Volume 1(3) May-June 2013 Page 371

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1 ISSN: (Print) INVESTIGATION OF IN-VITRO ANTI-OXIDANT, ANTI-INFLAMMATORY AND ANTI-ARTHRITIC ACTIVITY OF AERIAL PARTS OF SECURINEGA LEUCOPYRUS (WILLD.) MUELL T.K.Gopal*, T.Sheela, D.Chamundeeswari, C.Umamaheswara Reddy Faculty of Pharmacy, Sri Ramachandra University, Chennai, India Corresponding author: E.Mail: ABSTRACT Securinega leucopyrus (willd.) Muell. (Euphorbiaceae) is a thorny, large shrub or small tree. In Ayurveda it has a lot of potential medicinal uses. The decoction of leaves are used to dress the cancerous wounds. The juice or paste of the leaves along with tobacco used to destroy worms in sores. Its leaf decoction useful externally in the treatment of piles. The present study is designed to carry out Anti-oxidant, Anti-inflammatory, Anti arthritic activity by Invitro methods. The Securinega leucopyrus was authenticated and the anatomical features were done which shows calcium oxalate crystals, fibres, glandular trichomes, sclereids, vascular bundles (secondary xylem and phloem). The microscopical evaluation also performed by determination of stomatal index and vein islet number. The In-vitro anti-oxidant activity revealed that the Alcoholic showed more activity as compared to Chloroform, Ethyl acetate, n-hexane. In DPPH (1,1-diphenyl-2-picrylhydrazyl) radical method Alcoholic showed more activity and in nitric oxide scavenging method Hydro alcoholic, Alcoholic s showed more activity. Chloroform showed more activity in hydroxy radical method. In Total anti-oxidant method Alcoholic showed more activity. The Anti-inflammatory activity also performed by Human Red Blood Corpuscle (HRBC) Membrane Stabilization Method. The results were tabulated and the values are represented in the figure. In this Ethyl acetate showed more activity. The Anti-arthritic activity is performed by inhibition of protein denaturation method. And the results are given in table and values are represented in graph. This shows Alcoholic gave more activity. The above results shows that the presence of phytoconstituents like Glycosides, Flavanoids, Alkaloids, steroids and Tannins may be responsible for Anti-oxidant, Anti-inflammatory, Ant arthritic activities. Key words: Securinega leucopyrus, Anti-oxidant, Anti-inflammatory, Anti-arthritic. INTRODUCTION Plants are used as medicine since time immemorial. Approximately one-third of the top selling drugs in the world are derived from natural products and their derivatives. The traditional approach makes use of material that has been found by trial and error over many years in different systems of medicine (Cotton, 1996). Plants are used for discovery of modern medicine in four basic ways, as a source of direct medicinal agents, serve as a raw material base for elaboration of more complex semi-synthetic chemical compounds, chemical structure derived from phyto constituents can be used as models for new synthetic compound (Anon, 1994), Plants can be used as taxonomic markers for discovery of new therapeutic compounds (Cox.P.A, 1990). According to the world health organization (WHO), (Murry et al.,) 80% of the world s population has faith in traditional medicine, particularly plant drugs for their primary healthcare. India is a goldmine of well recorded and traditionally well practical knowledge of herbal medicine. India officially recognizes over 3000 plants for their medicinal value. It is estimated generally that over 6000 plants in India are use in traditional, folk and herbal medicine, representing 75% of the medicinal needs of the third world countries. The frequency of life threatening infections has increased worldwide and it is becoming an important cause of morbidity and mortality in immuno-compromised patients in developing countries. Eg: Cancer, Cardio vascular diseases, Rheumatoid arthritis, Cataracts, Alzheimer s diseases and inflammatory disorders. (Dev,1999). Several anti-oxidants of plants origin are experimentally proved and used as effective protective against oxidative stress. They play an important role in major health problems like cancer. Free radicals are produced in large amount during metabolic disease conditions like diabetes, hypertension, atherosclerosis, urolithiasis, ulcers etc. This free radicals attack DNA, protein molecules, enzymes and cells leading to change in genetic material and cell proliferation (Cancer). Plants which contain carotenoids, flavonoids and Tannins can be utilized to scavenge the excess free radicals from human body. An inflammatory disease like Rheumatoid arthritis is one of the most common immuno inflammatory conditions affecting the population worldwide. About 1% of the worldwide adult population is affected by Rheumatoid arthritis with above twice as many females suffers as male. In recent years, attempts have been made to investigate the indigenous drug against Arthritis. Research in the field of indigenous plant is a significant aspect of developing a safer anti arthritic principle through isolation, Volume 1(3) May-June 2013 Page 371

