ANTI-OXIDANT AND FREE RADICAL SCAVENGING ACTIVITY OF ASHWAGANDHA (Withania somnifera L.) LEAVES

Abstract

Ayurvedic medicines have been continuously using Ashwagandha as one of the active ingredients for centuries due to its pleotropic effects namely anti‑inflammatory, immuno‑modulatory and antistress properties. The present investigation aims to evaluate the antioxidant and free radical scavenging activity of Ashwagandha leaves. To begin with, the various Ashwagandha leaf extracts were prepared in different solvents of varied polarity starting from non‑polar to polar in a sequential manner. The maximum yield of 13.7% was obtained in methanol and the same extracts was found to contained maximum total polyphenolic compounds (TPC, 43.93 μg GAE/mg extract).The methanolic extract of Ashwagandha leaf was found to be appreciably effective in scavenging DPPH radical (EC50=197.50 μg), metal chelation (EC50=76.09 μg), hydroxyl radical (EC50=790.63μg), super oxide radical(EC50=117.70 μg) and inhibition of lipid peroxidation (EC50=536.43 μg). Seven polyphenols viz. gallic acid (0.17 μg/g), chlorogenic acid (0.70 μg/g), caffic acid (0.57 μg/g), sinapic acid (1.60 μg/g), rutin hydrate (0.17 μg/g), quercetin‑3‑rhamnoside (1.61 μg/g) and quercetin (0.27 μg/g) were identified and quantified using Reverse Phase‑High Pressure Liquid Chromatography. In conclusion, the study suggests that Ashwagandha leaf extract is of great use for the preparation of antioxidant rich nutraceuticles.

