Chemical and Biological Investigations of Cerbera Odollam Gaertn
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Chemical and Biological Investigations of Cerbera odollam Gaertn Monica Sharfin Rahman1, Adnan Faisal2, Choudhury Mahmood Hasan1, Monira Ahsan1 and Mohammad Mehedi Masud2 1Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Dhaka, Dhaka- 1000, Bangladesh 2Uttara Adhunic Medical College, Uttara, Dhaka, Bangladesh (Received: August 24, 2017; Accepted: September 20, 2017; Published (web): December 23, 2017) ABSTRACT: The present article describes the isolation and characterization of secondary metabolites from the leaves of Cerbera odollam as well as bioactivities of the crude methanolic extract and its petroleum ether (PE), carbon tetrachloride (CTC), chloroform (CF) and aqueous (AQ) soluble fractions. In this investigation, successive chromatographic separation and purification yielded β-amyrin (CO-01), lupeol (CO-02), β-sitostenone (CO-03) and triticusterol (CO-04). The study also indicated the antioxidant, cytotoxic, antimicrobial, membrane stabilizing activities and poor thrombolytic activities. Thus, the present study endeavors to provide scientific basis to establish Cerbera odollam as a potential medicinal plant. Key words: Cerbera odollam, triticusterol, antioxidant activity, cytotoxicity, antimicrobial activity, thrombolytic activity, membrane stabilizing activity. INTRODUCTION To date, plants have always been useful to us not mostly in the southern hemisphere, where only in the treatment of various diseases, but also as a approximately 80% of the population rely on a potential lead for discovering bioactive molecules in traditional system of medicine based on herbal drugs drug development process. The scientific exploitation for primary healthcare.1 Despite such high demand of of plants that are traditionally used as folk medicine this traditional medicine, it is observed that only 1% for pain relief, wound healing and abolishing fevers of them are known by the scientists and accepted for resulted in the identification of a wide range of commercial uses.3 Therefore, in this article, an compounds and consequently paved the way of new attempt has been described which was taken to therapies for cancer, hypertension, diabetes and isolate and characterize the chemical constituents of 1 infectious diseases. A dramatic increase is also the medicinal plant Cerbera odollam. In addition, observed in last decade in the demand of medicinal efforts were taken to evaluate the possible biological plant as new potential source for both traditional and profiles of the plant. contemporary medicine in both developing and C. odollam, sometimes called C manghas L, 2 developed countries. It is estimated that, world’s belongs to the poisonous Apocynaceae family. The population will be more than 7.5 billion in the next plant is also known as pong-pong, buta-buta, nyan or 10 to 15 years. This increase in population will occur yellow-eyed cerbera tree in various regions.4 It Correspondence to: Mohammad Mehedi Masud mainly grows in coastal salt swamps and creeks in Email: [email protected] south India and also along the river banks in southern and central Vietnam, Cambodia, Sri Lanka, Myanmar 5 Dhaka Univ. J. Pharm. Sci. 16(2): 179-186, 2017 (December) and Malaysia. The seeds have a long history as a 180 Rahman et al. poison, particularly in Madagascar, due to the evaporator was used at low temperature and pressure presence of highly toxic cardiac glycoside in them. to provide about 36.00 gm crude dry extract. Apart from poisoning, the plant is also used Isolation of chemical constituents. For the traditionally as emetic, cathartic, in curing initial rapid fractionation, the extract was subjected to 6 hydrophobia and rheumatism. Previous vacuum liquid chromatography (VLC).13 The whole phytochemical investigations of C. odollam showed crude extract was fractionated over silica gel powder the presence of triticusterol, 2,6-dihydroxy-4- (Silica gel 60H) by using different solvents of methoxybenzoic acid, 2-hydroxy-4-methoxy-6- increasing polarities. Initially, 100% pet ether was 7 methyl benzoic acid, vebioside, deacetyl-tanghinin, used as the solvent system, which was followed by a 8 neriifolin, tanghinin, monoacetylneriifolin, and mixture of pet ether and ethyl acetate and then cardenolide glycoside 3 beta-O-(2’-O-acetyl-l- ultimately 100% ethyl acetate. At the end, small thevetosyl)-15(14-->8)-abeo-5 beta-(8R)-14-oxo- amount of methanol was also used in combination 9 card-20(22)-enolide (2’-O-acetyl cerleaside A). with ethyl acetate for better separation of the crude Biological activity evaluation also revealed its extract. A total of 23 fractions (each 100 ml ) were 10 potential in anticancer, antinociceptive, anti- eluted . bacterial, diuretic,11 cytotoxic,12 