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BIOACTIVITY GUIDED ASSESSMENT OT' SOME SELECTED MEDICINAL PLANTS OF MALAKAND DIVISION Department of Botany Facult5r of Sciences Pir Mehr Ali Shah Arid Agriculture University Rawalpindi GUL RAHIM l0-arid-72 Pakistan 2014 {F&f, + .+ -, t t99a 1
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Transcript of {F&f, - Pakistan Research Repository
BIOACTIVITY GUIDED ASSESSMENT OT' SOME SELECTED
MEDICINAL PLANTS OF MALAKAND DIVISION
Department of Botany
Facult5r of Sciences
Pir Mehr Ali Shah
Arid Agriculture University Rawalpindi
GUL RAHIM
l0-arid-72
Pakistan
2014
{F&f,+.+-,
t t99a
1
BIOACTI!'ITY GI]IDED ASSESSMENT OF SOME SELECTED
MEDICINAL PLANTS OF MALAKA}ID DI\,IISION
By
GUL RAIIIY10-arid-72
A thesis submitted in paftial fulfillment ofThe requirements for the degree of
Doctor ofPhilosophy
1I
Botany
Department of Botany
Faculty of Sciences
Pir Mehr Ali Shah
Arid Agriculture University Rawalpindi
Pakistan
2014
1l
4
THEfHE P,NOr
111
CERTIFICATION
I hereby udertake that this research is an original one and ro part ofthis thesis
falls under plagiarism. If found otherwise, at any stag€, I will be responsible for the
consqluences.
ft,xV.Y_v_Student Name: Gul Rahim
Registation Number: 10-arid-72 /t- t-z't\Signature:
Date:
Certified that the cotrtents and fonn of the thesis entitled "Bioactivity guided
Assessment of some selected mediciral plants of Malakand divisiou" submitted by
Mr. Gul Rahim have beetr found satisfactory for the requircment ofthe degree.
Supervisor
Mernber:
Member:
(Dr. Rahmatullah Qureshi)
Director Advanced Stridies:
C-=-\B7
DTDIC.XIED
r'o
n4y ?arents
To toif and sweet of affectionate
? arents as moraf supy mt,
Enshrinefan{grafie{Inme untiring zeaf
lo get on the
S{igher ifeafs
of frfe
CONTENTS
List ofAbbreviations
List ofTables
List ofFigures
Acknowledgement
ABSTRACT
GENERAL INTRODUCTION
ETHNOBOTONY
2.1 INTRODUCTION
2.2 REVIEW OT LITERATURE
2.3 MATERIALS AND METHODS
2,3.1 THE STUDY AREA
2.3.1.1 Climate
2.3,2 COLLECTION OF ETHNOBOTANICAL INFORMATION
2.3.3 COLLECTION AND IDENTIFICATION OF PLANT
SPECIMENS
2.4 RESULT
2.4.1 Medicinal plant inventory
2.4.2 Fidelity percentage
2.4.3 Disease treated
Page
xvii
xx
xxi
I
5
t7
17
r9
22
22
23
25
25
26
26
26
27
2.4.4 Mode of application
2.4.5 Parts used
2.5 DISCUSSION
CONCLUSIONS
PHYTOCIIEMICAL SCRNENII{G
3.1 INTRODUCTION
3.2 REI'IEW OF LITERATURI,
3.3 MATERIALS AND METHODS
3.1.1 Medicinal Plants Selection
3.3.2 Preparation of plant extracts
3.3.3 CRUDE EXTRACTS YIELD
3.3.4 PIIYTOCHEMICAL SCREENING TEST
3.3.4.1 Detectior of Alkaloids
3.3.4.1.1 Mayer's reagent
3.3.4.1.2 Wagtrer's reagent
3.3.4.1.3 Hager's reagent
3.3.4.1.4 Dragen&off's test
3,3.4,2 Determination of Amino acids
3.3.4.3 Detection of Ca$ohydrates
3.3.4.3.1 Molish's test
3.3.4.3.2 Fehling's test
3.3.4.3.3 Barfoed's test
27
27
28
47
47
48
46
53
53
58
58
59
59
59
59
60
60
60
6l
61
61
61
v11
3
3.3.4.3.4 Benedict's tesr
3.3.4.4 Detection of Flavonoids
3.3.4.5 Detection offixed oils and fat
3.3.4.6 Detection of glycosides
3.3.4.6.1 Bortrager test
3.3.4.6.2 Legal's test
3.3.4.7 Detection of gum and mucilage
3.3.4.8 Detection of phenolic compounds
3.3.4.8.1 Gelatin test
3.3.4.8.2 Lead acetate test
3.3.4.9 Detection of Phltosterols
3.3.4.10 Detection of proteins
3.3.4.10.1 Millon test
3.3.4.10.2 Biuret test
3.3.4.11 Detection of saponins
3.3.4,12 Detection of tannins
3.3.4.13 Test for telpenoids
3.3.5 QUANTITATIVE PHYTOCHEMICAL SCREENING
3.3.5.1 Alkaloids
3.3.5.2 Flavonoids
3.3.5.3 Tannins
3.3.5.4 Saponins
62
62
62
63
63
63
64
64
64
64
65
65
65
65
66
66
66
67
67
67
68
71
VIII
3.3.6 STATISTICAL ANALYSIS
3.4 RESULTS
3.4.1 PHYSICAL PROPERTIES
3.4.2 QUALITATIIT PHYTOCHEMICAL SCREENTIIG
3.4.3 QUANTITATIVE PHYTOCHEMICAL ANALYSIS
3.5 DISCUSSION
CONCLUSION
4 PROXIIVLA.TE A}[D MINERALS ANALYSIS
4.1 INTRODUCTION
4.2 REVIEW OF LITERATURE
4.3 MATERIALS AND METHOD
4.3.1 PREPARATION OF PLANT I\4-4.TERIALS
4.3.I.1 PROX]MATE ANALYSIS
4.3.1.1 .l Detennioation of Dry Matter
4.3.1.1.2 Determinatioo of Moistue Content
4.3.1.1.3 Deteminatiotr of Ash Content
4.3, 1 . 1 .4 Determination of Crude Protein
4.3.1.1.5 Determination of Fat Content
4.3. 1.1.6 Determination of Crude Fiber
4.3.1.1.7 Detemination of Carbohydrate Content
4.3.2 MINERAL ANALYSIS
4.3.3 STATISTICAI ANALYSIS
'73
'75
'76
87
88
88
'71
72
72
89
9l
91
91
91
92
93
93
94
95
96
96
97
1X
4.4 RESULT
4.4.1 PROXIMATE ANALYSIS
4 4.2 MINERAL ANALYSIS
4.5 DISCUSSION
CONCLUSIONS
ANTIMICROBIAL ACTIVITIES
5.1 INTRODUCTION
5.2 REVIEW OF LITERATURE
5.3 MATERIALS AND METHODS
5.3.1 COLLECTION AND IDENTIFICATION OF PLANT SAMPLES
5,3.2 PREPARATION OF PLANT EXTRACTS
5.3,3 1N Z17RO ANTIBACTERIAL ACTIVITY
5.3.3.1 Prcparation of Inocula
5.3.3.2 Antibacterial Susceptibility Test
5.3.3.3 Selected Bacterial Strains
5.3.3.4 Prepamtion of sample dilution
5.3.3.5 Media for bacteria
5.3.3.6 Turbidity standards
5.3.3.7 Physiological saline
5.3.3.8 Tissue Equivalent vs activity
5,3,4 IN VITRO AN'IIFLINGAL ACTTVITY
5.3.4.I Media preparation for antifungal assay
97
97
98
99
104
t05
r05
107
113
113
1i3
tt4
I t4
tt4
116
116
tt'7
tr7
118
it8
118
I l9
x
5.3.4.2 Preparation of samples
5.3.4.3 Assayprocedure
5.3.5 STATISTICAL ANALYSIS
5.4 RXSULTS
5.4.i ANTIBACTERL{L ACTIVITIES OF MEDICINAL PLANTS
5.4.1.1 Antibacterial ac,oity of Monotheca burifolia
5.4.1.1.1 Inhibition Zone (mm) at 1Smg/ml
5.4.1.1.2 Inhibitiotr Zoae (mm) at 12mglm1
4.4.1.1.3 lnhibitiotr Zone (mm) at 10mg/ml
5.4.1.1.4 Inhibition Zone (nrm) at 7.5mg/ml
5.4.1.1.5 Inhibition Zone (mm) at 5mg/m1
5.4.1.2 A.Dtibaclerial activily of Myrtu5 communis
5.4.1.2.1 Inhibition Zone (mm) at 15mg/m1
5.4.1.2.2 Inhibition Zone (mm) at l2.5mg/ml
5.4.1.2.3 Inhitition Zone of (mm) at lOmg/ml
5.4.1.2.4 Intribition Zone (mm) at 7.5mg/ml
5.4.1.2,5 Inhibition Zone (mm) at 5 mg/ml
5.4.1.3 Aatibacterial activity of Teucium stocl$ianutu leaves
5.4.1.3.1 Inhibitiotr Zone of (mm) at 15mg/ml
5.4.1.3.2 Inhibitiotr Zone (mm) at 12.5mg/ml
5.4.i.3.3 Irlibition Zooe (mm) at 1omg/r
5.4.1.3.4 Inhibition Zone (mm) at 7.5m9/m1
119
119
t20
121
121
121
l2l
1.22
123
124
125
126
126
127
r28
lro
130
131
131
t32
133
134
xl
5-4.1.3.5 hhibition Zone (mm) at 5mg/m1
5.4.1.4 Antibacte al a ctrvrty of Teuclium stocksianum fTower
5.4.1.4.1 Llibition Zone (mm) at l5mg/ml
5.4.1.4.2 Inhibition Zone (mm) at 12.5mg/ml
5.4.1.4.3 hhibition Zone (Dm) at 10mg/ml
5.4.1.4.4 lohibition Zone (mm) at 7.5mg/m1
5.4.1.4.5 lnhibition Zone (mm) at smg/ml
5.4.1.5 Antibacterial acti,lity of l/erbascum thapsus
5.4.1.5.1 Inhibition Zotre (mm) at 15mg/m1
5.4.1.5.2 Inhibitiotr Zotre (mm) at 12.5m9/ml
5.4.1.5.3 Inhibition Zone (mm) at lOmg/ml
5.4.1.5.4 Inhibition Zore (mm) at 7.5mg/ml
5.4.1.5.5 Inhibition Zone (mm) at smg/ml
5.4.1.6 Antibacterial activity of Zizyphus sativa
5-4.1.6.1 Inhibition Zone (mm) at 15mg/ml
5.4.1.6.2 Inhibition Zone (rtrm) at 12.5mg/ml
5.4.1.6.3 Inhibition Zone (mm) at 1omg/ml
5.4.1.6.4 Inlibition Zone (mm) at 7.5mg/m1
5.4.1.6.5 Inhibitioo Zone (mm) at 5mg/ml
5.4.1.7 Minimum Inhibitory Concetrtratioo (MC) ard Iohibition Zones
(ro-)
5.4.1.2.8 Tissue Equivalent vs activity
135
137
t37
138
139
140
141
143
143
144
144
145
145
145
145
146
147
148
149
150
xii
152
5.5 ANTIFUNGAL ACTIVITY OF MEDICINAL PLANTS
5.5.1 Methanolic plant extact
5.5.2 Ethanoiic plant extract
5.5.3 Water platrt extracts
s.6 DISCUSSTON
CONCLUSION
ANTIOXIDANT ACTI!'ITY
6.1 INTRODUCTION
6.2 REI'IEW OF LITERATURE
6.3 MATERIAIS AND METHODS
6.3.1 DETERMINATION OF TOTAL PHENOLIC CONTENT
6.3.2 ANTIOXIDANT ACTIVITY
6.3.2.1 Prcparation of Stock Solution
6.3.2.2 lree mdical scavenging activity
6.3-2-3 Reducing power assay
6.3.2.4 Metal Ion Chelating Activity
6.3.2.5 STATISTICAI ANALYSIS
6.4 RESI'LTS
6.4.I TOTAL PHENOLIC CONTENT
6.4.2 Antioxidatrt activities
6.4.2.1 DPPH ftee radical scavenging activity
6.4.2.2 Reducing power assay
152
153
154
171
172
1't'7
179
179
181
186
186
187
187
189
189
190
190
191
191
),92
1,92
196
xIu
6.4.2.3 Metal Ion Chelating Activity
6.4 DTSCUSSTON
CONCLUSIONS
7 GENf,RAL DISCUSSION
CONCLUSION AND RECOMMENDATIONS
SUM}LA.RY
LITERATURX CITED
APPENDICES
209
2t3
229
230
239
240
248
290
xiv
.c.
ASA
BHA
BHT
CFU
dH20
d1
DMSO
DPPH
G
GA
GAN
M
mg
MIC
m1
mln
IllM
N4r1
Mo
MPs
N
orOD
PBS
pH
QR
LIST OF ABBREI'IATIONS
Cetrtigade
Ascorbic acid
Hydroxyanisole
Butylated hydrcxyl toluene
Colony formiog unit
Distilled water
Deci liter
Dimethyl sulfoxide
I , 1 -diphenyl-2-picryl-hydrazYl
Gram
Gallic acid
Gallic acid equivalent
Molar
Milii gram
Minimum i.tribition concentration
Millimeter
Millimeter
Milli molar
Manganese
Molybdenum
Medicinal Plaats
Normal
Superoxide
Optical density
Phosphate buffer
Hydrogetr ioo concentration
Quercetin
xv
Rpm
TCA
TPC
mg
pl
Revoiution per minute
Trichloroacetic acid
Total phenolic contents
Micro gram
Micro liter
xvl
Table No.
2.1
3.3
3.4
3.5
3.6
3.'l
3.8
3.9
3.10
LIST OF TABLES
Native uses ofindigenous plants of Malakand Division' KPK'
Pakistan.
Common aliseases heated by the native species ofthe study arca
Profile of plarts used as e*uro medicine in Malakand division
Profile of plants used as ethno medicine in Malakaad division of
Pakistan.
Yield (%) of solvent extracts of various plant species
Phyochemical screening ot tt. butifolia.
Phfochemical screed!gof M. (o munis'
Phl4ochernical screening of T. stockstanum Leaves'
PhltocheErical screenii,g of T. stocksianun FLoYtet '
Phyochemical screentng of v - rhapsus
Phltochemical screedng of Z. sativa.
Quantification of ph,'tochemicals from selected medicioal
plants.
Proximate Analysis (Yo) ofplant samples'
Mineral anallsis of selected medicioal plant samples (ppm) and
percentage.
Zone of idribition (mm) at the concentration of 15mg/m1'
Zone of inhibition (mm) at the concentratiotr of 1 2 5mg/m1'
Page
30
42
54
55
.,7
3.1
3.2
78
79
80
81
82
83
84
85
4.1
4.2
5.1
5.2
100
101
155
158
xvlI
5.4
5.5
5.6
Zone ofinhibition (mm) at the cotrc€ntration of 10mg/m1 160
Zone ofinllibition (mm) at the concetrtation of7 Smg/ml' 162
Zooe ofinhibition (nrm) at the concentratiotr of 5mg/m1 164
Mioimum inhibitory concentation (mg/ml) and zone of 166
inhibition (mm) oftested Plants'
Tissue equivalent vs. activity of solve[t extracts of selected plant I 68
species.
Antifungal activity ofmethanolic extracts 169
Antifutrgal activity of ethanolic plant extracts 170
Antifungal activity of aqueous extracts' 174
Total Phenolic Content (mg GAE/g) of selected medicinal plants 1 98
of Malakand division.
Inhibition percentage of M. blLrifolia $nongb DPPH method 199
Percentage scavenging activities ofMylr''ls communis 200
DPPH scavenging activities of f. slockia'am leaves extacts 20L
DPPH scavenging activities of f. s,ocfs'a'xum flowei 202
DPPH scavenging activities ofthe ,a 'l'apJus 201
DPPH scavengitrg activities ofthe Z satrva 204
Reducing power ofthe leaves extact ofMonotheca bLlxifolia' 206
Reducing power ofth e exft^d of Myrtus com unis 20T
Reducing power of re ucriutu stacksiahum Leaves 214
Reducirg power of th e Extacr of Teucrium stock\ia um f7owet 275
5.1
5.8
5.9
5.10
6.1
6.2
6.3
6.4
6.5
6.6
6.'1
6.8
6.9
6.10
6.11
xviii
6.12
6.t3
6-14
6.15
6.16
6.17
6.18
6.19
Reducing power of Zerbascum thapsus.
Reducing power of Zr'a,p hus satira.
Metal Scavetrging Capacity of M. b*tfolia.
\letal Scavengiog Capacrq of lt''lyrrus comnunis.
Metal Scavenging Capacity ofT. stocL'sianum leaves extracts
Metal Scavenging Cap aclty of T. stocksianum llowet
Metal Scavenging Capactty of l/- Thapsus
Metal Scavengiag Capaclty of Z. sati.ra
216
2t7
2t8
219
229
221
222
223
xix
Fig. No.
2.1
2.2
2.3
3.1
6.1
LIST OF FIGURES
Location map of the study area.
Mode of application of vafious recipes.
Different plant parts used in folk recipes.
Calibratiotr curve for tannic acid.
Calibration cuve for Gallic Acid.
Page
24
44
45
'70
188
xx
ACKNOWLEDGEMENTS
For the corEpletion of this work, I am tia't'ful to Almighty Allah' whose
Grace and Mercy blessed me with good health and enthusiasm l offer my humblest
thanks from ay heart to Eoly Prophet Eazrat Muhammad (Peace be UPon IIim)'
who is forever a torch of guidance and knowledge for humanity as a whole'
My deep appreciatiol goes to my dissertatiol advisor Dr' Rahmatulhh
Qureshi, Associate Professor, Depatmeflt of Botany, Pir Mehr AIi Sha] Arid
Agriculture University Rawalpindi, Pakistan for his constait help, guidance and
countless hous of attetrtion he devoted thrcughout the course of this work His
priceless suggestion made this irteresting and leamilg for me'
I would like to place on record my great appreciation and indebted gratihrde to
Prof. Dr, Muhammaal Arshad, ChainDan, Depatunent of Botany, PMAS-AAU,
Rawalpindi, for his coopemtion and heb to complete my thesis'
I am especially thankful and grateful to Prof. Dr' Muhammad Gulfraz,
Departrnent of Biochernistry, Pir MehI Ali Shah Arid Agriculture University
Rawalpindi, Pakistan for lus co-supe isiotr and for his invaluable and helpful
suggestion atrd encouagement to accomplish this task'
I am also extrernely grateful to my friends and fellow studetrts that have
worked on Advise Aid with me over the years. I want to specificatty thank Mr' Sajad
A.ti and Mr, Masood Ahmad, who has made work and this whole process fun and
who has given great advice over the years, and I am also grateful to my Aiends Mr'
xxi
NasibZamatrwhohasgreatlyhelpedmeilmylesearchwoikbyclarifyingthebasic
concepts through discussion and he1pful cotunents'
My thanks are to my brother Mr' Sahib Rahim' and my fiiends Musa I(han
for thei cooperatiotr and help dudng field visits'
I feel geat pleasure in offering thanks to my coopemtive lab fellows Mr'
Muhammad Ilyas, Mr. Ahmad Ali, Ms' 1llutrashrah Munir' Ms' Eumrira
Shaheen, Mr. Waseem, Ms. Mehmooda Manazr' Mr' Muhammad Maqsood and
Mr. Faisal Mirz!, for their moral support and their efforts and prayels made it
possible for me.
Last but not the least; I am highly glateful to Universify of Malakand'
Khyber Pukhtonkhawa, Pakistan, Poultry R€search Institute Rawalpindi'
Pakistan, for providing Lab facilities for research atrd Iligher Education Commission
(HEC) Pakistan, for funding my project without that I would not be able to complete
this task.
Fhally, I wish to express my heartfelt gatitude to my parents for continuing
support and constants pmyen l owe a lot of thanks to my dear for thek exta patience
and motivation aDd made this possible'
May Atlahbless all these noble persotralities (Ameen)
xxii
Gul Rahim
ABSTRACT
Present study was undertakm to document medicinal uses of native plants,
ph},tochemical sqeetring as well as aDtimicrobial and antioxidant acti\'ities of some
potential species. Forty one plant species distributed aqoss 38 genera and 32 families
were in use by the natives in treahng 27 human diseases. Based on use reports and
fidelity percentage, some species llke Monotheca buxifolia, Myrtus communis
Teucrium stocl$ianum, I/erbascum thaps s ar.d Ziziplls sativa were selected for the
evaluation of bioactivity. Plant materials of these species were collected, 'lried,
powdered and extacts were prepaled by using three solvents viz., ettanol, methanol
atrd water.
The present research screened for 13 phyochsmicals in five selected medicinal
plants of the study area. Ia order to evaluate solvent effectiveness in detecting
chonicals, methanol, ethanol and water exhacts were tested. Results revealed that
methanolic extracts were the most effective in detecting phloconstituents frorE
selected species. This solvetrt isolated all chemicals ftom T. stocksianum leaves and
flowen ard Z. sattua. lt was followed by l/. thapsus (11)' M. buxifolia afi M'
com ni.s (70 eacQ. The ethanol extracts exfacted all phl4chemicals from M
co muni; T. stocksiakum leaves, followed by M burifolia, V' thapsus and Z' sati\)a
(12 each), while in T. stocksianum,lO phltochemicals were idetrtified ln the case of
aqueous exhacts, ma'<imr.rm phl4ochemicals were detected from M. bttxifulia (9),
i
2
followed by M. communis, T stocksianum leaves and Z' satitta (8 each\' T'
stoclcsidnutu llowet (7) a I/ thapsus (6)'
The selected plants were subjected to the quantificatioo of ph]'tochemicals that
revealed that highest amount of flavonoids was found il Z' sativa (093+00%)'
followed by M. communis (0 78+0 OO%) Alkaloids were richly pres€nt in T'
stocksianum leaves (0.81'10.00%) The highest percentage of taffihs was present in Z
saava (9,1l:0.31%), followed by M' eommunis {g '17+0 22%)' while saponins were
heavily found in T. stocksianum flower (2'21+0 00%) a\d T stocLsianwh leaaes
(2.13+0.01%).
The extacts of selected plants were subjected to i/,l vil''o atrtibacterial assay
against some gmm po slnve (Staphylococcus aureus, S epidermidis a\d StrePtococcus
pyogen) and grxt neganve (Echerichia coli, Klebsella pneumonia arrd Pseus'lomonas
aeruginosa) bact lLal shains at the concentrations of 5mg/El to 15mg/ml by using agar
welldifftrsionmethod.Thesolvent.basedextactspossessedantibacterialactivity
against all selected miclobes and at the lowest conceltration (i e' 5mg/ml)' '41
coilmunis ^nd.
T. stocktianltm extacts showed relatively high activity compared with
standad d g, while other three plants did not show effectiveness against the test
organisms. The ethanolic extact of ,'i{ communis ex\ibited best against 'S a'reas
(6.5+0.7) and S. pyrogen (6 1+0.64), while methanolic exttacts foutrd best effective
agaiIfJt S. pyrogek (7 6+0 69) and .t epidermidis (5'l+O'72) Ethanolic T' stocksianum
leaves extract ilhibited best S. arrerr (5.8'!1.03), S' epidermidis (5 5+0 62) and K
3
pneu oniae (5+0.45), whereas, methanolic exhact was found effective for s.
epidermidis (6.5t0.7). The aqueous extact eKhibited highest activiiy against '
coli
(5+0.0) and S. epidermidis (5+0 3)' f stock'sianutt methanolic flower exhibited best
activity against S. py,' ogen (6+0 67), K' pneumoniae (5 2+O'g) aIld S aureus (5+l O)'
The antifungal activily was carrie'l out against two fungal pathogens i e
Aspergillus higer, A. f migates through tube dilntion method at ihe coDcentatiotr of
12mg/m1 ftom methaool, ethanol and water extracts There was significant difference
in the activity of all solvent based extacts (p>0 01) and all plants possessed antifungal
activity against l. /, migatus and A. ,ige," with rcspect to solvent extracts' highest
antifirngal activity was showE by metlanolic, followed by ethanol and water'
Methanolic Z. sativa le.I\/es exhact iDhibited best A fumigatus (73 22o/o)' whlle A'
zigel was inhibited best at 68.65% by T. stock'sia um flower, while rcst of platrts
extracts had relatively low activity. From ethaoolic extacts, A' fumigatus \'/as
inlibited alrnost equally by Z. sati|a and M. bwcifolia wifr 62 89 and 6l '860/0 acnvity
respectively, while l. ,r?e/ was best controlled by T' stocksianum flower and leaves'
The aqueous extract also derived significalt activity against both pathogens Tn this
case, A. niger was susceptible against M bwifolia lea'tes extract (58 88%), whereas'
M. com nis leaves extract inhibited best I - funigatus '
The antioxidant activity of forrr solvents based extracts of selected plart parts
was determined at six various concentatio[s ftom l0pg/ml to 60 pglml by using two
methods viz., DPPH scavenging aDd reducing power assays Both assa)s showed
4
sigoificart activity in all selected concertratiotrs (P<0 01) All plants showed good
scavenging activity in comparison with reference standalds (ascorbic acid and BHT)'
Out of the five species, M. communis Ni T' stockianu/l (Flower) showed highest
antioxidant activity in all solvent extracts. In the case of 7 s'ocfr ianum' lCso (p/rnL)
value was measued as 28.2,19.4,21.2 and for methanol, ethanol and water extracts
respectively. All thes€ extracts exhibited antioxidant activity higher than Ascorbic acid
(29.2 pglml-), while ethanolic extact was closer to BHT (14 4 pglm]-) ln the ca-se of
former species, water extact showed best antioxidant activity (18 12 pglml,) that was
near to BHT (14.1045 Pg/mL).
The reducing power assay revealed that ethanolic extract of M communis had'
supeior activity (2.2710,026) at (Pl0.0I). Compared with refermce stanilards
(ascorbic acid: 2.64+0.02 and BHT:2.11t001). The methanolic exhact of Z
stocksianum showed the highest activity ICso (P'g/mL) (28 2) that was at par with the
drugs namely ascorbic acid and BHT)
This study validated the kaditional medicinal usage of tested plants in the area
and can be concluded that M/"la.t cofimunis and Teucrium stockianur leaves and
florrff extacts possessed antimicrobial and antioxidant activities that may be of great
idportance to the pharmaceutical industry
Chapter I
INTRODUCTION
Natwe has provided Plant resources to fulfill different fleeds of huma[ being
such as fuel, foo4 shelter and medicines (Bhatli et al' 2001) Use of plants as
mea[cament is as old as human civilizatiol and since ancient time, plants are used for
curing various diseases (Samuelsson, 2004). The haditional system ofmedicitre based
on herbal fomulatiom has been used as pimary health care in rural communities
across the world. Therefore, a large portion of the human population still relies on
native plants to ovelcome iheir health requirements (Griersotr and Afolayaq 1999)'
The indigenous ktrowledge pertaidtrg to the speciftc use of plants and the
method of applicatiol for a particular aiiment was passed on omlly ftom getreration to
geaeration. This pnctice through updation and lefinement led to developing he$al
pharmacopoeias (Balunas, 2005). Local herbs as a soulce of secondary plant prcduct
have provoked more interest in sqeedng herbs for their poteEtiality itr curitrg various
infectionous cliseases ancl preserving foods ftom deletedous effects of oxidants
(Paoovska e, aL., 2005). Study of medicinal plants is providing a source for the
production of valuable compounds (Black, 1996)'
6
The potential of higher plants arc as source of drug is still oot fully explored'
The floweriag plants are estimated to be 250,000 to 400,000 species distributed in
about 415 families. Of ihem, more than 50000 are reported as medicinal one
(Govaerts, 2001). It has been rq)orted that a very small Aaction has been studied
phl,tochernically and even a smaller number for biological or pharmacological
screening. Conseque[tly, phyochemical analysis of a plaat will only expose spectum
of its chemical constituents. Tladitionally, the screening of bioactive compouods of
s,,nthetic or tratural dsrivation has still one of the sources of beyond measure healing
agents. The phltochemicai screenings ofplants have been identified as cleative means
in the discovering of novel biologically active compounds in the field of antibiotics
(Gerhafiz et al.,1985; Kroschwitz arrd Howe-Grant, 1992) Now a day, drugs ftom
higher plants also occupied a sigoificant position in the modern medicine Worldwide'
130 drugs arc isolat€d ftom higher plants or synthetic derivatives thereof (Newmatr el
aL.,2000).
There is steady increasing demand of medicinal plants in developing countries
especially in poorer due to less annual income lt has been estimated that 80% of the
human population are partially or wholly dependent on plant-based drugs (Kuete er al''
2010). This figure demands to carry out research emptrasizing on the field ofmediciral
plants. Plant based drugs are normally believed to be safe and effective against certaio
diseases. Recently, there is inqeasi[g use of herbs in dweloped countiies (Indu e/ al '
2006) and various studies reported high consumption of vegeiable aod fruits for the
prevention ofcbrooic diseases (Lee e, al, 2003; Alviano and Alviano, 2009)'
'7
Plant based medicines got recognition today, due to limited health facilities of
Eodem medicines in the developing countries However, due to availability of
spomdic scientific inforrnatiotr, such medicaments may cause severe adverse iffects'
Thereforc therc is a need to anallze plant extracts for l/? 'ilro alrd ft vivo activities aod
their phannacologicat properties that may facilitate developitrg less expensive
therapies to be used in the developing countries (de Sousa et al, 2004)'
Plants arc natual sources and several important biologically active compounds
have been discovered tlrough isolation and bioactivity fiactioaation These
biologically active compounds are mostly the secondary platrt products, after proper
processing to pure compounds these secondary plant metabolites become medicine'
Accordhg to survey caEied out in 2001 and 2002, around 1/46 dlugs in the globe were
secotrdary plant products or their derivatives (Butter, 2004)' Another survey ahout 28
percent of the NCES bet\reeo 1981 a.'rd 2002 are secondary plant prcducts and their
derivatives (Ne.?vman el a1.,2003) arrd another slrrvey was caried out in this period
considered 20 percent of the NCEs as secondary plant product mimics' which mean
that most of s)'nthetic biologically active compounds wele also based on secoodaqr
metabolites (Newman e/ al, 2003). During the period ftom 1981-2002' now it was
understooal that research work otr plant pro'lucts account for just about 48 percent of
the NCES reports.secondary plant metabolites also provide a fundameotal point for the
laboratory s)'ntheses having various structues by means of numetous stereo centers
which oan also be difEcult sFthetically (Petefion ard Overman' 2004; Clardy and
Walsh, 2004; Nicolaou atrd Stryder, 2004; Koehn and Carter' 2005) Secondary plant
8
products also show most of the stuctural featues which '!re
common and more
significant for drug discovery (Lee and Schneider, 2001; Feher and Schmidt, 2003;
Clardy and Walsh, 2004; Piggott and Karuso, 2004; Koehn andcarter, 2005) Several
rcsearcherc have started v/orking for the development ofderivaLives ofsecondary plant
product(Hall el al, 2001a; El&idge er a1.,2002; Tar! 2004; Ganesan,2004; Burke er
al.,2004).
Medico-botanicals are being evaluated globally for searching new drugs that
cue human and animal diseases. A lot ofwork has been done by several scientists for
tlxe search of antimiqobial activity from plants (Lall et al-,2006) There are still round
about 120 discrete chemical substances which are derivative Aom the plant species atrd
are used as alrugs tlrcughout the world. Few of them sold now a day are synthetically
modified or copies of the natually substances.
The European countries like Gemany and various pharmaceutical companies
are lagely prcparing herbal products as drugs by extacting active constituenLs from
plants. For example, a plant substance Claadn found ia common arttcioke (Cynara
scolyz,s) and is sold for liver problems and h)?ertensiol in GermaEy (Taylor' 2000)'
Various modem drugs are derived ftom plants based on their potential
applications available in tuaditional pharmacopeias These plants contair a vast array
of bio-compounds acting as driving force in treatitrg chrotric as well as infectious
diseases (Yogeshkuoar and Chand4 2007; DulaiPandiyan et al', 2006) Today'
9
pharmaceutical industy is mostly retying on these natural compounds derived *om
plants (Baker el al, 1995). These active irgredients are admitted by treating diseases'
Even in westem medicine, substances obtained ftom higher plants rq)resent about
25% of prescribed medicitres. Io addition, abo'tt 74o/o of the 121 bioactive plant-
derived compounds currently in practice worldwide were identified from
ethnobotanical source (Anyinam, 1995).
In ancient days people extracted drugs Iiorn the medicinal herbs which were
safe because they have no adverse effects but they belefited the people due to their
combioatiotrs v/ith vitamins and minemls (Ahmad and Husain, 2008)' Plants have been
used for medicinal purposss for the past 60,000 years. About 80% of world population
depends on uaditional herbs to cure their diseases (Dwsum el al, 2004)'
Due to side effects aEd at the same time resistance to microorganisEs against
antibiotics, a large interest has been given to bioactive compounds extracted ftom
botanicals which arc utitized in the traditional system of medicine (Essawi and Srour,
2000). The antimicrobials ftom plant odgin are massive available soulce for medicine'
These agelts have massive restorative potential and also effectively used in cuing
contaglous dis€ases, while at the same time reduce numerous adverse effects which are
ftequently allied with the slnthetic antimicrobials (lwu et 41.. 1999) A 1ot ofofthem
are also used for the cure of skin disease fol centuries (Schempp et al',7999; Avalos
and Maibach, 2000; Augustin and Hoch, 2004) Because of regularly increasing
resistaoce to antibiotics, plant €xtracts and active compounds from plants ale of new
l0
ilrterest as antimicrobial agents and antiseptics in dermatology (Norton, 2000;
Blascl\ek et al., ZO04l Augustin and Hoch, 2004)
Il the 19s centuy antioxidants were used by some engi[eers to avoid
corrosion of metals and wlcanization of rubber. By doiDg this practice they saved
many dollars in different materials every year. This principle was not successful in
biotecbnology until the 2Od century (Gutteridge and Halliwell, 2000). During the 20d
century, scietrtists thought to lengthem the life of foods. They used foods which have
high content of uNaturated fat in them and applied antioxidants to thern. They found
that foods were saved ftom rancidity. As people continued their research they found
that, some of the key and vital vitamins, also essential for the human diet, were able to
be classified as antioxidaDts (Gufteridge and Haliwell, 2000)
After these great rcsults, seve@l docto$ and physiciaN focused od the use of
antioridants in industry and they also made great efforts to apply these principles on
the human body atso (Gutteridge and Halliwell, 2000).
Altioxidatrts are those compounds which protect the living cells ftom oxidative
damage origined ftom ftee radicals. The antioxidants obstuct the oxidation reactioD ty
terminating the change reaction resultaotly catal)tic metals act as oxygen quencher
(Habila et a1.,2010). In livirg system, the Aee radicals mostly genemting as pafi of
body's metaboiic process and ploduce the chain reactions and this chain reactiol] in
tum are prcduced in liver mixed function oxidases, the Ditochondrial respiratory
11
chain, by bacterial leucoc)'tes, atrnospheric pollutaots' tkough xanthine oxidase
activity, and &om transitioflal catalysts metal and drugs (Atawodi' 2004)
Antioxidants protect the human body fiom the pathological effects of ischemia'
asthm4 mongolism, arthritis, neuro-degeneration, Parkinson's diseases' inflammation'
aging process anal dementias (Polterat, 1997) digestive syst€m disorders including
ulcer and gashointestinal ilflammation and autoimmune pathologies (Aruoma' 2003;
Reppeto and Llesuy, 2002).
Antioxidatrts, exogenous ol en'logenous, whether oatural or s1'nthetio are very
effectiveinthepreveotionoffteeradicalsformationandsuppressingsuchkindof
disorders (Soud et al., 2009). Antioxidant compound functions as reducing agetrts'
radical scavengers and oxygen formation Oxygen is very important for our
physiological and metabolic processes but during these reactioN about 59lo oxygen is
abridged to rcactive species (radical and non-radicals) known as reactive oxygen HzOz
(ROS) (Kumar, 2005).
During normal cellular metabolism reactive oxygen species (RoS) are
gensrated but when they are in excessive co[centation they start producing oxidative
damage, Most of the human diseases are caused by ROS' which includes arthritis'
neurodegenerative diseases, hfections and acquired immunodeficiency slodrome
(?atel et a1.,2010). To teat a1I these disorders' antioxidarts play vital role'
Antioxidants mostly inhibit the process
(Butukokuoglu, 200 1 ).
t2
of oxidation when they rcact with ROS
The disparaging role of ROS is interlinked to the task of another Aee radical
nitric oxide, which belongS to a gpup containing reactive nitrogen species (RNS)'
This group also possesses a strong oxidative potential (Zorcv et al ' 2003) Tnere are
celtainflavonoids,alkaloidsandflavoneswhicharewidelydistribrrtedamongpla[ts
which possess antioxidant and afltiradical activities and also thet effects on human
health and nutrition are significant (Nakayoma and Yamada' 1993) The antioxidants
Aom platrt origin such as vitamin C (Ascolbic acid) and flavonoids are steadily
popularizing as significant part ofhumatr diet (Ferguson' 2001; Cantuti-Castelevetri el
al, 2000). Accordirg to Aqil e' al, (2006), the antioxidant activity is due to presence
offlavonoids, flavones, isoflavotres, coumains anthoclanins' lignans' isocatechins and
catechins in different parts of plants Antioxidant based drugs formulated dudng the
last 3 decades are also useal to teat stroke, atherosclerosis' Alzheimer's' cancer afld
diabetes (Devasgayam et al.' 2004)'
Phlochernicals nahually present itr plants such as te4 berry crops' herbs'
beans, oilseeds, vegetables and fruits have a world' There are a lot of species ofherbs
andsornespiceshaveantioxidantactivityeg'thyrne'rosemary'sage'nutmeg'
turmeric, chili pepper, white pepper and gin gers (Lee et al '
2003)'
13
S),nthetic afltioxidants are added in preparation of foods because they are good
radical scavengers, but now it is reported that they are failed against the metabolic
problems so, researchers are concetrtrating to identifo and isolate Batural drugs having
high antioxidant activity (Agbor er a/., 2006) Moreover itr rccent times' some negative
effects of the frequently used slnthetic antioxidants have been documented Reports
also revealed that these active corDpoun'ls may also be concemed in many health risks'
including carcinogenesis (BEn, 1975; Assimopoulou et al ' 2005 Goli el al' 2005)'
Synthetic compoutrds are also quite expensive and not affordable (Kirby' 1996)'
Therefore, hence is a propensity towards the use ofplant origin natual alrtioxidants to
rcplace these s)'othetic antioxidants (Rahimlpanalt et al'' 2010)'
It is usually ixaplicit that everyday consumption of the plant derived
phltoconstituents from fruit, vegetables, tea and herbs may contribute to shift &e
balatrce towald srrq)lus antioxidant status (Halliwell, 1996). Thus, interest in recent
years has greatly hcreased in natural atrtioxidalts' to prctect humans from the
oxidativeeffectsproducedbyRNSandRosspecies(JayaprakashandRao'2000;
Goncalvaes et al., 2005).
Plants exhibited huge number ofbiologically active compounds which are used
mostly in medicine or in the novel drugs preparatioo about 30 percetrt of global drugs
are secondary Plant products and obtained from plants extracts (Grabley and Thiericke'
1999).Many attempes have been ma'le by scientists to extract novel biologically active
compounds fiom differetrt plant extracts, Systematic screening of plant extacts may
14
also result itr the detection ofnew active compoulds According to the report of WHO'
the majority ofdiseases have been cotrtrolied and about 40 percent deaths occurred due
to microbial hfectious in the developing countries Maintenance of food stuff is still a
chief problern at the moment, since novel foodstuffs are intoduced in bazaar which
wants more ennchment (Madno el al, 2001). Consequently, the rcsealchers are paying
more attention to fitrding out biologically active comPounds from the extacts of
medicinal plant to contol miqooiganisms (Mitscher et at. 1987) Some foodstuff
iagredieots and biologically active compound of medicinal plants and vegetable
exlensir ely useil as antimicrobial (Bagamboula er al'' 2003 )'
Medicinal plaots possess [umerous active constituents comprised phenolic
compounds which are found significant undfl stress condition induced by the ftee
radicalsanddisplayantimicrobialproperties(Hata-Kldoetal-'2004).Plantsplaykey
role to improve the human health and axe comprised of some active phltoconstituent
and perform different molecular aad physiological actions in the living organisms
(Hi11, 1952). Nowadays, trumercus secooddy plant products ale tested for the
occurelce of [ovel drugs exhibited new pharmacological activity- Padicularly plants
have their ability to proaluce trumerous secondary piant products (Caslello et al '2002)'
Cunent research is focused mostly on identificatiol and isolation of novel
compounds ftom higher plants for the cure of a panicular disease (Ernrk et al ' 2006;
Mohanta e, at, 2007). Biologically active compounds comprised alkaloids' tal]nins'
flavonoids, cardiac glycosides, tdterpenes, anthraquinones and sterols obtained liom
15
Eedicinal plants play key rote in physiology, nutritiotr and contl'ol ofdifferent diseases
(Sofowar4 1993). Tanrins betolg to the goup ofphenolic compourd responsible for
various phamacological properties such as spasmolrtic actioo oa smooth muscles
(Tona et a1.,2000), body protection fiom various oxidative sbess Aoshizawa et al '
1990). Saponins contol Nal*-Ca2* balance and Na* eff1ux in our body aad thus
minimizingthecardiacfailueandoxidativestress(SchleiderandWolfling,2004).
The glycosides participate in hpoglycemic dysfunction diseases (Athtar e' 41' 1984)'
Few of the fat soluble secondary plants obtained Aom extract of p1atrts are
excellent afltioxidants comprised vit&'nins and the phenolic constituents The
interaction of these compounals with the fiee radicals is also aftaid in frustration'
Current reports present that use of fat soluble rrot protect cels Aom the oxidative
damages but encourage the antitumQr agent in animal and apoptotic cell loss in the
cancerous cell lioes (Henry et al ' 2OO5) About 4000 flavonoids are used as an
element of humar foodstulls (Hemy et al ' 2005) At presetrt' due to antioxidant
properties of the phenolic compounds and flavonoids' their study become more
interestiflg. Some ofthe phenolic compouads which are isolated from medicinal plants
exhibit various biological activities fol instatrce enz)anatic hhibitroy activity. reactive
oxyge[ species scavenging, g€fiomic regllation and prophylaxis agaillst number of
human ailments (Sriaivasatr e' al , 2005)'
ln PakistarL about 6000 species of flowering plants are repoded and of which
400-600areofmedicinallyimportance(NasirandAli'19'72)However'Doeaiier
t6
rcports arc available r€garding ethnobotany' phltochemical screeoitrg' altibacterial
activity and antifirngal activity Keq'ing in view' the present stlrdy was designed with
the following objectives:
.! Inventorying medicinal plants from the study area'
.i. Screening importalrt ph]'tochemicals ftom selected medicinal plants'
1. Assessment of antimicrobial activity of some selected plants
.l Determioi[g aotioxidalt activity of some medicillal plants'
2.1
Chapter 2
ETIINOBOTANY
INTRODUCTION
The use of plants as dedicament is a very old traditiotr date back to human
civilization. Documentation of the most pimitive civilizations demonstates that plant-
based medicines woe formerly utilized by Roman' Eg]?tian' Greek' Chinese'
Babylonians, peoples of India and Pakistan (Khan' 1991) Etlrnobotany is a wide telrll
often referring to interrelations of humans a'nd plant species The fundamental faith of
human being on plant life for their live was principally started with domestication that
wasdatedbacktol0,000yeals(Martin,1995)'Theveryearlydocumetrtationofflora
mostly used by the human show their worth in health' shelter' clothing' economy and
food. Ftom platrt species, humans also obtained thek medicines' food' pesticides'
fodder, tools, constniction materials, fuel, and also their aesthetic and as well as sacred
fulfillments.ThereforenativeiDfomationonmedicinalplantspeciesappearedwherr
th€ humans being leamt and sta ed to utilize the plants species (Posey' 1999)'
Etbnobotany works principally to provide the starting method for the purpose
ofresearch activities, suitable for collectitrg information on the different uses ofplaDts
species and show once more the medical krrowledge handed down by the ordiaary
peoples make up the sources of j formation which is also valuable for rcsearch
activities and for several plant species utilized itr special traditions' when revealed
t'l
t8
beneath systematic examinations, it is valuable for special region industries'
coosequently the traditions and science have a firm association with one another_ The
science has frequently traditional derivation (Letrtifli' 2000) Nowadays' ethnobotatry
is generally acklowledgeil as a science of human being interaction with plant species
atrd its ecosystem. Etlnobotany is the combination of multiple discipline such as
ecology, antlropology and botany (Bhatti e' aL' 2001)'
Etlmobotadcal wisdom is part of cultuIal ecology of the area that shows
associations of the communities with their envtonmetrt This knorvledge has great
significance for the continued existence of the Peoples in a natural ecos)rstem'
Therefore ethnobotanical case stu'lies involve the associatioD between peoples and
plants. In the center ofthese things, the managemeflt of the plant species diversity by
indigenous people is made though the conventional use of medicinal plant species'
Plants also offer health secudty to the people in mral area al1 over the planet'
According to the report ofthe WHO, in the developing countries more than 80 percent
ofthe peoples mostly useal the cooventional medicine for their main health necessities
(Famswo*h and Soejarto, 1991)'
Ethnobotany is somewhat recently introduced to Pakistan' The counky hosts
about 6000 plant species olthe flowering plalts and amongst thetrT ' about 2000 species
have medicinally imPortant (Sher et at ' 2011)' Rural communities piacticed such
plaots thrcugh herbal practitioners (Hakeems) for teating various diseases The
presetrce of few of allopathic doctors itr the inaccessible regions and reservations of the
I9
side effects ofcurrent medicines forced the local citizens towards traditional healthcare
systern. These inaccessible areas possessed the old tradition of the use of local plants
forteatingvariousdiseases(Shinwari&Gilani,2003;Battachadee,2004;Pralapatiet
al, 2004). The young generations are no more inierested in indigenous knowledge and
as a result, such folk knowledge is decliing Studies urged tlat such valuable
traditional knowledge of plant species should be doqrmented before it's completely
lost (Reddy el al., 2OO5). Keeping this into consideration, the current study was
designed to document the ethnomedicinal uses of medicinally impoftant plant species
of Malakand Division, and to use this information for the evaluation of aotimicrobial
aod antioxidant activities.
2.4 RXI'IEW OF LITERATURE
Ethnobotany is more than simply a study of plants usefu1 to people' It is also
devotcd to unilerstaDd the limitation and behavioural consequence of the hman
population\ actiotr on their platrt envionment (Blattj' et al ' 2001) Medioinal plants
and plant-derived medicine are widely used in traditional cultures all over the world
and they are becoming increasingly popular in modem society as natural altematives to
s),lthetic chemicals (Wyk and Wink, 2004)'
An ettrnobotadcal suvey wals carried out ftom Camaguey' Cuba and reported
111 platrt species belotrging to 96 genera and 55 families from the study area- These
species are used in the teatmetrt of 173 local health problems in the study area (Belra
et al, 2004). Different ethnobotaDical studies have been carried in differctrt part ofthe
world. Qureshi and Bhatti (2008) conducted out an extensive ethnobotanical survey
ftom Nara Desert, Pakistan. They rq)orted 51 plant species belonging to 43 genera and
28 families which are being used medicirally by local inhabitants of the Nara Desert'
They reported 21 species possessilg trew uses not being recorded in the Indo_Pak
medicinal literature.
Ethnobotanical field is well recognized throughout the world and a 1ot of work
has been repo ed. Rasool (1998) repoded 15 medicitrally importatrt plants from
Saltanat of Oman. He described various parts of species which are used for the
treatmentofvadousdiseases'TheethrromedicinalpropertiesofplatrtsftomTianMu
Shafl Biosphere Reserve of Zhejiang provitrce, Chioa has been repofted by Chudary u
'
al. (2006). They discovered various folklore recipes of67 medicinal plants'
Purkayastlia et al (2005) carried out a survey of medicinal plants of Dibru-
SaikhowaBiosphereReserveofNortheastlndia.Theyreported55plantspecies
belotrging to 34 families and 52 different genera Ji el al ' (2004) reported the medico-
ethnobotany ofNajiatrg, Nothwest Yun-oan, and China They described 52 medicinal
plant species belonging to 32 families used for the teatment of vaious human
ailments. Among thern, 11 species werc repolte'l as rare and 16 werc commercially
utilized. Botrdya and Shama (2004) conducted a survey of medicinal plants used in
diabetes in Jhar}:laad and collected 11 plant species with rernarkable uses'
2A
The folk uses of medicinal plants of Calalabrin were lecorded by Passalacqua
et al. ir 2007. They identified 104 taxa distdbut€d into 42 families The ethnobotaDy
ofNaldiforest,Kenyawasreportedbylerutoetal.(2007).Tleyiderrtified35
medicinal plants which are use'l for the control of various human ailmellts- Mohan e'
al (2008) suweyed Tirunelveli dish'ict and thus recorded a total of 80 species
belooging to 72 genera and 46 families Likewise' Rabram and Raju (2008) studied the
therapeutic properties of 2l dude drugs ottained Aom plants which are used for bone
Aactue by the natives of Eastem Ghats Vidyasagar and Prashafltkumar (2007)
reported the medicinal ptants ofBidar distdct Kamataka' lndia and identified 18 plants
species belonging to 13 families and 18 genera salazar e' a/ (2008) recorded he6a1
remedies used fol gastoointestinal diseases ftom Maxico They identified 85 different
species which belong to 3 8 families Likewise, Ugurlu and Seoman (2008) itrvestigated
tho medicinal plants of the Yunt Mountains They idetrtified 52 mediciMl plants
belonging to 32 families. Of them, 41 species were wild and 13 species *ele
cultivated.
OlijviiK et at.. (2013) conducted field survey for the extraction of traditional
medicinalusesofplantsforthetreatmentofdiabetesfiomDhaka,Bangladesh.Theil
results showed the presence of 37 medicinal plants belonging to 25 families wbich are
being used for the treatment of diabetes in Bangladesh They discovered that most
Aequently used speci es werc Coccinia indica, Azadirachta indica' Trigonella foenum-
graecum, Sltzygium cumini, Teflninalia chebula' Ficus racemosa' Mornord'ica
21
chatantia and Swiete a ahagoni' Wet et al' (2013) caried out an ethnobotanical
survey oflorthem Maputaland, South Aftica They reported 47 plant species belonged
to 35 families for the treatment of 11 different skin disorders including abscesses'
acne, bums, boils, iDcisions, nngworm, rashes, shingles' sores' wounds and warts'
The etbnobotany in Pakistan is going to be matued with the passage of time
aodvadousstudieshavebeenreported.Matin€1,/.,(2oo?)studiedtheShogranvalley
(Hazara) and identified 7? species of herbs' 12 species of shrubs and 18 species of
tees, which are used for medicinal purposes Khan and Khatoo[ (2004) studied the
etbrobotany of Harartrosh and Bogrote valleys of Gilgit Agency' and Northem Areas
of Pakistan. They identified 260 species aod described 20 species of ethnobotaoically
importance. Khan et al. (2011) cotrducted a study of Khunjerab National park'
Pakistan and reported 43 plant species betongilg to 40 genera ard 28 families- out of
these Asteraceae (11.63 %) is the leading family followed by Fabaceae' Lamiaceae and
Rosaceae (9.3070), Chenopodiaceae and Elaeagnaceae (465 %) These species were
used by local people for curing different twes of diseases'
2.3 MATERIALS AND METIIODS
2.3.1 TEE STUDY AREA
Matakand division is situated in the Nofthem Areas' Pakistal lt lies at 34' 33'
56" North, 71" 55' 52" East (Figure 1) The population of Malakand divisioo is
heterogeneous, wiih etlnic divercity inlabiting in 7 district having population of about
23
5.5 milliotr. Most ofthe people speak Pashtu in plain areas, while Kohistani and Gujur
languages spoken in the hilly areas. Elevated MountaiD ranges, glaciers, sllow roofed
peaks, and lush green forests distiaguish the region. Elevation of Malakaod ranges
from 770 to 2705 feet above mean sea level. The area is bound by the River Indus in
the east, Bajaur agetrcy in the West and Afghanistat in the North West as well
Northem arcas in the North. Agriculture is the main occupation (607o) in the Malakand
divi'sion. Yousafzai pathans tribe is the most prominetrt trib€ in the Malakand division,
followed by Syed, Miagan, and Gujarc.