Full text

Research Paper
ANTI-OXIDANT AND FREE RADICAL SCAVENGING ACTIVITY OF
ASHWAGANDHA (Withania somnifera L.) LEAVES
Ajay Pal
1
, Mukesh Kumar
2
, Vinod Saharan
3
and Bharat Bhushan
4
1
Department of Chemistry and Biochemistry,
2
Department of Genetics & Plant Breeding,
CCS HAU, Hisar, Haryana, India
3
Department of Molecular Biology and Biotechnology,
Maharana Pratap University of Agriculture and
Technology,Udaipur, Rajasthan, India
4
Central Institute of Post Harvest Engineering and Technology,
Abohar, Punjab, India.
Abstract
Ayurvedic medicines have been continuously using Ashwagandha as one of the
active ingredients for centuries due to its pleotropic effects namely
anti‑inflammatory, immuno‑modulatory and antistress properties. The present
investigation aims to evaluate the antioxidant and free radical scavenging
activity of Ashwagandha leaves. To begin with, the various Ashwagandha leaf
extracts were prepared in different solvents of varied polarity starting from
non‑polar to polar in a sequential manner. The maximum yield of 13.7% was
obtained in methanol and the same extracts was found to contained maximum
total polyphenolic compounds (TPC, 43.93 μg GAE/mg extract).The methanolic
extract of Ashwagandha leaf was found to be appreciably effective in scavenging
DPPH radical (EC50=197.50 μg), metal chelation (EC50=76.09 μg), hydroxyl
radical (EC50=790.63μg), super oxide radical(EC50=117.70 μg) and inhibition
of lipid peroxidation (EC50=536.43 μg). Seven polyphenols viz. gallic acid (0.17
μg/g), chlorogenic acid (0.70 μg/g), caffic acid (0.57 μg/g), sinapic acid (1.60
μg/g), rutin hydrate (0.17 μg/g), quercetin‑3‑rhamnoside (1.61 μg/g) and
quercetin (0.27 μg/g) were identified and quantified using Reverse Phase‑High
Pressure Liquid Chromatography. In conclusion, the study suggests that
Ashwagandha leaf extract is of great use for the preparation of antioxidant rich
nutraceuticles.
Key words: Ashwagandha, Antioxidant, ROS, Polyphenolics, Oxidative stress.
INTRODUCTION
Oxidative stress plays an important role in the pathogenesis of ageing, inflammation and cancer
[1]. Free radicals such as superoxide, hydroxyl radical (OH
·
), nitric oxide, hydrogen peroxide, etc
collectively known as reactive oxygen species (ROS) are generated through oxidative stress and
have been implicated in etiology of various age related diseases such as atherosclerosis, asthma,
stroke, vasospasms, liver damage and Alzheimer’s disease etc [2]. Exposure to ionizing
radiations also generates ROS, which have been identified as important chemical mediators that
regulate signal transduction. These free radicals also affect mitochondrial membrane potential
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(MMP) and thus causes apoptosis [3]. The growing number of evidences indicates that these
ROS are also responsible for exercise‑induced protein oxidation and contribute significantly to
muscle and mental fatigue. It has been postulated that treatments that reverse these ROS
generated injuries may be acting through mechanisms that scavenge these species [4] .
The Indian medicinal plant Withania somnifera (L) Dunal (family‑ solanaceae), commonly
known as Ashwagandha, is widely used in herbal medicine for stress, arthritis, inflammations,
conjunctivitis and tuberculosis.[5‑9] The active principles of Ashwagandha, consisting of
sitoindisides VII‑X and withaferin‑A have been shown to exhibit significant antistress and
antioxidant effect in rat brain frontal cortex and striatum[10]. Keeping in view of its beneficial
antioxidative properties and worldwide consumption present study was conducted to evaluate
the antioxidant properties of Ashwagandha leaf based on its ability to scavenge various free
radicals.
MATERIALS AND METHODS
Preparation of sample extract
Leaves of Ashwagandha were shade dried, powdered and the antioxidant compounds were
extracted by adding solvents namely hexane (H), chloroform (C), ethyl acetate (EA), acetone (A),
methanol (M) and water (W) in a sequential manner in increasing order of their polarity. After
filtering through folded Whatman No. 1 filter paper, the filtrates in different solvents were
recovered and this process was repeated thrice with each solvent. Then, the respective solvents
from the filtrates were evaporated in a vacuum rotary evaporator to obtain the yield of different
extract. For checking the antioxidant activity, each extract/fraction was dried and re‑dissolved
in dimethylsulfoxide (DMSO).
Determination of total phenolic content (TPC) and total flavonoids (TF)
The TPC of different extracts was determined by the method of Folin‑Ciocalteu using gallic acid
as the standard [11]. A calibration curve was made for gallic acid and the results were
determined from the regression equation of this calibration curve, which was expressed as
gallic acid equivalent (GAE) in µg/mg extract. In brief, to 3 ml of appropriately diluted extract
was added 0.5 ml of (50%) Folin‑Ciocalteu reagent, followed by incubation at room temperature
(10 min) and addition of 2 ml of 7% Na
2
CO
3
solution. The mixture was boiled for 1 min, cooled
and the absorbance was measured at 650 nm.
The determination of TF was carried out according to the method of Delcour and Varebeke [12].
One ml of appropriately diluted extract was mixed with 5 ml of chromogen reagent (0.1%
cinnamaldehyde solution prepared in a cooled mixture of 25 ml of concentrated HCl and 75 ml
of methanol). After an incubation of 10 min, the absorbance was measured at 640 nm. The TF
content was expressed as catechin equivalents (CE) in µg/mg extract.
DPPH radical scavenging assay
All the extracts were evaluated in terms of their hydrogen‑donating or radical‑scavenging
ability using the stable radical, DPPH
*
, following the method of Blois [13] with slight
modifications. Briefly, the reaction mixture contained 3 ml of appropriately diluted extract in
methanol and 0.5 ml of 500 µM methanolic solution of DPPH
*
. The reaction mixture was allowed
to stand in dark at room temperature for 45 min and absorbance was recorded at 515 nm
against the methanol blank. A control was taken without plant extracts under identical
conditions. The percent free radical scavenging capacity (%RSC) of the extracts was calculated
from control and IC
50
from linear regression analysis. BHA was used as a standard antioxidant.
Metal chelation assay
The chelating effect of various extracts on ferrous ions was determined according to the method
of Dinis et al.[14] with some modifications. To appropriately diluted extract (2 ml) was mixed
0.05 ml of 2 mM FeCl
2
followed by addition of 0.2 ml of 2 mM ferrozine. The mixture was left to
react at room temperature for 10 min before determining the absorbance of the mixture at 562
nm. The inhibition percentage of ferrozine–Fe
2+
complex formation was calculated from control
without sample under similar conditions. The results were expressed as IC
50
calculated from
linear regression analysis. EDTA was used as a standard antioxidant.