neuropharmaco- For the assessment, elutes were spotted on Thin 6 7 logical and antioxidant activities of this plant. Layer Chromatography (TLC) plates . Based on the The main objective of the present study was to TLC plate behavior, some of the fractions were then isolate and characterize secondary metabolites of the subjected to gel permeation chromatography over leaf of C. odollam and evaluation of antioxidant, lipophilic Sephadex (LH-20). cytotoxicity, antimicrobial, thrombolytic and A total of 121 fractions, each 100 ml were membrane stabilizing activities. collected. Then the fractions were examined using TLC and on the TLC features, potential fractions MATERIALS AND METHODS were selected for further purification. At this stage, Chemical investigation the fractions were subjected to TLC over silica gel Collection and preparation of plant material. PF254 and the developed plates were then examined For the investigation, leaves of the C. odollam were under UV lamp (both at 254 nm and 366 nm) and collected from the Sunderban area, Bangladesh. also by spraying the plate with vanillin-sulfuric acid Identification was confirmed by an expert taxonomist reagent followed by heating at 110ºC for few minutes of Bangladesh National Herbarium (Acc. No. 31302). to locate promising spots. Further purification of After collection, the leaves were sun dried for eight fractions 11, 101, 02 and 107 by preparative TLC days. For better grinding, the leaves were then provided CO-04, CO-01, CO-02 and CO-03 subjected to oven drying for about 24 hours at respectively. considerably low temperature. A high capacity NMR spectroscopy. A Brucker (400 MHz) grinding machine was used for crushing the dried instrument was used for recording 1H NMR and 1H- 1 leaves into a coarse powder. H COSY spectra in CDCl3 and the chemical shift Extraction. About 600.0 gm of powdered plant values were determined with respect to the residual material (leaves) was soaked in about 2.00 liter of non-deuterated solvent signal. methanol in a 4.00 liter capacity amber glass Biological investigation of Cerbera odollam. container. It was kept for two weeks with occasional For biological investigation, the crude methanolic shaking and stirring. At the end of two weeks, the extract of C. odollam was subjected to solvent- whole mixture was subjected to filtration through solvent partitioning using the protocol designed by cotton plug and then through Whatman No. 1 filter Kupchan and modified by Van Wagenen et al.14 paper. For evaporating the solvent a Buchii Rota According to this method, about 5 mg crude extract Chemical and Biological Investigations of Cerbera odollam 181 was dissolved in 10% aqueous methanol. Then the subjected to biological investigations such as methanolic solution was extracted with petroleum evaluation of antioxidant, cytotoxic, antimicrobial, ether followed by carbon tetra chloride and finally thrombolytic and membrane stabilizing activities. with chloroform. Then each of the fractions were 1 CO-01(β-amyrin): White amorphous solid; H NMR (400 MHz, CDCl3), δ 0.77 (3H, s, H-24), 0.78 (3H, s, H-28), 0.85 (6H, s, H-29, H-30), 0.92 (3H, s, H-25), 0.95 (3H, s, H-23), 0.98 (3H, s, H-26), 1.23 (3H, s, H-27), 3.21 (1H, dd, J= 10.8, 5.2 Hz, H-3), 5.11 (1H, dd, J= 3, 6 Hz, H-12). 1 CO-02 (Lupeol): White amorphous solid; H NMR (400 MHz, CDCl3), δ 0.77 (3H, s, H-28), 0.78 (3H, s, H-23), 0.81 (3H, s, H-24), 0.94 (3H, s, H-25), 0.99 (3H, s, H-26), 1.05 (3H, s, H-27), 1.66 (3H, s, H-30), 3.20 (1H, m, H-3), 4.54 (1H, br, s, H-29), 4.66 (1H, br, s, H-29). 1 CO-03 (β–sitostenone): White amorphous solid; H NMR (400 MHz, CDCl3), δ 0.67 (3H, s, H3-18), 0.80 (3H, d, J = 6.8 Hz, H3-27), 0.82 (3H, d, J =6.4 Hz, H3- 26), 0.82 (3H, t, J = 7.6 Hz, H3-29), 0.90 (3H, d, J = 6.4 Hz, H3-21), 1.16 (3H, s, H3-19), 5.70 (1H, s, H-4). CO-04 (triticusterol): 0.89 (3H, s, H-1), 0.91 (3H, d, J=6.4 Hz, H-7), 0.98 (3H, s, H-8), 0.98 (3H, d, J=6.4 Hz, H-9), 1.01 (3H, d, J= 6.4 Hz, H-11), 1.02 (3H, d, J= 6.4 Hz, H-16), 3.20 (1H, m, H-18), 4.69, 4.64 (2H, s, H-24). Evaluation of antioxidant activity. For the discs and standard antibiotic discs (Ciprofloxacine 30 evaluation of antioxidant activity, DPPH assay, µg/disc) were as negative and positive controls designed by Brand-Williams et al.15 was selected for respectively. The test samples were loaded on the the current investigation. In this investigation, discs at a concentration of 400.0 µg/disc. The evaluation of scavenging activities of the stable 1, 1- antimicrobial activity of the test samples were than diphenyl-2-picrylhydrazyl (DPPH) was used as the evaluated by measuring the zone of inhibition with a basis for assessing the antioxidant activities of the digital slide calipers.17,18 test samples taking butylated hydroxyl toluene (BHT) Evaluation of thrombolytic activity.