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Academic Journal of Plant Sciences 2 (2): 60-64, 2009 ISSN 1995-8986 IDOSI Publications, 2009

In-Vitro Antioxidant Activity of Leaves of Ipomoea fistulosa Linn.


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Kalpesh Gaur, 1M.L. Kori, 1L.K. Tyagi, 2R.K. Nema, 3C.S. Sharma and 4Priyanka Tripathi Geetanjali College of Pharmaceutical Studies, Udaipur, Rajasthan, India S.D. College of Pharmacy and Vocational Studies, Muzaffarnagar, Uttar Pradesh, India 3 Bhupal Nobles College of Pharmacy, Udaipur, Rajasthan, India 4 Shri R.N.S. Mahavidhyalaya (Pharmacy), Bhind, Madhya Pradesh, India
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Abstract: Free radicals are implicated for more than 80 diseases including Diabetes mellitus, arthritis, cancer, ageing etc. In treatment of theses diseases, antioxidant therapy has gained an utmost importance. Current research is now directed towards finding naturally occurring antioxidant of plant origin. In Indian system of medicine Ipomoea fistulosa is an important medicinal plant. The in-vitro antioxidant activity of various extracts of Ipomoea fistulosa leaves was investigated. The extracts and the reference standard, butylated hydroxyl toluene (BHT) were evaluated for DPPH, nitric oxide, superoxide and hydroxyl radical scavenging activity. The methanolic extract exhibited significant antioxidant activity but petroleum ether and chloroform extracts of Ipomoea fistulosa did not show any significant antioxidant activity in comparison with standard (BHT). Key words: DPPH, Nitric oxide, Butylated hydroxyl toluene, Superoxide and hydroxyl radical scavenging activity INTRODUCTION Majority of the diseases/disorders are mainly linked to oxidative stress due to free radicals [1]. Free radicals are fundamental to any biochemical process and represent an essential part of aerobic life and metabolism [2]. The most common reactive oxygen species (ROS) include superoxide (O2.) anion, hydrogen peroxide (H2O2), peroxyl (ROO.) radicals and reactive hydroxyl (OH.) radicals. The nitrogen derived free radicals are nitric oxide (NO.) and peroxynitrite anion (ONOO.). ROS have been implicated in over a hundreds of diseases states which range from arthritis and connective tissue disorders to carcinogenesis, aging, physical injury, infection and acquired immunodeficiency syndrome [3]. In treatment of these diseases, antioxidant therapy has gained an immense importance. Current research is now directed towards finding naturally occurring antioxidants of plant origin. Antioxidants have been reported to prevent oxidative damage by free radical and ROS, and may prevent the occurrence of disease, cancer and aging. It can interfere with the oxidation process by reacting with free radicals, chelating, catalytic metals and also by acting as oxygen scavengers [4-5]. Plant and plant products are being used as a source of medicine since long. The medicinal properties of plants have been investigated in the recent scientific developments throughout the world, due to their potent antioxidant activities, no side effects and economic viability [6]. Flavonoids and phenolic compounds widely distributed in plants which have been reported to exert multiple biological effect, including antioxidant, free radical scavenging abilities, antiinflammatory, anticarcinogenic. etc [7]. They were also suggested to be a potential iron chelator [8-9]. Novel natural antioxidants from some plants have been extensively studied in the past few years for their antioxidant and radical scavenging properties. In view of this and the present understanding about ROS-induced multiple diseases, we have selected one of such ayurvedic herb, Ipomoea fistulosa L. (Family: Convolvulaceae) is a large, diffuse or straggling shrub with milky juice, leaf ovate cordate, entire, acuminate, flower large campanulate, pale rose, pink or light violet in lax, dichotomously branched axillary and terminal, pedunculate cymes; Fruits glabrous capsule; Seed silky [10-12]. It is well distributed in India and found particularly in Chhattisgarh and Madhya Pradesh [13-15]. The plant is commonly known as Besharam, Behaya and used for skin troubles successfully. The milky juice of Beshram is used for the treatment of Leucoderma. The juice is collected and applied externally on affected parts, anti-inflammatory. It is used to decrease the teratogenic effect resulting from cyclophosphamide [16]. Aqueous extract of Ipomoea fistulosa shows neuromuscular

Corresponding Author: Kalpesh Gaur, Geetanjali College of Pharmaceutical Studies, Udaipur, Rajasthan, India

