Experimental studies on antioxidant properties of calotropis gigantea

International Journal of Advanced Research in Engineering and Technology (IJARET), ISSN
0976 – 6480(Print), ISSN 0976 – 6499(Online) Volume 4, Issue 4, May – June (2013), © IAEME
168
EXPERIMENTAL STUDIES ON ANTIOXIDANT PROPERTIES OF
CALOTROPIS GIGANTEA SOLVENT EXTRACTS
David Mandepudi1,2
, Bharath Kumar Ravuru3
, Bhavani Mandepudi2
1
Faculty of Biotechnology, Jawaharlal Nehru Technological University-Hyderabad,
Hyderabad-500085, Andhra Pradesh, India
2
Department of Biotechnology, School of Engineering, Sir Padampat Singhania University,
Udaipur-313601, Rajasthan, India
3
School of Biotechnology, Vignan University, Guntur – 522213, Andhra Pradesh, India
ABSTRACT
The active metabolites from plant sources had been explored and extracted to serve
the societies as part of the medicine either internally or externally for the treatment of
ailments. In the similar lines, the Calotropis gigantea, has been investigated for antioxidant
properties using ethanol, methanol, chloroform and aqueous solvent extracts of leaves, buds
and flowers with the help of popular antioxidant testing methods of DPPH and hydrogen
peroxide free radical scavenging activities and an additional reducing power test. In the
results, many of the solvent extracts were reaching to variable saturation levels beyond the
respective extract concentrations of 5µg/ml. Further, comparable free radical scavenging
activities for some of the solvent extracts like, ethanol and methanol leaves, buds & flowers
extracts in addition to methanol, chloroform and aqueous extracts of leaves, buds and
flowers were observed with respect to free radical scavenging activities of the standard
antioxidants, ascorbic acid and quercetin in the tests. However the reducing power of these
solvent extracts of Calotropis gigantea leaves, buds and flowers were very low in comparison
to that of the standard antioxidants, ascorbic acid and BHT. So, the observed free radical
scavenging activities of the solvent extracts may be due to the presence of the detected active
metabolites like, alkaloids, tannins, polyphenols etc. Though these results favor the use of
Calotropisgigantea as part of the traditional medicine due to their auxiliary antioxidant
properties, similar studies may be carried out for the better utilization and management of the
Calotropis species.
INTERNATIONAL JOURNAL OF ADVANCED RESEARCH IN
ENGINEERING AND TECHNOLOGY (IJARET)
ISSN 0976 - 6480 (Print)
ISSN 0976 - 6499 (Online)
Volume 4, Issue 4, May – June 2013, pp. 168-180
© IAEME: www.iaeme.com/ijaret.asp
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169
Keywords: Antioxidant, Ayurveda, drugs of abuse, Prodrug, Polyphenols, Calotropis
gigantean
1. INTRODUCTION
The molecular agents that prevent the oxidation of other molecules either by stopping
the transfer of electron or hydrogen are known as the antioxidants. But then, these antioxidant
molecules get themselves oxidized often acting as reducing agents [1]. While the oxidation
and reduction reactions are very important set of reactions in biological systems, the soldier
like antioxidants play a vital role in the sustenance of the life [2-3] on the planet, Earth. On
the other hand, the reactive species and free radicals were known to cause severe damage to
the cellular components of the life systems especially of human and may lead to various
ailments like early ageing, oxidative stress related with Alzheimer's disease,Parkinson's
disease,diabetes, rheumatoid arthritis, and neuro degeneration in motor neuron diseases
[2,3,4-13]etc. Since, these oxidation and reduction reactions were found to have the greater
significance in the realm of biological systems with antioxidant mechanism, plants and
animals established complex systems of antioxidants like glutathione, vitamins – C, D, A, K
& E; enzymes like catalase, superoxide dismutase, peroxidases, β-carotene, lutein, lycopene,
vitamin A, vitamin C, vitamin E, flavonoids, polyphenols, ascorbic acid [14-17] etc. to
regulate and check the unnecessary burst of any reactive species [11] like hydrogen peroxide
(H2O2), hypochlorous acid (HClO) and free radicals like hydroxyl radical (.
OH) and
superoxide anion (O2
-
). Owing to these facts, synthetic antioxidants like
butylatedhydroxytoluene (BHT) and buytlatedhydroxyanisole (BHA)were also developed or
derived in the form of health supplements or drug molecules to serve the purpose by various
pharmaceutical and health care companies [7,18]. However the natural antioxidants are the
best means for the human consumption and in the lines of acceptability for the environment
without the creation of any xenobiotic compounds [19] by the synthetic processes. So, these
facts elucidated the need to search for the novel and bioactive compounds [20] like
antioxidants directing the scientists to investigate the plant based solvent extracts to isolate
and identify the antioxidants that may be used as dietary supplements [21-23] or the drugs
that can save the life. In such scenario, many plants like, Azadiractaindica, Meliaazedarach,
Digitalis purpurea, Digitalis lanata, Daturastramonium, Atropa belladonna,
Papaversomniferum, Catharanthusroseus, Colchicum autumnale, erythroxylon coca etc.
were investigated for potential active metabolites likeazadiractin, cardenolides,
parasympatholytic agents, analgesics, anticancer agents, antigout agent, local anesthetics etc.
for the use of medicine and food supplements [20,24]. In the similar lines, the Calotropis sp.
known for its poisonous nature for centuries and for medicine in the traditional medical
practices [25-27] of several ethnic groups has been investigated for the possible active
metabolites using the residual solvent extracts of Calotropis gigantea leaves, buds and
flowers.
2. MATERIALS AND METHODS
2.1 Plant material & solvent extraction
The Calotropis gigantia leaves, flowers and buds were collected [28] and processed
to obtain the respective dry powders and these dry powders were added to the solvents of
ethanol, methanol and chloroform and extracted the active metabolites by solvent extraction