2 characterization, identification and biological studies. (Pratt, 1992). Securinega leucopyrus (willd.) Muell, belonging to the family Euphorbiaceae commonly known as Bushweed, Indian snow berry, is a thorny woody shrub or a small tree distributed in different parts of India (Ayensu, 1996). Its leaves and fruits are edible. The berry is sweet. The slender branches are reported to be utilized for preparing wicker-baskets and for thatching (Kirtikar, 1918). The leaves of the plant contain germicidal properties. In Chattisgarh, the decoction of leaves is used to dress the cancerous wounds. It is used in combination with tobacco. The juice or paste of the leaves along with tobacco used to destroy worms in sores. Its leaf decoction useful externally in the treatment of piles and it is used to wash the wounds of cattle. It is used as popular veterinary medicine (Bakshu, 2001). The other uses are sweet, cooling, diuretic, aphrodisiac, tonic and are useful in vitiated conditions of pitta, burning sensation, strangury, seminal weakness, general debility, larvicide, paralysis, paresis, piscicide, insecticide. The anti-microbial properties of securinega leucopyrus is already reported (Bakshu, 2001). The leaves are set as a disinfectant and its paste is used to the extraneous materials from body tissues without surgery. Even though Securinega leucopyrus has lot of potential medicinal uses, the study on this plant is very less. Considering the importance of this plant, the present study was undertaken. Plant collection: It consists of dried aerial parts of Securinega leucopyrus (willd.) Muell. (Euphorbiaceae) is a thorny, large shrub or small tree. The plant Securinega leucopyrus were collected from Tribal Women s Welfare Trust s Herbal Garden, Thandarai, Chengalpattu (Dist), Tamilnadu in the month of December and authenticated at National Institute of Herbal Science, West Tambaram, Chennai (PARC/2009/289). Fig 1: Morphology of Securinega Leucopyrus (willd.) Muell METHODS AND MATERIALS Collection and processing of plant material: Then the aerial part of plant material was cut into small fragments and dried in shade until the fracture is uniform and smooth. The dried leaves, stem portions were separately powdered by using a blender and sieved in sieve No 60 (Gahan, 1984). The coarse powder 500 gm was subjected to maceration for 72 hours, followed by exhaustive maceration for 48 hours by using various solvents of increasing polarity [n- hexane> Chloroform> Ethyl acetate>alcohol> Water] by decanting and drying the marc after each ion. The solvents were recovered by distillation of the s at 75 0 C to 80 0 C. The s were dried under desiccator and the percentage yield was calculated. The residues obtained were used for the screening of the phytochemical analysis. These n-hexane, Chloroform, Ethyl acetate, Ethanol and Hydroalcoholic s were used for the preliminary phytochemical investigation, chromatographic analysis and its pharmacological evaluation (Sundaresan, 1991). In-Vitro Pharmacological Evaluation: The aerial part of the plant of Securinega leucopyrus (willd.) Muell had been stuied for its anti-oxidant activity by DPPH method (Yohozawa, 1998), nitric oxide scavenging activity (Alderson, 2001), determination of total anti-oxidant activity (Prieto, 1999) scavenging of hydroxyl radical deoxyribose method (Elizabeth, 1990), in-vitro anti-inflammatory activity by human red blood corpuscle (HRBC) membrane stabilization method, in-vitro anti-arthritic activity by inhibition of protein denaturation method. RESULTS AND DISCUSSION Anti-oxidant activity by DPPH radical scavenging method: The chloroform and ethyl acetate, alcohol, hydro alcohol s showed a dose dependent increase in anti-oxidant activity in DPPH method. About 82.5%, 88.42% were observed in Chloroform and Alcoholic s of Securinega leucopyrus. But in Hexane the effect was decreased when dose increases. The presence of flavanoids, Alkaloids, Tannins and Steroids in these s may be responsible for free radical scavenging activity. From the results it is made clear that Securinega leucopyrus possess free radical scavenging property and the order was, Alcohol > Chloroform > Ethyl acetate > Hydro alcohol >Hexane. DPPH is a relatively stable free radical. The assay is the measurement of scavenging ability of Antioxidants towards the stable radical DPPH. The plant containing Volume 1(3) May-June 2013 Page 372

