Isolation of β-Amyrin from Plant Parts of M. barteri

isolation of -Amyrin from topical anaestheticize Parts of M. barteriISOLATION AND BIOLOGICAL ACTIVITY OF THE TRITERPENE -AMYRIN FROM THE AERIAL PLANT PARTS OF MAESOBOTRYABARTERI(BAILL).AbstractMaesobotryabarteri(Baill), belonging to the family EUPHORBIACEAE, is a medicinal full treatment growing widely in tropical Africa. The Aerial plant part of Maesobotrya barteri (Bail) were self-contained fresh from Orokam, Ogbadibo local Government of Benue State, Nigeria in July, 2013. Taxonomical identification was done by Mallam Musa Abdullahi at the Herbarium unit of biologic sciences Department, ABU, Zaria, Nigeria. Pulverized aerial parts of Maesobotryabarteri (960g) was exhaustively extracted successively victimization petroleum ether, chloroform, ethyl groupacetate and wood alcohol and concentrated in the rotary evaporator at 40oC. The ethylacetate fraction having the highest activity against runnel microbes from preliminary crude microbic screenings was subjected to phytochemi cal studies, antimicrobial depth psychology and column chromatography (CC). The column chromatography yielded fraction EN, which was push purified using preparative thin point chromatography to surpass EN1. The structure of the marooned flux was established using 1-D proton magnetic resonance and 2-D NMR spectroscopic analysis and by direct comparism with data reported in literature was confirmed to be -amyrin. The bioactivity of this compound was carried kayoed using some clinical pathogens and the activity comp atomic number 18d with standard drugs and this was plunge to be same with the standard drug.Keywords Maesobotryabarteri medicative plant bioactivity Ethylacetate extract -amyrinIntroductionMaesobotrya sp is a variety of flowering plant belonging to the family Phyllanthaceae, or by some authors classified in Euphorbiaceae. The Euphorbiaceae plants are shrubs, trees, herbs or rarely lianas 1. Plants of the Euphorbiaceae are cognise to be rich in terpenoids (69.5%) 2.In Nigeria, the species M.barteri is beneath-exploited although the tree is of both medicinal and nutritional importance 3. Maesobotrya species are utilize medicinally in different regions in Africa 4. It bears succulent black-purple fruits that are edible and stain the tongue. The alimental values of the fruits and seeds have been studied in Southern Nigeria 5. Thus, this study aims at validating the antimicrobial personal effects of the aerial plant parts of Maesobotrya barteri utilise in conventional medicine in Orokam town of Benue State,Nigeria, as well as potent compounds that can be used as precursors for synthetic drugs.The presence of secondary winding metabolites in plants generally has been confirmed by 6, 7 and 8 through useful procedures and suggestions to get to targeted precursors which are of useful support in the formulation of modern drugs.Plant MaterialThe aerial parts of Maesobotrya barteri were collected from Orokam in Ogbadibo local government area of Benue State in the month of July, 2013. They were properly identified at the herbarium, Department of Biological Sciences, Ahmadu Bello University, Zaria. The whole plant was sorted, air dried under shade, segregated and pulverized by mechanically power hammer them using wooden mortar and pestle. The pulverized plant material was stored away from moisture.Extraction980.54g of the pulverized plant materials were carefully weighed and loaded into a Soxhlet extractor. It was extracted successively with Petroleum ether (60-80oC), Chloroform, Ethyl acetate (76-78oC) and Methanol by hot continuous percolation method in the Soxhlet apparatus for 72 hours respectively. Solvents used were those of JHD and general settle reagents The extracts were concentrated in vacuo at 40oC using rotary evaporator and subjected to air drying to give dried crude extracts.IsolationThe ethyl acetate extract was the most sensitive extract from the antimicrobial screening and was subjected to column chromatog raphy (CC) for fractionation. Solvents used to run the column chromatograghy were sealed and are products JHD with 98% purity. Fifteen grams (15g) of the extract was dissolved in ethyl acetate and preadsorbed on 10.0g silica gel (qualikens 60-120 mesh). The dried pre-adsorbed extract was transferred to a mortar and ground to give a fine powder and was added at the uniform layer on top of the column. The petroleum ether descended on a horizontal line indicating that the column was well packed. A total of 105 fractions (50mls) separately were initially collected using gradient elution with a solvent combination of petroleum ether and ethyl