South Indian Journal Of Biological Sciences 2016; 2(1); 119 124 ONLINE ISSN: 2454 4787 Research Article Study on the phytochemical, antibacterial and antioxidant activities of Simarouba glauca Sneha Kochath Santhosh 1, Aswathy Venugopal 1, Meera Chakrappully Radhakrishnan 1, * 1 Department of Microbiology, St. Mary s College, Thrissur, Kerala, India * Corresponding author: Meera Chakrappully Radhakrishnan; E mail: meera_mahes@yahoo.com; Tel.: +919495369274 Received 31 July 2015; Revised 22 September 2015; Accepted 28 September 2015; Published 2 January 2016 Abstract Since time immemorial Simarouba glauca has been used as a natural medicine in tropics. Crude petroleum ether and ethyl acetate extracts from dried leaves of S. glauca were tested for the antibacterial and antioxidant activity. Preliminary screenings for the phytochemicals were done using the standard qualitative methods. Antibacterial activity was studied using disc diffusion assay against the organisms like Staphylococcus, Salmonella, Bacillus, Klebsiella, Pseudomonas sp and Escherichia coli. Antioxidant activity was evaluated using total antioxidant assay, Hydroxyl radical scavenging assay and 2,2, diphenyl 1 picrylhydrazyl (DPPH) scavenging activity. Phytochemical screening revealed the presence of phenol, terpenoids, proteins, steroids, and carbohydrate in the ethyl acetate extract and terpenoids and steroids in the petroleum ether extract.both extracts showed significant antioxidant activity in a dose dependent manner. However, ethyl acetate extract was found to be more effective as compared to petroleum ether extract. Antibacterial activity was not found to be significant. The results suggest that S. glauca has promising antioxidant activity and could serve as a potential source for natural antioxidants. Keywords: Simarouba glauca, Antibacterial activity, Antioxidant activity, Phytochemicals 1. Introduction The use of higher plants and their extracts to treat infections is an age old practice. The medicinal value of these plants lies in some chemical substances that produce a definite physiological action on the human body. The most important of these bioactive constituents of plants are alkaloids, tannins, flavanoids and phenolic compounds (Hill 1952). Simarouba glauca is a rain fed wasteland evergreen edible oil tree, commonly known as Laxmitaru or paradise tree. Belonging to the plant family Simaroubaceae, S.glauca bears various groups of chemical components (Harbone 1973). The main active group of chemicals in Simarouba is called quassinoids, which belong to the triterpine chemical family. Simarouba glauca has several uses, mainly as herbal medicine. All parts of the plant namely seed, shell, fruit pulp, leaf, leaf litter, unwanted branches, stem, bark, and root generate products that are useful in the production of food, fuel, manure, timber, medicine Antonisamy et al., 2015; Barathi and Agastian 119
2015; Rathi et al., 2015; Nandhini and Stella Bai 2015)..Chemical constituents of simarouba extracts exhibit antimicrobial, antifungal, antiprotozoal and antiviral activity. Components with antimalarial, and antileukemic activities have been also isolated from Simarouba sp. The plant is considered to have antiseptic, analgesic, astringent and vermifuge properties (Collins and Lyner 1987). Simarouba bark is a natural remedy against chronic and acute dysentery. It is taken internally for diarrhea, dysentery, malaria and colitis. The tree is also called as dysentery bark. The chemicals present in leaf, fruit, pulp and seed of S. glauca are known to posses the medicinal properties such as antimicrobial, antiviral, astringent, emmenagogue, stomachic, tonic, vermifuge. The free radicals are species with very short half life, high reactivity and damaging activity towards macromolecules like proteins, DNA and lipids. These species may be either oxygen derived (ROS, Reactive Oxygen Species) or nitrogen derived (RNS, Reactive Nitrogen Species). In recent years, many studies evidenced that plants containing high content of antioxidant phytochemicals can provide protection against various diseases induced by free radicals (Sofowora 1993). Large number of scientific studies also revealed that the plant extracts are rich source of natural antioxidants (Trease and Evans 1989). The present study was focused on the evaluation of the antibacterial and anti oxidant properties of different extracts isolated from S. glauca. Preliminary photochemical evaluation of the isolated extracts was also carried out. 2. Materials and methods 2.1. Collection of plant materials Simarouba