Department of Biology,Faculty of Sciences, Hassan II University, El Jadida,Morocco.The fresh aerial parts were air-dried in shade at room

temperature and powdered. From the obtained powder,100 g were macerated during 48 h at room temperature with500ml of mixture of distilled water-methanol (3/2). Thecrude preparation was filtered and concentrated underreduced pressure to provide a crude extract which wasstored at �20 1C until use. This crude extract was dissolvedin water and the obtained aqueous phase was furtherextracted with hexane and the aqueous phase subjected toextraction through a SPE column. Elution was performedsuccessively by H2O, MeOH 10%, MeOH 40%, MeOH50%, MeOH 70% and MeOH 100%. The MeOH 50%fraction, which was shown to exhibit the most potentantioxidant activity was thereafter explored by semipre-parative HPLC using a C18 10 mm column and eluted withthe following solvents A: acetonitrile and B: water with0.5% acetic acid eluting from 5% to 30% A in 120minfollowed by a washing and a reconditioning of the column.After several successive injections, samples correspondingto the same chromatographic peaks were controlled byanalytical HPLC, concentrated under reduced pressure andlyophilized. The structures of the isolated compounds wereelucidated through spectroscopic methods as previouslydescribed (Es-Safi et al., 2005, 2006).

2.3. Reduction of 2,2-Diphenyl-1-picrylhydrazyl (DPPH1)

radical

The antioxidant activity of the isolated compounds wasevaluated through specrophotometric technique according

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to the method previously reported by Bors, Saran, andElstner (1992). Briefly, 50 ml of a methanolic solutioncontaining the compound to be tested were added to 5mlof a 0.004% MeOH solution of DPPH1. The studiedcompounds were tested with MeOH as control and BHTand quercetin as antioxidant references and absorbance at517 nm was determined after 30min. The absorbance (A) ofthe control and samples was measured, and the DPPH1

scavenging activity in percentage was determined as follow:

DPPH� scavenging activity ð%Þ

¼ ½ðAcontrol � AsampleÞ=Acontrol� � 100.

The data are presented as mean of triplicate and theconcentration required for a 50% reduction (IC50) ofDPPH1 radical was determined graphically.

3. Results and discussion

3.1. Antioxidant activity of the extracts

In continuation of our work on the search for newnatural antioxidants, we sequentially extracted the aerialparts of G. alypum with various solvent mixtures, and thereduction of DPPH1 was used to evaluate the antioxidantactivity of the obtained extracts. The results showed thatthe water–methanol (60/40) extract demonstrated a notableantioxidant activity. From its further fractionation on aSPE column, we get the methanol 50% fraction aspresented the strongest efficiency. In the present work, weinvestigated for the phytochemical composition of thisfraction. Analysis through analytical HPLC showed thepresence of several compounds belonging to differentfamilies as demonstrated by their UV-visible spectraobtained through the used DAD detector coupled to theHPLC apparatus. A typical HPLC chromatogram is shownin Fig. 1. Isolation of these compounds was achievedthrough successive injections on the semipreparativeHPLC apparatus. The obtained fractions were controlled

0

0.1

0 10 20 30 40

Time (min.)

AU I5

I6

I4

P4

SF1 F2

F3

I1

I3

P1

P2

P3

F4

I2

Fig. 1. Analytical HPLC chromatogram recorded at 280 nm of the 50%

methanolic fraction showing the presence of various compound families

(S ¼ syringin, F ¼ flavonoids, I ¼ iridoids, P ¼ phenylethanoids).

for their purity by analytical HPLC and pure fractions werefreeze-dried and their structures elucidated throughESI–MS, CID MS, tandem MS–MS, 1D and 2D homo-nuclear and heteronuclear NMR analysis (Es-Safi et al.,2005, 2006). The phytochemicals isolated from G. alypum

include flavonoids F1–F4, iridoids I1–I6 and phenyletha-noids P1–P4 in addition to syringin (S) (Fig. 2).

