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Original Article

Influence of agro-climatic conditions on antioxidant potentialof Mentha species

Bisma Malik, Neeta Raj Sharma, Giridhar Soni*

Department of Biochemistry, School of Biotechnology and Biosciences, Lovely Professional University, Phagwara 144806, India

a r t i c l e i n f o

Article history:

Received 30 January 2013

Accepted 24 May 2013

Available online 15 June 2013

Keywords:

Antioxidant potential

Flavonoids

Mint

Mentha longifolia

Mentha spicata

* Corresponding author. Tel.: þ91 9888483754E-mail address: giridhar.soni@lpu.co.in (G

0974-6943/$ e see front matter Copyright ªhttp://dx.doi.org/10.1016/j.jopr.2013.05.014

a b s t r a c t

Background: Mentha species are known to be important sources of antioxidants. The anti-

oxidant potential of different species may be affected when grown in different agro-

climatic conditions. So the effect of location on the antioxidant potential of two Mentha

species has been studied.

Methods: Mentha species (Mentha longifolia and Mentha spicata) were raised at two different

locations i.e. in Lovely Professional University (Punjab) and in Kashmir University (Srina-

gar). The first and second generation leaves of these Mentha species were plucked at after

one month interval from the plants and extracted with different solvents. The extracts

were analyzed for total phenols, flavonoids and antioxidant potential using various in vitro

models.

Results: M. spicata had a relatively higher total phenols, flavonoids and antioxidant prop-

erties than those of M. longifolia raised at either of the locations. M. spicata and M. longifolia

raised at higher altitude hadmuch higher content of total phenols and flavonoids as well as

antioxidant potential than the respective species raised in plains of Punjab during same

period. Moreover, the leaves of second generation had higher content of total phenols and

flavonoids but had lower antioxidant potential than that of first generation leaves.

Conclusion: Antioxidant potential of Mentha species raised at higher altitude was far better

than the corresponding species raised in plains. Moreover, first generation leaves had

better antioxidant potential. No direct correlation between total phenols, flavonoid content

and antioxidant potential of these species could be established.

Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights

reserved.

1. Introduction (SOD), catalase (CAT), glutathione peroxidase (GPx), gluta-

In biological systems, the reactive oxygen species (ROS) form

naturally during many metabolic processes. Cells have

developed several protective mechanisms to prevent ROS

formation or detoxify ROS. These protective mechanisms

include antioxidative enzymes such as superoxide dismutase

(mobile).. Soni).2013, JPR Solutions; Publi

thione reductase (GR), glutathione-S-transferase (GST) and

non-enzymatic antioxidants that repair oxidative cellular

damage. A disturbance in the balance between ROS produc-

tion on one hand and ROS removal and repair of damaged

complex molecules on the other results in oxidative stress.1,2

Environmental pollutant chemicals, drugs and food

shed by Reed Elsevier India Pvt. Ltd. All rights reserved.