2.3.1.1 Climrte:
The summers are mild, while wint€rs are harsh and long. The highest
maximum temperatue was recorded dudng the month of June was 33'C, while the
minimum was in January (i.e. ll"C and -2"C). The average means annual rainfall
reccrded as 1416 mrn. Precipitation is in the form ofrarnfall and healy snow fall starts
by the end of November on high peaks and descend downwards as temperature falls
till March. The snowfall usually starts ftom the month of December to the month of
March and remaios up to the months of Jutre and July on elevated mountains,
inqeasing the beauty of the valley (Anonl,mous, 1998).
21
lr,tf lluhmAm
lttqab
Sampling
sites
Figure 1: Location map of the study area.
,, ':j
The whole study area was visited for the documetrtatiotr of traditional
knowledge of medicinal plants by using semi-structured questioDnaire and oral
discussion (Qureshi and Bhatti, 2008). Local people including elder people and herbal
practitioner (Hakeems) were interviewed for the record of medicinal uses of native
plants. FurtherDore, for cross-verification of medicinal uses, vadous villages were
visited. Twenty informants between ages of 30-80 were randomly selected for
acquiriag medicinal uses of native plants. The importance of plant knowledge was
quantified through fidelity level (FL) for each species. In this case percentage of
itrformant's coNensus about the use of a particular species was calculated for the same
use by given below lormula:
)5
2.3.2 COLLECTION OF ETIINOBOTANICAI INFORMATION
FL (o/o) = Np X 100
2.3.3. COLLECTION AND IDENTIFICATION OF PLANT SPECIMENS
lf, = number of informants that reported a particular use of a species in the
same disease, and -l/ = total number of informants that codfirmed its medicinal use in
different diseases (Alexiades and Sheldon, 1996).
Duing the suvey, voucher specimens were made and mounted on standard
herbaium sheets. These were identified by Dr. Ratmatullah Qureshi and coafirrned
26
through flodstic material Q\lasir and Ali 1970-1989; Ali aod Nasi 1989-1992; Ali and
Qaiser, 1993-2009), The fully determined vouche$ were placed in the Deparbaent of
Botany, Pir Mehr Ali Shah Arid Agriculture University, Rawalpindi for turther
reference.
2.4 RXSULT S
2.4.1 MEDICINAL PLANT NVENTORY
The ethnot otanical knowledge of plants revealed that native people use 41
platrts species belotrging to 38 genera and 32 families to heat various human diseases.
Results of ethnobotadcal inventory are proyided in Table 2.1 that shows alphabetical
order of platrts, Vemacular names, family, parts used and medicinal uses. Lamiaceae
was the leading family that co[tributed 5 species, followed by Moraceae (3 spp.) and
Astemceae, Euphorbiaceae and Solanaceae (2 spp. each), while the rest of families
shared each a single species.
2-4.2 Fidelitypercentage
With references to fidelity percentage, Zi.iphus sativa was suPposed to be a
highly used medicinal plant havng 96%o FL, followed by Teucrium stocksianum
(92%FL), Citrullus colocythis & Myrtus communis (88Yo FL each), Medicago
27
polymorpha & Verbascum Thapsus (68%oFL each), Mentha spp. (64Yo FL), Monotheca
bwifol;a (56%FL) and, Melia azedarach (52%FL), while the rest ofspecies valued less
tlan 50% fidelity by the natives (Table 2.1).
2.4.3 Disease treated
Altogether 27 diseases were mostly common in the area and likewise keated by
41 native plants by the inlabitants ofthe study area. Cotrstipation was supposed to be
?revalent in th€ area and likely the highest number of plant species (9 spp.,13.24%)
were experimented and utilized to cule the said ailment (Table 2.2). constipation was
followed by abdominal pain (1 spp., 10.29%), diarrhea (6 spp., 8.82%), indigestion
Pain & Iaflammation (4 spp., 5.88%), throat paii,/tonsillitis, dysetrtery, vomiting, boils
and cough (3 spp., 4.41%).
2.4.4 Mode of application
Altogether 10 methods of applicatiotr were used by the natives while treatitrg
common diseases in the area (Fig. 2.2). Juice and paste were mostly used as
applicatiotr (18% each), followed by powder and vegetable cooking (14% each),
decoction (11yo), eatm raw (9%) and poultice (7%), while rest are scarcely used.
2.4.5 Parts used
Local people and Hakeems of the study area consumed every part of the Plaat.
However, a specific palt of plant is used as per need of the user. In all, 10 parts of
plants were utilized for maki[g recipes fo! the teatment ofvarious diseases (Fig. 2.3)'
28
It rcveals that leaves were highly preferred by the natives (44%), followed by whole
plant (20%) and fruits (18%), while the rest ofparts were of limited use.
2.5 DISCUSSION
Present survey revealed that natives arc very much knowledgeable in teEns of
the use of plants and repoded 41 plarts species (Table 2.1) for teating 27 various
acute and chronic ailments (Table 2.2). According to plant families, Lamiaceae was
fouod to be the most important that had 5 medicinal species, followed by Moraceae (3
spp.) atrd Astemceae, Euphorbiaceae and Solanaceae (2 spp. each). The current work is
ir endorsemert of the study canied out by Qureshi and Bhatti (2008), who rcported
about 51 plants belonging to 43 genera atrd 28 families, which were used for curing
various human diseases by local people of the Nara Desert. Their work supports the
way of usage of prcseot findings in addition of some more knowledge about the
medicinal plants from this area.
The area is scarce ro terms of moistule and resultantly constipatioo was
predomhant itr the area and similarly large number of platrt species (i.e. 9 spp.,
13,24%) were tested and us€d to treat this complaint (Table 2.2). This was followed by
abdominal pain (7 spp., 10.29%), diarrhea (6 spp., 8.82%), indigestion pain &
inflammation (4 spp., 5.88%), thoai pain/tonsillitis, dysentery, vomiting, boils and
cough (3 spp., 4.41%)- This qle of studies hase been reported by various sceintists
29
such as Kumar et al., (2004), Snvastava and Pandey, (2006), Pradhan and Badola
(2008), Rout and Thatoi (2009).
Various methods were used for making traditiooal recipes by the inhabitants
for teating corunon diseases in the area (Fig. 2.2). Juice and paste were preferably
used as application (18% each), followed by powder and vegetable cooking (149lo
each), decoction (1 1%), eaten raw (9%) and poultice (7%), while the rest arc scarcely
used. The findings ofpresent study are in lines with various studies such as (Saikia e,
al., 2006; Qureshi and Bhatti, 2008; Martirez and Ba6oza, 2010).
The people of the area and herbalists (Hakeems) used every bit oppotunity to
utlize natural resouces (Bhatti et al.. 1998). The people of the study area consumed
every part ofthe plant, though; a particular part ofthe plaot was plefened to prepare of
recipes. In all, 10 parts ofplants were utilized for maki[g rccipes for the teatment ol
various diseases. Results revealed that leaves were used as first hand for making
indigetrous recipes (44%), followed by whole platrt (20%) and Iiuits (18%), whereas
the remaining parts were scarcely used (Fig 2.3). Results presented here endorsed by
various sciedtific studies carried out across the world (Saikia el al, 2006; Martinez and
Barboza, 20 I 0; Aderutu e/ a/.. 201 I : ocvirk er al.. 2013; W a et al., 201 3 1
There are no organized shops for purchasing and selling of medicinal plants,
therefore n]ost people go to the field for ftesh collection as well as the nealest market
for selling plant products. Interestingly, the resrrlts of the interviews correlated well
between the different villages, the same species being used in similar ways in diflerent
villages.
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42
Table 2.2: Cornmon diseases treated by the tratiye species of the study area.
Sr. No. Disease treated Plant species Percentage
I Constipation 9 t3.24
2 Abdominal pain 7 t0.29
Diarrhea 6 8.82
4 Indigestion 4 5.8 8
5 Pain & Inflammation 1 5.88
6 Throat pai tonsillitis 3 4.41
7 Dysentery 3 4.41
8 Vomiting 4.4t
9 Boils 4.41
l0 Cough 3 4.41
11 Blood ptrrification 2 2.94
12 Jauldice 2 2.94
13 Nausea 2 2.94
14 Typhoid fever 2
15 Tonic 2 2.91
16 2 2.94
t7 Iever 1 L.47
18 Stomach ulcer I 1.47
19 Appetizer I 1.47
20 Johts pain I 1.4',1
2l Anti-dandruff I 1.47
22 Ringworm I 1.4'7
23 Pesticide 1.t.4'l
24 Diabetes 1 1.11
25 Asthma 1 1.47
26 Flu 1 1.17
27 Skin irritation 1 t.47
63 100.00
11
ct Latex
Figure 2,2: Mode of application ofvarious recipes.
:15
2
Lrt"r Resin Root
2 -"'
Aerial part4Yn
Figure 2,3: Different plant parts used in folk recipes.
16
CONCLUSIONS
The study area sustaim variety of medicinal platrts which are customary used
by the natives to fuIfill their medical requirement. The inhabitants especially herbalists
(lldfeem) possess indigenous knowledge regarding medicinal plants and their
taditional way oftheir use for curing various diseases. However, it has been observed
that this knowledge is retained amongst few people atrd not all of familiar about their
uses, pafis used as well as their time&ame collection and storage. In the case of
professiotral pmctitioner (Hakeem), t}Iey were properly awate about their collection
time and herbal use. The indigenous knowledge is very rapidly at decline due to
availability ofmodern medicine as well less interest taken by the young getreration in
leaming indigenous knowledge. The ethnobotanical leaming is rhe need of time in
order to document the native infomration ofthe plants ftom the local inhabitants ofthe
area.
Chapter 3
PIIYTOCIIEMICAL SCREENING
3.1 INTRODUCTION
It has been estimated that about 250,000-500,000 plant species exist on the
earth aDd a very small fraction has been scientifically tested for ph),tochemical,
biological and phanaacological screening. pharmacological activity was sought from
ethnobotanical rq)orts of valuable plant species and the[ compounds isolated and
tested that rcsulted into sources of healing. However, tatrdom selection ofplants for
determinitrg new active compounds has also been found the area of research in
afltibiotics (Gerhartz e, al, 1985; Kroschwitz and Howe-Gmnq 1992).
About 35000 plant species are traditiotrally used thoughout the world as
medicaments. There is increasing tend amongst the researchers to find natwal
products especially for cancer, viral and miclobial diseases (Hof6rann et al., 1993,
Harvey, 1999; Sdnivasan e1 al.,2001). Globally, nea y 20% plants are tested for
pharmacological or biological activities. Based o11 results it is reported that
biocompounds isolated from plants can be used as drugs, dyes, fiagrance and
pesticides (Hamburger ard Hostattman, 1991). Currently, there is steady increase in
demand for plant based products that has resulted in manifold increase in trade of such
products a6oss the world. Hence, today researchers are interested in isolateilg
41
48
secondary metabolites ftom plants and their pharmacological activities are being
itrvestigated that could Iead as a source ofmedicament (K-rishnaraj.u et al-,2005).
It is an estallished fact that medicinal virtues of plants govem by some
important chemical constituents of plant body Of them, the most leading ones are
phenolic compounds, tafifns and alkaloids (Edeoga e, a1, 2005.Considering
significance of phyochemicals, some selected medicinally important plant species
\/i2., Monotheca buaifolia, Myrh6 communis' Teuc um stocksiakum (L)' Teucriun
stoclisianufi (F). Vefiascutu thapsus and Zi4phus satil'a were selected for
ph),tochemical screedng to establish their antimicrobial and antioxidart activities.
Prcviously, no reports are available for the establishment of aforesaid activity except
the author (Rahim e, a 1.,2012 alJd 2Ol3).
REI'IEW OF LITERATURX
A wide range of mediciIral platrts is used for the extraction of raw drugs having
medicinal properties.Differetrt parts ale used including rcot, steE, flower, Iruit,
exudates and other modified plant organs. For local use, plants are collected in smaller
quantities by the local communities and folk healers; however these drug platrts are
collected in larger quantities and haded in the market as the lav/ material for maIly
herbal industries ojniyal et a1.,2006).
49
Plants have beetr exploited for treahnent of human diseases by differetrt ethnic
groups in different parts of the world since the dawn of civilization These traditional
cultures use such resources without proper scientific evidence for keahnent of humatr
diseases. Therefore, to ascertai[ the medicinal value of the ph]tochernicals,
pharmacological studies have been can'ied out around the world (Prusti et aI ,2008)'
These phyochemicals from medicinal plants also sefle as lead compounds in drug
discovery and design (Ebi and Ofoefule, 2000).
In recent yeaN, the growing dernand for herbal products has led to a quantum
jump in volume of plant materials traded within and across the counkies. In lecent
yea6, secondaxy plant metabolites (phytochemicals), previously with uaknown
pharmacological activities, hav€ beetr extetrsively investigated as a source ofmedicinal
agerts 0GishnaEju et a1.,2005).
In herbal medicine, crude plant extracts in the form of infusiotr, decoction,
tircture or herbal extract arc traditionally used by the population for the treatment of
diseases, hcluding infectious diseases. Although their efficacy and mechanisms of
action have not beeo tested scieltifically h most cases, these simple medicinal
prepamtions ofteD mediat€ beneficial responses due to their actlve chemical
constituetrts (Bames et a1.,2001).
50
Nafural products perfom various functions, and many of them have interesting
and useful biological activities (Harvey, 1999)' Planrderived products contain a great
diversity of phltochenicals such as phenolic acids, flavonoids, tannins, lignin, and
other small compounds (Cowaa, 1999). These compounds possess nui)erous health-
related effects such as antibacterial, antimutagenic, aiticarcinogenic,antitllrombotic
a$d vasodilatory activities (Bidlack et a1.,2000). A wide range of ph)'toclemicals
present in plants are known to inhibit bacterial pathogens (Cowai , 1999l, Medina et al ,
2005; Romsro et al.,2}Os) Successful determrnation of such biologically active
compounds from plant material is largely dependent on the t)?e of solvent used in the
extraction prccedure. Organic solvents such as ethanol, acetone, and methanol arc
olten useal to extract bioactive compounds (Elof[ 1998).
Alkaloids are naturally occurritrg chemical compounds containing basic nitrogetr
atoms. The name alkaloid is derived from the word alkaline and is used to describe any
Ditrogen containing base. Alkaloids can also be defioed as heterocyclic nitrogen
compounds that are rcported to be useful against Huma[ lmmunodeficiency Virus
(HM inl'efiion (McMahon et al. I995).
The medicinal value of plants lies in some chemical substances found in the
platrt body. The most important of these bioactive compoutrds of plaats are alkaloids'
tannins and phenolic compounds (Edeoga et a1.,2005). Medicinal plants represent a
rich source of antimicrobial agents. Plants aie used medicinally in diffelent countdes
and are a source ofmany potent atrd powerful drugs (Srivastavaet al'' 1996)'
5I
Plants possess potent biochemical and are integral part ofph)4omedicine si[ce
times imm€morial. Man has ability to derive/extract useful chemicals from any part of
plant i.e. bark, leaves, flowe$, roots, fruits, seeds, etc (Makad e' al' 2008) Many helb
species such as thlme, sage, oregano showed antioxida[t activity itr their extracts
(Panovska €/ al., 2005).The ph)toconstituents like phenols, antlraquinone' alkaloids'
glycosides, flavanoids and saponins are antibiotic principles of plants Frcm these
phltoconstitueflts, saponins have been repoted to exhibit hemol]tic and foaming
activity, antifungal, anti inflammatory, fungistatic, rrolluscidal properties (Ajay et al''
2011). Most antimicrobial secondary metabolites have relatively broad spectrum of
activity. The specificity is deterrnined to whether the pathogen has the erzlttes
necessary to detoxiry a particular host produ ct U atBtter, et al', 1994)'
Flavonoids are importallt atrd largest group of natural compounds with v'ide
distribution in plant kitrgdom. Thousands of flavonoids have been isolated and
characterized ftom various organs of medicinal plants and ale a valuable source of
protection against the pathogens and damaging radiation such as ultraviolet mdiation
(Harbome and Williams, 1998).
Ilavonoids are polJphenolic compounds present in all foods of plant origio They
have various effects on mammaiiao cellular systems and stmctues (Melzig, 1996)' and
52
protect biological membrares against Aee radical-induced oxidative damage
(K a}awa et al., l9g2). The strong antioxidant effects of flavonoids have been
higl ighted by several studies (Katan et al., 1998). Flavonoids may have therapeutic
efects on disease co[ditions caused by oxidative strcss, such as coronary
aiherosclercsis, ischernic damage, diabetes mellitus, aging processes atrd cancel
(Haragpchi et al., 1996).
Flavonoids are constitutiv€ compounds but are also s}'ntbesized by plants in
rcsporse to microbial infection (Dixol et al., 1983). Nearly half of the 200
phltoalexins charactedzed up to now belong to the flavonoids (Harborne, 1988).
Flavonoids have been found to show ir? vil'ro antimioobial activity agaiNt a wide
mtrge of microorganisms, some showing potent activity against MRSA (Iinuma er al,
1994). Their activity has been attributed to thei ability to complex with extracellular
and soluble proteins and to complex with bactedal cell walls (Cowan, 1999).
Accordhg to tlis brief history it is very much clear that human belngs are
familiar twith platrts alrd use tiem for their medication and dietary supplements. Their
bioactivity Aom the last decade have beeo widely explored and recogDized tlroughout
the world. Irts of work have beetr done in the past to fitrd out their phal[racogical
effects, ftee radical scavenging activities, phyochemical screetring etc. But there is
need to evaluate the relationship between i,, virro and ettrno botanical usage of a plants
53
for the welfare of humarity. So, the present research is designed to contdbute
something new in this field of research.
3.3 MATERIALS AND METIIODS
This piece of research work was undertaken in the Depaftrent of BotaD, Pir
Mehr Ali Shah Arid Agriculture U ve.sity Rawalpindi, Pakistan.
33.1 Medicinal Plants Selectiotr
Five medicinal plants compdsing Monotheca bu:tiJolia, Myrtus communis,
Teucrium stocksia utt, Verbascum thapsus, aad Zizipus sativa were utilized for
ph)4ochemical sdeening, proximate and mineral analysis, atrtimicrobial and
antioxidant investigation. The detail of place of collection, Vernacular lames,
coordinates, pictures and other relevant information is provided under Table 3.1 and
54
Table 3.2: Prolile ofplants used as ethno medicine itr Malakand divisiotr.
Sr.
No
Botadcal Platrt
habit
PartStation Attitude Longitude Latitude
1 Tree Leaves Talash Tangi 1483 N34'46.701 E071'56.924
2Myrtus
Stnb Balu 116,1 N34049.698 E071'57.110
3 HerbFloweIs,
Osakai Ghat Now 1053 N34.42.169 E0? l" 5',7 _',7',1 6
Herb Leaves Talash Gumbatkai 1365 N34'46.175 E071'56.081
5
ZizipusTala..h Tesi 1483 N34"46.701 E07t'56_924
55
Table 3.t: Profile ofplants used as ethno medicine in Melakand division of
Pakistan.
.:. BotanicalName:
btLxifuliu
Family:
Sapotaceae.
vernacular
name: curgura
English Name:
But berries:
.:. BotanicalName:
Myrtus
Family:
M)rtaceae
Vernacular
name; Mano
English name:
Myrtle
56
Botanical Name:
Teucriu l
Family:
Lamiaceae
Vernacular
name: Spare
botay
English Name:
Mountain
Celmander
I
Botanical Name:
Family:
Scrophulariaceae
Vernacular
name: Gidar
Tambaku
English Nalnc:
Great Mullei
57
Botanical Name:
Zizyphut
Family:
Rhamnaceae
Vernacular
name:
Unab/Markhanai
English Name:
Jujube l'ruit
58
PREPARATION OF PLAIiT EXTR.ACTS
Fresh plant flatei.als of Monotheca buaifolia, Myrtus cofimunis, Teucrium
stocksianum (L), Tellcrium stocksianum (F), l/erbascum thapsus and Zizyphu\ sativa
were collected Aom gowing localities of the study area that were washed thoroughly
with distilled water atrd desiccated under shade. They were powder (80 mesh) through
grinder and kept for futule analysis in refrigerator. The resultant powder samples were
soaked io methanol; ethatrol and water and vigolously shaken fo! 24 hours at 37'C It
was passed through Whabaan filter paper No. 1. The obtained filkates were evaporated
at 40"C under by using rotary evaporator till the menstrum dispersed atrd tumed to a
greenish color concenbate. Finally through water bath, lest of solvetrts traces were
evapomted. The final obtained residues (extracts) werc afterward subjected to
phltochernical screenin g.
3.3.3 CRUDE EXTRACTS YIELD
ln order to obtain the percentage of crude extacis yietd, following formulae
was used to calculate the same by followhg the protocols of Dellavalle et al- (2O11)l
Yield% = Weight oflyophilized extact x 100
Weight of powdered smple
3.3.4
59
PIIYTOCIIEMICAI SCREENING TEST
The phltochemical screening was PerforEed otr Aom olganic solvent exhacts
such as methanol, ethaool atrd water, \/hich wele prqrared for detecting alkaloids,
amino acids, carbohydrates, fixed oils and fat, flavoooids, glycoside, gum and
mucilage, phenolic compounds, phltostercl, protein, saponins, ta nins and terpenoid
by following statrdard methods of Harborne (1973), Trease and Evatrs (1989) and
Sofowom (1993). The results of tests tests articulated as positive @) or negative (N)
qualitatively.
3.3.4.1 Detection of Alkaloids
33.4.1.1 MaYer'sreagent
A little amount of crude extact was liquefied in 5ml of 1% hydrochloric acid'
It was then filtered and tested with Mayer's reagent. The appearaice ofprecipitation or
tEbidity with the reagents indicated the presence of alkaloids (Harbome, 1998 and
Sazada et a1.,2009). For positive control, berberine was employed and the negative
3.3.4,1.2 Wagner's reagent (Iodire in potassium iodide)
Few drops of Wigner reagent was poured in test tube containing few ml of
extact. Appearatrce of reddish-brown precipitate revealed the hcidence of alkaloids
60
(Wagner, 1993). For positive control, Berberine was employed and the negative one
3-3.4,1.3 Hager'sr€agent
Few ml of extract werc mixed with 1 or 2 ml of Hager's reagent The presence
of yellow precipitation was indicative of positive test (Wagner et at, 1996)' Fot
positive cotrtol, berberitre was employed and the tregative one v/as water'
3.3.3.1,4 Dragendroff s test
This method is defined by Waldi (1965) in which few ml ofextact was mixed
with 1or 2ml of Dragendrofls reagent. The occurence of prominent yellow
gecipitatiotr indicated the presence of alkaloid. For positive conhol, Berberirre was
employed and the negative one was water.
3-3.4-2 Determination of Amino acids (Ninhydrin test)
Two drops of ninhydrin solution was mixed io 2 ml watsr extract The
occulrence of purple color was indicative of amino acids in extract (Yasuma and
Ichikawa, 1953). For positive contrcl, tyrosin€ was employed and the negative control
was water.
61
3-3.4.3 Detectionof Carbohydrates
The I OO mg platrt extract was liquefied in 5m1 water and filtered The same was
used in followiDg tests (Ramlcrishran e/ al, 1994):
3.3,4.3,1 Molish'stest
Alcoholic solution of alpha-naphthol was taken in 2 drops and poured in 2 ml
water extract. It was shakeo and supplimeflted with 1 ml concentrated sulphuric acid
along test tube until settle down' The appeaxance ofviolet ring signified the occurrence
of carbohydrates. for positive control, glucose was employed and the negative conhol
3.3.4.3.2 Fehling's test
One ml filtrate was poured in a test tube and 1 rnl each of Fehling solution A
and B was supplimented and boiled on water bath The red precipitation indicated the
presetrce of sugar in the sample. For positive control, glucose was employed and the
oegative control was water.
3.3.4.3.3 Barfoed's test
One ml filterate was added ia I ml Badoed's rcageot atrd boiled ofl the water
bath for 2 minutes. The formation ofred Ptecipita on showed ftee\isteoceofsugarin
62
the plant sample. For positive conhol, glucose was employed and the negativ€ control
was water.
3.3.4.3.4 Benedict'stest
The 0.5 ml filterate \r-as mixed *ith 0.5 ml Benedict's reagents and warmed on
a water bath for 2 minutes. Appeaxance of red color precipitate marked the incidence
of sugar. For positive cotrtrol, glucose was employed and the negative one was water-
3.3.4.4 Detection of FlaYonoids (Alkaline reagent test)
Tw6 ml of water filterate of plant extract was keated with l0oZ ammonium
hy&oxide solution, The presence of yellow fluorescence showed the prcsences of
flavonoids. For positive contIol, quercetio was employed atrd the [egative otre was
3.3.4.5 Detection of fLxed oils and fat (Saponification test)
Few drops of alcoholic potassium hydroxide (0.5 N) mixed with little quaotity
of plant extract and phenolphthalein. It was then warmed on watet bath for 2 lrours'
Froth formation or partial treutralization showed the occurrence of fixed oils and fats
(Kokate, 1999). For positive contlol, balsam was emPloyed and the negative one was
water.
63
33.4.6 Detection of cardiac glycosides
The plant extract taken iII 50mg quantity was boiled on water bath for 2 hours
with concentated hydrcchloric acid and then filtered. This hydrolysate was used in the
following tests:
3.3.4.6.1 Bortrager test (Evans, 1997)
Two mt filtered hydrolysate was dissolved in 3m1 chloroform and was shaketr
vigorously. The chloroform layer was sepaeted and then 10% ammonia solution was
added. The occurence of pink color indicated glycosides in the extract For positive
control, digoxin was ernployed and the negative one was water'
3.3.4.6.2 Legal's test:
The plaot exhact in 50 mg quatrtity v/as suspended in p,Tidine Few drops of
sodium troprusside solutiotr Eixed in it and 10% sodium hy&oride was adde'l to
make alkaline mixture. The prcseoce of pink color exhibited the occurrence of
glycoside. For positive control, digorin was employed and the negative oDe was watel'
was useal as positive aod water as negative contol'
3.3.4.7
64
Detection ofgum and mucilage
The 100 mg exfact was suspended in 10 ml distilled water. It was then added
with 25mt absoiute alcohol by regular moving. The occurrence of white or cloudy
precipitate was marked the presence of gums and mucilage (Whistler and Be Millet
1993). For positive con ol, acacia gum was employed and the negative one was \'ater'
3.3.4.8 Detection of phenolic comPounds
3.3.4.8.1 Gelatin test
The extract (50ng) was suspended in 5ml distilled water adding with 2ml of
l% gelatin solution contai trg 107o sodium chloride. The white prccipitate exhibited
the preselrce of phenolic compotnals (Evans, 1997). For positive control, gallic acid
was ernployed and the negative oDe was water.
3.3.4.8.2 Lead acetate test
The extract taken io 50mg quantity was suspended in water followed by 3 ml of
1070 lead acetate solutiotr. A massive white precipitate showed the existence of
phetrolic codpounds. For positive control, gallic acid was ernployed and the negative
one \{as water.
65
3.3.4.9 Detectiotr of Phltosterols
Fifty mg exhact was poured in test tube and suspended in 2ml acetic anhydride.
Then 1 to 2 drops of concenhated sulphudc acid was amalgamated in it. The reactiotr
in terms of change color showed the presence of phltosterols (Finar, 1986). For
positive conkol, betulenic acid was ernployed and the negative olle was water'
3.3.4.10 Detectionofproteins
_ The presence of protein was detemined by following the methodology of
Fisho (1968) and Ruthmarur (1970). For this purpose, 100 mg plant extract was mixed
in 10 ml water atrd passed through whatmana No.1 filter paper' The following tests
were pedolmed for protein detection:
3.3,4.10.1 Millon test
Few drops of millon reagent mixed in 2 ml plant filtate. Appearance of white
precipitate showed the existetrce of proteins (Rasch and Swift, 1960). For positive
control, bovine serum albumitr (BSA) was employed and the oegative one was water.
3.3.4.10,2 Biuret test
The 2 ml filterate was added with 1 drop of ?o/o copper sufuhate. Then, 1ml of
ethanol (95%) was mixed in it that was followed by adding potassium hydroxide that
66
tumed into pellet formation_ The formation of pilk color in the ethaoolic layer
signified the occulence of proteins (Gahan, l9g4). For positive control, BSA was
employed and the negative one was water.
3.3.4.11 Detection of saponins
The plant extract in 50 mg quantity was dissolved io distilled water atrd raised
up to 20 ml. The solution was stirred vigorously for l5 mitrutes. The development of2
crn layer foam exhibited the occurence of saponins (Kokate. 1999). For positive
control, powder of soapberies was employed and the negative one was water.
3.3.4.12 Detection of tannhs
The extnct (50mg) was suspended in 5 ml distilled water. Few drops of 5%
ferric chlodde solution were mixed itr it. Appeamce of dark green color showed the
incidence of taDnins (Egwaikhide and Gimba, 2007). For positive conhol, tarmic acid
was employed and the negative one was water.
3.3,4.13 Test for terpetroids (Salkowski test)
The 0.2 g exkact was dissolved in 2m1 chloroform and subsequently with 3 ml
concentrated sulphuric acid (HrSOr). The formation of reddish brown color sigEified
the presence of terpenoids. Ior positive control, linalool was employed and the
negative one was water.
61
3.3.5 QUANTITATI\'EPEYTOCIIEI{ICALSCREENING:
Total alkaloids, flavonoids, tannios and saponins of selected plants wele
estimated through following standard flethods:
3.3.5.1 Alkaloids
The quatrtificatiotr of alkaloids was done by following the protocols of
Harbo,Jrc et al. (2005) with minor alteration. 10 I powdered plant sampl€ was added in
200 ml of 109/0 acetic acid in ethanol and left for 4 h. The filtrate was condensed on the
water bath till remained l/46 volume. Drcps of cotrcenhated ammonium hydroxide
were added to the filterate till the appearance ofprecipitation ald then allowed to stay'
The precipitate was washed with dilute ammonium hydroxide atrd filtered The same
was dried and weighed. The aikaloid content was estimated by using following
formula:
Final weight ofsarnpleAlkaloids (9/o) = x 100
lnitial weight of exttacts
3.3.5.2 I'lavonoids
Flavotroids wele determioed by following ihe work of Edeoga and Okwu
(2005) after minor amendment. Ten gram plant sampl€ was extracted repetitively with
100 ml of 80% aqueous Eethaool. This mixture was filtered through whaftian filter
68
paper No 42 (125 mm). The fitkate was then shifted to a coltaiDer ihat was condmsed
on the waier bath arld weighed to a constant weight. The following formula was used
for obtainitrg flavonoid percentage:
Final weight of sample
Flavonoid (7o) :
Ta[trins
Idtial weight of extracts
3.3.5.3
The stock solutions having zeto to 10 ml of tannic acid were poured in eleveo
100 ml volumetric vessels and 1.5 ml Folill-Denise reagent, 10ml sodium caibonate
solution a8d adequate de-ionized watet were added to make 100 ml solution. These
samples were separately shaketr titt a light to dark blue color progressively emerged
and same were kept at room temperatue for 3 hours eoabling the complete color
process. The absorbance was measured at 760 nm through Shimadzu pvlvisible
spectrophotometer 2100 (Toko, Japan). The mean of 10 daia readings appended below
was used for obtaining calib.ation curve @asheed and Haider, 1998; Waterman and
Mole, 1999). The concetrhation of tannin from each sample was calculated through
regression equatio[ acquired from calibration (Fig. 3.1).
x 100
S. No.
I
2
3
4
5
6
7
8
9
10
69
Concentration (mg/100mI)
0.1
0.2
0.4
0.5
Absorbanc€ (O.D)
0.062i0.00
0.143r0.00
0.199+0.00
0.27'1+0.00
0.341r,0.00
0.415+0.00
0.487+0.00
0.564+0.00
0.641r0.00
0.711+0.00
0.6
0.'7
0.8
0.9
'70
U
Absorbance (pg/n )
y = 0.718x- 0.01I
Fig. 3.1: Catibration curve equatiotr for tannic acid'
'7t
3.3.5.4 Saponins
Quaotitative estimation of saponins was made after Obadoni and Ochuko
(2001). About 10 g Platrt sample was mixed with 100 ml of207o aqueous ethanol and
drawn in a shaker for 30 minutes. The same was heated on water bath for 4 h at 55"C
and therr filtered. This residue rc-exhacted with aaother 200 ml of 20yo aqueous
ethanol. Both extacts werc mixed atrd boiled on water bath at 90"C till remained 40
ml. This mixture was shifted to a 250 ml separatory funael and extacted twice with 20
ml diethyl ether. Then ether layer was discarded, while the aqueous layer was reiained
and 60 ml ,-butanol was added. It was then washed twice with 10 ml of 57o aqueous
sodium chloride. This solution was again heated on the water bath and the samples
were drie<l in the oven at 40'C to a cotrstant weight The saponitr content was
cooputed by using following formula:
Saponin (7o) :Final weight of sample
x 100
Initial weight of exffacts
3.3.6 STATISTICALANALYSIS
The data werc recorded in triplicates and expressed as mean+standard deviation
(SD) by applying software Statistk ver.8.1.
'72
3.4 RESI]LTS
3.4.1 PHYSICALPROPERTIXS
The ethanol, Eethanol and water based platrt extacts wele subjected to
percentage yeld for Monotheca buxifolia, Myrtus communis, Teucriunt stocksianum
(L), Teucrium stocksianum (F), l/erbascum thaPsus al,d Zizyphus satitta' It has been
observed that methanol exhibited supremacy in yielding maximum extract, followed
by ethanol and watel The highest extact yield was observed in methanol
(2 I .4+0. 1 0%), followed by ethanol ( 1 9.3+0. I 0olo), whereas, water exllacts yielded least
psrcentage (17.5+0.10). In the case of yield percentage of plant exf:,acl" Z' sati\)a
possessed highest yield extiact, wblle Y. thapsus had the lowest leld proportion
(Table 3-1).
The methanolic extracts yield highest yield tn Z. sati'ra (21-4+0'10%), followed
by M. buxifotia (1g.3+O.l'7%), M. commuhis (11.6+0.06%), T. stocksianum le ves
06.35+0,00%), T. stocl<sianum flowet (14 8+0.10%) ald V. thapsus (9'5+0 10o/o) The
ethanolic extacted the highest yeld ir, Z- sativa (19.3*0.700A), followed by M
b\/xifolia (l8.4rO.2OYo), M. communis (16.1+0.l'1%), T. stocksiahum +lo'rtet
(13.5+0.20%), T. stocksianum leaves (11.4+0.20%) aod V. thapsus (7 '2l.0'10/o) ln'the
case of aqueous extacts, .'l{ bwifolia Md Z. satita collecdvely yielded the highest
amount (17-5+0.06%), followed T. stacksianum leaves (11 3+0 17%), T' stocktianum
floweG (10.8+0.17%) and V. thapslts (8-3+O.l0yo)'
13
Amongst three solvent extacts, the highest extaot yield was obtained ftom
methanol, followed by ethanol ard water' Water exhibited less yield compared to
methanol and ethanol. The reason could be that some compounds such as terpetroids'
carotenoials as well as some phelolic compounds are insoluble in water comparcd to
the other two organic solvent such as methanol and ethanol After cellulose' lignin is
the seconal most abundatrt organic substance in higher pla ts' It comprises about 15-
25% of dry weight of plant species and insoluble in water'
3.4.2 QUALITATIVEPEYTOCIIEMICALSCREENING
Five platrt species M bwifulia, M- communis, T. stocksianum, V thapsu's Dr.d
Z. sattva werc sqeeaed for arious phltochemicals such as alkaloids, amino acids,
carbohydrates, fixed oils and fat, Flavonoids, glycoside, gum and mucilage, phenolic
compounals, phltosterol, prctein, saponins, tannins and terpenoid Aom ttrree solvent
based extracts. Results ofphlochemical screening are provided in Tables 3 2 to 3 6-
Sevea phltochemical classes viz., alkaloids, amino acids, carbohydrates,
flavonoids, phenolic compounds, protein and taEnins were detected by all the tests and
solvetrts based extact of M. btttifolia (Table 3 2). Fixed oils and fat ph]'tosterol and
terpenoid were detected only from methanol and ethanol, while gum atrd mucilage atrd
saponiDs were detected by ethaaol and aqueous extracts, however glycosides were
found abseDt in the piant extact. From M. communis, some ph)'tocheEicals such as
alkaloids, amino acids, carbohydrates, flavonoids, phenolic compounds' protein 3nd
74
tannins wete idetrtified through all the tests as well as solvents based €xtract (Table
3.3). Methanolic and ethanolic extacts traced fixed oils and fat phltosterol and
terpenoid, whereas, saponins detected by ethanol and aqueous extracts and g]m and
Eucilage atrd glycosides were d€tected onlyby ethanolic €xtract (Table 3'3)'
The T. stocksia um leaves had showed alkaloids, amino acids, carbohydmtes,
flavonoids, phenolic compounds, protein, saponins and tarmins by all the tests and
solvetrts based extract (Table 3.4). MethaDolic and ethanolic extructs traced fixed oils
and fat, cardiac glycoside, phltosterol and terpenoid (Table 3 4) In the case of I
stocksiahum flower, amino acids, carbohydrates, flavonoids, phenolic comPounds,
protein, sapo ns and ta.nins were haced by all the tests and solvents (Table 3 5)'
Fixed oils atrd fat, phltosterol and terpenoid were detected in m€thatrolic and ethanolic
extracts, while atkaloids and glycoside were only traced in methanolic exhact (Table
l.s).
Methanolic, ethanolic and aqueous extacts of Z ,raps,s detected amino acids,
carbohydrates, flavonoids, phenolic compounds, protein, saponins and tamins by all
the tests (Table 3.5). Alkaloids, fixed oils and fat, gum and mucilage, ph]4osterol and
terpenoid were detecied in methanolic and ethanolic extacts and cardiac glycoside
were only traced in ethanolic €xtacts, while sapodfls were absent a]ld all solvent
extracts detected that (Table 3.5).
The methanolic, ethanolic atrd aqueous z satit)a le ves extracts possessed
alkaloids, amino acids, carbohydrates, flavonoids, gum and mucilage, phenolic
comporuds, proteiq saponins and tannins as revealed by the tests (Table 3.6). Fixed
oils and fat, cardiac glycosides, phlosterol and terpeDoid were detected by methanolic
and ethanolic extracts; saponhs were found otriy in methanolic extract (Table 3.6).
3.4,3 QUANTITATT!'EPIIYTOCIIEMICALANALYSs
This study revealed the presence of active ph)'to-constituents present in the
studied plants. Therc was sigtrificant difference in amounts of crude phytochernicals
contetrts itr the plant samples collected from different areas of Malakand division. The
rcsult of total cootents of phltochemicals is showed il Tables 3.7. Higlest amount of
flavonoids was recorded io Z. sati'a (0.93+0-0oA), followed by M. coumunis
(0.78+0.00), T. stocl$ia um flower (0.77+0.00%) & leaves (0.71+0.0OYo), M. bwifolia
(0.64+0.00%), while least amourlt was recorded for Y. thapsus (0.33+0.02). In the
same way, alkaloids were dchly present in T. stocksianu,n leaves (0.81+0 00%),
followed by T. stocksianwh flowers (0.78+0.00%), Z. sativa (0-16+0.00%)' M.
b rifolia (0.61+0.00%), M. communis (0.43+0.00%) ard l/. thapsus (0.30+0.0.0).
The high percentage oftannins was present $ Z. sativa (9.7+0.31%), followed
by M. ammunis (9.1'tr}.22%), M. buxifolia (8.2+0.13%), T. stocJaianum leaves
$.910.21%), flowers (6.2+0.3870) alld v- thapsus (0.37+0 12). Saponins were richly
16
found in I stocl{sia um flower (221+000%) and I stocl$ianuh 7eales
(2.13+0.01olo), while ex cept l/. thaPsus, rcst of species possessed less than 0 57o'
3.5 DISCUSSION
The sqeenirg for phlto-constituents through qualitative and quantitative tests
Aom the selected plants demonstated that that all plaots possess important chemicals
such as alkaloids, flavonoids, tannins and saponins All these secotrdary metabolites
are well recognized to possess medicinal properties that exprcss physiological activity
(Sofovr'aia, 1993).
The preseot tesealch screened for 13 phltochemical classes in five selected
medicinal plants of the study area lo ordel to evaluate solvent effectiveness in
detecting chemicals, methanol, ethanol and water extracts werc tested The study
showed significant difference in chemicals in tems of solvents as well as plant extracts
(Tables 3.2 to 3.6). lt revealed that methanoiic extracts were the most effective in
detectitrg ph)4oconstitueots from the selected species' This solvent isolated all
chemicals from I slo ck'sia um le ves arr'd flowers and Z Ja"a' It was followed by ,.
thapsus (ll), M. buxifulia aILd M. communis (70 each) The ethatrol extacts detected
all phltchemicals ftom M. communis, T. stocksianum lea'res, followedby M' buxifolia'
V, thapsus and Z. sattua (12 each), wlttle in T. stock'siafium. l0 ph1'tochemical classes
were identified. In the case of aqueous extracts, maximum phlochemicals were
detected Aom M bu-tifotia (9), followed bv M communis, T stocksianum leaves and
Z. sa,r,a (8 each,), T. stocksianu fTo\\'er ('7) aid l/' thapsus (6)'
'77
T. stocl'iahum leaves aod flower aDd Z. satfua were ich in phytoconstituents
and all were detected in any of solvents (T able 3.7) Teucrium stocksianum was fo:und
highly important medicinal plant of the area that is being used by the natives in treating
various diseases. The highest amount of alkaloids forurd it T stocLsianum leaves
(0.81+0.00%), followed by f. srocft-s ianu/n fiowe$ (0 78+0 00%) and that reflected its
very common use (92% fidelity) for h:eating chronic fever, diarrhea and cough in the
uea.
Saponins were richly found in T. stocksianum flower (2 21+0 00%) and I
stocksianum leaves (2.13.!0.01%), while rest of species possessed l€ss than 0 5%n'
Saponins are lolo'r'[ to cause inhibitory effect on swellings/inflarnmations (Just el al'
1998). These chemicals are responsible in coagulating red blood cells that include
development of foams in watel solutions, hemol)tic activity, cholestercl binding
properties and bittemess (Trease and Evans, 1985; Lewis and Elvin-Lewis' 1995;
Sodipo el a/., 2000; Okwu, 2004).
't8
Table 3.3: Yield (7o) of solvent extracts ofvadous plant species'
Sr.
No.Phnt sample Solvetrts Yield (%)
IMonotheca
bwifolia
Ethanol i8.410.20
Methanol 19.3:0.17
Water 17.5+0.10
2
Myrtus
commufiis
Ethanol 16.1r0.17
Methanol 17.6+0.06
14.5:0.10
Teucrium
stoclaianum (L)
Ethanol 11.4+0.20
Methanol 16.35+0.00
Water 11.3i0.17
4
Teucrium
stocksianum (F)
Ethanol 13.5*0.20
Methanol 14.8+0.10
Water 10.8,r0.17
5
Verbascum
thaps s
Ethanol 7.2+0.1.0
Methanol 9.5+0.10
8.3+0.10
6 Zizyphus satila
Ethatrol 19.3+0.10
Methanol 21.4*0.10
17.5+0.06
79
Tabl€ 3.4: Ph)"tochemi.^l screelitg ol M. bttxifolit''
Phltoch€micals Tests
Solvents
trth.
Altzloids
1) Mayer's lest
b) Drangandroff s test
)) Hager's resr
, Ni*ydrjn iest
CarbohydEtes
) Mol;sch's test
) Fehling's test
:) Benedjct's test
t) Barfoed's test
Fixed oils ed fat ) Saponification test
,With aqueous NaOH solution
clvcoside1) Bort'.ger test
r) Legal's iesl
Gun and nucilage a) With 90% alcohol
1) celadn rest
r) Lead acelat€ test
a)Libernarn burchaid tes!
, Biuret tesl
r) Millon s test
Sapoains ) Foam tesr
Ia ils a) FeCL test
r) Salkowski test
Legenal: Meth. = Methanol, Eth.: Ethanol, Aqu.: Aqueous, (*) Present and (-)
Absent.
80
Table 3.5: Phytochemical screenirg of ,|4. commuttis.
Legeod:
Meth. = Methaool, Eth.= Ethanol, Aqu.= Aqueous, (*) Present and (J Absent'
T€stsSolvetrts
Meth. tr"rh-
r)Mayer's test
b)Drogan&off s rest
c)Hager's test
r)NiBhydrin tesi
Carbohydrates
r)Molisch's iest
b)Fehling's rest
c)Benedicts test
l)Barfoed's iest
Fixed oils atrd fal 1) S apodficalion test
Flavonoids a)With aqueous NaOH solution
Glycoside a) Boreager test
r) Legal's iesi
SuE and mucilage 1)With 90% alcohol
Pheoolic compouds a) Gelalio rest
Ph)iosterol 1) Libermam burchard tesl
Proteinr)Biulet test
b)Millon's test
iaponirs
Ialnins i) FeCL test
Ierpenoids ) Salkowski test
81
Table 3.6: Phytochemical screening of 7. srocksianu, Le ves'
Legend:
Meth. = Methanot, Eth.= Ethanol, Aqu.= Aqueous, (*) Present and (-) Absetrt'
IestsSolvents
Meth. Eth.
Alkaloids
a)Mayer's test
b)Drdgandroi?s tes!
,Hager's lest
l)Wagner's test
b)Ninlydrin test
Carbohydtues
n)Molisch's test
))Fehling's test
l)Barfoed's test
Fixed oils ad fat a)Saponification test
,With aqueous NaOH solulion
3lycoside 1) Boft'ager test
r) Legal's test
Gum and nucilage r)With 90% alcohol
Phenolic compoundst) Gelalin test
r) Lead aceraE tesi
Phlaosterol ) Lihemam burchard test
1)Biuret test
))Millon s test
Saponitrs ,Foamlest
Iandns r) Feclr test
Ierpenoids ) Salkowski t€Sr
82
Table 3.7: Phytochemical screening of 7' stocksianu't| Flower'
SoIYentr
Phytoch€micals IestsMeth. Eth.
r)Mayer's test
b)Drangandroffs rest
:)Hager's test
C)vagner's test
,Ninh-vdrin iest
r)Molisch's lest
b)Fehlins's test
;)Benedict's test
l)Barfoed s test
Fixed oils and fat a) Sapo!.ification test
--=- Eiwirh aqueous NaoHFlavonoids Il\.luhon
Glycoside
)) Legal's test
Gum atrd mucilage ,Wiih 90% alcohol
Phenolic compoundst) Gelatin test
r) Libemann burchard test
r)Biuret test
r)Millon's test
Sapoains a)Foamtesr
r) FeCL tes!
) Salkowski tes!
Legend:
Meth. = Methanol, Eth.= Ethanol, Aqu.= Aqueous, (*) Present and (-) Absent'
Table 3.8: Ph,'tochenicd screenitrg of z "oPszs'
Phytochemicals T€sts Meth. Eth.
Aikaloids
a)Mayer's rest
r)Drangandrofs test
;)Hager's iest
l)Wagner's tesr
c)Ninhydrin rest
a)Molisch's test
r)Fehlins's test
r)Benedict's rest
d)Barfoed s test
Fixed oils and fat i)Saponification rest
a)With aqueous NaOH solution
Glycosid; l) Boft-ag€r tesl
c) Legal's test
3um and mucilage a)With 90% alcohol
Phenoiic r) Gelatin rest
Ph,,tosterol a) Libelmam bwchard test
1)Biuret test
b)Millon's iest
) FeCIi €st
I€rpenoids a) Salkowski test
83
Legetrd:
Meth. = Methanol, Eth.= Ethatrol, Aqu.= Aqueous, ( | Present and (-) Absent'
84
Table 3.9: Ph)'tochemical screening ofZ' salua'
)Benedict's test
Legend:
Meth. = Methanol, Eth.: Ethanol, Aqu.= Aqueous, (')Present and ( ) Absellt'
Solvents
Phltochemicals IestsMeth.
AIkaIoids
I)Mayer's test
,Drangandroffs test
r)Hager's t€sr
))Ninhydrin test
larbohydntes
DMolisch's test
))Fehling's tesr
I)Barfoed's iesl
lixed oils ed fat , S aponilicalion test
t)With aqueous NaOI
tolulion
llycoside r) BorFager test
, Legal's tesr
ium atrd mucilage ,With 90% alcohol
r) Gelatm test
lh),tosterolI) Libermann burchar
l)Biulet i.sl
))Millon s lest
;aponins ,Foam test
lanniff L) Feclr test
rerp€noids r) Salkovski test
85
Table 3.10: Quautification of ph)'tochemicals from selected medicinal plants'
S. No.Plant
codeFlaYotroids (%) Alk.loids (7") TaoDins (%o) Saponins (Yo)
8.2+0.13 0.4710.001 MB 0.6.1L0.00 0.61,r0.00
2 MC 0.78i0.00 0 43+0.00 9.17*a.22 0.53+0.00
3 TCSF 0.7?+0.00 0.7810.00 6.2+0.38 2.21r0.00
4 TCSL 0.71+0.00 0.81:r0.00 6.9+0.21 2.13a0.0i4
5 VT 0.33+0.02i 0.30+0.0.0 0.0+0.0.0 0.3'7+0.t2
6 ZS 0.93+0.0 0.7610.00 9.7+0.31 0.29r0.00
@ anatysis showed that aII the values are
significantly differ at (P < 0.05).
}1iC= Myrtus communis, W= l/etbascum Thapsu' MB= Mof'otheca bwifulia' TCSL:
Teucriutu stock;ianum leaves, TCSF= Teucli rt stocksianutu lTower aIId ZS= Zizipus
sati'na.
86
All piant extracts irdicateal the preseace of flavonoids (Table 3 7) The highest
amouflt of flavonoids was ilocumenteil iL Z sativa (0-93+0 0%)' followed by '14
communis (0.78+0.00%) and T stocLsianu flower (0'?7+0'00%) and leaves
(0.71+0.00%).
Flavonoids are watel solubl€ antioxidants that diminish the development of
carcinogenesis (Okwu and Okwt, 2004)' In response to microbial infection' flavonoids
are also slarthesized by mediciral ?1ants (D ixot et dl '
1985) Except M bttxifolia (ooly
methanolic), all platrt extracts showed flavoooids These compoulds dimitrish
carcinogenic process and iDhibit oxidative cell from damage and possess anticancer
and other biological activities (Ok!v'u, 2004, Lewis and Elvin-Lewis' 1995) such as
antimicrobial and anti-itrflammatory actvities (Cushnie and Lamb' 2005)'
' Present study also exlibited the existence of cardiac glycosides in the platrt
o(tract. Except M. btt:tifolia, the rest of examined plants possessed glycosides- This
gou? ofph)tochemicals is used to stimulate cardiac failure (Olail*.d et al ' 1992) aid'
lower blood pressure (Nyarko and Addy, 1990)'
Present study detected vaious phetrolic compounds such as flavonoids' tatrnins'
etc- Phenolics are the biggest and most abundatrt group of ph)4ochsmicals (Singh el
al.,2007). bia$e et al, (2009) reported same kind of rcsults Aotn A hispida znd A'
Terpenoiils were foud itr all methaoolic and ethaaolic extracts and found absent
in aqueous extract because these chemicals are lipophilic aod caDnot be detected in
87
water. Terpenes derive aroma in plants due to presence of esseDtial oil They are
manufactuedfiomisoprenoidunits,resernblinginfattyacidsinitsproperty(Cowan,
1999).
Tannins were richly present in inZ salivd (9 ?+0 31%)' followed by M communis
Q.17,r0.22%), M. buxifotia (8.2+0 13%), T stoclisianum leaves (69+021%) and
flowers (6.2+0.38%), while they were absetrt in L rfidpsl$ This phltochemical class
can act as aotidiarrheal, antiflrngal, antihemorrhoidal aad antioxidant agents (Asouith
and Butter, 1986). Furthermore, this possesses birier principle of drinks and foods
(Chikezie e, al-, 2008).