3.2. Antioxidant activity of the isolated compounds

The isolated compounds which were obtained with agrade of purity above 95%, were tested for theirantioxidant scavenging effects on DPPH1 radical and theiractivity was compared to the synthetic antioxidant BHTand quercetin used as antioxidant references. The resultsobtained at a concentration of 10 mmol/l are given in Fig. 3and expressed as the percentage of the remaining DPPH1.It showed that flavonoids and phenylethanoids exhibitedmuch stronger DPPH1 radical scavenging activities thaniridoids and are as effective as quercetin and they are allsignificantly better than BHT.While the antioxidative activity of natural compounds is

widely described; little information is reported on theirkinetic behaviour in the oxidation process. This representshowever an important factor in the antioxidative activityprocess. In terms of reaction kinetics Yen and Duh (1994)postulated that the more rapidly the absorbance decreased,the more potent were the antioxidant activity of thesample. Halliwell (1990) reported that the antioxidantpower results first from the capacity to preventthe autoxidation of free radical-mediated oxidation of thesubstrate in low concentration and second, that theresulting radical after scavenging must be stable. Fig. 3also showed the evolution of the remaining DPPH1 withtime of each compounds family. It showed that thecompounds have a slow kinetic behaviour, thus the rankof efficiency is quite the same at any time. This promptedus to express the scavenging activity by the Percentage ofDPPH1 reduction after only 30min of reaction, as it is lesstime consuming than waiting for the plateau.Fig. 4 showed the DPPH1 free radical scavenging activity

of each compounds family at different concentrations anddemonstrated that all the tested fractions showed a notlinear dose-dependant increase in activity. The free radicalscavenging activity is usually expressed as percentage ofDPPH1 inhibition but also by the antioxidant concentra-tion required for a 50% DPPH1 reduction (IC50). Theobtained results with the isolated compounds are summar-ized in Fig. 5. The results show that F1 (6.6), F2 (7.1) and F3

(7.8) were the most potent of all the compounds, near ofquercetin (7.8). F3 (12.2) and the four phenylethanoids P

(11.8–15.5) also showed strong activity compared to BHT(30). Finally, iridoids exhibit moderate activity, similar toBHT for I1 (28.2) and I2 (29.0) while I3, I4, I5 and I6 werepoor antioxidants, as syringin, derivatives I1–I6 exhibitedmoderate to weak DPPH1-scavenging activities comparedto the tested flavonoids and phenylethanoids.

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OH

OCH3

H3CO

O

O

OH

OH

OH

R1

OHO

HOOH

O

OH

S

R2O

6'

5'

4'3'

2'

1'2

3456

78

9

10

2

131'

4'

1"

O

O

OH

OH

OH

O

HO

O

O

OH

O

HOHO

OH

HO

HO

F4

2

36

71'

1"

1"'

2"

OH

OH

O

R1O

OH

O

OR3

R2O

P1:R1= H, R3= Rhamnosyl, R2= E-Caffeoyl

P2:R1= Rhamnosyl,R3= H, R2= E-Caffeoyl

P3:R1= Rhamnosyl,R2=H, R3= E-Caffeoyl

P4:R1=R2=H, R3= E-p-Coumaroyl

12

3

45

6

1'2'

3'

4'5'

6'

F1:R1= OH, R2= Glucosyl-(1→3)-glucosyl

F2:R1= OH, R2= Glucosyl

F3:R1= H, R2= Glucosyl-(1→2)-glucosyl

ROOH

OH

O O

I4

1

34

5

98

7

6

101'

2'

3'

4'5'

6'

34

5

98

7

6

O

HO

O

OH

HO

O

O

OH

OH

O O

O

HO

O

OH

HO

O

O

OH

OH

O O

O

HO

OH

HO

R1

R2

I3:R= Z-Cinnamoyl

I5:R= E-Cinnamoyl

I1:R1= OH, R2= OH

OH

OH

I2:R1= OH, R2=

I6:R1= Cl, R2=

α

β1'

2'

1"

5

9

α

α

β

β

1"4" 1'

1

1

34

2'

4"

4

98

10

10

7

Fig. 2. Structures of the compounds isolated from the hydro-methanolic extract of G. alypum (S ¼ syringin, F ¼ flavonoids, I ¼ iridoids,

P ¼ phenylethanoids).

