CHEMICAL INVESTIGATION OF SOME INDIAN MEDICINAL PLANTS AND BIOSYNTHESIS OF 7-ACETOXY-4- METHYLCOUMARIN

Abstract

Importance of investigations on natural products for the development of useful drugs is immense. Inspite of spectacular advances in synthetic drugs in the 20th century, many of the plant drugs like morphine, quinine, colchicine, atropine, caffeine, emetine, reserpine and vinblastine have stood the strictest test of scientific evaluation. Such investigations besides providing new drugs also continue to contribute effectively in drug development programme by providing valuable structural leads to the medicinal chemists, whereas many other compounds isolated during these studies offer potentiality of being used as raw materials for the synthesis of existing drugs. Inspite of all the efforts carried out so far, majority of the terristorial flora remains to be explored to unveil the vast potential of the vegetable kingdom. The extreme variability of the climatic and geographic conditions has provided India with one of the rechest vegetations comprising approximately 15,000 species of plants belonging to about 340 families. Out of these, about 2500 plants have provided the basis of Ayurvedic and Unani systems of medicines. Another important aspect of natural product chemistry is 'Biosynthesis', the way by which nature makes these molecules efficiently in laboratory. The importance of biosynthesis in the study of alkaloids, coumarins is well recognised. Concept of biosynthetic processes not only helps in solving complex structural problems but also gives stimulus in searching .. 11 such compounds as predicted by theory, thus resulting in the discovery of new and novel structures. Understanding of biosynthetic processes also helps in designing new and novel synthetic routes to complex natural products. In view of the informations gathered from the local people and the subsequent literature survey on a large number of plants, it was found that certain species of Aristolochiaceae, Legum inosae and Menispermaceae are medicinally important. Moreover, there are a number of plant species of these families on which either a very little or no chemical work has been done. Thus, chemical examination of three local plant species namely Aristolochia indica Linn. (Aristolochiaceae), Abrus precatorius Linn. (Leguminosae), Stephania hernandifolia (Willd.) Walp. (Menispermaceae) and the biosynthesis of 7-acetoxy-4-methylcoumarin were undertaken. The first chapter is an introductory one dealing with the importance and development of plant chemistry in India, general important techniques employed for the isolation, purification and characterization of plant constituents, a review of medicinal importance and chemical work done on some important plants of Aristolochiaceae, Leguminosae and Menispermaceae families and a brief ;note on biosynthesis. The second chapter describes the biosynthesis of 7-acetoxy- 4-methylcoumarin derived from Triqonella foenumqraecum Linn. (Leguminosae). Chapters III, IV and V incorporate the results of the chemical investigation of Aristolochia indica Linn., Abrus precatorius Linn, and Stephania hernandifolia (Willd.)Walp. respectively. Ill Triqonella foenum qraecum Linn, is cultivated in many parts of India, mainly in Punjab and Kashmir. The seeds of this plant have been reported to be used as carminative, tonic, aphrodis and for dysentery. The plant, used in the Indian system of medicine, has been extensively studied for its chemical components. It has been reported to contain p-sitosterol, p-coumaric acid, quercetin, luteolin and 7-acetoxy-4-methyl coumarin. The occurrence of 7~acetoxy-4-methylcoumarin is of biogenetic interest as it emphasizes on the acetate route against the shikimic acid route so far known in the formation of coumarins. Trigonella foenumqraecum Linn. (Leguminosae) elaborates a coumarin with an extra methyl group at position-4. Its bio synthesis can not be explained on the basis of well known shikirr4c acid route. Feeding experiments with radioactive . L-[U- C]-tyrosine, [2-14C]-glycine, L-[l-14C]-methionine and ethyl-[3- C]-acetoacetate have been carried out in a cell free system of 3 month old T.foenumqraecum plants. The results obtained demonstrate that othylacetoacetate is a better precurssor with more than ten fold incorporation over methionine. The specificity of radiolabeled biosynthesised coumarin has been established which shows that Carbon-4 of coumarin ring originates from ethylacetoacetate in a manner analogous to Pechmann reaction. This is a first report of such a reaction in nature. Aristolochia indica Linn, is found in low hills and plains from Nepal and lower Bengal to Chittagong, in the Decoan Peninsula from Konkan southwards. The root of this plant was . . IV reported to be used as tonic, stimulant, emmenagogue, emetic and found effective in fevers and for leucoderma. The juice of leaves is found useful in snake-bite. The root is also found to possess antifertility activity. The plant has been reported to contain number of alkaloids, sesquiterpenes, fattyacids, steroids and aliphatic compounds. The air dried whole plant was extracted with petroleum ether, benzene and ethanol successively, The petroleum ether extract was fractionated into methanol insoluble and methanol soluble portions. Methanol insolubleportion on column chromatography over