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INTERNATIONAL RESEARCH JOURNAL OF PHARMACY

ISSN 2230

8407

Available online http://www.irjponline.com

Research Article

IN-VITRO ANTIOXIDANT ACTIVITY OF GOSSYPIUM HERBACEUM LINN.

Sharma Pravesh Kumar

1*

, Sisodia Sidhraj Singh

2

, Naruka Puspendra Singh

2

, Porwal Mayur

1

1

Sanjeevan College of Pharmacy, Dausa, Rajasthan, India

2

B. N. College of Pharmacy, Udaipur, Rajasthan, India

Article Received on: 13/05/11 Revised on: 25/06/11 Approved for publication: 06/07/11

*Pravesh kumar Sharma, Sanjeevan College of Pharmacy, Dausa – 303303 Rajasthan, India E-mail: pravesh_pharmacy@rediffmail.com

ABSTRACT

The present study investigated In-vitro antioxidant activity of hydroalcoholic leaves extract (70: 30) of Gossypium herbaceum Linn. to provide scientific basis for traditional usage of this plant. TheIn-vitro antioxidant activity was evaluated by determining reducing power, total flavonoids and total phenolic contents using standard assay methods. The ability of the extract to scavenge 1, 1-diphenyl-2-picrylhydrazyl (DPPH) was also assessed using spectroscopic method. Ascorbic acid was used as standard antioxidant and positive control. The DPPH radical scavenging activity of the extract was increased with the increasing concentration. The IC50 values for DPPH assay of

Gossypium herbaceum and ascorbic acid were found to be 44.69µg/ml and 13.80µg/ml respectively. The reducing power of the extract was

found to be concentration dependent. Total flavonoid content in the extract was found to be 410 ± 0.74 mg Quercetin equivalents/g of dry material. Total phenolic content in Gossypium herbaceum was found to be 5.86 ± 0.75 mg Gallic acid equivalents/g of dry material. The results obtained from this study indicate that Gossypium herbaceum is a potential source of antioxidants and thus could prevent many radical diseases.

KEYWORDS: Antioxidant, DPPH, Reducing power, Phenolic content, Flavonoid content, Gossypium herbaceum.

INTRODUCTION

Scientific evidence suggests that under oxidative stress conditions, oxygen radicals such as superoxide anion (O2●─), hydroxyl radical (●OH) and proxyl radicals

(H2O2) are produced in biological systems. These oxygen

radicals are called Reactive Oxygen Species (ROS) and they can lead to oxidative damage to cellular components such as proteins, lipids and DNA1. Free radicals are reactive molecules involved in many physiological processes such as atherosclerosis, ischaemic heart disease, ageing, inflammation, diabetes, cancer, immunosuppression, neurodegenerative diseases2,3. Although several synthetic antioxidants such as butylated hydroxyl anisole (BHA) and butylated hydroxyl toluene (BHT) are available they are quite unsafe4. Therefore, in recent year considerable attention has been directed towards identification of natural antioxidants that may be used for human consumption.

Gossypium herbaceum Linn. belongs to family Malvaceae, commonly known as Kapas or cotton5. Gossypium herbaceum occurs in Middle East countries, Central Asia and Western India. The area of its greatest variability is found in Baluchistan, Iran, Afghanistan and Russian Turkestan. The cultivated types are found in India, China and Middle East countries6. The leaves are emollient, mucilaginous, haematinic, diuretic, cooling,

constipating and used in gastric irritation, diarrhoea, dysentery, dysuria, rheumatoid arthritis and Otalgia7. Gossypium herbaceum is reported as traditional medicine plant with the unique properties like antifertility8, antispermatogenic, antitumor9, abortifacient, contraceptive10, antidibetic, antiviral5 and antibacterial activity11. It is reported in the treatment of tooth pain also12. In the current study, In vitro experiments were conducted to determine possible antioxidant effect of hydroalcoholic leaves extract of Gossypium herbaceum. MATERIALS AND METHODS