acetate, starting with 100% petroleum ether with an increasing polarity of 1% ethyl acetate. Similar fractions were pulled unneurotic based on monitoring from TLC. Fractions F20-F25 were pulled together at the ration of 82 and where subjected to further polish using Preparative thin layer chromatography to obtain a solid snow-covered amorphous philia which after several trails using different solvent combinations showed a single spot which was visible under the UV lamp and after spraying the TLC main office with 10% H2SO4 and oven dried for 5minutes at 60oC. The solid white amorphous substance produced was insoluble in ethyl acetate or petroleum ether save soluble in chloroform.Results and DiscussionFrom the preliminary antimicrobial screening of the crude aerial plant parts extracts of M. barteri, ethylacetate extract exhibited the highest activity against the test microbes used via their zones of forbidding. Therefore, an activity guided isolation was undertaken.Bioactivity of Compound EN1 Pure impoundThe antimicrobial activities of compound EN1 isolated from ethylacetate extract of the aerial plant parts of M. barteri was examined and agar disc dispersal method 13 was employed for the determination of the antimicrobial activities. The pure compound was determined using some infective microbes the microbes were obt ained from the department of Medical Microbiology Ahmadu Bello University Teaching- Hospital, Zaria, Nigeria.The determination of minimum masterory concentration was carried out using the nutrient bloodline susceptibility assay nimble according to the manufacturers instructions, as recommended by NCCLS 14. Minimum inhibition McFarland turbidity standard scale number 0.5 was prepared to give turbid solution. Normal saline was prepared and was dispensed into test tube and the test microorganism was then inoculated into the normal saline, incubation was at 37oC for 6hrs, dilution of the microorganism in the normal was performed until the turbidity marched that of the McFarland by visual comparison at this point the microorganism had a concentration of about 1.5108 cfu/ml.Minimum bactericidal and antifungal concentration of compound EN1 was carried out to check whether the test microbes were killed or only their growth was inhibited. Mueller Hunton agars were prepared according to t he manufacturers instruction, as recommended by NCCLS 14. They were boil to dissolve and were sterilized at 121oC for 15 minutes, the media were cool off to 45oC and the medium (20ml) was poured in to sterile Petri dishes, the plates were covered and allowed to cool and solidify. The contents of the MIC in the serial dilution was inoculated on to the media, the media were incubated at 37oC for 24hrs for the bacteria and at 30oC for 1-7 days for fungi, after which the plate were observed for colonies growth. The MBC/MFC was the plate with lowest concentrations of the extract without colony growth.Table 2 shows the Zones of inhibition (mm) of the pure the compound EN1 from the ethyl acetate fraction which showed remarkable activity against twelve of the sixteen organisms tested. Compound EN1 could not inhibit the growth of Corynebacterium ulcerans, Candida albicans, Candida tropicalis, Aspergillus fumigates, Aspergillus nigre. The various minimum inhibitory concentration (MIC) and m inimum Bacteriocidal concentration and fungicidal concentration (MBC/MFC) for the different microbes are as shown in Tables and the bioactivity of the aerial plant parts of M. barteri is comparable to the drugs Ciprofloxacin, Fluconazole and Fulcin used as positive controls.Table 2 Zone of inhibition of Compound EN1 against the test microorganism shield OrganismCompound EN1CiprofloxacinFluconazoleFulcinStaphylococus aureus323700Streptococus pyogenes303500Streptococcus feacalis323900Corynebacteruim ulcerans03200Escherichia coli323800Klebsiella pneumonia324000Salmonella typhi304200Shigella dysenteriae304000Candida albicans00350Candida krusei270370Candida tropicalis00320Candida stellatoidea250370Microsporum sp260038Aspargillus fumigates00032Aspargillus nigre00034Trichophyton rutarum280038Table 2 and 3 shows the activity of the compound EN1 which the spectroscopic analysis was proposed to be a -Amyrin or -Amyrenol, (C30H50O, 426.7 g/mol). Antimicrobial screening reported from other natu ral products has also confirmed the microbial properties of -Amyrin. -Amyrin was isolated from Ardisia elliptica 9, a medicinal plant used for alleviating chest pain, fever, liver poisoning and parturition complications. It was found that -Amyrin was six seasons as active as aspirin in inhibiting platelets aggregation. -amyrin was