170
procedure and named them as the solvent extracts of ethanol leaves (EL), ethanol flowers
(EF), ethanol buds (EB); methanol leaves (ML), methanol flowers (MF), methanol buds
(MB); Chloroform leaves (CL), chloroform flowers (CF), chloroform buds (CB). The
aqueous extracts were also prepared by boiling the plant material (powder) individually and
processed [28] to obtain the final extracts of Aqueous leaves (AL), Aqueous flowers (AF)
and Aqueous buds (AB). These extracts were stored in the refrigerator until use in sterile
bottles.
2.2 Antioxidant Activity of solvent extracts
The extracted solvent extracts of leaves, buds and flowers of Calotropis gigantea
were tested for the detection and estimation of antioxidant activity in terms of free radical
scavenging activity using DPPH [29-32] and Hydrogen peroxide [33-34] scavenging assay
methods.
2.3 The DPPH free radical scavenging activity
The DPPH solution (3ml) of 0.02mg/ml in methanol was taken in three sets of clean
& dry test tubes and the solvent extracts of 1 ml were added respectively with an increasing
concentration in the range of (0-25µg/ml) in multiples of 5µg/ml. Similarly a standard
antioxidant, ascorbic acid of 1 ml was also added separately to the 3 ml DPPH solution in
three sets of test tubes and used as a reference for the assay. All those test tubes with the
solutions were vortexes and incubated at room temperature for 30 minutes under dark
conditions. The absorbance of these resulting solutions were recorded at 517nm using UV-
Visible double beam spectrophotometer. So the assay was performed in triplicates and the
results were expressed as mean values and the standard derivations were also calculated.
The DPPH free radical scavenging activity of the standard, ascorbic acid and the solvent
extracts were calculated using the equation (1).
(% ) DPPH Free Radical Scavenging Activity (DFRSA) =
ሺ ୅ೞି୅೐ሻ
୅ೞ
X100 (1)
Where As is the absorbance of the DPPH in the absence of test solution (solvent
extract/ascorbic acid) and Ae is the absorbance of the test solution.
2.4 Hydrogen peroxide free radical scavenging activity
Estimation of antioxidant activity of enzyme, catalase [35] was adopted to detect and
evaluate the hydrogen peroxide scavenging activity of solvent extracts of Calotropis gigantia
leaves, buds and flowers. The solvent extracts were taken as test solutions of 0.4ml with
varying concentrations in multiples of 5µg/ml in the concentration range of (5-25) µg/ml
separately in three sets of clean and dry test tubes and then added 0.6ml of 40mM hydrogen
peroxide solution (in 0.1M phosphate buffer of pH 7.4) to each test sample and incubated
them for 15 min under dark conditions. Similarly, a standard antioxidant solution of quercetin
was prepared and used for the assay following the above standard procedure with varying
concentration in multiples of 5µg/ml in the concentration range of (5-25)µg/ml. After the
incubation period, 1ml of dichromate solution in acetic acid (5% potassium dichromate
aqueous solution mixed with glacial acetic acid in the ratio of 1:3) was added to each solvent
extract test solution and standard quercetin solution separately and mixed them well. These
test solutions were further incubated in boiling water bath for 10min, where the characteristic
green color appeared in the test tubes. 2ml of deionized water (MilliQ water) was added to
each test solution and the resultant solution was used for the measurement of absorbance at