L-ascorbic acid, 1, 1-diphenyl-2-picryl-hydrazil (DPPH), Quercetin, Gallic acid, Folin-ciocalteau reagent were procured from Sigma Chemical Co., USA. All other chemicals, reagents and solvents were of analytical grade available commercially. UV spectrophotometer (Shimadzu 1700), Incubator (Insif, India), Centrifuge (Remi, India) were the instruments used for the study. Plant Material

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Preparation of Extract

100 gm coarse powder of shade dried leaves of Gossypium herbaceum were extracted with ethanol: water (70: 30) using Soxhlet apparatus for 48 hrs. The obtained extract was concentrated under reduced pressure in a rotary vacuum evaporator and stored for further studies at 4˚C. The percentage yield of extract was found to be 25%.

Qualitative Phytochemical Investigation

The hydroalcoholic extract of Gossypium herbaceum leaves were subjected to qualitative chemical analysis to detect the presence of various Phytoconstituents13, 14, 15.

IN VITRO ANTIOXIDANT ACTIVITY DPPH Radical Scavenging Effect

In vitro DPPH scavenging activity was measured by the spectrophotometric method of Blois (1958). To a methanolic solution of DPPH (200 µM), 0.05 ml of the test compounds dissolved in water were added at different concentration (100-500 µg/ml). An equal amount of water was added to the control. After 20 min, the decrease in the absorbance was read at 517 nm and the percentage inhibition calculated by using the formula (1)16.

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Reducing Power Assay

The reducing power of the aqueous extract was carried out by adapting the method of Oyaizu (1986). About 2.5 ml of different concentrations of the plant extract (100-500µg/ml), were mixed with 2.5 ml of phosphate buffer (0.2 M, pH 6.6) and 1% potassium ferricyanide. The mixture was incubated at 50˚C for 20 min, then rapidly cooled, mixed with 2.5 ml of 10% trichloroacetic acid and centrifuged at 3000 rpm for 10 minutes. About 2.5 ml of supernatant was taken and 2.5 ml of distilled water and 0.5 ml of 0.1% ferric chloride were added to it, mixed well and allowed to stand for 10 minutes. The absorbance was measured at 700 nm17.

Total Phenolics Content (TPC)

Total phenolics were quantified and expressed as Gallic acid equivalants according to a method proposed by Singleton et al., (1999). About 3.9 ml of distilled water and 0.5 ml of Folin-ciocalteau reagent were added to 0.1 ml of the extract in a tube and incubated at room temperature for 3 min. 2 ml of 20% sodium carbonate was added to this and kept at boiling water bath for 1 min. The blue colour formed was read at 650 nm18. Total Flavonoids Content (TFC)

To 0.1 ml of the extract, distilled water was added to make the volume to 5 ml and 0.3 ml of 5% NaNO2 was

added. About 3 ml of 10% AlCl3 was added 5 min later

and after 6 min, 2 ml of 1 M NaOH was added to it. The

absorbance was measured at 510 nm. Quercetin (50-250 µg/ml) was used as a standard19.

RESULTS

The phytochemical screening of the crude powdered leaves of Gossypium herbaceum showed the presence of carbohydrates, tannin, phenolic compounds, flavonoids, saponins, glycosides and steroids.

Several concentrations, ranging from 50 to 250 µg/ml of the hydroalcoholic extract of Gossypium herbaceum were tested for their antioxidant activity in different In-vitro models. It was observed that free radicals were scavenged by the test compound in a concentration dependent manner in all the models.

The percentage inhibition showed decrease in concentration of DPPH radical due to the scavenging ability of extract and ascorbic acid (standard). The IC50

values of Gossypium herbaceum and ascorbic acid were found to be 44.69µg/ml and 13.80µg/ml respectively (Table 1 and Fig. 1).

In-vitro antioxidant activity of Gossypium herbaceum by reducing power assay indicated that increase in absorbance with concentration confirms the reducing power of Gossypium herbaceum and it was compared with the reducing power of ascorbic acid (standard) (Table 2 and Fig. 2).

