Evaluation of Antioxidant Activity of the Aerial Parts of the Abutilon indicum (Linn) Sweet (Malvaceae)

S.B. Datir¹*, A.M. Patel¹, A.K. Patel¹, P.P. Patil², F.I. Rohit², V.D. Thorat², D.K. Bharti² and A.B. Ganjare³

¹Department of Pharmacognosy, S. N. D. College of Pharmacy, Yeola, (M. S.)

²Department of Pharmacology, S. N. D. College of Pharmacy, Yeola, (M. S.)

³Department of Pharmacognosy, Pravara Rural College of Pharmacy, Loni, (M. S.)

ABSTRACT:

Abutilon indicum (Linn) sweet (Malvaceae) is a weed found on way side and at west place throughout the India, commonly known as Atibala. Phytochemical study of the plant revealed that the plant contains various phytoconstituent like flavonoids, tannins and phenolic compounds. Plant may give antioxidant activity as these phytoconstituents are reported to have antioxidant potential. Antioxidant activity of the various extracts of the aerial parts of A. indicum viz. petroleum ether extract, chloroform extract, methanol extract and aqueous extract was evaluated by using DPPH assay method, ABTS assay method and FRAP assay method. Total phenolic content of all the extracts was also determined. Results showed that methanol extract shows potent antioxidant activity and highest phenolic content.

KEYWORDS: Abutilon indicum, DPPH assay, ABTS assay, FRAP assay, total phenolic content

INTRODUCTION:

Abutilon indicum (Linn) sweet (Malvaceae) is a weed found on way side and at west place throughout the India, commonly known as Atibala. Phytochemical study of the plant revealed that the plant contains various phytoconstituent like flavonoids, tannins and phenolic compounds.¹

The biochemistry of oxidative stress and hydroperoxide metabolism in mammalian organs, have been a focus of research form last few decades.^{2, 3} The nature of various biological oxidants was found to cover large ranges in biological lifetime, in concentration, and in the occurrence in cells and organs. Aerobic metabolism entails the production of reactive oxygen species, even under basal conditions; hence there is a continuous requirement for inactivation of these reactive oxygen species. The steady-state of prooxidants and antioxidants may be disrupted. A disbalance in favor of the prooxidants and disfavoring the antioxidants, potentially leading to damage, has been called ‘oxidative stress’.^{4, 5} Such damage may afflict all types of biological molecules, including DNA, lipids, proteins and carbohydrates. Thus, oxidative stress may be involved in processes such as mutagenesis, carcinogenesis, membrane damage, lipid peroxidation, protein oxidation and fragmentation.⁶ In view of the variety in prooxidants, it is not surprising that nature has evolved a battery of different types of antioxidants.^{7, 8} Experimental studies revealed that cells and organisms require defense against oxidants, without which survival under aerobic conditions would be jeopardized⁹.

MATERIALS AND METHODS:

Plant material collection and authentication

Samples of the aerial part of A.indicum (Linn) sweet were collected from wild sources in September 2009 from Ahmednagar district (Maharashtra) shade dried at room temperature and authenticated by Mr. T. Chakraborty. Botanical Survey of India, Pune (Voucher Specimen No. ABIAM1).

Extraction

The powdered aerial part (200 g) was subjected for successive extraction in Soxhlet extractor using various solvents viz. petroleum ether (60-80oC), chloroform and methanol10. The residue of the plant material obtained after the successive extraction was extracted using water in reflux condenser. All the extracts were concentrated and vacuum dried for further screening. Percentage yield of all four extracts viz. petroleum ether extract (PEE), chloroform extract (CHE), methanol extract (ME) and aqueous extract (AQE) was found to be 2.14% w/w, 2.31% w/w, 2.52% w/w and 3.41% w/w respectively.

Chemicals Used.

Ascorbic acid, 1,1-Aza-bis-benzothiazole sulfonic acid (ABTS), 1, 1-Diphenyl-2-picrylhydrazyl (DPPH), 2, 4, 6-Tri (2-pyridyl)-s-triazine (TPTZ), Trolox, potassium per sulphate. All these chemicals necessary for the antioxidant activity were purchased from Sigma (USA).

