蔓荊子抗PMS物質基礎、種內變異和品質評價研究
 

作者:第二軍醫大學, 辛海量

關鍵字:蔓荊;蔓荊子;牡荊屬;PMS;植物化學;種內變異;品質評價;含量測定

學位:碩士

專業:生藥學

摘要:

經前期綜合征(Premenstrual Syndrome,PMS)是成年女性的常見病和多發病,本病發病機理復雜,目前尚缺乏有效的防治手段和措施。目前,國外已將馬鞭草科植物穗花牡荊 (Vitex agnus-castus L.)用於防治PMS,效果顯著。蔓荊子(Fructus Viticis)為馬鞭草科植物單葉蔓荊 Vitex trifolia L.var.simplicifolia,Cham.或三葉蔓荊 V.trifolia L.的干燥成熟果實,具有疏風散熱、清利頭目的功效,現代研究表明其具有解熱、鎮痛、抗菌、抗炎、降壓、抗氧化等作用。由於穗花牡荊我國不產,而單葉蔓荊主產我國,資源豐富,與穗花牡荊有一定化學相似性,紫花牡荊素等有效成分含量高於穗花牡荊,品質較穗花牡荊更優。因此研究蔓荊子抗PMS的物質基礎,以其為原料開發抗PMS新藥,具有較高的可行性。本課題主要涵蓋以下三個方面內容: 一、抗PMS物質基礎研究 1 採用系統溶劑法和色譜方法,從江西新建產單葉蔓荊子中共分得18個單體成分,鑒定了其中的13個,分別為蔓荊呋喃(rotundifuran)、紫花牡荊素(casticin)、蒿亭(artemitin)、β-谷甾醇(β-sitosterol)、豆甾醇(stigmasterol)、豆甾-3,6-二酮(5α-stigmanstan-3,6-dione)、硬脂酸(stearic acid)、對羥基苯甲酸(p-hydroxybenzcic acid)、香草酸(vanillic acid)、木犀草素(luteolin)、3,4-二羥基苯甲酸(3,4-dihydroxybenzoic acid)、穗花牡荊苷(agnuside)、5,7,2′,5′-四羥基黃酮(5,7,2′,5′-tetrahydroxyflavone)。其中, 豆甾-3,6-二酮和5,7,2′,5′-四羥基黃酮系首次從蔓荊子中分得,紫花牡荊素、蔓荊呋喃及穗花牡荊苷具有較強的藥理活性; 2 以小鼠扭體實驗和高泌乳素動物模型進行紫花牡荊素抗PMS藥理研究,顯示其具有較強的鎮痛作用(10mg/kg,ig)和降低高泌乳素動物血清泌乳素水平的作用(10mg/kg,ig),可以認為其是蔓荊子抗PMS作用的活性成分之一; 3 採用溶劑提取,大孔樹脂、聚酰胺工藝分離純化,小鼠扭體實驗和高泌乳素動物模型篩選出蔓荊子抗PMS的有效部位。並以總黃酮(UV法)和紫花牡荊素 (HPLC法)為指標,對蔓荊子抗PMS有效部位制備工藝中的提取、大孔樹脂步驟的工藝條件進行了優化,初步形成兩條較為簡便、經濟的有效部位制備工藝。第一條為95%乙醇熱回流提取,以AB-8大孔樹脂吸附,G%乙醇洗脫,洗脫液濃縮,以聚酰胺吸附,E%乙醇洗脫即得;第二條工藝將第一條工藝路線聚酰胺步驟改為以溶劑R等體積萃取3

Abstract:

