Evaluation of antioxidant activity of ethanol extract of roots
obtained from Asparagus racemosus by in vitro and ex vivo models
M. Kundu1*, R. Mazumder2, M. D. Kushwaha 3
1
Govt. Polytechnic Uttarkashi, Dist:Uttarkashi, Uttarakhand 249193,
India.
2
Department of Pharmaceutical Technology, Noida Institute of Engineering
and Technology, Greater Noida, Uttar Pradesh 201306, India.
3
Govt.P.G.College Uttarkashi, Dist: Uttarkashi, Uttarakhnad, India.
*Corresponding author:
Dr. Rupa Mazumder, Professor, Dean (R&D) & HOD Pharmaceutics, Noida
Institute of Engineering and
Technology, Greater Noida, Uttar Pradesh 201306, India.
E-mail: rupa_mazumder@rediffmail.com, Tel: +91 9871963644.
*Corresponding author:
Dr. Rupa Mazumder, Professor, Dean (R&D) & HOD Pharmaceutics, Noida
Institute of Engineering and
Technology, Greater Noida, Uttar Pradesh 201306, India.
E-mail: rupa_mazumder@rediffmail.com, Tel: +91 9871963644.
ABSTRACT
The objective of the study was to explore the antioxidant activity of
ethanol extract of roots of Asparagus racemosus by in vitro and ex vivo models to substantiate the folklore claim of the
traditional practitioners. The roots of the plant showed sufficient
antioxidant activity by in vitro (determination of total
antioxidant activity through estimation of the conjugated dienes and
thiobarbituric acid reactive substances formed, total phenolic content,
reducing power, free radical scavenging activity by DPPH method, nitric
oxide scavenging activity, hydroxyl radical scavenging activity and super
oxide scavenging activity by NBT and DMSO methods) and ex vivo
(inhibition of haemolysis of RBC induced by phenyl hydrazine, inhibition of
lipid peroxidation induced by ferrous sulphate and carbon tetrachloride)
models. The experimental data were compared with that of standard
antioxidant like ascorbic acid, alpha-tocopherol acetate, butylated
hydroxyl anisol (BHA) and butylated hydroxyl toluene (BHT). All observed
results indicated ethanol extract of roots of Asparagus racemosus
to possess antioxidant activity in a concentration dependant manner and the
activity of the extract was found to be very much comparable to that of the
selected standard drugs.
Keywords:
Asparagus racemosus
, Antioxidant, Ethanol, Reactive oxygen species. Free
Radical Scavenging.
INTRODUCTION
Reactive oxygen species (ROS) namely superoxide anions, hydrogen peroxide,
hydroxyl, nitric oxide and peroxy-nitrite radicals play a significant role
in oxidative stress associated with a number of disease complications [1]. In healthy individuals, the production of free radicals is
balanced with the antioxidative defense system. When equilibrium gets
disbalanced, it results in the generation of free radicals beyond the limit
and ultimately depletion of antioxidant levels. The oxidation of cellular
lipids, nucleic acids, proteins, carbohydrates and other biomolecules by
ROS is thought to be one of the major risk factors for cancer,
atherosclerosis, diabetes mellitus, coronary heart disease and various
other degenerative diseases[2,3]. On the contrary,
free-radical-scavenging antioxidants derived from dietary sources play an
important role in preventing oxidative damages. The flavonoids existing
abundantly in vegetables and fruits, are good radical scavengers. Many
methods to determine the radical scavenging activity of plant extracts have
been reported, all of which have different advantages and limitations [4-7].
Asparagus racemosus Willd
. (Family: Liliaceae) commonly known as satawar, satavari or shatavari is a
tall climber, undershrub, distributed in low jungles of tropical and
subtropical parts of India, Ceylon, Africa, Java and Ausrtalia. The roots
of the plant has been used as antidiarrhoeal, appetizer, stomachic,
expectorant, laxative, tonic, inflammations biliousness, antidysenteric and
diuretic. The plant contains a number of secondary metabolites namely as
sterols and saponins[8,9]. The present investigation is directed
to the exploration of antioxidant activity of ethanol extract of roots of Asparagus racemosus by in vitro and ex vivo
models keeping in mind that the plant contains sterol.
