【作者】 魏太明；

【导师】 阎玉凝； 邹节明；

【作者基本信息】 北京中医药大学 ， 中药， 2003， 博士

【摘要】 1. 目的：垂盆草来源于景天科（Crassulaceae）景天属（Sedum）多年生草本植物Sedum sarmentosum Bunge.新鲜或干燥全草。垂盆草始载于《本草纲目拾遗》，原名狗牙半支。我国分布广泛，性凉味甘淡，新鲜或干燥全草入药。商品药材均为干燥品。垂盆草具有清利湿热、解毒之功，临床用于湿热黄疸、小便不利、痈肿疮疡、急、慢性肝炎的治疗，民间用于治疗各种肝炎。该药是中国药典收载品种，是垂盆草冲剂的主要成分。以前有人用垂盆草苷做指标成分，垂盆草苷遇热、遇碱不稳定，目前国内外没有此对照品，所以不宜用垂盆草苷做对照品。本课题是旨在研究活性成分的基础上，找出能控制质量的指标性成分即对照品，从而为控制质量提供科学依据。2. 方法：全文共分六个部分：一、对垂盆草进行了本草考证和生药学研究。二、化学成分的研究，用95%乙醇提取3次，回收乙醇，加水悬浮后，分别用不同溶剂萃取，得到氯仿、乙酸乙酯、正丁醇部分，分别用硅胶、聚酰胺、Sephadex-LH-20、C18等柱制备色谱技术进行化学成分分离；用UV、IR、1HNMR、13CNMR等鉴定技术进行化合物鉴定。采用活性跟踪的方法，结合活性筛选的结果，对垂盆草进行了较为系统的化学成分研究；三、药理活性的测定研究，通过药材的功效来设计试验，通过动物或相应的体外模型来筛选、研究了化合物对小鼠脾脏T、B淋巴细胞转化增殖的影响、对四氯化碳损伤的原代培养大鼠肝细胞培养液中ALT含量的影响，从来确定有效成分。四、对照品的研究，根据药理活性的筛选结果和化合物的专属性来确定对照品，并对对照品的性质和稳定性进行研究。五、用TLC和HPLC法研究药材指标性成分的定性和定量方法。六、优先选用了HPLC法对垂盆草进行指纹图谱研究。结果：通过以上研究对垂盆草的质量控制方法取得了一定的进展。垂盆草95%乙醇提取物经不同极性的溶剂萃取，从氯仿、醋酸乙酯、正丁醇部分得到三萜类、黄酮类、甾醇类、硫醇类、硫醚类、有机酸类等共计三十一个化合物，经波谱解析、化学分析确定了其中二十个化合物的结构，它们分别为5,4′-二-羟基-3′，5′-二甲氧基黄酮-7-O-β-D-葡萄糖苷(tricin-7-O-β-D-glucoside I)、木犀草素(Luteolin II)、异鼠李素(Isorhamnetin III)、异甘草素(Isoliquiritigenin IV)、δ-香树脂酮（δ<WP=5>-Amyrione V）、3β，6β-豆甾-4，22-二烯-3，6-二醇（3β，6β-Stigmast-4，22-dien-3，6-diol VI）、槲皮素（quercetin VII）、β-谷甾醇（β-Sitosterol VIII）、胡萝卜苷（Daucosterol IX）、十五烷基硫醇（pentadecyl mercaptan X）、丁香酸（4-hydroxy-3,5-dimethoxybenzoic acid XI）、3,4,5-甲氧基苯甲酸（3,4,5-trimethoxybenzoic acid XII）、双十八烷基硫醚(dioctadecyl sulfideXIII)、棕榈酸(palmic acid XIV)、苯甲酸甲酯（benzoic acid methyl ester XV）、3-表-δ-香树脂醇（3-epi-δ-AmyrinXVI）,δ-香树脂醇（δ-AmyrinXVII），β-D-半乳糖-(3-1′)-β-D-葡萄糖-（4′-1〞）-β-D-阿洛糖（beta-D-galactopyranosyl-(3-1′)-beta-D-glucopyranosyl-（4′-1〞）-beta-D-Allose XVIII）和KNO3、NaNO3（化合物 A 、B）。其中化合物X、VI、VII、XII、XIII、XIV、XV、XVIII是首次在该植物中发现。另还有9个化合物尚在鉴定中。体外活性实验表明化合物I能明显抑制小鼠脾脏T、B淋巴细胞转化增殖。化合物V能使四氯化碳损伤的原代培养大鼠肝细胞培养液中ALT含量显著降低；垂盆草乙醇提取液（正丁醇、乙酸乙酯、水萃取部分）也有一定程度的作用。本研究就指标性成分进行了研究，发现tricin-7-O-β-D-glucoside具有很好药理活性（免疫抑制作用）和专属性，故我们认为tricin-7-O-β-D-glucoside可以作为质量控制的一个指标成分。δ-香树脂酮能使四氯化碳损伤的原代培养大鼠肝细胞培养液中ALT含量显著降低，有很好的专属性，也可以作为指标性成分之一。研究并优化了对照品tricin-7-O-β-D-glucoside的制备工艺路线，认为直接用60%乙醇提取，得率比较高，在柱分离过程中直接用洗脱剂洗脱，接近对照品洗脱比例的时候，用对照品TLC跟踪，收集流份，提高了分离速度。研究并确定了对照品tricin-7-O-β-D-glucoside TLC定性鉴别和HPLC含量测定的方法，tricin-7-O-β-D-glucoside含量不得低于0.01%。对照品tricin-7-O-β-D-glucoside进行了初步稳定性试验研究，认为tricin-7-O-β-D-glucoside在室温较稳定，超过90℃开始变化，230℃基本破坏。在酸性条件下较稳定（Ph=2～3）；而在碱性条件下较为敏感，当Ph＞5时明显不稳定。研究并确定了δ-香树脂酮（δ- Amyrin）TLC定性鉴别和HPLC含量测定的方法，加工方法对δ-香树脂酮含量有明显影响，且有一定规律性，即含量由高到低的顺序为新鲜药材＞自然晒干＞60℃烘<WP=6>3. 干＞水烫（95℃）＞微波3分钟。不同采收季节对δ-香树脂酮含量也有影响，一般含量由高到低的顺序是9月＞8月＞7月＞6月。从产地看四川峨眉山、北京香山的含量比较高。以上是考察了产地、加工方法、采收季节对δ-香树脂酮含量含量的影响。认为δ-香树脂酮含量不得低于0.5%，大于1.0%为较好。研究了不同产地、更多还原