j o u rn a l o f p h a rma c y r e s e a r c h 7 ( 2 0 1 3 ) 4 2 7e4 3 2428

contaminants add to the oxidative stress making exogenous

need for antioxidants.3 Antioxidants are molecules that slow

or prevent the oxidation of othermolecules by scavenging free

radicals that play a major role in the pathogenesis of many of

age related diseases.3 Synthetic antioxidants can be incorpo-

rated as supplements but such an approach is never free from

side effects. Natural sources of antioxidants are safe and

acceptable. Antioxidants in foods have recently emerged as

biomolecules of utmost interest to human health. Dietary

antioxidants inactivate ROS, reduce oxidative damage, lead to

improved immune functions and reduced risk of infectious

diseases. Increasing intake of dietary antioxidants may help

maintain an adequate antioxidant status and therefore, the

normal physiological functions of living system.4,5

Mentha a genus of aromatic perennial herbs belonging to

the family Lamiaceae, distributedmostly in temperate and sub-

temperate regions of the world and find their use in Ayurveda

for treatment of number of ailments.6 Most of the commer-

cially important mints are hybrids or amphiploids. Mentha

spicata, and Mentha longifolia are amongst the most important

aromatic cultivatedworldwide as a source for essential oil and

other bioactive compound, The antioxidant, cytotoxic, and

anti-inflammatory activities of M. spicata have also been re-

ported in a number of studies.6 Today, the Labiate family is

considered as one of the most important sources for extrac-

tion of compounds with antioxidant activity.7,8 The medicinal

value of herbal plants may change with the agro-climatic

conditions. In the present study, an attempt has been made

to evaluate antioxidant potential of twoMentha species namely

M. longifolia and M. spicata raised at two different altitudes.

2. Material and methods

2.1. Plant material

The Mentha species viz. M. spicata and M. longifolia, selected for

present study were obtained from the Department of Botany,

University of Kashmir Srinagar. These Mentha species were

grown in poly bags both at Srinagar and at L.P.U during the

months of DecembereJanuary (10e14 �C) and at K.U Marche

April (13e15 �C) respectively.Fresh and healthy leaves ofM. spicata andM. longifoliawere

collected at one month interval and washed thoroughly in

distilled water and the surface water was removed by blotting

in the folds of filter paper. The leaves were subsequently

extracted with different solvents.

2.2. Preparation of extracts

One gram of leaves of M. spicata and M. longifolia was crushed

and transferred with 25ml of sterile distilled water, methanol,

chloroform, or hexane into stoppered vials and kept in vortex

shaker for 2 h and kept overnight in cold conditions. The

macerate was first filtered through double layered muslin

cloth and then centrifuged at 4000� g for 30 min. The super-

natant was preserved aseptically in the sample vials at 4 �Cuntil further use.

Before using, a known volume of organic solvent extract

wasmade free of solvent and re-dissolved in the same volume

of volume of water.

2.3. Total phenolics and flavonoid content

Total soluble phenolic content was estimated by

FolineCiocalteu reagent method8 using Gallic acid as a stan-

dard phenolic compound. The total soluble flavonoid content

was estimated by colorimetric method9 using rutin as a

standard flavonoid.

2.4. Antioxidant activity

2.4.1. Total reducing powerThe determination of reducing power of different extracts was

performed by the method as described by Yen and Duh.10 Fe

(III) reduction is often used as an indicator of electron

donating activity, which is an important mechanism of

phenolic antioxidant action. Total reducing power of extracts

was determined by determining the reduction of Fe (III).

2.5. DPPH radical scavenging activity

The free radical scavenging activity of the leaf extracts was

assayed using a stable free radical, 1,1-diphenyl-2-picryl

hydrazyl (DPPH). The DPPH scavenging assay employed in

the present studywas amodification of the procedure ofMoon

and Terao.11 DPPH is a stable nitrogen-centered free radical,

the color of which changes from violet to yellow upon

reduction by either the process of hydrogen- or electron-

donation. The percentage of DPPH scavenging activity was

calculated using the following formula:

% Scavenging ¼ ��Acontrol �

�Asample �Asample blank

��Acontrol

�� 100

2.6. Ferrous reducing antioxidant power assay (totalantioxidant activity assay)

A modified method of Benzie and Strain12 was employed.

FRAP assay is based on the ability of antioxidants to reduce

Fe3þ to Fe2þ. In the presence of 2,4,6-tri (2-pyridyl)-s-triazine

(TPTZ) Fe3þ forms an intense blue Fe3þeTPTZ complexwith an

absorption maximum at 593 nm.

2.7. Lipid peroxidation inhibitory activity

To evaluate the lipid peroxidation inhibitory activity of the

leaf extract ofMentha species, a liposomemodel was used. The

lipid peroxidation inhibitory activity of the leaf extracts was

determined according to the method of Duh & Yen.13 Extracts

were added to lecithin emulsion and then lipid peroxidation

was induced by ferrous chloride plus ascorbic acid system.