CONCLUSION
The present study discovsred various phltochemical constituents ftom selected
medicinal plallts of Matakand Division. Several previous studies confirmed the
occufience of detected ph]'toconstituents that contfibute therapeutic properties in
treating various diseases. Shce different solvents exhibited diversity in detecting
ph)4ochemical, hence, such extacts could be used as a good medicament This study
alsovalidatedtheethnobotanicaluseofthesespeciesinthestudyaleaduetothe
presence of phltochemicals in aqueous solution Furthermore' it is suggested that the
identified ph)'to-constituents may be used as valuable leservoir of searching bioactive
compounds il these plants that loay prove to have significatrt medicinal merits'
Chapter 4
PROXIMATE AND MINERALS ANALYSIS
4.1 INTRODUCTION
During past decade, much interest has been found in nutaceuticals; however
there is meager information about the prcseoce of phlto-cotrstituents which derives
dwable health enhancing or medicinal properties' Nutritiotral compounds have an
important role in the diet that can benefit human health by continuing use of them
(Korver, 1998). Ploximate and minerals analysis of fruits and vegetables is playing an
important role that highlights their nutritioDal importance Many medicinal plants are
utilized as food due to their medicinal effects atrd their evaluation may lead to
significancant in understanding of their valu e (Pax'dey et al'' 2006) It is reported that
oedicinal platrts are still used ia primary lealth needs of rural people and suppod
about 807o of the marginal communities round the globe (Prajapati and Prajapati,
2}Oz,I-atif er al. , 2003).
Prcximate alalysis provides important information on moisture' ash and
mineral contents. The ash possesses inorganic 'esidue
and orgatric matter r€sulted after
removitrg by heating. Total ash may vary witbin species due to variability of nature or
physiology of ash. Minerals are normally present is ash and trot luhed by heating and
they also have relatively low volatility contastiag to rcst of food compone[ts'
Therefore,measurernentofmineralcontmtisimportant'sincetheymayexpress
pharmacological effecs (Lee, 2005)' Carbohydrates, fats aDd protein are important part
88
89
of complete diet of humatr being Protein is richly fou'd in seeds of plants and their
quality and. quantity assessment may lead in selection of plants for nutritive value'
systematic classification atrd plant imprcvernent progmms (Nisar et al''2009)'
Plants possess vanous amounts ofessential nutients, minerals and fibrc (Gafar
and ltodo, 2011). The leaves anal fresh fruits of Morikga oleif'zra arc cooked as
vegetables and supposed to enhance immune systems The plant was analyzed for
proximate anal)sis to seek.iustification ofits dietary use (Ogbe atrd AtEL-u' 201 I )'
In Pakistan, very scaoty information is available regalding net chernical
composition of medicinal odenlal plants, which must be known for the basis of dietary
constituents preserlt in foods. Although the country has 6000 ptant species and out of
which more than 1,000 species have been repofted having medicinal values (Latif e/
al, 2004; Mushtaq et al, 2009). Keeping in mind five plaots viz'' Mottotheca
brnifolia, Myrtus commu is, Teuct[um stocksianum' Verbascutt thapsus and Zizipus
sarva were collected and anal]zed for proximate and minerals analysis'
4.2 REVIEW OF LITERATIJRE
Proximate atralysis in plants gives valuable information and help to access the
quaLity ofthe samPle. It provides infomation otr moisture content' ash content' volatile
matter contentr ash, fixed carbon etc. Ash is the inorgaeic residue remaining afler
water aIId organic matter have been removed by heating, which provides a measure of
90
total amount of minerals within the food Minerals arc not destoyed by heating and
they have a low volatility as coflpared to othel food componetrts Total ash may vary
with in wide limits for specimen of genuine drugs due to variable natural or
phlsiological ash. Ashes give us an idea of the mineral matter contained in a plant'
Measuritrg it is importallt, because mineral matter may be the cause of a
pharmacological effect (Surgglu, 2005)'
Chemical compositions ofleaves ofneem (l zadiruchta indica)' sajna (Mo/i ga
oleifera), aryr (Terminalia aiuna)' tnlsi (Ocimu sahctum)' tnrrne{c (Curaoia
longa); thizomes of glnget (Zingibel ofrcinale) and tu:rmeic- k:uits of a$ld (Enblica
offcinatis), haitaki (Terminalia chebula), bohera (ferminalia belerica) and bulbs of
gaiic (Allium sati'twm) of indigenous origin wsre determined' Proximate and mineml
components [Calcium (Ca), Phosphorus (P), Sodium CNa), Potassium (K)' Maglesium
(Mg), Copper (Cu), Zinc (Zn), Manganese (N&r)l were determine (Bhowmik e' a''
2008).
Proximate analyses of two traditional gdn legumes consumed in Fastem
Nigeria- Bambara gro:rllLdirvl (Voadzeia subtetranean) and African yam heans
(sphetrcstylis stelNoca?a) were cadied out Bambara groundflut was found to contain
2.86L0.02% moisture, 32.4Gi0.02% protein, '7 35+ O'02% fat' 5 78+0 02% ash'
2.68LO.O2oh qude fiber and 51.78r'0 02% total carbohydrates African yam beans
contained the following: 1.96+ 0 02%o moisture, 31 '21+O Ozoh plotei'ls' 9 49+0 02%
fat, 5-35+0.02 Ash, 3.55+0.02 crude fiber alld 44 4t0'02Yo total carbohydiates The
resultindicatesthatthetwounderutilizedfoodlegumeshavehighproteitrcontentwith
91
Africatr yam beaos having slightly higher value of the protein trutdelt They also
contain varying levels of ash content, crude fiber and carbohy&ates- This reveals that
barnbara groutrdlut and Africatr yam beans are important food sources that can be
exploited paflicularly in the developing coutrdes where there is shortage in animal
protein and utrder nutrition facing many families in developing countries (Nwodo and
Nwinyi,2012).
4.3 MATERIALS AND METIIOD
PREPARATION OF PLANT MATERIALS4.3.7
The plant matedals were washe'l thoroughly and air dried in the laboratory at
rcola tempemtule for four weeks. Each plant sample was grinded to a coarse powder
alld stored in a glass container for further use'
4.3.1.1 PROXIMATE ANALYSIS
The prcximate alalysis of the powdered plant samples for dry matter' moisture
and ash c-ontent protein, fat, fibre, was undertaken by fotlowing the methodology of
Moses el al. (2012).
4.3.1.1 .1 Determinatiotr of Dry Matter
The pladt saEple was taketr in a bottle a1ld mired with water' The watel
cotrtetrt was determined by 1{eighing out 2 g of the sample into a silica dish which has
92
been previously heated aad weighed. The same was dried in the oven for 24 hrs at
100'C and then allowed to cool for 10 minutes in desiccators before weighing'
Following formula was used to calculate the dry matter'
Dry matter = 100 - % of moisture
4.3.1.1.2 Determination of Moisture Content
Two grams ofeach ofplant sample was weighed into dded weighed crucibles'
The sample was put into a moistule extraction oven at 105'C and heated for 3h- The
dried sample was hansferred into desiccatoN and then allowed to cool and reweighed'
This process was repeated until a constatrt weight was obtained The difference
between the weight was calculated a pelcentage ofthe original sample'
W2-W3 x 100
Moisture (Yo) - x 100
w2-w1
Where
Wl= weieht (epms) of emptY dish
w2= weicht (erams) of dish and sample before heating
W3= weight (grams) of dish and sample after heating
4.3.1.1.3
9l
Determination of Ash Content
Two grams of each sample were weighed into crucible, heated in a moisture
extaction oven for 3 h at 100'C before being transferred into a mume fumace until it
turled white and fiee of carbon. The sample was then rernoved fiom the fumace,
cooled in desiccators to a room tsmpemture and reweighed immediately The weight of
the residue was ihetr calculated as ash content expressed in percentage _
Weight of ash x 100
Percentag€ Ash Yo =
Wl= Weight of sample = 2 gln
W2= Weight of ash = x
Weight of sanlple
4.3.1.1.4 DeterminatioDofCrudeProtein
For determining crude prctein, fliffo Kjeldahl meihod was used foliowing the method
of Moses el al (2012). Two grams samples dissolved in 10 ml coDcentrated H2SOa in a
heating tube. It was added with I tablet selenium as catallst and heated inside a tume
cupboard. After that the mixture was shifted into a 100 ml flask and raised the volume
with distilled water. Ten milliliter mixture was mixed with equal volume of 457o
NaOH sotutioD atrd then traNferred to kjeidahl appantus' The obtained distillate
mixed with 470 boric acid containing 3 drops of methyl red as indicator' Fifty ml
distillatewastakenandtitrated.Thetestwasperfomedthreetimesanavetagevalue
94
was calculated. The calculation for nitogen coltent was done and multiplied by 6.25
to get qude prctein content.
(100xNx14xvDTPercentage Nitrogetrl00 7o -
100 x Va
Where; W = Weight of sample
N = Normality of the titrate (0.lN)
vf = Total volume of the digest : 1 00ml
T = Titre value
Va = Aliquot volume distilled
4.3.1.1 ,5 Determination ofFat Content
Two grams sample was loosely wrapped with a filter paper and put into the
thimble which is fitted to a clean rouad bottom flask, which has teen cleaned, dried
and weighed. The flask contained 120 ml of peboleum ether' The sample was heated
with a heating mantle and allowed to leflux for 5 h. The heating was then stopped and
the thimbles with the speot samples kept and later weighed The difference in weight
was receiveil as mass offat and is expressed percmtage ofthe satnple Tle percentage
fat cotrtent is calculated as follows:
PerceDtage fat 70 =W2 W1 x 100
w3x 100
95
wtere
W1: Weight of the empty extaction flask
W2= Weight ofthe flask and oil extracted
W3= weight of the sample
4.3.1.1 .6 Deterrrination of Crude Fibre
Two grams sarnple was put into 200 ml of 1 257o of HzSO+and boiled for 30
minutes. The solution and content then poued into Buch-ner funnel equipped with
muslin cloth and secured with elastic band This was allowed to filter and residue
washed with hot water to free it fiom acid The residue was then put into 200 ml
boiiing 1.25% NaOH and boiled for 30 min, then filtered lt was then washed twice
with alcohol. The matedal obtained was washed tfuice with petroleum ester- The
residue obtained was put in a clean dry crucible and dried in the moisture exhaction
oven to a constant weight. The dried cmcible was removed' cooled and weighed The
difference of weight (i e. loss in ignition) is recorded as crucible fibre and expressed in
perceotage of the original weight'
w2_wl x 100
Percentage crude Iibr€ 7o =
W1= weight of sample befole incineration
W2: Weight ofsample after incineration
W3= weight ofodginal sarnPle'
w3
43.1.1 .7
96
Determination of Carbohydrate Content
The carbohydrate content was established by using tritlog€n fiee method as described
by Ai- Khalifa (1996) and Moses el al (201,2). Tlr'e carbohydrate is calculaied as
weight by difference between IOO aod the summation of other proximate palameters
such as crude protein, crude lipid, crude fiber, ash and moisture The formula is given
below:
Carbohydrate cortent = 100- (% moisture + % protei +% crude lipid + crude 6b€I + % ash)
4.3.2 MINERA.LANAIYSIS
The mineral ion cotrteflts ofth€ species were determined through wet digestion
method as illustrated by Association of Official Analytical Chemists (1990) The
studied pammeters include sodium, calcium altd potassium One gram powdered
sample was assimilated itr 10 ml coocenhated nitric acid and heated until the
production ofred nihous oxide fumes stopped lt was allowed to cool and then l0 ml
perchloic acid was supplemented to it This mixtue was again heated and reduced to
small ftactioo. The digested filtate was dissolved in distilled watel and inqeased its
volume up to 50 ml. The readings ofmiaeral ions were obtained in ppm tbrough Flame
?hotometer (Sherwood model 360) ard converted into percentage by using following
formulae:
Reading xDilutiotr factor(% )=
10,000"Sample weiglt
97
(% ): Reading x 100
10,000"1
4.3.3 STATISTICALANALYSIS
The data were recorded in triplicates and expressed as mean*standard deviation
(SD) by applying software Statistix ver.8.1.
4.4 RESI]LT
4.4.1 PROXIMATEANALYSIS
The proKimate analysis of medicinal plants viz., M. bt-txifolia, M. cohtuunis, T.
stocJctianufi leaves, T. stocksianum ftowa, V- thaPsus and Z. satira i,s provided in
Table 5,1. It reveals that the high percentage of dry matter was preser't fi M. bux;folia
(92.3+0.10%), followed by l/. thapsus (92.3+0.10%), T. stocksianurn flowet
(91.8+0.10%), T. stocLsianum leaves (91.4%), Z. sativa (90.7+0.100/") at;d M.
cammunis (90.5!0.70%). The moisture contetrt was highest in M. communis
(9.5+0.10%), fotlowed by Z s(ltira (9.4+0.170 ), T. stocksianum leaves (8.6+0.20%),
T. stocksian m flower (8.2+0.10%), V. thapsus (8+1.00%) and M. bltxifolia
(1.7+-1oo/o). The maximum ash content was recorded ;1 T. stocl$ianum leales
(11+1.00%), followed by T. stocksianum flower (9t1.00%), Z. sativa (s+l.O}yo), y.
thapsus (6.5+0.lly.), M. b xifolid (6+1.00%) ar,d M. cotnmunis (4+7.00%).
98
The crude prctein content was found in optimum proportion it Z sativa
QO.12rO.Ol%). It was followed by M. buxifolia (7.88+0.02%), I/ thapsus
0 33,10.58%), T. stockj[anutu flower (6.i3+0.01%)' T. stockaianum leaves
(5.25+0.O2Vo) ajad M. communis (4.38+0.01%). In the case of cmde fat, T' stocktianum
nd M. buxifolia flower possessed the highest percentage of crude fat was documented
ir l/. thapnu Q4L1.OO%), foltowed by M communis and Z' sativa (2O+7 00Yo eacl)'
M. buxifolia (18+l.}Oyo) andT. stocksianum leaves (17+1.006 9%)'
Ma,.rimum crude fibre was estimat€d from T. stocksiahum l7o'$et
(8.57r=0.41olo), followedby M. buxifotia and M. coumunis (7.3*0'70%o each), Z sativa
and T. sioclaianum leaves (6.6+0.44yo each), wbile V. thapsus possessed least quantity
(4.G0.44%). All plaDt samples contained high amount ofcartohydrate conteot and the
Eaximnm proportion was detected it M. communis (53 12+0 02yA, followed by M
buxifolia (53.32+O.O2yo), T. stocksia um leaves (51.75+0 01%), T stocksianu flowet
(4g -7't +O .01%), Y. thdpsus (49 .6+0.100/o) dnd Z. satira (36 48+0 '07yo) '
4.4.2 MINERAL ANALYSIS
The results of mineral analysis are Prcserted in Table 5'2 The highest amoutrt
of calcium was presetrt n M. buxifulia (1 7+0 1070), followed by M' communis
(1j+0i6%), l/. thapsus aad T. stocksianum leaves (1 1+0 00% each), however it was
low i T. stacksnnum flower (0.7or0 U29lo) an d Z. sativa tO'1-O lO';\'
The sodium was found in the order of l/. thapsl$ (0 3+0 10yo) > M btrxifulia
(0.3+0-10%) > T. stocksianum leaves (0 2+0 06%) > M- communis, Z- sativa ar.d T'
stocksianum flowet (0.1L0.00% each)' Similarly, the highest amount ofpotassium was
dete(jte.d 1n V. thapsus ( 1 .6+0. 1 0%), followe dby Z' sativa (1 j+0 }Ooh)' T' stocks ianum
leaves (0.96+0.02%), M. burifolia (0.3+0.10%), T stocl'sia m flower (0 7+0'067o)
urd M. communis (0.6+0.06%). The Na,/K ratio was foutrd in the order of M' buxi'folia
stocktianum flowet (0.01) nd Z. sativa (O O5)'
4.5 DISCUSSION
It has been observed that moisture content was relatively low h anal)zed plant
sarnples that may be attributed - to habitat conditioos All these species have the
capability to survive in ha$h climatic conditions, since there is no proper soil plesent
in hilly areas that carr retai[ moistue for the availability to ?lants Otr the othq hatrds'
such samples would hinder the gro*th of microorganisms and shelf life would be
higher for looger period of time. The moisture content of tested plant samples is low
compared to that of,Yy lopia aethiopia (16 04) as reported by Abolaji et al (2007) and
Acaltpha hispida (11.91) by Iniaghe er al (2009)' This is good for the long
preservation and will prevent early spoilage'
2
Nt.
n
s
:
l-
5
IE
\H
:-H
b
I+ +
tr+
H l+ fti.r
i.rli ItJ1r
+
J+=
'|
H
Il+ H
:-to
+;
t+ir.t: H
L)f-L
Jll'fJ
o
ff l+ +t+l+
bt+
o
It9 +l+
aft ti
p\
+ lli-
tiP
l+ u-
Lt+
oo
:_l:lBI
>lols,IEI.<- l
LIxl
+lr,_ I
-l<l
G'
-=
rl
.?
r01
Table 4.2: Min€ral analysis ofselecteil mealicinal plant samples (Ppm) and
pdrcentage.
@ificantly differs at (P < o o5).
Leg€trd:+SD standard deviation
S. No. Plant sample Ca Na K Na/K
1 Monotheca buxifolia 1.7+0.10 0.3+0.10 0.8+0.10 0.3 6
2 M.Jrtus communi! 1.7*0.06 0.1r0.00 0.6*0.06 0.09
3
Teucrium stocksianum
(F) 0.'7'7+0.02 0.1+0.00 0.7r0.060.07
4Teucrium stocksiatum
(L) 1.1+0.00 0.2+0.06 0.97+0.020.21
5 Yerbascum thapsus 1.1+0.00 0.3+0.10 1.6+0.10 0.11
6 Zizipus sativa 0.7+0.10 0.1!0.00 1.1+0.00 0.05
t02
All tested plani samples possessed low ash content compaled to the leaves ofl-
ritidus (22.84yo) reported by Pardey et al' (2006)' Iponea batatas (11'l}y') and
Moringa oteiferc ('15.09%) reported by Anlia et al (2006) However' these results are
higher than that of A. sativuttl (4 84%) rcported by Hussain et al' (2009)' e\cepl M
communis (4.0Vo),
Various studies reported crude protein content irl many plants lsong and Idong
(1997) discovered lower crude protein content it Momordica balsania (1129%') ar,d
Telfaria occidentalis (7 00) compared with present study However' these results are
lower than those of PiPer guineensis Q9 78) and Talinum' triangularc (3100) as
reported by Akindahunsi and Salawu (2005) Nevertheless' present investigation is
comparable with the values of,4 virizlas (16 41) aad S oletaceae (23 74) documented
by Pandey et al. (2006) Plants having rich crude protein content are considered as
good source of protein because it provides more than 12o% of caloritic value from
protein @earson, 1976).
All the studied plant samples had tow proportion of crude libre compared to
that of P. thonni gii (35 03%) as reported by (Ene-Obong and Camovale' 1992)'
howeve{,higherthanthatofGnetumafricanum(4.60%)'M,reans(4.00yo)a;nd
Parinari Polya dra rcported by (Ekpo' 2007) The plants are good source ofcrude
frbre and when consumed that can lower blood cholesterol and may helP in recovering
variousailmentssuchasconstipation,diabetes,hypertensionaswellasbreastcancer
(tshida e/ a/., 2000).
101
The investigated species ha'l mo'lerate amount of fat ihat are compamble with
fifldings of Akiadahunsi and Salawu (2005) ftom Tolinum tiangubre (5'09)'
Aflaranthes hybtidus (4.80) and Abolaji er at (2007) repoded from Gnetu'n articanum
(3.15). Dietary fat increases the palatability of food by absorbing and retaioing flavor
accoraliDg to lhe report of (Anf\d et al ,2006) A diet comprising 1-200% fat required as
its caloric energy is said to be deficietrt fol human being as excess fat coNumption is
implicated in certain cardiovascular disorders (Ant:ft et al-' 2006)
Vadous studies reported carbohydmte conteni from plants Fol instatrce'
Asibey-Berko and Tayie (1999) rep orted' 55 6'1Vo carbohydrate content from f/i6ul"r
tetestris ajrd 54.20Yo froa spinach leaves which are closer to present investigation'
however lower than 750lo ftom sweet potato leaves and 82 8% fiotrl, Cotchorus tridens
leaves.
The results of mineral analysis are in lines with those of Ladan et al' (1996)'
who reported the oore or less same ploportion of calcium in some green leafy
vegetables atrd some wild edible leaves grow[ in Eastem Anatolia' Tukey Calcium is
accountablefordevelopmentandmanagemetrtofbones,teethatrdmuscles(okakael
a1.,2006). Na,4( ratio in the body is important since it is helpful in controlliug
hlpertension (Yusuf e, al, 2007) The present firldings showed values within the range
ofless thatr I (0.05 to 0.36) that signifies that all these plants would reduce high blood
104
pressure (FND, 2002) Some studies are h the line of present findings such as Ihedioha
and Okoye (2011) and Seal (2011)'
CONCLUSIONS
The data preselted revealed that that the studied plants such as Mo otheca
burifolia, Myrtus cotumunis, and Zizip'rs Jadvd contains higher nutritional value and
elemental composition. These species possessed crude fibre and minerals especially
calcium, magnesium and potassium, while, for medicinal p.,dJl,ose' Myrtus commu is
attdTeuciumstocksianu/narrdZ-sati|aevidentedbyprcsenceofashandmineral
coltents. Such species must be utitized ulder the consultatioa of professionals'
Furthermorc, prcximate and mineral composition should be carried for other species
which are codmonly used plaats by the local people of the area uoder investigation
and provided under chapter 2.
5.1
Chapter 5
ANTIMICROBIAL ACTI\'ITIES
INTRODUCTION
Plants arc natural sources and vadous active compounds have been iDvented
through bioactivity coupled with ftactionation aod compound isolation A survey
caried during 2001 to 2002 revealed that about l/4th ofdrugs in the market world were
either natural prcducts or their derivatives (Butler' 2004) Medicinal plants are
evaluated globally fol searching for new drugs and cures for human and animal
diseases. A Iot of work has been done by several scientists for the search of
antimicrobial activity from plants (Lall eI ai ' 2006)'
Various modem drugs are derived from plants based on their potential
applications available in traditional pharmacopeias These plants contain a vast array
of bio-compounds acting as driving force in treating ch'ronic as well as infectious
diseases (Yogeshkumar and Chanda, 2007: Duaipandiyan et al ' 2006) Today'
pharmaceutical irdustry is mostly relyi[g on these natural compounds derived from
plants (Baker eI al , 1995)'
About 20olo of world plant resources have been phamaceutically or
biologicaliy screened arl'l resultantly various new antibiotics are produced and
availableinthemarketareacquiredfiomnatualolsemisyntheticresources.Dwing
105
106
the years 1983 and 1994, the evaluation of plant extacts for antibacterial activity
resulted iII finding new compounds that can be used against Eulti-rcsistant hacteria
(Ctagg et a1.,1999). Common pathogens causing infection comprise Pseudomonas
aerugtnosa alrd S. aureus (Toshkova and A-nnemuller' 2001; Wysocki' 2002; Baggett
and Hennessy, 2004).
There are two logical rcasons for mifiobioiogists to look into plant extracts for
antimicrobiatactivity.ThefiIstofleisthepresenceofvariousphytochemicalsthat
could be used as antimicrobial agents aIId many are even ear]i€r tested in humaa body
(Clark, 1996). The second one is over-prescription and mthless use antibiotics ln the
late 1990s, the use of plant exhacts becartre popular for treatilg diseases- ln this
decade, about 1/3d ofpeople used at least otre alternative therapy in the United States
(Eisenberg e, al, 1993).
Antirnicrobial screening cleals with anitibactedal and antifungal activity of
se]ectedplantexfacts.worldwideithasbeenrecognizedthatsyntheticdrugsal€
exhibiting adverse effects coupled with resistance in microbes alongside such dmgs'
ThistrecessitatedinsearchilgcertaincompoundsftomnatualresoucesThough'
good piece of research work has been perfomed to detetuin€ the atrtimicrobial
activity ofmedicinal plants, br.rt in order to maximize the recovery ofmedicinal plant
antimicrobialsforhumanconsumption,establishmentofoptimalandspecific
extraction condition using solvents systefl is important. Keeping in view, current
research wolk aimed at detemining methanol, ethanol and watel extracts fiom
107
selected medicinal plaots to optimize their antiDicrobial activity against sevelal human
pathogenic microorganisms.
According to Farns\irorth and Soejalto (1991), selection of pla[ts for seeking
bioactive compounds may prcduce pronounced results based otr ethnobotanical data'
Baseal on collected ethnobotanical data (Chaptet 2) and fidelity of more than 607o'
vanous species such as Myrh,ls cofimunis, Verbascum Thapsus Teucrium stocksnnum
Monotheca bu.xifolia and Ziz[pus sativa werc selected to evaluate antimicrobial activity
which is heavily used in treatitrg infectious human diseases by the flatives'
RE!'IEW OF LITERATURE
Vaious groups ofpatients are suffering liom non recovery ofbacterial diseases
caused by multi-resistant strains. Consequently, new itrfections can occur in hospitals
r€sultitrg in high mortality. Natwe has provided plants as souce of medicinal agents
since times immemorial The importance of herbs in the management of human
ailmeflts cafiIot be over emphasized lt is clear that the plant kingdom harbors an
inexhaustibte souce of active ilgredieots invaluable in the matragement of many
intlactable diseases. Furthemore, the active compotretrts of hertral remedies have the
advatrtage of being combined with many othq substances that appear to be inactive'
However, these comPlementary components give the plant as a whole a safety and
efficiency much supedor to that of its isolated and pure active compotrents (Shariff'
108
2001). The screening ofplatrt extracts and plant products for antimiuobial activity has
shown that higher plants represent a potentral source of trovei antibiotic prototx)es
(Afolayan, 2003).
The expanding bacterial resistance to antibiotics has become a growing concem
worldwide (Gardam, 2000). Intensive cale physicians considei antibiotic-resistant
bacteria a sigdficant or major prcblem in the treatment of patients (Lepape et al '
2009). Increasing bacterial resistance is prompting a resurgence itr research of the
antimicrobial role of herbs againsi resistant shains (Alviano and Alviatro' 2009;
Hefiatswatya et a1.,2008). A vast oumber ofmedicinat plants have been recognized as
valuable resouroes of natural antimicrobial compounds (Mahady, 2005) Medicinal
platrt extracts offsr considerable potential for the development oftrew agents effective
against infections currently difficult to u€at (Iwu e' al, 1999) Additionally' the
bioactivity ofplaat extracts depends on the water atrd ethanol concentration used in the
extaction process (Ganor4 2008).
The antioicrobial and antioxidant propedies ofplast extlacts have beelr widely
explored and recogtrized throughout the world From last decade, there is leawakening
in search of medicine from plant base due to their possible culative and less harmful
properties. Lots of work has been ilone with refetence to their pharmacological and
chemical screening of platrts worldwide' Atl such studies shess the oeed to evaluate
corelationbetweeni,yitroacl.|vityandethnobotadcalusageofpla:rtsandtheir
products-
109
Various plant species atrd their different parts have beer investigated for their
antimicrobial activity throughout world, against a wide range of bacterial aud firngal
strains, employitrg a wide variety of techniques atrd different solvents fol extraction'
SiEmonefti er al, (2009) emphasized to assess arltimiclobial activity of plauts against
different kiads of fungi and other microbes. Antimicrobial actiyity ofplant extacts on
baoteria/microbes has been studied by various researcb goups throughout tle world
(Reddy e, a/., 2001; Erdoorul, 2002; Ate and ErdoIlrul 2003; Yogesbkumar and
Chanda. 2007). Lopes-Lutz et al., (2008) stated that the efficacy of plant extacts
against microbes is beyond any doubt.
Antimiclobial activity of various plant exhacts have been investigated against
some skin infectioN by numerous research groups (Pissed el a/, 2009; Ekpo and Etim,
2009; Simmonetti et al., 2OOg). Among various methods employed for assessment of
antimicrobial activity in plants include broth diiution, agar well diffirsion and disc
di$rsion methods (M a,:tinez et a1.,1996). A11 these methods are most commonly used
atrd generat€ reliable results (Schmourlo et al., 2OO5; Yogeshkumax and Chanda'
(2007).
It has been established fact that microorgaoism including Gram positive and Gram
legative bacteda including fungal pathogens cause vadous humatr as well and plant
diseases. Antimiqobial agents have been developed over the years for cuing such
110
types ofdiseases, however rccently these drugs are facing rcsistance against miqobes.
This has necessitated for developing new a.rtimicrobial drugs especially from plant
rcsources. Medicinal plants are gaiDing importance due to their active compounds and
some bio-compounds are now used to prepare va ous pharmaceutical products (Kuete
et aI.,2008).
A more detailed study on antibacterial activity was done evaluating extacts fiom
50 medicinal plants belonging to 26 families (Srioivasan et al,2001) Results showed
that among 50 plants tested, 72% ihowed antimicrobial activity . Anethum grareolens,
Foeficul th wlgare dnd Trachyspennum ammi were evaluated for antibacterial
properties using agar diffusion method. Hot water and acetone seed extracts had
rcasonable atrtibacterial activity against most of tlre bacteia except Klebsiella
pneumoniae and Pseudomonas aeruginosa. Furthermore, their minimum inhibitory
concefltratiotr for aqueous anal acetotre seed extracts was within the ratrge of 20-80
mglml and 5-15 mg/ml respectively (Kaur and Arora, 2009).
Suaeda fructicosa ax,d Limonium echioides wete sceened for chemical
compositiotr of the votatile cofftituents ftom the aerial parts ard reported 65
compounds. The oils also ilhitited the grovth of Staphylococcus aureus, S.
epidermidis, Micrococcus luteus, Escherichia coli atd Salmonella typhinur[utfl except
Pseudomonas aerugtnosa. Besides, both halophyic oils failed to exprcss artifungal
activity against all the test firngi (Sardana e, al, 2008). The antibacterial activity oftle
111
methanolic extracts of aerial parts of Datura inoxia and, D_ stramonium has been
carried out a9aiirsl Pseudomonas aeruginosa and Escherichia coli. Extt ct of Datura
inorio showed maximum activity at 2.5mgml agarnst Bacillus subtillis, Enterococcas
faecalis ajid Staphylococcxts aureus, whereas, D. stramonium exlibited least
atrtibactedal activity at 2.5mg/m1. Besides, both the plants exkacts showed little or no
activity against ,rcrerichia coli ard, Pseudomonas aeruginosa (Eftekhar e/ al, 2005).
Ali et al., (1998) examined the ethanolic and aqueous extacts of fwenty
Palestitrian plant species for their biological activities agahst flve bacterial stains
(Staphylococcts aurells, Escherichia coli, Klebsiella pneumoniae, proteus lulgais and
Pseudomonas aerugitxosa) and one yeast spec\es Candida a/ricans. The results
slowed that plant extracts had 90olo antimicrobial activily, with reasonable variations
ir activity among different plant species. Latha and Kannabian (2006) carried out
antimicrobial activity of aqueous, methanol and n-butanol extracts of aerial pa.rts of
Sola um trilobatum though disc diffirsion method. The exhacts of leaves, stem,
flowers and Auits exhibited antimiqobial activities against Gram positive and Gram
Negative bacteria. The methanolic stem extract showed maximal antibactedal
activities agaiflst Staphylococcus auteus. "lhe aqueous extract showed midmum
irhibitory cotrcentation (MIC) against tested bacteria rarged betweer 0.06-0.5 myml.
tt2
Antifungal activity of aqueous extacts of 22 Palestinian plants was evaluated
against nine isolates of Microspot"utt canis, Trichophytott mentagrophytes al,d.
Trichophytok violaceum. AII extracts considerable reduced colonies of these
dematophles by 36 to 100% compared with coltrolled treatment. Antimycotic
activity of extracts varied considerably (p<0.05) among plants extracts of Capparis
Spinosa aad Juglans regia completely inlibited the growtb of MiclosPorum canis and
Tichophyton violaceurn. The most active extracts were of Anagallis aruensis,
Capparis spinosa, Juglans regia, Pistacia lentiscus and. Ruta chalapewis againsl M
canus, Inula viscosa, J. regia ar,d P. lentiscus againsl T. mentagroPhytes. The MICs of
these active plants extacts were ranged fiom 0.6 to 40 pgl ml (A1r et al.,1999).
Sitrce the introductiod of atrtibiotics. in the 1950s, the use of plant derivatives as
antimicrotials has been almost nonexistent. The interest in usiflg plant extracts for
treatment of microbial infections has improved in the late 1990s as conventional
antibiotics become unsuccessful (Cowan, 1999) For instance, none of the convetrtional
altifungal drugs used to date seems to be ideal in efiicacy, safety and antifungal
spectum (Ablodepp ey et al., 1999). Even though some novel drugs have heen
introduced for the treatrnent of inflexible fungal iniections, such as caspofim8ne and
allylamines (yicefie et a1.,2003), and combitratiotr treattretrt is occasiotrally used to
make the treahnetrt more valuable, there is a actual need fol a next generation of safer
and more effective antifungal drugs (Bartoli e, al., 1998).it is prcgressively more
difficutt to ileliver new antibacterial leads by modifying hlown antibacterial
compounils. Therefore, the focal point on much antibacte al lesearch has moved to the
113
identificatiotr ofnew chemical classes (Barkeq 2005). Antimicrobial compounds Aom
plants origin may also i:rhibit fungiAacteria through various mechanisms tharl
syrthetic antibiotics, and thercforc be of clinical value in the treatment of resistant
microbes (Elo{ 1998a).
<1 MATERIALS AND METEODS
5.3.1 COLLECTION AND IDENTIFICATION OF PLANI SAMPLES
The selected plant species were collected from different locations of Malakand
division of Pakistan and brought itr the Ta,\onomy Lab., PMAS-AALJR. These were
ideltified By Dr. Rahmatullah Qureshi. Fresh material ofPlatrts was examined and the
old, insect and fungus-infected leaves wele removed. Al1 materials were washed with
tap water atrd all plant parts wete made into small pieces. These were dried in open
airlunder shade. The plant sarnples wele finely powdered using a laboratory grindiog
oill and stored in refiigerator until extaction ExPosue of plalt material to sulr light
was avoided in order to prevent the loss ofbioactive compounds.
5.3.2 PREPARATION OF PLANT EXTRACTS
Differetrt solvents viz. methanol, ethanoi and water was used for extraction of
various plant parts. lnitially the sample was extracted with n-hexane (1:10) by shaking
for 24 hours at 37'C followed by centrifugation at 10,000 rym for 15 minutes'
Supematant was transfered to falcoa tube and residue was extracted with next solvent'
The same proiocol was followed l{ith all solvents and extacts was subjected to
114
drjmess in hcubator/rotary evaporator. The rotary evaporator was also used in order to
cotrcenhate the quantity of extract. A 250 ml of extracted liquid was also subjected to
rotary evaporator in order to evaporate all the solvent from the liquid extract. Water
bath was used to remove the last taces of solvent ftom extract. The water bath
temperatue was also adjusted to round about 40 oC and complete evaporation of the
rclevent solvent the resultant exkact was also stored in fteezel at -8 oC fol the process
of bioactivity purposes. The resultant dried extacts were dissolved in
dimethylsulfoxide (DMSO) for antimicrobial activity (Al-Bakri e, al, 2007) various
concetrtrations of all plant extract (i.e. 15mg/ml to 5mg/ml) were prepared and kept in
refrigerator.
IN Z'TRO ANTIBACTERIAL ACTII'ITY
5.3.3.1 Preparation of Inocula
Bacterial culture was prepared in sterilized Lauia-Bertimi media gL-1 (10 glrt
twtopha4 10 gm NaCl, 5 g!1 yeast extact mixed with distilled water) in separate test
tubes which will then be placed in shaker incubator at 37oC for 24 hours'
5.3.3.2 Antibacterial Susceptibility Test
Antibacterial activity was undertaken by Agar-well diffrrsion assay
fHeEaiswarya et al., 2009). The Lauria Bertini agar media was Pepared (Bertani,
2004), and autoclaved at 121"C for 15 mi[utes which was then cooled and poured in
lI5
autoclaved petd plates utrder sterilized conditions ofthe safety charnber. Wells (6mm
diam.) were bored in each plate by a sterile borer. Bacterial hocula were prepared
Aom ovemight grown cultures (24 h) in Luria broth atrd the tubidity was adjNted to
about 1.2x108 CFU/ml (Luqman et a1.,2005). Each bacterial suspensiod was spread
over the surface of Lauria Bertini agar plates contaidng 4 wells. These wells were
fil1ed with different plant extacts concentatiors (5, 7.5, 10, 12.5 and l5mg/ml). The
.final volume for each cotrcentation was maintained at lml ftom which 100 pl poued
itr each well. Tle standard antibiotics viz., Chlorampherricol, Ciprofloxaci! and
Penicillin were made as stock solution (2 mg/mi for 15 and 12.5mg/ml ofplant extracts
and 1mg for rcst of plant extract concmtrations). For each of pla.t extact
conce[tratioD (i.e. 15, 12.5, 10, 7.5 and 5mg/ml extlacts), various dilutions such as 30
lglrLl,25 pgml,25 pg/ml,20 pglml and 15 pg/ml were taken ftom drugs stock
solution respectively. The Plates were also incubated overnight at 37'C for 24 h The
results were presented in the form of inhibitiotr zone (Hemaiswarya et a1.,2009).
DMSO and water were used as tregative codtrol and the above mentioned standard
drugs as positive controt. A11 the assays were done in triplicate and the rcsults wele
given in mean + S.D. For interpretation ofresults intemational criteria appended below
was used to seek significance of the p1a[t extacts in the form of zonatiotr (Ayatollahia
et al.,20lO):
116
Zone oflnhibiton (rrm) Level ofsignificarce
0 No act;viry
9-11 Nol sigdlicart
12'14 Low activity
15-17 Good activity
Above 18 SigaificaDt activity
5.3.3,3 SelectedBacterialStrains
Following six bacterial species were used itr antibacterial assay:
(i) Staphylococaa aureus (ATCC 6538)
(ii) Staphylococcas epidermidis (ATCC12228)
(ii, StrePtococcus pyogen (ATCC 8A.A-946)
(ir) Pseudomo as aerugtnosa (ATCC7221)
(r) Escherichia coli (ATCCI5224)
("i) Klebsiellapneumoniae (ATCCUC57)
These pathogens were prccuted ftom Quaid-i-Azam University lslamabad
Depadment of Biochemistry for the evaluatioD of antibacterial activity of selected
o:edicinal plants such as Myrrs communis, Verbascum Thapsus, Teucrium
stocksiahum, Mo otheca btalfulia ar.d Zizipus sativa'
5.3-3.4 Preparatiotr of sample dilution
Five concentrations vi2..15,72.5,10, 7.5 and 5 mg/ml ofplaDt extEcts wele used
to evaluate aotibacterial activity (Walter e, a/., 2011). For tlis pupose, sample extact
tt7
in 15mg quantity was dissolved in 10 ml of DMSO and further diluted up to five
concertratioN of the extact. Different dilutions ratio of plant extract along with
DMSO is given below:
S. No. Con.(mg/ml)
1 15
r l? 50
3 10.00
4 '7.50
5 5.00
Stock Sol. (mI)
1.00
0.833
0.666
0.500
0,344
Final Vol. (ml)DMSO
0.00
0.167
0.334
0.500
0.666
i
1
1
I
1
Besides, standard antibiotic (Chlommphenicol, Ciprofloxacin and Penicillin) and
DMSO ard {ater were used for positive and negative control'
5.3.3.5 Media for bacteria
Lauria bertini media 500m1 was used for media preparation'
5.3.3.6 Turbiility staBdards (McFarland)
The 0.5 McFadand was prepared by mixin g 99 5 ml of 1Yo (vhr) sulfuric acid
to 0.5 ml 1115% (*lt') barium chloride dihy&ate (BaClz'2HzO) solution The
turbidity standard is then further aliquoted to the test tubes rnatching to those used to
prepare the inoculum suspension. Then 01% sodium azaide was also added as
118
pteservative- The McFarland standard tubes were sealed with Parafilm to prevent
evaporation. White precipitate ofbarium sulfate was shaken in the tube before use. For
the accuracy of McFarland statrdard, the absorbatrce (0.08 to 0.1) was checked at a
wavelength of625 nm.
5.3.3.7 Physiological saline
Sodium chloride (NaCi) in 8.5g was dissolved in one lihe distilled water,
autoclaved and sotred with tightened caps at loom tempenture fol a period of 6
months in order to prevent from evaporation.
5.3.3.8 Tissue equivalent vs. activity
In order to seek tissues required for activity, following fomula was used:
Weight of raw materiavwt. of extract X MIC
5-3.4 ..Nr'
'?TXO ANTIFIJNGAL ACTII'ITY
The antifungal assay was undertaken through agar tube dilution method
following the procedures of Sultanova et al (2001)' Fatima et al (2009) after slight
modificatiotr. The isolates of fiugal pathogens i.e. Aspergillus niger (079) aad A'
fl)migcttus (0064) were obtained fiom Quaid-iAzam Uliversity and used for checking
aDtifungal activity of taken plant extracts.
i19
Plant extacts dissolved in DMSO were diluted in 1.5 ml of sterile Sabouraud
Dextose Agar (SDA) and solidified in Laninar Flow keeping test tubes in slarting
positions. The negative contol contained the solvent, while positive one was
Fluconazole. The fuogal cultues were inoculated on the slanting position ofthe media
in test tubes and then incubated at temperatules of28 to 30'C. The fungal groMh was
checked after 48 h (Hemaiswarya el al, 2009).
5.3.4.1 Media preparation for antifungal assay
For inoculums preparation, SDA was used to cultivate fungus. The ingredients
oftbis media are peptone complex (1og/L), glucose (40gll) and agar (15g,T-)'
3.3.4.2 Preparation of samples
For antifungal assay, plant samples were prepared from initial stock solution of
12 mg/ml to obtain a cotrcentotion of200 pglml.
5.3.4.3 Assay procedure
Media was prepared for fungus by dissolving SDA io distilled water at rate of
.6.5 9/100 ml. First of all, the Test tubes wele marked up to 10 cm mark The SDA
(N{ERCK) was placed into these tubes arld autoclaved 6T pl of sample pipette from
the initial stock solution and all the tubes were solidified at 25 oC in aogled position
120
Positive, negative control and 7 day old culture organisms were used. Their grou4l
was measured and after which their percent i libitiotr was calculated with reference to
positiv€ contiol (Fluconazole) and negative contol (DMSO) for each concentation of
plant extract. Percentage inlabitatioB of fimgal $owth was determined by using the
following formula.
Inhibition (%) = 100- linear growtl in test tubes (mm)
x r00
linear growth in control (mm)
The results of inhibitiotr percentage were classified by following the criterion
(Ayatollahia e, al, 2010):
Inhibition Perecentage Level of significance
o-39%
40-60%
60-'t0%
Low
Moderate
Good
70% and above Significant
5.3.5 STATISTICALANALYSIS
The diameter of zoue of inhibition (excluding well diameter) obtained from
t iplicates werc expressed as mean+standard deviation (SD) by using Statistix ver-8 1'
The data were analyzed by factor factorial design (ANOVA) ald P value < 0 001 was
12t
considercd as significant. The mean values were compared by usiog Least Significant
Diffoence (LSD).
5.4 RESULTS
5.4.1 ANTIBACTERIAL ACTTVITIES OF MEDICIIiAL PLANTS
Various solvent based extracts of Monotheca buxifolia, Myrtus commurus,
Teucrium stocksianum, Verbascum thapsus ar\d. Zizipus satiya were assessed for
antibacterial activity against selected bacterial pathogens by using agar well diffusion
method. Results are provided in Tables 5.1-5.6 and species-wise discussed as follows:
5,4.1.1 Antibacterial activity oI Mohotheca bu.xifolia
5.4.1,1.1 Inhibition Zone (mm) at 15mg/ml
Antibacterial activity of different extracts (Ethanol, Metlanol and Water) of
Monotheca bwifulia was checked for six bacterial pathogens. Inhibition zones of all
tested samples were compared to standard antibiotics (Cbloramphenicol, Ciprofloxacitr
and Penicillin). Results are provided in Table 5 . 1 .
Ethatrol extract was found more effective agahst all stairl ofbacteria. The highest
inlribition zone ofplant extract was observed in S. pyogen (17 am) as shown itr Table
122
5.1, followed by S. aureus (14 mm), S epidermidis (13 mm), K Pneumohioe (7.5
t rn), E. coli (7.4 'rfrrn) ^nd
P. aeruginosa (4.0 mm). Methanol exkact was found more
effeotive against all strains of bacteria. The highest iniibition zone was recorded in S
pyogen (18 mm), followed by S. auteus (16 mm), S ePidermidis (14 mm), r(.
Pne moniae (10 mm), E coli (6.5 mm) arrd. P. aentginosa (6 mm). Ia the case of
water extract, no inhibition zone was found (Table 5.1).
Comparing with standard drugs, highest zooes of inlibition was shown by
Ciprofloxacin, followed by Penicillin and Chloramphenicol (Table 5.1).
Clrlorampherricot showed inhibition zotre in the ordq of]. E.coli (26 mm), S
epidermidis (23 mm), S aureus Q.].rnm), K. Pneumokiae O6 tom), P- aeruginosa (74
dr]rI) utd S. pyogen (14 mm). Ciprofloxacin exhibited inhibition zone in the order ot
S.aureu$ (28 rn;rri, S. epidemidk (26 r:rrr'), S. Pogen (23 mm), A coh (21 tr,m), K
pneumohtae (20 mm), P aeruginosa (19 cj,rn), while, Penicillir inhibited the growth
of selected bacteria in the order of. S. epidermidis (26 rnm), S. aureus (24 o],,0])' S.
pyogen (20 mfl), K. pneumohkz (19 mm), E coli (17 mm) wrd. P. aeruginosa (16
mm).
5.4.1.1.2 Inhibition Zone (mm) at l2mgltnl
The antibacterial activily of various extracts (Ethanol, Methatrol and Water) of
Monothec:a buxifulia was evaluated at 12r48/ml agaimt selected bacterial pathogens.
IDhibitiotr zotre of aU tested samples were compared to standard antibiotics. Results are
expressed in Table 5.1. At the concentmtion of 12mgl10m1 ethanolic plant extract of
t23
Monotheca buxifulia effectively tnhibited the gro\4'th of selected bacetedal patiogens.
.t aoneu.r was prore to plant extact alld effectively conkolled at the zone inhibition of
1 3 . 1 mm, followed by E. coli (13 mrr,), S. pyogen (71 nm), K. Pneumoniae (10 mm),
S. epidetmidis (10mm)andP aentgtnosa (8.3 mm). ln the case of methanol extract,
highest inhibitiotr zone was noted against S. pyogen (14 mm) followed by S. auezs &
E. coli (73 mm each), S. epidermidis (10 mm), (. Pneltmoniae (10 mm) and P
ael gtnosa (7.5 mm), white water extact was unable to exprcss its activity agaimt
tested pathogens and no irhibition zone was recorded.
There was different trend of selected drugs in terms of their inhibitiotr zones
against selected bacteria (Table 5.1). Chlonmphenicol showed a inhibition zone
against pathogens that orderly include: E coli (23 lrm), S. pyogen (21ttm), S.
epidermidis (19 mr:n), S. a rcus (18 mm), K. pneumoniae (.5 mm) and P. aerugtuosa
(12 mm). Ciprofloxacin exhibited inhibition zone such as S aureus (24 IJ].Ier), S.
epidermidis (22 tlrlm), S. pyogen (21 mm), A coli (19 rnm), K. pneumoniae (.8 rlm)
atd P.aeruginosa (17 mm). Penicillin expressed inhibition zone in the order oi S.
epidermidis (23 mm), S aureus (22 n:rl), S. pyogen (18 mm), K pneumoniae (-7
rnm), tcoli (16 mm) and P.aeruginosa (15 $1\).
4.4.1.1.3 Inhibition Zone (mm) at lOmYBl
At the concentation of l0mg/ml, ethanolic platrt extact of Monotheca
burifolia evJlibited ir$ibition zone variously against individual bacterial shains. The
highest inhibitiotr zone was observed in S. aureus (12 mm), followed by S. pyogen
r24
(10 mm), r. coli (9 ntr]r), S. epidermidis (6'5 mm), K pneumo''oe (6 l mm) and P
Aerugtnosa (5.2 mm). Methanol exhact produoed inhibition zone in the order of: s,
aureus (11mm)> S. pyagen (ll t li>E. coli(8 5mm)>S epidetmidis (8rnm)> K'
pneur oniae Q .2 rrrm) :rrrd > P. aeruginosa (6 1 mm) Water extract did not produce
any activity as well zone idribition (Table 5 2)'
Aatibiotics had expressed various activities against the strains (Table 5'2)'
Cbloramphenicol efibited intribition zone in the order of: -E' coli Q0 mml S pyogen
(19 mm), S. epider tidis (18.5 mm), S ar'rrers (l8 mm), K' pneumoniae (13 mm) and
P. aerutinosa (10.3 mm). Ciprofloxacin expressed highest inhibition zone for S'
aureus (22 nm), followed by S. epidermidis (19 itnt), S pyogetu (20'4 rl,rn)'E coli (16
rnm), K. pneumoniae (13 mm) and P. aeruginosa (15 mm), whereas' Penicillin
showed inhibition zone io the order of: S. epideflnidis (22 rdtm), S auteus (21 5 mn)'
S. plogen (76.3 tlrrt), K. pneumoniae (15 mm), A coli (74'2 1r'r,o) and' P aerugi ostl
(14 mm).
5.4-l.l-4 Inhibition Zone (mm) at 7.5mg/ml
The antibacterial assaya of vadous solvent exhacts of Monotheca buxifulia wx
checked at a cotrcenhation of 7.5mg/ml against selected bacterial strains Results are
given in Table 5.3. Ethanolic extract showed activity agahst all bacterial strains- The
highest inhibition zone was observeil it S aureus (9'1 mm)' followed by r(
pneumo iae (3.0 mm), P aeruginosa (2 7 mm), S epidermidis & S pyogen (2 mm
each) and E coli (1.1 rnm) Methanolic extribited highest activity compared with
125
water arld ethatrol except against S. ar#ers Methanol showed activity in the order of
S. pyogen(6r ri> S. epidermidis (4mm)>P aeruginosa (3 1mm), E coli&S'
aureus (3 mm each) and ,(. pneumoniae (2.5 mm), however, in the case of water
extac! no inlibition zore was recorded against the selected bacterial pathogens (Table
5.3).
The standard antibiotics exhibited inhibition zone variousiy against the pathogens'
Chloramphenicol inlribited pathogenic colonies in the oider oi S' Wogen (7'7 ,]l1\), S
epidermidis (16 om),.E. coli (15 mm), S. aureus (73 lta), K'p eumoniae (12 5 mm)
arrd P.aeruginosa (10.0 mm). Ciprofloxaci[ exhibited inlibition zone in the order of:
S. aureus (20 mm), S. epidetmidis (17 mm), S pyogen (16 mm), P aeruginosa (14
rrrm), E. coli (13 mm) and f. pnermoniae (71 mm), while Penicillin showed highest
inhibition zone against S. ePi(lermidis (20 mm), followed by S auleus (18 5 mm)' S'
pyogen (74 m,rI), K pnewnoniae Q4 mm)' P. aerugihosa (13 4 mm) al.d E' coli (12
mm).
5.4.1.1.5 Inhibition znne (mnn) at 5mg/rDl
At the cotrcentatiotr of 5mg/ml, ethanol, methanol and water Mo notheca buxifolia
leaves exhacts were evaluated for antibactsrial assay aligned with the selected
bacteria. Results are Prcvided ir Table 5.4. No acivity recorded against the pathogeN
by all solveo based extracts.
