Soubhagya K B

Evaluation of anti-oxidant and antiinflammatory activity in leaf and flower ethanolic extracts of Nyctanthes arbor-tristis Linn. Cite as: AIP Conference Proceedings 2263, 030003 (2020); https://doi.org/10.1063/- Published Online: 08 September 2020 K. B. Soubhagya, and M. 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Anilkumar2,b) 1 Department of Botany, Mar Athanasius College (Autonomous), Kothamangalam India. 2 Department of Botany, Union Christian College, Aluva, India. a) Corresponding author:-b)- Abstract. Studies on natural compounds are of interest nowadays, due to their adequate health benefits with low toxicity. Herbs and spices have been added to varieties of food to impart flavour as well as to advance storage stability due to their anti-oxidant effects. Anti-oxidants play a major role in curing degenerative diseases. In current scenario many synthetic antioxidants are frequently used but due to their toxic and carcinogenic effects their use has been regulated by legislative rules. So the search for natural anti-oxidants is necessary. In addition to the body's response to injury, inflammation is a physiological mechanism underlying the progression of numerous diseases. Investigation for the discovery and development of anti-inflammatory agents from natural sources is a promising strategy for curing inflammatory diseases. Leukocytes are the key players of inflammatory response, can remove microbes and dead cells by phagocytosis, followed by their destruction in phagolysosomes. The destruction is due to the free radicals generated in activated leukocytes and lysosomal enzymes. The human RBC (HRBC) membranes are analogous to these lysosomal membrane components and most anti-inflammatory drugs are targeted on these pathways. Preventing the discharge of inflammatory mediators is a better option than blocking a specific mediator or its pathway. Generally, conventional anti-inflammatory drugs are used to treat inflammations but they have side effects. Traditionally, Nyctanthes arbor-tristis Linn. (Nyctanthaceae) is used against a wide range of ailments in Indian traditional medicine. Phytochemical analysis of Nyctanthes arbor-tristis Linn. revealed the occurrence of biochemical compounds such as terpenes, steroids, tannins, phenols, reducing sugars, flavonoids, proteins etc.. Triterpenoids and flavonoids have remarkable anti-inflammatory activity. Here, the ability of Nyctanthes arbor-tristis Linn. ethanolic leaf and flower extracts is studied by HRBC membrane stabilization. The inhibition of hypotonicity induced HRBC membrane lysis was taken as a measure of anti inflammatory activity. Diclofenac was used as reference standard. The percentage of membrane stabilization of ethanolic leaf extract, flower extract and Diclofenac were estimated spectrophotometrically at different concentrations such as 200 µL, 400 µL, 600 µL, 800 µL and 1000 µL. They were found to be effective in protecting HRBC membrane. Leaf and flower extracts has a percentage of stabilization less than that of the reference standard, Diclofenac. Among the plant extracts, leaf shows greater stabilization than that of flower. INTRODUCTION Oxygen consumption inherent in cell growth leads to the generation of a series of reactive oxygen species (ROS). They are continuously created by the body’s normal use of oxygen such as respiration and some cellmediated immune functions. ROS comprise of free radicals such as superoxide (O2), hydroxyl radical (OH), peroxyl radical (RO2) as well as non-radical species such as hydrogen peroxide (H2O2)[1]. Inflammation is a pathophysiological response of mammalian tissues to diverse hostile agents including infectious organisms, toxic chemical substances, physical injury, or tumour growth leading to local accumulation of plasma fluid and blood cells[2]. The exploration for new anti-inflammatory and analgesic agents from the huge array of medicinal plant resources is intensifying. This is because such taxa may hold assurance for the discovery of novel therapeutic agents International Conference on Science and Technology of Advanced Materials AIP Conf. Proc. 2263,-; https://doi.org/10.1063/- Published by AIP Publishing-/$30.00 - capable of suppressing, reducing, or relieving pain as well as inflammation3 .Non-steroidal anti-inflammatory drugs (NSAIDs) make up one of the largest groups of drugs used for pain and inflammation[4] and are associated with unwanted side effects and have