171
620nm using UV-Visible double beam spectrophotometer. The control sample was prepared
without any extract or standard antioxidant, containing only deionized water and the
hydrogen peroxide as well as dichromate solutions in parallel with test sample& standard
samples of quercetin. Hence the test was performed in triplicates and the measured
absorbance at 620nm for each sample was taken as mean value and used this data for further
analysis.
The (%) hydrogen peroxide free radical scavenging activity of solvent extracts & the
standard, quercetin were calculated as per the equation (2).
(% ) Hydrogen peroxide Free Radical Scavenging Activity (HFRSA) =
ሺ ୅ೃି୅೐ሻ
୅ೃ
X 100(2)
Where AR is the absorbance of control sample, and Ae is the absorbance of extract or
standard at 620nm.
2.5 Reducing power assay
The solvent extracts of Calotropis gigantea leaves, buds and flowers were evaluated
[36], where 1ml of the variable concentration of residual solvent extracts (0-1 mg/ml) were
mixed with 2.5ml of 0.2M phosphate buffer (pH 6.6) and 2.5ml of 1% potassium ferricyanide
(K3Fe CN6) solution respectively and then incubated the resulting solutions at 50o
C for 20
minutes. Added 2.5ml of 10% trichloroacitic acid to each reaction mixture in the test tubes
and then centrifuged at 3000g for 10 min where the supernatant of 1ml was withdrawn
carefully and mixed with 2.5ml of distilled water and further added 0.5ml of 0.1% ferric
chloride solution to get the final solution. The absorbance of the resulting solution for each
test sample was measured using UV-Visible double beam spectrophotometer at 700nm. The
ascorbic acid and BHT were taken as the standard antioxidants for positive controls following
the above procedure and the experiments were performed in triplicates. The mean absorbance
for each test sample was calculated and used the data for further analysis.
3. RESULTS
The free radical scavenging activity of Calotropis gigantea leaves, buds and flowers
were calculated using DPPH and hydrogen peroxide methods as the (%) reduction in the
absorbance and used them to plot the free radical scavenging activity profiles of Calotropis
gigantea leaves extracts, buds extracts and flowers extracts against their respective
concentrations as represented in the Figures 1-6. These profiles in the figures also include the
standard or reference antioxidant (Ascorbic acid for DPPH method and quercetin for H2O2
Method) profile, providing a means for the comparison of the solvent extract antioxidant
profiles of Calotropis gigantea leaves, buds and flowers. The saturation free radical
scavenging activities and the corresponding Effective Dosage for the 50% free radical
scavenging activities (ED50) were noted for all the solvent extracts and represented in the
Figures 7 & 8 with respect to DPPH and hydrogen peroxide methods. Similarly the reducing
power of the residual solvent extracts of Calotropis gigantea leaves, buds and flowers were
represented as the absorbance profiles against their respective variable concentrations as
shown in the Figures 9-11 along with the absorbance profiles of standards, ascorbic acid and
BHT. The reducing power of the standards were so high that the ascorbic acid test results
were diluted to 5% and the BHT results were diluted to 12% for the convenience. However
the data represented in the profiles and the figures were taken as the average of the triplicates.

