Phytochemical Analysis Of Myrsine Africana Land Maesa Lanceolata Forsk And Structure Activity Relationship Studies Of 2,5-dihydroxyalkylbenzoquinones
Arot, Lawrence M
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ABSTRACT Myrsine africana Land Maesa lanceolata Forsk are among the five medicinal Myrsinaceae plants found in Kenya and find a wide application in ethnopharmacology as anthelmintics and as anti-microbials. The plant parts namely; fruits, leaves, stem and root barks were-e~acted with non polar (petroleum ether, dichloromethane, ethyl acetate and dioxane) and polar (methanol) solvents. Chromatographic separation of these extracts on oxalic acid impregnated silica gel (soaking in 2-3% oxalic acid solution) and active silica gel using various solvents led to the isolation of an array of compounds whose structures were established by physical, spectroscopic (UV, IR, IH and 13CNMR and MS) and chemical properties. The classes of compounds isolated included flavonoids, terpeniods and/or steriods, anthraquinones, benzoquinones with long aliphatic side chains and a long chain aliphatic ketone. The level of benzoquinones was found to be high in all parts of the plants examined compared to other secondary metabolites with the highest concentration being realised in the fruits. Chromatographic separation of M. africana leaf extract resulted in the isolation of four novel flavonol glycosides which were; myricetin 3-0-3",4"-diacetylrhamnoside (171), 3'-methoxyquercetin 3-0-13-D-glucoside (173), quercetin 3-0-13-D(6-0-a-L-rhamnosyl) galactoside (183), and quercetin 3-0-13-D-(6-0-a-L-rhamnosyl) glucoside 7-0-13-D-(4-0-aL-rhamnosyl)glucoside (184) together with known one~Kaempferol (102), quercetin (77), myricetin (100), myricetin 3-0-rhamnoside (170), rnyricetin 7-0-rhamnoside (172), quercetin 3-0-rhamnoside (lID, quercetin 3-0-6--D- glucoside (175), quercetin 3-0-6-Dgalactoside (178), myricetin 3-0-6-D-galactoside (179), myricetin 3-0-xyloside (176), myricetin 3-0-arabinoside (177), kaempferol 3-0-13-D-(6-0-a-L-rhamnosyl) glucoside (180), 3'-O-methylquercetin 3-0-13-D-(6-0-a-rhamnosyl) glucoside (liD quercetin 3-0-13D-(6-0-a-L-rhamnosyl) glucoside (182). Sitosterol (105), stigmasterol (148), stigmasterol 3-0-glucoside (190), taraxerone (187) and myricadiol (188) and spinasterol 3-0-6- D-glucoside (110) were the terpenes and/or steroids isolated from various parts of the plant. Four anthraquinone pigments, emodin (23), chrysophanol (25), physcion (197) and 2-hydroxychrysophanol (161), and nepodiri (162) a long with gallic acid (194) and a long chain aliphatic ketone, 2-triacontanone (196) were also isolated from the plant. XVI Apart from the compounds listed above several benzoquinones with long aliphatic side chains were isolated, including five considered to be obtained from the plant for the first time. These are: 2,5-dihydroxy-6-methyl-3-undecyl-l ,4-benzoquinones (muketanin) (202), methylvilangin (82), anhydromethylvilangin (,[l), methylenebis (2,5-dihydroxy-4- -~ tridecy 1-3,6- benzoquinones) (214) and 2,3 -dihydroxy -6- heptadecy 1-1,4-benzoquinones (206). Together with these, other known benzoquinones isolated were 2,3-dihydroxy-6-undecy-l,4benzoquinone (myrsinone) (129), 2-hydroxy-5-0-methyl-3-undecyl-l,4-benzoquinone (5-0methylembelin (130)) and myrsinaquinone (136). On the other hand a chromatographic analysis of M. lanceolata parts led t~ the isolation of three new benzoquinones 2-hydroxy-5-methoxy-6-methyl-3-undecyl-l,4benzoquinone (219), 2,5-dihydroxy-3-(nonadec-14'z-enyl)-1 ,4-benzoquinone (220) and the fourteen carbon chain linked bisbenzoquinone, lanciaquinone (222). The known benzoquinones from this plant were identified as maesaquinone (11), acetylmaesaquinone (94), 2,5-dihydroxy-3-pentadecyl-l ,4-benzoquinone (126), 2,5-dihydroxy-3-(pentadec-1O'zenyl)-I,4-benzoquinone (127) and 2-hydroxy-5-methoxy-3-(pentadec-1 O'z-enyl)-1 ,4benzoquinone(maesanin, 128). The other quinones isolated from this plant were emodin (23) and chrysophanol (m. Taraxerol (193) and spinasterol 3-0-glucoside (110) were the only terpenoids isolated and characterised from this plant. Apart from isolation and characterisation work, structural modification of known benzoquinones and synthesis of 2,5-dihydroxy-3-alkyl-f~-benzoquinones were attempted . ~ ... for structure activity relationship studies for their alkyl moiety. The compounds prepared were 2,5-dihydroxy-3-ethyl-l,4-benzoquinone (223), 2, 5 -dihydroxy-3-propyl-I,4benzoquinone (224), 2,5-dihydroxy-3-pentyl-l,4-benzoquinone (225), 2,5-dihydroxy-3heptyl-I,4-benzoquinone (226), 2,5-dihydroxy-3-nonyl-I,4-benzoquinone (227), 2,5dihydroxy-3-undecyl-l,4-benzoquinone (embelin, Q2), 2,5-dihydroxy-3-tridecyl-I,4benzoquinone (rapanone, 1Q) and 2,5-dihydroxy-3-pentadecyl-I,4-benzoquinone (126). Biological activity tests such as insect anti-feedant, anti-microbiols, phytotoxicity, acaricidal, contact insecticidal and nematicidal were carried out with crude extracts from various plant parts and also with some of the compounds isolated, prepared and/or structurally modified. Insect anti-feedant tests were carried out with Epilachna varivestis Muls on XVll Phaseolus vulgaris L using "choice" test in which one bean leaf with one half treated and the other half untreated is in one petri dish. Dichloromethane, ethyl acetate, and methanol extracts of both M. africana and M. lanceolata parts leaves, fruits stem and root barks in - - ~ three concentrations (O.S, 0.2 and 0.1 %, w/v) were tested. Similarly several compounds including the isolates, structurally modified ones as well as the prepared 2,S-dihydroxy-3alkyl-l,4-benzoquinones in up to five concentrations (0.1, O.OS, 0.02S, 0.02 and 0.01 % w/v) were also tested and results showed that both CH2Cl2 and ethyl acetate extracts of M. africana various parts gave less than 5% consumption by E. varivestis at all concentrations tested. However, extracts from M. lanceolata various parts did not deter the test animals from feeding. In this case the percentage consumption at all concentrations was over 90%. On the other hand several components of M. africana parts were tested at different concentrations and the activity of related chemical structures compared. Results obtained from the test showed that embelin (Q2) and rapanone eZID gave percent consumption as 15% and 2S% respectively when tested at 0.02%. Similarly 2,S-dihydroxy-3-nonyl-l,4benzoquinone (227) and 2,S-dihydroxy-3-pentadecyl-l,4-benzoquinone (126) afforded percentage consumption as 40 and SO respectively when tested at the same concentration. Phytotoxicity effects of benzoquinones has been demonstrated by measuring the inhibitory activity of these compounds at 1000, SOO and 2S0 ppm concentrations on seedling growth of Radish seeds (Scorlet globe variety). Bioassays showed that radicle elongation of seedlings tested was inhibited by most Qr the benzoquinones tested. For example, myrsinone (129), embelin (Q2), 2,S-dihydroxy-3-.pentadecyl-l,4-benzoquinone (126), rapanone (70), muketanin (202), 2,S-dihydroxy-3-alkyl-l,4-benzoquinones 223, 224, 22S, 226 and 227, maesaquinone ill), maesanin (128), and acetylmaesaquinone (2.1) inhibited radicle elongation at 1000 ppm with values ranging from O-S% and percentage germination in the range 0-30% after 72 hours. Nematicidal activity tests of benz oquin ones using Meloidogyn incognita revealed that at 100 ppm and 1000 ppm, the compounds, maesanin (128) and S-O-methylembelin (130) were 82% and 80.S% nematicidal respectively. Embelin (Q2), rapanone (70), methylvilangin (m, compounds 223, 224, 22S, 226, 227 and maesaquinone ill) gave values ranging from 52-73% with higher values recorded for embelin and rapanone. The latter compounds were considered not active as nematicides since the recorded values were less than 80%. Bioautographic technique for detecting fungitoxic substances on t.l.c revealed that XVlll at concentrations 50 ppm, 10 ppm and 1 ppm acetylmaesaquinone (.21), muketanin (202), compound (206), 2,5-dihydroxy-3-(nonadec-14'z-enyl)-1 ,4-benzoquinone (220), embelin (69), rapanone (70), anhydrovilangin (ru, methylvilangin (82), maesanin (128), maesaquinone (11) and 5-0-methylembe1in (130) were active against Cladosporium cucumerinum by displaying a white spot after incubation period. Anti-bacterial tests were conducted using a wide range of both Gram-positive and Gram-negative bacteria. Embelin (Q2), rapanone (1Q), 2,5-dihydroxy-3-alkyl-1,4benzoquinones (220) and (126) displayed strong antibacterial activity against Candida albicans and Streptococus faecacus but showed weak activity against Pseudomonas acidororous. On the other hand the compounds (223), (224), (225), (226) and (227) showed a weak activity against Candida and no activity against .s.. faecalus. Results of the effect of benzoquinone derivatives investigated on Boophilus microplus (Canestrinii) the southern cattle tick revealed that embelin (69), myrsinone (129), maesaquinone (11) and maesanin (128) were quite effective in inhibiting the hatching of this anthropod eggs. The order of activity in inhibiting eggs hatching at 8 ug per tick was: embelin 95.3% > maesanin 91.0% > maesaquinone 72.2% > myrsinone 45.5%. Finally contact toxicity effect of benzoquinone compounds on Cylas formicarius elegantulus, a sweet potato weevil showed that at 120 mg per insect, embelin (Q2), rapanone (1Q), embelin dimethylether (201), maesaquinone dimethylether (217) and 2,5-dihydroxy-3pentadecyl-l,4-benzoquinone (126) brought about 100~fnii.sect mortality 48 hours after treatment.