Chemical Study On The Torreya Nucifera (L.) Sieb Et Zucc. Barks

Torreya nucifera (L.) Sieb et Zucc., a Taxaceae plant, is an evergreen coniferous tree, which is common in Japan, korea China. The gene Torreya may be divided into six natural species and two artifactic species: T. califonica Torreya, T. taxifolia Arn., T. nucifera (L.) Sieb et Zucc., T. jackii Chun., T. grandis Fort. Ex Lindl., T. fargesii Franch., T. fargesii Franch. Var. yunnanensis (Cheng et L. K. Fu) N. Kang., T. grandis Fort. Ex Lindl. Var. jiulongshanensis Z. Y. Li, Z. C. Tang et N. Kang.The seeds of T. grandis Fort. Ex Lindl. and T. nucifera (L.) Sieb et Zucc. were widely uesd for the treatment of parasites, indigestion, consipation and so on. The seeds of T. nucifera (L.) Sieb et Zucc. have been used for the treatment of tapeworm infestation in Korea and to induce abortion in Japanese. As it is recorded in"the Dictionary of Traditional Chinese Medicine", the flowers of T. nucifera can be used to treatment parasites and tne roots of T. nucifera can be used to eliminate pathogenic wind and dampenss.There are few previous works about the chemical studies on the genes Torreya, mainly subjecting on the seeds and leaves of T. nucifera (L.) Sieb et Zucc., T. jackii Chun. and T. grandis Fort. Ex Lindl.. The seed is rich in fatty oil and sterol. The components in the leaves of the genes Torreya plants are sesquiterpenoids, diterpenoids and flavonoids. Most chemical studies on T. nucifera have been focused on its leaves and wood. A number of sesquiterpenoids, labdane and abietane diterpenoids, liganans and flavonoids were identified as active secondary matabolites. In order to found new compounds and to understand the structure-activity relationship further. we investigated the chemical composition of T. uncifera barks.Object: To develop the natural resources, enrich the diversities of Traditional Chinese Medicine and study the components of the barks of T. nucifera (L.) Sieb et Zucc., we use silica gel column chromatography, polyamide column chromatography, sephadex gel column chromatography, macroporous resin column chromatography, preparative TLC, HPLC and recrystallization to systemically isolated 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 estableish the structures of the pure compounds isolated from T. nucifera (L.) Sieb et Zucc.. To compare the differences in compounds between barks and other different parts of T. nucifera (L.) Sieb et Zucc. and the differences in compounds between the genus Torreya plants and the genus Taxus plants.Methods: The pulverized dried barks of T. nucifera (2.0 kg) were chipped and immersed in methanol for one week at room temperature, and residue was re-extracted for twice times. After filtered, the methanol extract was concentrated in vacuum to yield the total curde extract of 300.0 g. The crude extract was suspended in salt water and re-extracted with petroleum ether, dichloromethane, ethyl acetate and n-butyl alcohol in order. Three fraction were obtained: the petroleum fraction 5.1 g, the dichloromethane fraction 16.3 g, and the ethyl acetate and n-butyl alcohol fraction 39.3 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 several subfraction. Combined subfractions were subjected to silica gel column chromatography, polyamide column chromatography, sephadex gel column chromatography, macroporous resin column chromatography, preparative TLC and reversed phase preparative HPLC for further separation and purification to get pure compounds. The spectroscopic methods including variouse of 1D and 2D NMR methods were used for structural identification.Results: After investigation on extraction of the barks of T. nucifera, 21 compounds were isolated from the barks of T. nucifera. These compounds detected on the basis of spectral analysis and the structures of 9 compounds were established, including 12, 13-E-Methylozate (1),β-Sitosterol (2), 8, 11, 13-abietatriene-3, 12-diol (3), iso-Thujaplication (8), Thujaplication (14), Methyl-β-D-mannopyranoside (15), (-)-4′-demethyltraxillagene (18), (-)-Traxillagene (19), 3-Acetyloleanane (20).Conclusion: The results of our experiment indicated that the solvements and methods of extraction used in this experiment are practicable. Silica gel column chromatography, sephadex gel column chromatography, macroporous resin column chromatography, preparative TLC and reversed phase preparative HPLC were employed to isolate and purify the components of the barks of T. nucifera, and variety of spectroscopic methods were used to establish the structures of the compounds. We obtained 