MATERIALS AND METHODS
Plant Materials
Roots of Asparagus racemosus were collected in the month of April
and May from Purnia district, Bihar, India. The plant was authenticated by
the Botanist of Govt. P. G. College, Uttarkashi, Uttarakhand. A voucher
specimen of the herbarium was deposited in our laboratory for future
reference.
Preparation of the Ethanol Extract
Ethanol extract of roots was prepared in accordance to the method of
National Institute of Health and Family Welfare (NIHFW), New Delhi, India.
Dried matured leaves were crushed in an electrical grinder to fine powder
of mesh 40. The powder was then extracted with ethanol in a soxhlet
apparatus until the powder became exhausted totally. Resulting extract was
filtered with coarse sieve filter paper. The filtrate was dried under
reduced pressure with the help of rotary vacuum evaporator. The extract was
stored in a desiccator for use in subsequent experiments.
Chemicals
Ethylene di-amine tetra acetate (EDTA), phenyl hydrazine hydrochloride,
dimethyl sulphoxide (DMSO), thiobarbituric acid (TBA), ferrous sulphate
(FeSO4), trichloroacetic acid (TCA) and acetic acid were
procured from SD fine chemicals Ltd., India. Nitro blue tetrazolium
chloride (NBT) was purchased from Hi-Media Ltd., India, while ascorbic acid
and α-tocopherol acetate were procured from Cadila Pharmaceutical Ltd.,
India. All reagents used in the experiment were of analytical grade and
other reagents were obtained from Sigma (Sigma-Alrich GmbH, Sternheim,
Germany).
Determination of Total Antioxidant Activity
The antioxidant activity of the ethanol extract of the roots of Asparagus racemosus was determined according to the the
method using linoleic acid emulsion system[10]. Linoleic acid
emulsions were prepared by mixing 0.285 g of linoleic acid, 0.289 g of
Tween-20 as emulsifier and 50 ml of phosphate buffer (pH 7.2). The mixture
was homogenized for 5 min and the antioxidant was added at the final
concentrations of 25-800 μg/ml of the extract. The mixture was incubated in
an oven at 37oC for 24 hr and the course of oxidation was
monitored by measuring the formation of conjugated dienes (CD) and
thiobarbituric acid reactive substances (TBARS).
Estimation of CD Formation
Aliquots of 20 μl were taken every hour from the emulsion during 24 hr of
incubation. To each aliquot, 2 ml of methanol in deionized water (60 %)
were added and the absorbance of the mixture was measured at 233 nm by
UV-visible spectrophotometer.
Estimation of TBARS
Sample (100 μl) was taken every hour from the emulsion and the following
chemicals were sequentially to it, 100 μl BHA (3.6 %) and 2 ml of TBA-TCA
solution [20 mM TBA in 15% trichloacetic acid (TCA)]. The mixture was
heated in a water bath at 90oC for 15 min and cooled at room
temperature. 2 ml chloroform was added to it, the mixture was mixed and
centrifuged at 2000 rpm for 15 min. The chloroform layer was separated and
absorbance of the supernatant was measured at 532 nm agains a blank
containing 0.1 ml of double distilled water and 2 ml of TBA-TCA solution.
Lipid peroxidation was measured in terms of malondialdehyde (MDA) content.
The solutions without extract and containing equivalent volumes of solvent
were used as blank samples. All data about total antioxidant activity were
the averages of triplicate analyses. The inhibition of lipid peroxidation
in percentage was calculated by the following equation:
% Inhibition = (Ao – At)/Ao x 100,
where Ao = absorbance of the control reaction and At
= absorbnce in the presence of the sample of the extracts[11,12]
.