Total phenolic content in Gossypium herbaceum was found to be 5.86 ± 0.75 mg Gallic acid equivalents/g of dry material (Fig. 3).

Total flavonoid content in the extract was found to be 410 ± 0.74 mg Quercetin equivalents/g of dry material (Fig. 4).

DISCUSSION

Concerns over the safety of synthetic antioxidants have shifted the global interests towards exploration of antioxidant compounds from natural sources. A plethora of phenolic compounds extracted from several plant species have been reported to possess strong antioxidant activities. Phenolic compounds are ubiquitously present in plants, and when plants are consumed as foods, these phytochemicals contribute to the intake of natural antioxidants in the diets of human as well as animals. Out of all the phenolics, the flavonoids belong to a large family of compounds with a common diphenyl propane structure (C6C3C6) with different degree of

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Oxidative stress has been implicated in the pathology of many diseases and conditions including diabetes, cardiovascular disease, inflammatory conditions, cancer and ageing21. Antioxidant may offer resistance against the oxidative stress by scavenging the free radicals, inhibiting the lipid peroxidation and by many other mechanisms and thus prevent disease22. Free radicals are chemical entities that can exist separately with one or more unpaired electrons. The generation of free radicals can bring about thousands of reactions and thus cause extensive tissue damage. Lipids, proteins and DNA are susceptible to attack by free radicals23.

The 1, 1 - diphenyl –2– picryl hydrazyl (DPPH) radical was widely used as the model system to investigate the scavenging activities of several natural compounds such as phenolic and anthocyanins or crude mixtures such as the ethanol extract of plants. DPPH radical is scavenged by antioxidants through the donation of proton forming the reduced DPPH. The color changes from purple to yellow after reduction, which can be quantified by its decrease of absorbance at wavelength 517 nm. Radical scavenging activity was increased with increasing percentage of the free radical inhibition. DPPH is relatively stable radical. The assay is based on the measurement of the scavenging ability of antioxidants towards the stable radical DPPH which reacts with suitable reducing agent. The electrons become paired off and solution loses color stochiometrically depending on the number of electrons taken up16. DPPH was used to determine the proton radical scavenging action of extract of Gossypium herbaceum and ascorbic acid (standard), because it possesses a proton free radical and shows a characteristic absorbance at 517 nm. From the present results, it may be postulated that Gossypium herbaceum reduces the free radical to corresponding hydrazine when it reacts with hydrogen donors in antioxidant principle24. In the present study the IC50 values of Gossypium

herbaceum and ascorbic acid were found to be 44.69µg/ml and 13.80µg/ml respectively.

The reducing capacity of a compound may serve as a significant indicator of its potential antioxidant activity25. However, 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 abstractions and radical scavenging26. For the measurements of the reductive ability we investigated the Fe3+ to Fe2+ transformation in the presence of hydroalcoholic extract of Gossypium herbaceum17. The reducing power indicated that increased in absorbance with concentration showed that Gossypium herbaceum have reducing power

and compare with ascorbic acid (standard) as shown in Fig. 2.

The present investigation provides a comprehensive profile of the antioxidant activity of hydroalcoholic extract of Gossypium herbaceum leaves; with respect to its phenolic content. Total phenolic content in Gossypium herbaceum was found to be 5.86 ± 0.75 mg Gallic acid equivalents/g of dry material. Total flavonoids in the extract were found to be 410 ± 0.74 mg Quercetin equivalents/g of dry material.

CONCLUSION

There is a strong need for effective antioxidants from natural sources as alternatives to synthetic food additives in order to prevent deterioration of food, drug and cosmetics. The results of the present study showed that the leaves extract of Gossypium herbaceum Linn. contains phenolics which correspond to antioxidant activity. A search for its active constituents would be interesting and deserve further studies to discover isolated chemical constituents for antioxidant activity. ACKNOWLEDGEMENT

The authors are thankful to the authority of B.N. College of Pharmacy, Udaipur, Rajasthan for providing necessary facilities for the present study and also thankful to Dr. S. S. Katewa, Professor and Head, Department of Botany, Mohanlal Sukhadia University, Udaipur (Rajasthan) for his guidance in identification of G. herbaceum.

