Dissertation
Dissertation > Medicine, health > Chinese Medicine > Of Pharmacy > Traditional Chinese medicine chemical

Continued with the sub - seed and chemical composition of Inula

Author SuXiaoHui
Tutor ShiQingWen
School Hebei Medical University
Course Medicinal Chemistry
Keywords Euphorbia Euphorbia lathyris seed chemical constituents structure identification diterpenes coumarin steroidal glycoside Inula Inula japonica flowers structure identification sesquiterpene lactones triterpene steroid
CLC R284
Type Master's thesis
Year 2008
Downloads 355
Quotes 2
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Euphorbia lathyris, one of the plants of genus Euphorbium, is widespread in Europe, North Africa, Central and East Asia, and America. Cultivation of E. lathyris had a long history in China since it was introduced to China. E. lathyris grows on the sunny side of mountains. The seeds are oval-shap and about 5 mm long, which have the black and brown stripes on the surface. The oil of seeds usually is up to 50% of the dry weight of the seed[1]. The seeds of E. lathyris have been used to remedy hydropsy, ascites, coprostasis, amenorrhea, venous stasis, terminal schistosomiasis, and scabies[2]. The components of E. lathyris include diterpenes, triterpenes, coumarins and volatile oil. Diterpenes are most important and active components. E. lathyris is well known to possess the unique constituents of lathyrane diterpenes-L-factors. Besides many pharmacological activities, the structures of L-factors are endowed with a remarkable potential to generate complex polycyclic structures by transannular cyclization, it was considered as an important precurce for the biosynthesis of many Euphorbia diterpenoids[3]. As part of an investigation on the construction of libraries of natural products-like compounds and further screenning them against useful biological targets, large amounts of the macrocyclic L-factors were needed[3]. So it is of great significance to find out new compounds in E. lathyris and further study the structure-activity relationships on this basis. We systemically investigated the chemical composition of the seeds of E. lathyris, and established the structures of the isolated pure compounds on the basis of spectroscopic techniques including 1D and 2D NMR methods.Objective: In order to develop the natural resources, enrich the diversities of Traditional Chinese Medicine and find out new compounds from the seeds of Euphorbia lathyris, we use the technologies of Silica gel column chromatography, polyamide column chromatography, sephadex gel column chromatography, preparative TLC, HPLC and recrystallization to systemically isolate and purify the compounds of this plant, and use the spectroscopic methods including UV, IR, MS, 1H-NMR, 13C-NMR, 1H-1H-COSY, HMQC, HMBC and NOESY to characterize the structures of the isolated pure compounds. To check up the effect of the pure compounds isolated from I. japonica on human tumor cells.Methods: The pulverized dried seeds of Euphorbia lathyris (4 Kg) were extracted with 95% alcohol for three times. After filtered, the alcohol extract was concentrated in vacuum to yield the total crud extract of 986 g. The crud extract was suspended in water and re-extracted with petroleum ether, dichloromethane and ethyl acetate in order. Three fractions were obtained: the petroleum ether fraction 760 g, the dichloromethane fraction 2.5 g, and the ethyl acetate fraction 2.7 g. The three fractions were applied to silica gel column chromatography for preliminary fractionation in turn, each fraction was monitored with TLC and combined the similar fractions to give seven subfractions. Combined subfractions were subjected to Silica gel column chromatography, polyamide column chromatography, sephadex gel column chromatography, preparative TLC and/or reversed phase preparative HPLC for further separation and purification to get pure compounds. The spectroscopic methods including various of 1D and 2D NMR methods were used for the structural identification of these compounds.Results: Systemical separation of the seeds of E. lathyris yielded 8 compounds. Of them, the structures of 5 compounds were identified on the basis of chemical and spectral analysis, including 3 diterpenes, a coumarin, and a steroidal glycoside' dissertation">steroidal glycoside. They are Euphorbia Factor L1:(2S*, 3S*, 4R*, 5R*, 6R*, 11S*, 15R*)-5, 15-Diacetoxy-3-benzoyloxy-14-oxolathyra-6(17), (12 E)-diene-6(17)-epoxide (1), Euphorbia Factor L2:(2S*, 3S*, 4R*, 5R*, 7R*, 9S*, 11S*, 15R*)-5, 15-Diacetoxy-3, 7- dibenzoyloxy-14-oxolathyra-6(17), (12 E)-diene (2), Euphorbia Factor L3:(2 S*, 3 S*, 4R*, 5R*, 9S*, 11S*, 15R*)-5, 15- Diacetoxy-3-benzoyloxy-14-oxolathyra-6(17), (12 E)-diene (3), 6, 7-dihydroxy-coumarin (5), and (3β)-stigma-5-en-3-O-β- glucoside (6). Compound 6 was isolated from the plants of Euphorbium for the first time.Conclusion: The results of our experiment indicated that the solvents and methods of extraction used in this experiment are practicable. Silica gel column chromatography, sephadex gel column chromatography, preparative TLC and