their own limitations. About 34—46% of the users of NSAIDs, usually sustain some gastro-intestinal damage due to the inhibition of the protective cyclooxygenase enzyme in gastric mucosa[5].The added advantages of indigenous medicinal treatment would include its complementary nature to the conventional treatment, making it a safer well tolerated and economical remedy for acute and chronic inflammatory conditions. Nyctanthes arbor-tritis Linn. is a small sacred ornamental tree. It is native of India, distributed wild in subHimalayan regions and southwards to Godavari[6]. It is having white fragrant flowers commonly known as night jasmine [7, 8] .Different parts of Nyctanthes arbor-tritis Linn. are known to possess various ailments by tribal people of India especially Orissa and Bihar along with its use in Ayurveda, Sidha and Unani systems of medicines. Nyctanthes arbor-tristis Linn. leaves are known to exhibit analgesic, antipyretic, ulcerogenic, Anti-stress, anxiolytic, tranquilizing, antihistaminic and purgative property and flowers are used as carminative, astringent to bowel, antibilious, expectorant, hair tonic and in the treatment of piles and various skin diseases 9 and in the treatment of ophthalmic purposes[10]. Nyctanthes arbor-tritis Linn. contain chemical constituents like polysaccharides, iridoid glycosides, phenylpropanoid glycoside (nyctoside A), ß-sitosterol, ß-amyrin, hentri-acontane, benzoic acid, glycosides, nyctanthoside-a iridoid, nyctanthic acid, Friedelin and lupeol and oleanolic acid and 6ß-hydroxylonganin [11] and iridoid glucosidesarborsides A, B and C, alkaloids, Phlobatanins, terpenoids and cardiac glycosides. Iridoid glucosides (arbortristosides-A, B, C) and 6ßhydroxyloganin, 4-hydroxy hexahydrobenzofuran-7-one tertiary alkaloids mainly 7-(alpha-anilino-p-nitrobenzyl)-8-quinolinol and quarternary alkaloids belonging to protoberberines and aporphines [12] has also been isolated from this plant. The present study aims to evaluate the anti-oxidant and anti-inflammatory potential of Nyctanthes arbor-tritis Linn. leaf and flower ethanolic extracts using standard procedures. MATERIALS AND METHODS Collection and Authentication of Plant Material Leaves and flowers of Nyctanthes arbor-tritis Linn. were collected from the botanical garden, Union Christian College, Aluva, Ernakulam district. Taxonomically identified and herbarium specimen was prepared and depicted in the department herbarium. Method of Extraction The collected N. Arbor-tristis Linn.leaves and flowers were dried at 700C for 72hrs and 40hrs respectively and was powdered. The powders were subjected for extraction by cold percolation method in ethanol. The extracts were filtered and the filtrates were stored in refrigerator. Determination of In-Vitro Anti-Oxidant Activity The ethanol extracts of leaves and flowers of N. arbor-tristis Linn. was determined for their reducing power modifying the method of Hanato et al. [13]. Reaction mixtures were prepared by adding 1 ml of phosphate buffer (0.2 M, pH 6.6), 1 ml Potassium ferric cyanide (1%) and varying concentrations of extracts separately (100 – 500 µl). After, the reaction mixtures were incubated at 500C in water bath for 30 minutes, allowed to cool at room temperature (280), and 2.5 ml of 1% TCA (trichloroacetic acid) were added to each reaction mixture, and then centrifuged at 2000 rpm for 10 minutes. The supernatants (2.5 ml) were separated in the test tubes and added with 3 ml of distilled water and 0.5 ml ferric chloride (1%), and then allowed to react for 10 minutes at room temperature and the absorbance was measured at 700 nm. Ascorbic acid solution (10%) was used as standard. - Determination of in-vitro Anti-inflammatory Activity Anti inflammatory activity of the ethanolic extracts of leaf and flower of Nyctanthes was carried out as described by Oyedapo et al [14] .The blood was collected in heparinised tubes from weeks prior to the experiment. The blood samples were centrifuged at 3000 rpm for 10 min at room temperature. The supernatants (plasma and leukocytes) were carefully removed while the packed RBC was washed in fresh normal saline (0.85% w/v NaCl). The process of washing and centrifugation were repeated five times until clear supernatants were obtained. From this a 1% (v/v) human erythrocyte suspension was prepared using isosaline. Diclofenac was used as standard drug. The assay mixtures consisted of 2 ml of hyposaline (0.25% w/v) NaCl, 1.0 ml of 0.15 M