N.-E. Es-Safi et al. / LWT 40 (2007) 1246–1252 1249

3.3. Structure–activity relationship

Many attempts have been reported in the literatureto explain the structure–activity relationship of somenatural antioxidant compounds. It has been reportedthat the antioxidant activity of phenolic compounds mayresult from the neutralization of free radicals initiatingoxidation processes, or from the termination of radicalchain reactions, due to their hydrogen donating ability(Baumann, Wurn, & Bruchalausen, 1979). It was alsoproposed that the higher antioxidant activity is related tothe greater number of hydroxyl groups on the flavonoid

nucleus (Cao, Sofic, & Prior, 1997). Foti, Piattelli, Baratta,and Ruberto (1996) proposed that the antioxidant activityof flavonoids was dependent on the presence of orthophenolic functions. These hypotheses can explain why F1,F2 and F3 with, respectively, 4, 4 and 3 OH groups showedthe strongest activity. This finding is in accordance withthe results reported by Bors, Heller, Michel, and Saran(1990) and confirms that the o-dihydroxybenzene (cate-chol) structure is an important feature for enhancedradical-scavenging activity. The flavonoid hydroxy-luteolinderivatives (F1, F2) showed stronger free radical-scavengingactivity due to the presence of two o-dihydroxyl groups

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0

20

40

60

80

100

0 20 40 60 80 100 120

Rem

aini

ng D

PPH

(%

)

F1 F2 F3

F4 BHT Q

0

20

40

60

80

100

0 20 40 60 80 100 120

Rem

aini

ng D

PPH

(%

)

P1 P2 P3

P4 BHT Q

0

20

40

60

80

100

0 20 40 60 80 100 120

Time (min)

Rem

aini

ng D

PPH

(%

)

I1 I2 I3 I4I5 I6 BHT Q

Fig. 3. Remaining DPPH1 as a function of reaction time for flavonoids

(top), phenylethanoids (middle) and iridoids (bottom) isolated from

G.alypum and tested at a concentration of 10mmol/l (F ¼ flavonoids,

I ¼ iridoids, P ¼ phenylethanoids, Q ¼ quercetin, BHT ¼ butylated

hydroxytoluene).

0

20

40

60

80

100

0 20 40 60 80 100

DPP

H R

educ

tion

(%)

F1 F2 F3

F4 BHT Q

0

20

40

60

80

100

0 20 40 60 80 100

DPP

H R

educ

tion

(%)

P1 P2 P3

P4 BHT Q

0

20

40

60

80

100

0 20 40 60 80 100

Concentration (μM)

DPP

H R

educ

tion

(%)

I1 I2 I3 I4

I5 I6 BHT Q

Fig. 4. DPPH1 reduction measured after 30min of reaction as a function

of concentration of flavonoids (top), phenylethanoids (middle) and

iridoids (bottom) isolated from G.alypum (F ¼ flavonoids, I ¼ iridoids,

P ¼ phenylethanoids, Q ¼ quercetin, BHT ¼ butylated hydroxytoluene).

N.-E. Es-Safi et al. / LWT 40 (2007) 1246–12521250

(one in the ring B and the other in the A ring) which areimportant for radical scavenging activity (Rice-Evans,Miller, & Paganga, 1996). It is noteworthy to mentionthat the flavonoids F1 and F2 were more antioxidant thanquercetin which is widely recognized as a potent antiox-idant. The enhanced antioxidant activity observed incompounds F1 and F2 is probably due to the additionalhydoxy group in ring A which significantly improvesthe radical-scavenging activity as compared to luteolin7-diglycoside F3. This indicated that the hydroxyl group in

position 6 of ring A could increase the antioxidantcapability of parent flavonoids. So it is obvious that forflavones with 5,6-dihydroxyl substitution in ring A andwith a free 30, 40 hydroxyl groups, a more importantantioxidant activity would be expected in agreement withpreviously reported data (Lu & Foo, 2001). In other words,the greater the number of hydroxyl groups, the greater theradical scavenging potency of flavonoids which is increased

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6.6 7.1 7.812.2 11.8 12.1 12.2

15.5

28.2 29.0

7.8

30.0

70.0

76.0

66.0

44.7

85.0

F1 F2 F3 F4 P1 P2 P3 P4 I1 I2 I3 I4 I5 I6 S Q BHT

Compounds

Fig. 5. Free radical scavenging activity of the isolated compounds

measured after 30min of rea. The results represent the concentration

IC50 (mmol/l) needed to decrease by 50% the initial DPPH1 absorption at

517 nm (S ¼ syringin, F ¼ flavonoids, I ¼ iridoids, P ¼ phenylethanoids,

Q ¼ quercetin, BHT ¼ butylated hydroxytoluene).