Premenstrual Syndrome (PMS) is a disorder characterized by a set of hormonal changes that trigger disruptive symptoms in a significant number of women for up to two weeks prior to menstruation. Characteristically symptoms may be both physical and emotional. Over 150 symptoms have been attributed to PMS. They may include physical symptoms as headache, migraine, fluid retention, fatigue, constipation, painful joints, backache, abdominal cramping, heart palpitations and weight gain. Emotional and behavioral changes may include anxiety, depression, irritability, panic attacks, tension, lack of coordination, decreased work or social performance and altered libido. Often symptoms tend to taper off with menstruation and women remain symptom-free until the two weeks or so prior to the next menstrual period. Nowadays, treatment of PMS focuses on relieving symptoms and involves exercise, dietary changes, and medication. Different treatments aim at different causes of PMS, and different approaches may relieve some symptoms but not others. Medication mainly includes anti-inflammatory drugs, diuretics, anti-anxiety drugs and antidepressants drugs, and all these medications only take a small part in treating PMS because of their specificity and severe side effects. Therefore, it is very necessary to investigate much more effective and safe medications. Chastetree and its extract have long history in treating PMS and PMS related complaints, and its newly preparation named Ze440 is getting more and more popular in Europe and America. But the R&D of chastetree is somewhat unpractical in China because of the scarcity of its resource. Fructus Viticis as a classic species is recorded in the pharmacopeia of the PRC and commoly used in clinical practice. It is classified in the same genus, its chemical constituents are similar with those of Chastetree and total flavonoid and casticin contents are even higher. So the R&D of anti-PMS drug from Fructus Viticis is very practical and meaningful. The studies on Fructus Viticis were mainly as following three aspects: 1. Active chemical components of anti-PMS effect1.1 18 single chemical constituents were isolated from Fructus Vitcis with systemic solvent and various chromatography methods, and 13 structures of them were elucidated on the basis of spectroscopic analysis as following: rotundifuran, casticin, artemitin, (3-sitosterol,stigmasterol, 5α-stigmanstan-3 , 6-dione,stearic acid, p-hydroxybenzcic acid,vanillic acid, luteolin, 3,4-dihydroxybenzoic acid,agnuside and 5 , 7 , 2 \' 5 \' -tetrahydroxyflavone. Especially, 5a-stigmanstan-3 , 6-dione and 5 , 7 , 2 \' 5 \' -tetrahydroxyflavone were firstly isolated from Fructus Vitcis. These three components, rotundifuran, casticin, and agnuside have a significantly effect related to the PMS;1.2 The anti-PMS activity of Fructus Vitcis was screened with acetic acid-induced writhing animal model and HPRL animal model, which was prepared with macroporous resin and polyamide methods;1.3 Casticin was also tested as one of important active chemical constituents with these same animal models (lOmg/kg, ig);1.4 Some key conditions in the preparation process of the active anti-PMS part of Fructus vitcis were optimized carefully with the content of toal fiavonoid and casticin, and two convenient and economical processes came into being.2. Intraspecific variation2.1 The originl plant of Fructus Viticis is identified as Vitex trifolia h.ax.simplicifolia Cham but not Vitex trifolia L. through herbalogy study. The Fructus Viticis simplicifoliae is somewhat different from Fructus Viticis trifoliae in such aspects as chemical constituents, pharmocology, herbalogy and quality. Therefore, it is somewhat questionable that the fruits of them are recorded as Fructus Viticis without any differences in the pharmacopeia of the PRC (from 1963 edition to 2005 edition) ;2.2 The geographical distribution of Vitex trifolia L.var. simplicifolia Cham., V. trifolia L., V. negundo var. cannabifolia (Sieb.et Zucc.) Hand.-Mazz. and V. negundo L. was clarified in general, which is in some degree different from that recorded in some florae;2.3 The intraspecific variation of Vitex trifolia L.var. simplicifolia Cham, of different populations was investigated with numerical taxonomy method, NIRDS and RAPD analysis. Vitex trifolia L. var. simplicifolia Cham, distributed in various populations was not only influenced by latitude related conditions but also many other factors. All populations of Vitex trifolia L. var. simplicifolia Cham, could be classified 3 types with numerical taxonomy method, type -1 distributed in low latitude regeons, complexing type-1 , complexing type- II. And 3 types were also classified with NIRDS method, distributed in low latitude regeons, type - I distributed in mainly producing area, complexing type-1 . Vitex trifolia L.var. simplicifolia Cham., V. trifolia L., V. negundo var. cannabifolia (Sieb.et Zucc.) Hand.-Mazz. and V. negundo L. could be specifically identified with RAPD

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