Studies And Development Of TCM Antian Capsule As Well As Separation And Studies Of The Potential Components From Wild Plants

Headache, including migraine and tension-type headache, is one of the most common medical disorders and constitutes a major public health problem because of its deterioration of the quality of life. Migraine is a complex pathophysiology in which both central and peripheral components of the trigeminal pain pathway probably play a significant role, both in the symptoms and signs of the attack and in the mechanisms of action of antimigraine compounds. Despite the high frequency of migraine in the general population, the pathogenesis of this disorder is still unclear. Unfortunately, at present there is no complete, miracle cure for migraine, but migraine patients can control their condition by minimizing symptoms, reducing disability and improving quality of life with pharmaceuticals. There are several types of acute medications, and by far the most widely used are still the analgesics or pain-killing drugs, ergotamine, and serotonin blockers. Chinese traditional herb has been used in medical treatment for several thousand years. The Antian capsule is composed of Xuanfuhua and Fructus arctii from Taipingshenghui prescription which has been applied in the treatment of migraine for more than 100 years in China. In the study, the extractant of the TCM shows obvious analgesic activity by the abdominal constriction (writhing) test. On the basis of the studies and manufacture on TCM in China, the study focused on the studies and development of the potential components in TCM, established and optimize with modern advanced techniques.Inula, from Compositae, in traditional Chinese medicine, both I. britannica and I. japonica are called"Xuanfuhua". The flowers from these plants several steroids, sesquiterpenes, kaurane glycosides, and some flavonoids were isolated. The studies on the bioactivities of these components show that the TCM can be used for the treatment of digestive disorders, bronchitis, hepatitis, inflammation, antitumor activities and so on. Among them, various sesquiterpene lactones are reported to have anti-inflammatory, antitumor activities. However, there are no any researches working on the analgesic activity of I. britannica for treatment of migraine. Inula britannica is a wild plant found in China, including the northeast of China, Inner Mongolia, and Xinjiang province. The herb as TCM in traditional prescription has not found. The previous research has indicated that the total extractant of the plant by alcohol had potential dose-dependent analgesic activity in some pharmacological tests, such as the abdominal constriction. For the purpose of development of the wild plant, using phenol-sulfuric acid method, GC-MS analysis, and silica gel column chromatography, the studies worked on the preparation, determination and separation of polysaccharides, essential oil, and the potential fraction as well as components with analgesic activity. This is the first time to find the potential analgesic activity from this kind of herb. The results will be benefit for the further studies and development of the plant as new analgesic agents.One of the main strategies for the treatmnt of cancer is chemotherapy. About sixty drugs (mostly chemical synthetic products) are available today to fight against various types of cancer. Some of them, however, are natural products or semi-synthetic analogs of natural products. The vast diversity of chemical structures and the promising biological activity of many natural products have captured the attention of the chemistry and drug discovery communities for decades. Among the natural anticancer products, Paclitaxel (taxol) is one of the best anticancer agents in clinical use today for treatment of ovarian cancer. It has been approved by the FDA for the treatment of advanced ovarian cancer since 1992. Now it is extensively used against skin, lung, brain and neck carcinomas. There are some major problems associated with taxol. One of them is only very small amounts of taxol present in the plants (the initial yield of taxol from T. brevifolia bark was only about 0.007%), although this has now been doubled by a combination of improved isolation methods and improved bark-collection method. Because of taxol's limited availability from natural sources, many strategies have been investigated in order to produce large enough quantities of taxol for chemotherapy treatment. A major advantage of the semi-synthesis approach, compared to direct isolation, is that it permits the creation of taxol analogs as well as taxol. This has made the genus of Taxus one of the most intensely investigated of all the plant genera. Chemical studies on the constituents of the Japanese yew began in the 1960s. Approximately above 200 taxanes, including taxol and 10-DAB, have been isolated from the Japanese yew. In 1992 the major taxane, 9-dihydro-13-acetylbaccatin III, was isolated from the needles of Taxus canadensis (a ramping evergreen bush, commonly found in shady woods in