博士 / 國立臺灣大學 / 藥學研究所 / 87 / ABSTRACTI. Studies on the Production of Secondary Metabolites by the Tissue and Cell Cultures of Cryptocarya chinensis, Angelica dahurica and Lycium barbarumPart IStudy on the Production of Secondary Metabolites by the Callus Cultures of Cryptocarya chinensis The lauraceous plant, Cryptocarya chinensis Hemsl., is a perennial woody plant widely distributed in Taiwan. Past studies on the constituents of this species have revealed it to be a rich source of pavine alkaloids. The basic skeleton of pavine alkaloids is very similar to that of MK-801, an NMDA receptor antagonist and a potential brain cell activator, and the electrophysiological effect and antiarrhythmic potential on rat cardiac tissues of (-)-caryachine. Based on these findings, it is worthy of investigating the medical application of this type of compounds. Because the plants used for the isolation of (-)-caryachine is at the age more than six years, this study is aimed at seeking other source of pavines through the exploration of plant tissue culture techniques. The production of pavine alkaloids by the use of tissue and cell culture of Chinese cryptocarya is of great interest and, to our knowledge, it has never been investigated. In the present study, the callus cultures were induced from young leaves of the plant. Following the modifications of basal media as well as growth regulators, the optimal growth medium was found to be 1/2 MS supplemented with 8 ppm NAA, 1 ppm BA, 2 g/l casein hydrolysate, 3 % sucrose and 0.9 % agar. The callus biomass was harvested after culturing for several generations, and was then subjected to extraction, fractionation, isolation and identification of the constituents. The main pavine alkaloid, caryachine N-metho salt (2) was produced by the culture as expected, accompanied catechin (1), neocaryachine N-metho perchlorate (3), crychin N-metho perchlorate (4). The yield of caryachine N-metho salt was up to 0.08 % dry weight, and compound 3 is the first occurrence from natural product. The effect of various culture media components on the growth of callus, and the production of caryachine N-metho salt (2) determined by reverse phase HPLC were also investigated. The higher yield of compound 2 was achieved from 1/2 MS basal medium containing IAA ( 4 ppm ) or low concentration ( 1 % ) of sucrose.Part IIStudy on the Production of Secondary Metabolites by the Cell Suspension Cultures of Angelica dahurica The Chinese traditional herb, Bai-Zhi ( Angelica dahurica (Fisch.) Benth. et Hook.) has been listed in Herbals for the treatment of headache. Recently it has also been used in chinese medicine for the skin disease, called Yin-Hsieh Ping (Psoriasis). The chemical constituents of Bai-Zhi were reported to contain furocoumarins. Among them, imperatorin is the major bioactive ingredient. The cell culture of A. dahurica was established, based on the interst to study the secondary metabolites. The biomass from the cell cultures of A. dahurica was examined for its constituents and the result suggested the differences in the biosynthetic products between cell cultures and the whole plant. In the present study, light-yellowish soft callus of A. dahurica was successfully induced on the solid medium containing MS basal salts supplemented with 1 ppm 2,4-D, 0.5 ppm kinetin and 3 % sucrose. The rapidly growing and finely dispersed cell suspension culture was successively obtained by transferring the callus to the liquid medium containing MS basal salts, 1 ppm 2,4-D, 0.5 ppm kinetin and 3 % sucrose. In the analysis of secondary metabolites, fractionation of the ethanolic extract of the cultured cell, further isolation and purification through repeated chromatography, using silica gel, CPC, Sephadex LH-20 and preparative HPLC, resulted in the isolation of 8 compounds. They were identified to be veratraldehyde (6), p-anisaldehyde (7), vanillic acid (8), tryptophan (5), p-hydroxy-benzoic acid 4--D-glucoside (9), -D-glucosylvanillate (10) and stigmasterol (11) and stigmasterol--D-glucoside (12) based upon the NMR spectroscopic analysis. However, coumarins was not detected from the cultured biomass.Part