The antibacterial activity of aDtibiotics is leflected in Tatle 5 4 Chloramphenicol
effectively cortrolled best S. pvogen (76 mm), followed by E coli (14 mo), S
126
epidelfiidis (13 ,nrrt)' S aureus (13 'Dt\)'
K' pneumoniae (lO 5 fim) s\d P aet'uginosa
(9,5 mm). Ciprofloxacir exhibited iEhibition zon€ in the order of: S' pyogen (75 mm)'
S.arlers(l4mm),5-epidernidis(l3mm)'P'aeruginosa(72mm)'Ecoli(70n1\t)'
K. pneumoniae (9 mm), while Penicillin showed inlibition zone in the order of: S'
epiderrnidis(l7mm),saureus(16nrrt)'S'pyogen(12mm)"(pneuoniae(72
rrrm), P. aeruginosa (11 om) and Ecol' (10 mm)'
5.4.1.2 Antibacterial activiry of Mrttus cofim"/'l7
5.4.1.2.r Inhibition Znne (mm) at 15mg/n
The altibacterial activity of solvent leaves extfacls of Myttus communis was
tested agaitrst selected bacterial pathogens The extracts activity is given in Table 5 1 '
Ethanol extact exhibited highest activity against pathogens and found more effective
against all strains except K pnettmonia (Table 5 1) At the concentration of
15m!1oml, S. auteus was inhibited best (21 mm)' followed by s P/ogen (11 rnm)' S'
epidetmidis (12 am), E coli (11mm) and P aerugihosa (8'2 rrtm)' while no activity
wasrecordedinthecaseofK.pneumoiae(Table5.l).Thisactivitywasatparwith
the standatd aotibiotic (Chlorarnphenicol) The methanol extact exhibited same trend
in activity and except K. pneumonia, rest of pathogens effectively conholled'
Maximumilhibitiolwasobservedagaiosts.aureus(lg'njr}),followedbySpyogea
(18 mm), S epidermtdis (74 run)' E' coti (73 mm) and P aeruginosa (8 mm), and to
activity l,as shown by K. Pneumonia Watet exiact showed the highest inlibition zone
127
agalnstS. aurcus (13 mm), followed by S epiderlnidis (10 5 mm)' E coll(10rnm)'P
aeruginosa (9.5 rrrm) and S. pyogen (9 mm), while no activity was observed in the case
of K. pneumonia.
The standard aatibiotic (chloramphenicol) showed inhibition zore in the order of:
E.coli (26 rnm), S. epidennidis (23 mm), S aureus (2llr;Lm)' K pneumoniae (76 mm)'
P. aerugttxosa (14 mm) and S pyogen (14 mm) Ciprofloxacin exhibited irhibition
zone in the order of: S. aureus (28 rrra), S epidermidis (26 mm)' S pyogen (23 mm) E'
coli (21 mm), K pneumoniae (20 mm), P aerugtnosa (19 mm)' while' Penicillin
inhibited the grot'th of selected bacteria in the order of: 'S ePide flidis (26 mm)' S
aureus Q4 mm), S pyogen (20 mm), K pneumoniae (19 rnm)' E col' (17 mm) atrd P
aeruginosa (16 rl:,rr.).
5.4.1,2.2 ItrhibitioE Zone (mm) at 12'5mg/m]
Various solvetrt based Myrtus communis leaves extracts were evaluated for
aotibacterial activity agaiost the selected bacterial strains All extacts werc foutrd
effective against pathogeff except K. pneumoniae; however there was diversity in
activity agaitrst the tested bacterial strains (Table 5 1) Ethatrol extract was fouod more
effective than methanol and watel against all strains except K p eumoniae' At the
concenkation of 12mgl10m1 ethaoolic plant extact, the highest inhibition zone was
obse*ed ia S. pyogen (15 mrn), followed by S aureus & P aeruginosa (74 mm each)'
S. epidermidis (11 mm) aad E colt (10 mm) Methanol extact was second drug of
choiceafterethanolforinhibitingtheSrowthofselectedpaihogens(Table5.2)'The
128
highestinhibitioozonewasrecordedagainsts.aureas(i6mm),followedbyS.pyogen
(13 rnm), -E coli (9.5 mm), S' epidermidis (9 1 mm) and P aetuginosa (8'2 mm)'
watelextlactwasfourr.dmoleeffectiveagailsts.aureusthatproducedhighest
bhibitionzone(l3mm),followedbyP.ael"uginosa(11.1mm),s.epidermidis(10
mm), e coli (9.5 mm) and S p,ogen (7 mm)'
There was different trend of selected drugs in tertrs of their inhibition zones
against selected bacteria (Table 5 2) Cbloramphenicol showed a inhibition zoDe
againstpathogensthatorderlyinclude:'coli(23mm)'S'pyoge\(zlnl,ln)'S'
epid.etmidis (19 rr:rrl), S. azreus (18 mm)' K paeumoniae (15 mm) and P aeruginosa
(12 mo). Ciprofloxacin exhibited inhibition zone such as: S aareus (24 mm)' S'
epidennidis (22 ma), S- pyogen (21 mm) 'E col;(19mm)'K pneumoniae 08lrrlr)
ard P.aeruginosa (17 mm). Pedcillin expressed itrhibition zone irr the ordet of: S'
epidel idis (23 mm), t aureus (22 mm)' S pyogen (18 om)' K pneumoniae fll
mm), E coli (1 6 nlm) arrd P aeruginosa (15 rr.:rl)'
5-4.1.2.3 Inhit ition Zone of (mm) at lomg/rnl
The antibacterial assay of various solveots extracts of Myrtus contttunis leaves
was tested against tested organisms' Results of activity are provided in Table 5 3'
Altogether, eihanol exuact was found more thao methanol and water for checking
bacterialgovr'thofselectedstrains.ExceptK,pneunoniae,ethanolicplaotextr.act
inhibited best S. epidermiclis (lO ma), followed by P aeruginosa (9 mm} S aureus (8
t29
ruin), S. pyogen (7.5 rI1rr,) alld E coli (7 '4 rrlm) Methanol extmct Produced inhibition
zone ain the order of S. pyogen (10 mm) > E coli(7 5mm)>S aureus(7 3mm)> S'
epidermidis (6 mm) aad > P aeruginosa (5 mm)' however' K' pneumonia'e was
seemingly resistant anal IIo activity was recorded' In the case ofwater extlact' except 'S'
arler,s (5 mm), rlo inhibitio[ zotre was docume ted in rest ofmicrobes'
Antibiotics had exprcssed various activities against the strains (Table 5 3)'
Chloramphenicol exhibited inhibition zone io the order of: E coli (20 ro:,m)' S pyogen
(19mm),s epidetmidis (18.5 mm), S aureus (18 mm)' K' pneumoniae (13 mm) aod
P. aeruginosa (10.3 mm) Ciprofloxacin express€d highest iohibition zooe for S
aureus(22am),followedbyS.epidermidis{19rrl:r),5'pyogen(204rtm)'Ecol;(16
fi$'i, K. pneumoniae (13 r!m) and P' aeruginosa (15 mm)' whereas' Penicillin
showed inlribition zone in the order of: S epidelruidis (22nm)'s. aureus (21'5nm\'
S. pyogen (16,3 tnnt), K. pneumoniae (15mm),E coli (142mrn) and P aeruginosa
(14 mm).
5-4.1.2.4 Itrhibitiotr Zor€ (mm) at 7'5mgiutl
Ethanol, methanol and water leaves exttacts of Myrtus communis were tesied
agaiDst selected miqobes for the determination of atrtibactenal activlty and results are
given in Table 5.4. The highest inhibition zone was shown by ethanolic extract atrd
excepl K. pneumo iae, lest of migobes were effectively controlled At the
cotrcentatiotr of 7.5mg/10m1, the highest inhibition zone was observed against S
epidermidis (7.4 mm), followed by S aureus & S pyogen (7 mm each)' A coli (5 ,l]n)
130
and P. aerugihosa (4.9 mm). Methanol exkact was found effective agaiDst all skairls
except K pneumoniae and at the concenhatioD of 7.5mg/1Oml, the inhibition zone was
in the order ol E pyogex (8 mm) > S epidermidis (6.3 mnt) > S. aurew (5.6mm) > E.
coli (3.5 mm) alrd. > P. aeruginosa (3.3 mm), while water extract only i.hibited ,S.
aureus (Table 5-4).
The standard antibiotics exhibited bhibition zone variously against the pathogens.
Chlommphenicol inhibited pathogenic colonies in the order of: S. pyogen (17 rr-m), S.
epidermidis (16 mn), E coli (15 mm), S. aureus (13 rrn), K.pneumoniae (12.5 mrl)
a'I1d P.aeruginosa (10.0 mm). Ciprofloxaci]l exhibited inhibition zone in the order ol
S. aureus (20 m:m), S. epidermidis (17 mm), S. pyogen (16 mm), P aeruginosa (14
t JJJt), E. coli (13 mm) a]Ld K pneumoniae (77 mm), while Penicillin showed highest
inhibition zooe against S. epidermidis (20 mm), followed by S. aureus (18-5 mm), S
fi)ogen (74 tr.rJt), K. pneumoniae 04 ,x,/li,), P. aeruginosa (13.4 o]Ix,) and E. coli (72
rDm).
5-4.1.2.5 Inhibition Zone (mm) at 5 mg/ml
The antibacterial assay of various solvents solvent based extacts of Myrtuj
com nis was tested agaiNt the selected bactedal pathogens. Ethanol extract
exhibited highest activity and effectively idribited all strains except K pneumoniae
(Table 5.5). At the conceDhation of 5mg/10m1, highest inhibition zone was observed
against .S. aa'er6 (6.5 mm), followed by S- Wogen (6-1 mm), S- ePidenbidis (5 ilr:t),
E. coli (4,5 mm) and P aeruginosa (4.0 mm). Methanol extract was also found
t3l
effective against tested microbes except K. pneumoniae. At the coDcentration of5mg/loml, highest inhibition zotre was noted agatnst S. pyogen (7-6 rnm), foltowed by
S. epidermidis (5.1 mm),s aureus (4.3 mm),p. aeruginosa (3.3 mm) and A col, (3.0
mm), however water extract failed to erpress any activity against all tested pathogens.
The antibacterial activity ofantibiotics is rcflected in Table 5.5. Chloramphenicol
effectively cotrbolled best S. pyogen (16 mm), followed by E. coti (14 mm), .S.
epidermidis (13 mrn), S. atreus (13 rlfi), K. pneumohiae (10.5 ram) arrd. p.aeruginosa
(9.5 mm). CiprofloxaciD exlibited inhibition zotre in the order of S. pyogen (15 on),
S. aweus (l4 r[m), S. epidermidis (13 mm), P ae, uginosa (12 mm), A coli (10 mm),
K pneumoniae (9 mm), while Penicillin showed inhibition zone itr the order of: S.
epidermidis (17 mm), S. aurew (16 nlrrt), S. pyogen (12 mm), r( pneumoniae (12
nml. P. aeruginosa( II mm ) and E coli tl0 mm).
5.4.1.3 Antibacterial activity ol Teucrium stocksianum leates
5.4.1.3.1 Inhibition Zone of (mm) at lsmg/lDl
The antibacteial assay of various solvelts leaves exttacts of Teucrium
stoclTianutl was evaluated against selected bacterial pathogens. The activity in the
form of inlibition zone of all tested samples is provided in Table 5.1. Etharol extact
was formd the best for inlibiti[g the gro1ath of all strains. At the coocentration of
15mg/10m1, ethanolic extact produced inlfbitiotr zone in the order ofP aeruginosa
(21.6mm)>S aureus (20.4 rr-rt) > E. coll(16.5mm)>S. epidermidis (16.8 am)> K.
132
prleltmoniae (15.4 mm) and > S. pyogen (13 6 mm) In the case of P aeruginosa ar,d
E. coli, the antibacterial activity of ethanolic leaves extract was superior comparcd
with all standard drugs, while rest of pathogens were closel to &ugs activity'
Methanolic extract was also found effective and inhibited significantly all pathogens'
The highest inhibition zotre was observed in E. coli (21 5 mm) that is higher to drugs
(Chloramphenicol: 16mm, Penicillin: 17 mm, Ciprofloxacin: 21 mm) It was followed
by P. aeruginosa (18.4 mm), S. auteus (17.3 nlil), K. Pneumoniae (16 2 mm), S'
epidermidis (14.1$ra), and S- Pyogen (17.5 mm). In the case of water extract, highest
iohibition zone was observedin K pneumoniae (13.6 niLr,i,), followed by E' Coli (11"5
turn), P. aeruginosa (11.3 mm) S- auteus (8.9 tum)'l. epidermidis (7 0 mm), and S'
pyogen (7 .0 run).
The statrdard antibiotic (chloramphenicol) showed inhibition zooe in the order ot
E.coti Q6 tu$\ S. ep{dermidis (23 mm), S. auteus (27n:;rrt), K. pneutho iae 06 lrrm),
P. aerug;nosa (14 mm) and S. pyogen (14 mm). Ciprofloxacin exlibited inhibition
zone in the order of: S.aure* (28 mm), S. epidermidis (26 rnm)' S. Wogen (23 c\n\)'E'
coli (21 mm), K. pneumoniae (20 mm), P aeruginosa (19 mm), while, Penicillin
bhibited the gro{th of selected bacteria in the order of: S. epidetmidis (26 mm), S'
aureus (24 mm), S. pyogen (20 mm), K pneumoniae (19mm) .E coli(17mm)andP
aeruginosa (16 mm).
5.4.1.3,2 Inhibition Zone (mm) at 12.5mg/ml
Ethaool, methanol ald water leaves extract of Teucrium stocksia 'lm
was assessed
for antibactedal actiyity against the selected bacterial straiN Results are provided in
133
Table 5.2. Ethanol extract was found best in contolling populations of selected
pathogens. At the concetrtration of 12.5mg/ml, bighest irlibition zone was observed
for S. aureus (16.0 mm), followed by S epidermidis (14.5 mm), E. coli (14.0 lr.m),
O22 Infr), S. pyoge (1 1.3 mm). Methanol extract was also found effective against all
shains. The highest inhibition zone was observ ed for K. pneumoniae (I4.2), followed
by S. auretas (14.1 mm), P. aeruginosa (14 mm), E coll (13.5 mm), S. epidermidis
(i2.1 mm) atd S. pyogen (11.5 mm). Water extact expressed activity aod at the
concetrhation of 12.5mg/1ml, the inlibition zone was in in ihe order ol K. pneumoniae
(1 1.6 mm) > r. coli (1 1.0 mm) > P aeruginosa (9.3 rJ],l]i,) > S. epider idis (8 r,x,j:,],) > S.
aurens (7.5 mm) and > S. pyogen (5 mm)
There was different tend of selected drugs in terms of their inlibition zones
against selected bacteria (Table 5.2). Chloramphenicol showed a inhibition zone
against pathogeN that was orderly include: E coli Q3 I[jlE]), S. pyogen (21mm), S.
epidermidis (19 mm), S. aureus (18 fii\), K p eu ohiae (15 mm) ^nd.
P. aeruginosa
(12 mm). Ciprofloxacin exhibited inhibition zone such as: S. aureus (24 nrn) S.
epidermidis (22 rlorrr), S. wogen (21 mm).e coli (19 mm), K. pneumoniae (,8 uw,)
ar.d P.aeruginosa (17 mm). Penicilin expressed inhibition zorle in the ordsr of S.
epidetmidis (23 mm), S. aure s (22 mm). S. pyogen (18 mm), K pneumoniae (1,7
mm) E coli (16 mm) and P.aeruginosa (15 mm).
5,4.1,3.3 Itrhibition zone (mm) at 10mg/ml
Antibacterial activity of ethanol, methanol and water extlacls of Teucrium
stocksianum \eaves was tested against selected bacterial strai[s. Activity of all solvent
t34
based extEcts is shom in TablE 5.3. Ethanol extract showed highest activity agaiDst
bacterial colonies (Table 5.3). At the concentration of 10mg/1m1, ethanolic extacts
produced i::hibition zone variously and highest inlibition zone was observed against P
aerugihosa (11.3 mm), followedby S epidermidis (t0.5mm),S aureus (10. mm), E.
coli & S. pyogen (8.3 mm each) and K. pneumonia (5.3 mm). Methanol extact was
also found effective against all shains and the highest inlibition zone was observed
agailrsi S. aureus (11.1 mm), followed by S. epidetmidis (9.0 ntm), K. pneumoniae
(8.4 nrr\), S. pyoge ( 8.0 mm), E. coli (6.5 mm} P. aeruginosa (6.1 mm). Water
extract exhibited the highest inhibition zone agarnst P aeruginosa (9.3 mm), followed
by E. coli ('7.5 'rni), K. pneumoniae (5.3 mm) and S. aureus & S. pyogen (4.5 mm
each).
Antitiotics had expressed vadous activities agaiNt the strains (Table 5 3)'
Chloramphenicol exlribited intribition zone in the order of: -E. coli (20 ,Irrt), S Pyogen
(19mm),S epidermidis (18-5mm),S aureus (18 Inm), K. pneumoniae (13 mln) axd
P. aeruginosa (10.3 mm). Ciprofloxacin expressed hiShest inhibition zone for S
aurew (22 mm), followed by S. epidermidis (19 mrJj,), S. pyogen (20.4 mlJ].) E coli (16
fitu), K. pneumoniae (13 mm) aad P. aeruginosa (15 mm), whereas, Penicillin
showed inhibition zone ir the order of: S. epidermidis (22 mm} S. aureus (21 5 rDm),
S. pfogen (76.3 t)m), K. pneumoniae (15 mm), E coli (74-2 mm) Ni P-aeruginosa
(14 mm).
5.4.1.3.4 lohibition Zone (mm) at ?.58g/ml
Teucrium efiar'ol exh_act showed effectiveness against all straitrs at the
cooceatation of 7.5 mg/loml (Table 5.4). The highest in-lribition zoae was observed
135
agallst S. epidermidis (8.5 mm), followed by P. aerughosa (7 -5 rr,.o;,)' S. aureus (6.3
mtu), K. Pneu oniae (5.0 mm), .E coli (4.'7 $n) attd S. Wogen (4.0 mm). Methanolic
leaves exhact possessed the same trend and all strains were effectively controlled The
inhibitioo zone is provided in Table 5.4. The highest inhibition zole was documented
it S. aureus (7.5 mm), followed by S. epidermidis (7.0 mm), -K. Plleumoniae (6.2
nm), P. aeruginosa (6.7 rrrm), S. pyogen (5.0 mm) and E coli (4.0 mm). In the case of
water extractr the achvity is sho$tx by the leav€s exhact and inlibited the colonies of
all pathogens (Table 44). At the concentration of 7.5mg/10m1 aqeous extacts of
Teucrium stocksiaturrt leaves produced inhibition zone at optimum level against S
epidetmidis (5.0 mm), followed by P. aeruginosa (5.0 mm), E col; (5 0 mm), K'
p eanoniae (4.6 mm), S aureas (4.0 mm) and S pyogen (3.5 mm).
The standard antibiotics exhibited i ibitiotr zone variously against the pathogens'
Chto€mphenicol inlibited pathogenic colonies in the order of S. pyogen (17 mm), S'
epidetmidis (i6 mm), E col; (15 mm), S. aureus (73 tunt), K.pneumontae (12 5 mm)
arld P.aerugihosa (10.0 lr]m). Ciprofloxacitr exhibited inhibition zone in the order of:
S. aureus (20 ma), S. epidermidis (17 lnrLa), S. pyogen (16 mm), P aeruginosa (74
mri, E. coli (73 nrn) ad K. pneumoniae (ll mm), while Penicillin sh6wed highest
inhibition zone ag &st S. ePidermidis (20 mm), followed by S. aureus (18 5 mm), S
Wogen (14 $i\), K. pneutuoniae fla mm), P aerugi osa (13'4 rnla) ar:d E coli (12
om).
5.4.1.3.5 Inhibition zone (Em) at sEg/n
136
Antibacterial activity of ethanol, methanol aod water exf:racls of Teuctiuttt
stocksiahu leaves was checked. The iDhibition zone of the said extacts is provided in
Table 5.5. The ethanolic extact was found effective against all straiff. At 5mg/10m1
coacentration, ethaoolic exkacts exhibited inhibition zone in the order ofS. azreus (5.8
rnm) > S. epidennidis (5.5 mm) > K. pneumoniae (5.0 ram) > S. pyogeh (3.5 rrrrn) > P.
aeruginosa (3.5 mm) and > E. coli (3.5 mm). Methanol extract effectively inhibited
best S. epidenltidis (6.5 mm), followed by S. aureus (5.5 mm), E co,/i (4.0 mm), K
pneumoniae (3.2 f.;m), P. aeruginosa (3.1 mm) and S pyoge, (3.0 mm) Water extBct
expressed pronounced €ffects that inhibited variously all pathogens except P
aeruginosa. The highest inlibition zone was observed it E. coli aatd S. ePider,,lidis
(5.0 rnm each), fotlowed by K p eumoniae (4.6 mm), S aureus (4.0 mm) and S.
pyogen (3 .0 rrrlan).
The antibactedal activity of atrtibiotics is reflected in Table 5.5 Chloramphenicol
effectively- controlled best S. pyogen (16 mm), followed by E. coli (14 mm), S
epidennidis (13 m:rn), S. aureus (13 mm), K. pneumoniae (70-5 t\r,t) and P.aeruginosa
(9.5 mm). Ciprofloxacin extribited inlibition zone in the order of'. S. pyogen (15 nm),
S. aureus (14 lrttrl), S. epidermidis (13 Inm), P aerugilosa (121\n)' E coli (10 mm),
K. pneumoniae (9 mm), white Penicillin showed io.hibition zone in the order of: S
epidermidis (17 mm), S aureus (76 mm), S. pyogen (12 mm), K pneumoniae (12
mm), P. aeruginosa (11 nrm) ard acoli (10 mm).
3.4.1.4
5.4.1.4.1
137
Antibacterial activity of Teucri u stocksianum flo\yer
Inhibition Zotre (mm) at lsmg/ml
Vadous solvents such as ethanol, methanol and water flower extacts of Teucrium
stocl<$;anum were screened for antibacterial activity against microbes. Results are
shorn by Table 5.1. Ethanolic extract de ved highest infribition zone amongst all
solvents agaiNt bacterial colonies. The maximum inhibition zone was observed
against S. aureus (26 rnm), followedby S. pyogen (16 mm), P aeruginosa (14 rlrr;'), K.
pneumoniae (10 mm) and E. coli (7 mrn). In the case ofP aeruginosa arld. E. coli, rJ;.e
afltibactedal activity of ethanolic leaves exhact was superior compared with all
standard drugs, while rest ofpathogens were closer to drugs activify.
Methanol extract was also showed effectiveless after ethanol. At the
cotrcentation of 15mg/1m1, methanolic extact produced highest iDhibitiotr zone
agalnst S. aurets (22 mm), followed by S. pyogen (18 mm), P aeruginosa & K
p eumoniae fl6mmeach)andE coli (13 mm), while no activity was,ecorded for S.
epidermidis. Water extact was effectively controlled pathogenic colodes except S.
epidermidis. The highest inhibition zore was observed in S aurezs (23 mm), followed
by E. coli (12 mm), S. pyogen (9 mm), K. pneumoniae (8 mtr) ar.d P. aeruginosa (7.0
mm).
The standard antibiotic (chloramphenicol) showed inhibitiol zone in the order of:
E.coli (26 l:om), S. epidennidis (23 mrtr), S. aureus (2lrrrrtr), K- pneumoniae (1'6 mm),
138
P. aet"uginosa (14 mm) and S. pyogen (14 mm). Ciprofloxacir exhibited inhibition
zone in the order of: S aureus (28 mm), S. epidenhidis (26 r:trr), S. pyogeh (23 tu:,l).E.
coli (21 mm), K. pneumoniae (20 rnm), P aeruginosa (19 mm), while, Peniciliir
intribited the glollth of selected bacteda in the order of: S. epidetmidis (26 mm), S.
aureus (24 mm} S. pyogen (20 rnrl:.). K pneumoniae {19 rlll,), E. coli (l'7 ll;,rla) arld, P.
aeruginosa (16 mm).
5.4.1.4,2 Inhibition Zone (mm) at 12.5mg/ml
The antibacterial assay of various solvents flower extracts of Teuctium
stocksianum was scteened against selected bacterial pathogeDs. Results arc compiled in
Table 5.2. Ethanol extact was found effective for inhibiting the bacterial stlaills excq)t
S. epidetmidis. At the concentation of l2mglml, maximum inhibition zone was
recorded against s. aureus (15 n.,m), followed by S. p.yoger, (11.5 mm), P aeruginosa
(1 1 mm) and -g. coli (7 .0 mm). Methatrolic exh act had highest potertial for inlfbiting
bacterial cell e\c?t S. epidermidis. lt produced highest itrhibition zone against s
aureus (16 mm), followedby K. pneumoniae (74 rffn), S. pyogen (13 mm), E. coli (12
mm) and P aeruginosa (10 rEm). Watel extract was also established activity against
all strains except S epidermidis. The inhibition zone was in the order of E. coli {9.1
mm) > S. arre,6 (8.9 mrt), K. pneurnoniae (8 mm) and S pyogea (7.3 mm).
There was different tend of selected drugs in terms of their inhibition zones
against selected bacteria (Table 5.2). Chloramphenicol showed a inhibition zone
139
against pathogens that orderly include: -E coli {23 rfinj,), S. pyogen (21mm), S.
epidermidis (l9ltnti, S. aureus 178 mmj, K. pneu*oniae 05 rntn) attd P. aeruginosa
(12 mm). Ciprofloxacin exhibited inhibition zone such as: S. aureus (24 rllrn)' S.
epidermidis (22 rnm), S. pyogen (21 mm).E coli (19 mm), K pneumo iae 08 slm)
and. P.aentginosa (17 mm). Penicillin expressed irlhibition zone in the order ol S
epidermidis (23 mm), S auleus (22 mm), S. pyogen (18 mm), K pneumoniae Q7
mm), E coli (16 mm) and P.aeruginosa (75 mm).
5.4.1.4.3 Inhibition Zone (mm) at lomg/ml
Various solvetrt based viz., ethanol, methanol atrd water extacts of Teucriu '
stocksianum flower were evaluated for antibacterial activity against sel€cted bactedal
strains. The intribition zone of all tested samples is provided in Table 5 3 Ethanolic
extact showed good activity against all pathogens except S epidermidis' At lhe
concenhation of 10mg/1m1, ethanolic extracts established inlibitioll zone in tle order
of: S aueus (6mm) > K. pne moniae f5 rnm) >P. aeruginosa (4'5 mm) > S pyogen
(4. 1 mm) and > E. coli (3 .g mm), while and no activity was shown against 'S'
epidermidis. Methanolic extract exlibited activity agaiNt all strains except S'
epidet"rnidis. The highest intribition zone was observed against S A/oge' (8 1 mm),
followed by S aureus (6.1t n), K. pneumoniae (5 am), P aeruginosa (4 3 mm) aad
E. coLi (4.1mm). Water exhact was also fouad effective against all strains except S'
epidermidis. S. aureus was found prone to water extract and intribited best (6 mm),
140
followed by S pyogen (5.1 rrl,r.l,), E coli (5 mm), f. prteumoniae (4 5 mm) and P
aertginosa (3 .7 mm).
Antibiotics had expressed vadous activities against the straiN (Table 5.3).
Chloramphenicol exhibited inhibition zore in the order of: E. coli {20 mm)' S. pyogen
(19 flnl), S. epidermidis (18.5 mm), s' aureus (18 mm), K. pneumoniae (13 mm) and
P. aeruginosa (10.3 mm). Ciprofloxacin exprcssed highest iDhibition zone for S'
aureus (22 r:nm), followed by S. epidermidis (19 mrn), S. pyogen (20.4 rrtm).E- coli (16
mnt), K. pneumoniae (13 mm) ald' P. aeruginosa (15 mm), whereas, Penicillin
showed inlribition zone in the ordel oi S. epidermidis (22 mm), S. au\eus (21'5 mm)'
S. pyogen (16.3 mm), K. pneumoniae (15 mm), ,. coli (14.2 mm) alld P.aer\gi osa
(14 mm).
5.4.1.4.4 IBhibition zone (mm) at 7.smg/ml
Ethanol, methanol and water extracts of Teucrium stocksianum flower were
assessed for the establishment of antibacterial activity agaiDst selected midobes The
results of fuhibition zone are glven in Table 5.3. Ethaool exhact was found effective
agaiDst all strains except S. ePidetmidis. At the concentration of7 5mg/10m1, ethanolic
extracts exhibited inhibition zone variousty and highest otre was observed agaifft r<'
pneumoniae & S. pyogen (4.5 mm each), followed by S aureus & P. aer ginosa (4
mm each) and -E coti (3 n$\ The methanolic extact showed suPremacy in activity,
however like previous solvent extract, it was failed to exPress actiyity against s
14I
epidermidis. The activity in the folm of inhibition zotre was in the order of: J. pToger
(8 mm) > .1 aureus (6 lllm) > K. pneumoniae (5 mm) > E coli and P. aeruginosa (4
mm). Water extract was also fouad effective against all stmins except S. eptdermidis.
At the cotrcertation of 7.5mg/10m1, aqeous extacts produced inhibition zoDe against
the selected strains. The highest inhibition zone was observed in A coli & S. pyogen (5
mm each), followed,(. pneumoniae (3.5 mm), S auretu (3 mm) atd, P. aeruginosa
(2.5 mm).
The standard antibiotics exhibited inhibition zone variously against the pathogens.
Chloramphenicol ifibited pathogenic colonies in the order of: S. pyogen (17 mm), S.
epidermidis (16 mm), ,E coli (15 mm), S. aureus (73 rnrl), K.pnewnoniae (12.5 mm)
aad. P.aeruginosa (10.0 nlm). Ciprofloxacin exhibited iriibition zone in the order of:
S. aureu.s (20 mm), S. epidermidis (7'7 rr,Il]), S. pyogeh (16 mm), P aeruginosa (74
m:tri), E. coli (13 mm) and f. pneumon[ae (l I mm), while Penicillin showed highest
inhibition zone dgaitrst S. epidermidis (20 mm), followed by S. aureus (18.5 mm), S.
pyogen (14 rnj't), K. pneumoniae 04 rr,ni.), P. aeruginosa (13.4 mm) ard E. coli (72
mEl).
5.4.1.4.5 Inhibition Zone (mm) at smg/mt
Solvent based flower extacts of Teucrium stocksianufi was tested agahst
microbes for antibacierial activity and results are compiled in Table 5.5. Ethanol
extact demonshated good activity against a1l strains except S. epidermidis. At the
142
concentration of 5mg/1 0m1, ethanolic exh acts reflected highest iDhibition zone against
K. p eumoniae (4 mm), followed by P. aet'ugihosa (3.6 mm), S. aureus & S. pyogen
(3.5 mm each) atd E. coli (3.3 mm). Methanolic extract was superior in impede and
found more effective against all strains excepl K. pneumoniae. The highest iDlibition
zone was observed againsl S. pyogen (6 mm), followed by K. pneumoniae (5.2 mm),
S. aureus (5 mJn), E. coti artd P. aeruginosa (3.5 mm each). In tbe case of water
extract, except S. epidermidis, rest of pathogens inbibited effectively. At the
cotrcent ation of 5mg/loml, aqeous extracts showed inhibition zone and it was in the
order of: E coli & S. pyogen (5 mm each) > r(. prt eumoniae (3 .5 mm) > S orireas (3
mm) > ardP. aeruginor@ (2.1 mm).
The antibactedal activity of afltibiotics is reflected in Table 5.5 Chlorarnphenicol
effectively controlled best S. pyogen (1,6 mm), followed by E. coli (14 nrm), S'
epid.ermidis (13 :irrrn), S. aureus (13 mm), K. pneufioniae (10.5 mll) anJ P aeruginosa
(9.5 mm). Ciprofloxacin exlibited inhibition zone in the order oft S Wogen (15 i]f,n),
S. aureus (14 mm), S. epidermidis (13 mm), P aen'tginosa (12 mm), A coli (10 mm),
K. pneumoniae (9 mm), while Penicillin showed ilhibition zone in the order of: S'
epidermidis (17 mm), S aureus (16 mm), S. pyogen (12 mm), f pneumoniae (12
n$), P. aeruginosa (11 mm) and E coli (10 mm)'
5.4.1.5
143
Antibacterial activity ol Verbascum lhapsus
5.4.1.5.1 Inhibitior Zone (mm) at l5mgE
Altibacteriat activity of various solvent extracts i.e. €thatrol, methanol a[d water
of I/erbascum thapsus was evaluated against selected bactedal strains. Results in the
form ofinhibition zone of all tested sarnples are showed in Table 5 1 Ethanol extact
was found effective against all strains excepl E. coli, K. pneumoniae and P'
aeruginosa. At the coocentration of 15mg/10m1, ethanolic leaves exhact unveiled
highest inhibition zone against S arzrer.r.s (4 mm), followed by S pyogem (3'2 mm) and
S. epid.ermidis (1.2 fim). Methanol extract expressed inhibition zo\e excelrt E' coli, K'
pneumoniae and P. aeruginosa. The highest inhibition zone was recorded against S
aureus (6 rnm), followed by S. epidermidis (1 .8 $m) ar.d S Pyogen (1 '5 i:l\), however
Do acti\.ity was reflected by water exkact.
The standad atrtibiotic (chloramphenicol) showed hhibitior zone in the order of:
E.coli (26 mm), S. epidel'nidis (23 mm), S aureus (27mm), K. pneumoniae (16 mm),
P. aeruginosa (14 mm) and S. pyogen (14 mm). Ciprofloxacin exhibited inhibition
zone in the order of: S aureus (28 r.lllrl), S. epidermtdis (26 mm} S pyogen (23 mm)'E'
coli (21 mm), K. pneumoniae (20 mm), P aerugi osa (19 mnl), while, Penicillin
inhibited the gro&th of selected bacteria in the order ol S. epidermidis (26 mm), S'
auleus (24 mrl), S. pyogen (20 ti$r), K. pneumoniae (19 mm), E coli (17 mm) and P
aeruginosa (16 mm),.
t44
5.4.1.5.2 Inhibition Zone (En) at 12.5m9/ml
Antibacterial activity ethanol, methanol atrd lvater extacts of yetbascum thaPsus
was assessed agaiDst bacterial stlains. Results are provided in Table 5.2. Ethanolic
extract effectively inlibited pathogens except E. coli, K- pneutllo iae x\d P'
aeruginosa. At the concentration of 12.5mg/1m1, ethanolic plant extract Produced
highest inlibition zone against S aureus (3.5 mm)1, foliowed by S. pTogez (3 mm) and
S. epidermidis (2.1 mm). Methanol exh:act significantly inhibited S. aureus (4.2 mm),
followed by S. epidenntdis (1.5 mm) and S. PyoAez (1.1 mm), however water exkact
was unable to express activity against the tested pathogens (Table 5.2).
There was different tretrd of selected drugs itr tems of their inhibitiotr zones
agahst selected bacteria (Table 5.2). Chtorumphenicol showed a inhibition zone
against pathogens that orderly inctude: E coli (23 mm} S pyogen (2lmm)' S'
epid.ermidis (19 mm), S aureus (18 mm), K pneumoniae (15 mm) and P aetuginosa
(12 mm). Cipofloxacin extribited inhibitiotr zone such as: .S. aureus (24 mm)' S.
epidermidis (22 mm), S. pyogen (21 nm), E. coli (19 rnr::), K. pneumoniae (-8 nm)
ard P.aeruginosa (17 mm). Penicillin expressed inhibitioo zone itr the order of: 'S
epidermidis (23 mm), S aureus (22 $rn), S. pyogen (18 mm), K pneumoniae (77
mm) E coli (16 mm) and P.aeruginosa (15 r-r:t).
5.4.1.5.3 Inhibition Zone (mm) at lomg/ml
Ethanol, methaool and water based Verbascam thapsus leaves exhacts were
evaluated for antibacterial activity against various microbes. A11 solvent based exhacts
failed to show inhibition zotre against the selected pathogens (Table 5.3).
115
5.4.1.5.4 Inhibition Zone (mm) at 7.5mg/ml
Antibacterial activity of ethaool, methanol aDd water based l/etbascum thapsus
leaves extracts were evaluated at 7.5mg'ml against various miqobes No activity
exhibited by the exhacts (Table 5.4).
5.4,1-5.5 Inhibition Zone (mm) at smg/lrl
Ethanol, methanol and water based Verbascum thapsus leaves exbacts were
evaluated for antibacterial activity against vatious miqobes Like previous
cotrceotration, no activity rccorded by the tested extacts (Tabl€ 5 5)'
5.4.1.6 Atrtibacterial activity of Zizyphus satita
5.4.1-6.1 Inhibitiotr Zone (mm) at 15mg/n
The inhibitory effects by different solveot extract of Zizyphus sativa was
established against the selected microbes (Table 5.1) Ethanol extract efficiently
intribited all strains aod the highest inhibition zotre was noted equally against S aureus
& S. pyogen (74 mm each), followed by S. epiden idis (13 mm), P aerugtnosa (72'3
nfi), K. pneumoniae (lt rnm) aod E. coli (9.5 mm) Methaaol extract exhibited
almost leading effects ofthe inlibitiofl ofpathogens The highest inhibition zone was
t46
found in S. pyogen (18 mm), followed by S auleus & S. epidermidis (76 mm each), E'
coli (13 mm), P aeruginosa (8.5 mm) and K. pneufioniae (8 mm) These rcsults were
found comparable/nearcst to the standard drugs (Table 51)' Water extact was found
effective against only two strains such as S aureas (14 mm) and S epidermidis (12
mm).
The standard antibiotic (chloramphenicol) showed inhibition zone in the order of
E.coti (26 rnm), S. epidermidis (23 nm), S aureus (27mm), K. Pneu oniae (16 nr,Ir),
P. aen)ginosa (14 mm) and S pyogen (14 mm). Ciprofloxacin exhibited inhibition
zotre in the order of: S. aureus (28 n)m), S epidermidis (26 rnn), S pyogen (23 mm)' E'
coli (27 mm), K pneumoniae (20 mm), P aetuginosa (19 mm), while, Penicillin
inhibited the grou'th of selected bacteria in the order of: S ePidermidis (26 mm), S'
aureus (24 mm), S. pyogen Q0 mm) K. pneumoniae (19 mm), E coli (17 mm) and P
aeruginosa (76 firt).
5.4-1.6.2 Inhibition Zone (mm) at 12.5mg/ml
Ethanol, methanol an d slatet extacts of zizyphus sat[\)a leaves wete evaluated for
antibacterial assay on some microbes. The inhibition zotre of all tested samples is
givetr ir Table 5.1. At the concentration of 12.5mg/ml, all extracts exhibited almost
same results as that of 1 5mg/m1.
t47
Ethaool extract successfi ly inhibited the bactedal got\th of all strains. The
highest inhibition zone was documented against S auteus (\6 mm), followed by S
epidermid.is (15 r;Llri, P. aeruginosa & S. pyogen (14 mm each), ,. coli (11 mm) and
K. Phe oniae (6 mm). Methanol extract inhibited best S. pyog€,x (17 mm), followed
by S. aureus (16 mm), A coli (13 mm), S. epidermidis (70.5 r\m), K. pneumoniae (7.1
mm') and P. aeruginosa (6 mm). The watff exbact significandy inhibited s- a?lreud and
S. epidermidis \\lrhthe inhibition zones of 12 mm and 10 mm respectively (Table 5.2).
Therc was different kend of selected drugs itr telrns of their inlibition zo[es
against selected bacteria (Table 5.2). Chloramphenicol showed a inhibition zone
against pathogem that ordelty include: Z. coli (23 mm} S pyogen (27mm), S'
epid.ermidis (l9mm),S. aureus (18 itfi), K pneumoniae (15 mm) andP aeruginosa
(12 mm). Ciprofloxacin exhibited inlibition zone such as: s. aureus (24 ntD)' S.
epidermidis (22 urm), S pyogen (21 rtrirt).E coli (19 mm), K. pneumoniae (18 mm)
alrd P.aeruginosa (17 mm). Penicillin expressed inhibition zone in the order of S
epi.dermidis (23 mm), S aureus (22 n\n), S. pyogen (18 mm), r<. pneumoniae (.7
rnm), E coli (16 rnm) and P.aeruginosa (75 mm).
5-4.7.63 Inhibition Zone (mm) at lomg/ml
The antibacterial assay of vadous solvents exhacts of Zi?phus sativa was
determined against some selected microbes. The irllilition zone is compiled ir Tahle
5.3. At the concentration of lOmg/ml, ethanolic plant extmct expressed inhibition zone
148
against the selected strains of bacteria and highest o[e was rccorded against 'S'
epidennidis (12.5 mm), followed by S. Pyogen (11 lrllr,)' E coli (9 mm)' P
aeruginosa (7 .4 m.rr.), S. aureus (6.2 mm) and K pneufioniae (5 4 mm) T}re methatrol
extract also inhibited the gro*th of pathogens and t}1e inlibition zones were in the
ordet of: S. pyogen (70 mm) >e coll (8 9 mm) > S aurezs (8 5 mm) > S epidermidis
(8 mm) > P. aerugi osa (6.2 mm) > and K p/Leum<)niae (6 1 mm) However' water
extact was only foutrd effective aga:lirst S awe s, S epidetmidk with the inhibitiotr
zones of l0 mm atrd 8 mm respectively'
Antibiotics had expressecl vaaous activities against the strains (Table 5'3)'
Chloramphetricol exhibited inhibition zone in the order of: e coli (20 rom), S pvogen
(19 mm), S. epiderttidis (18.5 r.Lrl), S aureus (18 rrtrt) K pneumonide fl3 mm) and
P. aeruginosa (10.3 rrlln). Ciprofloxacin expressed highest hhibition zone for S
aureus (22.an), followed by S epiderilidis (19 lrrlm)' S pyogen (20 4 mrt)' E- coli
(1,6 r \), K. pneunoniae (13 nrn) arLd P. aeruginosa (15 mm), whereas, Penicillin
showed hhibition zone in the order of: s. elidermidis (22 mm), S aureus (21 5 mm)'
S. pyogen (16.3 i\m), K. pneumoniae (15 mm), E coli (74'2 r,lra) aod P aerugtnosa
(14 mm).
5.4.1.6.4 Inhibition Zone (mm) at 7'5mg/ml
Ziuphus sativa leaves were employed for the extract preparatio' by usiag ethanol'
methanolatrdwatelsolvents.Theseexkactsweleevaluatedforantibacterialactivity
t49
against some selected microbes. Inhibition zone of all tested samples was recorded and
rcsults are compiled in Table 5.4. Ethanol extract revealed highest inlibition zone
agzil:nst S. epidel idis & S. pyogen (12 mm each), followed by ,. coli (9.5 mm), ,(.
p euhoniae (6mm), P aeluginosa (4.4 mm) a]ldS aureus (3.5mm)- In the case of
methanol extract, S. epidermidis was found highly susceptible afld inhibited best with
the bhibition zone of 13 mm, followed by S. pyogen (11 mm), E col{ (7.4 mr.r.), K
pneumoniae (6.5 mm), P. aeruginosa (5 fim) atd S auteus (4 mm). Water extract was
found effective only against .S. arrer6, S. epidermidis havlng l0 mm and 7 mm zone
inhibition respectively.
The standad antibiotics exldbited inhibition zone variously against the pathogens
Chloramphenicol inhibited pathogenic colonies itr the order of: S- pyogelx (17 mmI S.
epitlermidis (16 mm), E coli (15 mm), S. aurcus (13 mm), K.pneumoniae (12 5 mm)
and P.aeruginosa (10.0 mm). Ciprofloxacin exhibited inhibition zone in the order of
S. aureus (20 1mm), S. epid.ermidis (17 mm), S. pyogen (16 mln), P. aeruginosa (14
rnm), E. coli (73 mm) and K. pneumoniae (17 m), while Penicillin showed highest
inhibition zone against S. epidermidis (20 mm), followed by S. aweus (18.5 mm), S.
pyoge (14 mm), K. pneumoniae 04 mrn), P aeruginosa Q3 4 mm) ar,d E. coli (12
mm).
5.4.1.6.5 Inhibition Zone (mm) at smg/ml
The antibacteriat assay of various solvents extracts of Zizyphus sati\'a was
esiablished against some misobes. At the concentation of smg/ml, all extacts failed
to inhitit the growth ofbacterial strains (Table 5.5).
150
Minimlrm Inhibitory Concentration (l[C) !trd Inhibition Zones5.4.7.27
(m-)
The results of MIC and Inhibition zones are provided in Table 5.6. it revealed
that T. stoclrsianum leaves exkacts fulibited all bactedal strains at minimum
concentration (5mg/ml) varying with the inhibition zones r.mging Aom 3.G10.0.2 to
6.5+0-7, however, aqueous extact iniibited P aeruginosa at 7.5mg/m1. In additioD,
except S. epidennidis, flower extacts checked al1 bacterial stains at smg/ml
cotrceot ation- M. communis was also another potential plant which derived activity
against all microbes exce,pl K. pneumoniae at the concenkation of 5mg/ml. Ovemll, Z
stoelsla ufi leaves and floweN atrd methanolic as well as ethanolic extiacts of M
cofimu .is i)hibited. S. aurelJ at mioimum concentration (j.e. smg/ml), followed by Z
sativa (1.5mgy'ml), methanolic aIId ethanolic extacts of ,4l buxifulia and aqu'eo:Jrs
extract of M. communis (7.5m9/ml each), while V. thapsus inhibited relatively at
higher corcetrtation (Table). No activity was recorded against the said bacteria by
aqueous exhacts of M. bwifuha alrd V thapsus. T. stocksialxum leaves extact
effectivety checked the bacterial growth of S. epidemidis at minimum concetrtation,
bowevq flower exracts ofthe said species lailed to express activity oD it.
Ir1 the case of,44 communis, ett.ar,ol afid methalol extacts exhibited activity at
5mg/ml and aqueous extract at 12.5m9/ml. The M. bux{folia (ethanol and methanol)
dr,d Z. satira extracts expressed activity at the cotrcetrtration of 7.5mg/ml, followed by
151
ethanol and methanol extracts of Z Thapsus (l2jrr.dtul). S. 1ryogen was inhibited by
T. stocksi@ um leaves, floweE and ethanol and methanol exkacts of ,ll communis zt
5mglml, followed by etharol, methanol extracts of M bttxifulia, Z. sativa (1.51ngl,J]l
each), while aqueous extract of ,,ll communis and eth?rrrol and methanol extracts of Z
,raprur express activity at 12.5mg/m1 (Table 5.6).
E. coli 'was inhibited best at 5mg/ml concentation by all extacts of 7.
stocksianum leaves, flowers and ethanol, methanol extmcts of M- communis leaves,
followed by ethanolic and methanolic extracls of M. bwcifulia ard Z. sattua (7.sl,]gn\l
each) and aqueous extact of M. communis (12.5ng/mt), while no activity
demonstrated by the exhacts of V. thapsus and \\at$ extracts of M br,uifulia arjd Z.
sattua. Exoept M. communis and V. rftaPs,.t, all plant extacts exhibited activity against
K. pneumoniae in either of solvent. Leaves and flowe$ extracts of T. stocktianufi
showed activity against the said microbe at the lowest cotrcentration (5mg/ml),
followed by ethanol and methanolic extracts of M. btuifulia afi' Z. satila at'7.5m9/n\1,
while no activity demonstrated by aqueous extracts of ihese plant extracts. For P
aeruginosa, all plant exracts established aciivity, except V. thtipsus. All exhacts of 7.
stocksia um flower showed actiyity at 5mg/ml, while leaves extacts also inhibited at
the same concentration except watel which inhibited it at 7.5mg/ml M. communis also
proved to be the best choice of drug against tlre same bacteia which inhibited at
5mg/El by ethaDol ard methanol extracts and aqueous at 12.5mg/ml. in the case of M'
bltxifolia ajnd, Z. satira, C'thalrol and methanol extracts inhibited at 1.51lrdlltl
coDcentration (Table 5.6).
t52
5.4.1.2.8 Tissue equivalent vs. activity
The highest activity was drawn by M communis tl7at was almost half of the drug's
inhibition zoDe against ali bacterial stains except K. pneumonia (Table 5.7). The tissue
equivalent activity is showo in Table 5.7 revealing that for triggering activity, how
much tissues of piant required. The lowest tissue co(esponds to the highest activity
atrd vice versa. The highest activity was exhibited by methanolic exkact of .41
coflmuhis a1ainst S- aureus S. epidermidis, S. pyogen, E. coli, K. pneumonia and, P
aeruginosa (28.41), followed by methanolic T stocT.sianum teaves (30.58), etlanolic
M. conmu is (31.06) and methanolic 7. stoclisia m tTower (33 78). Wlile in the case
of K. pneumonia and. P. aeruginosa, it was followed by methanolic extract of U
stoclcsianum flowet Amoflg the selected plants ,both M. communis and T- stocl',sianum
showing good activity in compadsioo of tle commercial antibiotics such as
Chloramphenicol, Ciprofloxacin aad Penicillin.
^ nrrr Fr _\rr:
^ r a(-TrvITv oE' !IEn!(!rg.4l PL.A-\TS
The autifungal actlily of Monotheca btaifolia, Myrtus commuhis' Teucriun
stocksianl,tm, Verbascum thapsus urd Zizipus sativa was tadertaken against two futrgal
strains (i.e. Aspergithts tiger and. A. fumigatus) through tube dilution method at a
coocetrtation of 12mglml Aom water, methanol aad ethanol extracts Significant
difference observed iII the activity Aom solvent based extracts fu>0.05) and the
activity was itr the order of meihanol > ethanol a]ld > water. The detailed observaiions
are described as under:
153
5.5._t Methanolic Plant Extract
Results are depicted in Table 5.7. Methanolic plant extract ofMonotheca buxifolia
exhibited 60 mm linear gowth and inhibited 49.159,o fungal gowth compared with the
control (1 18 mm LG) ag inst AspergilLus niger. ln tl.e case of Aspergillus llmigatus,
75 mm grofth inhibition with 36.0470 antifungal activity was recorded by the sam€
extract. Methanolic plant exkact of Myrtus cammunis showed 42.3770 antifungal
activity with 68 mm linear growth compared with the standard (118 mm) against
Aspergillus nige4 whlle 58.04% activity and 45 mm linear recorded against
Asperyilus fumigatus.
Methanolic Teucrium stocksianum Ieaves extract resulted in 61.58% antifungal
activity with 45mm linear gro$'th against Aspergilllts fiiger', howevet, Aspergillus
furkigatus itfii[.ited. with 50.60% with 55 mm linear grouth. Froln Teuciutt
*-,1"--^r:^ o^..ar avkz.r AP A<q^ '.;wiR, *,,. dndrmentcd h:tino 't7
mm linear growth against Aspergillus niger ar.d 56.25o/o as well as 49 mm linear
gowth against lspergi I lus fuikigatus.
Methanolic leaves extract of Verbascum thapsrr exhibited 15 90% hlibition with
80 mm thear growth agaillst AsPergillus niger and 37.50oA a(,iiry with 70 mm linear
grow+h agaiost Aspergillus fumigatus. In the case of Z. sativa,.4. n4ger was formd
highly susceptible and best inhibited (63.88%) with the linear gro*'th of 35 mm.
followedby A. fumigatus rravsrg'l 3.22yo ilhlbition and I0 mm lLnear glouth Ctable 5 7).
154
Ethatrolic Plant Extract
Results are provided in Table 5.8. Ethanolic leales extact of Monotheca buxifulia
expressed 38.68% antifungal activity with 65 mm linear gro*th agaitst Aspergillus
niger aofiparcd to positive control (106 mm). The same effectively controlled at
27.84% inhibitioo with 70 mm linear in the case of Aspergillus fumigatus (Table 5.8).
Myttus communis ethanolic extact showed 46.23% irlLibition with 58 mm linear
grorth agaiDst Aspergillus niger ai],d 61.86%o $'ith 37 mm linear growth against
As p e r gi I lr.ts fumi ga tus.
Ettlalaolic Teucium stocksiakutt leaves extract established 63.21% antifungal
activity having 39 mm linear groMh against Aspergillus niger compared with positive
control (106 mm), wlile Aspergillus fumigatus was inhibited at 31.96% with 66 om
linear by the same extract. In the case of ?eudium stoclgianum flower, ethnolic extract
exhibited 68.87% activity with 33 mm linear grou'th agaiNt AsPergillus niger
compared to cotrtrol (106 mm), while the same showed 41.240% activity with 57 mm
linear growlh against l sPeryillus fumigotus.
Yerbascum rrapsu ethalolic exhact delineated 43.40% antifi:ngal activity with
60 mm linear gowth against Aspergillus niger with rcspect to positive cotrtol
(106mm), while Aspergillus J nigah,6 lrtibited by the same exllact at 24-15yr
inhibition with 73 mm linear grou4h. 1^ lhe case ol Zizyph,6 Jaliva, ethanolic exkact
showed 62.27yo activity with 40 mm linear grouth with agaist Aspergillus niger
compareal to the standard (106 mm), while 62.89% iniibition and 36 mm linear gor,th
obserevd against lspe rgi llus fun iga tus.
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{38 l3= t3E
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5F5 !,+*:.
+!-z;
9.
s
l+tr H- tr.
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+ t++l++
in9E
l- Pr H- !*
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t+ H-l+ t+ t:-
bx2
t t+
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+ H-
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9<
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2?
