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Phytochemical Analysis, Antioxidant Assay and Antimicrobial Activity in Leaf Extracts of Cerbera Odollam Gaertn

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Phytochemical Analysis, Antioxidant Assay and Antimicrobial Activity in Leaf Extracts of Cerbera Odollam Gaertn Pharmacogn J. 2018; 10(2): 285-292 A Multifaceted Journal in the field of Natural Products and Pharmacognosy Original Article www.phcogj.com | www.journalonweb.com/pj | www.phcog.net Phytochemical Analysis, Antioxidant Assay and Antimicrobial Activity in Leaf Extracts of Cerbera odollam Gaertn Abinash Sahoo, Thankamani Marar* ABSTRACT Introduction: In the current study, methanol and aqueous extracts of leaf of Cerbera odollam Gaertn were screened for its antibacterial, antifungal, phytochemicals and antioxidant ac- tivities. Phytochemical constituents were investigated both qualitatively and quantitatively. Methods: The leaf extracts of Cerbera odollam Gaertn were prepared by drying and extracted using Soxhlet apparatus into methanol and aqueous media, which were subjected to phyto- chemical screening. Total phenols, tannins, flavanols, alkaloids and its antioxidant activity were determined using spectroscopic techniques. Antimicrobial activity were determined using well diffusion method. Results: Aqueous extract exhibits higher content of phenols, tannins, flavanols and alkaloids, whereas methanol extract exhibits higher content of anthocyanin and cardiac glycoside respectively. Aqueous extract exhibits higher inhibitory concentration (IC %) value for DPPH (2, 2-Diphenyl-1-picrylhydrazyl) and H2O2 radical scavenging assay and reduc- ing power (RP) assay. The methanol extracts exhibited higher inhibitory concentration (IC %) value in SO and NO radical scavenging assay, exhibiting antioxidant properties in five antioxi- dant models that were investigated. The methanol extract showed some antibacterial activity against Bacillus subtilis, Staphylococcus aureus, Salmonella typhi and Escherichia coli with inhibitory zone ranging from 2 mm to 3 mm, whereas the aqueous extract showed no activity. Abinash Sahoo, High antifungal activity was found against Saccharomyces cerevisiae and Candida albicans for methanol extract and moderate for aqueous extract with inhibitory zone ranging from 9mm Thankamani Marar* to 26 mm. Conclusion: The finding of our study have suggested that the extracts ofCerbera odollam Gaertn, possesses a significant amount of phytochemicals and exhibits antioxidant School of Biotechnology and and antifungal activities. Bioinformatics, D.Y.Patil Deemed to be Key words: Cerbera odollam. G, Phytochemicals, Antioxidants, Radical scavenging, Antibacterial, University, Plot no. 50, Sector 15, CBD Antifungal. Belapur, Navi Mumbai, Maharashtra, INDIA. Correspondence Dr. Thankamani Marar INTRODUCTION School of Biotechnology and Cerbera odollam Gaertn is a tree belonging to the Medicinal properties of C. odollam are associated Bioinformatics, D.Y.Patil Deemed to be family of Apocynaceae and widely distributed on the with various phytochemicals found in the plant. University, Plot no. 50, Sector 15, CBD Belapur, Navi Mumbai, Maharashtra, coastal area of South East Asia and around the Indian Different plant species have shown cytotoxic activity INDIA. Ocean. The fruits and seeds ofC. odollam are highly against bacteria, fungus, and virus. It also possesses Phone No: +91- 9892455625 toxic, containing cardiac glycoside like cerberin, the property to scavenge reactive oxygen species neriifolin, odollin, etc. responsible for 10% to 50% of (ROS) which is the major source of primary cata- E mail Id: [email protected] total poisoning cases in the state of Kerala, India. It is lysts that initiate oxidation in vivo and in vitro and History create oxidative stress which results in numerous • Submission Date: 03-12-2017; used widely for suicide and homicide and hence the 1 12,13 14 • Review completed: 19-12-2017; name “suicide tree”. The tree is also known for various diseases and disorders such as cancer cardio- 15 16 • Accepted Date: 10-01-2018 medicinal properties. The latex is known in India for vascular disease, neural disorders, Alzheimer’s 17 18 its emetic, purgative and irritant effects.2 The oil from disease, mild cognitive impairments, Parkinson’s DOI : 10.5530/pj.2018.2.50 19 20 21 the seeds as a cure for, itching or applied to the hair disease, ulcerative colitis, ageing and atheroscle- 22 3 rosis. Article Available online as an insecticide. The bark and leaf of the plant are http://www.phcogj.com/v10/i2 traditionally used as emetic and cathartic; kernels are Phytochemicals are natural bioactive compounds used as an emetic; fruit is used as a cure for hydro- widely distributed in plants which have been Copyright phobia.4 Its bark and fruits are purgative and used for reported to exert multiple biological effects, including © 2018 Phcog.Net. This is an open- 5 access article distributed under the terms the treatment of rheumatism. There are reports of antioxidant, free radicals scavenging, anti-inflam- of the Creative Commons Attribution 4.0 the anti-nociceptive and sedative effects of its barks.6 matory, anti-cancer, antibacterial, antifungal