Evaluation of antioxidant potential

DPPH Assay^{9, 10}

7.886 mg of DPPH was accurately weighed and dissolved in 100ml methanol to obtain 200 µM solution of DPPH. Different concentrations of extracts (25-100 µg/ml) were prepared. To 2 ml methanol solution of DPPH, 2 ml of sample solution was added. The mixture was incubated in dark at room temp for 15 min. The degree of free radical scavenging activity in presence of different concentration of extracts and their absorbance were measured colorimetrically at 517 nm. The degree of free radical scavenging activity was expressed as percentage inhibition.

% inhibition = {(A control – A sample)/ (A control)} X 100

A control – Absorbance of DPPH alone

A sample – Absorbance of DPPH along with different concentrations of extracts.

IC50 was calculated from equation of line obtained by plotting a graph of concentration (mcg/ml) verses % inhibition.

ABTS Assay^{9, 11}

ABTS^•+ radical cation is generated by reacting 7 mM ABTS and 2.45 mM potassium peroxodisulfate via incubation at room temperature (23 ⁰C) in the dark for 12–16 h. The ABTS^•+ solution was diluted with 80% HPLC-grade ethanol to an absorbance of 0.700 ± 0.040 at 734 nm and equilibrated at 30 ⁰C. Plant extracts were diluted with distilled water or 80% methanol, such that after introduction of a 30 µl aliquot of each dilution into the assay, it produced from 20% to 80% inhibition of the blank absorbance. To 3 ml of diluted ABTS^•+, 30 µl of the plant extract solution was added and mixed thoroughly. The reactive mixture was allowed to stand at room temperature for 6 min and the absorbance was recorded immediately at 734 nm. Trolox standard solutions (concentrations from 0 to 2.5 µg/ml) in 80% ethanol were prepared and assayed using the same conditions. Appropriate solvent blanks were run in each assay. The percent of inhibition of absorbance at 734 nm was calculated and plotted as a function of concentration of Trolox for the standard reference data. The absorbance of the resulting oxidized solution was compared to that of the calibrated Trolox standard. Results were expressed in terms of Trolox equivalent antioxidant capacity (TEAC, µM Trolox equivalents per g dry weight of plant)

Ferric reducing antioxidant potential assay^{9, 12} (FRAP)

An aliquot (200 µl) of an extract (with appropriate dilution, if necessary) was added to 3 ml of FRAP reagent (10 parts of 300 mM sodium acetate buffer at pH 3.6, 1 part of 10 mM TPTZ solution and 1 part of 20 mM FeCl₃•6H₂O solution) and the reaction mixture was incubated in a water bath at 37 0C. The increase in absorbance at 593 nm was measured at 30 min. The antioxidant capacity based on the ability to reduce ferric ions of the extract was expressed as µM Trolox equivalents per gram of plant material on dry basis.

Total phenolic content¹¹

An aliquot of 100 µl of an extract was mixed with 2.5 ml of Folin–Ciocalteu phenol reagent (10 x dilutions) and allowed to react for 5 min. Then 2.5 ml of saturated Na₂CO₃ solution was added and allowed to stand for 1 h before the absorbance of the reaction mixture was read at 725 nm. The total polyphenol contents (TPC) of the extract was expressed as mg gallic acid equivalents per gram of plant material on dry basis (db).

Phytochemical screening of the crude extracts.

Various Phytochemical studies including test for carbohydrates, proteins, alkaloids, glycosides, steroids, flavonoids, tannins and phenolic compounds were carried out.¹³

RESULTS AND DISCUSSION:

In DPPH assay methanol extract followed by aqueous extract shows better percentage inhibition (fig.1) than other extracts. IC50 value shown by the methanol extract was found to be lowest as compared to other extracts (fig.2; Table 1). Results of ABTS assay depends on the percentage inhibition shown by the different concentration of trolox, which is used as a standard in this assay. Depending upon the percentage inhibition shown by the various extracts (Table 2) and comparison with the percentage inhibition shown by the various concentration of trolox, trolox equivalent antioxidant concentration (TEAC) of the extracts was determined. It was found that in ABTS assay, TEAC of the methanol extract was highest (1.339 µg/ml trolox equivalent per g dry weight of plant) than other extracts of the A. indicum (fig.3; Table 3). In FRAP assay also, TEAC of the methanol fraction was found to be highest (1.090 µg/ml trolox equivalent per g dry weight of plant) than other extracts of the A. indicum (fig.4; Table 5). The TEAC for each extract were calculated by taking absorbance of each extract at 593 nm (Table 4) as per the procedure described. Higher the absorbance higher will be the TEAC value. Highest TEAC is related to the highest antioxidant potential. Thus methanol extract of the aerial parts of A. indicum has highest antioxidant potential than the other extracts. Total phenolic content estimation shows that methanol extract followed by aqueous extract showed highest phenolic content (Fig. 5, Table 6.). Thus, we can conclude that due to highest phenolic content methanol extract showed strong antioxidant activity.