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=
!s i:E: !{
=!q'
q:
+ +t+t+ THL"
=;
lrPr+ I
t!:t e:i:
t++ll h€Fi
+ ++tr. t5:-o,q?
trH+ h€t
ti t+ lr H-++ Pr+
Q?i=
+H-+
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t:-u
"-!G
z
g d!l
zg9-
9.
o o
+H +
ii tr +:-
++ +lr ll
tr
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:.
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q1
3E' :t:; c
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1a I =
71
4 I z
:
sl,' ; 3
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t
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?P
E9
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Y F E!.J
:
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4
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159
Table 5.8: Antifungal activity of methanolic extracts'
Legetrd: m€an+SD; values with different superscripts in the same column differ
significartly (P < 0.05); L.G.C=linear gowth in conhol, L G T' =linear growth in test
tubes, MB= Monotheca buxiJolia MC= Myrtu: conmunis TCSL Tcucrium
stocA.sianu t leaves, TCSF= Teucrium stocksianum ftowet W= Verbascuru thapsus'
andZS= Zizipus sLt;Ya.
Platrts
A. niger A. fmigatus
L.G.C
(mm)
L.G.T.
(mm)Inhibition 7.
L.G.C
(am)
L.G.T.
(mm)Inhibition Yo
MB I l8+0.00 60+0.0.05 49.L5cD 112r0.00 75+0.10 36.041
MC 118+0.00 68r0.10 42.3 i L2+0.00 47*0.01 58.04cu
TSL l18ro.0o 45+0.10 6i.5 8B 11210.00 55!0.00 50.60F
TSF 1I8+0.00 37r0.00 68.65A 112+0.00 49.r0.10 56.25Er
VT 118+0.00 80+0.0.02 15.90c 112+0.00 70+0.0.07 37.50HI
7,5 1I8+0.00 35+0.00 63.8848 112r0.00 30,r0.0.01 73.2),^
t70
Table 5.9: Antifungal actiYify of ethanolic plant extracts
@erscripts in the same column differ
significantly @ < 0.05); MB: Monotheca bu-xi,folia, MC: M!'/tus communis, TCSL:
Teucri m stocksianulh leaves, TCSF= Teucrium stocksianum llower W: Yetbasatm
thopsus, al.d ZS= ZiziPus sativa.
Plant
A. niger A.fu islttus
L.G.C
(mm)
L.G.T.
(mm)Iubibitior (%)
L.G.C
(mm)
L.G.T.
(mm)Inhibitiotr (%)
MB 106+0.00 65+0.10 3E.68E97n0.00 70r0.0.10 27.84K
MC 106,10.00 58a0.07 46.23.D 9?+0.00 37r0.05 61.868c
ISL 106+0.00 39+0.10 63.21A897+0.00 66+0.02 3t.961
TSF 106a0.00 33+0.10 68.87A97!0.00
5710.10 4l.24GE
w 106+0.00 6Gr0.0.10 43.40DE97+0.00 73+0.10 24.'75K
ZS 106+0.00 4010.0.03 62.2'?^B 97+0.00 3610.03 62.898
\'7 t
5.5-3 \Yater plant extracts
Results of antifungal activity of aqeous extracts are shown in Table 5 10. Water
extrad of Monotheca butifulia revealed 58.887" antifungal activity with 41 rrrm linea]
go\r4h against l.spergz llus niger compared to positive contol (87 mm linear gro*4h).
For Aspergillus fumigatus, 36.23Yo inhibition and 65 mm linear gro*'ih expressed by
the aqueous extact compared to cotrtrol (Table 5.10) The aqeous extact of M./r s
co munis l:nhibited Aspergillus niger \lirh 27}9% exhibiting 63 mm lioear gro*th
compared with positive control (87 m linear'gowth). In the case of Asperyillus
ful igatu.s, this extract showed 52.43% inhibitioD arld 49 mm linear go''rth with
rcspect to conhol ( I 03 mm).
Teucriutt stocksianum leaves extract established 18.40% aDtifungal activity
along with 71 mm linear glowth again sl Aspergillus niger, wbile Aspelgillw fumigdtus
irlibited with 15.54% having 87 mm linear $oPth (Table 5.10) In the case offlower
extacl of Teucrium stocksianum,2l.64Yo ilrhibition recorded along with 68 mm linear
Ero,h,Lh ag?ilnst Aspergillus niger cor\pared lo positive conhol (87 m$). wlile 26'22V'
vrith 76 mm linear growth was recorded fot Aspergillus fumigatus compared to contlol
(101 mm).
The aqeous extract of yerbascufi lraPsus expressed 38.32% activity witl 53
mo linear grow'th against Aspergillus niger with respect to conftol (87 mm). For
Aspergiltus fumtgatus, this extact exhibiied 18.45% inhibition with 84 mm growth
172
(Tab1e 5.10). Frorl Zi4phus sativa aqueous extact, 36.79% antifungal activity witn 55
exhibited by positive contlol. ln the case of Aspergillus fu tgatus, 43 07% antifungal
activity and 59 mm growth was documented compared to control (103 mm)'
5.6 DISCUSSION
The herbal treatnent is cheapel and simple to Procue and manage ln the
westem world, exploratiotr of plant based medicine began with interpretatio[ on ihe
traditional usage, followed by extraction, identification of active principles and fitrally
formulation and clinical trials (Hextable, 1993). Vaious data claimed that majority of
the people in the world are rellng on the phltomedicine, lather than commercial
products (Barret and Kiefer, 1996). Developing counties like Pakistan are dep€ndetrt
o[ natural plant resouces for medicinal, food, forage, construction of dwelling,
making homes and household goods and for fuel. Pakistan is endowed with variety of
medichal plants. Many ofthese are being used for therapeutic purposes without proper
information about their active agent. Medicinal plants are requted to investigate for
the purposes ofdrug development.
Plant extracts arc screened for the establishment of biological activities based
on ethnomedicinal uses of the plants. A number of infectious diseases are ktro*'n to
have been culed by using hobal remedies tbroughout the history of human being'
173
Despite ofpast, ir preseDt da)rs, plant remedies are playing a major role in basic health
care. Itr the study area great diversity of medicinal plants is found, these plant are
available from the nearby hilly area. Local people residing village trade these
medicinal plants to he.bal stores. Medicinal plants, which are growing raturally iD
different seasons of study are4 are used locally in various forms to treat many
ailments. People use plant as extract, powder atrd condiments. Biological activity is a
face tetling the valuable or helpful possessions of crude drug or living substance.
When the medicille is a multifaceted chemical combination, then its action is exerted
by the compound irgredients but can be customized by the other constituetrts. The
chief category of biological activity is a compound toxicity. The acriviry is usually
dosage depetrdent and it is not rmusual to have special effects ranging as of
encouEging to undesirable for otre material as available as oflow to high to doses.
The present research work was cooducted to evaluate the antimicrobial activity of
five highly trsed medicinally plants s\ch as Monotheca buxifalia, Myrtus communis,
Teucrium stocloianum, Yerbascum thapsus a,]d Zizipus satiya by using methanol,
ethanol and water solvent extacts. These plants are frequently used as indigenous
remedies by the tratives. Native uses of these species are well defined itr Chapter 2.
Plants which have better biological activities especially antibacterial activities are
easily and efEcietrtly soluble in ethanol and methanol ard extact m&\ioum ingredient
ftom platrt material.
171
Table 5.10: Antifungal activity of aqueous extracts.
Plant
A. fiiger A. fumigatus
L.G.C
(-m)
L.G.T.
(mm)Inhibition (Yo)
L.G.C
(Dln)
L.G.T.
(mm)Inhibition (%)
MB 87+0.00 41+0.0i 52.88c 103i0.00 65+0.10 36.231
NIC87+0.00 63+0.2 2'7.59F 103+0.00 49+0.10 52.$EF
TSL87+0.00 71!0.I0 18.40c 103i0.00 87+0.15 15.541
TSF87i0.00
6E+0.05 2t.61Fc103+0.00
76t0.4'7 26.22K
VT87r0.00 53r0.10 38.32E
i03+0.00 8410.I0 18.45K
ZS87r0.00 55+0.10 36.79E
103+0.00 59+0.05 43.A74
Legend: mean+SD; values with different superscripts in the same column differ
significartly (P < 0.05); NIB= Monotheca buxifulia, MC= Myrns communis, 'ICSL=
Teucrium stocksianut l leaves, TCSF= Teucrium stocl<rianum flower I/?= Verbascum
thapsus, and ZS= Zizipus sativa.
t75
In the present study, organic solvelts i.e. ethanol and mefhanol extracts
exhibited maximum antitactedal activity, while aqueous extnct showed relatively low
activity against the tested bacterial stains. Various studies signified the importance of
ethanol, methanol and water as solvents for bioactivity of various plants (Bisignino €l
al, 1999j Loureff e/ a1.,2004i ParcVJr et al.,2005; Rojas et a1.,2006). However, it is
evidetrt that herbalists (Hakeems) use aqueous e{tract to treat various diseases from
trative platrts, while scientifically it is confirmed that organic solvents are more reliable
for antimicrobial activity (Parekl et a1.,2005). Results of this study clearly showed
that gram Degative bacterial strains were inhibited best by Teucriu t stocl$ianufi
leaves, whercas, feucrium stocksiahu flower mostly inlibited gram positive bacterial
straiDs. Organic solvetrt based Mrrl[r commutT[s leaves extacts showed antibacterial
activity agahst all strains exceF)t K pneumoniae, whlle aqueous extact did not target
any microbe.
The antibacterial assay exhibited good activity against the test orgadsms.
Compared with solvents, orgaDic solvents viz., methanol and ethanol extracts showed
relatively higher antibacterial activity against multi-drug resistant clinical isolates. It is
motivating to lrote that trvo ofthese five platrt specles, Myrtus communis urd Teucrium
stocJsianum were highly active against bacterial. These results were found
comparable/nearest to the statrdad drugs (Table 5.1). Even at the lowest concentration
(i-e. Sm{ro,l\ Myrtus cammunis .rrd Teucium stocksiahuh extacts showed relatively
higher activity compared with standaid drug, while other thee plants did not show any
activity at this conceatratioo against the tested organisms.
1',76
It can be concluded that these extracts hoid potential for further work agaiflst
these bacteria through toxicity testing. The usefuhess of antibacterial assay has also
been hcknowledged by vadous scientists fiom various species s'ach as Adhatoda
zqtlanica (llango et a1.,2009), Holarrhena antidysenterica, Leucas aspera (Prcethi et
aL,2010) and Trianthema decandra (Geethalaksbmi er al, 2010), Argemone mexicana
(Rahman el al., 2011), Tinospora cordiftlia and Cassia fstula QJpadhyay et al.,
2011).
The study also revealed that aqueous extract showed moderate activity. The
finditrgs of present study are in line with those of Murugesan e/ al (20 1 I ) based on his
work on Memecyloh umbellatum. Besides, wat leaves extract of Pterospermum
acerifolium wu fottrd to possess good antibactedal effects against a number of human
gram positive aod glam tregative bacteial strains (Thatoi el al, 2008).
Aspergillus fu igatus carses Aspetgillus infections (Hedayati et al.,2OO7t
Alessandro, 2008). On the other hand, l. n iger is food bome pathoge[ that causes a
seious lung disease, aspergillosis, otomycosis pain iflarge amoutrts of spores inhaled
(llema et a1.,2009). These two imgal pathogens are responsible for causilg skin
diseases acting as dermatoph)4es. Keepirtg importance of both the organisms,
antifuogal activity of ethanolic, methanolic and aqueous extracts of Monotheea
btuiftlia, Myrtus comhuni& Teucriur stocksianum, Verbasatm Thapsus alJd Zizipus
sahra was carried out. Results reveaied that all plants possessed antifungal activity
t7'7
agafist A. fumigatus arld A. niger. With respect to solvent extracts, the highest
antifungal activity was shown by methanolic, followed by ethanol and water.
Methanolic Z saliva leaves extract inhibitedbest A. fumigatLls (13.22%)''whlLle
l. ,iger was inhibited at'70.34% (T^ble 5.7), while rest ofplant exh-acts had relatively
low activity. From ethanolic ex,tracts, A fumiSalrs was inhibited almost equaliy by Z
sattua and M. b*r{otia 62.89 and 61.86% respectively, while l 'igel was best
conholled by T. stocksianun flower & leaves (Table 5.8). The aqueous extnct also
derived significant activity agaiNt both pathogens (Table 5.10). In this case, A. niger
was susceptible a+arnst M. buxifolia leaves exhact (58.88%), whereas, M. communis
leaves extract intdbited best l. rDtlgatus. Similar findings are rcported by Naz and
Bano (2012) froan Ricinw cot munis &d revealing the supremacy of methanolic
extract followed by water.
CONCLUSION
The extacts ofselected plants were subjected for in vito antibacterial activity
agai[st some gram positive (i.e. S. auteus, S. epidermidis atd Streptococcas pyogen)
and grarn negative (i.e- Echerichia coli' Klebsella pneumonia af.d Pseusdomonas
aeruginosa)bacteial strans at ihe cotrcenhations ranging ftom 5 to 15mg/ml by using
agar well diffusion method All these plants showed significant differences in
i.libiting bacterial gro*th (P!0.01). Gram positive bacteria wele found mole
susceptible than glam negative. The extracts expressed good antibacterial activity
178
against selected bacterial strains eveo at the lowest concentration (i.e 5figt1l), Myttus
communis and Tel-tcrium stocksianum extracts showed relatively higher activity
compared with standad drug, while othel thrce platrts did not show any activity
against the tested organisms at this concedtation. A fumigatus afj A. nige|vlerc
inlibited best (13.22% ad 70.34% respectively) by methanolic Z. sativa led\es
extract, while rest of plant extracts had relatively low activity. The ethanolic extracts
inhlbited A. fumigans somewhat equally by Z. sat]a and M. buxifolia (62.89 aid
61.86% respectively) , while A. niger was best controlled by T. stocksianum flowet &
leaves. The aqueous extact of M. butifolia leaves irlfbited l. niger witt. 58.88o/o,
whereas, M comhunis leaves exhact iDhibited best A. fumEatus (52.43%) From the
results, it oatr be concluded that present itrvestigatiotr validated scientific ground for
thei! ethnobotanicat use in the study area. Out offive, M communis' T- stocksianum
and Z. sativa possess strong a ibacterial and antifungal activity that hhibited the
growth of pathogens even at lowest concentation. It is evident from the results that
reported species can be exploited for pharmacological soeening, isolation and
identification of active principles and ultimately il drug developmelt programs in near
future.
Chapter 6
ANTIOXIDANT ACTIYITY
6.7 INTRODUCTION
Free radicals produced inside the ccll are responsible for oxidative stress that
are responsible for the development of various diseases such as Palkinson's disease'
Alzheimer's disease, rheumatoid arthdtis, Pathologies caused by diabetes aod
neurodegradation in motor neuron disease. Antioxidants are specified molgcules that
inhibit formation of free radicals by removing their intermediates Today, developed
colrntdes are manufacturing various formulations of antioxidants and sell them as
dietary supplements by nutraceutical and health food companies (Radimer e' a/,
2OO4). The antioxidant activity of a Plant may be attributed due to existence of
phenolic compor.mds such as phenolic terpenes, flavonoids, tannins and pollphenols
(Rahaman and Moon, 2007) Chelation derived Aom "chelos", the Greek word for
claw, invoives the incorpomtion of a mineml ion ol cation into a comPlex ring
structure by an organic molecule, the chelating agent. T)?ically, electron-donor atoms
on the chelating molecule include sulphur, nitrogen' and/or oxygen The stength ofthe
chemical bonds are formed between chelato6 and metal ions depends upon the
elements involved and details of the stereochemistry. The identity of the metal
predominately bound by a chelating agent depends both upon accessibility of the
chelator to the tissues (Apostoli er a/., 2006).
t'/9
i80
Chelators have the effect ofmobilizing metals fioD tissues ard maintaidng the
chelate moiety dudng circulation to the kidneys for excretion id the urine, aIId to the
liver for excretion in the bile. There are significant concems related to enterchepatic
rcckculation and re-absorption in the kidney (Rooney, 2007).
Despite medicinal use of antioxidants, they are heavily used in cosmetics and
food prese(ation. Various antioxidants such as ascorbic acid, propyl gallate,
tocopherols and tertiary butylhydrcquinone prevent spoilage of frozen or refrigerated
food by inhibiting oxidation (Zallen et al., 1975). Some ofthe antioxidants are also
used as stabilizers to prevent pol).merization of fuels alld lubricants atrd ratrcidity in
cosmetics (Boozer et al., 1955\. Amongst antioxidants, phenolic compounds are
leading group of compoutrds and have beeo recomrnended by many workers (Gulcin er
al., 2007, Prukash et al., 2007).
Plants aie endowed with lumerous phenolic compou:nds used in different
functions and are thus a rich source of phenolics. Pakistan is blessed with the rich
flodstic diversity consisting ofplants growing in vaded ecological zones mnging ftom
hot and dry deserts to humid rainforests and cold Mouataias. Like various rich cultual
hedtage, Malakand division is the one which possess nch heritage of plant use as
medicametrt since ancient times. This study extracted valuable plaots and their
taditional uses as provided in Chapter 2. It revealed that people are still relfng on
medicinal rcquirement from the native plants. Based otr the ethnobotanical
information, a study was designed to carry out systematic study for antioxidant and
18r
fiee mdical scavenging activity of highly reported plant species such as M//rr..t
communb, Verbascum thapsus, Teuciut l stocksianum, Monotheca buxifulia aDj.
Zizyphus sativaby *\e inhabitants of Malakaad division.
6.2 REYIEW OF LITERATURE
Plants possess potent biochernicals and are integral part of phytomedicine srnce
times immemorial. Man has ability to derive/exkact industrial chemicals ftom any part
of plant i.e, bark, leaves, flowers, roots, fruits, seeds, etc (Makari et a1,2008), Mary
herb species such as thyme, sage, oregano showed antioxidant activity in their exftacts
@anovska et al., 2005).'feas, wines, fruits, spices, vegetables are already exploited
either as atrtioxidant additives or nutritional supplements (Schuler, 1990).
The metha]lolic ieaves extact of Mimusops elengi el.ai]oaied by DFPH assay,
reducing powo and total antioxidant capacity. They coflclud€d that the metanolic
extract of this plant species may act as a chemoPrcventative agent providing
antioxidant Foperties a:rd offering effective protection from free radicals (Saha et al,
2oo8).evaluated antioxidant activity of methanolic and hexatre exhact of two plants
Cordi wallichii and Celasb-us peniculala through DPPH assay. They concluded that,
with the increase in the concetrkation of exkact scavenging activity inqeases aod they
founded that methanolic extract of C wallichii was Inorc effective than hexatre extracL
t82
Niranjatr and Tewad (2008) examined the rcots and aenal pdrts of Des o'liuh
gangeticl)m though DPPH and reducing power assay. They concluded that trot only
roots but aerial parts of this plant also cotrtaio phenols and phltochemicals which are
responsible for antioxidant activity.
!a;i et al. (2007) concluded that the methanolic extacts of Leptadenia
pyrotechnica contain some specific active compounds which can deqease liPid content
of serlrm aod reduce the chances of athercsclerosis in those rabbits which are
cholesterol fed.
Panovska e, al (2005) evaluated tbe 14 vitro atrtioxidant aciivity of Teucrium
species. They used three assays i.€. DPPH assay, hydrcxyl radical assay and beta-
carotenes-linoleic acid. The result showed tiat Teucium species possess fiee radical,
hydroxyl radicals and antioxidant activity
Ara and Nur (2009) d etefiined irl vitro anlioxidant activity of methaDolic extracts
of leaves and flower of Lippia atba lhrottfi, DPPH method. They reported that natual
aotioxidant compoulds can lead us to intoduce new drugs which are helpfirl in
aotioxidaot therapy. In medicinal field plants can be considered as a good source of
Datural aatioxidants. If we increase antioxidant rcserves ofan organism, we can reduce
oxidative stress.
Ravishankara et al. (2006) evaluated the antioxidant potential of the methanolic
183
extract of root bark o f Hemideenus i dicw in different i' vi"'o and ex viva models by
using different assays. The extact was folnd to have different levels of antioxidant
propeIties in the models tested. They concluded that the fiee radical scavenging
propedy of this plant may be one ofthe mechanisms by which this drug is effective in
seveml free Edical mediated disease conditioos'
A\y), et al. (2010) evaluated the i,, virlo atrtioxidant activity of Anisopus manii
N.E. Br. The crude methanol extract, ,-butanol and ethyl acetate Aactiofls were
investigateil for ftee radical scavenging activity of the DPPH, total phenolic contents
and reducing power assay, They cotrcluded that the crude methanol extract and solvent
fractions of l. mannii have irrdlcated strong antioxidant activities, ir, vi'lo The platrt
cofltained phenolic compounds which can serve as natual souces ofantioxidants'
Basniwal et aL (2009) ilvestigated the aqu€ous (hot) exkact of Helicteres sora
Lirn. fruits in various in itto fiodell They compared differeot standards and
cotrcl'rdeC that llsol4 also coltain phellols and flavonoids, which are important in
controlling oxidation.
Za\ir et al. (2009) studied iz vitro antioxidant activity and total phenolic contetrt
of four medicinal plants. They used diffeient plant parts like root, stem, rhizome and
bark ofthese four plants atrd they used different assays such as TBA, FTC and DPPH
assays. Finally they concluded that taditional medicines play important role in
discovery of naural antioxida[ts.
184
Khalaf et al. (2008) screened out the methaaolic crude extacts of some
commody used medicinal plants for their free radical scavenging ploperties usirg
ascorbic acid as standard antioxidant. Free €dical scavenging activity was evaluated
using DPPH free radical. The study revealed that the consumption of these spices
would exert several beneficial effects by vitue oftheir antioxidant activity.
Fiipa et al. (2009) conducted leseaJch to investigate in virro antioxidant activity of
chloroform exhact of flowel of Saccharum sponta eum. He observed that crude
extract of chloroform showed standard antioxidadt activity. Thakal et al- {2010)
aoallzed methanolic extract of the aerial par:ts of Convolvulus orver?s}J to obtain the
antioxidaot potential. Different concenhations methanolic extract aod its ethyl acetate
Aaction were subjected to antioxidant assays by DPPH method, reducing power assay'
Finally they suggested that this plant have antioxidant activity due to the presence of
flavoaoids.
Alierc et al. (2008) ilvestigated the methatrolic and acetone extract of S4,,sevielra
h)acilthoides leaves and roots. The extract exhibited DPPH Iadical scavenging
activity. From the result it was suggested ihat the said plant can be used as a source of
ratural antioxidant. Zabi et al. (2008) determined the presence of flavonoids in the
Ieaves anal ste|lrr oi Secatuone afzetii. Flavottoids presetrt in this plalt have seveml
structural shapes. All these flavonoids could act in slnergy io order to ircrca-se the
af,tioxidant property of S. Afzelii planl
185
Atfit et al. (2010) the etlanolic extact of Calotropis gigahtea LiI]n was
investigated for its antioxidant activity by reducing power, DPPH and nitric oxide
method. They resulted that this plant has been used in several disease conditions such
as leprosy, tumour, ulcers and piles. Various pharmacological activities reported
includitrg antifertility activity, anti-inflamatory activity, antimycardial inAaction and
antidiarhoeal activity.
IGishnaraju el a/. (2005) assessed ir? ritlo ar,d i vivo artioxidant activity of the
methanolic and water extracts ofbark oflphanamixis polystachya using NBT, DPPH,
ABTS and FRA? assa)s. They concluded that due to the natual odgin aod potetrt ftee
radical scavenging abtlity A. polystachya could be used as a potential preventive
inten entioo for free radical-mediated diseases.
Oyederni et al. (2010) investigated the in vitro ar'd il, vivo atrtioxidant activity of
aqueous extract of Srry chnos henningsii planl using spectroscopic method. Frce mdical
saa./enging activity of r.he plant extact against differeEt assays was cooceit€tion
depetrdent. Total phenols, flavonoid, flavonol and proanthoclanidia were also
determined to assess their effects otr the antioxidalt activity of this plant. They
demonstrated that aqueous bark extmct of s. henningsii has both in 'ri'ro and i 'ritto
antioxidant activities due to the presence ofphenolic compounds.
Kttmar et al. (2008) studied antioxidaat activity of some specific Iudian medicinal
platrts named as .4lDizia annra, Achyrahthes aspera, Cassia fstula' Cassia auriaiata
and, Datura stramoniurr. They observed that a platrt named ,{ lbzzia amara was i\osl
186
effective ir hhibition of lipid per oxidation and plant could be used in dmg
formulation.
Paril et al. p010) evaluated the antioxidant activity of aromatic plants herbage.
They plepared the leaf extracts of the plants and concluded that these plants protect
skin ftom harmful effects of sun aDd are also helpful itr anti-aging cosmetics
formulation.
6.3 MATERIALS AND METHODS
6-3.1 Determination of Total Phenolic Contetrt
Total quantity of phetolic coltetrt in leaves extracts of Myrtw communis,
I/erbasctm thapsus, Teucriu stocksiahwn, Monotheca buxifulia utd ZiziPus satira
was measured by Folin-Ciocalteu rcagent in telm of Gallic acid equivaleot in
mg/g of the exhact using spectrophotomefy after Kiihkdnen et al- 11999) and Wolfe
er a/. (2003). Various dilutions such as 1.0, 2.5, 5.0, 10, 25, 50 and I C0pg/ml of gallic
acid was mixed with 5.0 ml ofFolin-Ciocalteu reageot (l:10 v/v) and 4'0 ml of sodium
carbonate solution (75gn). The tubes were mixed tlrough vortex mixer for 15 seconds
and left to staod for 30 minutes at 40"C for colol development. The absorbance was
measrred at 765 nm using the AJI-Co3 UV-VIS spectrophotometer appetrded
below. The amount of phenolic conteot was calculated with the help of
calibGtion cuve (Fig. 6.1). Results wele expressed as mg/g of Gallic acid
equivalenl using the calibrarion cune:
187
Cotrcertration(pglml) Absorbance(O'D)
100 I.122i0.00
50 0.524+0.00
25 0.275t0.00
10 0.153+0.00
5 0.116,10.00
2.5 0.105t0.00
1 0.033t0.00
6.3.2 ANTIOXIDANT ACTI\'ITY
In order to investigate antioxidant capacity of the plant extracts, tlree
antioxidatrt assays were selected such as Antioxidant activity through DPPH, reduchg
power aIId metal ion chelating agent as follows:
6.3.2,1 PreparationofStockSolution
Stock solution of various solvent based plaDt extacts (i.e. methatrol, etharol
aod water) was freshly prepared by dissolving 0.02 g of plant extract itr 20 ml
of methanol to make a coocentration of 1 mg/ml. Six concentrations viz..10' 20,
30, 40, 50 and 60 pg/ml) were prepared ftom the stock solution to give the
primary staadard. Ascorbic acid and butylated hy&oxfoluene (BHT) were used as
standard aatioxidaots.
183
t.2
I
0.8
i o.e
0.4
0.2
10 60 30
Concentrrlior (pgn )
Fig. 6.1: Calibration curve for Gallic Acid.
wlere .r is the absorbance and I is the Gallic acid conc.
6-3.2.2
189
Free radical scavenging actiYity
The activity was undertaketr by using protocols of Blois (1958) and
Yildirm eral (2000) with slight modification. One ml 1, 1-diphetryl-2-picrylhydrazy
(DPPH) radical solution was fieshly prepared in methaaol. About 0.3 mM DPPH was
mixed in 100m1 methanol and kept in the dark. Various concentatiotrs viz., 10-60
pg/ml were prepared Aom stock solution by putting 5 ml of the plant extract in each
colcentration. ODe ml ethanol was used as negative control and lml DPPH dissolved
in 5ml methanol as positive control. The tests were taken thr:ice. The rcactiotr mixtures
were kept for 30 minutes in a dark room and the absorbance was taketr at
517nm. The fotlowing formula was used to calculate the percentage scavenging
activities of each extract.
DPPII Scavenging Capacity (7o) - Control absorbance - sample absorbance x 100
Cotrtol absorbance
6-3.2.3 Reilucing power assay
The assay was conducted after Oyaizu (1986). The test solutiotr along with 2ml
quantity of each phosphate buffer (pH 6.6, 0.2 14) and potassium fenicyanide
(lOmg/ml) was mixed and incubated for 20 mitrutes at 50"C. After that, 2ml
Trichlorcacetic acid (100 mg,4) was poured to the said mixtwe. In a test tule, an
amount of 2 ml of mixture was combined with 0.4 ml of 0 1% ( v) ferric chloride and
190
2ml distilled water. The absorbance was taketr after 10 minutes specbophtometrically
at a wave length of 700nm. A high reducing power indicated by increased absorbance
of tested sample. The experimetrts were performed in thrice. Butylatedhydroxltoluene
(BHT) and ascorbic acial were useal as refermce standard, while Phosphate buffer fuH
6.6) used as negative control.
6.3.2.4 Metal ion chelattug activity (MICA)
This assay was carried out after Kumar et al. (2001).In this method, 0.5 ml of
extract was added ia 1.6 ml ofde-ionized water along with 0.05 ml ofFeCl2 (2 mM).
After 30 seconds, 0.1 ml of ferrozine (5 mNO was poued into it. The mixture was
shaken vigorously and allowed to stand at rcom temperatuie for l0 min. The test was
prepared in kipticates. The absorbatrce of Fe2*-Ferozine was measu€d at 562 nm.
The chelating activity ofthe extmcts for Fe2+was calculated as:
AO-A1Chelatitrg rate (%) = x 100
Where A0 is the absorbance ofthe blark (without the extract) and Al is the
absorbance in the preseoce of the exEact.
6.3.2.5 STATISTICAL ANALYSIS
All experiments for total phenolic compoutrds, DPPH and reducing power
assay were coDducted in triplicates aod thcir mean standard deviations were calculated'
AO
19i
The data were anallzed by factor factorial design (ANOVA) and P value < 0.001 was
coosidered as significant. In the case ofDPPH the values arc presetrted ia the fomr of
IC5o value that was calculated by using Graph prism pad and biostat 200 softwares.
The mean values. Foa reducing power assay, the data were recorded thrice aod mean
was calculated with +standard deviatio[ (SD) by using Statistix ver.8.l. The mean
ralues uere compared by using Least Significant Difference (LSD)
6.4 RESULTS
6.4.1 TOTAL PIIENOLIC CONTENT (TPC)
The results of total phenolic content (TPC) are presented in Table 6.1- In
present study, TPC of three solvetrt based extlacls of Monotheca bu:tifulia Qea'tes)'
Myttus communis (leav es), Teucrium s to cks ianum (leav es, llow et), Ver bas cum thapsus
(leaves) and Zizipus sativa (leaves) was deteDrined. The ethanolic extacts yielded
TCP as 60.54,10.50, 72.07,11.08, 66.07+0.53, 89.50+0.0.10, 44.73t0.42 and
71.4Gr0.0.34mg GAE/g respectively. The methanolic extracts possessed TPC of
Mytlus communis (lea./es), I/erbascltm thapsu.s Qea!es), Teucrium stocksianum
(flower, leaves), Mo otheca bu-tifolia (leaves) &d Zizip s sanla (leaves) as
80.07+0.42, '77.78+0.91, 89.21+0.19, 102j4+0.62,65.88+0.25 and ?6.83+0 34mg
GAE/g respectively. In the case of aqueous extracts, the TPC was found to be
50.45*0.25, ',72.35+0.50,68.64+0.25,83.88+0.99, 53.30+0.0.25 and 67.78t0.83 tr1g
GAE/g respectively for aforemertioned plant exhacts.
192
6-4.2 Antioxidantactivities
The present research wotk was conducted to assess antioxidant activity of
five highly medicinally plants such as Monotheca bu:xifolia, Myrtus communis
Teucrium stocksianum, Yerbascum thapsus and Zizipus satfua belolging to five
different families Sapotaceae, M),rtaceae, Lamiaceae, Scrophulariaceae and
Rhamnaceae respectively. Various plant parts such as leaves and flowers were useil for
the preparation ofplant exkacts using three solvents such as methanol, ethanol and
water. The activities are as follows:
6.4.2.t DPPII free radical seavenging activity
Various solvent based extracts were analyzed for free radical scavenging
activity ofM buxifulia at differett concentrations (i.e. 10pg/ml, 20tLg/ml, 30pg/ml-,
40pg/ml, 50prg/ml and 60 pg/ml-). The results are tabulated and provided Table 6 2'
A11 extracts were found significant compared to contol (PS0.01). There was positive
corelation between activity and coocentation that was directly proportional io each
other. The lowest concentlation (i.e. loFg/mL) exhitited good activity that was in the
orderly marurer as 1'7.44+0.008,23.6'710.005 and 24.21+0.002 for water, ethano! and
methanol, extacts. At the higher colcentration (60 pglmI-), methanol showed the best
results (78.36+0.004), foilowed by ethanot (67.90+0.0 001) and water (55 5510 002)'
The standards were higher itr activitl however, methalolic leaf extract was near to
ascorbic acid (93.63+0.01) aod BHT (96.58+0.004). The ICio (pg/ml-) values of '42
193
bldifolia leaf exhact are 31.81, 36.98 and 55.88 for methanol, ethanol and water
extracts respectively (Table 6.2). Based on ICso value, methanolic extact showed the
best antioxidant activity that was compamble to ascorbic acid (17.83).
Free radical scavenging activities of methanol, ethanol and water qude
exhacts of 1,{ communis 'e'as measured by ernplolng differcnt concentratioEs (i.e.
l0pg/ml-, to 60 pg/ml) and results are presented in Table 6-3. Results were found
significant compared to control (P50.0t). There was increasing lrend in activily that
was diiectly prcportional to exhact concentrations. At 10 Fglml conceotation, the
activity was recorded as 29.43+0.1045, 33.39+0.030 and 43.70+0.0.003 for methanol,
ethaDol and water extracts respectively. The water exkact was closer to the Ascorbic
acid (45.0410.007). The highest concenhation (60 Fglm1,) exhibited maximum activity
for all solvent extacts; however, ethanol was superior in activity (91.63t0.003),
followed by water (89.74t0.006) and methanol (87.73+0.002). These results wsre
found closer to Ascorbic acid and BHT especially of ethanol aad water. The lCso
(pglml-) value of M. communis leaves was measrred as 26.78,24-23 and 18.12 for
methaaol, ethanol and watet extracts respectively (Table 6.2). Water exftact showed
the best antioxidant activity (18.12) that was comparable to ascortic acid (12.25).
Antioddant activity of methaool, ethanol and water leaves exhacts of Z
stocl<sianum was documented at concentrations (i e l0pg/mt. 2oPglml-, 30pLg/rnl,
4opg/ml, sopg/ml and 60 pg/rnl) and provided in Table 6 4. The results of the
activity \iEre found significant (P:!0:01) compared to contol. Overall, the activity was
I94
directly propoftiotral to exkact concenhations. Starting from lower concentratioa (10
pg/ml), the actiyity was in the order of 17.87+0.005, 23.98+0.004 and 25 82+0 002
for ethalol, methanol and water extracts lespectively At the highest concentration (60
pglm]-), the activity was found optimlm in all solvent extracts; however, meihanolic
extact govemed supeior activity (86.4+0.003), followed by ethaool (83 89+0 001)
alld water (78.82 +0.001). The standards viz., ascorbic acid ard BHT ex]ibited almost
equal antioxidant activity (96.37i0.002 altd 97.47+0.001 respectively) and methanolic
leaf exhact was found closer to them (86.4+0.003). The leaves extracts of Z
stocksianum ex7)rbited good ICso (pglml) values that was in the order of 32'6'1,34 65,
38.25 atrd for watet, methanol and ethanol extacts respectively (Table 64) Water
extract sho ed the best antioxidalt acti\trty (32.67) that was comparable to BHT
(14.04).
Antioxidant property of methatro1, ethaDol and water based flower crude
extracts of Z srockridnurz was detemined by using different extraction cotrcentations'
Results are given in Table 6.5. Results were found sigoificant compared to control
(P<0.01) and directly proportional to extact concetrtrations At l0 pglml-
conceotration, the activity was in the order of 18.57+0 007, 1177t0'00360 and
10.48+0.007 for methanol, ethaiol and water exhacts. At 60 pglm]- conce tation,
maximum activily was lecordd in methanolic extract (78'25+0 004), followed by
ethanol (76.35 t0.005) and water (73.95+0 002). The former extract was found closer
to the stardard (ascorbic acid) for antioxidart activity.
195
The IC50 (pglml) valte of Teucrium stockstanum flowet was measureil as
28.2, 19.4,21.2 and for methanol, ethatrol aad watet extracts respectively (Table 6.5).
Ethatrolic exhact showed the best antioxidant activity (19.4) that was comparable to
BHT (14.4).
Free radical scavenging activity of Z /rapsr6 leaves extracts werc evaluated
at different concentrations (i.e. l0pg/ml,, 2opglml,, 30pg/ml, 4opg/ml, 50pg/ml and
60 pg/ml) and results are presented in Table 6.6. Results were found significant
compared to control (P!0.01). At lowet concentration (10 Fg/mL), water extract
exhibited highest activity (21.45+0.002), followed by ethanol (19.29+0.001) and
methanol (16.92+0.010). The activity was gradually increased with increasing extract
concentratio[s and this trend was found directly proportional to exEact concentrations.
At maximum corcentatio! (60 pglml-), methanolic extract exldbited highest activity
(83.79+0.004), followed by ethanol (76.8G10.0.001) and water (68.80+0.0.003). Ia this
case, methanolic extract was found closer to ascotbic acid (96.38*0.001) add BHT
(97.4s-0.002).
The ICm (pg/ml) value of Z ,iapsrn leaves was measued that was in the
order of 33.88, 34.50, and 44.15 for ethanol, methanol aDd water extacts respectively
(Table 6.6). Etharolic extract showed the best antio)ddant activity (33.88) that was
near to ascorbic acid (11.42).
196
Free radical scavenging activity of methanol, ethanol and water cmde extracts
of Z. satit)a was measured by using various concentations (i.e. l0pg/ml-, 20pglml-,
30Fg/mL, 40pg/ml, 50pg/ml and 60 pg/ml-). The compiled results arc presetrted in
Table 6.7. Results were significant compared to control @10.01). The activity was
fould directly proportiotral to exkact concentrations. At 10 pg/ml concentation, the
activity was itr the order of23.31+0.005, 19.58+0.003 and 11.95+0.008 for methanol,
ethaool and water extracts respectively (Table 6.7). The highest co[centration (60
pglml) showed maximum activity in all solvent extacts; however, methanol derived
the optirnum activity (83.8Gt0.0.002), foliowed by ethanol (72.62+0.003) and water
(53.84t0.001). Both the standards expressed maximum activity i.e. 98.46+0.003 and
91.42+0.003 for Ascorbic acid and BHT respectively, however, methanolic extact was
found closer to them.
The ICso (pg/ml) valne of Z. satira lea\es revealed that methanol extract had
lowest value (28.54) that express potential antioxidant property (Table 6.7), followed
ethanol (40.39) and water (54.27). Methanolic extact showed the best a.ntioxidant
activity (28.54) that was comPa€ble to ascorbic acid (15.93).
6.4.2.2 Reducitrg power assay
Reducing power assay of M. buxifolia was conducted for antioxidant activity and
rcsults are prcvialed in Table 6.8. There was signifrcant diffelence ir activity in all
selected concetrtntioBs amongst the extracts at (P=0.01). lt revealed that at 10 prml-
t9'7
concetrtratioq methaDol exhibited the highest activity (0'392't0 001), fo owed by
etharol (0.36110.002) and water (0.321*0.001). In this case, methanolic extract was
found cioser to BHT (0.423+0.003). Approaching to higher concentration (60 pg/ml),
methanolic exhact had supreme aciiyity (1 76+0 036), followed by Ethanol
(0.875+0.005), water (0.509+0.002). With reference to stardards, Ascorbic acid
showed 2.64i0.02 and BHT 2.11+0.01 values. The methanol extract showed the
highest activity that was although less tban standard antioxidants'
fhe results of M. communis leaves extracts for reducing power assay ale
compiledinTable6.9.ltrcvealedthatatlowerconcentration(10pg/ml)'highest
activity was govemed by ethanol (0.488+0 005), followed by methanol (0 299+0 002)
and water (0.213+0 006). Ilterestitrgly, the activity of ethanolic extact was higher thatr
BHT expressed (0.423+0.003). At higher concenhation (60 Uglml-)' ethatrolic extract
cotrsistently maintained its supreoacy in a cn\ity Q"27rO'026)' followed by methanol
(0.395+0.001) and water (0.397+0.002) The result of this solvent extlact was at par
withthestatrdald:Ascolbicacid(2'64+0.02)andBHT(2.11+0.01).Theotherexti-acts
shgwed lower aciiyity than BHT and ascorbic acid standard antioxidaot that may be
due to presence of trace amount of phltochemical compoutrds rcsponsible for such
activity. There was significaot difference in activiiy in all selected cotrcentratiotrs
amongst the extacts at (P=0.01).
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205
T. stocltsianum leaves were subjected to reducing powel assay and results are
presented in Table 6.10. There was significant difference in activity iD all extract
concenhations amongst the extuacts at (P<0.01). At the lower concentration (10
prglml-), the activity ofmethanolic exkact was at Par (0.522+0.002) with the standard
(BHT: 0.423+0.003). It was followed by ethanol (0.301+0.001) and water
(0-211+0.003). The activity was steadily indeased up to the higher concenhation (60
pglml-) in which methanolic extract consistently maintaired its supremacy in activity
(2.38t0.034), followed by ethauol (0.965+0.006) and watcr (0.409+0.004). The
activity of methanolic extact was more or less equal to the standard antioxidants
(Ascorbic acid: 2.&+0.02, BHT: 2.11t0.01).
The reducing power activity of T. stocksi1fium flower was carried out in
differetrt solvent exhact cotrcentation and lesults are given in Table 6 ll There was
sigdficant differetrce in activity in all extracts concentations at P<0.01 A1 initial
concentation (10 frg/ml), the highest activity was articulated by methanol
(0.511+0.002), followed by ethanol (0.381+0.001) and water (0 208+0.002). The
activity of methanolic exhact was comparable with Ascorbic acid expressed
(0.562+0.002). At higher concenlration (60 pglml), metha.nol exhact showed the
highest activity (1.91+0.043), followed by ethanol (0 955,t0'001) and water
(0.533+0.003). The activity of former extact was closq to tle staDdard (BHT:
2.11+0.01).
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208
Various extracts of Z ,?dps!rJ werc evaluated for reducilg power propeLy and
tesults are presented in Table 6.12. There was significant difference in activity in all
selected concentrations amongst the extncts at (PS0.01). It has been observed that at
lower cotrcetrtation (10 pglml-), methanolic extact exptessed the highest activity
(0.393 +0.002), followed by ethanol (0.331+0.003) and water (0.301+0.001).
Methanolic extract was found closer to BHT expressed (0.433 +0.002) itr terms of its
activity. At higher conceotration (60 pglml-), again methanolic exhact was consistent
in activity (0.503+0.002), fottowed by ethanol (0.465+0.004) and water (0.409+0.001).
These results are compamtively less than Ascorbic acid and BHT standard antioxidants
that may be attibuted to the compoutrds concentation in the plart extracts.
Results of reducing power assay of Z. sativa lea.ves extacts are
arraoged in Table 6.13. A1l extracts had significant differetrce in activity at various
coocentJations (P<0.01). At low concentration (10 pg/ml), methanol extract showed
the highest activity (0.401+0.001), followed by ethatrol (0.381+0.001) and water
(0.371*0.002). The activity of Eethanolic extract was almost equal to BHT
(0.42310.003). At higher cotrcenhation (60 pglml-), methanol kept its supremacy and
the highest activity was shown by it (1.16+0.01), followed by ethanol (0.975,10.001)
aad water (0.519+0.002). Both the standards i.e. Ascorbic acid and BHT had shown
2.64x0.02 ad 2.11+0.01 activity rcsPectively. The methanol extact showed the
highest activity closer to the standaids.
209
Free radical scavenging activity of methanol, ethanoi and water crude extacts
of Z. sativa was measwed by using various concentrations (i.e. l0pg/rnl, 20pg/ml,
3Opg/ml, 40pg/ml, sopg/r[ and 60 fg/ml). The compiled results are presented in
Table 6.7. Results were signihcant compared to cont ol (P:0.01). The activity was
found directly proportional to extract concenhatioN. At 10 pg/ml conce[hation, the
activity was ir the order of 23.31+0.005, 19.58+0 003 and 11.95+0.008 for methanol,
ethanol and water extacts rcspectively (Table 6.7). The highest concenkation (60
pglml-) showed maximum activity in all solvent extacts; however, methanol derived
the optimum activity (83.8Gt0.0 002), followed by ethanol (72.62+0.003) and water
(53.8440.001). Both the standards expressed maximum activity i.e. 98.46+0.003 3nd
91.4210.003 for Ascorbic acid and BHT respectively, howevel, methatrolic €xtract was
found closer to thern.
The lCso (pglml-) value of Z. sativa leaves revealed that methatrol extract had
lowest value (28.54) that express potential antioxidant property (Table 6 71. followed
ethanol (40.39) a , walet (54.27). Methanolic extact showed the best artioxidant
aaivity (28.54) that was comparable to ascorbic acid (15.93).
6.4.2.3 Metal lon Chelating Activity
Vaious solvelt based exbacts werc anallzed for Metal 1on Chelating Activity
of M. buxiJolia at different concetrtrations (i.e. l0pg/ml, 20pglmI-, 3opg/ml-,
40pg/ml, 5Opg/ml and 60 pyml). The results are tabulated and provided Table 6 14'
2t0
All exhacts were fouad signifrcant compared to control (PS0.01). There was positive
corelation between activity and concentration that was directly proportional to each
oiher. The lowest concentration (i.e lolrg/ml) exhibited good activity that was in the
orderly manner as 10.51t0.00, 19.21t0.01land 14.5L.0.054 for water, ethanol and
methanol, exbacts. At the higher colcentation (60 pg/ml), water showed the best
results (44.15+0.00), followed by ethanol (42.01+0 001) aad methanol (31.57+0.00).
The standards were highq in activity; however, watq leaf exhact was near to BHT
(58.12+0.045). When the chelating capacity (Table 6.14) is calculated usiDg equation
A, portrays an increasing capacity (Table. 6.14). A11 the extacts show itrGeasing
capacity with increasing concentatioo although; none of them possess capacity higher
than the stardard antioxidant (BHT).
Metal Iotr Chelating activities of methanol, ethanol and watsr clude extacts
of M. communis was rlreasured by employing different concentrations (i.e. lopg/n[, to
60 pg/ml) and results are presented in Table 6.15. A11 the extracts show increasing
capacity with increasing coDcenkation Results were fonnd sigtrifioant compared to
control (PS0.01). There was incieasing trend in activity that ra'as diectly proportional
to extract cotrceltratiotrs. At 10 pglmI- concetrtration, the activity was recorded as
15.44+0.046, 16.21+0.016 and 15.5G-0.036 for methanol, ethanol atrd water extracts
respectively. The ethanol exhact was closer to the BHT (22.5310.032) The highest
coDcentration (60 pglml-) exhibited maximum activity for all solvent extacts;
however, methanol was superior in acrivity (47.24+0 011.), followed by water
(48.13a0.067) and etianol (45.21+0.046). These results were found closer to BHT
2tr
especially of metianoi and ethatrol. No[e of them possess capacity higher than the
standard antioxidant (BHT).
The chelating effect was shown by the reduciag absorbance of the plant
samples with increasing concentation of which, when the chelating capacily (Table
6.16) is calculated using equation A, porhays al increasing capacity. Metal Ion
Chelating Activity of methanol, ethanol and water leaves extacts of Z stocLsianum
was documented at concentrations (i.e. l0pg/ml, 2\pg/mL, 301lg/mL, 4}p,g/mL,
50pg/ml and 60 pglml) and provided in Table 6.16. The results ofthe activity were
found significant (P10.01) compared to contuol. Overall, the activity was directly
proportional to extract concenhations. Starting from iowel concentation (10 pg/ml),
the activity was in the oder of 18.42*0.043, 14.4 0+0.031 and 13.51+0.013, m€thanol
water and ethanol extracts respectively. At the highest concentration (60 ,rglml), the
activity was found optimum itr all solvent extracts; howevel, methanolic extact
govemed supedor activify (49.21+0.012), foltowed by ethatrol (47.24+0.042) and
wato (45.13*0.068). The standard BHT exhibited almost comparable atrtioxidant
activity (58.1210.045). And metha:rolic leaf extact was found closel to theE
(49 .21+0.012).
Metal Ion Chelating properly of oethaool, ethanol and water based flower
crude extracts of T. stockstanum was determin€d by using different extnction
2t2
concentations. All the extracts show inueasing capacity with increasing concentration
Results are given in Table 6.17. Results were found significant comparcd to control
(P<0.01) and directly proportional to extact concenhations. At l0 pg/ml
concetrtation, the activity was in the ord,et of 75.22t0.044, 13_4 0j0.00 and
11.41+0.014 for methanol, watff and ethanol extacts. At 60 Ug/mL concetrtratiotr,
maximum activity was recorded in methanolic extact (53.64+0.047), followed by
ethanol (51.24+0.043) and water (49.13+0.061). The methanolic extract exhact was
found closer to the standa.d (BHT) for antioxidant activity. None of them poss€ss
capacity higher than the standard antioxidant.
Metal Ion Chelating Actiyity of V. rraprE leaves extacts were evaluated at
differedt concenhations (i.e. l0pg/n[, 2opg/ml, 30pg/ml, 40pg/ml, 50pg/ml and
60 pg/ml) and results are presented in Table 6.18. Results were found significant
compared to control (PS0.01). At lower concentration (10 pg/ml), watsr extract
exhibited highest actiyity (13.22+0.043), followed by ethaaol (12.38+0.012) aad
methanol (10.22+0.00). When the clelating capacity (Table 6.18) is calculated usiog
equation A, portrays ao increasing capacity (Table. 6.18). All the extacts show
increasing Capacity with hcreasing cotrcentation although; none of them possess
capacity higher than the standard antioxidatrt (BHT). At maximum conceDtration (60
pglml,), methanolic extract exhibited highest activity (29.33+0.037), followed by
ethatrol (27.73+0.030) and water (25.16t0.037).
213
Metal Ion Chelating Activity of methanol, ethanol and wate! crude extracts of
Z. satiya was measued by using various concentrations (i.e. l0pg/ml, 20pglml-,
30pg/ml, 40pg/ml. 50pg/ml and 60 pg/ml-). The compiled results are presented in
Table 6.19. Results were significant compared ro control (P<0.01). All the extracts
show increasing capacity with iDcreasing concetrtration. At 10 pglml- concentation,
the activity was in the order of 16.41+0.014, 13.2A.0. OO and,72-51r0. 00 for ethanol,
methanol, and water exlracts respectively (Table 6.19). The highest concetrhation (60
pglml-) showed maximum activity in all solvent extracts; however, water dedved the
optimum activity (45.15+0.00), followed by ethanol (41.01+0.00) and methanol
(36.50+0.00). A11 the extracts show increasing capacity with inoeasing concentration
although; none of thern possess capacity higher than the standard antioxidant (BHT).
6.4 DISCUSSION
With the passage of time, utrderstanding the mechanism of action of
medicinal plants reported by taditional heal€N has been increased. Vadous
ph,,tochemicals are reported to derive medicinal activity and phenolics arc the most
abundantly found in plants. These compounds are recognized as natural antioxidaot
due to their radical scavenging properties. This study evaluated total phenolic contents
(%) of three solvent based extiacts ofMorotheca bluifolia Qeayes), Myrtlts communis
(lea,tes), Teucrium stocksianum (leaves, flower), Verbascum ,lrapsr.r (leaves), and
Zizipus sati\)a (leaves) presetrted itr Table 6. 1 .
ElE!
l+
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224
It is estimated that the aDtioxidant activity is mostly related with the phenolic
compouDds havitrg rcdox prcpeties, hydrogen donorc and oxygetr scaveoger (Rice-Evans
et al, 1995). These compounds recogl zed as natural antioxidants that showed substantial
scavenglng activity against radicals. Consequently, antioxidant properties arle due to
occrurence of phenolic contetrt (Shahidi atrd Naczk, 1995; Zheng and Wang, 2003;
Cbinnici et at.,2004; H!^ng et al., ZOO9).In the lille of these studies, M- communis aad, T.
stocksianum possessed good antioxidant capacity due to the possessing phenolic
compounds in thern. Similar results ale reported by Proestos et al. (2006)' Turkmeq e' al,
(2006), Viuda-Martos er al (2009) aDd Kiihtooei et al. (1999).