Melondialdehyde formed is reacted with thiobarbituric acid

and a colored florescent product is formed. Percentage radical

scavenging was calculated using the following formula:

% Inhibition ¼ ��Acontrol �

�Asample � Asample blank

��Acontrol

�� 100

j o u r n a l o f p h a rm a c y r e s e a r c h 7 ( 2 0 1 3 ) 4 2 7e4 3 2 429

2.8. Superoxide radical scavenging activity

The scavenging activity of the different extracts toward su-

peroxide anion radicals was measured by the method of

Nishimiki14 with slight modifications. The superoxide radical

generated from dissolved oxygen by PMSeNADH coupling

measured by their ability to reduce NBT. The decrease in

absorbance at 562 nm with the plant extracts indicated their

ability to quench superoxide radicals in the reaction mixture.

The % inhibition of superoxide anion generation was calcu-

lated using the following formula:

% Scavenging¼ ��Acontrol �

�Asample �Asample blank

��Acontrol

�� 100

3. Results and discussion

In this present study the antioxidant activity of various ex-

tracts of Mentha species have been investigated. Initial studies

revealed only aqueous and methanolic extracts exhibited

reasonable antioxidant activity, so the work was carried out

with these solvents. These extracts were assayed for their

total phenolic and flavonoid content and antioxidant activ-

ities by using different in vitro models.

3.1. Total phenolic and flavonoids

It is evident from the results (Table 1) that the leaves of

M. spicata had a higher content of total phenols and flavanoids

in plants raised at either of the altitudes as compared to

M. longifolia. The results also revealed that the total phenolic

and flavonoid content of both the species was higher in sec-

ond generation leaves as compared to the respective first

generation leaves of plants raised at either of the locations.

Moreover the total phenolics and flavonoid content of both

the species of Mentha raised at K.U Srinagar was much higher

than the corresponding species raised at L.P.U Phagwara.

3.2. Antioxidant activity

3.2.1. Total reducing powerFe (III) reduction is often used as an indicator of electron

donating activity, which is an important mechanism of

Table 1 e The total phenols and flavonoid content ofMentha spUniversity.

Total phenolics (mg/g)

Mentha spicata Mentha longifolia

First generation leaves

Extract L.P.U K.U L.P.U K.U

Aqueous 4.7 9.4 1.5 3.8

Methanol 2.0 6.0 1.9 3.4

Second generation leaves

Extract L.P.U K.U L.P.U K.U

Aqueous 5.5 10.0 1.6 4.1

Methanol 9.2 11.8 2.6 6.1

K.U: Kashmir University, Srinagar.

L.P.U: Lovely Professional University, Phagwara.

phenolic antioxidant action.15 Reducing power is associated

with its antioxidant activity and may serve as a significant

reflection of the antioxidant activity.16 Compounds with

reducing power indicate that they are electron donors and can

reduce the oxidized intermediates of lipid peroxidation pro-

cesses, so that they can act as primary and secondary anti-

oxidants.10,17 Their studies have indicated that the

antioxidant effect is related to the presence of reductones.10

Reductones are reported to be terminators of free radical

chain reactions,18 thus, the antioxidant activity of extracts

observed may be related to its reductive activity.

Total reducing power of different solvent extract is shown in

Table 2. The results that the total reducing power of M. spicata

was substantially higher in both the extracts at both the alti-

tudes as compared toM. longifolia. The results also revealed that

the total reducing power of first generation leaves of both the

specieswasmuch higher than second generation leaves except

M. longifolia which showed higher total reducing power in

aqueousextract raisedatK.U.Moreover theresultsalsorevealed

that the total reducingpowerofM. spicataandM. longifolia raised

at higher altitude i.e. at K.U. Srinagar was much higher in both

the extract than the same species raised at plains of Punjab.

Thus it appears that total reducing power of Mentha is greatly

affected by the soil and environmental conditions.

TotalantioxidantactivitywasalsodeterminedusingFerrous

reducing antioxidant power assay (FRAP assay) based on the

ability of antioxidant to reduce Fe3þ to Fe2þ in the presence of

2,4,6-tri-(2-pyridyl)-s-triazine (TPTZ). Fe3þ forms an intense

blue Fe3þeTPTZ complex has been utilized for the assessment

ofantioxidantactivity.Theabsorbancedecrease isproportional

to the antioxidant.12 The results of FRAP assay (Table 3)

strengthened the view that the antioxidant power of Mentha

species raised at K.U is higher at higher altitude. Moreover

M. spicata is a better source of antioxidants thanM. longifolia

3.3. The DPPH radical scavenging activity

The stable radical DPPH has been used widely for the deter-

mination of primary antioxidant activity.19,20 The DPPH anti-

oxidant assay is based on the ability of DPPH a stable free

radical, to decolorize in the presence of antioxidants.21 The

model of scavenging stable DPPH free radicals has been used

to evaluate the antioxidative activities in a relatively short

ecies raised at two different altitudes i.e. L.P.U and Kashmir

Total flavonoids (mg/g)