silica gel afforded n-heptadecane, n-triacontane, methylester of 12-nonacosenoic acid (New natural product), palmitic acid, hexacosanoic acid, hexadecane, octadec~ne, stearylalcohol, stearic acid, pentatriacontanoic acid (New natural product), and linoleic acid while the methanol soluble portion afforded stigmast-4-en-3- one and lupeolacetate including methylester of 12-nonacosenoic acid, palmitic acid, hexacosanoic acid, stearylalcohol, stearic acid, pentatriacontanoic acid and linoleic acid. The benzene extract was chromatographed to obtain friedelin and P-sitostercl, The ethanolic extract was further extracted with petroleum ether, ethylacetote and n-butanol successively. The latter two extracts wore combined and resolved into chloroform and methanol soluble portions. The chloroform soluble portion on chromatography afforded p-sitosterol and cycloeucalenol while the methanol soluble portion yielded two compounds which were characterised as rutin and p-sitosterol-p-D-glucoside. A part of the alcoholic residue was further extracted with Et»0, which was then shaken with 5 percent aqueous NaHC03 and the aqueous layer was acidified with HCL whereupon a solid separated. The solid residue on crystallisation from ethanol afforded yellow needles of aristolochic acid. Abrus proc-torius Linn, is found throughout the greater part of India, ascending the outer Himalayas to 3,500 ft, sometimes planted in gardens. The seeds of this plant have been used as purgative, tonic, emetic, aphrodis and in nervous disorders. Poultice of seeds have been reported to cause abortion. This plant has been reported to contain steroidal oil, ketosteroid, cholesterol, campesterol, stigmasterol, (3-sitosterol, squalene, flavones, anthocyanins, lectins, alkaloids and amino acids. The air dried and powdered seeds were extracted with petroleum ether and ethanol successively. The petroleum ether extract was fractionated into methanol soluble and methanol insoluble portions. Methanol insoluble portion on column chromatography over silica gel yielded n-hentriacontane, pentacosanoic acid, octacosancic acid along with two new natural products, 7, 9, 15-trimethyl heneicosane and 16, 17, 18, 19-tetramethyl-l-heneiccsanol, the structures of which are also established by spectral data while the methanol soluble portion yielded (3-amyrin, cycloartcnone and p-sitosterol including pentacosanoic acid, octacosancic acid and 16, 17, 18, 19- tetramethyl 1-heneicosanol. The ethanolic extract was extracted with petroleum ether, ethylacetate and n-butanol successively. The latter two fractions were combined and resolved into chloroform soluble and methanol soluble portions. The chloroform . . VI soluble portion on chromatography afforded only p-sitosterol while the methanol soluble portion yielded two compounds identified as stigmasterol and campesterol. Stephania hernandifolia (Willd.) Walp. is a menispermaceous slender twining shrub found in India on the west and east coasts, in Cachar, Sikkim, East Bengal and Assam. The roots are reported to have use in the treatment of fever, diarrhoea, dyspepsia and urinary diseases. The roots, rhizomes and leaves of this plant were reported to contain number of alkaloids. In addition, steroids and fats were also reported to be present. The air dried whole plant was extracted with petroleum ether and ethanol successively. The petroleum ether extract was fractionated into methanol insoluble and methanol soluble portions. Methanol insoluble portion on chromatography afforded n-hentriacontane and 1-triacontanol while the methanol soluble portion yielded two compounds characterised as 1-triacontanol and aknadinine The ethanoiic extract was extracted with petroleum ether, ethylacetate and n-butanol successively. The latter two fractions were combined and resolved into chloroform soluble and methanol soluble portions. The chloroform soluble fraction on chromato graphic separations afforded p-sitostencne and P-sitosterol while the methanol soluble portion yielded a compound identified as ^-sitosterol. The isolated compounds were characterised by spectroscopic methods, such as IR, UV, NMR and MS. Chemical methods, such as methylation, acetylation were also used for structural elucidation. The known compounds were compared with authentic samples, wherever possible. LISJ_OF PUBLICATIONS AMD RESEARCH_PAPFRS 1. Biosynthesis of 7-acetoxy-4-rnethyl coumarin, Accepted for presentation at the ACS Meeting in Anaheim, Division of Organic Chemistry, American Chemical Society, September 9 - 12, 1986. 2. Isolation of methylester of 12-nonacosenoic acid frc i£i£l2i£chia jndica, Indian Journal of Chemistry, Accepted for publication. 3. Constituents of Stephania hernandifolia (Willd.) Walp., Die Pharmazie (BERLIN), (Communicated). 4. Hydrocarbons, fatty acid ,triterpenoid and sterols in the seeds of Abrus precatorius Linn., Science and Culture (CALCUTTA),(Communicated). 5. Chemical investigation of Indian Medicinal Plant, Aristolochia indica Linn. (Aristolochiaceae). presented at the 73rd session of the Indian Science Congress Association held at New Delhi on Jan. 3-8,1986 ; Proc. 73rd Ind.Sc.Cong. jPart III, 141 (1986). 6. Chemical investigation of Indian Medicinal Plant, ^rus precatorius Linn. (Leguminosae) : presented at the 55th annual session of the National Academy of Sciences, India held at Gwalior on November 3-5,1985.