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blocking activity [17]. It used as aphrodisiac, purgative and cathartic. The leaves of Ipomoea fistulosa contain 1-3 flavonol glycosides and Ergine (D-Lysergic acid amide). Polyhydroxylated alkaloids were isolated from the leaves, flowers and seeds [18]. Chromatographic separation of the leaf extract resulted in the isolation of swainsonine, 2-epilentiginosine, calystegines B (1), B (2), B (3) and C (1) and N-methyl-trans-4-hydroxy-l-proline and beta sitosterol [19-21]. MATERIALS AND METHODS The chemicals used DPPH (1,1-diphenyl-2picrylhydrazyl), TBA (thiobarbituric acid), Griess reagent and NBA (nitrobluetetrazolium) were obtained from Sigma Chemicals Co, St Louis, Mo, USA. Trichloroacetic acid (TCA) and potassium superoxides were obtained from Merck, KGOA, Germany. All the other chemical and reagents were used of analytical grade. Collection of the Crude Drug and Preparation of Extracts: The leaves of Ipomoea fistulosa were collected in the month of August from Pharmacognosy garden of Department of Pharmaceutical Science, Dr. H.S. Gour University, Sagar (M.P.) India. The plant was identified with the help of available literature and authenticated by Prof. V.K. Dixit, Department of Pharmaceutical Sciences, Dr. H.S. Gour University, Sagar (M.P.) 470003. The powdered leaves (500 g) were packed in soxhlet apparatus. The drug was defatted with petroleum ether (60-80C) for about 30-35 complete cycles. Defatted material was extracted with two liters of petroleum ether by soxhlet apparatus and then extracted material successively extracted with methanol and chloroform followed by maceration at room temperature and then these extracts were dried by rotary vaccum dryer. The petroleum ether, methanol and chloroform extracts of Ipomoea fistulosa was designated as PEEIF, MEIF and CEIF and the percentage of yield was found to be 6.98, 11.85, 5.68% respectively DPPH Radical Scavenging Activity: The free radical scavenging activity was measured in terms of hydrogen donating or radical scavenging ability, using the stable radical, DPPH [22]. A 0.1 mM solution of DPPH in methanol was prepared and 1 ml of this solution was added to 3 ml of control i.e. standard butylated hydroxyl toluene (BHT) at different concentration (25-100 g/ml) and test solutions at different concentrations (5-100g/ml) in different test tubes. Thirty minutes later, the absorbances were measured at 517 nm. 61

Nitric Oxide Scavenging Activity: Nitric oxide scavenging activity was measured by the spectrophotometric method [23]. Sodium nitroprusside (5 mM) in phosphate-buffer saline was mixed with a control without the test compound, but with an equivalent amount of methanol. Test solutions at different concentrations (5-100 g/ml) were dissolved in methanol and incubated at 25C for 30 min. After 30 min, to 1.5 ml of the incubated solution was diluted with 1.5 ml of Griess reagent (1% sulphanilamide, 2% phosphoric acid and 0.1% naphthyl ethylenediamine dichloride). The absorbance of the chromophore formed during the diazotization of the nitrile with sulphanilamide and the subsequent coupling with naphthyethylene diamine dihydrochloride was measured at 546 nm. Superoxide Scavenging: Superoxide scavenging was carried out using the alkaline dimethyl sulfoxide (DMSO) method [23]. Solid potassium superoxide was allowed to stand in contact with dry DMSO for at least 24 hrs and the solution was filtered immediately before use; the filtrate (200 l) was added to 2.8 ml of an aquous solution containing nitroblue tetrazolium (56 M), EDTA (10 M) and potassium phosphate buffer (10 M, pH 7.4). Test solutions at different concentrations (5-100 g/ml) were added and absorbances were recorded at 560 nm against the control. Hydroxyl Radical Scavenging Activity: The scavenging capacity for hydroxyl radical was determined according to the modified method [24]. The assay was performed by adding 0.1 ml of EDTA, 0.01 ml of ferric chloride, 0.1 ml of hydrogen peroxide, 0.36 ml of deoxyribose, 1.0 ml of test solutions (5-100 g/ml) in distilled water, 0.33 ml of phosphate buffer (50 mM, pH 7.4) and 0.1 ml of ascorbic acid were dissolved in sequence. The mixture was then incubated at 37C for 1 hr and 1.0 ml portion of the incubated mixture was mixed with 10% TCA and 1.0 ml of 0.5% TBA to develop the pink chromogen and measured at 532 nm. Statistical Analysis: The results are presented as mean SEM. All parameters were analysed using Students t-test. P<0.05 was considered as significant. RESULTS Inhibition of DPPH Radical: The potential decrease in the concentration of DPPH radial due to scavenging property of MEIF and BHT showed significant free radical

Academic J. Plant Sci., 2 (2): 60-64, 2009


Table 1: Free radical scavenging activity of various extracts of Ipomoea fistulosa Drugs Petroleum ether extract of Ipomoea fistulosa (PEEIF) Concentration (g/ml) 5 10 25 50 100 5 10 25 50 100 5 10 25 50 100 25 50 100 DPPH radical inhibition (%) 03.650.121 05.240.124 10.090.014 15.450.007 48.650.005 05.140.101 11.410.121 21.260.006 23.260.009 33.590.004 11.240.517 22.360.453 51.440.004* 72.640.005** 86.440.002*** 83.120.141 86.940.125 91.860.156 Nitric oxide 04.990.244 12.740.411 32.100.011 38.140.008 48.250.005 02.540.103 05.680.138 13.990.004 25.290.007 43.810.021 10.590.112 22.720.451 51.140.009* 72.920.001** 74.120.001*** 76.690.054 80.121.215 82.140.512