Mentha spicata Mentha longifolia

L.P.U K.U L.P.U K.U

1.2 2.3 0.3 3.2

2.6 2.7 1.6 2.4

L.P.U K.U L.P.U K.U

1.5 3.6 0.4 3.3

3.3 3.9 2.2 4.1

Table 4 e DPPH radical scavenging activity of Menthaspecies grown at two different altitudes i.e. within L.P.Uand Kashmir University.

%age DPPH scavenging activity of Mentha species

Mentha spicata Mentha longifolia

First generation leaves

Extract L.P.U K.U L.P.U K.U

Aqueous 30.9 49.3 14.9 32

Methanol 62 68 21 38

Second generation leaves

Extract L.P.U K.U L.P.U K.U

Aqueous 12.7 36.1 12.2 19.9

Methanol 33.8 42.6 20.5 26.7

Table 2 e The total reducing power of Mentha speciesgrown at two different altitudes i.e. within L.P.U andKashmir University.

Total reducing power of Mentha species (DA)

Mentha spicata Mentha longifolia

First generation leaves

Extract L.P.U K.U L.P.U K.U

Aqueous 0.248 0.937 0.042 0.418

Methanol 0.177 1.267 0.032 0.616

Second generation leaves

Extract L.P.U K.U L.P.U K.U

Aqueous 0.050 0.890 0.030 0.438

Methanol 0.109 0.990 0.026 0.545

j o u rn a l o f p h a rma c y r e s e a r c h 7 ( 2 0 1 3 ) 4 2 7e4 3 2430

time. Antioxidant activities of aromatic plants are mainly

attributed to the active compounds present in them. This can

be due to the high percentage ofmain constituents, but also to

the presence of other constituents in small quantities or to

synergy among them. The DPPH radical scavenging activity of

Mentha species leaf extract is presented in Table 4. Among the

extract tested, methanol extract had better scavenging activ-

ity when compared with aqueous extract. It is evident from

the result that the first and second generation leaves of

M. spicata had much higher DPPH radical scavenging activity

in both the extracts at both altitudes as compared to M. long-

ifolia. The results also revealed that the DPPH radical scav-

enging activity of both the species in both the extracts was

much higher in first generation leaves than second generation

leaves at either of the altitudes. The results also shows that

the DPPH radical scavenging activity of M. spicata and

M. longifolia raised at K.U in both the extracts wasmuch higher

than the same species raised at L.P.U.

3.4. Superoxide radical scavenging activity

The superoxide radical generated from dissolved oxygen by

PMSeNADH coupling was measured by their ability to reduce

NBT. Although superoxide anion is a weak oxidant, it gives

rise to generation of powerful and dangerous hydroxyl radi-

cals as well as singlet oxygen, both of which contribute to

oxidative stress.22 It is evident from the result (Table 5) that

both generation leaves of M. spicata had much higher

Table 3 e Total antioxidant activity (FRAP assay) ofMentha species grown at two different altitudes i.e. withinL.P.U and Kashmir University.

Total antioxidant activity of Mentha species (DA)

Mentha spicata Mentha longifolia

First generation leaves

Extract L.P.U K.U L.P.U K.U

Aqueous 1.179 1.225 0.352 0.461

Methanol 1.394 2.026 1.380 1.419

Second generation leaves

Extract L.P.U K.U L.P.U K.U

Aqueous 0.525 1.049 0.152 0.417

Methanol 0.355 1.227 0.211 0.872

scavenging activity in both the extracts at both altitudes as

compared to M. longifolia. The results also indicated that the

superoxide radical scavenging activity of both the species was

higher in first generation leaves than that of second genera-

tion leaves. The results also revealed that the superoxide

scavenging activity of M. spicata and M. longifolia raised at

higher altitude is higher than that raised in the plains.