Determination of Total Phenolic Content
Total soluble phenolic compound in the ethanol extract of the roots of Asparagus racemosus was estimated with Folin-Ciocalteu reagent by
the method[13]. Gallic acid was used as a standard phenolic
compound. 1 ml of extract solution (containing 1mg extracts) was kept in a
volumetric flask diluted with water (46 ml), 1ml of Folin-Ciocalteu reagent
was added and the content of the flask was mixed thoroughly. After 3 min,
3ml of sodium carbonate (2%) was added, then the mixture was allowed to
stand for 2 hr with intermittent shaking. The absorbance was measured at
760 nm in a spectrophotometer. The concentrations of the total phenolic
compounds in the extracts were estimated as equivalent microgram of gallic
acid by using an equation that was obtained from standard gallic acid
graph:
Absorbance = 0.0053 × total phenols (gallic acid equivalent µg ) – 0.0059
Reducing Power
The reducing power of ethanol extract of roots of Asparagus racemosus was determined according to reported method [14] with little modifications. The three different
concentrations of extract (20μg/ml, 40μg/ml and 80μg/ml) were mixed with
2.5 ml of 200mM sodium phosphate buffer (pH 6.6) and 2.5 ml of 1% potsssium
ferricyanide. The mixture was mixed properly and then centrifuged at 650
rpm for 10 minutes. The upper layer 5 ml was mixed with 5 ml of distilled
water and 1 ml of 1% ferric chloride and absorbance was measured at 700 nm
in a spectrophotometer. Higher absorbance of reaction mixture indicated
greater reducing power.
Free Radical Scavenging Activity by DPPH Method
This assay was based on the measurement of the scavenging ability of
antioxidant test substances towards the stable radical. The free radical
scavenging activity[15] of the ethanol extract was examined in vitro using DPPH radical. Different concentrations (25-800
μg/ml) of the test extract was used in the study. The reaction mixture
consisted of 1 ml of 0.1mM DPPH in ethanol, 0.95 ml of 0.05M Tris-HCl
buffer (pH 7.4), 1 ml of ethanol and 0.05 ml og the herbal extract. The
absorbance of the mixture was measured at 517 nm exactly 30 sec after
adding the extract. The experiment was performed (in triplicate) and %
scavenging activity was calculated using the formula
% Scavenging = ( Ao – At )/Ao x 100,
where Ao = absorbance of the control reaction and At =
absorbance in the presence of the sample of the extracts.
The activity was compared with ascorbic acid, which was used as a standard
antioxidant.
Nitric Oxide Scavenging Activity
Sodium nitroprusside[16] (5 µM) in standard phosphate buffer
solution was incubated with different concentration of the test extracts
dissolved in standard phosphate buffer (0.025M, pH 7.4) and the tubes were
incubated at 250oC for 5 hr. After 5 hr, 0.5 ml of incubation
solution was removed and diluted with 0.5 ml Griess reagent (prepared by
mixing equal volume of 1% sulphanilamide in 2% phosphoric acid and 0.1%
naphthylethylene diamine dihydrochloride in water). The absorbance of
chromophore formed was read at 546 nm. The control experiment was also
carried out in similar manner, using distilled water in the place of
extracts. The experiment was performed (in triplicate) and % scavenging
activity was calculated using the same formula, as mentioned above.
The activity was compared with ascorbic acid, which was used as a standard
antioxidant.
Hydroxyl Radical Scavenging Activity
The hydroxyl radical scavenging activity was measured by studying the
competition between deoxyribose and the extract for hydroxyl radicals
generated from the ferric ions/ascorbate/EDTA/hydrogen peroxide system. The
reaction mixture contained deoxy ribose (2-8mM), Fecl3 (0.1mM), EDTA
(0.1mM), hydrogen peroxide (1mM), ascorbate (0.1mM), KH2P04-KOH buffer
(20mM, pH 7.4) and various concentrations (25-800 ug/ml of extracts and
5-80 ug/ml of the standard drug) in the final volume of 1 ml. The reaction
mixture was incubated for 1 hr at 37oC, deoxyribose degradation
was measured at 532 nm[17].
