IJPRD, 2015; Vol 6(11):January-2015 (033 – 039) International Standard Serial Number 0974 – 9446 -------------------------------------------------------------------------------------------------------------------------------------------------FREE RADICAL SCAVENGING POTENTIAL OF 'THULASI VETTILA', AN INDIGENOUS CULTIVAR OF BETEL VINE (PIPER BETLE L.) IN KERALA D K Sathish1*, Vrinda1, Thomas Bennans2 1 Dept. of Pharmaceutical Chemistry, Padm. Dr. D. Y. Patil College of Pharmacy, Akurdi, Pune-411044, India ABSTRACT Betel Vine (Piper betle L.), commonly known as ‘paan’, is Correspondence Author a plant of antiquity with its global spread in terms of distribution, its acceptance by diverse cultural groups and known for the ethnomedicinal properties. The cultivar ‘thulasi’ is indigenous to Southern Kerala and is well known for its traditional medicinal properties and rarely cultivated for commercial purposes. The present study attempts to assess the antioxidant potential of the cultivar in relation to its phytochemical composition and major phenolic compounds.Total carbohydrates, proteins, flavanoids, lipids, D K Sathish phenolic compounds, chlorophylls, vitamins etc were analyzed Dept. of Pharmaceutical Chemistry, and quantified from the aqueous and methanolic extracts of Padm. Dr. D. Y. Patil College of the plant material in order to assess the phytochemical Pharmacy, Akurdi, Pune-411044, composition. Antioxidant potential of a plant is measured in India terms of its ability to scavenge free radicals. So super oxide anions and hydrogen peroxide were localized and quantified. It Email:- [email protected] was followed by the localization of the major indicator enzymes associated with free radical scavenging such as catalase and peroxidase. Lignin was also localized to verify the end impact of free radical scavenging. The investigation showed that P.betle cv ‘thulasi’ constitutes a significant level of major phenolics and antioxidant phytoconstituents that account for the anti oxidant potential of the plant. Both DPPH and FRAP assays revealed a high degree of free radical scavenging ability of betel vine. The investigations clearly suggest that the organic and aqueous extract of P.betle could be further exploited in the future as a source of useful phytochemical compounds for the pharmaceutical industry owing to its strong anti oxidant potential. Keywords- Betel vine, Piper betle, ‘thulasi’, antioxidant potential, free radicals, DPPH, FRAP. 33 International Journal of Pharmaceutical Research & Development ISSN: 0974 – 9446 southern regions of Kerala and is not usually INTRODUCTION cultivated for commercial purposes as its Human dependence on plants as a chewing quality is poor because of its very source of medicine dates back to prehistoric stringent taste. However, thulasivettila has been times. Piper betle L., commonly known as betel traditionally used for religious and medicinal vine or Paan, is one of the important plants in the purposes and is grown as a household plant. Asiatic region which ranks second to coffee and Hence the present investigation attempts to tea in terms of daily consumption. Use of Piper assess the antioxidant potential betle leaf was known for centuries for its curative properties such as to reduce or prevent body MATERIALS AND METHODS odour and bad breath, throat and lung problems, Plant Material cough prevention and healing (1). In Peninsular Betle (Piper betle) is a tropical shade-loving Malaysia, the leaves of betel vine are used by the perennial evergreen vine cultivated mainly for rural population as an antimalarial remedy. The leaves. Development of the leaf beyond a liquid extract of the plant has been used particular level is of no commercial value because traditionally in curing inflammation and infection of the increased production of undesirable of the respiratory tract, cough, indigestion, phenolic compounds. These leaves were taken for diphtheria, hysteria as well as general and sexual the whole biochemical and antioxidant analysis. debility. A concoction of indigenous Indian drugs For experimentation, the plant species was grown containing P. betle dry extract was found to be an in two environmental conditions, namely the effective long-lasting oral contraceptive. The normal and drought stressed conditions. parts of P.betle utilized are leaves, roots, stems, Analytical Studies stalks and fruits. The plant has got large number The total protein was estimated as per Peterson of biomolecules which show diverse (3). The total Carbohydrates was estimated by pharmacological activity. Medicinally