【Abstract】 1.Objective: Chuipencao (Herba Sedi) originates from the fresh or dry herb of Sedum sarmentosum Bunge, which exists extensively, being widely used as a folk TCM for kinds of hepatitis and other diseases. Its formal name is Gouyabanzhi, recorded by the book "Compendium of Materia medica". People consider it cool and sweet, then apply it to clearing heat, detoxifying, excreting dampness. In addition, in clinic , it can cure suelling, gynogenic infection, ulceration of skin, jaundice due to damp-heat, difficult dysuria and less urine as well. So, Herba Sedi is the main component of Chuipencao powder preparation, which is to be taken after being infused in boiling water. However, it’s reported that its glycosides is not suitable to be as a contrast because of its instability when meeting hotness, acid and alkaline compounds. On the basis of the research on active constituents, the purpose of our study is to find out a contrast to be as a norm controlling the quality.2. Method: Our studies included six parts as follows: (1) Herbal verification and pharmacognosy study of Herba Sedi. (2) Study on chemical constituents. Extract with 95% ethanol 3 times, recycle ethanol, and after being suspended with water, extract with chloroform, ethyl acetate and n-butanol, isolate chemical constituents from these extractions with silica gel column, polymide column, Sephadex LH-20 column, and C18 column, and lastly identify them with UV, IR, 1HNMR, 13CNMR. Using the method of activity track and the result of activity sifting, we made a system isolation study on the chemical constituents of Herba Sedi. (3) Discussion about pharmacological activity measure. According to the activities of the herb, effects of the compounds on the transformation and proliferation of T、B lymphocytes of mice spleen, and both on the ALT content in the culture media of liver cell injured by CCl4 were studied in vitro and in-vitro. All these work is to define the effective compound. (4) Contrast research. Define the contrast on the basis of the specialization of constituents and the result of sifting. Also, do research on the quality and stability of contrasts. (5) Research on the methods of qualitative analysis and quantitative analysis with TLC and HPLC. (6) Preferably choosing HPLC, make a discussion on the fingerprint graph of Herba Sedi.<WP=8>3. Result: By means of those works, we have made a progress on quality control of Herba Sedi, and 31 compounds were isolated from chloroform extract, ethyl acetate extract and n-butanol extract, 20 of which were defined by spectrum analysis and chemical analysis:tricin-7-O-β-D-glucoside(Ⅰ)、Luteolin(Ⅱ)、Isorhamnetin (Ⅲ)、Isoliquiritigenin (Ⅳ)、δ-Amyrione (Ⅴ)、3β，6β-Stigmast-4，22-dien-3，6-diol（Ⅵ）、quercetin (Ⅶ)、β-Sitosterol (Ⅷ)、Daucosterol (Ⅸ)、pentadecyl mercaptan (Ⅹ)、 4-hydroxy-3,5-dimethoxybenzoic acid (Ⅺ)、3,4,5-trimethoxybenzoic acid (Ⅻ)、dioctadecyl sulfide (ⅩⅢ)、palmic acid (ⅩⅣ)、benzoicac acid methyl ester (ⅩⅤ)、3-epi-Amyrin(ⅩⅥ)、δ-Amyrin(ⅩⅦ)、beta-D-galactopyranosyl-(3-1′)-beta-D-glucopyranosyl-（4′-1）-beta-D-Allose ⅩⅧ 、KNO3 ( compound A ) and NaNO3 ( compound B ). Among them, compound Ⅵ、Ⅶ、Ⅹ、Ⅻ、ⅩⅢ、ⅩⅣ、ⅩⅤandⅩⅧ were discovered in this herb for the first time, and other 9 compounds are still being identified. Furthermore, in-vitro activity experiment showed that compound Ⅰ can obviously restrain the transformation and proliferation of T 、B lymphocytes cell in mice spleen, injured by CCl4. Also, the ethanol extract of Herba Sedi had certain effect, and so does ethyl acetate extract, n-butanol extract, and water extract. Depending on the experiment of index constituents, tricin-7-O-β-D-glucoside possesses its specialization and superior immunity-controlled effect, so that it could be an index. Besides, δ-Amyrin has the same effect as tricin-7-O-β-D-glucoside,so it may become another index. Study and optimize the making process of tricin-7-O-β-D-glucoside, and believe that its productivity would be higher when extracted by 60% ethanol, direc更多还原