0976 – 6480(Print), ISSN 0976 – 6499(Online) Volume 4, Issue 4, May
Fig.1 DPPH free radical scavenging activity p
6499(Online) Volume 4, Issue 4, May – June (2013), © IAEME
172
DPPH free radical scavenging activity profiles of Calotropis gigantea leaves extracts
DPPH free radical scavenging activity profiles of Calotropis gigantea buds extracts
DPPH free radical scavenging activity profiles of Calotropis gigantea flowers extracts
June (2013), © IAEME
leaves extracts
buds extracts
flowers extracts

Fig.4 Hydrogen peroxide free radical scavenging activity of
Fig.5Hydrogen peroxide free radical scavenging activity of
Fig.6 Hydrogen peroxide free radical scavenging activity of
173
Hydrogen peroxide free radical scavenging activity of Calotropis gigantea
extracts
extracts
Calotropis gigantea leaves
buds extracts
Calotropis gigantea flowers

Fig.7 Variations in the saturation of DFRSA of
radicals and the corresponding ED50 concentrations
Fig.8 Variations in the saturation of HFRSA of
peroxide radicals and the corresponding ED50 concentrations
Fig. 9 Comparative reducing power of
standard antioxidants, ascorbic acid and BHT
174
Variations in the saturation of DFRSA of Calotropis gigantea extracts on DPPH
radicals and the corresponding ED50 concentrations
Variations in the saturation of HFRSA of Calotropis gigantea extracts on hydrogen
peroxide radicals and the corresponding ED50 concentrations
Comparative reducing power of Calotropis gigantea leaves extracts along with the
extracts on DPPH
extracts on hydrogen
leaves extracts along with the

standard antioxidants, ascorbic acid
4. DISCUSSION
The free radical scavenging activity of many plant based extracts and the standard
antioxidants like ascorbic acid and quercetin were dependent on their concentration [37
however such free radical scavenging antioxidant properties of
extracts on free radicals like that of DPPH were found to be saturating (for concentrations
beyond 5µg/ml) as shown in the Figures 1
follows.
4.1 Antioxidant properties of Calotropis gigantea
The leaves extracts of Calotropis gigantea
values of free radical scavenging activity by 5µg/ml concentration whereas the standard
antioxidant, ascorbic acid followed a gradually increasing free radical
the saturation level at around 15µg/ml concentration. Conversely, the quercetin, the other
standard antioxidant was found (Figure 4) to reach the saturation levels of free radical
175
Comparative reducing power of Calotropis gigantea buds extracts along with the
standard antioxidants, ascorbic acid (AA) and BHT
Comparative reducing power of Calotropis gigantea flowers extracts along with the
antioxidants like ascorbic acid and quercetin were dependent on their concentration [37
however such free radical scavenging antioxidant properties of Calotropis gigant
beyond 5µg/ml) as shown in the Figures 1-6. The details of the results were discussed as
Calotropis giganteaLeaves extracts
Calotropis gigantea (Figures 1 & 4) reached the saturation
antioxidant, ascorbic acid followed a gradually increasing free radical scavenging activity to
buds extracts along with the
flowers extracts along with the
antioxidants like ascorbic acid and quercetin were dependent on their concentration [37-41],
Calotropis gigantea solvent
6. The details of the results were discussed as
(Figures 1 & 4) reached the saturation
scavenging activity to