N.-E. Es-Safi et al. / LWT 40 (2007) 1246–1252 1251

by the existence of the 6-hydroxyl group. In the opposite towhat was reported (Cotelle, 2001; Rusak, Gutzeit, &Muller, 2005) our findings pointed out that the C-6hydroxyl group is favourable structural feature regardingthe antioxidative effects of flavonoids supporting pre-viously published data (Lu & Foo, 2001).

The difference between compounds F2 and F1 is that thelast one had an additional glucoside moiety at the sevenposition. It appears that glycosylation slightly influence theantioxidant activity of the host molecule as previouslyreported (Plumb, De Pascual-Teresa, Santos-Buelga,Cheynier, & Williamson, 1998). This influence is dependingon the glycosylation site being more pronounced for thecatechol B than the phloroglucinol A ring positions.

The luteolin glycoside F3 also showed potent antioxidantactivity due to the presence of a catechol group in ring Bwhich has better electron-donating properties in additionto a 2,3-double bond conjugated with the 4-oxo groupresponsible for electron delocalization. This result confirmsthe fact that luteolin and its glycosides fulfil all criteria forstrong radical scavenging activity (Bors et al., 1990). Theflavanone F4 exhibited less activity than the flavones due tothe lack of conjugation provided by the 2,3-double bondwith the 4-oxo group which is an important determinantfor free-radical scavenging activity. Even though, due tothe presence of the catechol system, it is not unexpectedthat this flavonoid expressed the observed radical scaven-ging activity.

The structure–activity relationship conclusions concern-ing the strong radical scavenging activity of phenyletha-noid compounds P1–P4 is consistent with the fact thatcinnamic acid derivatives have superior antioxidant activityto the benzoic acid analogues. Thus, the derivatives P1–P3

which possess a caffeoyl moiety in their structures showedsimilar and higher radical scavenging ability than P4

consistent with the chemical criteria applied to diphenolics.This is due to the presence of the conjugated unsaturation

that facilitates the delocalization of the resulting freeradicals. The presence of additional o-dihydroxy moietiesconferred higher stability to the radical form andparticipated in the electron delocalization (Pietta, 2000;Rice-Evans et al., 1996; Rice-Evans, Miller, & Paganga,1997). Compound P4 was slightly less active becausehaving the p-coumaroyl group instead of the caffeoylmoiety, the p-coumaroyl group being less efficient for theradical scavenging activity (Rice-Evans et al., 1996).The moderate radical scavenging activity of I1–I6 is due

to their poor hydrogen-donating ability. Increasing thenumber of hydroxyls, results in a more efficient radicalscavenging effect as suggested by comparing the resultsobtained for compounds I3, I4, I5 and I1, I2, I6 in agreementwith previously published data (Castro-Gamboa & Castro,2004; Harput, Saracoglu, Inoue, & Ogihara, 2002). Amongthis family the antioxidant activities decrease in thefollowing order: I1, I2, I6, I5, I4, I3. The relative highactivity of compound I1 and I2 compared to the otheriridoids is due to the presence of three hydroxyl groups intheir structures. The substitution of an hydroxyl group by achlorine atom slightly reduces the antioxidant activity asobserved in the case of compound I6. The presence of adouble bond in the iridoid skeleton did not seem toincrease the antioxidant activity. Thus compounds I3 andI5 which presents a 3,4 double bond were less antioxidantthan compound I4 which does not.

4. Conclusion

The antioxidant activity of G. alypum compoundsconsisting of four flavonoids, four phenylethanoids, sixiridoids, and syringin was evaluated for their capacity toscavenge the stable DPPH1 free radical. The resultspresented in this paper show the significant antioxidantactivity of G. alypum hydroalcoholic extract in vitro assay,where all the isolated compounds showed strong tomoderate antioxidant activity. The obtained results showedthat the flavonoids have a potent antioxidant activityfollowed by phenylethanoids, while the studied iridoidsshowed the less antioxidative activity. Within the samefamily, the antioxidative activity was found to beinfluenced by the structure of the tested compound and astructure–activity relation is discussed on the basis of theobtained results.In light of our findings, the hydroalcoholic extract of

