Evaluation of In-vitro Anti-oxidant
and Free Radical Scavenging activities of Withania somnifera and Aloe vera
Patel
D.S. 1*, Shah P. B. 2, Managoli
N. B.3
1 Department of Pharmaceutical Sciences, Jodhpur National
University, Jodhpur, Rajasthan
2 Shri B. M.
Shah Diploma Pharmacy College, College Campus, Dhansura
Road, Modasa
3 Sahajanand Life
Sciences Pvt. Ltd., Surat, Gujarat, India
*Corresponding Author E-mail: gajeradipak@gmail.com
ABSTRACT:
In this study,
In-vitro Antioxidant and free radical scavenging activity of Withania somnifera (WS)
and Aloe vera (AV) extract were evaluated in
series of in vitro assays. Anti-oxidants are vital substances which
possess the ability to protect the body from damage caused by free radical
induced oxidative stress. In this present study we investigated anti-oxidant
and free radical scavenging activity of Withania
somnifera and
Aloe vera by Hydroxyl radical
scavenging activity, Hydrogen peroxide‐scavenging assay,
Super oxide scavenging activity, Reducing power, Chelating capacity, Total
antioxidant activity. The results showed that both the plant possesses
excellent anti-oxidant and free radical scavenging activity. Screening of both
the plant at different doses (100, 150 and 200 µg / ml) help to reveal the
potential of individual plants. Withania somnifera shown better hydroxyl radical scavenging
activity, reducing power and superoxide radical scavenging activity compare to Aloe
vera. While Aloe vera
possesses better chelating power then Withania
somnifera. Both the plant possesses almost
equivalent hydrogen peroxide scavenging activity while the total antioxidant
capacity was found much better in Withania somnifera as compare to Aloe vera.
The antioxidant activity of both the plant might be attributed to its polyphenolic content and other phytochemicals constituents.
The findings of the present study suggest that all extracts could be a
potential source of natural antioxidant that could have great importance as
therapeutic agents.
KEYWORDS:
Hydroxyl radical scavenging activity, Hydrogen peroxide‐scavenging
activity, Super oxide radical scavenging activity, Reducing power, Chelating
activity, Total antioxidant activity
INTRODUCTION:
Free radicals
are reactive oxygen species (ROS) or reactive nitrogen species (RNS) generated
in the body during normal metabolic activities or by environmental conditions1.
The most common ROS include superoxide anion (O2), hydrogen peroxide
(H2O2), peroxyl radicals (ROO)
and reactive hydroxyl (OH) radicals. RNS includes nitric oxide (NO) and peroxynitrite anion (ONOO)2,3.
Excess
production of these free radicals leads to cause cellular damage by reacting
with various bio molecules of body such as membrane lipids, nucleic acid,
proteins and enzymes. Antioxidants have been reported to prevent oxidative
damage by free radical and ROS, and may prevent the occurrence of disease,
cancer and aging.
Recently, the
biological and pharmacological properties of herbs have begun to receive more
attention in the scientific community and have become a very important research
focal point5,6. Withania somnifera has analgesic, mildly sedative,
anti-inflammatory and anabolic activities and it is useful in stress, strain,
fatigue, pain, skin diseases, diabetes, gastrointestinal disease, rheumatoid
arthritis, and epilepsy, chronic fatigue syndrome and even during pregnancy
without any side effects20–23. Aloe vera is acclaimed to cure ailments ranging from
mild fever, wounds and burns, gastrointestinal disorders, diabetes, sexual
vitality and fertility problems to cancer, immune modulation and AIDS24–26.
This is
attributed to the fact that they contain phytochemicals, such as antioxidants,
and other bioactive compounds. In view of this, in the present investigation an
attempt will be made to study of antioxidant activity and free radical
scavenging activity of extract of Withania somnifera and Aloe vera through
hydroxyl radical scavenging activity, hydrogen peroxide‐scavenging
activity, super oxide radical scavenging activity, reducing power, chelating
activity, total antioxidant activity.
MATERIALS AND METHODS:
Both of the standardized extracts (WS, AV), chemicals,
and solvents were of A. R. grade and provided by Sahajanad
Life sciences Pvt. Ltd., Surat. All the standardized
herbal extracts passed through 80 # sieve.