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Hydroxyl radical scavenging assay
The assay was performed following the method of Halliwell et al.[15] with slight modifications.
The reaction mixture contained 0.1 ml of 28 mM 2‑deoxyribose, 1 ml solution of various
concentrations of extracts, 0.2 ml of mixture solution containing equal amount of 200 µM FeCl
3
and 1.04 mM EDTA, 0.1 ml of H
2
O
2
(1.0 mM) and 0.1 ml ascorbic acid (1.0 mM). The mixture was
then incubated at 37
o
C for 1 h and boiled for 20 min with 1 ml of 1% TBA in 50 mM NaOH and 1
ml of 5% trichloroacetic acid in a water bath. After cooling, absorbance of the mixture was
measured at 532 nm. The % inhibition was calculated from control without sample under
similar conditions. IC
50
was calculated from linear regression analysis
.
Gallic acid was used as a
standard antioxidant.
Superoxide anion radical scavenging assay
The measurement of superoxide anion scavenging activity of different extracts was based on the
modified method of Liu et al.[16]. The method is based on the generation of superoxide radicals
in PMS–NADH system by oxidation of NADH and subsequent assay of reduced NBT. In this
experiment, the superoxide radicals were generated in 3ml of 16 mM Tris–HCl buffer (pH 8.0)
containing 1ml of 150 μM NBT solution, 1ml of 468 μM NADH solution and the sample/extract.
The reaction was started by adding 1ml of 60 μM PMS solution to the mixture. The reaction
mixture was incubated at 25
o
C for 5 min and the absorbance at 560 nm was measured.
Decreased absorbance of the reaction mixture indicates increased superoxide anion scavenging
activity. The inhibition percentage of superoxide anion generation was calculated using control
without sample under similar conditions. IC
50
were calculated using regression analysis. L‑
ascorbic acid was used as the standard.
Lipid peroxidation inhibition activity
A modified thiobarbituric acid reactive species (TBARS) assay [17] was used to measure the
lipid peroxide formed using egg yolk homogenates as lipid‑rich media [18]. Briefly, 0.5 ml of
10% v/v egg homogenate and 0.1 ml extract were taken in a test tube and made up to 1 ml with
distilled water. Then, 0.05 ml of 0.07 M FeSO
4
was added to induce lipid peroxidation and the
mixture was incubated for 30 min. It was followed by addition of 1.5 ml of 20% acetic acid (pH
3.5) and 1.5 ml of 0.8% (w/v) thiobarbituric acid in 1.1% sodium dodecyl sulphate (SDS). The
resulting mixture was vortexed and heated at 95
o
C for 1 h. After cooling, 5.0 ml of n‑butanol was
added and the content was centrifuged at 3,000 rpm for 10 min. The absorbance of the upper
organic layer was measured at 532 nm. Inhibition of lipid peroxidation (%) was calculated using
control without sample under similar conditions. IC
50
was calculated from linear regression
analysis. BHA was used as a standard antioxidant.
Characterization of methanol extract
The most potent methanolic extract was examined for the identification and quantification of
individual polyphenols. An analytical HPLC system consisting of a JASCO high‑performance
liquid chromatograph coupled with a UV/VIS multi‑wavelength detector was employed for
analysis of this extract. The mobile phase consisted of 0.1% formic acid (solvent A) and
methanol (solvent B). The programme/gradient used was: 85% A/15% B (0 min), 20% A/80%
B (55 min), 85% A/15% B (60 min). The flow rate was 0.8 ml/min and the injection volume was
20 µl. Absorbance was monitored at 270 nm and the identification of each polyphenol was
based on retention time [19].
RESULTS AND DISCUSSION
Ashwagandha has been used since centuries in the Ayurvedic system of medicine to enhance
longevity and vitality. The plant is reported to contain several alkaloids, withanolides, a few
flavonoids and reducing sugars besides rich in iron [20]. The roots, leaves and fruits of this plant
have high antioxidant potential. The involvement of a very complex chemistry of oxidation and
anti‑oxidation processes clearly suggests that a single testing method is inconclusive to gain
comprehensive picture of the antioxidant potential of a given herb. In the present work,
therefore, we have used a multi‑method approach to judge completely the antioxidant potential
of various leaf extracts.