isolated for the first time fromLaurencia microcladia, marine algaedistributed widely in Egypt found to have antibacterial activity against Staphylococcus aureus, Bacillus subtilis, Salmonella typhi, Escherichia coli, and genus Pseudomonas aeurginosa 10.Table 3a Minimum Inhibition Concentration (MIC)Test Organisms3.12g/ml 6.2g/ml 12.5g/ml 25g/ml 50g/mlConcentrationStaphylococusAureus o* +StreptococusPyogenes o* + +StreptococcusFeacalis o* +EscherichiaColi o* +KlebsiellaPneumonia o* +SalmonellaTyphi o* +ShigellaDysenteria o* +CandidaKrusei o* + +CandidaStellatoidea o* + +MicrosporumSp o* + +TrichophytonRubrum o* + +KEY = No turbidi ty (No growth), o* = MIC,+ = Turbid (Growth)Table 3b Minimum Bactericidal Concentration andMinimum Fungicidal Concentration (MBC/MFC)Test Organisms3.12g/ml 6.2g/ml 12.5g/ml 25g/ml 50g/mlConcentrationStaphylococus aureus o* + + +Streptococus pyogenes o* + + +Streptococcus feacalis o* + + +Escherichia coli o* + + +Klebsiella pneumonia o* + +Salmonella typhi o* + + +Shigella dysenteria o* + + +Candida krusei o* + + +Candida stellatoideao* + + + +Microsporum spo* + + + +Trichophyton rubrum o* + + +KEY = No turbidity (No growth), o* = MBC/MFC+ = Turbid (Growth)Spectra resultsThe structure of compound EN1 was elucidated using Nuclear Magnetic Resonance Spectroscopy (NMR), 1-DNMR and 2-DNMR and also by comparing the obtained data with already active literature. The results obtained are as shown in the table.H1NMR7.2401, 5.2346, 3.2100, 3.2021, 3.1911, 3.1836, 2.3241, 2.1768, 2.1585, 2.0742, 2.0181, 2.0023, 1.9942, 1.9793, 1.9727, 1.9067, 1.9012, 1.8923, 1.8865, 1.8621, 1.8455,1.8379, 1 .8227, 1.7276, 1.7067, 1.6857, 1.6669, 1.6529, 1.6353, 1.6302, 1.6127, 1.5952, 1.5890, 1.5669,1.5463, 1.5230,1.4987,1.4836,1.4667,1.4579,1.4402,1.4276,1.3730,1.3429, 1.3195,1.2990, 1.2780, 1.2630, 1.2329,1.1813,1.1514,1.1169,1.0870, 1.0639,1.0316, 1.0195,0.9989,0.9759, 0.9681, 0.9404, 0.9322, 0.9217, 0.9088, 0.8927, 0.8841, 0.8680, 0.8591,0.8450 0.834113CNMR 137.9624, 125.9002, 79.0731, 77.2169, 77.0054, 76.7938, 55.2568, 52.7408, 47.9239, 47.5759, 42.0388, 39.5194, 39.0811, 38.8495, 38.7715, 38.6441, 37.0200, 36.7105, 33.0001, 30.6267, 29.6969, 29.3557, 28.1492, 28.0359, 27.2530, 24.2027, 23.5770, 23.3098, 21.1608, 18.3150,17.0954, 16.9846, 15.5986, 15.4777.The 13C NMR spectrum (Figure 3) showed thirty (30) major recognizable carbon signals, eight methyl radical group groups at 37.0200 (C-22), 28.0359 (C-23), 15.5986 (C-24), 15.4777 (C-25), 16.9846 (C-26), 24.2027 (C-27), 17.0954 (C-28), 28.1492 (C-29), 23.3098 (C-30) and a secondary hydroxyl bearing carbon 79.0731 at (C-3). It also showed some recognizable signals at 125.9002 and 137.9624 ppm which is assignable to the double bond at C-12 and C-13. In addition, ten methylene groups, eight methyl groups , six quaternary carbons atoms from DEPT experiment were observed. The chemical shift at 137.962, 125.9002 which are C-12 and C-13 and the consistent flow of methyl groups from C-23 to C-30 were characteristic peaks for a - Amyrin type of skeleton 11 and 12.1HNMR, 13C NMR and DEPTH spectra of the proposed compound -amyrinFigure1 1HNMR Figure 2 13C NMRFigure 3 DEPTTable 4 Comparison of 13C NMR spectrum data of -amyrin Obtained from the Aerial parts of M. barteri with literatureCarbon Position13CNM ppm -amyrin, Experimental 13CNMR ppm -amyrin, Literature 1213CNMR ppm -amyrin, Literature 111 38.849538.737.32 27.253027.228.28379.073179.371.854 38.771538.537.30555.256855.136.816 18.315018.621.127 38.644132.442.358 39.519439.845.909 47.923947.650.1810 36.710536.936.5511 23.577023.624.3312 125.9002121.7121.7013 137.9624145.2140.8114 42.038841.744.3615 29.355726.226.1316 21.160826.123.1117 29.696932.648.4818 47.575947.245.891939.081146.839.822030.626731.036.4921 33.000134.734.002237.020037.131.712328.035928.029.32415.598615.419.8425 15.477715.419.102616.984616.818.902724.202725. 918.3028 17.095428.419.202928.149233.836.183023.309823.719.42Proposed structureEN1-(C30H50O, 426.7 g/mol)Name -Amyrin or -AmyrenolConclusionThe isolation of -Amyrin from the aerial plant parts of M. barteri, whose bioactivity was established from this work by its zone of inhibition, is comparable to the drugs of Ciprofloxacin, Fluconazole and Fulcin. This justifies why the plant really serves as a general purpose antibiotic in traditional medicine in our society. The preliminary phytochemical screening also shows that the ethylacetate extract contains other classes of compounds that can be further isolated and tested for microbial activities and this can lead to chains of discoveries as the quest of precursors for m odern drugs are continually on course.