Preparation of sample:
Aqueous extract–10gm mixture of both the extract
individually extracted with distilled water by cold extraction process for 24 h.
After completion of the extraction, the solution was recovered by filtration.
Fresh sample was prepared and used for each test. Both the extracts (WS, AV)
were tested at the concentration of 100μg/ml (Low dose), 150μg/ml
(Medium dose), and 200 μg/ml (High dose).
1. Hydroxyl radical scavenging activity6:
The scavenging activity for hydroxyl radicals was
measured with fenton reaction. Reaction mixture contained
60 μL of 1.0 mM FeCl2,
90 μl of 1mM 1,10-phenanthroline, 2.4 mL of 0.2
M phosphate buffer (pH 7.8), 150 μL of 0.17 M H2O2,
and 1.0 mL of extract at various concentrations. Adding H2O2
started the reaction. After incubation at room temperature for 5 min, the
absorbance of the mixture at 560 nm was measured with UV visible spectrometer
Shimadzu, UV-1800, Japan. The percentage inhibition of hydroxyl scavenging
activity was calculated using the following formula,
Where, Absorbance (Test): Absorbance of the test (With
extract) and Absorbance (Blank): Absorbance of the control (Without extract)
2. Hydrogen peroxide‐scavenging
activity7, 8:
A solution of hydrogen peroxide (2 mmol/l) was prepared in phosphate buffer (pH 7.4).
Extract at various concentrations were added to hydrogen peroxide solution (0.6
ml). For each concentration, a separate blank sample was used for background
subtraction. Absorbance of hydrogen peroxide at 230 nm was determined after 10
min against a blank solution containing phosphate buffer without hydrogen
peroxide by UV visible spectrometer Shimadzu, UV-1800, Japan. The percentage
inhibition of H2O2scavenging activity was calculated
using the following formula,
Where, Absorbance (Test): Absorbance of the test (With
extract) and Absorbance (Blank): Absorbance of the control (Without extract)
3. Superoxide radical scavenging activity9, 10:
100 μl Riboflavin
solution (20 μg), 200 μl
EDTA solution (12 mM), 200 μl
methanol and 100 μl NBT (Nitro-blue tetrazolium) solution (0.1 mg) were mixed in test tube and
reaction mixture was diluted up to 3 ml with phosphate buffer (50 mM). The absorbance of solution was measured at 590 nm
using phosphate buffer as blank after 5min. This is taken as control (Blank).
Different concentrations of extracts were diluted up to 100 μl
with methanol, to each of this, 100 μl
Riboflavin, 200 μl EDTA, 200 μl
methanol and 100 μl NBT
were mixed in test tubes, further diluted up to 3 ml with phosphate buffer.
Absorbance was measured after 5 min. at 590 nm was measured with UV visible
spectrometer Shimadzu, UV-1800, Japan. The percentage scavenging activity was
calculated using the following formula,
Where, Absorbance (Test): Absorbance of the test (With
extract) and Absorbance (Blank): Absorbance of the control (Without extract)
4. Reducing power11:
In this assay the ability of the extract to reduce Fe3+
to Fe2+ was estimated. Reducing power of the extracts was determined
by using potassium ferricyanide-ferric chloride
system. Briefly, 1ml of extract at different concentration was added with 2.5ml
of 0.2 M phosphate buffer (pH 6.6), mixed with 2.5ml of potassium ferricyanide (0.1%) and the mixture was incubated at 50°C
for 20 min. 2.5ml of trichloroacetic acid (10%) was
added to the reaction mixture and centrifuged at 8000g for 10 min and 2.5ml of
supernatant was mixed with equal volume of distilled water & 0.5ml of 0.1%
ferric chloride was added and the absorbance was measured at 700 nm using UV
visible spectrometer Shimadzu, UV-1800, Japan. Increased absorbance of the
reaction mixture indicated the increased reducing power.
5. Chelating activity12:
To 1 ml of different concentration of extract was
treated with an equivalent amount of reaction mixture which contains 1 ml,
0.05% ortho–phenanthroline in methanol, 2ml ferric
chloride (200 mM). The treated compound was incubated
at ambient temperature for 10 min and the absorbance of same was measured at
510 nm.