Super Oxide Scavenging Activity
Super oxide scavenging activity by NBT model:
Super oxide scavenging activity was measured using reported method [18] with little modification. The reaction mixture consisted of
50mM sodium carbonate buffer (pH 10.2), 24μM NBT, 0.1mM EDTA, 1mM
hydroxylamine and 0.03% Trifonx-100 along with the compound to be tested in
a total volume of 1 ml incubated for 20 minutes at 37°C and the absorbance
was measured at 560 nm with the help of spectrophotometer. Ascorbic acid
was used as standard antioxidant.
Super oxide scavenging activity by alkaline dimethyl sulphoxide
(DMSO) method
:
Superoxide scavenging activity was determined by using alkaline DMSO method [19] with minor modifications. Solid potassium sulphoxide was
kept in contact with dry DMSO for at least 24 hours and the solution was
filtered immediately before use. Filtrate (200 µl) was added to 2.8 ml of
an aqueous solution containing NBT (56μM), EDTA (10μM) and potassium
phosphate buffer (10mM). Ethanol extract of the leaves of 1 ml at different
concentrations were taken and the absorbance was observed at 560 nm against
a control in which pure DMSO was taken instead of alkaline DMSO. Ascorbic
acid was used as standard antioxidant.
Inhibition of Haemolysis of RBC Induced by Phenyl Hydrazine
20% packed cell volume (PCV) of RBC suspension (from human blood) was
prepared according to the procedure described by Hill and Thornalley. The
assay was carried out according to the method[20] with certain
modifications. The incubation mixture comprised of 1 ml of phenyl hydrazine
hydrochloride (0.5mM), different concentrations of the sample extracts and
0.1 ml of 20% RBC suspension made to a total volume of 3.0 ml with
phosphate buffered saline (PBS) solution. The mixture was incubated at 37°C
for one hr and centrifuged at 100 rpm for 10 min. The extent of haemolysis
was measured spectrophotometrically by recording the absorbance of the
supernatant at 540 nm. Suitable controls were kept to nullify the effect of
solvents and inherent haemolysis. α-tocopherol acetate was used as a
positive control for the inhibition of phenyl hydrazine induced haemolysis
of RBC.
Inhibition of Lipid Peroxidation
Inhibition of lipid peroxidation induced by FeSO4
Animals:
Male Wister strain albino rats (160-180 gm) were procured from M/s B N
Ghosh Co. Kolkata, India. Rats were housed in standard polypropylene cages
(two animals per cage), maintained under standard laboratory conditions ( i.e. 10:14 hour light and dark order; at an ambient temperature of
26 ± 2 ºC; 40-55% of relative humidity); the animals were fed with standard
rat pellet diet (Hindustan Liver Ltd. Mumbai, India) and water ad libitum. Animals were allowed to be acquainted for a period of
a week in our laboratory environment prior to the experiment.
Preparation of rat liver homogenate: Rat liver homogenate was prepared by
the following method[21]. Randomly selected 6 rats were fasted
over night and were sacrificed by cervical dislocation, dissected and
abdominal cavities were perfused with 0.9% normal saline. Livers of
sacrificed rats were separated out and visible clots were removed. Then 10%
liver homogenate was prepared in cold phosphate buffer saline (pH 7.4)
using glass teflon homogeniser and filtered to get a clear homogenate.
Assay method:
The degree of lipid peroxidation was assayed by estimating the
thiobarbituric acid reactive substances (TBARS) by using standard method [22] with minor modifications[23]. Briefly, different
concentrations of extract (50-300 μg/ml) were added into 10% liver
homogenate. Lipid peroxidation was initiated by adding 100 μl of 15mM FeSO 4 solution to 3 ml of liver homogenate (final concentration was
0.5mM). After 30 min 100 μl of this reaction mixture was taken in a tube
containing 1.5 ml of 10% TCA. After 10 min tubes were centrifuge and
supernatant was separated and mixed with 1.5 ml of 0.67% TBA in 50% acetic
acid. The mixture was heated in a hot water bath to complete the reaction.