etc. International license. Other research works have reported cytotoxic activity,7 Therefore, the need arises to evaluate and quantify its effect on the central nervous system,3 purgative and these phytochemical constituents. antirheumatic activity,8 cardiac stimulant activity,9 The present study was designed to determine the neurological activities,10 and cardiotoxic activity.11 phytochemical constituents, antioxidant activity, Cite this article: Sahoo A, Marar T. Phytochemical Analysis, Antioxidant Assay and Antimicrobial Activity in Leaves Extracts of Cerbera odollam Gaertn. Pharmacog J. 2018;10(2):285-92. Pharmacognosy Journal, Vol 10, Issue 2, Mar-Apr, 2018 285 Sahoo and Marar.: Phytochemical analysis, antioxidant assay and antimicrobial activity in leaf extracts of Cerbera odollam G. antibacterial and antifungal activity in methanol and aqueous extract Test for Amino acids -Ninhydrin test29 from the leaves of Cerbera odollam Gaertn through a number of testing Two ml of the filtrate was treated with 2-5 drops of 1% ninhydrin solu- methods. tion placed in a boiling water bath for 1-2 min and observed for the for- METHODS mation of purple color. Collection and extraction of plant material. Test for Sterols - Liebermann-Burchard test28 Fresh leaf of Cerbera odollam Gaertn collected from Mumbai, Maharashtra, One ml of the extract was treated with drops of chloroform, acetic anhy- India and authenticated by Blatter herbarium, St Xavier‘s college, matching dride, and conc. H2SO4 and observed for the formation of dark pink or with the Blatter herbarium specimen NI 2084 of N.A. Irani. Leaves were red color. cleaned and dried at room temperature for a period of 25 days under shade. Finely ground dried leaf, were weighed and extracted using Soxhlet Test for Terpenoids - Salkowki’s test23 apparatus by two different solvents methanol and distilled water, 150 ml One ml of chloroform was added to 2ml of each extract followed by a each for 30 g of powder. The solution of each extract was then sub- few drops of concentrated sulphuric acid. A reddish brown precipitate th jected to rotatory evaporator and reduced to 1/8 volume. The solution produced immediately indicated the presence of terpenoids. obtained was stored at 4°C and appropriately diluted for further studies. Test for Quinones30 Phytochemical screening (qualitative study) To 1 ml of extract, 1 ml of conc. H SO was added. Formation of red color 23 2 4 Test for Alkaloids - Mayer’s Test. indicated the presence of quinones. Few mg of the residue of each extract was taken separately in 5 ml of 1.5 % v/v hydrochloric acid and filtered. These filtrates were treated Test for Oxalate28 with Mayer’s reagent (1.36 g mercuric chloride and 5 g of potassium To a 3ml portion of extracts were added a few drops of ethanoic acid iodide dissolved in 100 ml distilled H O). The formation of a yellow glacial. A greenish black colouration indicates the presence of oxalate. cream precipitate indicates the presence of alkaloids. ₂ Test for Proteins. - Biuret Test31 Test for Flavonoids - Alkaline reagent test.24 Extracts were treated with 1 ml of 10% NaOH solution and heated. To Two ml of extracts was treated with few drops of 20% sodium hydroxide this, a drop of 0.7% CuSO4 solution was added. Formation of purplish solution. Formation of intense yellow color, which becomes colorless on violet color indicates the presence of proteins. the addition of dilute hydrochloric acid, indicates the presence of flavonoids. Test for Phlobatannins32 Test for Saponins - Foam test.25 About 2 g of powdered sample was boiled with 1% aqueous hydrochloric About 2 ml of distilled water and 1 ml of extract were mixed and shaken acid for 5 min. A positive test result was confirmed by deposition of a vigorously. Formation of a stable persistent froth indicated the presence red precipitate. of saponins. Test for Betacyanins32 Test for Tannins - Potassium Dichromate Test.26 To 2 ml of plant extract, 1 ml of 2N NaOH was added and heated for The test residue of each extract was taken separately in water, warmed 5 min at 100 C. Formation of yellow color indicated the presence of and filtered. If on an addition of a solution of potassium dichromate in betacyanin. test filtrate, dark colour develops, tannins are present. ⁰ 33 Test for Carbohydrates - Molisch’s test.23 Test for Fatty acids Extract (0.5ml) was mixed with 5 ml of ether and allowed for evapora- Few drops of Molisch’s reagent were added to a 2ml portion of the tion, on filter paper and dried. The appearance of transparent spots filter extracts. This was followed by addition of 2ml of conc. 2H SO4 down the side of the test tube. The mixture was then allowed to stand for 2-3 min. paper indicates the presence of fatty acids. Formation of a red or dull violet color at the interphase of the two layers Estimation of total phenols was a positive test for the presence of carbohydrates. The total phenol content was determined using the Folin-Coicalteu Test for Phenols - Ferric chloride test.27 method.34 Extract and 0.1ml of Folin Coicalteu reagent (0.5N) were One ml of the extracts in 3ml of distilled water was treated with aqueous mixed and incubated at room temperature for 15 min.
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