3 flavanoids, Alkaloids, Tannins and Steroids reduces the radical to the corresponding hydrazine when it reacts with a hydrogen donors in the anti-oxidant principle. In DPPH method results are highly reproducible. The active constituent present in the plant donates an electron to reduce the DPPH radical to its corresponding hydrazine. The chemical constituents present in this plant like Flavanoids, Tannins, Alkaloids and Steroids may be responsible for this activity. They shows the anti-oxidant activity by inhibition of enzymes involved in oxidation systems. Anti -Oxidant Activity by Nitric Oxide Scavenging Method: All s of Securinega leucopyrus shown a dose dependent increase in Nitric oxide scavenging property.about 85% inhibition was observed by Hydroalcoholic. But in Hexane and Ethyl acetate s the effect was very less compared to standard. The presence of flavonoids Tannins and steroids in these s may be responsible for Nitric oxide scavenging activity. Nitric oxide is a radical produced in mammalian cells, involved in the regulation of various physiological processes. In the present study the nitrite produced by the incubation of sodium nitro prusside in standard phosphate buffer at 25 c was reduced by plant. This may be due to the antioxidant principles present in the plant which compete with oxygen to react with nitric oxide, there by inhibiting the generation of more deleterious products such as Nitric anhydride (N 2 O 3 ) and perhydroxy nitrite (ONO --- ) (Chen et al 2001). This activity is due to the presence of flavonaids, Tannins and steroids. They inhibit the free radicals by inhibition of enzymes involved in oxidation systems (5- Lipoxygenase, cycloxygenase, mono oxygenase, xanthine oxidase. Determination of total anti-oxidant activity: The Ethanolic, Hydro alcoholic s showed maximum effect due to the presence of flavanoids, Reducing sugars, Alkaloids, Tannins and steroids. The values were expressed as equivalents of Vitamin E. From the results it is made that Securinega leucopyrus possess free radical scavenging activity through anti-oxidant property. Anti-oxidant activity By Hydroxy radical scavenging method: All the s of Securinega leucopyrus shown a dose dependant increase in nitric oxide scavenging property. About 87.11% inhibition was observed by Alcoholic. The Chloroform and Hydroalcoholic showed 78.56%, 78.52% inhibition. But in Ethyl acetate and Hexane the effect was very less compared to standard. The presence of flavanoids Tannins and steroids in these s may be responsible for Hydroxy radical scavenging activity. Ferrous salts can react with H 2 O 2 and form hydroxyl radical via Fention s reaction. The iron required for this reaction is obtained either from the pool of iron or the heme containing protein. The hydroxyl radical (OH) - thus produced may attack the sugar of DNA deoxy causing ribose fragmentation, base loss, and DNA strand breakage. The generation of (OH) - in fenton reaction is due to the presence of iron ions. When the Fe 2+ /Fe 3+ redox couple is bound by certain chelators, the OH fragmentation is prevented, whereas the increased colour formation in the absence of crude s were observed in deoxy rebose assay. In this, the act as a chelator of iron ions, binding to them, & preventing the formation of free radicals, though the s not directly involved in the OH scavenging. The results indicates that the s of plant plays a major role in the inhibition of ribose fragmentation and hence the decreased color formation in the deoxy rebose assay. The free radical scavenging property of the crude s of plant against DPPH, Nitric oxide, Hydroxy radicals and the Total anti-oxidant activity is clearly understood from the results of this chapter. The phytochemical screening of the revealed the presence of flavonoids, tannins which is responsible for anti-oxidant property. Anti-inflammatory activity by HRBC membrane stabilization method: The all s shows a dose dependent increase in Anti-inflammatory activity in HRBC membrane stabilization method. About 89.66%, 87.42%, 72.32% inhibitions were observed in Ethyl acetate, Chloroform and Alcoholic s. But in Hexane and Hydro alcoholic s the effect was very less when compared to standard Diclofenac sodium. This anti-inflammatory activity may be due to the presence of flavonoids, Tannins and Alkaloids. Flavonoids may produce their Anti-inflammatory effect by a multitude of ways to inhibit the inflammatory processes. Formation and release of various mediators of inflammation like Histamine and Prostaglandin are affected by Flavanoids. They inhibit the increased capillary permeability during inflammation. The adhesion of leucocyte to endothelial surface and subsequent migration is influenced by flavanoids. They inhibit the prostaglandin and Leucotriene C 4 in human platelet. They were investigated for lipoxygenase inhibitory activity. They inhibits cytokine release from cells. Anti-Arthritic Activity by Inhibition of Protein Denaturation Method: The results of anti-arthritic activity of all s of Securinega leucopyrus on inhibition of protein denaturation method as shown in table18 and figure 5.6. The Ethanol, Chloroform, Hydro alcoholic s showed maximum activity when compared to n-hexane and Ethyl acetate s. The effect is represented as, Ethanol > Water > Chloroform > Ethyl acetate > Hexane s. The effect may be due to Alkaloids, Tannins, Flavanoids, Triterpenes and Reducing Volume 1(3) May-June 2013 Page 373