Plants extracts are being explored tkoughout the world for searching new
coflpounds that can used to prctect fiom various diseases lelated to oxidative stress and
ftee radical-induced damage. The compoutrds are supposed significant in defensive
mechanisrn of human body in reactive oxygen species (ROS), responsible for creating
harmful effects during cell aerobic respiration (Gutteridge and Halliwell, 2000) The Aee
radical compounds havitrg an unstable and unpaired electon that would be stabilized by
steeling an elechon fron other Aee ndical that cause cell damage and involved in parasitic
aod bacterial infectioas and aging (Roy et al.' 1994; Rao et a1.,2004) A large number of
assays are coNialered to aletermine antioxidant activity in indicating capacity of host to
endure skess generated by fiee radicals.
Amongst all assays, DPPH method is the best one for determining high number of
plzrnt samples based on various polarities (Katerere and Elofq 2005) This method is widely
used for the estimatiot of natual antioxidants aom plants including dietary items (Brand-
2)5
Williams er al., 1995; Iao and Ko, 2002). This method is mostly associated with the
bhibition ofthe lipid peroxidation (Rekka aad Kourounakis 1991) and also frequetrtly used
to assess the antioxidant actiyity of plant extracts (Bhuiyatr e/ a 1.,2009; Tata Chatd et al-,
2012). Furthermore, antioxidants doDate an electron to DPPH ftee ladical and therefore
neuEalize its ftee rudrcal state ean et a1.,2008). This bioassay is principally used due to its
speedy, constant and repeatability parametel to soutinize lhe in rito antioxidant activity
extracts as well as pure compounds (Koleva et aI.,2002).
The antioxidatrt activity of three solvents based exkacts of selected plant parts was
determined at six concentrations ,anging fiom l0pg/ml to 60ug/ml by using tlvo methods
viz., DPPH scavenging and reducing power assays. Both assa)ts showed significatrt activity
itr all selected concenkations (P<0.01). The absorbance was found directly proportional to
extract concentatioos. All plants extacts showed good scaveoging activity in comparison
with standard antioxidant agent (Ascorbic acid and BHT)
In order to seek atrtioxidant properties, ICso values of methanol, ethanol and water
exlr^ct of M)rtus communis, Verbascutu thapslLs' Teucium stockianum, Monotheca
btflifolia and Zizyphus srrva alotrg with standards wele calculated and provided in Tables
5.2-5.'t, AJt plaDt extacts showed good mdical scavengiflg activity and the IC5o values
were rangerl ftom 18.12-55.88pg mlt. The highest scaveoging activity was governed by 'lZ
comfi nis i^ watet (18.12pg tr -l) that was closer to Ascorbic acid (12.25pg ml-t) It was
followed by ethanol (24.23pg mlr) and methanol (25.78pg m1-1). T. stocksianum was
second drug ofchoice that also showed higher.activity than rest ofplatrts The activity ofin
the order of 32.67, i4.65 and 38.25 [g mll for water, methanol and etlanol exhacts
226
respectively. Frcrn Z. sativa, methanolic aad ethanolic extacts had significa4tly activity
with IC5o values of28.54 and 40.39 pg ml-t respectively, while water exhact had least otre
(54.27 pg nn't). The methanolic M. buxifolia had' considerable activity with IC5o value of
31.81pg ml-r, followed by ethanolic (36.98 pg ml-r); however water exkact showed the
loi{est activity (55.88 pg mlr). In l/. thapsus, ethanolic exhibited good activity (33.88 pg
mll), foltowed by methaaolic (34.50 $g n f), while aqueous exhact showed less activity
(44.15 pg rnl'I).
Overall, methatrolic plant exhact possessed considerable scavenging effect on the
DPPH and the activity was found concentation dependent, however, the scavenging
activity of plant extacts was less thal1 that of standard. A higher DPPH scavenging
properties was associated with a lower IC5o value. DPPH is a frequetrtly used subshate for
rapid assessment of antioxidant properties (Bozin et a1.,2008). This bioassay gives reliable
results relating to the antioxidant capability of the biocompounds present in the extract
(Huarg and Prio, 2005).
Each individual antioxidant compound derives total antioxidant activity of the
extacts. These compounds exPress their properties thrcugh various meatrs like radical
scavengiag, metal chelating, tipid peroxidation irlibition and oxygeo reducing' If one
sample demonstates elevated action in one technique, it does not reveal same results in the
course of all methods. Thelefore, it is mandatoly to assess tests through various methods'
Keeping in view, the antioxidant properties of three solvent based five plant extracts werc
determine-d via DPPH and reducing power assays. The results of attioxidant properties of
the present study are comparable to that of l1-vp ericutu perforatunl (Hunt el al, 2001; Silva
22',7
et at., ZOO5 Be\edi et aL,2004; Conforti et al., 2005) and with the BHT, a well-known
s,'nthetic antioxidant.
The reducing power assay of five plants 'tiz., M. communis, Y. thapsus' T.
stocksianu , M. bttxifulia a]Ird Z. sativa 'was carried out ftom methalol, ethatrol and water
based extracts Gnging from lopg to 60pg concentratiolls. The activity was found
prcportionate to extract conceutration. Ail extEcts exlibited high absorbance. In addition
to this the reducing power activity of standard, ascorbic acid and BHT was far higber than
[he €xkacts of all the selected medicinal plants. Methanolic exkact of five plants viz.,
Mlrtus comfiunis, Verbascum thapsus, Teucrium stocksianum, Monotheca buxifulia afi
ZiVphus sativa exhibited good reducing power \ii'as summadzed in (Tables). The highest
absorbance indicated maximum rcducing power activity in these plants; however, the same
was fouDd conc€ntation dependent. These results substatrtiate the findings of Giilgin e/ aL
(2003) atrd Noriham e/ al. (2004) lhat confirmed antioxidative properties ot Pimpinella
arisrm seeal exhacts and four Malaysian medicinal plants respectively The reducing power
activity ofplant extracts was in the order of methanol > ethanol > water' Nevsrtheless, the
reducing capacity of BHT was found to be higher than the extBcts at each concentration
points. Some studies i .e. Gao et al-, (2000), Amarcwicza et al.' (2004) reported ihat there is
direct association between reducing capacity aDd antioxidant activities itr selected plants
The rcducing powet assay is usualty linked with that of reductones (Pin-Der- Duh,
1998). Itr this study, the extracts possessed a concentratioo-dqrendent antiradical properties
by standard BHT exhibit ef6cient reducing capability at all the concentration The leducing
capability of the compourds could also serve as Earkff of potential antioxidaflt activity
@l.,r et a1.,1995). Besides, the reducing power is mostly associated to the electron transfer
228
capacity of the compoutrd \rrhich could also lead to neutralize the free radical (Zht et al ,
2001)- The increase in reducing potential of tie plant extBcts which dependent on the
concentationsuggeststhattheyalealsogoodelectloadonols.Researchhasshomthatthe
reducing ability of a bioactive compound may lead to arl impo'ant marker of its covert
altioxidant property (Sofidiya et a1.,2006). Several studies confirmed antioxidant activity
Aom vadous plants su ch as Angelica sinensis {WE et al ,2004)' Mimuscops elengi (Asltok
et at.,2O7O), Cuminum cyminum (El-Ghorab et al.,2O1O)' Foeniculum vulgare vat- dulce
(Malo et at-. 2017), Mesua Jerrea (Yadav and Bhahragar, 2070), Myristica fragrans
(Akioboro el al, 201 1), Cuminum c1'minum (El-Ghorab et al ' 2010) and. Artemisia annua
tSing.h eI al. 201 l)
TraNition metal species such as ferous iron (Fe2+) can facilitate the production of
ROS within animal and human systems, the abitity of substaflces to chelate irotr can be a
valuable antioxidant capability (Hauiwell atrd Gutteridge, 1984) Iron, in natEe' can be
found as either ferrous (Fe2+) or ferric ion (Fe3+), with the latter form of ferric ion
predominating in foods. Ferrous ions (Fe2+chelation @ay render impotant antioxidative
effects by rctarding metal-catallzed oxidation Ferrous ioos (Fe2+) chelating activities of
methanol,ethanol anal water extract of teaf and flower of Myrtw communis' yerbaacum
thapsus, Teucrium stocksianwll, Mo otheca bwifotia an.d Zizyphus sativa are shown in
Table. The chelating effect of ferrous ions @e2+) by ofmetbanol,ethanol and water extact
ofleafatrdfloweraodstan.IaldswasdeteminedaccordingtothemethodofDinisQ994).
This study established grounal that various extracB of Myrtus communis' Verbascum
thapsw Teucium stocksianum' Monotheca bwifulia alrd Zizipus Jaf'd possess excellent
229
activity aod there is still much to elucidate of biological activity of crude extracts and
active compounals fiom these plants. New techniques for bioactivity guided isolation of
active compounils, such as miuo-ftactionation, may enlance the finding of new
compounds with new biological activities fiom this selected plants group The investigation
of the biological effects to be just in its begiining, but has exparded duing the beginning
of ihe new millennium with many new investigations- The antioxidant capacities of the
compounds show concentration dependence, because of the solubility problems the
r€ducing capacity of compoDe[ts could be measured in a lower concentration mrlge as seen
in graphical veNions.
CONCLUSION
The Fesent resealch work suggests that each plant extact possessed valuable
atrtioxidatrt activity, however, leaves exhacts of Myrtus commlt is and Teucrium
stocl,ia um possessed higher antioxidant activity compared with the rest of species and
their activity was found alElost equal to the statrdards These plants exhacts possessed
highest antioxidaDt properties that can be used to develop the ftee radical scavengen and
antioxidant agents. Nevertheless, the mechanism of action ofthe pl3trt exkacts is unknown'
Consequently, these species should be fully evaluated for isolation afld characterization of
biocompounds lesponsible for such kind of activities'
Chapter 7
GENERAL DISCUSSION
The present shrdy revealed that natives ofthe area are very much knowledgeable in
ethnobotanical use of plants and reported 4l plants species (Table 21) for treating 27
vadous acute and chronic ailments. Constipation was Predominant in the area and likewise
highest number of plants (i e. 9 spp., 13.24%) was used to treat this complaint (Table 2'2)'
This *as followed by abdominal pain (7 spp, 10.297o)' dianhea (6 spp' 8 82%)'
indigestion, pain & inflarnrnation (4 spp , 5 88% each)' throat pair/tonsillitis' dysentery'
vomiting, boils and cough (3 spp., 4.41% each) This type of studies has been reported by
vadousscientistssuchasKumare'a/',(2004),sdvastavaandPandey'(2006)'Pradhanand
Badola (2008), Rout and Thatoi (2009)
The screening for phlto-constituents thrcugh qualitative and quantitative tests tom
the selected plants exhibited that all Plants possess important chemicals such as alkaloids'
flavonoids, tannins and saponins All these secondary metabolites are well recognized to
possess medicinal Properties that exprcss pharrnacological activities (Sofowara' 1993)'
Several studies reported that ph)4ochemicals derive medicinal effects of va ous nature'
Plant species are normally screened for detecting ph)4ochemicals that are acknowledged to
reveal pharmacological properties (Sofowra, 1993)'
230
231
The present study screened for 13 phytochemical classes in five selected medicinal
plants of the study area- In order to evaluate solvent effectiveness in detecting chemicals,
methanol, ethanol and water extracts werc tested. The study showed siglificant difference
in chemicals in terms of solvents as well as plant exhacts (Tables 3.2 to 3.6). It revealed
that methanolic exkacts were the most effective in detecting Phltoconstituents fiom
selected species. This solvent isolated all chemicals ftom Z sroc,irtiazum leaves and flowers
ajrd Z. sattua.It was followed by V. thapsus (71), M. bttxifulia atd M- communis (10 each).
The ethanol extracts detect€d all phltchemicals ftom M communis' T. stocksianum leaves.
followed by M btaifolia, V, thapsus al,d Z. sativa (12 each), while io I stocksianum, l0
ph)dochemicals were identified. ln the case of aqueous extracts, maximum phlochemicals
were detected ftom i4 bttxifulia (9). followedby M. communis, T. stocksi1num leaves ar,d
Z. sativa (8 each), T. stocksianum flower (7) and Y. thaPsus (6).
stocksianum leaves and flower and Z. sativa werc ich in
phytoconstituents and all were detected in all of solvents (Table 3.7) Teucrium
stocksianum \r/as fo'nnd highly important medicinal plant of the area that is being used by
the Ilatives itr treating various diseases. The highest amount of alkaloids found in 7-
stoclcsianum leaves (0.81t0.00%), followed by T. stocksiatum flowe6 (0.?8+0.00%) and
that reflected its very corlmotr use (92% fidelily) for treatiag ckonic fever, diarrhea and
cough in the area. Such kind of activity may be attributed dug to presetrce of variety of
ph]4ochernicals in it. Being astringelt nature, alka]oids possessed atrti-diarIheal activity
that would act on intestitral tact atrd may demonstate aatimicrobial, antih)lertensive
antifutrgal, antifibrogeoic and anti-inflammatory properties (Ghosal et al., 1996) Various
studies reported that alkaloids possess analgesic (Antherdetr, 1969; Harbome, 1973),
Teucrium
232
atrtispasmodic and antibacteriat (Stray, 1998; Oloru and Okwu, 2OO4) properties of
alkaloids. Another study also reported the impofiance of some alkaloids against htestinal
and as well as HIV infection which is mostly related with AIDS (McDevith e' al, 1996)'
AII plant extncts indicated the presence of flavonoid (Table 3 7)' The highest
amount offlavonoids was documented in Z satiw (0.93+0 0y'), followed by M communis
(0.78+0.00%) and ?. r tocksianutt flower \0.77+0 OO7o) and leaves (0 71+0 00%)'
Tamins were dchly present il in Z. satila (9.'1+O 31%), followed by M communis
(9.17+0.22yA, M. b adolia (8.2+0.13%), T. stocl$ianum leaves (6 9+0 21%) and flowe$
(6.2]0.387o), while the same was absent itr Z tiapsus. This phyochemical is responsible to
act as antidiarrheal, antifungal, antihemorrhoidal and antioxidant agents (Asquith and
Butter, 1986). Furthermore, this possesses bitter p:inciple of ddnks and foods (Clikezie et
a\.,2008).
The prcsent tesearch work was conducted to evaluate the antimioobial activity of five
highly medicilally plants such as Monotheca buxifolia, Myrtus communis' Teucrium
stocksianum, Verbascum thapsus and Zizipus sariva by using methanol, ethanol and water
solvent extracts. For comparing antibacterialbial activity of plant extiact, chloranphenicol,
peniciliin and ciprofloxacin were used as a positive control, while for atrtifUlgal activity;
Fluconazole was used as a positive conkol For negative control, pure DMSC) and water
were used in both the cases.
233
ln the present srudy. organic 60l\mls ie. ethanol aod metha'ol extracls exlibited
maximum antrbacterial activity, while aqueous extact showed relatively low activity
agaitrst the tested bacterial straiff. Various studies signified the importance of ethanol'
methanol and \rater as solvents for bioaaivity of various plants (Bisignino el al, 1999;
Louens e, al, 2004; Pareki et at., 2OO5 Rojas e, al, 2006) However, it is evident that
herbalists (-Fla,teears) use aqueous extract to treat vadous diseases from native pla]lts, while
scientifically it is confirmed that o!8anic solvents are more reliable for antimicrobial
activity (Parekh et al.,2OO5). Results of this study clearly showed that gffn negative
bacterial skains were iDtribited best by Teucr[um stocksianum leaves, wh*eas, Teuctium
stocksianum lTowet rnostly iDhibited gram positive bacterial strailN Organic solvent based
Myrtus colnmunis lea]ves extracts slowed atrtibacterial activity against all strains except K
pneafioniae, whtle aq'reous exhact did not target any microbe'
The antibaoterial assay exhibited good activity agafust the test olgaoisms CornPared
with solvents, organic soivents viz., methatrol and ethanol extacts showed relatively higher
antibact€rial activity against multi-drug resistant clinical isolates lt is motivating to note
that two of these five platrt species, Myrtus com unis and Teucium stocl<siakum $tere
highly active against bacterial These results werc found comparable/nearest to the standald
drugs (Table 5.1). Even at fhe lowest concentation (i e 5mg/m1), Myltus com nis and
Telrcrium stocl$ianwn extacts showed relatively higher activity compared with standard
drug, while other three plants did trot show any activity at this conceDtration against the
tested orgarrisms.
234
It can be concluded that these extracts hold potential for further work against these
bacteria tbrough toxicity testing. The study also revealed that aqueous extract showed
moderate activity. The findings ofpresent study are in line with those of Murugesatr el al
(2011) based on his work on Memecylon umbellatum. Besides, water leaves extract of
Ptetospermum acerifoltum was found to possess good antibactedal effects against a
number ofhuman gram positive and grarn negative bacterial strains (Thatoi el al, 2008)
Aspergillus fumigatus canses Aspergillus infections (Hedayati et al., 2001;
Alessatrdro, 2OO8). Ol the other ha.rd, l. zrger is food bome paihogen that causes a senous
lung disease, aspergillosis, otomycosis pain iflaige amounts ofspores irhaled (Hema el dl,
2009). These both fungal pathogens are responsible fol causing skin diseases acting as
d€rmatophltes. Keeping importance of both the orgadsms, antifungal activity of ethanolic,
methanolic and aqueous extralcts of Monotheca buxifulia, Myrttts communis, Teuciufi
stocksianum, Yerbascum Th(lpsus and Zizipus soriva was caried out. Results revealed that
all platrts possessed antifungal activity agalnsl A. fumigotus aj,,d A. higer. With respect to
solvent exhacts, highest antifungal activity was shown by methanotic, followed by ethanol
and water.
Methanolic Z saliva leaves extract inhibited best A. f miSatus (73.22%), wnile A'
,jter was inhibited at 10.34V. (Table 5.7), wlile rcst of plant extracts had relatively low
activity. From ethanolic exkacts, A. fumigatus was inhibited almost equally by Z Jarrna
a;nd M. buxifotia 62.89 ar,d 61.86y. rcspectively, while l. ,Iger was best contolled by f'
stocksianum flower & leaves (Table 5.8).
235
The aqueous extact also derived significant activity against both pathogens (Table
5.10). In this case, A- niger was susceptible agairnst M. bur;folia leaves extract (58.88olo),
. whercas, M communis lea.ves extract inlibited best A. fumigatus- Similar findings are
reported by Naz and Bano (2012) ftom 'Rrclrrus communis ar.d revealing the suprernacy of
methanolic extract followed by water.
With the passage of time, understandilg the mechanism of action of medicinal
pla[ts repofted by kaditionat healers has been increased. Various phyochernicals are
reported to derive medicinal acti!'ity and phenolics are the most abutrdantly fou[d in plants'
These compounds are recognized as natural antioxidant due to their radical scavenging
properties. This study evaluated total phenolic contents (%) of thee solvelt based extacts
of Monotheca buxifolia (lea'ves), Myrtus communis (leaves), Teucrium stocksianum (leaves,
fTowet\, Verbasc m thapsus (leaves), arrd Ziz?rrs saliva (leaves) preseoted in Table 6 l '
It is estimated that the atrtioxidant activity is mostly related with the phenolic
compounds having redox properties, hydrogen donors and oxygen scavatrger (Rice-Evans
et at.,1995). These compoutrds recognized as natural antioxidants that showed substantial
scavenging activity against radicals. Consequently, antioxidant prcperties ale due to
occulrence of phenolic content (Shahidi and Naczk, 1995; Zheng and Wang, 2003;
Ciiaicj, et aI.,20041Hnar'g et a1.,2009).
236
In the line of these studies, M- communis and T. stocksianum possessed good
antioxidant capacity due to the possessitrg phenolic compounds in them Similal results arc
reported by Proestos et al. (2006), Tuknen et a/, (2006), Viuda-Martos et al' (2009) aad
Kiibkitren ",
dl (1999).
The antioxidant activity of three solvents based extracts of selected plant parts \tas
determined at six concentrations ranging ftom 10pg/m1 to 60pg/ml by using two methods
viz., DPPH scavenging and reducing power assays. Both assays showed significant activity
in all selected concentrations (P50 01). The absorbance was found directly proportional to
extract concetrtations. All plants extracts showed good scavenging activity in comparison
with standard atrtioxidant agent (Ascorbic acid and BHT)'
In oraler to seek antioxidant propefiies, IC5o values of methanol, ethanol and water
ettract of Myttus commu is, Verbascum thapsus' Te crium stocksianum' Monotheca
butifotia and Zizphus.tdrivd along with standards were calculated and plovided in Tables
5.2-5.7. AJI plant extacts showed good Edical scavengiflg activity and the lCso values
were ranged ftom 18.12-55.88pg m1-1. The highest scavetrging activity was govemed by M
communis in'walet (18.12pg rnl'r) that \las closer to Ascorbic acid (l2 25lrg r[r) It was
followed by elharol (24.23Pg ml-r) and methanol (26 78pg ml-t) T stocksianum was
secoDd drug of choice that also showed higher activity thall rest of plalts The activity of in
the order of 32.67,34.65 aad 38.25 pg ml-l for water, methauol and ethanol extracts
respectively.
237
Ftofi Z. satt|a, methanolic and ethanolic extracts haal significandy activity uith
lCso values of 28.54 and 40.39 pg m1-1 respectively, while water extract had lea-st one
(54.27 pg nn'r). The methaoolic,4Z bttxifulia had, considerable activity with ICso value of
31.81pg ml-r, followed by ethanolic (36.98 pg ml-l); however water extract showed the
lowest activity (55.88 pc rrn-t). ln V. thapsus, ethanolic exlibited good activity (33 88 pg
ml1), followed by methanolic (34.50 pg mll), while aqueous extact showed less activity
(44.15 pg mlr).
Overall, methanolic plant extract Possessed considerable scavenging effect on the
DPPH and the activity was fomd concenhation dependent, howeveq the scavenging
activity of platrt extracts was less than that of standard A higher DPPH scaveDging
properties was associated with a lower ICto vaiue DPPH is a fiequetrtly used substrate for
rupial assessment of antioxidant propefiies (B omt et al',2008) T\is bioassay gives reliable
results relathg to the antioxidant capability of the biocompounds prcsent in the extract
(Huang aod Prio. 2005).
The reducing power assay of compounds may sewe in prospective antioxidant
Foperty (Meir e/ 41, 1995).The reducing power assay of five pl arrls \iz'' M communis' l/'
thapsus, T. stocksiahum, M. bttxifulia arrd Z. so,i'a was caried out from methanol' ethaDol
and \rater based extracts raoging ftom 1opg to 60Fg conceltrations' The activity was found
proportiooate to extract co[centratioD. A11 extracts exldbited high abiorbance ln addition
to this the reducing power activity of standard, Ascorbic acid and BHT was far higher than
the exhacts of all the selected medicinal plants'
238
Methanolic extact of five plants viz., Myrtus cot fi is, tserbasculh thapsus,
Teucrium stockstanum, Mohotheca buxifolfu ar,d Zizyphus saliv4 exhibited good reducing
power was summarized in (Figure). The highest absorbance indicated maximum reducing
power activity in these plants; however, ihe same was found concentation depetdent.The
reducing power activity ofplart extracts was h the order of: methanol > etharol > water'
Nevertheless, the reducing capacity of BHT was found to be higher than the extracts at
each concentation Points.
239
CONCLUSION & RECOMMENDATIONS
. Ph)'tochemical sqeening and bioactivity of some plants soch as Monotheca
burifolia, Myrtu.t commuhis, Teucium stocksianum, Verbascum thapsus and Zizipus
scttiva wete undertakm using different solvents (i.e. methanol, ethanol and water)'
Various ph)4ochemicals viz., alkaloids, flavonoids, tan.rlins, saponins and phenols
were detected through this study. These extracts also 'lemonstrated
broad spectrul
atrtirnicrobiat activity atrd exh.ibited strong activity against grarn+ve as well as gram-
ve bactena atrd fungal skains The activity was almost equal to standard drug'
Furthermore, present study also provided scientific validation for indigenous use of
the plants against various ailments as reported by local irhabitants Based on the
present study, following parameters need to be studied itr order to develop new drug
from the plant:
Isolation, characterization and puification of compounds responsible for
aotimicrobial and anrioxidant properties.
In vivo study.
Toxicological study of plant extracts.
Teucrium stocksianunr leaves are employed in jauldice by indigenous
communities; this aspect of the plant should be explored'
Myrtus cofimunis alld Teucriuth stocksianutu possessed highly mediciaal
anal aromatic plants and cotrtaining huge amount of ph)4ochemicals so
further work is required to fuIfi1l the demand for essential oils'
Zizipus sativa leaves are employed in antidiabitic by irdigenous
communities; this aspect of the plant should be explored'
SUMMARY
Use of plants as a source of medicine is as old as human civilization Today,
medicinal plants have atkacted tesearchers to discover antimicrobial and antioxidant agents
due to increased tesistance in microbial stains against synthetic antibiotics coupled with
adverse effects by chemical antioxidants. Keeping in view, present study was designed to
document medicinal uses of native plants in order to select few species for phytochemical
screening, antimicrobial and antioxidant activities. Allogether, 4i plants distributed across
38 genera and 32 families wele used by the local inhabitants of Malakand Division to treat
27 human diseases. Based on highest fidelity percentage, some plants snch as Monothecd
bwdolia, Myttus cofimunis, Teucfium stocLsianum, Verbascum thapsus ar'd Zizipus sat a
were selected for evaluation of aforcsaid activities. Plant mate als of these species were
collected. dried, powdered and extracts were prepared by using tbree solvents viz ' ethanol'
methanol and water.
The present rcsearch screened for t3 ph)'tochemicals in five selected medicinal
plants of the study area ln order to evaluate solvent effectiveness in detecting chemicals'
methanol, ethanol and water extracts were tested Results revealed that methanolic extracts
were the most effective in detecting ph)'toconstituents ftom selected species This solvent
isolated all chemicals from T- stocksianum leaves and flowers and Z satira ll was
followed by V. thapsus (ll), M. bttxifulia alrd M' communis (10 each) The ethanol extracts
detected all phytchemicals fro,n M- cornmunis, T stocl"sianum leaves' followed by M
b xifolio, V, thapsus and Z. sa,i,4 ( 12 each), while i\ T stocksianllm' 10 phltochemicals
were identified
240
241
In the case of aqueous exhacts, maximum phlochemicals were detected Aom M
buxifolia (9), followed by,44 commun{l T. stocksianum leaves and Z sarva (8 each), Z
stocksianum floyret (7) zud' r/. thapsus (6).
The ethanol, methanol and water based plant extracts were subjected to percentage
yteld for Monotheca buxifolia, Myrtus comnunis, Teucrium stocksia um (L), Teucrium
stoclrsianum (F), Verbasatm thaPsus aad Zi4phus sativa. It has been observed that
methanol exhibited suprernacy in yielding fiaximum extact, followed by ethanol and
water. The highest extact yield was observed itr methaool (21.4+0 10%), followed ethanol
(19.3+0.r0%).
The methanolic exhacts yield highest yield t\ Z. satita (21.410 70%), followed by
M. buxifutia 09.3+0.17%), M. commu/tis (l'7.6+0 06%), T. stocksian m leaves
(16.35+0.00%), T. stocksianum flower (14.8:0 10%) al.d V. thapsus (95+0 10%) The
ethanolic extracteal the highest Yield ln Z. satira 09.3+0.10%), followed by M bwifulia
(18.4*O.ZO%), M. communis (16.1t0.17%), T. stocksianum flower (13 5+020%), I
stocksianum leives (11.4+0.20%) alrd Y. thapsus (7 2+0]0%)' In the case of aqueous
exhacts, M. bw;fulia afi Z. sativa collecli\ely yielded highest amount (17 5t0 06%),
followed L stocksia um lea1rcs (11.3+0.1'7yo), T. stocksia um flower(10 8+0 17%) and trl
thapsus (8.3+0.10%o).
The present research screened for 13 phlochemicals in frve selected medicinal
plants of the stualy mea. In ortler to evaluate solvent effectiveness ia detecting chernicals,
methaool, ethanol and water extacts werc tested. The study showed significant difference
242
in chemicals in terms of solvents as well as platrt exhacts. lt revealed thal rnethanolic
extacts were the most effective in detectiDg ph,'toconstituents fiom seiected species This
solvent detected all chemicals from T stocksianum \eaves and flowers and Z sa'va It v/as
followed by V. thapsus (ll), M. blLrifolia aIId M. communis (10 each)"[\e ethanol extracts
detected all phytchemicals from M. communis' T. stocksianutrl leaves, followed by 'lZ
buxifulia, V, thapsus alrrd Z. sath'a (12 each), while in ?. stocktia um' l0 phltochemicals
were identified. In the case of aqueous extracts, ma.ximum phytochemicals were detected
ftoim M. buxifotia (9), followed by M communis, T. stocksianum leaves ar,d Z' sativa (8
each), T. stocksianum flower(7) and Z thapsus (6)'
Teuciut l stocksiakutt was found highly important medicinal pla[t of the area that
is being used by the oatives in keating vadous diseases Highest amourt of alkaloids found
in T. stocks,anum lea\/es (0.81+0.00%), followed by Z stocksia um flowers (0'78+0 00%)
and that reflected its very common use (92% fidelity) for teating chrodc fev€r' diarhea
efld cough in the alea.
All plant extacts indicated the presence of flavonoid The highest amount of
flavonoiilswasrecordedinZ.satiya(0.93+0'07"),followedbyMcommunis(0.78+0.00),
T. stock:ianum flower (0.77+0.00%) & leaves (0 71+0 000/0), M buxifolia (0 64+0 00%)'
whileleastamoutrtwasrecordedforV.thaPsus{0.33L0.02).Inthesameway,alkaloids
wsre dchly present it T. stocl<sianum leaves (0 81+0 00%), followed by T' stocksianu
flowers (0.78r0.00Y"), Z. sativa (0.76a0.00%), M. bwcifulia (0 61'10 00%)' M co munis
(0.43+0.00yo) and V. thapstts (0.30+0 0 0)'
243
The high percentage of tannins was present in Z. sativa (9.'l+0-31%), followed by
M. communis (9.17+0.22%), M. bttxifolia (8.2+0.l3yo), T. s tocksianum lea'res (6.9+0.27yo),
flowers (6.2+0.38%) and V. thapsus (0.37+0.12). Saponins were richly fomd in 2
stockianum lTowet (2-21+0.00%) and I stocksianum leaves (2.13+0.019/o), while the same
was absent iD V. thapsus.
The extracts of selected platrts were subjected for i, virlo antibacterial activity
against some gram positive (S arre i, S. epider idis and Sveptococcus pyogen) atd gram
]negative (Echerichia coli, Klebsella pteumonia a^d Pseusdomonas aeruginosa) bacteljal
stlaim at the cotrcetrtrations of 5mg/m1 to 15mg/m1 by using aga.r well diffi:sion method.
All these plants showed significant differences in checking bacterial grou'lh (PS0.01).
Gram positive bacteria were foutrd more susceptible thaa gram negative. There was good
inhibitory activity aligoed with tested orgadsms and at the lowest cotrcetrtration (i.e
Sm/mll M. communis and T. stocksianum extacts showed relatively high activity
compared with stardard drug, while other three plants did not show effectiveness against
the tested orga sms.
The ethaoolic extact of M. communis exblbited best against S. autew (6 5+0,7)'
followedby S. pyrogen (6.1+0.64), E. coli (4.5+0.95) ar:i. P. aerzrgizosa (4.G10.0.0). While
methaoolic exkacts ofthe said piant found best against S. pyrogen (7.6+0.69), followed by
S. epidetni.dis (5.1!0.'72), S. aureus (4.310.60), P. aerugtnosa (33+001) and E- coli
(3.C,t0.0.0). This plant did not show atry activity agaitrst K. pneumoniae ln the case of 7'
stocksiahum leaves extact, ethanolic ones was superior in inhibiting various bacterial
244
straios followed by ethanolic and water, however, flower extracts of the said species
exlibited opposite trend i(I actiyity with respect to solvents.
Etharolic Z stocksianuh leaves extract iniibited best S azreta (5.8+1.03),
followed by S. epidermidis (5.5+0.62), K. pheumoniae (5+0.45), E col, (3.5+0.55), .'.
pyrogen (3.5+0.45) al.d P. aeruginosa (3.5+0.62). From methanolic exhacts, S. epidelmidis
(6.5+0.7) was irftibited best, followed by s. aureus (5.5+1.01), K pneltnoniae (5.2+,0.73),
S. aureus (510.75). as E. coli (4.0+0.0.5), K. pneumoniae (3.2+0.2), P aeruginosa
(3.110-51) and S. pyrogen (3.0+0.0.6). The aqueous extact checked best e col, (5*0.0)
and S. epidermidis (5t0.3) followed by,K. p eumoniae (4.6j0.95), S. aureus (4.0+0.0.0)
ad S. pyrogen (3.0+0.0.2).
The inhibitory activity of methanolic extracts of T. stocksianum flower against
various bacteria was in the order of S. pyrogen (6+0.61), K pneumoniae (5.2't0.9) > S.
dureus (5+l.O), P. aeruginosa (3.510.55) atrd .E. col, (3.5+0.06), while no activity recorded
for S. epidermidis.
The antifungal activity was carried out against two fungal pathogetrsl.e. Aspelgtllus
iiger, A. fuhigates through tube dilution method at the concenhation of l2mglml ftom
Eethanol, ethanol and watet exhacts. Therc was significant difference in the activity of all
solvent based exkacts (p>0.01) and all plants possessed antifungal activity against l.
fumigdtus alld. A. niget. Wrlfi respect to solvetrt extracts, hi+est antiirngal activity was
shown by methaDolic, followed by ethanol and waier. Methanolic Z. sativa lea'/es axtracl
inhibited best l. /am igatus ('13.22%), \ tbite A. niger was hhibited best at 68 65% by T'
245
stocksian)m fTowe\ wblle rest ofplants extacts had relatively low activity. Frcm ethanolic
exttacts, A. fumigatus was inhibited almost e qnally by Z. sativa aud M. blltifolia with 62 Ag
a 61.86% activity respectively, whlle A. nige.was best conkolled by T. stocksianum
flower and leaves. The aqueous extract also derived significant activity against both
pathogens. In this case, ,4. r?ige," was susceptible against M bllatfolia leaves extrdct
(58-88%), whereas, M. comtt%nis leaves exttactinlibtledbest A. futuigatus'
Methanol exhact of Z sativa was found best for inhibiting fungal strains followed
by T. stocksianum (flower and leaves respectively), M. communis' M- btt'tifulia and Y'
,raprur. With reference to fungal pathogens, l. /,iger was inlibitedbeslby T' stoct-\ianilm
flower (68.65%), leaves (61.58%), Z. sativa (63.88yo), wlnlle' A fumigatLls was checked
bestby Z. sativa (73.22Yo), followed'by M. communis (58.04Yo) artd T' stocksianum llower
(s6.25v4.
The etharol extract of T. stocksianum leaves showed the highest activity against l'
niger (63.27), followed by Zizyphus sativa (62.27Yo)' M communis (46'23yo)' V thapsus
(43.4'/o) and M. btaifolia (38.68Yo) Fot A. fumigatus, Z. sattua exlibited highest activity
(62.89%), followed by M. communis (61.86%), T. stockstanum flower (41'24Vo)' T'
stocksianum leaves (31.96%), M. bLaifolia (27 .84oA) arLd V thapsus Q4'7 5Yo)'
The water extacts of a1l selected plants also inhibited vadously for both fungal
pathogens. The highest inhibitory activity was showa by M btaifulia (52'88Yo) agairst A'
niger, follo,Nedby I/. thapsus (38.32%), Zi?Jphto satita (36 79Vo), M communis (27 '591/o)'
216
T. stocl$ialum flowet (21.64Yo) ar.d T. stocksianum leaves (18.40%). In the case of I
fumigatus, M. communis st,ownltig}est inhibitory activity (52.43%o), follo*edby Zi4phus
sativa (43.0'7yo), M. bwifolit (36.23%), Y. thapsus (18.45o/o), T. stocl',sianwrl flowet
Q6.22oA) ar.d T. stocl<sianum leaves (15.54Vo).
The antioxidant activity of four solvents based exhacts of selected plant parts was
determined at six different cotcentratiols ranging ftom lopg/ml to 60pg/ml by using two
methods viz., DPPH scavenging and reducing power assays. Both assays showed
significant activity in all selected concentrations (p<0 001). The absorbance was found
directly proportional to extract cotrcentrations. A11 plants showed good scavenging activity
in companson with standard antioxidant agent (Ascorbic acid and BHT) The DPPH
activity in the form ICso value was calculated by gaph prism pad software The ICso
(pglml-) value of M. comtuunis leaves was measured as 2678' 24'23 and 1812 for
methanol, ethanol and water extracts respectively. The aqueous extract showed the best
aotioxidant activity (18.12) that was closel to Ascorbic acid (12.25)' In the case of
Teucrium stocksianum (flower), the ICso (pglml-) value was measured as 28'2, 19 4,21 2
aod for methanol, ethanol and water exkacts respectively All these extracts efibited
afltioxidant activity higher thao those of Ascorbic acid (29 2), while ethatrolic extract was
closer to BHT (14.4).
Compaitrg \rith the standards (Ascorbic acidt 2.64+0 02 and BHT: 2 11+0 01) aU
plants extracts alemonstrated significant activity (P<0.01) even at the highest concentration
(60 lrg/ml-). The reducing power assay of M. buxiJolia at the concentration of 60 pglml
indicated that methanolic extract had superior activity (1.76+0 036), followed by ethanol
241
(0.875+0.005) and water (0.509+0.002). I-eaves extracts of M commltrtis had established
activity in the order of ethanolic (2.2'/+0.026) > methatrolic (0.895+0.001) > and water
(0.397t0.002). The ethanolic extract possessed activity that was at par with the standards'
Leaves exhacts of V. thapsus ex.hibited highest activity by methatrol (0 503+0 002),
followed by ethanol (0.465+0 004) and water (0.409+0.001). Z stocfrta'uD' leaves extacts
gave optimum result by methanol (2.38+0.034), followed by ethanol (0 965+0 006) and
r/ater (0.40*0.004). The activity of methanolic extract was equal to the standard
atrtioxidants. The reducing power assay of T. stocksian m flower revealed tiat methanol
extract had the highest activity (1.91+0.043), followed by etharol (0 955+0 001) and water
(0.533+0.003). The activity of fomer extract was closer to the standard ln tle ca'se of Z
sariva, leading reducing power activity was showed by methanol (1 1610 01), followed by
ethatrol (0.975+0.001) and water (0.519+0.002). The methanol e)(tact showed the hiShest
activity closer to the standaJds.
Results revealed t\4t Myttus communis Md Teucrium stockia um leaves extacts
possessed atrtimicrobial activity that may be of great use by the phamaceutical industry to
develop a therapeutic agetrt against vadous diseases Fulthelmorc, results of the present
study support the folk claim of both the plants in the study area Presence of phenolic
compormds in both plant exkact demonstated antioxidant activity that can be employed as
a potential tratural antioxidatrt agent which may be used for mitigating oxidative stress'
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APPENDIXA
APPENDTX. FACTOR FACTORIAL ANOVA FOR RESULTS OF ANTIBACTERIAL
Appendix i. Analysis of variaaee Table fo! E. co77
SourceRepsamplesSolConcsanples*So1sanples*ConcSoI*Concsamples * So1*ConcErrorTotaI
Grand Mean 8.7094
SourceRepsanplesSo1Concsamples*Solsamples*ConcSoI*Concs amples + SoI +Conc
ErrorTota 1
Sou!ceRepsampfesSo1Concsamples*So1samPles*ConcSoI*Concs amPIes * So1*Conc
cv 8.30
of valiance fable for
MS
L.461538.03
127 - 95434 .',l 2
34 - 61-
35 -8211.515-750-'74
MS
2.901186.86225.43551- 60
32.15
10 .17
K- pneurfionio.e
E'
2080.01
587.9146.8148.4415.56
'7 .'71
DF2
2
4
1632I
64268404
0.912428 .8
369 .92959 .5
860.5L254.6
744.',7
140.118142.9
ltff;4.4'7
1553.60184.94739.88
53.78
18.098.50a .52
F
29'72 . 6-t353. B7
1415.69)-42 .9L
34-6rL6.26
P
0.00000.00000.00000.00000.00000.00000.0000
P
0.00000.00000.00000.00000.00000.00000.0000
Appendix ii. AnaLysis
DF28
2
4
16
8
64268404
ss2.9
L23a4.2243 .9
l-738.9553. B
l!46 .292.4
3 6'7 .'7L98.2
L664'1 .8
Grand Mean 7.2741 CV
AppeD4ix iii. Analysis
7L.82
of Valialrce Ta.b1e fo! P. eetuginosa
DF2
I2
4
L6
8
64
5.8
450.12246.4
514.4),a2'7 .8
81,4568.3
F
2866.45
'7 -t -6677.5824 -5121- .45
P
0.00000.00000.00000.00000.00000.00000.0000
297
ErrorTotal
Grand Mean 7.6?98
268 1114a4 14464
0.41
Tab].e fo!
MS
a.a22012 .44294.48
7'729 .lL53.07
23.89
0.71
Tab].e for S.
MS
2 .3'72915.00
161 .44'7 39 .39
33 .0038.28I .'716.341.09
0
1
Appendi.x iv. Analysis
0.0332316A99 .2588.9566916 .45849.1861719 . B7
191 .099
190.5132'7 098 .3
S. aureus
F
2830.00474 . 1-2
243L.62'74.6475.58
Sou!ceRepsamPlesSo1Concsanple s * Solsanples *ConcSof*Concsamples * Sol*ConcErrorTotal
Gr:and Mean 11.4f1
Appendix v. Aia].ysis
SourceRepsamplesSolConcsamPles*So1samples *Conc
samples * Sol*Conc
10car
Grand Mean 9. 9331
SourceRepsamplesSolConcsamples*Sols amPles *Conc
Sol *Concs amples * Sol*ConcErro r
cv 7 .35
of variance
DF SSa a1B 23320.02 334 .94 2951 .6
16 528.032 L225.AB '70.2
64 406.0268 292.94A4 29739.3
cv 10.53
P
0.00000.00000.00000.00000.00000.00000.0000
eFidex,,-idiE
2666.88 0.0000153.19 0.00006'7 6.46 0.000030.19 0.000035.a2 0.0000
8 -A2 0.00005.80 0.0000
S - E yogien
F
'7 A1 .'7 3832.08105.32
46.3453.0110.96
P
0.00000.00000.00000.00000.00000.00000-0000
DF28
2
4
1"6
32
64268444
Appendj-x vi. ADatysis of variance
DF SS
2 14.9B L3'744.32 l3'7 4 .34 2925 .6
16 L48l.232 1303.4B 3'7 2 ."7
64 616. 5268 235 .6
Table fo!
MS
5.451718.03
6B-t .74731.39
92 . 5'7
4A.1346.59
9.630.88
29a
Totaf 4A4 22A64.3
crand Mean 9.9630 CV 9-41
4)pendix vii-.LSD AlL-Pailtise ComPalisons Test of Ec for samPles
sa.mples l4,ean EoDoqetteous GrouPs7 17.700 AB 15.667 B
9 13.840 C
3 9-0800 D
4 6.5044 E
2 6.2314 E
6 5 -4422 F
1 3.9133 G
5 0.0000 H
Atpha O.O5 Standard Error for Comparisona.1524Critical T value 1.969 Critical vafue for Comparison0.3001Error term usedi Rep*samples*so]*Conc, 268 DE
There are I groups (A, B, etc.) in which the means
are not significantly different frorx one another'
LSD Af1-Paj.rwise ComPalisons Test of Ec fo! So1
So1 ltban Eomogeneous GlouPs3 9.5333 A2 9.2252 B
1 'l .3696 C
Alpha O.O5 Standard Error for Compar:ison 0'0880Critical T Va]ue 1.969 Critical Value for Comparisona .7132Error term used: Rep*samples+Sol*Conc, 268 DE
AL1 3 Eeaas ale sj.grrifieant]-y different flos one another'LSD A1l-Pairwise CodParisons Test of Ec fo! Conc
Conc Mean Honogeneous Groups2 7L.617 A1 11.664 A3 8.757 B
4 6.494 C
5 4.956 D
Alpha O.O5 Standard Error for Comparison 0'1136critical T Va]ue 1'969 Criticaf value for CofiLparison
0 .223'7Error term used: Rep*samples*so1+Conc, 268 DE
There are 4 gro,.lps (A, B, etc.) in which the means
are not significantly different from one another'
293
tSD A].].-Pairl,.ise ComParisons Test of Ec for saeples*sol
saEP1es So1 !4eaD EomogeDeous GrouPs1 1 17.700 A'1 2 11 .'7AA A
? 3 17.700 AB 1 15.66? B
B 2 75.661 B
B 3 15.661 B
I 1 1.3.840 c9 2 t3.a4A C
9 3 13.840 C
3 3 9.9067 D
3 2 9.4000 D
6 3 8.526'7 E
3 1 7.9333 F
6 2 '1 -B)aa EG
2 2 1-58A0 EGH
4 3 '7 .326'7 GH
A \ 1.286'7 GH
2 3 '1 -2333 H
1 2 6.1400 r1 3 5.6000 J4 2 4.9AAA K
2 1 3.9000 L
1 1 0.ooo0 M
5 1 o.o00o M
5 2 o.O00o M
5 3 o.ooo0 M
6 1 o.0oo0 M
Alpha C'05 Standard Error for compalison
0.2644irtii..,- T value 1'969 critlcal value for comparison
0.5191gtto. a"t^ used: Rep*samples+So1*Conc' 268 DE
;;;t; "t. 13 groups (A, B, etc') in which the means
,."-".i-"lo"iii.u"tty diff"t"t''t fron one anolher'
tSD A].L-Pailwise Cooparisoas test of Ec for saep].es*conc
sabples Coac !!ean Eomogeneous GlouPs1 2 23.400 A
B 1 21.000 B
I 3 20.000 cB 2 18.333 n
9 1 17.000 E
7 1 16-500 EF
294
38
1
1
8
3
9
2
4
B
9
2
4
6
1
6
3
6
1
6
2
1
3
4
3
4
4
2
21
1
55
55
56
1 16.4003 16-0002 16.0004 15.0003 14.2005 14.0004 13.0002 L2 .8334 12.000L Lt.2221 l0 .'7 615 10.0005 10.0002 9 .666'72 9.41'782 8.00002 '7.'71'78
1 7.50003 7.43333 6.07f B
3 5.80004 5.63333 4 .966'71 4.58894 4 .5661
5 4.766't4 4.01115 3.93334 2.83335 2.50004 1.40005 0.00001 0.00002 0.00003 0.00004 0.00005 0.00005 0.0000
EEE
FG
H
H
(K
LLLt
M
M
M
M
R
R
TTTTTTT
Alpha O'05 Standard Error for Comparison 0'3408
i.iti"-l T Value 1.969 Critical value for Comparison
0.6710Er!or tern used: Rep*samples*So1*conc' 268 DE
Th"t" .ta 20 groups (A, B, etc') in which the means
a.. not siqr'tiiicantly different from one aoother'
NNNO
OPPO
PQ
PQ
o0o
295
I,SD Al].-Pailwise ComParisons Test of Ec fo! so]-*conc
sol Conc l"treatr Eoeogeneous erouPs3 ! 13.46'7 A3 2 72.815 B
2 2 72.5a9 B
2 L :,r.'785 C
2 3 9.'756 D
1 1 9.-t4l D
1 2 9.696 D
3 3 9.548 D
2 4 7.033 E
1 3 6.96'7 E
3 4 6.893 E
1 4 5.556 r2 5 5.033 G
2 q A qAt G
t" 5 4.889 G
Alpha0-1968Critical T Value0 .3A',7 4
standard Error for Compari son
Critical Value for ComParison
saEP].es Sol11'72't33381
B3'7],12'7381
839I929332'77'72
0.05
! .969
CoDc l4,eaa2 23.0042 23. AA02 23.044
1 21.0001 21.0001 21.0003 20.0003 20.0003 20.000
2 18.3332 18.3331 17.0001 1?.0001 17.0001 16.5001 16.5001 16.500
i omogefleous s! euPsAAA
BBC
BCBC
C
C
C
D
D
D
E
E
E
E
E
E
Error term used: Rep*samples*Sol*Conc, 268 DE
There are 7 groups (A, B, etc') in which the means
are not slgnificanlly different from ooe anothe!'
LsD AAl-Pai:.IIise ComPalisons Eest of Ec fo! samples*So1*cone
296
't391929381a283'7 I'72'739192
3217'72'7333t243638182
63234l-4391-9293312231621327818283
222l2362624!