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Taking zero percent inhibition in the assay mixture without extract, linear regression equations
were developed from a plot between the extract concentration and percentage inhibition of free
radical formation/prevention. These equations were employed for the calculation of IC
50
values
(concentration of sample required to scavenge 50% free radicals). Lesser the IC
50
values more is
the antioxidant activity of extract.
Extraction efficiency
Starting from non‑polar (hexane) to polar (water), six solvents were used sequentially to extract
compounds from Ashwagandha leaves and the corresponding yield is presented in Fig. 1. The
experiment revealed that maximum migration of molecules took place in methanol and it was
the best solvent for the extraction of compounds as it recorded a maximum yield of 13.7%. On
the contrary, a minimum yield of 1.05% was recorded in ethyl acetate fraction. Because
methanol is a relatively polar organic solvent compared to other extracting solvents like hexane,
chloroform, ethyl acetate and acetone, it can be concluded that compounds present in
Ashwagandha leaves are most likely polar in nature. Methanol has earlier also been reported as
better extractant for the extraction of compounds since methanol is more efficient in cell wall
degradation as compared with other solvents [21].
DPPH scavenging activity
The capacity of a test compound to scavenge free radicals can be judged by its ability to bleach
DPPH absorption (517 nm). Hence, DPPH‑scavenging activities of the extracts were taken as the
parameter to check their antioxidant potential. A dose dependent increase in quenching of free
radical was observed for all the extracts. Table 1 summarizes the linear regression equations
used for calculation of IC
50
values of different extracts which are shown in Fig. 2. Methanolic
fractions (IC
50
value 197.5 μg/ml) showed the highest free radical scavenging activity followed
by water extract (IC
50
value 231.3 μg/ml). The hexane fraction was found to least potent as
indicated by its highest IC
50
value (713.24 μg/ml).
Metal chelating activity
Iron is essential for life because it is required for oxygen transport, respiration and the activity
of many enzymes. However, it is an extremely reactive metal and causes the oxidative damage
to lipids, proteins and other cellular components [22].
Therefore, ability of different extracts to
chelate/bind metal ion
was tested by a method which is based on chelating of Fe
+2
by the
reagent ferrozine, which is a quantitative formation of a complex with Fe
+2
[14]. Formation of
this complex is disturbed by the other chelating agents, which result in the reduction of
formation of red‑coloured complex. Therefore, measurement of the rate of reduction of the
color allows the estimation of chelating activity of the co‑existing chelators. In the present study,
all the extracts intervened with the formation of red‑coloured complex suggesting that it has
chelating effects and captures the ferrous ions before ferrozine. The absorbance of Fe
+2
‑
ferrozine complex linearly decreased with the increase in concentration of extracts. The
regression equations (Table 2) were generated for the calculation of IC
50
values (Fig. 3). The
result shows that the methanolic extract was most potent as evident by lowest IC
50
value (76.09
µg/ml). It was followed by ethyl acetate fraction (IC
50
value 98 µg/ml) and acetone fraction (IC
50
value 116.4 µg/ml). Like DPPH free radical scavenging activity hexane extract also showed least
metal chelation activity with IC
50
value of 600 µg/ml. The data clearly demonstrate that the
methanolic extract of leaf possesses effective capacity for iron binding.
Hydroxyl radical scavenging activity
Among the reactive oxygen species, the hydroxyl radicals are the most reactive and
predominant radicals generated endogenously during aerobic metabolism [23].
This radical is
an extremely reactive oxygen species capable of modifying almost every molecule in the living
cells. It can cause strand damages in DNA leading to carcinogenesis, mutagenesis, and
cytotoxicity. Hydroxyl radicals are also capable of quick initiation of lipid peroxidation process
by abstracting hydrogen atoms from unsaturated fatty acids [24‑25].
However, due to their high reactivity, the radicals have a very short biological half‑life thereby
demanding its effective scavenger either to be very efficient towards these radicals or to be
present at a very high concentration. In the present study, the ability of all extracts to scavenge
these radicals was evaluated by the 2‑deoxyribose assay. Hydroxyl radicals were generated by