Where, Absorbance (Test): Absorbance of the test (With
extract) and Absorbance (Blank): Absorbance of the control (Without extract)
6. Total antioxidant activity13:
The assay is based on the reduction of Mo (VI) - Mo
(V) by the extract and subsequent formation of a green phosphate/Mo (V) complex
at acidic pH. Different concentration of (0.3 ml)
extracts was combined with 3 ml of reagent solution (0.6 M sulfuric
acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). The tubes
containing the reaction solution were incubated at 95°C for 90 min. Then the
absorbance of the solution was measured at 695 nm using spectrophotometer
against blank after cooling to room temperature. Methanol was used as blank. Ascorbic acid
equivalents were calculated using standard graph of ascorbic acid. The experiment was conducted in triplicates
and values are expressed as equivalents of ascorbic acid in μg
per mg of extract.
Statistical analysis:
All assays were carried out in triplicates and results
are expressed as mean ± SD.
RESULTSAND DISCUSSION:
Hydroxyl radical is highly reactive oxygen centre
radical formed from the reaction of various hydroperoxides
with transition metal ions. It attacks proteins, DNA, polyunsaturated fatty
acid in membranes, and most biological molecule it contacts 7, 8 and
is known to be capable of abstracting hydrogen atoms from membrane lipids and
brings about peroxidic reaction of lipids. Both the
extracts exhibited concentration dependent scavenging activity against hydroxyl
radical generated in a fenton reaction system. Activity
of the extracts on hydroxyl radical has been shown in Figure I (It was also
found that low to medium dose of Withania somnifera is more effective then Aloe vera as hydroxyl radical scavenging activity).
Hydrogen peroxide can cross cell membranes rapidly,
once inside the cell, H2O2 can probably react with Fe2+,
and possibly Cu2+ ions to form hydroxyl radical and this may be the
origin of many of its toxiceffects14, 15. As shown in Figure II, Withania somnifera
shown better hydrogen peroxide scavenging activity at low dose but at high dose
Aloe vera found better than Withania somnifera.
As per the assay, Figure III presents the reductive
capabilities of both the extracts. The reducing power of extracts might be due
to their hydrogen‐donating ability. Possibly, both the
extracts could react with radicals to stabilize and terminate radical chain
reactions. The data suggest that reducing power of Withania
somnifera was better than Aloe vera and shown dose dependant free radical scavenging
activity.
Figure IV shown Aloe vera
had marked capacity for iron binding compare to Withania
somnifera. The effect was may be due to the
presence of polyphenols which has potent iron chelating capacity. The reducing
power of a compound is related to its electron transferability and may serve as
a significant indicator of its potential antioxidant activity14, 15.
Superoxide has been observed to directly initiate
lipid peroxidation14, 15. Effect on superoxide scavenging activity
was shown in Figure V. It was observed that Withania
somnifera possesses better dose dependent
superoxide scavenging potential than Aloe vera.
Ferrous iron can initiate lipid peroxidation by the fenton
reaction as well as accelerating peroxidation by decomposing lipid hydroperoxides into peroxyl and alkoxyl radicals 16, 17, 18.
The total antioxidant capacity18, 19was
concluded from Figure VI. Again both the extracts had shown the dose dependant
efficacy among which the maximum total antioxidant potential was found with Withania somnifera.
The antioxidant potential of the extracts was increased markedly with the
increase in concentrations of Withania somnifera.
CONCLUSION:
In present study, antioxidant activities of Withania somnifera and
Aloe vera were investigated. The results obtained
in the present study indicate that Withania
somnifera exhibits powerful Hydroxyl radical
scavenging activity, superoxide radical scavenging activity, reducing power and
total antioxidant activity as compare to Aloe vera.
It was also observed that Aloe vera possesses
better chelating power as compare to Withania
somnifera while hydrogen peroxide scavenging
potential found better at high dose with Aloe vera
but at low dose Withania somnifera possesses better activity. The present
findings of the study suggest Withania somnifera could be a potential source of natural
antioxidant that could have great importance as therapeutic agents when compare
to Aloe vera.
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