1 16.5002 16.0002 16. AAA
2 16.0003 16.0003 16.0003 16.0004 15.0004 15.0004 l-5.0003 14.2003 74.2443 14.2002 14.0005 14.0005 l-4.0005 14.0002 13.500
1 13.0002 13-0004 13.0004 13.0004 13-0001 13.000\ 12 .6611- 12.0002 12.a)a4 12 - 0004 12.0004 12.0001 11.16?1 11.0002 11".0002 11.0002 10.0001 10.0005 10.0005 10.0005 10.0005 10.0005 10.0005 10.0002 10.0002 9.50002 9.50001 9.50004 9.50002 9.4333
EEFEF
EEEEEE
iG
!'G
GHGH
GHIGHIGH1GHIHIJHfJ
IJKIJK
IJKIJKIJK
JKKLKLKLKLKL
LLMLM],M
MN
MNMN
MN
MN
MN
MN
MNMN
NONONONO
NOP
297
63l26213322331226342l24213332231314L41-4L3222434333
4223324342132342a21111111111721321-2L2l
515151
3 9.23333 9.00003 9.00003 8-40003 8.30003 7.50003 7.50003 7.40004 7.4000t 7.30001 1.266-t2 7.0000l- 6.50003 6.50004 5.00004 5.00005 5.00003 5.00004 5.00005 5.00004 4.?0005 4. s0003 4.10004 4.03334 4-00005 4.00003 3.90004 3.50005 3.50005 3. s0005 3-30004 3.10005 3.00004 3.00004 1- 10001 0.00002 0.00003 0.00004 0.00005 0.00005 0.00005 0.00003 0.00004 0.00005 0-00001 0.00002 0.00003 0.00004 0.0000
NOPNOP
NOPoP0
PQ
OR
QRQR
QRR
R
R
sssssS
STSTU
STUSTU
TUTUTU
U
U
U
U
298
5 1 5 0.00005 2 1 0.00005 2 2 0.00005 2 3 0.00005 2 4 0.00005 2 5 0.00005 3 1 0.00005 3 2 0.00005 3 3 0.00005 3 4 0.00005 3 5 0-00006 1 1 0.00006 L 2 0.00006 1 3 0.00006 1 4 0-00006 1 5 0.00006 2 5 0.00006 3 s 0.0000
Error for Compari son
value for Corpari son
Alpha 0
0-s903Ctiticat T Value 1.L.1622
05 Standard
Crltlcal969
Error term used: Rep*samples*So1*Conc' 268 DF
Th.t. u." 22 qta:ups (A, B, etc') in which the means
.re ,rot "iqtrliicantly different from one another'
Appendix wiii-LsD Al1-Pailwise Comparisods Test of KP forsamples
sadples lllean Eo&ogeneousGlouPs9 15.400 A8 14.200 B'7 72 .561 C
3 8 --1022 D
4 6.8861 E
1 3.9778 E
6 3.7333 E
2 0.0000 G
5 0.0000 G
Alpha O'05 standard Elror fol Comparison
0 . 1813c.tai""f T value 1.969 critical value for Comparrson
0.3s69eitot t..^ used; Rep*samples*SoI*Conc' 268 DE
? oro-rDs (A, B, elc J in hnich lhe means
.aa "oa "in"ificantly different from one another'
299
LSD Aal-Pairrtise CoEPalisons Test of KP for sol
so]. lYieaD EoEogeneous Gloups3 8.0333 A2 '7 .580'7 B1 6.2487 C
Alpha O.O5 standard Error for Compalison
0.1047Crltical T Value 1.969 Criiical Value for Comparlson
a -24 61Error term used: Rep*samples*So1*Conc' 268 DE
aii : ^"un" are significantly dlfferent from one another'
ISD Aal-Pairwise CosPalisoDs Test of Np for Conc
Conc lllean Eomogeneous GrouPs1 9.9346 A2 9.32'72 B
3 6 -'7153 C
4 5.8889 D
5 4.4444 E
Alpha O'05 Standard Error fol Comparison
0.1351i.iti"uf T Value 1.969 Critical Value for Comparason
a.2664E.ror te.. used: Rep*san''pfes*So1*Conc' 268 DF
;i;-; ;.;;. a.e sisniricintlv different from one another'
,,SD Aa1-Pai.llrise ConPalisons Test of Kp fo! saePtes*So].
samp].es So1 !4ean EoEogeneous GlouPs
9 1 15.400 A
9 2 1s.400 A
9 3 15.400 A
B 1 14.200 B
I 2 14.2A0 B
I 3 14.200 B'7 1- 12-56'7 C
1 ? L2.56'7 C
'7 3 12 .56't C
3 3 9.6133 D
4 3 9.0600 DE
3 2 8.5533 EF
3 1 ?.9400 E
7 2 5-9e61 G
a 3 5.946'l G
4 2 5-8333 6
300
4
6
6
1
222
55
5
6
1
2
3
1
1
2
3
1
2
3
t-
sarPles ConcBl-91-B29231'l 2
9394'7383'7495'7 1
418475tu85L2613311433413
5."/ 6615.68675-51330.00000.00000.00000.00000.00000.00000.00000.0000
G
G
G
Alpha0.3140Critical T Valuea.6182
H
H
H
H
H
H
H
H
Standald Error for Compari son
Error term used: Rep*sampfes*Sol*Conc, 268 DE
There are I groups (A/ B, etc.) in Lnhich the rneans
are not significantly dj.fferent from one another'
LSD All-Pairwise ComPalisons Test of Kp fol saePles*Colre
Critlcal Value for Compar.i s on
0.05
llrean20.00019.00018.00017.00015.067l-5.00015.0001,4-00013.00013.0001,2 .5-1812.50012.00011.83311.33311-00010.5009 .22229.0000'7 .'71',7 B
6.34446.33335.83335 . 31115,266',74 - 4333
Eoroogieneous GrouPsA
B
C
D
E
E
E
FG
G
GH
GH
HIHIIJ
JKK
LL
M
NN
NOoo
P
301
6 2 4.366'74 4 4 .33333 5 4.266'74 5 4.23336 4 4.05566 3 3.90007 4 a.84441 5 0.00002 l- 0.00002 2 A.AAaA2 3 0.00002 4 0.00002 5 0.00005 1 0.00005 2 0.00005 3 0.00005 4 0.00005 5 0.00006 5 0.0000
Alpha 0.05 standard0.4054
Critical
P
P
P
PPP
R
RR
RR
RRRR
RR
R
Error for ComPari son
Value for Comparisonclitical T value 1.9690.7981Error term used: Rep*samples*SoI*Conc, 268 DE
There are 18 groups (A, B, etc') in which the means
are not significantly different from one another'
LSD ALl_Pairflise CoalPalisolrs Test of KP fo! So].*Conc
So1 Conc l'raan Eooogeneous GlouPs
3 1 11.226 A
3 2 70.5'74 B
2 1 10.530 B
2 2 9.6'74 C
1 1 8.048 D
L 2 '7 --133 DE
3 3 1.556 E
2 3 6.9'7a F
I 3 4 6.3'18 G
2 4 6.222 GH
1 3 5.793 H
1 a 5.06? t
2 5 4.500 J3 5 4.433 'l1 5 4.400 'l
Alpha0.2340
0.05 Standard Error for Conparison
302
Critical T Value 1.969 Critical Value for ComParison
0.4608Error term used: Rep*samples*S01*Conc, 268 DE
There are 10 grorrps (A, B, etc') in whlch the means
are not siqnificantty different from one another'
iSD A].l-Pailrrise CoEparisons fest of Kp for samPles*So1*Conc
sa.aples Sol Conc ltlean Eomogeneous GlouPsB 1 1 20.000 A
a 2 1 20.000 AB 3 1 20.000 A
9 1 1 19.000 AB
9 2 1 19.000 AB
9 3 1 19.000 AB
I 1 2 18.000 BC
I 2 2 18.000 BC
8 3 2 18.000 Bc9 1 2 17.000 cD
9 2 2 1'1 .AA0 CD
9 3 2 17.000 cD
3 3 1- 16.2A0 DE
4 3 1 16.000 DE
3 2 1 15.400 EE
9 I 3 15.000 EEG
9 2 3 15. 000 ErG
9 3 3 15.000 EEG
1 L 2 15.000 EEG.7 2 2 15-000 EFG
1 3 2 15.000 EFG
3 3 2 14.A6'7 EGH
g 1 4 14.000 GH
9 2 4 14.000 GH
9 3 4 14-000 GH
4 3 2 14.000 GH
3 1 1 13.600L 2 2 13.333I 1 3 13.000I 2 3 13.000I 3 3 13.000'7 \ 3 13.0001 2 3 13.000'7 3 3 13.000'7 1 4 12.500'7 2 4 l2.5aa'7 3 4 12.500? ) 2 12-4619 1 5 12.0009 2 5 12.000
HIHIJHIJKHIJKHIJKHIJKHIJKHIJK
IJKLIJKLIJKT,
JKLMJKLMJKLM
303
93'7712'l 3
3162
B2B311-'72
131342B182B3334l6341t21363633363t2626241-42623132434332324243313142424l4133
5 12.0001 11.8331 11.8331 11.8332 11,.6001 11.0334 11.0004 11.0004 11.0005 10.5005 10.5005 10.5001 10.0002 10.0001 10.0005 9.00005 9.00005 9.00003 8.40002 8.00001 8.00001 8.00001 1.50003 7.20002 7.10003 6.30004 6 -2A004 6.L66'73 6.10002 6.00004 6.00003 5.83332 5 .66613 5.40003 5-30003 s.30005 5.20003 5.10004 5.00005 5.00003 5.00004 5.00004 4.60005 4.60004 4.50005 4.00004 3.s0005 3.5000s 3.2000
JKLMKLMNKl,MNKLMN
lMNMNO
MNO
MNO
MNO
NONONO
OPOPOP
PQ
POPO
QR
OR
QRQR
RSRSTRST
STUSTUSTU
TUTUTUTUV
UVUVUVW
UVW
UVW
uvwUVI,IUVW
UVW
U!{^1
w{XVWXVWXY
wx-rzX-l ZaX!Za
YZa
304
11
1
1
1
1
11
1
2
2
2222
22
2
2
222
225
5
55
5
55
5
55
5
5
5
6
6
66
6
6
6
2
1
1
1
1
1
2
1
1
1
11
22
22
23
3
3331
1
1
1
1
22
2
22
3
33
3
1
t-
1
1
1
2
3
4
4
1
2
34
55
5
1
2
34
51
2
3
4
5
1
23
4
51
234
5
12
3
4
51
2
34
5
t-
2345
55
3.0000
0.00000.00000.00000.00000.00000.00000.00000.00000.00000.00000.00000.00000.00000.00000.00000.00000.00000.00000.00000.00000.00000.00000.00000.00000.00000.00000.00000.00000-00000.00000.00000.00000.00000.00000.00000.00000.00000.00000.00000.00000.00000.00000.00000.0000
0.05
Zaa
bbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbb
Standard Error for ComParis onALpha
305
Critlcal T Value 1.969 Critical Value for ComparisonL.3823Error term used: Rep*samples*So1*Conc, 268 DE
There are 28 groups (A, B, etc') in whlch the means
are not significantly different from one another'
Appendix ix.LsD Al].-Pailwise ComParisons Test of Pa fol sampfes
samPle s !{ean EoEogeneous GlouPs8 15.400 A
9 13.880 B7 11.160 C
3 9.1800 D
4 6-4844 E
2 5-9L33 E
6 4 -24AA G
1 2.8600 H
5 0.0000 r
Alpha O.O5 Standard Error for Comparison
0.13s6iritical T Value 1.969 Critical value for ComPall-son
0 - 26'71Error t"rm used: Rep*sampLes*Sol-*Conc' 268 DE
;ii-; ;;;"" are significantrv different from one another'
r-sD Al].-Pai!!.ise ComPalisons Test of Pa for SoL
so1 Mean Eottrogierteous GlouPs2 I .'7222 A
3 8.0815 B
L 6 -2356 C
Alpha O.O5 Standard Error for Compar:ison
0.0?83iriti.u:- T value 1.969 critical Value for comparl son
a -L542i..o. tetrn used: Rep*sanpfes+sol*Conc' 268 DE
;ii-a ;""" are significantly different from one another'
LSD All-Pairwise Conpatisons Test of Pa for Conc
Mean Homogeneous GrouPSConc1 10.805 A2 9.'799 B
3 't .22L C
4 6.100 D
5 4.4'74 E
305
Alpha0. t-011criticaf T Value0.1991Error telm used:A11 5 rLeans are
Standard Error for Comparis on
Criticaf value for compari son
0.0s
Rep*samples*Sot*Conc, 268 DF
significantly different from one another.
LsD Afl-Pailwise coeParisons fest of Pa for salnples*so1
salsp].es SoL Mean Eomogeneous GlouPsB 1 15.400 A8 2 15-400 A8 3 15.400 A9 1 13.880 B
9 2 13.880 B
9 3 13.880 B
7 1 11-160 C't 2 !1 .!60 c? 3 11-160 C
3 2 11.000 c3 3 9.5600 D
2 2 8.02AA E
6 2 1 .6461 EE
4 3 1.5600 EE
4 2 1.3533 EG
3 1 6.9800 G
2 3 5.5600 H
6 3 5.0733 r4 1 4.5400 ,l
1 3 4.5400 J2 7 4-1'60a JK1 2 4.A4AA K
1 1 0.0000 L
5 1 0.0000 T
5 2 0.0000 L
s 3 0.0000 L6 1 0.0000 L
Alpha O'05 Standaid Error for Cor(parison
a.2349Ciitical T Value 1.969 Critical Value for Comparason
0 .4626Error term used: Rep*samples*So1*Conc' 268 DF
th"ta ut. 12 groups (A, B, etc') in which the means
.r. ,tot "iq,riiicantly different from one another'
307
I,sD A11-Pailuise coEPalisons Test of Pa fo! samPles*conc
salrpfes Collc l'teatt Eomogeneous GlouPs8 1 19.000 A3 1 17.033 B
8 2 17.000 B
9 1 16.000 c9 2 15.000 D
I 3 15.000 D
7 1 14.000 E
8 4 14.000 E
9 3 14.000 E
9 4 13.400 E
4 1 12-333 G'7 2 72 .1oa GH
8 5 12.000 GH
3 2 11.533 Hr2 2 LL .16'1 r9 5 11.000 r7 3 10.300 J7 4 10.000 JK4 2 9-666'7 K'7 5 9.5000 K
3 3 8.8667 L2 1 8-566'7 L
6 I 6.9'718 M
6 2 6.5556 MN
3 4 6.2000 N
7 2 5.2661 02 3 4.666'7 P
6 3 4-5333 P
4 3 3.8556 o! 3 3.'?66'7 04 4 3.5000 QR
1 1 3.3333 QR
6 4 3-1333 RS
4 5 3.0661 RS
2 4 2.'7333 sr2 5 2.4333 ru3 5 2-2661 TU
1 4 1.9333 u
1 5 o.o0o0 v5 1 0.oo0o v5 2 o.0ooo v5 3 0.0000 v5 4 o.o00o v5 5 o.oo0o v6 5 o.0oo0 v
308
Alpha 0.05 Standard Error for Comparison0.3033Clitical T Value 1.969 Critical value for Coinparisona.5912Error term used: Rep*sampfes*So1*Conc, 268 DF
There are 22 qtonps (A, B, etc.) in lihich the meansare not significantly different from one another.
LsD Al.f-Pailltise corlPalisotrs Test of Pa for so1*Conc
so1 Conc l,Iean goEogeneous GrouPs2 1 12.115 A3 I 1L.'76'1 B
2 2 ).1.400 C
3 2 9.930 D
1 1 8.533 E
2 3 8.485 E
1 2 8.06'7 E
3 3 '7.433 G
2 4 6.'76'7 H
3 4 6.544 H
1 3 5.'744 r! 4 4.989 J2 5 4.844 J3 5 4.133 J1 5 3-844 K
Alpha 0.05 Standard Error for Comparison0.1751Critical T Value 1.969 Criticaf Value for Comparison0.3448Elror tern used; Rep*samples*SoI*Conc, 268 DF
There are 11 groups (A, B, etc') in which the means
are not significantly different from one another'
I,sD A].l-Pailwise CoEPalisons Test of Pa for saEPles*So1*conc
saEples So1 Conc l4ean Eomogeneous GrouPs3 2 1 21.400 A
8 1 1 19-000 B
I 2 1 19'000 B
8 3 l- 19.000 B
3 3 1 18-400 B
8 1 2 )-1 .OOO C
B 2 2 17.OOO C
8 3 2 l'7.400 c4 3 1 16.000 cD
I 1 1 16.000 cD9 2 1 16.000 cD
309
93B182839192932262-1
1-
'72'738182B3919293334291
936211'72'73818283322L
324291
93'7 1-
12-l 3
43'7 I'72132l11'7273
1 16.0003 15.0003 15.0003 15.0002 15.0002 15.0002 15.0002 14.0002 t4.AAA1 14.0001 14.0001 14.0004 14.0004 14.0004 14.0003 14.0003 14.0003 14.0002 L4.0001 14.0004 13.4004 13.4004 13.400| 72 .4332 12 -AAa2 12.A002 72 .0005 12.0005 12.0005 12.0002 1,1.3002 11.3001 11.3003 11.3002 11.0005 11.0005 11.0005 11.0003 1-0.3003 10.3003 10.3002 10.0004 10.0004 10.0c04 10.0001 9.50005 9.50005 9.50005 9.s000
CDDEDEDE
DEDE
DE
EFETEEEEEEEEEEEEEFEFEEEEEE
EG
EGEG
GH
HIH]HIHIH1H1IJI.'TJIJIJKIJKIJKIJK
,rK1,JKLJKL
K1.M
KLMK],MK',M
LMNLMNI,MNLMN
310
313122637222232347l-332
4t631333331363t2
31632262434212224342423243232333134lL241-471111111111t213
2 9.30003 9.30003 9.00001 8.50002 8.30001 8.20002 B.2aA01 8.00002 8.00002 7.50004 7.50003 ?.40001 7.00003 6.20003 6.10004 6.10003 6.00001 6.00002 5 .66613 5-20003 5.00004 5.00004 5.00004 4.90004 4.40003 4.30003 4.r66'71 4.00005 4.00004 4.00004 4.00005 3.60005 3.50005 3.50004 3.30005 3.30005 3-30004 3.10003 3.10004 2 .10044 2.50005 2.10001 0.00002 0.00003 0.00004 0.00005 0.00005 0.00005 0.0000
LMNOLMNO
MNOPNOPQ
OPORPQR
PQRPQRSPQRS
QRSQRS
RS
TUTIJ
TUTUVTUV
UV!{UVWX
VWXYWXYV!{XY
WXY
xvzYt zaXYZa
YZabYZabYZabYZab
Zabczabcdzabcdabcdabcdabcdbcdebcde
cdede
efffffff
22235
5
5
55
5
55
5
55
5
6
6
666
6
6
1
1
1
L
1
1
1
1
1
2
2
22
233
33
3
1
1
1
1
1
2
3
3
4
1
2
34
5
1
23
4
51
2
34
51
2
3
4
5
5
5
0.00000.00000.00000.00000.00000-00000.00000.00000.000c0-00000.00000.00000.00000,00000.00000.00000.00000.00000.00000.00000.00000.00000.00000.00000.00000.0000
Alpha 0.050 .5254Critical T Va1ue 1.9691.0343Error term used: Rep*samples*So1*Conc, 268 DE
There are 32 groups (A, B, etc') in which the means
are not significantly different from one another'
Appendix x.LSD AL1-PairY.ise ComPaliso s Iest of Sa fo! sa]Bp1es
sa8Ples tlleaa goEogieDeousGlouPsB 21.600 A9 2A .4AA B
1 76.240 C
4 10.324 D
2 9.580 E
3 9.491 E
6 8.341 E
r 6.1,24 G
5 1.1,80 H
311
ftffffffffttfffffttfffffff
Standard Error for ComPari son
Critical value for comParison
AIpha 0.05 Standard Error for ComPari son
372
0.L1'7BCritical T Value 1.969 Critical Vafue for Comparison0.3500Error term used: Rep*samples*Sol*Conc, 268 DEThere are B groups (A, B, etc.) in \,.7hich the meansar.e not s-lgnificaotly different from one another.
ISD Afl-Pairwise Compalisons test of Sa for SoI
So1 Mean Eomogefleous Groul)s2 L2.384 A3 1-2 .262 Ar 9.161 B
Alpha 0.05 Standard Error for Comparison0.1a26Critical T Value 1.969 Critical Value for Comparisona.202tError term usedr Rep*samples*So1*Conc, 268 DF
There are 2 groups (A and B) in which the meansare not significantly different from one another.
,,SD All-Pairwise Comlralisons Test of Sa fo! CoDc
conc It ean EomogeDeous GlouPs1 17-533 A2 1-4-495 B
3 10.880 C
4 8-463 D
5 5.985 E
Alpha O.O5 Standard Erro! for Comparison0.1325Critical T Value 1.969 Critical value for Conparisona.2609Error term used; Rep*samples*Sol*Conc, 268 DE
A11 5 means are significantly different fron one another'
LsD AJ,l-Pairlrise comPalisons Test of Sa fo! saEP1es*Sol
sasPles SoI ltlgaD Eoaogeneous CrouPsB 1 21.600 Aa 2 2a.6aA AB 3 21.600 A9 1 20.400 B9 2 24.404 B9 3 20.4A4 B1 I 16.240 C
1 2 L6.20A C
'7 3 76.2403 2 1-L .64-/2 2 tl.50a3 3 11.1134 3 11.0004 2 aO .96'12 3 1A .440I 2 9.'7A6'14 1 8.99336 3 8.90001 3 8.666?6 1 8.20006 2 '7.94002 L 6.80003 1 5 - 71335 3 2.04005 2 1.s0001 1 0.0000s 1 0.0000
313
H
H
HIIJ
JK
LM
M
N
N
cD
DE
DE
EEEE
F6
Alpha 0.05 standard Error for Comparison
0.3079iriti"uf T value 1.969 critlcaf Va-lue for comparison
a .6A62Error term used: Rep*samples*S01*Conc' 268 DE
There are 14 grouPs (A, B, elc') in lrhich the means
.ra ,rot "igrtiiicantly different from one anothel'
LSD ell-Pailwise Compalisons Iest of Sa for saEPLes*colrc
sanp].es colrc !!ean Eosogeneous GloupsI 1 28.000 A
a 2 24.AAA B
9 I 24.000 B
4 L 23 .66'7 B
I 3 22.000 c9 2 22-OaA c9 3 2l--500 cD
7 1 21.000 D
8 4 20.000 E
g 4 18-500 E
1 3 18.000 EG
7 2 18.000 EG
2 L 11 .66'l G
9 5 16.000 H
3 1 15.467 H
6 r 14.661 r6 2 a4 .66'7 r2 2 14-333 r
314
I 5 14.000 rJ4 2 13 .433 JK7 4 13-000 Kr,3 2 L2.533 L7 5 11.000 M
1 1 10.000 N
L 2 8 -92223 3 L 42221, 3 '7 .666'l6 3 7.56612 3 6.'76614 3 6.00003 4 5.93332 4 5.2A003 5 5.10006 4 4.83334 4 4 .666'7L 4 4.03332 5 3.93334 5 3.8333
5 2 2 .566'71 5 0.00005 3 0.00005 4 0.00005 s 0.00006 5 0.0000
oOP
PQ
oR
RSST
TUU
U
UVV!'VW
W
WX
xY
YYYY
Alpha O.O5 Standard Error fol Contparison0.3975Critical T Value 1.969 Critical Value for Comparison0 .'7 82'1Error term used: Rep*sampfes*soI*Conc, 268 DE
There are 25 groups (A, B, etc.) in which the means
are not significantly different from one another'
LSD A11-Pai!r,ise CorPalj.sons Test of Sa fo! Sol*Conc
So1 Cotrc l4,eatr Eomogeneous GrouPs2 1 79.126 A3 1 18-819 A
3 2 15.959 B
2 2 75.807 B
1 1 1,4-656 C
L 2 ll.-ll9 D
3 3 11.111 D
2 3 LL.522 D
1 3 9.407 E
2 4 9.A44 E!'
315
EG
H
H
1
0.05 StandardAlphaa.2295Critical T Value0-4519
Error for Conpara son
Value for: Compari son
3
1
23
1
4
4
5
5
B .622"7 .1226.4226.2405.333
I .969
Erro! term used: Rep*samples*Sol*Conc, 268 DF
There are 9 groups (A, B/ etc.) in which the means
are not significantly dafferent from one another.
I,SD Al-]--Pairwise Coepalisons Test of Sa for sadples*So].*CoDc
sa$pIes So1 Conc ltlean Eomogreneous GrouPsI l, 1 28.000 AI 2 1 28.000 AB 3 1 28.000 A4 2 1 26.000 B
8 1 2 24.0A0 ca 2 2 24.AAA C
8 3 2 24.A00 C
9 1 1 24.000 c9 2 1 24.000 cI 3 1 24.000 c4 1 1 23.000 cD
8 1 3 22.AAA DE
B 2 3 22.400 DE
8 3 3 22.AAA DE
9 1 2 22-AA0 DE
9 2 2 22.000 DE
9 3 2 22.044 DE
4 3 I 22.A00 DE
9 1 3 21.500 E
9 2 3 21.500 E
9 3 3 21.500 E
2 2 1 21.000 EF'7 7 1 21.000 EF'7 2 1 2l-.000 EE'7 3 1 21.000 EF
3 2 L 2A.L33 EG
8 1 4 20.000 FG
B 2 4 20.000 EG
8 3 4 20.000 EG
2 3 l, 19.000 GH
9 1 4 18.500 Hrg 2 4 18.500 Hr
Critical
316
934'7 l2113'1 22123'732-1 33331322622631
925935t-312324326324223321,21222611621815a25835L22132271'1 L4'724134
723612333'7L5'725'7353231244L2311633223613225
18.50018.00018.00018.00018.00018.00018.000L'7 .36116.00016.00016.00016.00016.00016.00016.00016.00016.00016.00015.33314.10014.00014.00014.00014.00014.00014.00014.00013.43313.33313.00013.00013.00013.00013.00012.00012.00011.10011.00011.00011.00011.00010.0009.1000a.96618.90008.50008.00008.0000t.5000
HIHIHT
H1HIHIHII
JJJJJJJJ,l
JJK
KLK],KLKLKLKLKLK1,
LLMLMLM1,M
LMLM
MN
MN
NONONO
NONO
OPPOPQ
PQ
QRQRSQRS
377
31332261326241-434353423223332143235331313142524163425262132L41-111111111172132l51515151
5252
53
2 7.50004 7.50003 7.30004 7.00004 7.00004 6.30003 6.2AAA3 6.00003 6.00004 6.00001 6.00003 6.00005 5.80004 5.60005 5.50003 s.00005 5.0000s 4.30002 4.2AAa3 4 . L66'74 4.00005 4.00004 4.00001 4.00004 4.00004 4.00005 3.50002 3.50004 3.50004 3.00004 3-00005 3.00001 0.00002 0.00003 0.00004 0.00005 0.00005 0.00005 0.00005 0.00001 0.00002 0.00003 0.00004 0.00005 0.00003 0.00004 0.00005 0.00003 0.0000
RSTRSTRS TU
STUVSTUV
TUVW
TU\'I{UVW
UVW
UVI^i
UVI{UVW
VW
WX
wxYWXYZWXYZ
XYZaYZaYZa
Za
ZaZa
aaa
aabbbbbbbbbbbbbbbbb
5 3 4 o.oo00 b
5 3 5 0.o0oo b
6 1 5 o.0ooo b
6 2 5 o.o00o b
6 3 5 0.oooo b
Alpha 0.05 Standard Error for Comparjson
0.688sCritical T Value 1.969 CriticaL Value for Comparlson
1.3556E!ror term used: Rep*samples*So1*Conc, 268 DE
there are 28 groups (A. B, etc') io which the neans
are ,rot "iqttiiicantly different from one another'
Appendix xi.tsD Aa1-Pairwise coePalisons Test of se fo! samples
saaPles l'trean EoBogeneous GlouPs9 21.600 AB 19.400 B-t l'1 .91,3 C
6 9.4689 D
3 9.1044 D
2 1-L333 E
1 4.3333 F
5 0.4444 G
4 0.0000 H
318
SoI lrean Eomogeneous GrouPs
Alphaa -224 4
0.05
Crlticaf T Vafue 1.9690.4340
"rr"t aut. used: Rep*samples+So1*Conc' 268 Df
ii"r. ".u B groups (A, B, etc ) in whlch the means
.r. ""t "iq"itic;ntLy different from one another'
LSD AL}-Pairrise CosrParisons Test of Se for So]
Standard Error for Comparis on
Critical Value for Comparison
2 70.694 A
3 10.450 A1 8.655 B
Alpha O'05 standard Error for Comparison
a.'213i.iai.., T Value 1.969 critical Value for Comparlson
0.2505urr"t a.t. '.lsed: Rep*samples*Sof*Conc' 268 DE
in... u." 2 groups (A and B) in which the means
...-""i "iq"iftcantly different from one another'
319
LSD A1l-Pairwise cotlparisons Test of Se for conc
Conc Meaa EoDogeneous GlouPs1 13. 7 93 A2 \1.64a B
3 9.816 C
4 8.614 D
5 5.781 E
Alpha 0.05 Standard Error for Comparison0.1643Critical T Value 1.969 Critical Value for Comparisona.3234Errol term used: Rep*samples*So1*Conc, 268 DF
Af1 5 means are significantly different from one another'
LSD A1t-Pairwise ComPalisons Test of Se for saEP1es*sol
saoPfes So1 !!ean Eomogeneous Gloups9 1 21.600 A9 2 2)- .600 A9 3 21.600 AI 1 19.400 B
I 2 L9.400 B
8 3 19.400 B'M.1.9L3 C
1 2 7'7 .9L3 C'7 3 l'7 .913 C
3 2 11.160 D
6 2 10. 50 0 D
3 3 9.6133 E
6 3 9.506? E
2 2 9.2t33 E
6 1 8.4000 E
2 3 8.0861 E
1 3 '/.20AA G
3 1 6.4800 GH
1 2 5.8000 H
2 1 4-1000 r5 3 0.6733 J5 2 0.6600 J1 1 0.0000 J4 1 0.0000 J4 2 0.0000 J4 3 0.0000 J5 1 0.0000 J
Afpha 0.05 Standard Ertor for Comparison
320
0.3818Critical T Value 1.969 Critical Value for Compar.ison0.7516Error term used: Rep*salnples*sof*Conc/ 268 DF
There are 10 groups (A, B, elc.) in which lhe means
are not significantly different from one another'
LsD ALl-Pairr,.ise coBPalisons Test of se fo! samP].es*conc
sa4Ples Conc lilean Eomogeneous GrouPs8 1 26.000 A9 1 26.000 A'7 I 23 .461 B
9 2 23.000 B
9 3 22.aAA C
I 2 22.A04 C
9 4 20.000 D'7 2 19.000 E
8 3 19.000 E
? 3 18.500 E
9 5 1?-000 E
I 4 17.000 E
7 4 16.000 G
6 L 13.661 H
7 5 13.000 HI8 5 13.000 Hr3 r 12.989 Hr2 1, 12.389 TJ6 2 11,.833 J6 4 \a.66'1 J3 2 11.533 J6 3 10.178 K
2 2 10.011 K
1 l- 9.0000 L
3 3 8.s000 L
3 4 6.8333 M
1 2 5.8333 N
3 5 5.666't NO
c ??11 NOP
1 3 4.8333 0P
2 4 4.5661 P
2 5 3-366'7 01 4 2.OOOO R
5 2 1.2aoa RS
5 I 7.A222 s
1 5 0.0000 r4 1 o.oo0o T
4 2 o.0o0o T
4 3 o.ooo0 T
321
4
4
5
5
5
6
4
53
4
5
5
Alpha0 .4928Critical T value0 - 9703
TTTTTT
Standard Error for Comparison
Critical Value fol ComParis on
0.00000.00000.00000.00000.00000.0000
0.05
)- .969
Error term used: Rep*samples*SoL*Conc, 268 DE
Theae ale 20 groups (A, B, etc.) in which the means
are not significantly different from one another'
LSD All-Pailwise ComParisons Test of Se fo! So1*Conc
sol conc Mean Eomogeireous Groups3 1 14.967 A2 I 14 .63'7 A2 2 12.6'78 B
3 2 1t.9A4 C
\ 7 LL.114 C
2 3 11.000 D
1- 2 l0 -222 E
3 3 10.059 E
3 4 9.256 E
2 4 9.27L E
1 3 8.389 G
L 4 7.556 H
3 s 6.a61 r2 5 5.944 r1 5 5.333 J
Error term used: Rep*sampfes+s01*Conc, 268 DE
There are 10 groups (A, B, etc') in which the means
are not signiiicantly different from one another'
,.sD All-?ailrtise CooParisons Test of se fo! saEples*So1*Conc
Alpha 0
o.2845Critical T Value 1.0. s 602
sanrples SoL91
9381
.05 Standard
Critical
Err.or for Cotnpari son
value for ComPari son969
Conc lvlean Bomogeneous GlouPs1 26.000 A1 26.000 A
1 26.000 A1 26.000 A
B2B3'7 I'72'7391
93B1a28391
93919293'7L'72't 381a2'7 I72'7381a28391
933263'7 I'72'736232132333L26317'7213B1a2
1 26.0001 26.000L 23.06'7I 23 .0 6'7
L 23 .0 6'7
2 23 . AAA
2 23 . AAA2 23 . AAA2 22. AAA
2 22. AAa2 22.0043 22 .0043 22 .0AA3 22 . AAA4 20.0004 20.0004 20.0002 19.0002 19-0002 19.0003 19.0003 19.0003 19-0003 18.5003 18.5003 18.5004 17.0004 11 .0004 17.0005 17-0005 17.0005 17.0001 16.8001 16.0004 16.0004 16.0004 16.0002 15.0002 14 - 5A01 14.0001 14.000L L3 .'7 611 13.0004 13.0005 13.0005 13.0005 13.0005 13.0005 13.000
AA
322
B
B
BBBBB
B
B
B
B
BC
C
C
cC
C
C
cC
CDCDCD
DE
DE
DE
DE
DE
DE
E
EEEEEF
FG
EGH
GHIGHIJ
HIJKHIJKHIJKH]JKHIJKHTJKHlJK
323
XxxXYXY
YYYYYY
B3622262
616222273263132J-2261616123333231631331L222
L233
3123
232231311352L2
5352111111111112
5 13.0001 r-3.000L 12.66'73 12.5002 12.raa1 12.0004 72 . AA02 11.0001 10.5003 10.5002 10-5002 10.0002 10.0003 10.0002 10.0003 10-0004 10.0002 9.03333 9.00004 8.50001 8.40003 8.03333 8.00002 8.00002 ?.50004 7-40004 ?.00003 6.50005 6.50004 6.30003 6.00003 6.00005 5-50005 5.10005 s-00004 5.00005 5.00004 4.00002 2.7A404 2.00001 1.86672 1.5000r- 1.20001 0.00002 0.00003 0.00004 0.00005 0.00005 0.0000
HIJKHlJK
]JKLIJKl,
JKLMKLMKLM
LMNMNO
MNO
MNONOPNOPNOPNOPNOPNOP
OPQ
OPQPQRPQR
QRSQRS
QRSQRSTORST
RSTUSTUVSTUV
TUVTUVTUV
U!'I1]VW
!n'tVW
vww
324
1
2
2
24
4
4
4
4
4
4
4
4
44
4
4
4
4
5
5
5
5
5
5
5
6
6
6
3
1
11
1
1
1
1
1
22
2
22
333
3
31
1
11
1
22
2
3
33
1
23
5 0.00003 0.00004 0.00005 0.00001 0.00002 0.00003 0.00004 0.00005 0.00001 0.00002 0.00003 0.00004 0.00005 0.00001 0.00002 0.00003 0.00004 0.00005 0.00001 0.00002 0.00003 0.00004 0.00005 0.00003 0.00004 0.00005 0.00003 0.00004 0.00005 0.00005 0.00005 0.00005 0.0000
YYYYYY
Y
YYYYY
YYYYYYYYYYYYYYY
YYYYYY
Atpha O'05 Standard Error for Comparison0.8536Crrtical T Value 1.969 Critical Va1ue for Comparison1 .6AA'7Error telm used: Rep*samples*Sol+Conc, 268 DE
There are 25 groups (A, B. etc') in which the means
are not significantly different from one another'
]\)peadix xii.LsD A1].-Pairiiise ComPalisons Test of SP for saEples
sa.sPles llean Eomogeaeous GlouPsB 19.140 A7 r1 .333 B
9 16.193 C
2 8.3L6 D
325
4 8.296 D
6 ?.133 E
3 6.'736 F
1 5.933 G
5 0.587 H
Alpha 0.05 Standard Error for Comparason0 .19'77Critical T Value 1.969 Critical Value for Comparison0.3891Error ter:m used: Rep*samples*SoI*conc, 268 DE
There are I groups (A, B, etc.) in which the means
are not significantly different from one another'
aSD Al-l-Pairw1se CoEPalisons Test of sP for so1
So1 ltleatr Eomogeneous GrouPs3 11.5?8 A2 7a.926 B
1 7.385 C
Alpha O.O5 Standard Error for Comparison0 . 1141Critical f Value 1.969 Criticaf value for Compari son
0 .224-lError term used: Rep+samples*S01*Conc, 268 DF
A11 3 means are significantly different from one another'
ISD A1l-Pair!.ise coBPalisoDs aest of SP for conc
Conc l"treaD EoEogeneous GrouPs1 13-385 A2 12 .464 B
3 9.16'7 C
4 8.037 D
5 6.165 E
Alpha O.O5 Standard Error for Comparison
0.14?3Critical T Value 1.969 Criticaf Value for Comparison
0.2941Error term used: Rep*salxples*Sol*Conc' 268 DF
oir s *.u.r" are significantly different from one anothel'
326
T.SD All-Pai:.rdise CoDpalisous Test of SP for samPl'es*sol
samples So1 l"rean Eodrogeneous GlouPs8 1 19.140 A8 2 19.140 A8 3 19.140 A7 1 17.333 B'1 2 1,'7 .333 B
7 3 1?.333 B
9 1 16.193 C
9 2 16.193 C
9 3 16.193 C
2 3 LL.32A D
6 3 11.200 D
4 3 10.687 DE
2 2 1a.42'7 EE
6 2 la.2aa EF
I 3 9.8000 F
3 3 8-0067 G
1 2 8.0000 G
4 2 '7 .9200 G
3 2 7.8800 G
4 1 6.280A H
3 1 4.3200 r2 I 3.200a J5 2 1-24aA K
5 3 0.5200 L
1 1 0.0000 L5 1 0.0000 L
6 1 0.0000 L
0.05 standard
1.969 Crit ica I
Error term used: Rep*samples*Sol*Conc, 268 DF
There are 12 groups (A, B, etc') in which the means
are not signliicantly different from one another'
tsD A1l-Pailrrise coEPalisons fest of sP fo! salrples*conc
sasP]'es Conc ltean Eomogeneous GloupsB 1 23.000 A
8 2 2L.333 B'1 2 21.000 BC
8 3 20.400 cD
9 1 20.000 D
9 2 18.667 E
1 3 78 .66'7 E
Alpha0 .3423Critical T Value0.6740
Error for ComParison
Vafue for ComParison
3)1
1 4 17.000 E9 3 16.300 EG1 5 16.000 G
8 4 16.000 cB 5 74 .96'/ H
2 L 14 .66'7 HT4 L 14.333 Hr? 1 14.000 r9 4 14.000 r9 5 12.000 J1 1 1t .66'7 J2 2 77,661 J6 1 7A-66'7 K4 2 10-600 K3 1 10 .5 67 K6 2 10-333 K3 2 8.8444 L1 2 8.3333 LM6 4 1.6661 MN6 3 7.0000 N
1 3 7.0000 N3 3 6.9333 N
2 3 5.8333 04 4 5.8333 04 3 5 -'7 66'7 0P2 4 5.0000 oPQ4 5 4.9444 P02 5 4.4LLt 03 4 4.L661 03 5 3.1667 R| 4 2.6661 R
5 1 1.56675 2 a -366'71 5 0.00005 3 0.00005 4 0.00005 5 0.00006 5 0.0000
TTTTT
Alpha 0.05 Standard Erlor for Comparisona .4424Criticaf T value 1.969 Critical Va-Iue for Comparison0 .8'7 A2Error term used: Rep*samples*SoI*Conc, 268 DE
There are 20 groups (A, B, etc.) in which the meansare not significantly different from ooe anothen.
328
LsD All--Pailwise CosPalisons Test of sP fo! So1*conc
Sol Conc I'tean Eomogreneous GlouPs3 1 15.889 A2 ! 15.31"1 B
3 2 14.515 C
2 2 L3.944 D
3 3 11.385 E
2 3 7A.696 F
3 4 9-444 G
1 1 8.956 GH
1- 2 8.922 H
2 4 8.500 Ir
1 3 1-219 r3 5 6-656 J2 5 6.118 JI 4 6-76'7 ,l
1 5 5.663 K
Alphaa.2552Critical I Value0.5024Er!or term used: Rep*sanples*SoI*Conc, 268 DF
There are 11 groups (A, B, etc') in which the meaos
are not significantfy different from one another'
I,SD A1l-Pailwise ComParisons Test of SP fo' saEPles*So1*Conc
saaP].es So1 CoDc lYtearr Eomogerreous ClouPs8 1 1 23.000 AB 2 1 23'000 A
I 3 1 23.000 AB 1 2 2t.333 B
8 2 2 21'.333 R
8 3 2 27.333 B'7 | 2 21.004 B
1 2 2 21.A40 B
1 3 2 21.004 B
8 1 3 20.400 B
I 2 3 20.400 B
B 3 3 20.400 B
9 1 1 20.000 BC
9 2 1 20.000 BC
9 3 1 20.000 BC'7 | 3 18.667 CD'7 2 3 18.667 CD
1 3 3 78.66'7 CD
9 1 2 L8.66'7 cD
0.05
I .969
Standard Erlor for ComPari son
Critical Value for ComParis on
329
929313234363L22263'7 1'72'7391929342-1
1
12738182
22
B2B313'7 1
72'739192936262324323
629l92933342L2136263
2 78 . 66'72 1A - 66'11 18.0001 18.0001 18.0001 18.0001 17.0001 17.0002 L1 .0004 17.0004 17.0004 1?.0003 16.3003 16.3003 16.3001 16.0005 16.000s 16.000s 16.0004 16.0004 16-0004 16.0002 15.0005 14.9615 L4.96'75 14 .96'72 14 - AA01 14.0001 14.0001 14 - 0004 14.0004 14.0004 14.0001 14.0002 14.0001 13.6002 13.0002 13.0001 12-5004 12.0005 12.0005 12.000s 12.0002 11.5332 11.5002 11.0003 11.0003 11.0004 11.000
CD
CD
DEDE
DE
DE
EE
EEEEEEEE
FGEG
FGEG
EG
EG
FG
EG
FGGH
GH
GH
GH
HIH1HIH]HIHIH]H]HIHIIJIJlJK
JKJK
JKJKJKL
KLKLKLKI,
330
7223326321-4732
2343332322412L224313223147313341-413142
32313242
3133
t2
531111111111l2132L21-2l
3 10.0003 10.0002 10.0003 10.0001 9.00001 9.00003 8.30003 8.10004 8.00004 8.00003 8.00005 7.60003 7.s0002 '7.30442 7.00004 7.00005 6.33334 6.00005 5.63331 5.60003 5.l-0002 5.00004 5.00004 5.00005 5.00003 4.50004 4.50003 4.10004 4 - 00004 3.50005 3-50005 3.50001 3.20005 3.00005 3.00002 3.00004 2-00001 1.50002 1.10001 0.00002 0.00003 0.00004 0.00005 0,00005 0.00005 0.00003 0-00004 0.00005 0.0000
IMLMLMLM
MN
MN
NONO
NONONONOPNOPQ
OPQOPOROPQR
QRSTRSTRSTU
STUVSTUVVJSTUVWSTUVWSTUVW
TUVWXTUVWX
UVWXVWX
WXYWXY
wxYXYXYZxYzxYz
Yzazab
abbbbbbbbbbb
331
5
5
5
5
5
5
5
5
6
6
6
6
6
6
6
11
1
1
1
22
2
3
3
3
1
1
1
1
1
2
3
1 0.00002 0.00003 0.00004 0.00005 0.00003 0.00004 0.00005 0.00003 0.00004 0.00005 0.00001 0.00002 0.00003 0.00004 0.00005 0.00005 0.00005 0.0000
bbbbbbbbbbbbbbbbbb
Alpha 0.05 standard Error for Comparison 0'7655 Critical T
vafue 1,g69 Crj-licat vafue for Comparison 1'5072 Eiror ternused: Rep*samples*So1*Conc, 268 DE There are 2Bgroups (A' B'
etc,) ln which the means are not significantly different from
one another -
Appendj-x xij'i.Ana1Ysis of variance Iab]-e for AN
P
0.0c0c0.00000-0000
Source DE
rep 2
plant 5
sol 2
plant*sof 10Error 34Total 53
Grand Mean 45.547
Appendix xiv.Analysis
Sortrce DE
rep 2
plantsoIplani*sol 10Erlor 34
Total 53
Grand Mean 41.895
SSMSE'/4.A 36.98
3i99 .3 639.A6 3r- ' '45
4634.6 231-'7 -28 135 .64'7258 .2 '125.82 42 .49580.9 1r.08
l5'7 46 .9
cv 9.07
of valiance Table fo! AI'
SSMSEP13.3 36 .6'7
84'7 6.g L695.3'/ 305.13 0 ' 0000
3584.9 7'792.4'7 322.61 0.00001863.3 186.33 33.54 0.0000188.9 5.s6
14187.3
cv 5.63
332
AppenAix xw.LsD Al1-Pairl,rise ComParisons Test of All and AF forsahPles
Tukey ESD All-Pairsise ComPalisons Test of AN for samP]-es
saeples llrean EomoqeneousGrouPs6 54.313 A4 53-056 AB3 4'7 .133 BC1 46.94',7 C
2 38.132 D
5 32.540 E
0.05
Critical Q Value 4.2685.8806Error term used: rep*samples*s01, 34 DE
There are 5 groups (A, B, etc') in which the means
ar:e not significantly different from one aoother'
Tukey EsD AL1-?airwise CoEPalisons Test of Alil fo! so1
so1 ltl,ean Eomogeneous GlouPs2 53.'t'78 A
1 s0.258 B
3 32.6A4 C
A.Lph a1,9484
AIphaI .3't'7 B
Standard Error for Compari son
Critical Value for Compari son
siandard Error for ComPaiis on
criticat value for ComParis on
0.05
Critical Q Value 3.4663 .3'7 69Erro! te!m used: rep*samples*s01, 34 DF
oii : ^."rl" are significantly dlfferent from one another'
Iukey ESD A].l-Pai!1,ise CoEpalisons fest of AN for saDP]-es*so].
saapfes sol. an gomogeneous Groups4 2 6A.8]3 A4 1 68.650 A6 1 63'880 AB
3 2 63 .213 AB
6 2 62.2'74 AB
3 1 61-581 ABC
1 3 52.880 BCD
1 1 49.L5'72 2 46 -2305 2 43 .404
CDEDE
DE
sa.top].es !4ean Eooogeneous GlouPs
333
standar:d Error for Comparis on
crj-tica1 vafue for Comparisoo
Standard Error for Compari s on
critical Value for Compari s cn
2 I 42.31'7 DE
1 2 38.683 EE
5 3 38.320 EF
6 3 36 --190 EE
2 3 21 -590 rG4 3 21.643 G
3 3 18.400 G
5 1 15.900 G
Critical Q Val-ue 5.32212 .141Error term used: rep*samples*s01, 34 DE
Th"." ".a 7 groups (A, B, etc') in which the neans
"ra ,roa "igrriflcantty
different from one anothel'
Tukey BSD Aa].-Pairwise ColParisoIls Test of .A! fol samPfes
Alpha3 .3-1 48
0.05
0.05
0.05
3 .466
6 59.718 A2 5-1 .443 A4 4L.239 B
1 33.370 c3 32.'7 AA C
5 26.90r D
Alpha1-LL72Critical O VaIue 4.2683.3536Error term used: rep*safiIp1es*sol, 34 DE
There are 4 groups (A, B, etc') in which
are no! signiticanLly diffe-e_t Iron one
Tukey EsD Al.l_Pailitise Comparisons Test
so1 l4ean
2 4l .'7 5-l3 31.986
the meansanother.
of atr' fo! so1
Eomogeneous GrouPsA
B
c
Alphaa -1451Critical Q Value1.9258Error term used:A1I 3 means are
Standard Error for ComParison
Criticaf Vafue fol ComParj- son
rep*sampfes*s01,34 DE
signific"trtty different fron one another
fukey BSD Aal-Pairwise ComPariso[s Test
samples sol Mean EoDogeneous GrouPs6 7 -t3.21--t A6 2 62.894 B
2 2 61.860 B
2 1 58.040 BC
4 1, 56.251 BCD
2 3 52 .434 CD
3 l- 50.600 D
6 3 43 .44'7 E
4 2 41,.240 E
5 I 37.503 EE
I 3 36 .230 Ff1 I 36.040 EE
3 2 37.96A EG
7 2 2-t .840 G
4 3 26.22A G
5 2 24-'75A GH
5 3 18.450 Hr3 3 15.540 r
of A'r fo! samples*soI
Standard Error for Compari son
Critical Value for Comparison
Alpha 0.05L .9246critical O Value 5.322'7.243LError term used: rep+sampfes*soI, 34 DF
There are 9 groups (A, B, etc') in which the means
are not siqnificantly diffeLent from ooe another'
AppeDd.ir. xvi.Ana].ysis of Varia[ce TabJ'e for DPPE
Sou!cerepsamples
sols amples *concsamples*so1
s ampfes * conc* so1
Total
Grand Mean 58.109
DE SS
21L5 613405 1089824 158339
25 114 952A 33-12120 10 411
100 10643358 '7L10
539 4A2l2A
cv 7.70
MS
72268 .1-
2t'7 96 .539584.8
459.81686.4520.5106.420.0
F
672 .531088 .2'71,9't 6 .42
22 .9684.2425.995.31
P
0.00000 - 00000.00000.00000.00000.00000.0000
33s
Apl)endix xvj.i.LSD A1].-Pail1lise ComPalisolts Test of DPPE fo!satEPl-es
sampLes Mealr EoErogeneous GlouPs2 69.910 A3 62.L'73 B5 62.034 B
6 59.551 C
a 59.291 C
4 35 .626 D
Alpha0 .66'7 \
0.05
Critical T VaIue 1. 9671.3120Error term used: rep*samples*conc*sol' 358 DF
There are 4 groups (A, B, etc') in i'hich the means
ur. ,'rot "igtrificantly different from one aoother'
LSD All-Pairwise cofirParisons Test of DPPH for conc
Mean Homogeneous GrouPs
Standard Error for ComParj- son
Critlcal Vafue for ComPari son
standard Error for ComParison
Crltical Value for ConParison
conc605040302A10
76.145 A1A .110 B
6s.012 C
56.141 D
44 -699 E
35.884
AIpha0-66'7L
F
0.05
Critical T Value 1.96f1.3120irror ter^ used: tep*sanples*conc*soI' 358 DE
iii a ^.u"" are sig;ificantry different from one another
LsD AaL-Pairtrise CoePalisons Test of DPPB for sol
sol
4
123
Alpha0.6090
liiean EoDogeneous GlouPs8l .008 A67.888 Baa .622 C
46.6L4 D
40.415 E
0.05 Staodard Error fol Comparison
336
Critical T Value 1.96f critical value for Comparison
I -191'7Error terrn used: rep*samples*conc*sol, 358 DE
A11 5 means ar:e significantly different from one another'
LsD Al1-Pai! ise coePalisons fest of DPPE for samP].es*conc
samples coDc l"rean EomogeDeous GlouPs2 6A 91.973 A5 60 84.649 B
2 4A A2.8'7 9 BC
2 50 82.048 BCD
6 60 80.036 CDE
3 60 -19 .466 DE
1 60 r8.r18 E
5 50 78.111 E
3 50 't'7 -293 Er6 5a '7 4 .'759 EG
L 50 -72-1'15 GH
3 40 69 -024 Hr5 40 6'1 .9'79 I6 40 6'1 -493 1
2 3A 66 -649 I1 40 65.9'77 r3 30 60.'734 J5 30 60-306 J6 30 58.131 Jr 3A 5'7 .'123 J
2 2A 52.544 K
3 20 48.595 L
I 2A 45 .492 LM
5 2A 44.'7'70 M
6 2A 44.101 M
2 la 43.12'7 M
4 60 42 -328 MN
4 50 40.177 No
3 LA 3"7 .925 0P
4 40 36.'724 P
5 1-O 36.328 PQ
1 10 36.006 P0
4 30 33.305 QR
6 10 32.'7 85 R
4 2A 32.694 R
4 10 28.531 s
o'0r srandard E'ror ror compa-ison
t.6342
".iii".l T value 1'96? criticaf value for comparlson
3.2738
337
Error term used: rep*samples*conc*soI, 358 DE
There are 19 groups (A, B, etc.) in which the means
are not significantly different from one another'
LSD A]'l-Pairrtj,se ComPatisons Test of DPPE fo! samPles*so1
sadl)les so]. I'llean EoEogeDeous Groups1 5 88.684 A3 5 87-911 AB4 5 87.91-1 AB
2 5 A6.727 I\B5 5 86.108 AB6 5 85.313 B
3 4 78.993 C
5 4 78.839 CD
2 4 '16 .124 CD
6 4 76.049 D
1 4 71.605 E
2 2 64.3a2 E
2 3 62.994 FG
2 I 6A.3A9 GH
6 1- 58 -432 H
1 1 53.671 r3 3 53.628 I5 2 5t.813 r5 1 51.303 II 2 48 -33"7 J3 2 46.825 JK6 2 44.8'7A KL
3 1 43.507 T,
5 3 42-a46 L1 3 34.190 M
6 3 33.130 M
4 4 25.159 N
4 1' 24.5L0 N
4 2 23.453 N
4 3 1'6 .499 0
Alpha 0.05 Standard Error fol Comparlson
1.4918iritical T Value 1.967 Critical vaLue fc]. Comparrson
2.9331Error t.tm used: rep*samples*conc*sot' 358 DE
,h"ta .tu 15 gnoups (A, B, etc') in which the means
iie not siqnliicantly different from one another'
Appendix xwiii.Arralysis of variaDce Tal1e for Reduciag Power
ass ay
DE2
5
4
52A252A
100
539
cv 2.03
SS
0.004
62 .41-0'79-691
'1 . 6122 .6'71
0.096192.565
MS
0.0018a .5239
15.602515.9393
0.38360.10717 - 4'7250.01940.0003
E
1960.18
PS ou rceRepsampfes0.0000SolventConcSanPles*Solvents anples *Conc
Solvent *Conc
s amPles + Solvent * Conc
Er rorTotal
Grand Mean 0.8062
58376.3 0.000059636.3 0.0000
1435 -2'7 0.0000400.63 0.0000
5509.15 0.0000291 .L3 0.0000
Appendix xviv.LSD Af'1-Pai!!rise Compalisons Test of Reducing
POWer assay fo! saEp.Les
saaples lYtean EoEogelteorjrsGroups4 0.8533 A3 0.8448 B
1 0.8439 B
2 A.8235 C
6 0.8189 C
5 0.6528 D
AIpha O.05 Standard Error for Comparison
2 . A3'78-a3iriir..r-t value 1'96f Criticaf Value for Comparison
4.793E-03u.r"t-a"t. used: Rep*samples*Solvent*Conc' 358 DF
;;;;; .." 4 sroups (A, B, etc') in which the means
.r!-""a-"ir"ittcantly differeot fron one another'
LSD Aa]'-Pair1'ise Cooparisons fest of RPA fo! Solvent
Solvent llean EoEogeneous GrouPs
4 r.29'76 A5 1.0628 B
1 a.1446 C
2 0.5880 D
3 0.3380 E
AtPha2 -225E-A3
0.05 Standand Error foa ComPari son
339
Critical T value 1-967 Critical Value for Comparison4.37sE-03Error term used: Rep*samples*Sofvent*Conc, 358 DE
A1l 5 means are significantty diffelrent from one another_
I,sD A11-Pai!Il{ise CoEParisoas Test of RPA for conc
Conc ltlean Eomogeneous GrouPg60 1.5213 A50 1.0072 B
40 0.8648 C
30 0.5746 D
20 0.4515 E
10 0 .41? 9 E
Alpha 0.05 standard Error for Comparison2 - 43'78-03Cl:iticaf T Value 1-967 Crlt.rcal Value for ComParison4.793E-03Error tern used: Rep*samples*Solvent*Conc, 358 DF
el1 6 ^.ar'r" are significantly different fron one another'
LsD A].1_Pairwise CodparisonE Test of RPA fo! saInl'les*solvent
samples solweltt l{ean Eomogeneous GrouPs1 4 1.3563 A
2 4 1.3563 A
3 4 1.3563 A
6 4 r.3563 A
4 4 1.3295 B
1 5 1.0650 c2 5 1.0650 c3 5 1.0650 c4 5 1.0650 c6 5 1.0650 C
5 5 1.0517 D
5 4 1-0307 E
3 1 1.0168 E
4 7 0.9'743 G
2 2 A-932'7 H
I I 0.8502 r6 1 0.6891 J4 2 0.5912 K
6 2 0.5647 L
1 2 0.5400 M
3 2 a.5r22 N
2 L 0.4815 0
5 1 0-4495 P
6 3 A.4L94 o
Alpha5.4508-03Critical T Value0.010?