sodium phosphate buffer at pH 7.4 (25oC), 0.5 ml of 10% (v/v) HRBC suspension, 0.2-1.0 ml of standard drug (Diclofenac)/extract (concentrations ranging from 400µg - 2000µg) and final reaction mixtures were made up to 4.5 ml with iso saline. Extra care must be given to avoid any water content. Drug/extract were omitted in the blood control, while the drug control did not contain the erythrocyte suspension. The reaction mixtures were incubated at 40oC for 45 min on a water bath, followed by centrifugation at 3000rpm for 10 min at room temperature. The absorbance of the lysed haemoglobin was read at 560 nm using UV-Vis Spectrophotometer (Shimadzu, Japan). The percentage of membrane stability was estimated using the expression : Percentage of haemolysis = [ ] x 100 Percentage of membrane stabilization = 100 –[ ] x 100 Where, the blood control represents 100% lysis or 0% stability. Each experiment was conducted in triplicates and the average percentage of stabilization at each concentration was calculated. RESULT Determination of in-vitro Anti-oxidant activity Ferric chloride reducing ability test TABLE 1. In-vitro Anti-oxidant activity of Nyctanthes arbor-tristis Linn. ethanolic extracts flowers and leaves Concentration Nyctanthes leaf Nyctanthes flower Ascorbic acid (µl) (ethanolic extract) (ethanolic extract) (reference standard) 100 0.806 ± 0.023 0.879 ± 0.077 0.806 ± 0.011 200 0.819 ± 0.022 0.883 ± 0.076 0.814 ± 0.018 300 0.835 ± 0.032 0.893 ± 0.091 0.821 ± 0.016 400 0.846 ± 0.045 0.901 ± 0.088 0.864 ± 0.021 500 0.857 ± 0.057 0.911 ± 0.087 0.878 ± 0.029 The natural antioxidants might directly react with or quench the stable cation radical, which is reflected as their antioxidant activity. Nyctanthes arbor-tristis Linn. flowers and leaves with all the ingredients show an increase in total antioxidant activity with the increasing concentration from 100 – 500 µl. The anti-oxidant activity of the standard used i.e. ascorbic acid is greater than the flower extract at 400 µl but less at all other concentrations. The anti-oxidant activity of ethanolic leaf extract was found to be greater then the ethanolic flower extract and ascorbic - acid (Table 1). The result presented here indicates that the marked ferric reducing power activity of extract seem to be due to presence of polyphenols which may act in a similar fashion as reductones by donating the electrons and reacting with free radicals to convert them into more stable products and terminate free radical chain reactions. Determination of in-vitro Anti-inflammatory activity TABLE 2. In vitro Anti-inflammatory study in N. arbor-tristis Linn. ethanolic extracts flowers and leaves Leaf extract Concentration (µl) Flower extract Diclofenac % of haemolysis % of protection % of haemolysis % of protection % of haemolysis % of protection 200 89.02 10.98 89.14 10.86 87 13.79 400 75.94 24.06 78.28 21.72 73.78 26.22 600 61.92 38.08 65.91 34.09 59.87 40.13 800 47.37 52.63 56.15 43.85 45.79 54.21 1000 37.92 62.08 43.8 56.20 33.9 66.10 The results for anti-inflammatory activity of leaf and flower extracts in ethanolic and Diclofenac were estimated spectrophotometrically. Ethanolic leaf and flower extracts and Diclofenac were found to be an effective protection respectively. Leaf and flower extracts has a percentage of stabilization less than that of the reference standard, Diclofenac. Among the extracts, leaf shows higher stabilization than that of flower (Table 2). DISCUSSION Ferric reducing power ability study in ethanolic leaf and flower extracts of N. arbor-tristis revealed the greater anti-oxidant property of leaf than flower. The reducing power enhanced with increasing concentration of both leaf and flower extract. Studies of similar kind 15 also reported antioxidant activity from N. arbor-tritis Linn. The reducing capacity of a compound may serve as a substantial indicator of its potential antioxidant activity [16]. This strong reducing power of the plant can also be attributed to the occurrence of various anti-oxidants such as phenolics and flavonoid contents. The anti-oxidant property of N. arbor-tristis Linn. is also also due to the existence of carotenoids and lycopene which are potent anti-oxidants. Inflammation is a common phenomenon and it is a reaction of living tissues towards injury. Steroidal antiinflammatory agents will lyse and possibly induce the redistribution of lymphocytes, which cause rapid and transient decline in peripheral blood lymphocyte