G. alypum can be considered as a potentially active extractfor use in conditions where reactive oxygen species areimplicated. It seems plausible to assume that the studiedextract and the isolated compounds might be able to confersome health-benefiting values against oxidative damage.However, more scientific work needs to be done toestablish the usefulness of this natural extract and/or itsphytochemical compounds in the treatment of humandisease or as food additives. Clinical studies and researchregarding the bioavailability of the isolated bioactiveconstituents are still needed in order to further verify their

ARTICLE IN PRESSN.-E. Es-Safi et al. / LWT 40 (2007) 1246–12521252

antioxidant effects in in vivo conditions. This showed thatthe aspect of natural antioxidants and their use as foodadditives remains an open research area of a good interest.Given their importance in food industry, it is interesting toknow which factors can influence their use and in whichmanner this could be achieved. Questions regarding thecomposition of the extract and the contribution of eachcompound in the global antioxidant activity in addition totheir probable synergic effect must also be investigated. Itis noteworthy to mention that the possible use of thestudied extract or its phytochemicals could not beenvisaged without taking into account the toxicologicalaspect and current legislative rules, in addition to theinfluence of these compounds on the organoleptic proper-ties of food like taste, colour, odour, and stability.

References

Aligiannis, N., Mitaku, S., Tsitsa-Tsardis, E., Harvala, C., Tsaknis, I.,

Lalas, S., et al. (2003). Methanolic extract of Verbascum macrurum as a

source of natural preservatives against oxidative rancidity. Journal of

Agricultural and Food Chemistry, 51, 7308–7312.

Baumann, J., Wurn, G., & Bruchalausen, F. V. (1979). Prostaglandin

synthetase inhibiting O2-radical scavenging properties of some

flavonoids and related phenolic compounds. Naunyn-Schmiedebergs

Archives of Pharmacology, 308, R27.

Bellakhdar, J., Claisse, R., Fleurentin, J., & Younos, C. (1991). Repertory

of standard herbal drugs in the Moroccan pharmacopoeia. Journal of

Ethnopharmacology, 35, 123–143.

Bondet, V., Brand-Williams, W., & Berset, C. (1997). Kinetics and

mechanism of antioxidant activity using the DPPH1 free radical

methods. Lebensmittel–Wissenschaft und Technologie, 30, 609–615.

Bors, W., Heller, W., Michel, C., & Saran, M. (1990). Flavonoids as

antioxidants: determination of radical-scavenging efficiencies. In L.

Packer (Ed.), Methods in enzymology (pp. 343–355). San Diego, CA:

Academic Press.

Bors, W., Saran, M., & Elstner, E. F. (1992). Screening for plant

antioxidants. In H. F. Linskens, & J. F. Jackson (Eds.), Modern

methods of plant analysis. Plant toxin analysis. New series

(pp. 277–295). Berlin: Springer.

Brand-Williams, W., Cuvelier, M. E., & Berset, C. (1995). Use of a free

radical method to evaluate antioxidant activity. Lebensmittel–

Wissenschaft und Technologie, 28, 25–30.

Branien, A. L. (1975). Toxicology and biochemistry of butylated

hydroxyanisole and butylated hydroxytoluene. Journal of the American

Oil Chemists’ Society, 52, 59–63.

Cao, G., Sofic, E., & Prior, R. L. (1997). Antioxidant and prooxidant

behaviour of flavonoids: structure–activity relationships. Free Radical

Biology and Medicine, 22, 749–760.

Castro-Gamboa, I., & Castro, O. (2004). Iridoids from the aerial parts of

Verbena littoralis (Verbenaceae). Phytochemistry, 65, 2369–2372.

Cotelle, N. (2001). Role of flavonoids in oxidative stress. Current Topics in

Medicinal Chemistry, 1, 569–590.

Cuendet, M., Hostettmann, K., & Potterat, O. (1997). Iridoid glycosides

with free radical scavenging properties from Fagraea blumei. Helvetica

Chimica Acta, 80, 1144–1152.

Diplock, A. T., Charleux, J. L., Crozier-Willi, G., Kok, F. J., Rice-Evans,

C., Roberfroid, M., et al. (1998). Functional food science and defence

against reactive oxidative species. British Journal of Nutrition, 80,

S77–S112.

Es-Safi, N., Khlifi, S., Kerhoas, L., Kollmann, A., El Abbouyi, A., &

Ducrot, P. H. (2005). Antioxidant constituents of the aerial parts of

Globularia alypum growing in Morocco. Journal of Natural Products,

68, 1293–1296.