176
scavenging activity on hydrogen peroxide by 5µg/ml concentration, but then its saturation
free radical scavenging activity was only of about half the free radical scavenging activity
saturation values of ascorbic acid (Figure 1). Similarly the Calotropis gigantea leaves
extracts recorded the higher (twice) free radical scavenging saturation activities in case of
ethanol, methanol & chloroform leaves extracts on DPPHradicals compared to that of
hydrogen peroxide radicals; whereas the aqueous leaves extracts exhibited higher free radical
scavenging activity on hydrogen peroxide compared to that of DPPH radicals (Figures 1, 4, 7
& 8). These results were also supported by the increasing reducing power of Calotropis
gigantea leaves extracts as shown in Figure 9 where the leaves extracts reducing power
profiles against the concentration were gradually increasing but then these profiles were very
low in reducing power in comparison to that of the standard antioxidants, ascorbic acid and
BHT. The effective dosage for 50% saturating free radical scavenging activity (ED50) as
shown in the Figures 7 and 8 for various Calotropis gigantea solvent extracts, brought out
ethanol leaves extract with lowest ED50 concentration of 2µg/ml on DPPHfreeradicals and
aqueous leaves extract with highest ED50 concentration of 4µg/ml on hydrogen peroxide free
radicals.
4.2 Antioxidant properties of Calotropis giganteaBuds extracts
The free radical scavenging activity of Calotropis gigantea buds extracts were shown
in the Figures 2, 7 & 4, 8 with respect to DPPH and hydrogen peroxide free radicals where
the methanol buds extract recorded higher percentage of free radical scavenging activity on
DPPH radicals (80%) and the least ED50 concentration was found as 2.2µg/ml with respect
to hydrogen peroxide radicals. However the ethanol buds extract has shown decreasing
activity on DPPH radicals beyond 5 µg/ml concentration whereas its converse profile was
observed on hydrogen peroxide radicals, otherwise all other buds extracts exhibited the
saturating free radical scavenging activity for the respective concentrations (>5µg/ml). These
results were also supported by the moderate and decreasing reducing power of the Calotropis
gigantea solvent extracts of leaves, buds and flowers as shown in the Figure 10.
4.3 Antioxidant properties of Calotropis giganteaFlowers extracts
The Calotropis gigantea flowers extract free radical scavenging activity with respect
to variation in their respective concentrations in the solvents were shown in the figures 3 & 6
where ethanol flowers extract exhibited higher and lower scavenging activities on DPPH
(76%) and hydrogen peroxide (15%) radicals respectively. On the other hand aqueous
flowers extracts had the least scavenging activity on DPPH (10%) free radicals and the higher
scavenging activity on hydrogen peroxide (41%) free radicals. Conversely, the chloroform
flowers extracts recorded moderately saturating free radical scavenging activity on both
DPPH and hydrogen peroxide free radicals whereas these chloroform flower extracts
reducing power profiles were found to exhibit lowest saturating reducing powers as shown in
the Figure 11. In another interesting finding, the methanol flowers extracts recorded
increasing free radical scavenging activity on hydrogen peroxide free radicals beyond the
concentration of 5 µg/ml. However the reducing power of methanol flowers extract was
found to be decreasing with increasing concentration whereas the reverse effects were found
in case of aqueous flowers extracts. These observed free radical scavenging activities of the
solvent extracts of Calotropis gigantea leaves, buds and flowers may be due to the bioactive
compounds like tannins, polyphenols and alkaloids [42-43] etc. present as the residual
components in these solvent extracts.

177
5. CONCLUSIONS
The study has provided an evidence for the antioxidant free radical scavenging
activities of Calotropis gigantea leaves, buds and flowers extracts of ethanol, methanol,
chloroform and aqueous solvent extracts. These results also demonstrated the comparable
free radical scavenging activities for some of the solvent extracts like, ethanol leaves extract,
methanol leaves extract, ethanol buds extract, methanol buds extract, ethanol flowers extract
and methanol flowers extract on DPPH radicals with that of the standard antioxidant, ascorbic
acid. Similarly, methanol, chloroform and aqueous extracts of leaves, buds and flowers also
exhibited the comparable free radical scavenging activity on hydrogen peroxide radicals with
that of the standard antioxidant, quercetin. However the reducing power of these solvent
extracts of Calotropis gigantea leaves, buds and flowers were very low in comparison to that
of the standard antioxidants like ascorbic acid and BHT. So these results support the use of
Calotropis as part of the traditional medicine to a considerable extent as these extracts
recorded the moderate antibiotic and wound healing properties [44-48] that may aid as part of
the treatment to terminate the pathogens and their effects on animals and humans.
ACKNOWLEDGEMENT
Dr. M Lakshmi Narasu, Professor & Dr. ArchanaGiri, Assistant Professor, Faculty of
Biotechnology, Jawaharlal Nehru Technological University, Hyderabad, Dr.
ArchanaGajbhiye, Head, Department of Biotechnology, Sir Padampat Singhania University,
Udaipur were acknowledged for their suggestions and critical comments on the research
work. Mr. Ashok Ghosh, President, Ms. Rinu Ghosh, Vice President, Prof. P.C. Deka, Vice
Chancellor and Prof. AchintyaChaudary, Dean, School of Engineering of Sir Padampat
Singhania University, Udaipur were also acknowledged and thanked for their financial
support and approvals for the research work.
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Experimental studies on antioxidant properties of calotropis gigantea

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