Chloroform extract of Ipomoea fistulosa (CEIF)

Methanolic extract of Ipomoea fistulosa (MEIF)

Butylated hydroxyl toluene (BHT)

Values are mean SEM, 6 independent analysis, P<0.05*, P<0.01**, P<0.001*** as compared to standard (Students t-test) Table 2: Free radical scavenging activity of various extracts of Ipomoea fistulosa Drugs Petroleum ether extract of Ipomoea fistulosa (PEEIF) Concentration (g/ml) 5 10 25 50 100 5 10 25 50 100 5 10 25 50 100 25 50 100 Superoxide inhibition (%) 02.450.184 04.460.514 13.430.128 15.940.052 48.560.119 04.120.846 09.550.532 22.120.641 23.460.684 36.460.214 11.870.016 26.690.592 68.460.158* 70.430.364** 75.500.654*** 75.480.784 76.020.887 81.871.246 Hydroxyl radical inhibition (%) 06.820.125 15.340.279 35.330.357 37.990.549 48.870.217 04.110.526 06.760.548 18.960.643 29.350.351 44.560.386 11.040.135 24.580.876 54.630.745* 61.930.984** 68.840.647*** 68.650.386 71.880.423 73.560.368

Chloroform extract of Ipomoea fistulosa (CEIF)

Methanolic extract of Ipomoea fistulosa (MEIF)

Butylated hydroxyl toluene (BHT)

Values are mean SEM, 6 independent analysis, P<0.05*, P<0.01**, P<0.001*** as compared to standard (Students t-test)

scavenging activity viz. 86.44 and 91.86%, respectively at 100 g/ml. The IC50 of MEIF was found to be 24.56 g/ml whereas PEEIF and CEIF did not show any significant activity (Table 1). Nitric Oxide Scavenging Activity: The scavenging of nitric oxide by MEIF and BHT was concentration dependent. There was a moderate inhibition of nitric oxide formation with the maximum inhibition being 74.12 and 82.14% respectively at 100g/ml MEIF and BHT. The IC50 of MEIF was found to be 24.45 g/ml. Similar results were not found in case of PEEIF and CEIF (Table 1). 62

Superoxide Radical Scavenging: The MEIF and BHT showed a moderate inhibition of the superoxide radical 75.50 and 81.87% respectively at 100 g/ml. There was no significant inhibition of superoxide radical by PEEIF and CEIF (Table 2). Hydroxyl Radical Activity: The effect of MEIF and BHT on hydroxyl radical and iron (II)-dependent deoxyribose damage was protected significantly at all concentrations; the percentage of inhibition of hydroxyl radical being 68.84 and 73.56% respectively at 100 g/ml. No significant inhibition of superoxide radical by PEEIF and CEIF (Table 2).

Academic J. Plant Sci., 2 (2): 60-64, 2009

DISCUSSION Oxidative stress, in which large quantities of reactive oxygen species (ROS) like hydrogen peroxide, superoxide, hydrogen radical, singlet oxygen and nitrogen species are generated, one of the earliest responses to stress. These ROS have a role in disease and aging in animals [25]. The antioxidative system protects the organism against ROS-induced oxidative damage. There are restrictions on the use of synthetic antioxidants such as BHT, as they are suspected to be carcinogenic [26]. Natural antioxidants therefore have gained importance. DPPH is a stable free radical at room temperature and accepts an electron or hydrogen radical to form a stable diamagnetic molecule. The reduction capability of DPPH radicals was determined by the decrease in its absorbance at 517 nm, which is induced by antioxidants. The significant decrease in the concentration of DPPH radical is due to the scavenging ability of MEIF. Nitric oxide was generated from sodium nitroprusside and measured by the Greiss reduction. Sodium nitroprusside in aqueous solution at physiological pH spontaneously generates nitric oxide, which interacts with oxygen to produce nitrate ions that can be estimated by use of Greiss reagent. Scavengers of nitric oxide complete with the oxygen, leading to reduced production of nitric oxide [27]. The MEIF was shown significant scavenging activity. The potentially reactive hydroxyl radicals can cause oxidative damage to DNA, lipids and proteins, the effect of MEIF and BHT on the inhibition of free radicalmediated deoxyribose damage was assessed by means of iron (II)-dependent DNA damage assay, which showed significant results [28]. The MEIF has potent antioxidant and free radical scavenging effects in different in-vitro systems, but PEEIF and CEIF showed no significant effects as compared to standard BHT. Further work is necessary to elucidate the mechanism involved in the antioxidant activity MEIF. ACKNOWLEDGMENT The authors are grateful to Mr. J.P. Agarwal, Chairman, Geetanjali University Trust, Udaipur (Rajasthan) for providing laboratory facilities throughout the study. 15. 1.

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