Fig. 1. Percentage inhibition of various extracts of the aerial parts of A. indicum at different concentrations.

DPPH Antioxidant Assay:

Table. 1. IC₅₀ values for various extracts of A. indicum

Name of extract	IC 50 Values.(µg/ml)
Ascorbic acid.	11.25
Petroleum ether extract	96.25
Chloroform extract	83.75
Methanol extract	17.5
Aqueous extract	37.5

Fig. 2. IC₅₀ values for various extracts of the aerial parts of A.indicum

ABTS Antioxidant Assay:

Table. 2. Absorbance and % inhibition of various extracts of A. indicum

A .indicum extracts (30µl of 100 µg/ml )	Absorbance	% Inhibition
ABTS solution	0.720	0.00
Petrolum ether extract	0.557	6.9
Chloroform extract	0.706	12.5
Methanol extract	0.170	76.38
Aqueous extract	0.370	48.61

Fig. 3. TEAC, µg/ml trolox equivalent per g dry weight of plant by ABTS assay method

Table. 3. TEAC, µg/ml trolox equivalent per g dry weight of plant

Name of extract	TEAC µg/ml trolox equivalent per g dry weight of plant
Petroleum ether extract	0.125
Chloroform extract	0.205
Methanol extract	1.339
Aqueous extract	0.912

Ferric reducing Antioxidant Potential (FRAP) Assay:

Table 4. Absorbance of various extracts of the aerial part of A. indicum

A. indicum Extracts (200µl of 50 µg/ml )	Absorbance
FRAP solution	0.089
Petroleum ether	0.131
Chloroform	0.121
Methanol	0.240
Aqueous.	0.175

Fig. 4.TEAC, µg/ml trolox equivalent per g dry weight of plant by FRAP assay method

Table 5. TEAC, µg/ml trolox equivalent per g dry weight of plant

Name of extract	TEAC µg/ml trolox equivalent per g dry weight of plant
Petroleum ether	0.520
Chloroform	0.505
Methanol	1.090
Aqueous	0.556

Total phenolic content determination:

Table 6. Concentration of phenolic content of various extracts of the aerial part of A.indicum.

Concentration µg/ml	Concentration of phenolic content of various extracts of the aerial part of A.indicum. (µg/ml).
	PEE	CHE	ME	AQE
50	3.2	3.1	13.2	8.70
100	6.8	4.4	21.5	14.20

Table 7. Phytochemical screening of the various extracts of A. indicum

Test performed for	Petroleum ether extract (PEE)	Chloroform extract (CHE)	Methanol extract (ME)	Aqueous extract (AQE)
Carbohydrates	-	-	+	+
Proteins	-	+	+	+
Alkaloids	+	+	+	+
Glycosides	-	+	+	+
Steroids	+	-	-	-
Flavonoids	-	-	+	+
Tannins and phenolic compounds	-	-	+	+

Phytochemical screening performed on various extracts of A. indicum shows that methanol extract contains flavonoids, tannins and phenolic compounds (Table 7.) which are generally showing antioxidant property. Thus, we can conclude that the polar constituents in the plants like flavonoids, tannins and phenolic compounds may be responsible for the antioxidant activity of the plant.

Fig. 5. Concentration of phenolic content of various fractions of the aerial part of A. indicum (µg/ml).

CONCLUSION:

The result shows that the methanol extract of the aerial parts of A. indicum is having potent antioxidant activity. Phytochemical investigation reveals the presence of phenolic compounds in highest concentration in methanol extract itself. Thus we can conclude that higher the phenolic content higher is the antioxidant activity of the plant. Phenolic compounds like flavonoids and tannins present in the methanol extract of the plant may be responsible for the highest antioxidant activity shown by the methanol extract as compared to the other extracts of the plant.

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Received on 10.04.2010

Accepted on 31.05.2010

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Research J. Pharmacology and Pharmacodynamics. 2(5): Sept.-Oct. 2010, 324-327