P.betle leaf the method of Rose et al., (4). Total polyphenol is an aromatic, carminative, stimulant and content was measured by the method of Mayer astringent used as a preventive for worms and in et al., 1995 (5). Total flavanoids was measured by snake bite. the method of Chang et.,al (6). The lipid content The pharmacological profiling of betel vine in the sample was estimated by following the reveals its antimicrobial activity, protective and method of Bligh and Dyer (1959). Superoxide healing activity, antidiabetic and anion was quantified, following the method of gastroprotective activity, and hepato-protective Doke (7). H2O2 concentration of leaves was activity etc, owing to the predominant presence of essential oils, hydroxycatechol, chavicol, estimated as per the procedure of Bellincampi et Methyleugenol, caryophyllene, phenyl propane al (8). etc. Phytochemical studies have shown that Piper Histochemical Studies betle contains a wide variety of biologically active Superoxide anions were localized by the compounds whose concentration depends on the procedure of Frahry and Schofer (9). variety of the plant, season and climate (2). Histochemical localization of hydrogen peroxide Piper betle L., belongs to the family Piperaceae, is was done by procedure of Ros Barcelo (10). POX a dioecious, shade loving perennial Malay activity was localized by the procedure of creeper extensively cultivated in hot damp Bestwick (1998). Cytochemical localization of localities. Leaves usually opposite, cordate, catalase was carried out by the method of Fang petioled, all 4 to 7-ribbed and glabrous. There et al. (11). Lignins can be localized by the are about 125 to 150 cultivars of betel vine in procedure of Faulkner and Kimmins (12). India. In Kerala the most prevalent cultivars are Biochemical Analysis Venmani, Arikodi, Kalkodi, Karilanchi, Karpuram The amount of vitamin –E was determined etc. ‘Thulasivettila’ is a variety restricted to the following the method of Rosenberg (13). DPPH Available online on www.ijprd.com 34 International Journal of Pharmaceutical Research & Development ISSN: 0974 – 9446 (1, 1-Diphenyl-2-Picrylhydrazyl) radical Lignin, being the end product of this biochemical scavenging activity was estimated by the sequence of free radical scavenging, was also procedure of Saeed et al., (14). The FRAP localized which showed very distinct deep red procedure described by Benzie and Strain (15) colour of lignin deposition in the stress induced was followed for assessing the antioxidant leaves (Figure 4). Normally lignin deposition is activity. Antimicrobial activity and Antifungal comparatively higher in ‘thulasi’ cultivar as evident activity were analyzed following the procedure from the rough texture of the leaves. The increased production of super oxide anions of NCCLS,1993 (16). and hydrogen peroxide was further verified by localizing the enzyme catalase which is responsible RESULTS AND DISCUSSION for the conversion of hydrogen peroxide back into Phytochemical Composition molecular oxygen and water. The figures 5 shows Phytochemical composition is a significant factor in the localization of the increased production of both determining the antioxidant potential of a plant the enzymes which clearly justify the enhanced apart from the nutritional view point. Analytical production free radicals as revealed by the studies were conducted to estimate the histochemical data. phytochemical composition of the plant material The histochemical data of super oxide anions and and to relate their quantitative variation with the hydrogen peroxide is well supported by the antioxidant potential of the plant especially during quantification data which also show a substantial the stress conditions. Total carbohydrates, total increase in the production of both the substances proteins, phenols, chlorophylls, carotenoids, during stress induction in P.betle.( Figure 6). The flavanoids, vitamin C, vitamin E, total lipids etc reducing activity of NBT by superoxide anions were analyzed and estimated as per the standard present in the extract was taken as a measure of procedures. Pre-weighed fresh leaves were taken quantification. for the whole studies and either water or DPPH Assay – The Radical Scavenging Activity methanolic extracts were used for estimation The relatively stable organic radical DPPH has been purposes. Figure 1 shows the comparative widely used to determine the antioxidant activity distribution of the major phytoconstituents in Betel of different plant extracts (18). Table 1 shows the vine cultivar ‘thulasi’. Higher content of