Quebec). The structural similarity of 9-dihydro-13-acetylbaccatin III to the core structure of taxol and related taxanes makes it of considerable importance as a new starting material for the semi-syntheses of new taxanes In Canada, Quebec, there is also growing Japanese yew. To verify the yew tree as a new natural source, the study focuses on comparing taxanes of the needle of the T. cuspidata Sieb. et Zucc. in Canada to in Japan, and described the isolation and elucidation of taxanes form the needles of T. cuspidata in Quebec.PART 1 Study on Extraction Methodology for TCM Antian Capsule Objective: To establish the extraction methodology for Antian Capsule based on the tests of pharmacology, so as to get over the drawbacks in decoction.Methods: (1) Inula britannica L. and Fructus arctii were extracted by alcohol and water respectively, after precipitating in the mixture of alcohol and water at different concentrations, the filtrates were combined and evaporated to get dry powder. (2) Extraction methodology was determined by testing of extract yield, contents of potential components and the rate of the abdominal constriction. Orthogonal test was adopted to optimize the extraction methodology of extract solvents, distilling time and so on. (3) To test the stability and practicability of the extraction methodology a magnified manufacture had been done with quality control.Results: Inula britannica L.: refluxing with 10-fold 95% alcohol for 3 times and each for 1 h, the condensed extractant was mixed with water to precipitate the impurity at a concentration of 50% alcohol; Fructus arctii: refluxing with 10-fold water for 3 times and each for 1 h, the condensed extractant was mixed with alcohol to precipitate the impurity at a concentration of 95% alcohol.Conclusion: To find a technological process of the capsules preparation, a reasonable experiment design was used in the study. Using potential components and pharmacological test as control factors, the optimum procedures were determined and verified by the dimensional monitoring. The new technological process involved in a new methodology, the different TCM with different procedures, and realized the standardization of TCM preparation. The process which was undergone is economical, reasonable, and suitable for industrial manufacture. The quality of TCM can be controlled and determined by modern techniques under the process.PART 2 Studies on the Polysaccharides, Essential Oil, and Potential Component in Inula Britannica L.Objective: By preparation of polysaccharides, the essential oil, the potential fraction and component from Inula britannica L. which collected from Yuncheng area of Shanxi province, the determination of the polysaccharides, the chemical components of the essential oil analyzed by GC-MS, and the chemical structure of the potential component were undergone. The data will be a necessary supplement for the pharmaceutical studies of the herb and could be used in the further studies on the species.Methods: (1) After degreasing using 95% alcohol, the air-dried materials was extracted by water twice; the filtrate was concentrated and precipitated by adding ethanol. Then the polysaccharides were filtered, washed and dried. Finally, the content was determined by phenol-sulfuric acid method. (2) The essential oil was extracted from Inula britannica L. by steam distillation, the components were separated with the capillary chromatographic columns, and the amount of the components from the essential oil was determined by normalization method. The components separated were identified by data searches system. The chromatographic conditions were as follows: DB-5 (30m×0.25mm,0.25μm)capillary column; High purity helium was used as carrier gas, and the flow rate was 1.0ml/min; Column temperature: 70℃keeping 2min, from 70℃to 230℃at rising rate 10℃/min and keep 10min; Split ratio 15:1; Injector temperature 250℃. (3) Air-dried materials were smashed and immersed in 95% alcohol for two days at room temperature, the residue was re-extracted for three times. The combined filtrates were evaporated at reduced pressure. The dry residue was suspended in water and subsequently partitioned with petroleum, chloroform (CHCl3), ethyl acetate, and n-butanol, respectively. The potential fraction was determined by the abdominal constriction test. (4) The CHCl3 fraction was separated by flash silica gel column chromatography was used to preliminary fractionation; each fraction was monitored with TLC. Similar fractions were pooled and subjected to silica gel column chromatography with lower air pressure. Each subfraction was monitored with analytic HPLC. The products crystallized from the concentrated solvent. The structural identification was carried out by NMR.Results: The extract yield of polysaccharides with higher purity was 2.7%. The data showed that this species had a higher content of polysaccharides (20%). In the analysis of the essential oil, 63 peaks were identified representing 70.8% of the total contents. The main component was 2,3,4,5-tetrahydro-1-Benzoxepin-3-ol (4.276% ). By pharmacological test, the CHCl3 fraction (P<0.01) was the potential fraction and showed obvious analgesic activity comparing to aspirin (P<0.05). The subsequent separation indicated that 1-O-acetylbritannilatone was the potential component exhibiting an analgesic activity. Its chemical structure was identified by NMR, and the yield was 0.075% from Yuncheng area of Shanxi province.Conclusion: It is the first time to study on the preparation and analysis of the polysaccharides, essential oil and the potential component from Inula britannica L. The methodologies are reliable, stable, and good reproducible and the products of the polysaccharides and essential oil could be used in the further bioactivities study. As the first time to report the analgesic activity of Inula britannica L. and determine the potential fraction and component, the products of 1-O-acetylbritannilatone can serve as the index constituents in the quality control of the species. As the potential component, it has been used in the molecular biological studies on the anti-inflammation and applied for the national patent of China (No. ZL02117985.9).PART 3 Chemical Studies on the Taxane Components of the Taxus cuspidata Sieb. et Zucc. Needles from QuebecObjective: To study the components in the needles of Taxus cuspidata and search for new bioactive taxoids, strategies and modern techniques were used, including Silica Gel column chromatography to fractionate the crude extracts, preparative HPLC to separate and purify integrates, TLC and analytic HPLC to monitor the composition of fractions, as well as spectroscopic methods to determine the structure of isolated pure compounds. By comparing the differences in components between Taxus cuspidata in Quebec and in Japan, to find the feasibility about Japanese yew in Quebec could be a new natural source of taxol.Methods: Air-dried needles of Taxus cuspidata in Quebec were smashed and immersed in methanol for three times, each time 24h. The filtrates were combined and evaporated at reduced pressure. The residue was suspended in brine and subsequently defatted with n-hexane, then partitioned with dichloromethane (CH2Cl2) successively. The CH2Cl2 fraction was fractionated by silica gel column chromatography, each fraction was monitored with TLC. Similar fractions were pooled and checked by analytic HPLC, then subjected to preparative HPLC for further purification. The structural identification was carried out by NMR, and other spectroscopic methods. For the known compounds, the structural determination is greatly simplified by direct comparison of NMR with relevant publications.Results: After investigation on extraction of the needles of Taxus cuspidata in Quebec, 73 compounds were isolated. In these compounds 59 compounds were taxoids, among these taxoids, the structures of 31 compounds were established, including taxinine (1), 9-deacetyl taxinine (taxinine NN-3,2), taxinine B (3), 2-deacetoxy-taxinine J (4), O-cinnamoyltaxacin I triacetate (5), taxinine A (6), putative contracted taxinine A (7), 9,10-deacetyltaxinine (8), 5-cinnamoyl-9,10-diacetyltaxicin I (2-deacetyltaxinine) (9), 10-diacetyl taxinine (10), taxuspinanes H (11), yunnanxane (12), 5α-cinnamoyloxy-9α,10β,13α-triacetoxytaxa-4(20),11-diene (13), 2,10-diacetyl-5-cinnamoyl-phototaxicin II (14), taxuspines W (15), Taxine B (16), 2α,9α,10β,13α-tetraacetoxy-taxa-4(20),11-dien-5α-ol (17), 1-hydroxytaxuspine C (3,11-cyclotaxinine NN-2, 18), 10-deacetyl taxinine B (19), taxagifine (20), taxacine (21), 20-cinnamoyloxy-5-epi-canadensene (22), taxuspines J (23), Baccatin IV (24), 2α,10β-diacetoxy-1β,9α-dihydroxy- 5α-cinnamoyoxy-3,11-cyclotaxa- 4(20)-dien-13-one (25), taxuspines F (26), putative contracted tracetyl-5- decinnamoyltaxicin I (27), 1β,2α,9α,10β- tetrahydroxy-5α-cinnamoyoxytaxa- 4(20),11-dien-13-one (28), 7-epi-10- deacetyltaxol (29), Taxuspines (30), Taxezopidine G (31).Conclusion: There are 12 compounds were reported for the first time from Taxus cuspidata, including 2-deacetoxy-taxinine J (4), 9, 10-deacetyltaxinine (8), 5-cinnamoyl-9,10-diacetyltaxicin I (2-deacetyltaxinine, 9), yunnanxane (12), 5α-cinnamoyloxy-9α,10β,13α-triacetoxytaxa-4(20),11-diene (13), 2,10- diacetyl-5-cinnamoyl-phototaxicin II (14), Taxine B (16), 10-deacetyl taxinine B (19), 20-cinnamoyloxy-5-epi-canadensene (22), 2α,10β-diacetoxy- 1β,9α- dihydroxy-5α-cinnamoyoxy-3,11-cyclotaxa-4(20)-dien-13-one (25), 1β,2α, 9α,10β-tetrahydroxy-5α-cinnamoyoxytaxa-4(20),11-dien-13-one (28), 7-epi- 10-deacetyltaxol (29). The putative contracted taxinine A (7) was a new product from natural plants; The putative contracted tracetyl-5- decinnamoyltaxicin I (27) was a new taxoid. The further studies about the instability of both of them will need in the experiments follow-up. Therefore, the region and climate could be a factor in the chemical composition of wild plants. Since there were no taxol and its semisynthetic materials, 10-DAB and 9-dihydro-13-acetylbaccatin III found in Taxus cuspidata of Quebec, the needles of the yew tree cannot be a natural source of taxol.