counts to affect longer term response. Ethanolic leaf and flower extracts of N. arbor-tristis Linn. showed a potential in-vitro anti–inflammatory activity on HRBC in agreement with the former studies. The anti-inflammatory activity may be due to some chemical constituents present in the leaf and flower such as flavonoids, tannins, coumarins, phenols etc. that are biologically active whose properties might have been found in the ethanolic extract. Occurrence of flavonoids has been reported in Nyctanthes arbor-tristis and flavonoids are known to inhibit prostaglandin synthesis by inhibiting cyclooxygenase enzyme [17]. Since prostaglandins are involved in the late stage of acute reaction and are inhibited by flavonoids, it would be suggested that reduced availability of prostaglandins by flavonoids of Nyctanthes arbor-tristis might be responsible for its acute anti-inflammatory effect. - Oleanolic acid also has anti-inflammatory effect. They have been found to act on interferon of inducible nitric oxide synthase as well as strong effect on cyclooxygenase 2 enzyme. It has been reported that flavanoids exerts stabilizing effect on lysosomal membrane both in-vitro and invivo while tannins possess ability to bind to cations there by stabilizing erythrocyte membranes and other macromolecules [18]. It has been demonstrated that flavonoids are able to prevent both enzymes, as well as other intermediaries of the inflammatory process such as reactive c protein or adhesion molecules. The flavonoids have been found to have antimicrobial, antiviral, anti-ulcerogenic, cytotoxic, anti-neoplastic, mutagenic, antioxidant, antihepatotoxic, antihypertensive, hypolipidemic, antiplatelet and antipyretic anti-inflammatory activities. Flavonoids also have Biochemical effects, which prevent a number of enzymes such as aldose reductase, xanthine oxidase, phosphodiesterase, lipoxygenase, cycloxygenase, etc. Similar studies in N. arbor-tristis have been reported earlier that revealed the anti-inflammatory property of the plant. Arbortristoside-a from ethanolic extract of seeds of N. arbortristis have been isolated and proved to possess significant dose dependent anti-inflammatory activity [19]. Time has come to make worthy use of centuries old knowledge of Nyctanthes arbor-tristis Linn. through present-day approaches of drug development which has given encouragement among scientists in discovering more information about this medicinal plant. ACKNOWLEDGMENTS The first author greatly acknowledge the support extended by Dr. David Saj Mathew, Head of the Department of Botany, Union Christian College, Aluva for providing the necessary laboratory facilities and for his wise suggestions and encouragement. The author also express sincere thanks to all the teachers of the Department of Botany, Union Christian College, Aluva for the substantial help rendered to them for the successful completion of this work. REFERENCES-. P. A. Cerutti, Eur. J. Clin. Investig 21, 1-11 (1991). R. Sobota, M. Szwed, A. Kasza, M. Bugno, and T. Kordula, Biochem. Biophys. Res. Commun 1, 329– 333, (2000). K. M. Anil, Research Signpost 2, 267–293, (2010). V. M. Jose, T. T. Antony, Indian J Pharmacol. 35, 318-19 (2003). H. P. Rang, M M Dale, J. M. Ritter, R. J. Flower, Rang and Dale’s Pharmacology, Elsevier, 6, 226-45 (2008). K. Harleen, K. Mohanjith, Inter. Pharmaceutica Sciencia. 1, 28-35 (2011). I. Siddique, M. Anis, A. A. Jahan, World J. Agricultural Sci., 2, 188-192 (2006). G. R. Rout, A. Mahato, S. K. Senapati. Horticulture Sciences (Prague) 34: 84-89 (2007). N. A. Khatune, M. E. Islam, M. A. Rahman, M. A. Mosaddik, M. E. Haque, J. Medical Sciences; 3: 169-173 (2003). D. Sasmal, S. Das, S. P. Basu. Pharmacognosy Rev. 1, 344-349 (2007). S. R. Jensen, H. Franzyk, E. Wallander. Phytochem. 60, 213-231 (2002). M. Kannan, Singh,T. A. Kumar, P. Jegatheswari, S. Subburayalu. Afr. J. Microbiol. Res 1, 088-091 (2007). T. Hanato, H. Kagawa, T. Yasuhara, T. Okuda, Chemical Pharmaceutical Bulletin 36,- (1988). O. J. Oyedapo, O. Ekundayo, W. A. Koenig, Flav Fragr J 18,384-386 (2004). - 15. S. Meir, J. Kanner, B. Akiri, S. P. Hadas, J. Agric. Food Chem 43(7),- (1995). 16. W. Brand-Williams, M. Cuvelier, C. Berset; Lebensmittel-Wissenschaft and Technology 28(1), 25–30 (1995). 17. B. Havsteen, Biochem Pharmacol.32 (7), 1141-48 (1983). 18. B. H. Havsten, Pharmacol. Ther.; 96(2-3), 67-202 (2002). 19. S. Das, D. Sasmal, S. P. Basu, J Ethnopharmacol. 116(1): 198-203 (2008). -