preparative HPLC were employed to isolate and purify the components of the seeds of E. lathyris, and variety of spectroscopic methods were used to establish the structures of the compounds. We obtained 8 compounds in all, and the structures of 5 compounds were identified. Compound 3 showed strong activity in the test of primary bioactive screening on HEC-1, T-98 and tumor cells. Compound 5 showed strong active on KT tumor cells. Inula japonica is one of the plants of genus Inula. There exist about 100 species of genus Inula, which widely distributed in Europe, Asia and Africa, predominantly in Mediterraean, with more than 20 species being distributed in China. Many of these plants have long been used in Chinese folk medicine. Chemical investigation resulted in the isolation of many components from the plants of genus Inula. I. japonica is a perennial herbaceous plant which grows on hillsides and roadside wet land. The plant mainly distributed in the areas in Northeast, Northern, and Northwest of China, especially in Zhijing, Jiangsu, Sichuan, Guangdong, and so on. The first record of I. japonica is in‘Shennong’s Herbal’, and later developed in‘YaoXingLun’,‘BenCao TuJing’,‘Compendium of Materia Medica’, and‘The Dictionary of Traditional Chinese Medicine’. Since 1985,‘Pharmacopoeia of the People’s Republic of China’has included the dried flowers of I. japonica together with I. britannica as quality certified medicinal herbs resource. The flowers of I. japonica are used in Traditional Chinese Medicine for the treatment of diverse diseases such as cough due to wind-cold evil, vomiting and ructation [1]. The main components of I. japonica are sesquiterpene lactones. In 2003, Yang C et al. reported the cytotoxic activity of several sesquiterpene lactones against SMMC-7721 (human hepatoma cell) and HO-8910 (human ovarian carcinoma cell) [2]. The root and aerial parts of I. japonica also have shown antifungal and antibacterial activity [3]. In order to make a better use of the pharmaseutical value of genus Inula and provide information for exact pharmacological activity study, we systemically investigated the chemical composition of the dried flowers of I. japonica, and established the structures of the isolated pure compounds.Objective: In order to develop the natural resources, enrich the diversities of Traditional Chinese Medicine and find out new compounds from the flowers of I. japonica, we use the technology of Silica gel column chromatography, polyamide column chromatography, Sephadex LH-20 column chromatography, preparative TLC, HPLC and recrystallization to systemically investigate this plant, and use the spectroscopic methods including UV, IR, MS, 1H-NMR, 13C-NMR, 1H-1H-COSY, HMQC, HMBC and NOESY to establish the structures of the isolated pure compounds. To check up the effect of the pure compounds isolated from I. japonica on tumor cells.Methods: The dried flowers of I. japonica (10 Kg) were extracted with 95% alcohol for three times. After filtered, the alcohol extract was concentrated in vacuo to yield the crud extract total 1458 g. The crud extract was suspended in water and re-extracted with petroleum ether, dichloromethane and ethyl acetate in order. Three sections were obtained: the petroleum ether section 298 g, the dichloromethane section 137 g, and the ethyl acetate section 134 g. The dichloromethane section was applied to silica gel column chromatography successively for preliminary fractionation and elution was monitored with TLC. Similar fractions was combined into several subfractions, the subftactions were subjected to silica gel column chromatography, polyamide column chromatography, Sephadex LH-20 column chromatography, preparative TLC and/or reversed phase preparative HPLC for further separation and purification to get pure compounds finally. The spectroscopic methods including various of 1D and 2D NMR methods were used for the structural identification of these compounds.Results: Systemical separation of the dichloromethane section of dried flowers of I. japonica yielded 10 compounds. Of them, the structures of 4 compounds were identified on the basis of chemical and spectral analysis, including two sesquiterpene lactones, a triterpene, and a steroid. They are taraxasteryl acetate (2), eupatolide (3), 1-O-acetylbritannilactone (4),β-sitosterol (5), britannilactone (6) and ent-16β,17-dihydroxykauran-19-oic acid) (10). Compound 10 was isolated from the plants of genus Inula for the first time.Conclusion: The results of our experiment indicated that the solvents and methods of extraction used in this experiment are practicable. Silica gel column chromatography, Sephadex LH-20 gel column chromatography, preparative TLC and preparative HPLC were employed to isolate and purify the components in the flowers of I. japonica, and variety of spectroscopic methods were used to elucidate the structures of the compounds. The structures of 6 pure compounds were established. Compound 4 showed strong activity in the test of primary bioactive screening on Hela, HLE and HMV-1 tumor cells.

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