The intensity of pink coloured complex formed was measured at 535 nm in a
spectrophotometer. The percentage inhibition of lipid peroxidation was
calculated as per the following formula:
Inhibition (%) = 100 x (Control – Test)/Control
Inhibition of lipid peroxidation induced by CCl4
Inhibition of lipid peroxidation induced by CCl4 was measured by
the method[24]. Rat liver (30% w/v) homogenate in ice-cold 0.15M
potassium chloride was prepared in a homogenizer. Aliquots of 0.5 ml of
homogenates were taken in different small conical flasks. These were
incubated at 37ºC in a constant shaker bath (150 cycles/min) for 45 min
with 1.5 ml of potassium sulphate buffer (pH 7.4), 2 ml of 0.15M potassium
chloride CGM at (25- 800) μg/ml and ascorbic acid (5-100 µg/ml) in
different flasks and finally 10 µl of CCl4 was added. The
reaction was stopped by the addition of 4 ml of 10% w/v TCA and after
incubation. The contents were centrifuged at 4000 rpm for 10 min and about
2 ml of clear supernatant was transferred to a graduated tube. 2 ml of
0.67% w/v of TBA was added and heated in a boiling water bath for 15 min
the tubes were cooled bringing the mixture to pH 12-12.5 with potassium
hydroxide stabilized the colour developed and the absorbance was measured
at 543 nm. In case of control, only drug was excluded.
RESULTS AND DISCUSSION
Total antioxidant activity was estimated by calculating formation of CD and
TBARS, as reported in Table1.
Total phenolic content was 15.67 µg gallic acid equivalent of phenols as
was determined in 1 mg of the ethanol extract of roots of Asparagus racemosus, reported in Table 2. Phenolic compounds are
most vital constituents because of their scavenging ability due to their
hydroxyl groups[25]. The phenolic compounds may contribute
directly to the antioxidant effect. According to recent reports, a highly
positive corelation exists between total phenols and antioxidant activity
was found in many plant species[26,27]. In addition, it was
reported that phenolic compounds were associated with antioxidant activity
and play a vital role in stabilizing lipid peroxidation[28].
Reducing power of the extract and standards followed the order (BHA>BHT
>ascorbic acid> extract), as shown in Table 3. Reducing capacity of a
compound may serve as a significant indicator of its potential antioxidant
activity[29]. The antioxidant activity of putative antioxidants
have been attributed to various mechanisms, among which are prevention of
chain initiation, binding of transition metal ion catalysts, decomposition
of peroxides, prevention of continued hydrogen abstraction, reductive
capacity and radical scavenging activity[30].
The ethanol extracts of the leaves of the plant showed prominent free
radical scavenging effect of DPPH in a concentration dependant manner up to
a concentration
of 800 µg /ml. IC50 values of extract and ascorbic acid were
found to be 180.0 µg/ml and 11.6 µg/ml, respectively, by this method (Table
4). The free radical scavenging activity of the extracts was determined on
the basis of ability to scavenge the synthetic
DPPH. This method is a widely used method to evaluate antioxidant
activities in a relatively short time compared with other models due to
prominent discolouration from
purple to yellow in visible spectrum.
The ethanol extract showed significant scavenging activity against the
nitric oxide radical when compared with the standard ascorbic acid in a
dose dependant manner.
IC50 values of the extract and ascorbic acid were found to be 265.0 µg/ml
and 15.2 µg/ml, respectively, by this method (Table 4). Nitric oxide
scavenging activity was determined by the formation of the chromophore
during diazotization of the nitrite with sulphanilamide and subsequent
conjugation with napthylene diamine. The ethanol extracts significantly
scavenged the hydroxyl radicals when compared with the standard ascorbic
acid in a dose dependant manner. IC50 values of the extract and
ascorbic acid were found to be 235.0 µg/ml and 16.4 µg/ml, respectively, by
this method (Table 4). For tissue injury hydroxyl radical is the one of the
main factor. The extract scavenged the hydroxyl radical formed in the
Fenton reaction and it was quantified using 2-deoxy-D-ribose degradation.