IIIStudy on the Production of Secondary Metabolites by the Cell Suspension Cultures of Lycium barbarum Lycium barbarum L. (Kou-chi), a well known traditional Chinese medicine, has long been employed for the restoration of health in debilities caused by various pathogeneses and aging. The chemical constituents of Kou-chi were reported to contain alkaloids and glycosides. In the pharmacological studies, it possesses potent biological activities such as improving hematopoiesis, decreasing blood lipids, protecting function and immunostimulating effect. In the study of secondary metabolites, fractionation of the ethanolic extract from the cultured cells and further isolation, purification of the chloroform soluble part through repeated chromatography by using Sephadex LH-20 and silica gel, yielded scopoletin (13), ferulic acid (14), -sitosterol (15), -sitosterol-D-glucoside (16), stigmasterol (17) and stigmastanone (18).II. Study on the Chemical Constituents of Suriana maritima Suriana maritima L. (bay-cedar), a poly-branched shrub, grows widely along the coral coasts of the tropical region. Its taxonomic classification is still uncertain, although the recent reports placed it under a monotypic family, Surianaceae, which is allied to Stylobasiae and Simaroubaceae. The native inhabitants of several West Indian islands use the flowers of this plant in the treatment of blood disorders and employ the stems to relieve severe fever. In the course of biological activity screening, the ethanolic extract of the leaves of S. maritima exhibited inhibitory effect against the human immunodeficiency virus reverse transcriptase (HIV-RT) (40 g/ml). Based on the finding, the present work was aimed to study the bioactive ingredients of this plant. The ethanolic extract was fractionated into CHCl3-, n-BuOH- and H2O- soluble parts. Fractionation of the CHCl3-soluble part on Sephadex LH-20 and further separation on silca gel columns yielded 15 flavonoid compounds (19~32, 36), 4 cycloartenol compounds (41~44) and 2 phytosterol compounds (15, 16). Repeated fractionation and separation of the n-BuOH-soluble fraction over Sephadex LH-20 and CPC afforded additional 7 flavonoid compounds (33~35 and 37~40) and 1 phenolic glycoside (45). Their structures were elucidated by elaborated spectral analysis. They belong to 8 categories:1. Flavonols：isorhamnetin (19)、kaempferol 4’-methyl ether (20)2. 3-Methoxyflavones：ermanin (21)、isokaempferide (22)、quercetin 3, 3’-dimethyl ether (23)、flindulatin (24)、7-O-demethyl flindulatin (25)、herbactin 3,4’-dimethyl ether (26)、4’-O-demethyl flindulatin (27)、terantin (28)、isoterantin (29)、5-hydroxy-3,7,8,3’,4’-pentamethoxyflavone (30)、5,7,3’-trihydroxy-3,8,4’-trimethoxyflavone (31)3. Flavanones：5,4’-dihydroxy-7,8-dimethoxyflavone (32)4. Catechins：epiafelechin (33)、epicatechin (34)、epigallocatechin (35)5. Flavonoid glycosides：rutin (36)、rutin 3’-O-methyl ether (37)、8-methoxy-3’-O-methyl rutin (38)、8-methoxykaempferol-3-O-rutinoside (39)6. 3-coumaranone類：3’-dehydroxy-alphitoniin-6-O-β-glucoside (40)7. Triterpenes：cycloeucalenol (41)、cycloeucalenone (42)、4β-nor-cycloartane-3, 24-dione (43)、24-methylenepollinastanol (44)8. Phytosterols：β-sitosterol (15)、β-sitosterol-3-O-β-D-glucoside (16)9. Phenolics：acetophenone glucoside (45) Among the flavonoids obtained from the study, kaempferol 4’-methyl ether (20), 4’-O-demethyl flindulatin (27), isoterantin (29) and 5,7,3’-Trihydroxy-3,8,4’-trimethoxyflavone (31) are the natural products isolated for the first time from the plant. Literature search revealed that the anti-HIV-RT activity could be attributed to epicatechin (34). The detailed bio-assay is still in progress. The chemical investigation of S. maritima chemistry provided information to solve the conflicting taxonomic problem. The classifying of S. maritima under Surinanacea by Mitchell and Geissman (1971) was supported.
| Identifer | oai:union.ndltd.org:TW/087NTU01551021 |
| Date | January 1999 |
| Creators | Wen-Te Chang, 張文德 |
| Contributors | Karin C. S. Liu, Shoei-Sheng Lee, 陳瓊雪, 李水盛 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 265 |
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