1
5
4
23
samPles Conc3 602641 604646 601 5n
5 601 502 503 506 506 401 403 404442405 505 401 304 306 303 302305 30420L206246 105 20324220
3
2
33
33
0.40790.38130 - 3 5100.30040.2159a .2'7 31
0.05
L .9 6'7
340
Eomogeneous G!ouPsA
BC
C
D
E
ET
G
G
H
IIJ
JK
L
R
Ciiiicat Value fol ComPari son
VV
Standard Error for ComParis on
Error term usedl Rep*samples*Sofvent*Conc, 358 DF
There are 22 gr:au:ps (A, B, etc') in which the means
are not signif-lcantly different from one another'
LSD Aal-Pailwise CoIBPalisons Test of R?& for saEP]-es*cone
!Iean1.10017 .66241.57881.56961.48081.13941.13611.05901.0298\.4289l-.01000.93600 .929'7a.92410.88520.86490 .'71 620.64900.61ss0.60890.59140.56340.5454a .52280.49960.45540 . a5 430.44080.43580.4354a .4282
N
o0
P
0R
S
T
UU
VW
VW
WX
341
1 10 0.42503 10 0.41704 10 0.41702 t0 0 .41425 10 0.3972
Alpha 0
5.970E-03Critical T Value 1.0 . 0117Error term used: Rep*samples*Solvent*Conc, 358 DE
There are 26 groups (A, B/ etc') in which the means
aae not significantly different from one another'
LsD a-U-Pairwise ComParisons Test of RPA for SolveEt*Conc
(15 Slandaro
CriticaL
WX
XYXY
YZ
Error for Compari son
Value for ComPari son9 6'7
Solvent Conc lYlean4 6A 2 - 53225 60 2.09334 40 1.61334 50 1.55175 50 1.44001 60 1-43475 40 L.22AA2 6A L.08421 50 0.90624 3A A.82922 50 a .'7 6'155 30 0.75631 40 0.6838
. 4 20 0.65884 10 0.60031 30 0-55162 40 a .4'7 64
L 20 a .4'7 763 60 0 .46275 2A 0 - 44232 30 a .42835 LA 0 .42411 10 0.41972 2A 0.39782 1A 0.37383 50 0.37083 40 0.33043 30 0.30?43 2A A .28 6'7
3 10 a .2'7 A8
Boeogeneous GrouPs
Bc
D
E
E
E
G
H
L
JK
LM
N
oP
PQ
0R
S
S
S
TU
U
Vilj
xY
AIPha 0.05 Staodard Error for Comparison
342
5.450F-03a.iti""f T Value 1.967 Critical value for Comparison
0 - 0107irror te.^ used: Rep*samples*Solvent+Conc' 358 DF
Th.tu .t. 25 groups (A, B, etc') in which the means
u.. ,roa .ig"iit""rrtly diff"t"t't from one another'
lSD Alt-Pailwise Compalisons Test of RPA fo!sampies * so].went*conc
sanlples Solvent Collc l"Ieanr 4 60 2.64002 4 6A 2.64003 4 6a 2 .64446 4 6A 2 .64003 1 60 2.38004 4 60 2.34005 4 60 2.29332 2 6A 2.2'7001 5 60 2.11002 5 60 2.11003 5 60 2.11-004 5 60 2.11006 5 60 2.11005 5 60 2.01004 I 60 1. 910 0
4 4 50 1.91001 4 40 1.82002 4 40 1.82003 4 40 1-82006 4 40 1.82001 1 60 1.76004 4 40 1.5800t 4 50 1.56002 4 50 1.56003 4 50 1.56006 4 50 1.56001 5 s0 1.44002 5 50 1.44003 5 50 1.44004 5 50 1.44005 5 50 1.44006 5 50 1'44001 5 4A L-22442 5 40 1.22043 5 4A L.224A4 5 40 7.22045 5 4A 1.22406 5 40 1.2204
Eoloogeneous GlouPsAAAA
BC
D
D
E
E
E
E
EE
G
G
H
H
H
HI
JJJJ,]
KKKKKK
LLLLLL
343
1 50 1.21002 50 1 .164 0
4 50 1.16001 60 1 1600l, 50 1.13s01 50 1.11502 60 0.91502 60 0.96502 60 0.95501 60 0.89502 60 0. B?504 30 0.86501 40 0.85404 30 0.84204 30 0.84204 30 0.84204 30 0.84201 50 0. B 3501 40 0.82302 50 a -82204 40 0.82001 40 0.'76445 30 0.75805 30 0.75805 30 0.15805 30 0.75805 JU2 5A 0.75601 40 0.?5405 30 0.74804 30 0 .14202 50 0.'72142 50 a.'72144 2A A.-12a41 30 0.71101 20 0.68101 50 0.66104 20 a .6484a 2A 0.64804 2A a .64804 2A a .64801 30 0.64434 20 0 .6aLA1 30 0.63404 10 0.62804 10 0.62804 10 0'62804 10 0.62801 20 0.5760
L4
2
5
6
316
34
214
31
236
6
4
4
5
6
1
2
3
4
6
61
5
5
1
3
4
4
4
2
1
23
6
L53t-
236
3
M
MN
MNNO
oP
P
P
oQR
RS
RSTSTUSTUSTUSTU
TUU
U
U
vt{WX
WX
WX
xY
\z
Za
ZaZa
aa
aa
b
344
2
24
6
4
24
5
364
1
5
2
6
4
5
1
5631
2
5
11
2
3
4
61
5
6
4
1
2
34
6
56
31
6
6
2442304 101 302442203 604 101 103 601 103 601 602la1 s02442642301 403 505243 502 4a2401 401 30234520524520524520L205 10L202245 1,0
5 105 105 105 102541242343 603 603 403 401 10
0-57400.56400-56200.54400.53700.53600.53300.5280a .52240.51900 . 51100.50900.50300.48800.48100.47300.46500.46100.46100.45900.45900.45900.4s330.4500a . 44100.4444a - 44200.43900.43900.43900.43900.43900.43600.43300.4303a .4214a.4230a.42300.4230a.42340.4230a . a,2la0.4150a. a1200.40900.40510.40430.40300.4010
bbcbcd
cdedef
etg
efgh
fghishihiji j klkrkIm
klmnImnolmno
ImnoplmnoplmnoPmoopq
moopqrmnopqrs
nopqrsnopqrs
nopqrstnop qrs tnopqrstnopqrs tnopqrstopqrs tuPqrstuvqrs tuvl^Irstuvwxstuvw)<YstuvwxystuvlrxYstuvwxystuvwxYstuvwxYtuvwxYzuvwxyz
vwlyzwxyzA
wxyzABxyzABxyzAB
345
6
25
1
11
6
4
6
55
566
1
1
5
3255
214
5
3
2
2
33
34
2
4
324
23
23
4
Alpha0.0133Ciiticala.a263
xy zABYZABCZABC
ZABC
ZABCZABCABCDABCDBCDCDECDECDEDEE
DEFEEG
EEGHFGHl
GHI,]GH].]
HTJKI,]KJKL
JKLMKLMK],M
KLMNLMNO
LMNOMNOPNOPQOPQR
POR
QRSRSTSTUSTUSTU
TUU
U
U
30 0.401060 0.397010 0.393010 0.392020 0.391030 0.391020 0.391010 0.381010 0 .381050 0 . 319040 0.373040 0.371010 0.371020 0.363320 0.363010 0.361020 0.351030 0.3 4 9030 0.342030 0-332010 0.331030 0.331050 0.324010 0.321050 0.31502o 0.313010 0.301010 0-301010 0.299020 0.29)-a2A A.297050 0.2887Aa a.2'73040 a -26644A a.263330 a.249330 0.241030 0.231020 0.23102A 0 .22'7 o2A A.2234t-0 0.213010 0 . 211010 0.2080
0.05 Standard Errolr
33
112
3
22
233
2333222
21233
3
33
23
1
1
23
3
333
33
3
3
33
33
for Compari s on
for Compari sonT Value 1.967 Ciitical- Valrre
346
Error term used: Rep*sampfes*Solvent*Conc' 358 DF
ifr"." u." 73 groups (A, B, etc') rn which the means
u." ,toa "ig"iiia",',tIy difftt"''tt from one aoother'
Rep stands for rcplication, samples for plants and antibiotics' sol for solvents' sa:lplesxSol
for intenction betweeo samples and solvents' samples*Conc for interaction between samples
and concentations , samples+Sol*Conc for interaction arnong samples' solvents and
concetrtrations, CV for coefficient of variation and the Conc for concentrations where dF
Degrees of Freedom, SS= Sum of Squares, MS= Mean Squares and P-Value for Probability
value. Steric sign (*) denotes significant difference and (trs) denotes [on-significant
difference.while E. coli, K. pneumoniae, P, aeruginosa, S- auteus' S, ePidetmidis' S, p}ogen
for Echerichia coli, Klebsellt pneumonia, Pseusdomonas aeruginosa,staphylococcas aureus'
Staphylococcus. epidermidis arrd Streptococcw Pyoge rcspecti\tely
347
Trica.horyli. a.ld cY.le
APPENDIX B
IN I!-RNA IIoNAL JoURNAL oF AGR lcuLTURr & RIol 'oGY
tssN Prinl: 1560-8530;ISSN onl;ne: l8l4 9596
t2428 I 2013 / I 52 i7 7 -38 t
h11p://w w.fspublishers org
Phytochemical AnalYsisstocksianun Blower from Malakand Division, Pakistan
T" .it. ihl. p.p.t, Rrh*Jt, R ,iJ"l at.ua -a Fc,ttuz :ol: r't'ltchedical flalsn t'd dlloxidml poPerties of li:'t"' s'd*'r'"Dr
and Antioxidant Properties of Teucrium
CulRahinr, Rahmatullah Qnreshir', M. Arshad'and Muhammtd Culfrazlr;;;;;; ;f;;;",,y. i; ieh Ati ihah Atitt Asricuk*e u'1itdlitv Mutee Rocttt RLvatpindi P ktdn
*ror correspondence: [email protected]; rahmatullahq@uaar'edu pk
Abstmct
The flower extracts of Ie trctiun stoct\ionuh were screened for antioxidant and phltochemical conslituents by usjng nine
aife.art solvents suctr as acetone, butanol, chloro iorm, eth) I acerate, erlanol, merhan'l' n_he \ane petroleun ether and water'
B\ d,rn! rhese ej\rmc1.. t0 oh\aocon,lIUenr, $ere sc.eenei. saponin. der<.red b) dll sol\enl'. lollo$ed b) Lrnnin reducing
"i.; (.J;,,';;;['iij, ,".p.""iJ or, alkaroids, anthraquinone (4 each), -l,vhereas' .sleroids
detected bv 3 and
nhtdh.rannin as well as elvcoside isotared by 2 solvents. ihree sivents viz., chlorofonn, ethyi acetate and water erttcl€d
iiJi.t ".*-r,i "i"; ,r'ti;aifLso 91, rottoi'ea uv peiroleum ether ( 130 s), n-hexane,.acetone ( 120 s each)' methanol (l l8
gi]li-r,"r(--. yi"ra"a r'igrr"tt.i..l iiigt't 1ze gI followed bv bulanol (20 g), methanol (19'3 g)' water (17 g)' acetone (14 5
ii, "r,i"i"r.# iu.s el,"p"tro1eum 1t z.: -g)
ana Jtiranot ( I L4 s). rhe percentage yield of er:ract was varied ransjng fiom L73
f'zs.ot *itl, d,.,,;nrum value recorded from n-hexane (tI.6%).lt qas follosed by butanol (20.3%), methanol (16.370),
acetone (12.08%). water (l1.33%), petroleun ether (9.46i0) and chlorolbrm (9%) The plant possessed good antjoxidant
p.p".i,ira "r
ni a"*.i too rc,.ripps vatue or nower was 412.82+0.003 thar is near to rhe satndard (440.54+0.00 r ). rte
ii,"i,"ril "".p"*a, *er. atso iiiected in plant that
'"as about half of the quantiry of standard (gallic acid)' This studv wil
serve as benchmark for turther detailed analrsis ofplanl extraci prjor to drug developmenl and its riliTation in tutu€. O 2013
Friends Science Publishers
Keydords: Terczir- rtocfu,?zzr; Traditional medicine; Phltochemical consliluents; Antioxidant
rnrroduction ylif*.f.hT:ffii;,si,;fffflJl;J:'J,,::Recently it has been obsefled that there is steady increase in @ ojab €; 4/ , 2003)' The plant mav reach up to 1 5 30 cm
human infections, especially in tropical and subtropical t\irh dense branching pattem and gey-gr€en leaves As
i"""i"pirg """"i1""
ie"rr; d dt., 2005\. ftis mai te rnedicament, mostl] leales as trell vouns bmnches arc used
Atner duJto a,bitrary use of antibiotic drugs or inue;e in to treat various ailments such as gastro-intestinal, diabetes
resistance to these s),nthetic dmgs (Muheiee €, ,/.. )002). and as sell-as rnflammat'ory condilions also (Radhakishnan
Medicinal plants p;ssess certain biocornpounds, *hich are c/ al, 2001)'-Another srud). reponed its use to tr€at feet
ir,"*p""ir"lrry ;ilpon*t * precursors for lielding s),ndrome and diabsles me irus (Barkatulah and Hussain,
;;#,; ,lligr lSoro*ora :06s1. rhe phyocliemicali 200gl Experimentallv' e:\tracts of this plant were also
p.Jr"", ;. *"fr- pfira p*duce certain physiological action investigared asanri-ulcerogenic and cltopmtective (Islam el
on r}e human body and relieve disease (tqbal and uamal trn, a/" -2002)'
Besdes exftcrs ol this plant is reported having
2oo4;Akinmoladtm e, d1,2007). analgesic and anri-inflammaory activities (Radhakrishnan'pnyt*r,"lnt""t"
ar€'natu;lty found in plants_ These et at.,2oo1). orher studies discovered its use as a blood
are deriving color, flavor ana smeil ofplants. eesides, thel puifier, elile?sy and ]rpeflension (Ahmad el d'' 2002) and
*" pily*gLy.i" * *tural defense mechanism in planrs lhroar pain (Iqbal and Hama)ur 2004)' Keeping into its
"g"i"i a;-""*L- They are w€lt recognized as thempeLuic rnedicinal use lor larious.purposes, present study therefore
fr".ii"ii" r,r-* ,rr"rirg and dise; (okw1], 200aj. The was carried-out for screening phltochenical and anlioxidant
li""i p-".mng phyJchemicals inclxde: ak;loids, activities of Zercltu D-sro.ilia,rm flower locally growing
tu*i,,s, nuuoooia",-and phenolic compounds, which in Malakand division Pakistan'
possess bacteriostatjc and bactericjdal effects. Most ofdreseLiocompoundr other than antimicrobials can also ac1 as Materials and Methodseffective antioxidants (Venkata et dL.,2012).
Telrcrium stoclsianum Boiss. is a member of PlantMaterials Collection and Identilication
bmraceae tamil). This aromaric perennial and $ood) herb
is nostly found inthe hilly area ofnorthem oman a. ieX as frucriun :tack'ionun plant samples were gaihered at
nowdnonMalrddDvision.P*jsrd. Lt. J,lgti.. Bial. t5: 317 381
Raiifler al lInt-J. Agric Biol, Yol 15'No 2'2013
n^.erinc slase ftom rhe glo*,lng aJeas fl ala:h)' Dir louer'
i,Y,1,-LTa'oi"i,;"q ru>tir Pakhrun Khwa (KPK '
Pakr(tan
]J-Jni. *o rue.20rr. voJcher 'p{imeD wb al'o
::--: ;- ,he ftesh couec oo od 'detrtiG<d thoush
ililJlq-G llora of Pakisun (Hedge. Ieao'' rbe
;:il-_** mainra,ned in rle depanmenl of Bounv'
iffi- *;a Agriculrue uni\er.jrv Ra$alpind for
Pr€parrtion of th€ Extracts
Flo\len wete sepal"red 6om fie platrr collec$on and
".iJ z t : umes iluougb rap $arer and subtque'dv
.*" L,lr"a *r*' * ,sed lo renove impuitv ed Lhen
.i^J. *t*a. Tbe dried malerial was blesded to fi'e
""*a.r leO ...hf wrth he help of a labomrory grindrg
L.r,i""'-a "*.a in reFjgeraror' Tbe oblamed poeder
'ilral **,r,." ""n*'"a h acetone butanol cbloroforo
.'i,i *.t"r.. .,1-"1 ..,1-ol. !-be\de petoltu eLhtr
^.i *r.t tv sbakhs for 24 h ar lr"C' The re5uiunr
macerate were tne" sie"ea using Wlatman filter paper No'
i-Ji o*" solvat ext.cis were evaponred and
".o"**t"a *ao ..a*"a pr€ssure at 40"C with the help
oi."t) .*porato.. these e\tracb sere fieo emplo)'ed for
the scr€etrils of Ph}aochedicals'
trvaluation oI Phvtochemicats Screenhg Test
The chemical tests were canied out from nirc diferenl
';re 5 ba-'ed exracts of $e r"ucnua stockanun fiorct*i* r"ra.a prmedures for $e rdenn6catioo or planr
*r,s-tin.os Uy tollowing the qorL' of fgwrkhjde alld
Gimba t2007).iet for alk-aloids: ExEacr in 0.2 mg sa' healed wrtb 2";
Johuric acia for r*o arLrles. Few drop' of &agendor6's
*"1*t *. added with fie filmre An emergence of
o.uie" ,.d pr."lpilut" was indicarive of alkaloids
rEswaili&lde and Cimba. 2007)'
T-eit for rsorins: A ponioD of Plant e\ract was di:solved
in aoua aod mrmed b1 usng $aler barh Tbe !e(r sample
*.'ttrm filtered atrd ferric cbloride $as added to iL
Presence of dark geen color indicates sigD maik fot qnnins
rEewaikhide and Cinba, 2007)
i;r ror anthraquiootres: Al1 amount of 0'5 s ol the re)t
(dDle r"5 heaied wilh looo hvdrocbloric acid for soDe
rime ir *arer bari and *41 filEdred- cooled ard r'he same
"oru-e .f
"r o.ofom was mixed. Few &ops of 10%
enlronia Ngr) 'ms mixed ad heated Ar emergence of
rose-Dink color was marked as pre*nce of anthraqutnone
/FMn<hde ano ulmDa, 2u! /1.
Test for gllcosides: Pla.El exEact [€s m!\ed widr HCI and
,r,* f'"5i *t,,;"" Subsequeldv some drops of the
Ienlinq's solurioD A dd B $ere \uPplemented' 4!,"*,i.o.. or *tai"t p,."ipiL{e rcv€aled fte i!'ideDce ofsiwosides (EgM*thide ad Glnbl 2007 r'
?i.t ro. .*au"i"g susars: Planl exo-acr *a! agruting ro
distiled water. filtered and simmered with some drops
oi tl" f"nf;ngt soluiioD A and B for some time-
i*.*."t "r ui o*.e. ^ seu a" red rype preciprmre
iJ.r" ,1. i".ia*.i oi reducing su-qa's rEgwail-hide
dd Gr[lba.2007).Telt tor sapodrs: The pLai! exo-act in 02 g $as
i*,-i.ul".iur* *;,r, s m.L ofdrsLilled uarer and boiled
.ippearoce of bubble is e\idenr of saponins tEgwaLkhjde
and Gimba. 2007).i*t ro. n."onoia", pt*t sample extmct in 0 2 g quadq
was mi{ed in diluted sodiM hvdroxide and after which HCI
Ms DouinE inro ir. Preseoce of )ellow solunon atrd drm
tminp rnri colorles< Mlnin lew Dinures sbosed rbe
ocorirrce of 0avonoid. (Eg\t dihide and Girr ba 2007)'
Test for Dhlobaudns: An amounl of 0'5 g e\racr was
lmuefi€d h rcme distilled s€Ier and then 6lteted'
sutseouenrlr jr was bealed wift 200 bvdrocbloric acid
.a*i*. rU. emersence of red precipiutioD e\pored fie
occurcnce ofpl obatanins (Eewaiklide and cimbq 2007)
Test for steroids: About 0 5 g plal extract was added with
2 ml- acetic arnydride along wrth 2 mL of HrSOa Th€
Edsfomation of colo! fioE violet to glee or blue showed
lne occurrence ofsteroids (Eewaikhid€ and Gimba,2007)''Ierr for leroenoids (ralkoslki t€sl): The planl exD:act
rakeo ir 0.2 e wa" mired wil}l 2 rnt chloroform and Lheo 3
mL concenEated sulphuric acid (i{rsoa) An appearance ofreddish broim color was iDdicated the occurerce of€rDenords. 'I he perenuge ofcrude e\trac6 vield %) was
a.imined bv rhe tonnula siven below (Deuaralle et a1"
2011):
Yiel.I - Wershr o I lvophiLzed ermct r 100
weight ofdried flo\i€r
Antioxidant Bioassay (D?PE Radical-scavenging
ActiYity)
The evalurion of DPPH (SigEa-Aldricb, German)a N{W'
394.32) quenching acrivity was canied out according to the
Drourcol. oefined by Yrldr-rm eI al (200J) wih a slighr
amendmeDL For *is purpose. iD mefianol lmM DPPH
radical soiution uas prepared. One IDe/mL of plani extsact
$as prepared l-o merlaDol Besides. 6 r I0']mol I DPPH iD
me*rdnol was also prepared. One mI quantiD was laken
ftom the said solurion that *as amalgamale with 3 tr[ test
solutions h me6mol laviog ditrqeni dilutions in rle 'ange
of (20, 40, 60, 80,100 [g) and Positive control (Asco$ic
Acid: Merck M.W. 176.13). AI the test saEple dilution
and posiiive control were kePt in a dark rooo Aftd l0 nin'al ordinary room temperature- The test samples 2nd stzn&rd
absorbance was rccorded at 517 nm spectlophotomeldcallyin triDlicate dd convened into the perce age antio da[tu"ti"ity *iog the fottowing perced ndical scaveoging
378
% DPPH (rq,$s op:cit!=
Phytoche@ical A@bsis ad Antioxiddt Properties of Teucriuhl sto'kiatu IInt J Astic Biol ' l/ol !5 No 2' )013
The suNdcal dalv{s *?s dohe ior oedns ud Stardnrd
i-i"t t. u.* (SEN0 ror utioKidad and Phmolic
acEviries bv usi.g $c following fo6uld
wlrcrq s : sanple sland{d dwiation ard n: siz€
(number ofsaoPles).
Assessmetrt of Totd Ph€nolic Content
Th..omoEarioo! of phenolic compounds Rere Deasted
roUo*ire the protcoL dc"eloPed b' L: e! dl (2007)' Tbe
air,i"o i.pr" i" o.so o,r was Dixed to 2'5 oL or I:10
Ii"i"a r".i, "r Acrl rcasenl Afrd r0 6rq 2 o! of6dr@ dboBte sMted solutioD (abod ?5 g'll) was
loaded to il After 2 hours ofintelval a! no.trlal temperatu€'
;e nixture sp6t'olhotoEenic absoltrece *?s rcco'ded at
'rro * fo, comparison wilh plet exFact Gallic acd a
-a"reoc" stand".t $a, ard aI lhe obBired resulls s'tre
shom as me ga[c acid equiqldt (Bg CArVsr wi dlv
wEight ofplad lested oateriaL
Results
Physicll ProPstie.
AI solvetrt ba6ed *racE poss.'ised bmwD color uth rone
dc of vuiatioor l?ngire ftom Blackisb to light bmu'o
.Tablc l). Mosr of rbese .xtracB wne s.Eisolid ald ooD'
sdck howEver rcsiroEs !o sricky aid povder/ 'hecter(
waa atso recoded fton petroleu etber, ethvl acetate ard
butaool, rEspectiv.lv. The bighesl weight of raw mataial
rl5o c) used for chlorcfoE\ qte! ud edvl acetate'
i"ir.*ii t, **t* .fier (r30 s,. acltooe '-hsane'
riio e *.il;,."'n-a rlr8 8) wherea tor erhan 'l ddi"*ir r"" ** mnterial (10 g eacn) uts used' with
relerence lo exE-acis qeighq n'bexae yielded hiebesl
*"i'l r:t nt. fouo*"a tihutuol (20 g) delhanol (19 3
nl. -*,.t lir er. aceon; (1a.5 sr' chlorcfom (ll5 g)'
i!rcreu ti:.i d ud elheol (r l a er' rbil' rcsr ori.r,*".*.""a r"^t vield. fte Petdrage vield oiextract *a5 ried rusing Aom 1 73 lo 39 08 Mth the
ma\dum value recorded 6'od n-hexane t2l'6"/r' It was
foltoweo bv buBnol t20.le0r. mefianol (161"6), 'ce6oe
ii:.0:s"1. *u* (ll3lo/"1. petroleum ether (9 a6"6) and
hlorcfor (gqo). lmBestilglv the ledt valLes Er€dctected tm ethyl acetate (1.73%)
Phytoctemicd Constituentr
ReruLB of oht'tocbemrcal s.ree rs of floweE of Isbch,ian,h'ut !rcvided ia Table 2. MaIEuo Plant
.miituetrts wrc dctected bv m€thanol, fotlow€d bv
butarol t9), edanol (,7), acemo.. shvl &eate (5 each)'
mEoleM eter. chlorciorEl (a each). $ater (3) and rileroe (2r Saoonis deteced bv aI solveds fo[owed bv
t:!.il! redeG ssd (erh 7). thrceids (6). t6?ooid{51. alkaloids. ;lbraquitrole (a eeh). wbeEas- st"oidsd;cEo bv 3 &d plnobaEmi! 6 *eU 6 tlltoside isolatcd
by 2 solvedts (Fi8. 1).
A.utioxidart Activit
Methanol sot@i detected atl slected Plet co.stit€un6 ed&@forE tzkm for andondart rrcPerties vdiou!corcatratioDJdilutions were @de ftoD the flolE e'Iha'tand used io assess etioxidart poPeitv DPPH vEs us€d fot
'EM -_
l50118
100l]0150
120
13.519J26tL4t2J2.674314.5lr,0
16.3
I t,4
1,7320312,08l].33
T5ble 1: Color, co,sisierxcy ed perceMge vi€ld of *kads Aom 1%riou soh€Drs
Table 2: Pbytochefiicat scleoiDg of I rrr.ktia,!, flower usils differdt solve[ts
-9!!
P
EP
i-
P
!g
PP
P
379
F]zhim et al. / lnt. J. Agtic. Biol.. t/al. 15, No. 2,2013
the evaluation ofantioxidant activity. Result ofthe assay is
expressed in Table 3 and Fig. 2. Th€ flo$'er extmct ofTeucium stack'idnum \|erc diluted in the range of 20 Fg to
l0O rrg 1() seek the optimum level of activity. The plant
extl€ct exhibited activiry in the range of 222.5810.001 to412.82+0.003 a120 pgto 100 llg concentntion respeclivel),.
while control had 213.33+0.001 to 440.54+0.001 at the
same concorlrations. Cornparing with control (Ascorbic
acid). flower extracl parallely exEessed good activiq'(Fig. 2). At the dose of 100 [g, the DPPH va]ue of flowerwas 4i2.82+0.003 that is near lo the satndad(440.54+0.001).
The result of antioxidant properties of Teucti nl
flower was due to the presence ofphenolic compound in lhe
plant. Table 4 revealed that phenolic compoMds were
present in plants sample about half of the quantjty ofstandard (gallic acid).
Discussion
Nine solvents were used for the evaluation of yield ofextacts, del€ction of ph)1o€hmicals and antioxidant act;vityof th. T. stoc*sianu fiower. The higlesl weight of extracl
was obtained by n-hexane (21 g), fouotred by butanol (20
s), methanol (19.3 g), water (17 g), acetone (14.5 g),
cl orofom (13.5 g), petoleum (12.3 g) ard ethanol (11.4
g). On the o0ler hand, high percentage yield of extmct ahorecorded from n-hexane (21.6%), followed by buiznol(20.37"). methanol (16.3%), aceione (12.08%), water(l1.33%), peboleum e$et (9.46%.) and chloroform (9%).
The etbyl acetate yielded the leasi percentage yield
0.73%).Tlis stud/ discovered varjous important
ph)tochemicais m T. stocl<iidru flower like alkaloids,
tannin, saponins, anthmquinone, steroi4 phlobatannin,
terpenoid, flavonoids, glycoside and reducing sugar (Table
I ). These chemjcals were detected variously by the selected
solvents. The presenc€ of alkaloids flower ex.racts ofTdcrium stock\imum makes them recommendable forpatient as alkaloids posses a significant phamacologicalprop€rry. This study reported presence of alkaloid in the
plant and endorse the findings of Awoyin-ka er dl (2007)
who detect€d alkaloid in C,id.solus aconitifutius.
With referenc€ to efficacy of individllal solvents forthe detection of ph)4ochnicals, methanol was the most
efficient one that detected all phltochenicals (Table 2) Ilwas followed by butanol (9), ethanol (7), acetone, ethyl
ace6e (5 each), petroleun ether, chlorofon4 (4 each),
water (3) and n-hexane (2). With reference to chemicah,
saponins detected by aU solve s, followed by tannin,
reducing sugar (each 7), flavonojds (6), terpenoid (s),
alkaloids, anthraquinone (4 each), whereas, steroids detected
by 3 and phlobataff n as well as glycoside holated by 2solvenrs (Fig. 1).
For analyzing optimun a ioxidant activity, the
flower extract was dilut€d in various concentation.
Table 3: Antioxidant property of fercl,rn flowet extract
?ri r*u,,,r,,g] escod,ctid Fl.*er
250 8711) 001
41282+0003
Table 4: Total phenolic compound obtained from pla as
compared silh the standard
6080 {2 3t 0001
T.tc|iun s1@kt hthldtd l 142 0850100 2 74
\ 162t90
[ig, 1: Detection of selecGd ph]to-constiteunts by nine
solvents from Terclir flower
Fiq. 2: Antioxidant Eopenies of flower o(tract by DPPH
*
380
Phltocledicat Analysis dd Altioxidant P.oPcnies of Teuctiuh stockianu lInt J.,a$ric BioL. yol. 15.No'2 2013
The plMi €xe.at exhibiled pamrlely g@d activiry wi& the
$andard (Fig. 2). At 100 Fg, L\e DPPH value of flower
was 412.82 that i5 ned to 6e contol (440.54). Resutts
re\ealed IIdr oowr exnac:s af Teuc'1ud sto.kiatunconiain€d flavonoid" These comPomds are \rater soluble
ddoxidats a wel d ft.c Edica] scaveneen responsjble
for oIfveoDne otidarjve cell damse ud posses uncancerpro;mies Md Educc the p,ocets of ceinogmdis (Oke:OOzt. t}r" prcs*r stud) er.bib,rcd Lhar stsoids ale
Eequeody lrestu! ir rhe floqq e\EcL! of f.,.ausroitu,ar- Tts .oopouds dhece ssr ed a,e ofsear impoflalce in Pbmcy (Oklq 2001). Ow resdl!ir" i, ,t" ti,. ot e$lLs b) IDd€he .r d/. (2009) ubo
su.€ned A- hispida Nd -1. .acenow and Elon &e
Fesence of pblobatzmh h these planls- The Eicrcbial infectios c& @u!. dtoive drmge
io cells dd tissues (KoUd .t a/., 2006). The detected
comDounds (Phedol) Aom pldt ex8cr exlxbired tudked
mdical stavoeilg acdury agai!$ tbe 'able DPPH free
ndical Clable 4). The activity of the phenolic compoud
,"! abnost ha]f of the Galic acid Althoueh the crude
exfact of this piet show€d a rc1ativelv low acliviry
comDared to lhf,L oi reL"rmce @rerial hokvr. L\is @vei&n be due to low coDcfftratioo of lhis coDlousd 6ed iD
the extact or io u olposed effect with other
Dhltocb@icals of tle drEct' ' It can be coocluded l,\dt difrerEo( solEls extr&Ls ofTeucriu stoc|sian$ flaw{ Posess suficient a6o!41 ofohuochemicaL such a5 flavono,ds. saporu$ and Phmolic
---r"a' rUr" phro-coEstiuenls demoEsE"te &ee
6d;al scavmsjig dd hiei antioxidar acrivities. The D
vi,ro dsay rcv$led 6at oow* cxtrace can b€ lsed as a
natual dtiovjd@t that Eav hclp in Pr€vdting ondative
<l]6. Thjs is prclimilarv studv ilat €quiB a delailed
mdv on bacd-outioo codPotrmls csPolsible for ficc
adical sca"mgug dd edoti.tafl aciiviry. Tb6, oorc
sudy is reqrred !o segregae and to kmw aboui Lhe
uootidaar compouas rcmalv PlEsenl i.o lhe Plarextracl Otr the other !an4 m v,,o antioxidant activity
should also be evaluted lrior to its clioical Ese Thes
resdts also odoised the ethnoboonical use of thi' plant
froE the cotlecled lerrilorv due rc pres€ace of vdious
Acloowledgemefts
Th. filst two autho6 arE indEbted to Higha Educatiotr
CoE@jssio!, Pakistao for tusdins the project uddtr IIEC
Iddise&us 5OO0 PhD. f.Iowship Pmgrala Baich'\'l
RefereBces
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Alimolade, C.. EO. Ibuhn\ EM. Obuob &d EO Fmtbi. ?007.
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K:6hq 2ooo. rph"{l46ide Ehlbibry mt}Eoh'k;[email protected] a<, 6-om th. ffi ttr ot Hh"Ena'[email protected] NaL Pba
' 6q:723'2l\
ri ll8.. K*. ch6a C C. woa Kw Fh F. cha md Y &n& 200_.
EEluum of_rdidar' dDarrv fd 6El phdolic mht oldfiiat n&d6 ofdor.d dicMlgae Food Cnem,102'111--116
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;l M d.FdEr l/,r.,im rp6i6 eil,bL i! hoia P'r'd'Pr
\ad.CS.K.S V.Al-FsiandS.A rt'{Rt?a . O.mpl6r Call'tua'drqn"e lrndFaraee d M.dnnol Pt@t Sp*i4 i \hah Onah'
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R dh.lar,l]@ L. V.N.M Zr*a!i4 M w. ls@ M. KmiL A Lsma L Kalhm a- al.Afu 2OOI. aEls6'c ad uE inn@rort &ti"iD6ot fa@i'n no.ktia"M. Phm Blol. 19:45!459
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va}l; s.P.. Mc MM! K PBmla R soqJ md B.A Rilq ?ol2
s]:r!a..e or AnMmbial sd Arhorddr Poratia! of 'l'd'i?Md@,
-Di!@i!t ,?dtos o tA B,imM tantu nas
M&.deln rld &lndii J Pr, tu R.t.. s' tl jLt-38Y dnfr A-. A Mau ed A,A f'.ra,2ool. AnLNdd md atihimbitl
d'idd of Pal!@nu- eE @ Mds .ec' J S<r tood
181
(Rdir.d 19 Srprenber 2Ot2; A.cePt d t, Nov.nn.' ?012)
,oLmalo' Med.cnal o'anG R'sea!- vol 6/5),Po 7O4aO1,oFea\a1 2412
av. ieb e ollir at http"r$/w.acaoemEloJnals.o_g/J[4oRDOr.'0.5897/JMPR1 1.1001
rsSN 1 996{875 O201 2 Academic JoLlrnals
Full Length Research PaPer
Preliminary phytochemical screening and
ethnomedicinal uses of Teucrium stocksianum lromMalakand Division
Gul Rahiml, Rahmatullah Qureshil*, Muhammad Gulfrazl, M Arshadl and Sahib Rahim2
!nan,dman, .r Botanv Prr Meh- A.i Sl'ai Ario Aqr'cu'tJre UriveFity MLaee Road Rawalpi'rdi' Pakrs'a''- -'-'olprnnr".t
"t 6"''o1rr€ 1Ial Sc ences, lnlernatlo'dl sla'r'ic U.1i!e-siry' lslamabad Pakisla'r'
Accept€d 22 N.vember, 2011
6t rcocium stocksanum were screen€d for secondary metabolites by using methaool'
:iiili;;; .-h",,"e sorvenrs. rhe methanoric exrracts save positive resurts ror arkaroids. tannins,
ir,,"ioias. sapo.ins, steroid, reducins sugar, rerpenoid, anthraquinone. phtobatannin and glycoside.ln
;1';;;-;i ;n;..i;;, atiatoios, tinnins, reducins susar, saPonins rravonoids' terpenoid were
Ill.".i "J ,.it'-..l"one. qlvcoside, phlobatannin, steroid were found to be absent' while n' hexane
:[#.;;-;iil i;;"i,", i"t*i,s !i,g*' flavonoids and failed to detect arkaloids' anihraquinone'Illl-^.i" -.ii"t,t,".1.
and lerD€n;id. This paper hishlights the significance of plsnt in traditional
iilii.]r" "ia Ji",.ir*i.n ofvari;us chemical consrituents with respect to Malakand Division. Pakistan.
Keywords: feucrium slockslanrm, secondary metabolites, iraditional medicine' phltochemicalconstituenls'
INTRODUCTION
For the existence of lile on the eadh, planls played very
imoonance rote frorn tfe t'me 'm-remoral Hunan being
i.'Ji"atv ,"raveo ol planls to fu'fill their daiy i'e
...r;r..6. ri"v a'e Lsed as iooo' 1-el, o'namenlals'n,ior and neoicine. TnroughoLl lhe wodd higrer plarls
aie oerng enployed to treal va'ious inlectious d seases'
in.v or-or:oe ;umber o' natural prodLcts inclLd rgmeicaments to fiqht againsi drseases'io ma'ntan qu;ity oI d'ug, lhere is reed lo evalLale
acti;e consliluenls of raw material to ascedain thelr
tr.-.iaoeur c eftecrs. tt erables the phamacist to orescr be
..-Jrlca vatues and easure uniforriry of slandards(Arnen, 1996). Medical plant can be derned as any pai;ithe Dlant whicF contairs sLbstances tnat can be Lsed
ior theraper:trc purposes and iis orecursor for the
*nrnesrs oi usefu drugs. Thsy corlain nutr'erts and
o'r,rro"rern.*t lhat can hear ai'nenls o' the body
iT/;se ard Evans, 197s) P.anI maie'rai tnal has cellLLd'
-o-str.oi,--i@ +-;;;=;-,aii"-',ahmatul ahq@ uaar.edu Pk.
structurc is referred.as organized drug in pharmacy'whereas, non-cellular structure as unorganized or a
cellular drug (Akinpelu and Onakoya, 2006).lvledicinal and aromauc planis (l\rAP) are a large group
of economically impoi(ant Planis ihat provide rawmaienals for pharmaceuticals, Perfumery, flavor and
cosmetic industries. Teucriun stocksianum Boiss-(Lamiaceae) locally known as Speer bolay is a perennial'
woodv, aron'allc herb whicl' 's nalive lo the mountainoLSreoio;s of Ihe lJniled Arab Em'rales (UAE), norilernO;ar {Wesle.n 1989. Nadar et al., 2OO3), Pakistan(Al-'rad et a'., 2oa4 and van (Mo)ao et al. 20ol). Thrs
o.anr ahairs a heignl of 10 to 25 cm havrng densely;acked st€m and qrev-qreen leaves. Tne leaves andyoung shoois of I slockslanum arc commonly used forihe preparation ol traditional medicines to treat severalailments including diabetes, gastro_intesiinal ailmentsand inflammatory condliions (Radhakrishnan et al.,
2001r. l1 addilon to fe gastro prolective e'ect.o'..od,on of T. stac(stanum nas beer Jsed 'or lhetreatment of diabetes and buming ieet syndrome(Barkalullah and Hussain, 2009).
Secondary meiabolites are chemicals produced
745
tSroJqh seconodry reaclrors resJllrrg i o pr ma-y
l-.""i'a,.r.s. ani'. acds ard lip'ds (Tirq, 1982)' Ineir
L","li ii,""lio" " p'""t
"etabohs'r rs not well recosnized
;;i;;i;. ii*";,. rieir -ore r ecosvsrems (Dev and
i.,.-.".i* rgszi, pan;"utarlv in pla'rt helbrvore i.1teractiol
1..--1"'" .qzs, S*ii". a979iaid chemora(ono-y (Gibbs.
l'ir-"".,. u""" weh known. Pra'rts conlail seco'dary
-'"til"i't". *"n as ar\aloids, saoonins ard tannins 3_e
".].JLrr, ,roio"o bv grazing arrmals a"d lea'feeding
i"L..i ir,"i, 0,"."i"" ," plrns a.rd intal'e al Lign reveL
l:::;. ;; ;uinenl ur. zat,or feed efrc'e-cv' a'riTal
.il;;i,,ty "* '" *." "ases mav caLse dearh (Mdkka'
,nd Goodchrld, 1996)'''r"i "rt." "' L sioc*s;anum nas beer foJnd io have
".i",r*r*""'" ard cyloprotedive p'ooedies when
I"orlo o"-"*p""."nt"lti-induced qasrrc lesio.rs (lslam
f,i'J.ioozt. ;r add,t,on. rr'e pra,rl exYact las also beer
ll,ii,.a tJ, aralsesic ard anrrnflarrato"/ acrv'ties'
il"i"ii ""oo"o r; tradidora use or lhe planr :1 iLre
i..rt."r'r ' "r pa 'lfLl irflammatory condiliols
ie'lJnar.risrnan et at.. zoo ). T\s olant's also 'rsed as a
il'^^.i "',;i., a renedv for rhe treatmert of hvoene'sion
].? ",Ii"p"l tan."o ,it
"'., 2002) and sore lh-oar (lqbal
and Hamayrn 2004)'" ii. ".oire
o 'lutit
and division l"ave good krowledqe
,oori ir'tii pru,l fo' hs 'ned;cina use' Based on rhis
i.li*nou" ino*teoqe ot T- stocksianum p'esent siudv
*iil""i"o our ro iralrze pnvloctremrcals :n order to
see( scienhfic logrc lortheir use in folk'nedic'ne'
MATERIALS AND MEIHOOS
Cotleclion and id€nti,ication ol plant materlal'
rleaves. stem r@1, a"d fo'al head) ol I-.^--,li'-,,- l-..irt"aeo in t'e rond'or Mav lo June 20 1 rrctr'rI.i"r"a.*. + rr.*t tor'ower) Me.€40 oivso' Khvber
L,irin,i xr*a rxpxr, pa<.srar o'e ser ol speciFe-s (No 2211)
*I. i,a" *rio c;nLertol.r merod d o@55in9' dr)'ns and
::: -l:: ^" .'";",. nelbdium sreer The p'a-r was desclbedllf i"i,n"a r, o,. nar."rurrah ourosFi 'n
-axo'oFv Lab' bv
l-1h1-"i -ir.,. ol Pak6lan (Hedse' 19so)' rhe denr''ied
--*,'li,jj."** o"-"r* " tt€ depan;e1r or Bolarv Pr MeFr alr
i-nl'" irit &,1,.'* u",*""v. +@rindr rb' 'unle- record'
Prcp.Etion .l th6 .tt€cts
Thp nlantma erialwas hasred ftorcughy 2 kr ! n-es wii_ rulnrrg
;.i;; "*" wn sronlg d'str;d Mter imned ai6rv aler
::i:;;;; *^"," oin !rc, ro r1e orvns Prc@s' 'nd sr-ade
::;;-n.; *. he1 rnerv po&6r6 (80 resr) usre a
il.:^l-^^, "+";" hi, ..d s6,ed in rer-iqe,ator rhe Powderediitill i,'" *"* .. .an"1or chroo'or a'd
'-herane (1 r0)
ii,i.ii.- ro, z" r' "r
szc. rFe esads he-e lhe' I rered us:n!
ii^,i."i nr", p"p", ro. 1 -he fihrareq or trera'o ' ch o-o'om::- --;".,". ** combined ano .o^certaeo under redLced
I)il.,i" "i noc uenq rcrarv erapo*ror' Bacrish e''ads or
''i!i,1]:Jr,- "r,,";"'.-- ano --hera'e were oora'ed ror
phlochemie! scEening'
Phytochenical screening
chemical tests were caFied oui in metha'ol, chlorcfom a'd n_
;;;;;;""" or ri sbck',anLd Ls'.e sr.-oard proedures ro
o"",iti tn" i"*ur.*" r, "esc'beo
bv Soro@E (1991) Trcase
and Eians {1978)aid Hadohe (1973).
Abour o 2 o o' tle eir-ects @s wamed oirh 2% F7SO. for t'{o
--*i 1i. ntra" "* added wlh 'ew d'ops or D6qe'dolfs,.i.."t r."rr',""
"r o"'"t"u- bismuF
'odioel' Preselce ol orarq€
€d;ec,'pnate nd caled as Positrveness fora kaloids'
Lhi e a,nount oi enract was mixed wilh water and hoaled .n waler
o"in-, ri "
.r.t,. ** I t","a and le c chbnde was added to hefiliGie. The da steen soluiion indi=ted the presence orlanrins'
feslfo. aolhnquin.nes
a.ou! 0.5 o 6nEd was oo,ed with 1006 HCI for 'ew
m'nulec ir*r"i u.in.'rr" rr'o" *.
"llowed ro cool and equal volure a'
chloroiom was added.it Ms added with i@ drcps ol 1o% NH and lhe minu'e was
heated. The appearance of mse'pirk color indicatBd lhe presenc€
The extract was LYdrclr2eo ado:ng wm Hcl ard rcJtra':ed siinN,oF soulion. An;se fM orops or F€rn.gs sourion A a1n B
*.* "oo"o.
ri'" ocdtrence of .ed pecipilaio sho@d lhepresence otglycosides
r.st for r..lu.ing suga6
Tnp Erfi-r.l Ms shaken wt- disn'ed wal€r llrered a'o Doileo slhdrc;s or Fe\'nqi sol"ton A and B fo- oilres' Snw'1s or an
o6_se rcd pr€'pllal€'no caleo re occurerce ofredJciag sugars'
Abolt o 2 o of Ee enraci Ms shaken w'lh 5 rlo'd:stled Mter
".i *i. ri.r"a ro m'. rrothha {appearance or crearv mi$ or
small blbblea)shows Lhe Presence or saponlns.
The 02 o erhacl Ms dissolved i dilJred NaO! ard PCI was
,dd{ A €rlow solLfio. thai tLmeo co.dess wlhin lew oinut€s
i.dicaled tie presence of flavo.oids.
fesl fot phlobatanias
The 0.5 o extract was dissolved in d slilled water and lillerEd h was
r,"ii.a .-tr, Z"r Hq solllron The appea€nce of red precipitatio'
evealed lle preserce ol ol'obatal1s.
706
Table 1. Phyt@hemlcal screning of T. st@ksiaron usinO different solvenls.
, mr ,cerc anhvd ide was adoeo to 0.5 g p al erEcl alo'g wrr 2
ii ot rtso.. rr" cna-se or colq iiom voer lo brJe o'sree_
i^dicried the pesence of sleroids
tesr l te.,enoids (s.lkowski tesq
rh' 0 2 o Dla.l en?d w6 rred w n 2 d' chorolorr ard 3 rl."i"iir.ir& .t,so.. r"" to,ruron or redo'sh bryn Golo' lo the
I"i..riiJ-*i ti""",t a'"r"steo posir:ve sen ror reroplo'ds'
,-i;--^^',...mmun [es or Ma akeno DNc'or @re :nlPfriewed to
l-l'i^"-*"i ."*" a.d ..ecdota uses ot r' socksianud
L]r"*ni: t'e me0,oOotoqv ot oureshi and Bhafl i {2008)
RESULTS
The rcsu'Is of pl')4ochemical screering of I sl'cks'ianum
^.,.,"r"" -.ttrnot. cn oroform and '-hexane
sovents are
ii-."ir." ,. Tabe 1. The melhaqolic extracls
Iil;lir"t po"iri," for the presence of all screened
.i""ir,".i*t" such as alkaloids, lanrrns ravonods'
Irloi"., "".ia, reducing sJgar, leryenoid'
;.!h-laoll1.-", pnlooatarnn and glycosde T']e cl orG
iorm ertacts detecreo attaloios tannins, reducing sugal
er.onns, flavonoids, leQeno,d except arttraqu'nore'ii,i"o";0., p.tourt"nnir. steroid' similarlv' n- Fexane
iiir"i tl'"Lo tanntns. reducirg sugar' ravoro'ds ard
i"lLJ io a"t"a a'kaloids, anlhraqLinore grvcoside'
phlobatannin and terpenoid.
Ethnobotanical use
The Deoo.e ol Malakard D v:so' nave good knowledge
,.Jr[1ri.,rm for its ned'ciral Lrse' Ehnobotanical'v'
irl-lril" oi r "roc,t'ran,,,,r,
is sive' for lhe tleahent or
iaundice and as olooo pun'ier as well as cooling agent
ile oecoclio'1 o' prart is also prescrbed to Treal cl^-o''cfever. Leaves soiked in water ovemight and lhe iuice is.iven before breakfast lo diarrhea and abdominal pain'
Voung rea,.s are boiled in water and obtained juice is
used forcuring cough-
D IS C USSIO N
The oresent research wo-k reveals lhat there are various
inoo.ranl Dhuochemicals such as alxalords, 1annn,
saoonins. ;nthraqurnore, slero'd, phlobatarnin'
terDeno,d, flavonoids q ycoside ard reouong sugar we'euesenr in f stocks;num Clable 1). Besides, tannn'saoonins. favonoios and _educi']g sugar we'e unrfo_mlv
deieoed bv al solverG (rigure 1). The chemrcal
constrluerti e'dracted bv vanous solvenls were i'1 ihe
order of nrethanol> chloroform> n-hexane'It is an established fact that secondary metaboliles
oresent in ola,)ls der've therapeulic aclivities {Rabe'?nonl pariicLta v. a.<ato:ds a.e reported il.e most
inrDo;anl lheraoeutic siqn'ficanl sJbstance (N,okJ and
akumetuta- 2007). The presenl study stratified ihat
alkaloids were deiected by metl'ano and chlo'ofon1
ioverrs tnat mav signify'or'u1her iryest:gat'on frortrrese sofvent Uase exl'acts. Tann'ns erd flavorods are
also reported to possess biological activities which ars
;sed for tne Drelention alo maragement of many
diseases (Jan"es et al.. 2007). Our investigation from listocksia,rim reveals that tannins ard flavonoids are
iraced bv all so'vents and link loqical'y whh ethno_
."oi"in"i u." of lhts plant in tradiloral syslem of
m;dicine. 'lhe medicinal properties of the plant could be
ari.iouted to the o'esence of one or more of the delected
;ant natural o-douas These ,ind'ngs give credibiLity to
;e tredit,onar meo'onal releva.ce ol the plarl as
remeaies for abdominal pain, chroric fever, diarhea
brooo oLificat'on, laJrdce ard coJgr' Hodever' Ileplarracologica ,ctons of tre pa_t cannoi be
Rahim et al. 707
3
2.5
2
1.5
,1
0.5
0
ascertained by the resull of the phyiochemical analysisonlv. Results of this investigation offer a scientificfou;ctation for carrying out In vitro and rn vivo aclrvities. ln
conclusion, isolation and purification of the phllochemicalfollowed by a detailed study might resull in ideniificationlead compound for curing various diseases.
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