glauca was collected from outskirts of Thrissur, Kerala. The plant was identified at the Dept. of Botany, St. Mary s College, Thrissur. Fresh leaves of the plant Simarouba glauca were collected and were dried for six hours at 40 50 0 C. The dried leaves were then crushed into powder. The powdered samples were stored in bottles at room temperature till use. 2.2. Preparation of extracts The crude powdered sample of 200 g was extracted with petroleum ether and ethyl acetate in a soxhlet apparatus for 8 10 h, repeatedly thrice. Both solvent extracts were collected separately and filtered through Whatmann No.1 filter paper and evaporated to dryness. The dried samples were than stored in air tight bottles at 4 0 C. 2.3. Phytochemical screening Phytochemical analysis were carried out with petroleum ether and ethyl acetate extracts of Simarouba glauca, using standard procedure described by Sofowara (1993), Trease and Evans (1989) and Harborne (1973). The presence of alkaloids, tannin, anthraquinone, flavanoids, phenol, steroids, terpenoids, proteins and carbohydrates were tested. 2.4. Antibacterial activity Antibacterial test was carried out by disc diffusion method (Collins 1987; Balachandran et al., 2015) against Staphylococcus, Salmonella, Bacillus, Klebsiella, Pseudomonas sp and Escherichia coli. Briefly, 0.1 ml culture was uniformly distributed on to Muller Hinton agar plates. Whatsman No 1 filter paper disc of 4 mm diameter were made and sterilized. Then discs were placed on the surface of Muller Hinton agar 120
plates at a distance of 2 cm. Drugs of different concentrations (250, 500, 750, 1000 μg/ml) were added on each disc and incubated at 37 0 C for 16 18 h. After incubation zone diameter was measured. 2.5. Anti oxidant activity 2.5.1. Total anti oxidant capacity assay The total antioxidant capacity measured according to sepctrophotometric method. Each extract (10 mg/ml) upto a volume of 0.1 ml was dissolved in water and taken with 1 ml of reagent solution (0.6 M sulphuric acid, 2.8 mm Sodium phosphate and 4 mm Ammonium molybdate). The tubes were capped and incubated at 95 0 C for 90 minute. After cooling to room temperature, absorbance was measured at 695 nm. Vitamin C was used as the standard. 2.5.2. Hydroxyl radical scavenging activity Hydroxyl radicals generated from Fe 3 ascorbate EDTA H2O2 system (Fenton s reaction) was estimated by its degradation of deoxyribose that resulted in thiobarbituric acid reactive substance (TBARS) (Elizabeth and Rao 1990). The reaction mixture contained deoxyribose (28 Mm); FeCl3 (1 mm); KH2PO4 KOH buffer (20 Mm, p H 7.4); EDTA (1mM); H2O2 (1 Mm); ascorbic acid( 0.1 mm) and various concentrations of the extracts in a final volume of 1.0 ml. The reaction mixture was incubated for 1 hour at 37 0 C. The TBARS formed was estimated by TBA method (Ohkawn et al., 1979). The absorbance was measured at 532 nm. Vitamin C was used as the standard. 2.5.3. DPPH free radical scavenging activity In this method a commercially available and stable free radical 2,2, diphenyl 1 picrylhydrazyl (DPPH + ) soluble in methanol was used. DPPH in its radical form has an absorption peak at 515 nm, which disappeared on reduction by an antioxidant compound. An aliquot (25 μl) of the extracts was added to 1 ml of freshly prepared DPPH solution. The samples were kept for 20 min in darkness and the decrease in the absorbance was measured at 515 nm. Vitamin C was used as the standard. 3. Results and discussion 3.1. Phytochemical analysis Phytochemical screening revealed the presence of phenols, terpeniods, proteins, steroids, and carbohydrate in the ethyl acetate extract and terpeniods and steroids test in the petroleum ether extract (Table 1). Table 1. Phytochemical analysis of Simarouba glauca. Phytochemicals Ethyl acetate extract Petroleum ether extract Tannin Flavanoids Quinines Terpeniods + + Phenols + Steroids + + Alkaloids Carbohydrates + Proteins + 121
