文冠果中文冠果皂苷E含量的HPLC测定法的研究及对照品的制备

【作者】 李欣；

【导师】 李铣；

【作者基本信息】 沈阳药科大学 ， 天然药物化学， 2008， 硕士

【摘要】 文冠果（Xanthoceras sorbifolia Bunge）为无患子科（Sapindaceae）文冠果属植物,分布于我国辽宁、河北、陕西等省。民间多用于治疗风湿性关节炎和小儿遗尿症。现代医学研究表明文冠果具有抗炎、抗癌、抑制HIV蛋白酶、改善学习记忆等药理作用和生物活性,在医药、保健、化工等领域应用前景广阔。文冠果总皂苷是文冠果改善学习记忆障碍的有效部位,文冠果皂苷E（bunkanka saponin E）是其主要有效成分之一。本文以文冠果皂苷E作为对照品,首次建立了利用HPLC法测定文冠果各部位（果壳、果柄、种皮、种仁）中文冠果皂苷E含量的分析方法。测定条件为:HypersilODS₂（250mm×4.6mm,5μm）色谱柱,甲醇—水—磷酸（V:V:V=70:30:0.02）,检测波长210nm,柱温34℃,流速为1.0 mL·min^-1。文冠果皂苷E的保留时间在15min左右,与相邻峰分离度大于1.5,达到了色谱分离的要求。质量浓度在0.0098～0.1960 g·L^-1内与峰面积成良好线性关系。果壳、果柄、种皮和种仁的平均加样回收率在99.2～100.4%（n=9）范围内。结果表明,果柄中的文冠果皂苷E含量最高（0.45%）,果壳（0.20%）、种皮（0.15%）次之,种仁中未检测到文冠果皂苷E。此方法稳定可靠,快速简便,为文冠果药材的质量控制和其不同药用部位的合理利用和综合开发提供了科学依据。从文冠果果柄70%乙醇提取物中,通过系统溶剂萃取、反复硅胶柱色谱及反复重结晶等方法制备对照品文冠果皂苷E,经HPLC法测定含量达到98.6%,满足对照品的要求。在制备对照品的同时,共分离得到了12个化合物。通过理化性质和波谱数据分析鉴定了它们的结构。分别属于三萜类:玉蕊醇R₁（1）、21,22-二-O-当归酰基-玉蕊醇R₁（2）、3-O-（3-O-α-L-呋哺阿拉伯糖基-2-O-β-D-吡哺半乳糖基）-β-D-吡喃葡萄糖醛酸基-21,22-二-O-当归酰基-玉蕊醇R₁（文冠果皂苷E）（3）、21-O-当归酰基-玉蕊醇R₁（4）、3-羰基甘遂烷-7,24-二烯-21-酸（5）;甾醇类:β-谷甾醇（6）、胡萝卜苷（7）、α-菠菜甾醇（8）、α-菠菜甾醇-3-O-β-D-吡喃葡萄糖苷（9）;其他类:正三十一烷酸（10）、正二十七烷醇（11）、正丁基吡喃果糖苷（12）。更多还原

【Abstract】 Xanthoceras sorbifolia Bunge（Sapindaceae）is a plant widely distributed in Liaoning, Hebei and Shanxi Provinces of China.It is used to treat rheumatism and enuresis of children as a folk medicine in China.It has been reported that Xanthoceras sorbifolia Bunge showed good biological activities of anti-inflammation,anti-tumor,inhibition of HIV protease and improving memory,etc.The total saponins is determined to be the effective composition of Xanthoceras sorbifolia Bunge,and bunkanka saponin E is main effective component of the total saponins.It is the first time to establish the RP-HPLC method for quantitative determination of bunkanka saponin E content in Xanthoceras sorbifolia Bunge.The Chromsphere HypersilODS₂（250 mm×4.6 mm,5μm）column was used to determine the content of bunkanka saponin E.The mobile phase was methanol-water-phosphoric acid（70:30:0.02）. The flow rate is 1.0 mL·min^-1.Column temperature is 34℃and the detection wavelength is 210 nm.The retention time of bunkanka saponin E was about 15 minutes under its chromatograms conditions,and the R-value between the main peak and the nearby peak was bigger than 1.5,which means reaching the standard of chromatograms separation.The linear range of bunkanka saponin E was from 0.0098 g·L^-1to 0.1960 g·L^-1.The rang of average recoveries in husk,carpophore,tunic and kernel was 99.2%to 100.4%.The results indicated: the content of bunkanka saponin E in the carpophore,husk and tunic were 0.45%,0.20%and 0.15%respectively.The bunkanka saponin E was not determinated in kernel.The method is simple,accurate,sensitive and stable used for the quality control for Xanthoceras sorbifolia Bunge.The results provided scientific basis for comprehensive development and utilization of different parts from Xanthoceras sorbifolia Bunge.From the 70%ethanol extract of the carpophore of Xanthoceras sorbifolia Bunge,the reference substance--bunkanka saponin E was isolated by means of solvent extraction, repeated column chromatography on silica gel and recrystallization.Its content determined by HPLC was 98.6%which meets the requirement of reference substance.During this course,12 compounds in total were isolated.On the basis of physico-chemical properties and spectroscopic analysis,their structures were identified as follows.Tritepenes:R₁-barrigenol （1）,21.22-di-O-angeloyl-R₁-barragenol（2）,3-O-（3-O-α-L-arabinofuranosyl-2-O-β-D-galactopy ranosyl）-β-D-glucuronopyranosyl-21,22-di-O-angeloyl-R₁-barrigenol（3）,21-O-angeloyl-21 -angeloyl-R₁-barrigenol（4）,3-oxotirucalla-7,24-dien-21-oic（5）;Sterols:β-sitosterol（6）, daucosterol（7）,α-spinasterol（8）,α-spinasterol-3-O-β-D-glucopyranoside（9）;Others: n-hentriacontanic acid（10）,n-heptacosanol（11）,n-butyl-fructopyranoside（12）.更多还原