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 Mandepudi

2

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

Journal Impact Factor (2013): 5.8376 (Calculated by GISI)

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© I A E M E



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) =� ��

�� 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) = � ��

��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 50oC 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



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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



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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





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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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