4 sugars. Denaturation of protein is one of the cause of Rheumatoid arthritis. Production of auto antigen in certain arthritic disease may be due to denaturation of proteins. The mechanism of denaturation probably involves alteration in electrostatic hydrogens and hydrophobic di sulphide bonding. The present study, Securinega leucopyrus capable of controlling the production of auto antigens and thereby it inhibit the denaturation of protein in Rheumatic diseases. Tab 1: The Percent inhibition of different s of Securinega leucopyrus on DPPH radical scavenging activity Concentrations in μg/ml Hexane CHCL 3 EtoAc Alcoholic Hydoalcholic Percent Inhibition of Standard (Vit. E) Fig. 2: Effect of securinega leucopyrus on DPPH method Tab 2: Percentage inhibition of different s Securinega leucopyrus by Nitric Oxide Scavenging method. Concentrations in μg/ml Hexane CHCL 3 EtoAc Alcoholic Hydrialcocolic Percent Inhibition of Standard (Vit. E) ` , Volume 1(3) May-June 2013 Page 374

5 ISSN: (Print) Fig 3: Effect of Securinega Leucopyrus on Nitric Oxide Scavenging activity Determination of Total Antioxidant Activity: In this investigation the absorbance (Y-axis) of different s were extrapolated to the concentration (X-axis) of standard Vit E. Fig 4: Determination of total anti-oxidant activity Tab 3: Absorbance of Test s plotted on the above graph is as follows Extracts 200 µg/ml Hexane Chloroform Ethyl acetate Ethanol Hydroalcohol Tab 4: Estimation of Total anti-oxidant activity Concentrations of Equivalent to Standard Vit E in μg/ml s in μg/ml Hexane Chlorofom Ethylacetate Ethanol Hydroalcohol The above table shows the extrapolated values. Hence the effect of 200µg/ml of s is equivalent to the effect of µg/ml of Vit E with respect to the s. Volume 1(3) May-June 2013 Page 375

6 Tab 5: Percent inhibition of Securinega leucopyrus in Hydroxyl Radical Scavenging method Concentrations Hexane CHCl 3 EtoAc Alcoholic Hydro Percent Inhibition of in μg/ml alcocolic Standard (Vit. E) Fig 5: Effect of Securinega Leucopyrus on Hydroxy Radical Scavenging Activity Tab 6: Percent HRBC Membrane Stabilization of different s of Securinega leucopyrus Ethyl Hydro Percent Concentrations Hexane Chloroform Alcohol acetate alcohol stabilization of in μg/ml Diclofenac sodium Fig 6: Effect of Securinega leucopyrus on Anti-inflammatory Activity Volume 1(3) May-June 2013 Page 376

7 Tab 7: Percent denaturation of protein of different s of Securinega leucopyrus Concentrations Hexane CHCL 3 Ethanolic Alcohol Hydro Percent stabilization of in μg/ml alcohol Diclofenac sodium Fig 7: Effect of securinega leucopyrus on Anti Arthritic Activity CONCLUSION From this study we can conclude that Securinega leucopyrus showed the presence of Alkaloids, Sugars, Flavonoids and Tannins. This is identified by phytochemical analysis and confirmed by thin layer chromatography. The present study on Securinega leucopyrus showed that the plant has moderate anti-oxidant activity and it compared with standard vitamin E. The plant also showed anti-inflammatory and anti-arthritic activity and it compared with standard Diclofinac sodium. Chloroform, Ethyl acetate, Ethanol and Hydroalcohol fractions are having significant anti-inflammatory and antiarthritic activity may be due to the presence of alkaloids, Phytostrols, Flavonoids, saponins, Tannins, and Lignans. The future work will be the determination of Anti-oxidant, Anti-inflammatory and Anti arthritic activities by Invivo methods. REFERENCES Alderson WK, Cooper CE, Knowels RG, Nitric oxide systhesis, structure, function & inhibition, Journal of Biochemistry, 2001, 357: Anon K, Ethnobotany in search for new drugs. Ciba foundation symposium, John Wiley & sons, Newyork, 1994,188. Ayensu ES, World medicinal plant resourses. In conservation for productive agriculture (VL chopra and TN Khoshoo, edds) ICAR, 1996, New Delhi, India Bakshu LM, Ram AJ, Raju RBV, Fitoterapia, Volme-72, No-8, Dec 2001, Chen Y, Zhen R, Jia Z, Flavonoids as super oxide scavengers and anti-oxidants, Free radical Bio Med, 9, 1990, Cotton, 1996, The traditional approach makes use of material that has been found by trial and error over many years in different countries and systems of medicine Elizabeth K and Rao MNA, 1990, Oxygen radical scavenging activity of curcumin, Indian J Pharm, 58, 1990, 237. Volume 1(3) May-June 2013 Page 377

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