3.5. Lipid peroxidation inhibitory activity

The antioxidative action of Mentha species leaf extract in the

liposome model is shown in Table 6. It is evident from the

result that the first and second generation leaves of M. spicata

hadmuch higher %age of lipid peroxidation inhibitory activity

in both the extracts at both altitudes as compared to M. long-

ifolia in both of the extracts at both altitudes. The inhibition of

lipid peroxidation can be attributed to the scavenging of hy-

droxyl radicals at the stage of initiation and termination of

peroxyl radicals6 by phenolics and flavonoids present in good

amount in these species. The results also indicate that the

percent inhibition of lipid peroxidation of both the species

was much higher in first generation leaves in both of the ex-

tracts at both locations as compared to second generation

leaves in both of the extract at both locations.

Thus the present study revealed thatM. spicatahas a higher

antioxidant activity than that of M. longifolia raised at either of

the altitudes. The results also revealed that the antioxidant

Table 5 e Superoxide radical scavenging activity ofMentha species grown at two different altitudes i.e. withinL.P.U and Kashmir University.

%age superoxide radical scavenging activityof Mentha species

Mentha spicata Mentha longifolia

First generation leaves

Extract L.P.U Kashmir L.P.U Kashmir

Aqueous 11.9 50.6 3.4 50

Methanol 16.6 41.2 12.6 30.1

Second generation leaves

Extract L.P.U Kashmir L.P.U Kashmir

Aqueous 9.2 38 6.4 24.3

Methanol 8.9 15.6 5.9 11.0

Table 6 e Lipid peroxidation inhibitory activity of Menthaspecies grown at two different locations i.e. within L.P.Uand Kashmir University.

%age lipid peroxidation inhibition activity ofMentha species

Mentha spicata Mentha longifolia

First generation leaves

Extract L.P.U K.U L.P.U K.U

Aqueous 50.3 72 13.3 25

Methanol 55.6 76 32.9 39

Second generation leaves

Extract L.P.U K.U L.P.U K.U

Aqueous 23.1 29.3 3.8 16.9

Methanol 34.6 39.9 26.4 30

j o u r n a l o f p h a rm a c y r e s e a r c h 7 ( 2 0 1 3 ) 4 2 7e4 3 2 431

activity of both the species was much higher in first genera-

tion leaves than in the second generation leaves at both alti-

tudes. The results also showed that the antioxidant activity of

M. spicata andM. longifolia raised at K.U had higher antioxidant

potential than the same species raised at L.P.U.

Medicinal plants are an important source of antioxidant.23

Polyphenols are the major plant compounds with antioxidant

activity. Typical phenolics that possess antioxidant activity are

known to bemainly phenolic acid and flavonoids.24 Flavonoids

have been shown to possess various biological properties

related to antioxidant activity.25,26 Flavonoids are very effective

scavengers of peroxyl radicals and they are also chelators of

metals and inhibit the Fenton and HabereWeiss reactions,

whichare important sources of oxygen free radicals.27 Fromthe

present studies it appears that there is variation inphenolic and

flavonoid content in both of the species raised at two different

altitudes and there is also variation within species raised at

same location. There is an increase in total phenol and flavo-

noid content in second generation leaves over that of first

generation leaves of both the species but the antioxidant

properties of second generation leaves of both the species is

lower than that of first generation leaves. Therefore it appears

that there is nodirect correlationbetween the total phenols and

flavonoids content and the antioxidant properties. Earlier work

has also indicated no direct correlation between the total phe-

nolics and antioxidant potential.28 Since M. spicata had rela-

tively better total phenol and flavonoid content as well as

antioxidant potential than M. longifolia, it can the species of

choice for preparation of drinks rich in antioxidants. Since

higher levels antioxidants were present in first generation

leaves it is very important to use only first generation leaves for

this purpose. As the antioxidant properties were better in spe-

cies grown inKashmir, it appears that the bioactive compounds

can be best isolated from M. spicata grown at high altitude.

Conflicts of interest

All authors have none to declare.

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