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the Fenton reaction which attacks deoxyribose and degrades it into fragments that react with
thiobarbituric acid on heating at low pH to form a pink color. All the extracts were found to
show a dose dependent response towards chelation of hydroxyl free radical. The linear
regression equations were generated which are summarized in Table 3. The best results were
exhibited by the methanolic extract with minimum IC
50
value of 790.7 µg/ml (Fig. 4).
Superoxide radicals scavenging activity
Although by themselves superoxide radicals are less reactive but they can give rise to toxic
hydroxyl radicals thereby damaging cellular macromolecules directly or indirectly with severe
consequences [26].
The superoxide radicals have been proved to play crucial roles in ischemia‑
reperfusion injury [27]. These radicals are also involved in many pathological processes. Thus,
scavenging of superoxide radicals would be a promising remedy for this disease.
In this experiment, superoxide radicals were generated by a PMS/NADH system and the
scavenging activity of different extracts was assessed by measuring the absorbance at 560 nm
[28].
As in other assays, the IC
50
values were calculated using the regression equations listed in
Table 4. Fig. 5 summarizes the IC
50
values of various extracts against superoxide radical anions.
The highest scavenging activity was shown by methanolic extract followed by ethyl acetate
extract with IC
50
values of 119.7 and 138.7 µg/ml, respectively. Hexane extract was least potent
with highest IC
50
value of 531.5 µg/ml.
Lipid peroxidation inhibition activity
It has been suggested that antioxidant activity of an extract is its ability to delay the onset of
auto‑oxidation by scavenging reactive oxygen species, or its ability to act as chain breaking
antioxidants by inhibiting the propagation phase of lipid auto‑oxidation [29].
The inhibition of lipid peroxidation induced by FeSO
4
in egg yolk was assayed by measuring the
lipid peroxidation products such as TBARS. All the extracts of Ashwagandha leaves showed
inhibition of lipid peroxidation in a dose dependent manner. The regression equations
developed for the calculation of IC
50
values of all extracts is shown in Table 5 and a comparison
of IC
50
values is shown in Fig. 6. Results showed that all the extracts inhibited TBARS formation
in a dose dependent manner with maximum activity shown by methanol (536.4 µg/ml) followed
by water extract (707.8 µg/ml).
Total phenolic and total flavonoids contents
Plant phenolics are one of the major groups of compounds which act as primary antioxidants or
free radical terminators. It is therefore worthwhile to quantify their content in the extracts
under study. These compounds have been identified to possess a wide range of chemical and
biological activities including radical scavenging activity. Therefore, the total phenolic and
flavonoids content were determined in all the extracts of Ashwagandha leaves. It was also aimed
to establish relationships of different antioxidant activities with total phenolics and flavonoids.
The TPC values of the different extracts determined using the Folin–Ciocalteu assay, ranged
from 7.55 to 43.93 µg GAE/mg of extract (Table 6). This difference was observed due to the
varied polarity of the extracting solvent. Among all the fractions, methanol extract was found to
have the highest phenolic content (43.93 µg GAE/mg). The lowest amount of phenolics i.e. 7.55
µg GAE/mg was reported in the hexane fraction. The above findings clearly indicate that most
polyphenolics evaluated in this study are likely polar compounds.
The flavonoids, another class of secondary plant phenolics with powerful antioxidant properties
[30], were estimated in the different extracts under observation. The investigations
demonstrated the presence of maximum and minimum flavonoids in ethyl acetate (1.21 µg
CE/mg of extract) and hexane (0.18 µg CE/mg of extract) extracts, respectively.
Maximum radical scavenging activity was found in methanolic extract and the same extract also
contained maximum phenolic contents. Therefore, a positive correlation between the content of
antioxidant compounds and the RSC can be established [31]. The observed data are in
coherence with others reports, where it has been shown that high TPC increases the antioxidant
activity [32‑33]
and there is a positive correlation between phenolic content and antioxidant
activity [34].The antioxidant activity of phenolic compounds is mainly contributed by their
redox potential, which plays an important role in neutralizing free radicals [35].

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Characterization of methanol extract
The methanol extract of Ashwagandha leaf was found most potent and an attempt was made to
identify the main individual polyphenolic compounds present in it. Quantification was done
using RP‑HPLC through ‘external standard method’ via calibration with standards. Seven
polyphenols namely Gallic acid (0.17 μg/g), Chlorogenic acid (0.70 μg/g), Caffic acid (0.57 μg/g),
Sinapic acid (1.60 μg/g ), rutin hydrate (0.17 μg/g), Quercetin‑3‑rhamnoside (1.61 μg/g) and
Quercetin (0.27 μg/g) were identified and quantified (Fig. 7 , Table 7).However, few peaks could
not be identified due to the lack of standards.
Table 1. Regression equations and correlation coefficients for DPPH scavenging activity of
different extracts of Ashwagandha leaves.
Extract Regression equation Correlation coefficient (R
2
)
Hexane y = 0.0687x + 1 0.9884
Chloroform y = 0.1015x + 0.2333 0.9898
Ethyl acetate y = 0.1629x + 0.6857 0.9768
Acetone y = 0.1141x + 3.025 0.9899
Methanol y = 0.2407x + 2.4611 0.9703
Water y = 0.2028x + 3.0964 0.9832
Table 2. Regression equations and correlation coefficients for metal chelating activity of
different extracts of Ashwagandha leaves.
Extract Regression equation Correlation coefficient (R
2
)
Hexane y = 0.0774x + 3.5622 0.9875
Chloroform y = 0.2535x ‑ 0.0114 0.9906
Ethyl acetate y = 0.516x ‑ 0.5917 0.9887
Acetone y = 0.459x ‑ 3.4417 0.9875
Methanol y = 0.6837x ‑ 2.0286 0.9881
Water y = 0.4144x ‑ 3.2117 0.9888
Table 3. Regression equations and correlation coefficients for hydroxyl radical scavenging
activity of different extracts of Ashwagandha leaves.
Extract Regression equation Correlation coefficient (R
2
)
Hexane y = 7.1971x + 0.581 0.9868
Chloroform y = 15.737x ‑ 1.325 0.9859
Ethyl acetate y = 0.0366x ‑ 1.9714 0.9892
Acetone y = 0.041x + 1.8 0.9849
Methanol y = 0.0668x ‑ 2.8143 0.9913
Water y = 24.464x + 1.4321 0.9865
Table 4. Regression equations and correlation coefficients for superoxide radical scavenging
activity of different extracts of Ashwagandha leaves.
Extract Regression equation Correlation coefficient (R
2
)
Hexane y = 0.0934x + 0.3619 0.9917
Chloroform y = 0.2028x + 3.8057 0.9803
Ethyl acetate y = 0.3307x + 4.1138 0.9846
Acetone y = 0.2653x ‑ 1.26 0.9906
Methanol y = 0.3627x + 6.5838 0.9729
Water y = 0.285x + 3.9446 0.9835