Es-Safi, N., Khlifi, S., Kerhoas, L., Kollmann, A., El Abbouyi, A., &

Ducrot, P. H. (2006). Iridoid glucosides from the aerial parts of

Globularia alypum L. (Globulariaceae). Chemical and Pharmaceutical

Bulletin, 54, 85–88.

Foti, M., Piattelli, M., Baratta, M. T., & Ruberto, G. (1996). Flavonoids,

coumarins, and cinnamic acids as antioxidants in a micellar system.

Structure–activity relationship. Journal of Agricultural and Food

Chemistry, 44, 497–501.

Halliwell, B. (1990). How to characterize a biological antioxidant. Free

Radical Research Communications, 9, 1–32.

Harput, U. S., Saracoglu, I., Inoue, M., & Ogihara, Y. (2002).

Phenylethanoid and iridoid glycosides from Veronica persica. Chemical

and Pharmaceutical Bulletin, 50, 869–871.

Ito, N., Fukushima, S., Hassegawa, A., Shibata, M., & Ogiso, T. (1983).

Carcinogenicity of butylated hydroxyanisole in F344 rats. Journal of

the National Cancer Institute, 70, 343–347.

Ladikos, D., & Lougovois, V. (1990). Lipid oxidation in muscle foods: a

review. Food Chemistry, 35, 295–314.

Linderschmidt, R., Trylka, A., Goad, M., & Witschi, H. (1986). The

effects of dietary butylated hydroxytoluene on liver and colon tumor

development in mice. Toxicology, 387, 151–160.

Lu, Y., & Foo, L. Y. (2001). Antioxidant activities of polyphenols from

sage (Salvia officinalis). Food Chemistry, 75, 197–202.

Madsen, H. L., & Bertelsen, G. (1995). Spices as antioxidants. Trends in

Food Science and Technology, 6, 271–277.

Namiki, M. (1990). Antioxidants/antimutagens in foods. CRC Critical

Reviews in Food Science and Nutrition, 29, 273–300.

Pietta, P. G. (2000). Flavonoids as antioxidants. Journal of Natural

Products, 63, 1035–1042.

Plumb, G. W., De Pascual-Teresa, S., Santos-Buelga, C., Cheynier, V., &

Williamson, G. (1998). Antioxidant properties of catechins and

proanthocyanidins: effect of polymerisation, galloylation and glyco-

sylation. Free Radical Research, 29, 351–358.

Rice-Evans, C. A., Miller, N. J., & Paganga, G. (1996). Structure-

antioxidant activity relationships of flavonoids and phenolic acids.

Free Radical Biology and Medicine, 20, 933–956.

Rice-Evans, C. A., Miller, N. J., & Paganga, G. (1997). Antioxidant

properties of phenolic compounds. Trends in Plant Science, 2, 152–159.

Rusak, G., Gutzeit, H. O., & Muller, J. L. (2005). Structurally related

flavonoids with antioxidative properties diffentially affect cycle

progression and apoptosis of human acute leukemia cells. Nutrition

Research, 25, 141–153.

Sanchez-Moreno, C., Larrauri, J. A., & Saura-Calixto, F. (1998).

A procedure to measure the antiradical efficiency of polyphenols.

Journal of the Science of Food and Agriculture, 76, 270–276.

Schwarz, K., Bertelsen, G., Nissen, L. R., Gardner, P. T., Heinonen, M. I.,

Hopia, A., et al. (2001). Investigation of plant extracts for the

protection of processed foods against lipid oxidation. Comparison of

antioxidant assays based on radical scavenging, lipid oxidation and

analysis of the principal antioxidant compounds. European Food

Research and Technology, 212, 319–328.

Tappel, A. (1995). Antioxidants protection against peroxidation. Interna-

tional News on Fats, Oils and Related Materials, 6, 780–783.

Vagi, E., Rapavi, E., Hadolin, M., Vasarhelyine Peredi, K., Balazs, A.,

Blazovics, A., et al. (2005). Phenolic and triterpenoid antioxidants

from Origanum majorana L. herb and extracts obtained with different

solvents. Journal of Agricultural and Food Chemistry, 53, 17–21.

Yen, G. C., & Duh, P. D. (1994). Scavenging effect of methanolic extracts

of peanut hulls on free-radical and active-oxygen species. Journal of

Agricultural and Food Chemistry, 42, 629–632.