Super oxide scavenging activity was measured by in vitro assay
methods employing both NBT and alkaline DMSO models. The ethanol extract of
roots of Asparagus racemosus at a concentration range of 25-800
µg/ml significantly scavenged the superoxide radicals. The percentage
scavenging of superoxide radicals by the extract was increased in a dose
dependant manner in both NBT and DMSO models. IC50 values of
extract and ascorbic acid were found to be 130.0 µg/ml and 18.2 µg/ml,
respectively, as found in NBT model, whilst in DMSO method the IC 50 values were found to be 285.0 µg/ml and14.1 µg/ml for extract
and ascorbic acid, respectively.
The extract inhibited the haemolysis of RBC induced by phenyl hydrazine in
a dose dependant manner. The IC50 values were found for the
extract was 65.0 µg/ml, whilst standard drug, α-tocopherol acetate had
shown IC50 of 11.6 µg/ml.
The extract had shown a concentration dependant inhibition of FeSO4 induced lipid peroxidation in rat liver homogenates. The IC 50 values were found to be 175.0 µg/ml and 17.5 µg/ml,
respectively, for the extract and the standard antioxidant ascorbic acid.
Inhibition of CCl4 induced lipid peroxidation was also found to
increase in a dose dependant manner. The IC50 value was found to
be 152.5 µg/ml and 12.5 µg/ml, respectively, for the extract and standard
antioxidant ascorbic acid. The results of superoxide scavenging
activity with DMSO and NBT method, inhibition of haemolysis of RBC induced
by phenyl hydrazine and inhibition of lipid peroxidation induced by FeSO 4 & CCl4 model had been presented in
Table 5.
CONCLUSION:
The living cells producing ROS during several metabolic pathways lead to
oxidative stress, which is associated with several pathological
complications. Production of ROS depends upon rapid uptake of oxygen,
activation of NADPH oxidase and the production of superoxide free radical.
Oxidative stress can be prevented by different endogenous antioxidants like
superoxide dismutase, reduced glutathion, catalase and glutathion
peroxidase by escaping ROS and lipid peroxidation dependant injury.
Antioxidants work as radical scavengers, peroxide decomposers, hydrogen
donor, electron donor, enzyme inhibitors, singlet oxygen quenchers,
synergist and metal chelating agents. The requirement of antioxidants of
human beings is solely filled by dietary vegetables, which play an
important role in preventing oxidative damages. Plants also need to protect
themselves from free radical damage with the help of their own metabolites,
so they develop a number of different classes of antioxidant. The pigments,
flavonoids, coumarines, phytosterols, pro-anthocyanidines, tannins,
essential oils, resins and gums are responsible phytochemicals for
antioxidant activity. Thus it has fetched interest of researchers to
isolate novel antioxidant from plant sources.
Preliminary phytochemical screenng indicated the presence of sterols and
saponins in the extract. Sterols isolated from different sources are
reported to have antioxidant activity. So the lead compound may be sterol.
Now our intention is guided to isolate bioactive sterols from the extract