21 compounds in all, and the structures of 9 compounds were identified. In addition, we also studied the general activity screening of nine compounds. Rhaponticum uniflorum (L.) DC, which belongs to the tubular flower subfamily of Compositae, is distributed mainly in the tropical and subtropical regions, especially in Europe, Asia and Africa. It comprises of about 24 species all over the world and there are almost 10 species mainly distributed in Heilongjiang, Jiling, Liaoning, Hebei, Shandong, Shanxi in China. R. uniflorum (L.) DC is a traditional medicine of the Chinese minorities in Menggu, which has the effect of stopping pricking pain, clearing fever, detoxicating, diaphoresis relieving superficies and so on. It can be used for curing bowel twinge, distemper, generate sign, prominentia laryngea, measles, tox-fever, cardiac heat, heat in blood, heat in injury and so on in clinical[3]. Pharmaco-investigation shows that the plants of R. uniflorum (L.) DC has the effect of decreasing blood fat, antioxidation, antiatherosclerosis, for increasing immunity and so on as a traditional Chinese medicine [4]. The components of R. uniflorum (L.) DC include phytoecdysteroids, triterpenes and diterpenes, sesquiterpene lactones, phenolic acids and flavonoids and so on, Phytoecdysteroids are the most important and active components. In addition, studies indicate the plant of R. uniflorum (L.) DC are rich in triterpene. Triterpene has many effections, such as antiinflammatory, anti-tumor, depression blood sugar, immunoregulation, prevention and cure cardiovascular disease and so on[5]. It's very significance to found out more and more triterpenes from R. uniflorum (L.) DC and further study their acticities. We systemically investigated the chemical composition of the roots of R. uniflorum (L.) DC, and established the structures of the isolated pure compounds on the basis of spectroscopic techniques including 1D and 2D NMR methods.Object: To develop the natural resources, enrich the diversities of Traditional Chinese Medicine and study the components of the barks of R. uniflorum (L.) DC, we use silica gel column chromatography, polyamide column chromatography, sephadex gel column chromatography, macroporous resin column chromatography, preparative TLC, HPLC and recrystallization to systemically isolated 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 estableish the structures of the pure compounds isolated from the roots of R. uniflorum (L.) DC. To check up the effect of the pure compounds isolated from the roots of R. uniflorum (L.) DC on human tumor cells.Methods: The pulverized dried roots of R. uniflorum (L.) DC (9.7 kg) were extracted with 95% ethanol for there times. After filtered, the ethanol extract was concentrated in vacuum to yield the total curde extract of 300.0 g. The crude extract was suspended in salt water and re-extracted with petroleum ether, dichloromethane, ethyl acetate and n-butyl alcohol in order. Three fraction were obtained: the petroleum fraction 26.4 g, the dichloromethane fraction 6.8 g, the ethyl acetate and n-butyl alcohol fraction. Because of less time left, only petroleum ether and dichloromethane 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 several subfraction. Combined subfractions were subjected to silica gel column chromatography, polyamide column chromatography, sephadex gel column chromatography, macroporous resin column chromatography, preparative TLC and reversed phase preparative HPLC for further separation and purification to get pure compounds. The spectroscopic methods including variouse of 1D and 2D NMR methods were used for structural identification.Results: Systemical investigation on extraction of the roots of R. uniflorum (L.) DC yielded 10 compounds. These compounds detected on the basis of spectral analysis and the structures of 6 compounds were established, including: Arctinal (1) , Arctinone-b (2), 7-chloroarctinone-b (3), 5-methoxyl-5'-(1-propinyl)- 2,2'-dithiophene (4),β-Sitosterol (6), Stigmasterol (10).Conclusion: The results of our experiment indicated that the solvements and methods of extraction used in this experiment are practicable. Silica gel column chromatography, sephadex gel column chromatography, macroporous resin column chromatography, preparative TLC and reversed phase preparative HPLC were employed to isolate and purify the components of the roots of R. uniflorum (L.) DC, and variety of spectroscopic methods were used to establish the structures of the compounds. We obtained 10 compounds in all, and the structures of 6 compounds were identified.