phenolic free radical scavenging ability of both aqueous and compounds and other major antioxidants such as methanolic extracts of the plant material at carotenoids, flavanoids and vitamin C and E justify different concentrations. It is expressed as the the increased antioxidant capacity of the cultivar percentage inhibition of free radicals by the and is comparable that of many medicinal plants. antioxidants available in the extracts. The aqueous Phytochemical screening of cultivars from Sri lanka extracts showed a comparatively higher inhibition also revealed the same kind of phytochemical than that of both control and methanolic extract. composition in methanolic extracts (17). More over higher the concentration greater was Histochemical and Quantification Data the percentage inhibition may due to the higher Figure 2 shows the localization of super oxide quantity of antioxidants present in the extracts. anions in the leaves of P.betle cv thulasi. The FRAP Assay induction of water stress in the leaves has resulted The total antioxidant potential of the plant material in an increased production of superoxide anions as visible in the form of blue deposits. Since H2O2 is was also determined using ferric reducing ability of plasma FRAP assay based on the reducing power of derived from the molecular oxygen by the action of the antioxidant. Table 2 shows the free radical the enzymes NADPH oxidase and super oxide scavenging ability of both aqueous and methanolic dismutase (SOD) and super oxide anions as extracts of the plant material at different intermediate products, hydrogen peroxide was concentrations. Contrary to the DPPH assay, further localized in order to verify its increased methanolic extract showed a comparatively higher production during stress conditions ( Figure 3). Available online on www.ijprd.com 35 International Journal of Pharmaceutical Research & Development ISSN: 0974 – 9446 free radical scavenging ability especially at the domestic cultivar of P.betle, P.betle which has not yet been maximum concentration of the extract. exploited as a commercially or pharmacologically The antioxidant potential of P.betle as revealed by significant cultivar of the plant. Since P.betle also both the DPPH and FRAP assays ssays showed results offers an alternative drug delivery mechanism similar to that of many medicinal plants with high through buccal mucosa sa bypassing the gastric route antioxidant ability(14). owing to its chewable quality, its potential as an effective antioxidant agent can be utilized for CONCLUSION developing new medicines in the coming years. The present investigation substantiates the free radical scavenging potential of the ‘thulasivettila’, ‘thulasivettila’ a Concentration(mg/g FW) 8 7 6 5 4 3 2 1 0 -1 -2 Biochemical Compounds Figure 1:: Estimation of major phytoconstituents and antioxidants in Betel Vine ( Piper betle L.) a b Figure 2 :Localization of Superoxide anion in the leaves of Betel vine ((Piper betle.L) .L) cv Thulasi a) Normal leaf. b) Stress induced leaf. a b Figure 3:: Localization of Hydrogen Peroxide in the leaves of Betel vine (Piper (Piper betle.L) betle cv Thulasi a) Normal leaf. b) Stress induced leaf. Available online on www.ijprd.com 36 International Journal of Pharmaceutical Research & Development a ISSN: 0974 – 9446 b Figure 4: Localization of Lignin in the leaves of Betel vine (Piper betle.L) cv Thulasi a) Normal leaf. induced leaf. a b) Stress b Figure 5: Localization of Catalase and Peroxidase in the leaves of Betel vine (Piper betle.L) cv Thulasi a) Catalase. b) Peroxidase QUANTIFICATION OF H2O2 AND O2(Concentration µ/g.) 10 8 6 NORMAL 4 STRESS 2 0 -2 H2O2 O2- Figure 6: Quantification of Superoxide anions and hydrogen peroxide in the leaves of P.betle Sl.NO 1 2 3 Concentration Aqueous Methanol L-Ascorbic acid* (µg/ml) (%of inhibition) (%of inhibition) (%of inhibition) 10 78.7±0.75 47.35±0.50 51.12 ± 0.35 50 90.5±0.45 66.37±0.75 67.92 ± 0.25 100 96.8±0.76 91.05±0.58 84.38 ± 0.69 Table 1: In vitro Free Radical Scavenging Activity of Different Extracts of Piper betle L. by DPPH Assay. *Positive control (L-Ascorbic acid) Available online on www.ijprd.com 37 International Journal of Pharmaceutical Research & Development ISSN: 0974 – 9446 Sl.No Concentration Aqueous Methanol 1 20 125.88±0.65 161.82±.72 2 60 181.04±0.34 285.52±.54 3 100 611.65±0.34 465.99±.42 BHT* 100 190±0.02 Table 2:In vitro Free Radical Scavenging Activity of Different Extracts of Piper betle L. by FRAP Assay. *Positive control (BHT) Physiology and Plant Pathology. 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