【作者】 张筱；

【导师】 欧阳平凯； 卢定强；

【作者基本信息】 南京工业大学 ， 生物工程， 2004， 硕士

【摘要】 贯叶连翘 Hypericum perforatum Linn 为藤黄科 Garcinia 金丝桃属Hypericaceae 植物,是我国传统的中药材,在欧洲及美国被称为圣约翰草St.John’wort,用于治疗抑郁症有几百年的历史。近几年研究发现,贯叶金丝桃素是贯叶连翘中抗抑郁的主要活性成分。本文对贯叶金丝桃素的定性定量分析方法和提取纯化方法进行了研究。本文主要研究内容如下: 1)建立了一个新的贯叶金丝桃素的高效薄层色谱和薄层色谱定性分析方法,为国内首次报道。薄层色谱的最佳条件为:以薄层层析硅胶 GF254 为固定相,以正己烷-乙酸乙酯 (5:1,v/v)为展开剂,贯叶金丝桃素的 Rf值为 0.41。 2)建立了一个新的贯叶金丝桃素的反相高效液相色谱定量分析方法。高效液相色谱的分析条件为:固定相为 Alltima C18柱;A 溶液:80%甲醇+1%乙酸,B 溶液:甲醇,采用梯度洗脱:0min～10min ,0%B～100%B;10min～40min,100%B;流速为 1mL/min;柱温为 30℃;检测器为 UV 检测器(270nm)。贯叶金丝桃素可达到基线分离的要求,它的保留时间为14.0min。线性方程为:M=10.3238 A-0.01332,相关系数 r=0.9993,线性范围 0.5-10μg,精密度 RSD=1.14%,平均回收率为 98.34%,当信噪比为 3:1 时,最小检测限为 7.34×10-7μg。 3)进行了贯叶金丝桃素的提取和粗分离方法的研究。采用超声波强化提取的方法。采用薄层制备色谱对贯叶连翘粗提物中的贯叶金丝桃素进行纯化。最佳制备薄层色谱条件为:固定相为薄层层析硅胶 GF254,展开剂为正己烷-乙酸乙酯(4.5:1,v/v),纯化后贯叶金丝桃素样品的纯度为 76.64%。进行了用常压硅胶柱层析技术分离制备贯叶金丝桃素的研究,优化了分离条件,以柱层析硅胶为固定相,以正己烷-乙酸乙酯(6:1,v/v)为流动相进行洗脱,分步收集,合并相同组分。得到的贯叶金丝桃素样品的纯度为 61.14%。 4)首次采用干柱层析对贯叶连翘粗提物中的贯叶金丝桃素进行纯化,该方法国内外尚未见报道。固定相为薄层层析硅胶 GF254,流动相为正己烷 I<WP=7>-乙酸乙酯(4.5:1,v/v)。纯化后的贯叶金丝桃素样品的纯度为 72.13%。5)使用制备薄层色谱法和加压型制备液相色谱联用的方法制备高纯度贯叶金丝桃素样品。其纯度大于 98%。6)采用 ESI-MS 对样品进行分析鉴定。样品的 MS 谱与文献中贯叶金丝桃素 MS 谱一致,可确定制备的样品为贯叶金丝桃素,并且提出了贯叶金丝桃素分子的可能裂解过程。更多还原

【Abstract】 Hypericum perforatum Linn,a plant of Garcinia family, is one of Chinesetraditional medicines. It is also named St John’s wort in Europe and America. Ithas been used to treat depression for hundred years. Recently pharmacologicaland clinical studies in St.John’s wort indicated hyperforin is the ‘true’activecomponent with the antidepression properties. In this paper, the analyticalmethods and preparation of hyperforin were studied. The main contents of thispaper were as follows: 1) A novel high performance thin layer chromatographic (HPTLC) and thinlayer chromatographic (TLC) methods to qualitative analyse hyperforin weredeveloped novelly in China. The optimal conditions of TLC were as follows:silica gel GF254 as stationary phase, n-hexane-ethyl acetate (5:1, v/v) as mobilephase. The Rf value of hyperforin was 0.41. 2) A new method of the reverse phase high performance liquidchromatography (HPLC) determination of hyperforin was developed. Theoptimal HPLC conditions were as follows: Alltima C18 column as the stationaryphase, gradient elution, A solvent: 80%methanol+1%acetic acid,B solvent:methanol, 0min～10min,0%B～100%B, 10min～40min,100%B, 1mL/min offlow rate, 30℃ of column temperature, UV detector (270nm). Under theseconditions, a baseline separation of hyperforin was obtained and the retentiontime of hyperforin is 14.0 minutes. The calibration equation is M=1.3238A-0.013921. The related coefficient r is 0.9993, the linear range is 0.5μg to 10μg, when the signal-to-noise ratio was 3:1, the LOD was 7.34×10-7μg. The RSDis 1.14%. The average recovery is 98.34 %. 3) Extraction of hyperforin from Hypericum perforatum were studied. Theextraction was intensified by ultrasonic method. Then the crude extracts ofhyperforin was obtained. The separation of hyperforin from crude extracts bypreparative thin layer chromatography (PTLC) was studied. The optimal III<WP=9>conditions of PTLC were as follows: silica gel GF254 as stationary phase;n-hexane-ethyl acetate (4.5:1, v/v) as mobile phase. The purity of hyperforinwas 76.64%. The separation of hyperforin from crude extracts with silica columnchromatography was studied. The optimal conditions of silica columnchromatography were as follows: column chromatography silica gel as stationaryphase, n-hexane-ethyl acetate (6:1, v/v) as mobile phase, fractively collection,combining the same constituents. The purity of separated hyperforin was61.14%. 4)The separation of hyperforin from crude extracts with dry columnchromatography was studied firstly. The optimal conditions of dry columnchromatography were as follows: silica gel GF254 as stationary phase, n-hexane-ethyl acetate (4.5:1, v/v) as the mobile phase, the purity of hyperforin was72.13%. 5)By combination of PTLC and preparative high performance liquidchromatography (PHPLC), a high-purity hyperforin sample was obtained. Thepurity of sample was higher than 98%. 6)The product was identified as hyperforin by ESI-MS. The ESI+-MSand ESI--MS spectrum of the product was in accord with literatures. TheESI-MS was elucidated and the fragmentation pathway of hyperforin wassuggested.更多还原