REFERENCES

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6. Atal, CK, Kapoor, BM. Cultivation and Utilization of Medicinal Plants. New Delhi: CSIR Publication; 1982. P. 576.

7. Indian medicinal plants: a compendium of 500 species. 3rd ed. Orient Longman Ltd; 2001. P.100-103.

8. Garratt LC, Janagoudar BS, Anthony P, Davey MR, Power JB, Lowe KC. Hemoglobin-stimulated growth and antioxidant activities in cultured cotton cells. Free Radic Biol Med 2001; 31:1156-62.

9. Lee R, Lin JY. Antimutaganic activity of extracts from anticancer drugs in chinese medicine. Mutation Res 1988; 204 (2):229-234.

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11.Reddy UM, Reddy MM. Antibacterial activity of leaf extract of Gossypium herbaceum. Geo Bios 1981; 8 (6):277-278.

12.Hebber SS, Harsha VH, Shri Patni V, Hegde GR. Ethno medicine of Dharwad district in Karnataka, India: Plants used in oral health care. Jour of Ethnopharmacology 2004; 94:261-266. 13.Khandelwal KR. Preliminary Phytochemical Screening. In:

Practical Pharmacognosy, 10th Ed., New Delhi: Nirali Prakashan; 2003. P.149-156.

14.Pulok KM. Phytoconstituents and their Chemical analysis. In: Quality control of herbal drugs, 1st Ed., New Delhi: Business Horizons; 2005. P.305-312.

15.Kokate CK, Purohit AP, Gokhale SB. Pharmacognosy. 22nd Ed., New Delhi: Nirali Prakashan; 2003. P.108-109.

16. Blois MS. Antioxidant determination by the use of stable free radical. Nature1958; 81:1199-2000.

17.Oyaizu M. Studies on products of browning reaction: antioxidant activities of products of browning reaction prepared from glucosamine. Jpn J Nutr1986; 44:307-315.

18.Singleton VL, Orthofer R, Lamuela-Raventos RM. Analysis of total phenols and oxidation substrates and antioxidants by means of Folin-Ciocalteau reagent. Methods Enzymology 1999; 299:152-177.

19.Suseela V, Gopalkrishnan VK, Yadav RH. Free radical scavenging potential of aqueous extract of Artemisia nilagiria

(Clarke) PAMP. Leaves. Adv Pharmacol Toxicol 2009; 10:113-118.

20.Sharma RK, Chatterji S, Rai DK, Mehta S, Rai PK, Singh RK, Watal G, Sharma B. Antioxidant activities and phenolic contents of the aqueous extracts of some Indian medicinal plants. J Med Plant Res2009; 3:944-948.

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Table 1. In-Vitro Antioxidant Activity by DPPH Assay

Extract Inhibitory concenteration (IC 50 µg/ml)

Gossypium herbaceum 44.69

Ascorbic acid 13.80

Table 2. Reducing Power of Hydro-alcoholic extract of G. herbaceum

Concentration (µg/ml) Absorbance (700 nm)*

Ascorbic acid Gossypium herbaceum

50 0.38 ± 0.1014 0.12 ± 0.0733

100 0.68 ± 0.0838 0.36 ± 0.07

150 0.98 ± 0.1873 0.63 ± 0.0793

200 1.23 ± 0.2112 0.84 ± 0.0321

250 1.38 ± 0.0971 0.96 ± 0.1327

*Each value represents mean ± SEM

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Figure 2: Antioxidant activity of different concentration of hydroalcoholic extract of Gossypium herbaceum and Ascorbic acid in reducing power assay. Each value represents mean ± SEM

Figure 3: Standard curve of Gallic acid

Figure 4: Standard curve of Quercetin

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