and substantiate its antioxidant efficacy.
Table 1:
Determination of total antioxidant activity of ethanol extract of roots of Asparagus racemosus (EEAR)
|
Concentration of
EEAR(µg/ml)
|
|
% Inhibition
|
|
Conjugated dienes
|
TBARS
|
|
25
|
|
17.43 ± 0.65
|
23.67 ± 0.54
|
|
50
|
|
26.84 ± 0.23
|
37.40 ± 0.36
|
|
100
|
|
33.76 ± 0.19
|
45.62 ± 0.74
|
|
200
|
|
44.22 ± 0.11
|
58.32 ± 0.65
|
|
400
|
|
53.44 ± 0.23
|
66.82 ± 0.25
|
|
800
|
|
59.44 ± 0.54
|
71.07 ± 0.43
|
|
IC50 (EEAR) (µg/ml)
|
|
215.0
|
132.5
|
|
IC50 (Standard) (µg/ml)
|
|
46.9
|
34.2
|
Values are mean ± S.E.M. of 3 replications
Table 2:
Total phenolic content of EEAR
|
Parameter
|
Level (µg/g)
|
|
The extract (EEAR)
|
12.89
|
Table 3:
Total reducing property of ethanol EEAR
Sample
|
Absorbance
|
20 μg/ml
|
40 μg/ml
|
80 μg/ml
|
Ethanol extract (EEAR)
|
0.092±0.064
|
0.117±0.082
|
0.210.45±0.023
|
|
Ascorbic acid
|
0.190±0.016
|
0.365±0.015
|
0.543±0.013
|
Butylated hydroxyl anisole
( BHA)
|
1.040±0.025
|
1.756±0.017
|
2.810±0.028
|
Butylated hydroxyl toluene
( BHT)
|
0.546±0.016
|
0.996±0.012
|
1.640±0.015
|
Values are mean ± S.E.M. of 3 replications
Table 4:
In vitro
antioxidant effect of EEAR through superoxide scavenging activity
with DMSO and NBT method, free radical scavenging activity by DPPH method,
nitric oxide and hydroxyl scavenging method
|
Concentration of
EEAR (µg/ml)
|
|
% of Scavenging
|
|
SOD scavenging effect
|
Free Radical
|
|
DPPH method
|
Nitric oxide
|
Hydroxyl
|
|
|
|
|
|
NBT model
|
DMSO model
|
|
|
|
|
|
25
|
|
23.25 ± 0.12
|
27.45 ± 0.44
|
24.59 ±0.53
|
19.56 ±0.32
|
28.21 ±0.32
|
|
50
|
|
34.35 ± 0.54
|
31.76 ± 0.28
|
33.48 ±0.37
|
31.63 ±0.33
|
33.62 ±0.22
|
|
100
|
|
47.35 ± 0.38
|
38.95 ± 0.43
|
44.77 ±0.32
|
36.73 ±0.23
|
43.85 ±0.16
|
|
200
|
|
56.25 ± 0.39
|
45.63 ± 0.34
|
51.23 ±0.13
|
45.34 ±0.22
|
49.13 ±0.35
|
|
400
|
|
66.21 ± 0.63
|
54.65 ± 0.12
|
63.32 ±0.23
|
59.15 ±0.22
|
54.23 ±0.13
|
|
800
|
|
75.87 ± 0.23
|
73.54 ± 0.47
|
67.85 ±0.42
|
70..92 ±0.18
|
59.33 ±0.27
|
|
IC50 (EEAR) (µg/ml)
|
|
130.0
|
285.0
|
180.0
|
265.0
|
235.0
|
|
IC50 (Standard) (µg/ml)
|
|
18.2
|
14.1
|
11.6
|
15.2
|
16.4
|
Values are mean ± S.E.M. of 3 replications
Table 5:
Ex vivo
antioxidant effect of EEAR through inhibition of haemolysis of RBC induced
by phenyl hydrazine and inhibition of lipid peroxidation induced by FeSO 4 andCCl4 model
|
Concentration of
EEAR(µg/ml)
|
|
% Inhibition
|
|
RBC membrane stabilization activity
|
Inhibition of lipid peroxidation
|
|
|
|
|
|
FeSO4 model
|
CCl4 model
|
|
|
|
|
|
|
|
25
|
|
|
|
35.12 ± 0.16
|
|
27.54 ± 0.13
|
30.32 ± 0.14
|
|
50
|
|
|
|
44.98 ± 0.12
|
|
34.87 ± 0.34
|
37.01 ± 0.15
|
|
100
|
|
|
|
58.80 ± 0.32
|
|
42.62 ± 0.22
|
44.42 ± 0.32
|
|
200
|
|
|
|
67.52 ± 0.18
|
|
51.52 ± 0.32
|
55.37 ± 0.53
|
|
400
|
|
|
|
73.55 ± 0.23
|
|
55.32 ± 0.35
|
60.71 ± 0.28
|
|
800
|
|
|
|
76.75 ± 0.15
|
|
67.33 ± 0.43
|
68.32 ± 0.22
|
|
IC50 (EEAR(µg/ml)
|
|
|
|
65.0
|
|
175.0
|
152.5
|
|
IC50 (Standard) (µg/ml)
|
|
|
|
11.6
|
|
17.5
|
12.5
|
|
|
|
|
|
|
|
|
|
Values are mean ± S.E.M. of 3 replications
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