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Table 5. Regression equations and correlation coefficients for anti‑lipid peroxidation activity of
different extracts of Ashwagandha leaves.
Extract Regression equation Correlation coefficient (R
2
)
Hexane y = 19.629x + 1.9762 0.9858
Chloroform y = 0.0406x + 1.6286 0.9884
Ethyl acetate y = 0.0627x ‑ 0.0771 0.9897
Acetone y = 0.0508x ‑ 0.1714 0.9802
Methanol y = 0.0959x ‑ 1.4433 0.9883
Water y = 0.0677x + 2.0821 0.9877
Table 6: TPC and TF in different extracts of Ashwagandha leaf
Extract TPC TF
(µg/mg extract)
Hexane 7.55 0.94
Chloroform 16.97 0.98
Ethyl acetate 36.24 1.21
Acetone 20.28 0.33
Methanol 43.93 0.18
Water 26.48 0.13
Table 7: Individual polyphenol of methanolic extract of Ashwagandha leaf
Polyphenols Amount (µg/g)
Gallic acid 0.17
Chlorogenic acid 0.70
Caffic acid 0.57
Sinapic acid 1.60
Rutin hydrate 0.17
Quercetin‑3‑rhamnoside 1.61
Quercetin 0.27
Fig.1. Yields of Ashwagandha leaves compounds in different solvents
0
4
8
12
16
H C EA A M W
Solvent
Yield (%)

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Fig.2. Comparison of DPPH radical scavenging activity of different extracts of Ashwagandha
leaves
Fig.3. Comparison of metal chelating activity of different extracts of Ashwagandha leaves
0
160
320
480
640
800
H C EA A M W BHA
Solvent
IC
50
value (µg/ml)
0
130
260
390
520
650
H C EA A M W EDTA
Solvent
IC
50
value (µg/ml)

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Fig.4. Comparison of hydroxyl radical scavenging activity of different extracts of Ashwagandha
leaves
Fig.5. Comparison of superoxide radical scavenging activity of different extracts of
Ashwagandha leaves
0
1000
2000
3000
4000
5000
6000
7000
H C EA A M W GA
Solvent
IC
50
value (µg/ml)
0
150
300
450
600
H C EA A M W BHA
Solvent
IC
50
value (µg/ml)

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Fig.6. Comparison of anti‑lipid peroxidation activity of different extracts of Ashwagandha leaves
Fig.7. HPLC chromatograph of Ashwaghand leaf extarct
CONCLUSION
Solvents of varied polarity starting from non‑polar to polar were used sequentially to extract
the compounds from Ashwagandha leaves. The methanolic extract was found enriched with
total polyphenolic content. Among all the extracts, methanol extract was the most effective in
terms of its radical scavenging activities tested using an array of assays. The antioxidant activity
0
750
1500
2250
3000
H C EA A M W BHA
Solvent
IC
50
value (µg/ml)

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of various extracts can be directly related with the total polyphenolic compounds contain in
them. Overall, the present investigation indicates that the methanolic extract is most potent
regarding its antioxidant potential.
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