3.2. Antibacterial activity The antibacterial effects of ethyl acetate and petroleum ether extracts from Simarouba glauca were tested against Gram positive and Gram negative bacteria by disc diffusion method. Petroleum ether and ethyl acetate extract of Simarouba glauca showed no activity towards all the tested organisms. 3.3. Antioxidant activity Antioxidant acitivity of the petroleum ether and ethyl acetate extract from the plant leaf Simarouba glauca was done by total antioxidant assay, DPPH and hydroxyl radical scavenging assay. 3.3.1. Total antioxidant activity The ethyl acetate as well as petroleum ether extracts of Simarouba glauca showed significant antioxidant activity in a dose dependent manner. IC50 value of ethyl acetate and petroleum ether was found to be 1000 μg and 2000 μg respectively. IC50 value of Vitamin C was 700 μg (Fig.1). Petroleum ether Ethyl acetate Fig.1. Total antioxidant capacity of the Simarouba glauca. 3.3.2. Hydroxyl radical scavenging assay The petroleum ether and ethyl acetate extract of Simarouba glauca showed significant scavenging activity of hrdroxyl radical generated by Fenton s reaction. The graphical representation shows the increase in activity with response to the increase in drug concentration. The ethyl extract has more activity than petroleum ether. IC50 value of ethyl acetate and petroleum ether was found to be 1120 μg and 1320 μg respectively. IC50 value of vitamin C was 700 μg (Fig. 2). 122
Ethyl acetate Petroleum ether Fig. 2. Hydroxyl radical scavenging activity of Simarouba glauca. 3.3.3. DPPH free radical scavenging activity The ethyl acetate as well as petroleum ether extract of Simarouba glauca also showed profound free radical scavenging activity in DPPH assay. The graphical representation shows the increase in DPPH activity with response to the increase in drug concentration. IC50 value of ethyl acetate and petroleum ether was found to be 1200 μg and 1280 μg respectively. IC50 value of Vitamin C was 700 μg (Fig. 3). Ethyl acetate Petroleum ether Fig.3. DPPH free radical scavenging activity of Simarouba glauca. Phytochemical analysis showed the presence of a large number of biological active plant constituents in the extract of Simarouba glauca. The result showed variations in number of phytochemicals obtained in both the extracts. Compared to both the solvents extracts, more number of 123
metabolites was detected in the ethyl acetate extract. This may be due to high solubility of active compounds of Simarouba glauca with ethyl acetate using the extraction process compared to petroleum ether solvent. 4. Conclusion The antioxidant activity for both the extracts was conducted using total antioxidant assay, DPPH, Hydroxyl radical scavenging assay. Both extracts showed significant antioxidant activity in the dose dependent manner. Ethyl acetate extract was more active compared to the petroleum ether extract. It may be due to the presence of more number of phytochemicals in this extract. The results of the study revealed the antioxidant property of Simarouba glauca. Simarouba glauca can be a potential and effective resource for drug discovery. Further research may pave way to the development of effective antioxidant and antibacterial agents from Simarouba glauca. Conflict of interest statement We declare that we have no conflict of interest. References 1. Antonisamy P, Duraipandiyan V, Ignacimuthu S, Kim J H. (2015). Anti diarrhoeal activity of friedelin isolated from Azima tetracantha Lam. in Wistar rats. South Indian Journal of Biological Sciences, 1(1), 34 37. 2. Balachandran C, Duraipandiyan V, Emi N, Ignacimuthu S. (2015). Antimicrobial and cytotoxic properties of Streptomyces sp. (ERINLG 51) isolated from Southern Western Ghats. South Indian Journal of Biological Sciences, 1(1), 7 14. 3. Barathi KK, Agastian P. (2015). In vitro regeneration of a rare antidiabetic plant Epaltes divaricata L. South Indian Journal of Biological Sciences, 1(1), 52 59. 4. Collins C H, Lyner P M. (1987). Microbiological methods. Butter worths and co publisher, London. 5. Elizabeth K, Rao MNA. (1990). Oxygen radical scavenging activity of curcumin. International Journal of Pharmaceuticals, 58, 237 240. 6. Harborne JB. (1973). Phytochemical dictionary. A handbook of bioactive compounds from plants. Taylor and Francis, London. 7. Hill AF. (1952). Economic botany: A Textbook of Useful Plants and Plant Products, 2 nd edn. Mc Graw Hill Book Company Inc., New York. 8. Nandhini VS, Stella Bai GV. (2015). In vitro phytopharmacological effect and cardio protective activity of Rauvolfia tetraphylla L. South Indian Journal of Biological Sciences, 1(2), 97 102. 9. Okhawa H, Ohisimi N, Yagi. K. 1979. Assay for lipid peroxides in animal Tissue thiobarbituric acid reaction. Analytical Biochemistry, 95, 351 358. 10. Rathi MA, Meenakshi P, Gopalakrishnan VK. (2015).Hepatoprotective activity of ethanolic extract of Alysicarpus vaginalis against nitrobenzene induced hepatic damage in rats. South Indian Journal of Biological Sciences, 1(2), 60 65. 11. Sofowora A. (1993). Medicinal plants and Traditional Medicine in Africa. Spectrum Books Ltd (Pub.), Ibadan. 12. Trease GE, Evans WC. (1989). Pharmacognosy. 13th (ed). ELBS/Bailliere Tindall, London. pp. 345 346. 124