【作者】 王妍；

【导师】 王宝华；

【作者基本信息】 北京中医药大学 ， 中药制药学， 2015， 硕士

【摘要】 1.目的：对黄蜀葵花主要黄酮类成分含量及HPLC指纹图谱进行研究,完善其质量评价体系；对黄蜀葵花中的挥发性成分进行分析鉴定,为黄蜀葵花药材资源的进一步开发利用提供研究基础；对黄蜀葵茎叶中的黄酮类成分及主要黄酮类成分含量进行研究,揭示黄蜀葵茎叶中黄酮类有效成分的物质基础,为黄蜀葵茎叶药材的质量控制及其后续药理作用的研究提供参考依据。2.方法：(1)采用紫外分光光度法,建立黄蜀葵花和茎叶中总黄酮成分的含量测定方法。(2)采用高效液相色谱法,建立同时测定黄蜀葵花中指标性成分金丝桃苷、异槲皮苷、杨梅素、槲皮素含量的方法,并比较不同产地黄蜀葵花中黄酮类成分的含量差异。(3)建立黄蜀葵花HPLC指纹图谱,并运用指纹图谱相似度评价软件匹配共有峰,生成对照图谱,并应用SPSS分析软件进行聚类分析。(4)采用气相色谱-质谱(GC-MS)联用技术,分离鉴定黄蜀葵花中的挥发性成分。(5)采用高效液相色谱串联离子阱质谱(HPLC-ITMS),分离鉴定黄蜀葵茎叶中的黄酮类成分。(6)采用多种分离纯化方法和结构鉴定手段(如1H-NMR、13C-NMR等波谱学技术),对黄蜀葵茎叶黄酮类成分进行了研究。(7)采用高效液相色谱法,建立同时测定黄蜀葵茎叶中指标性成分芦丁、金丝桃苷、异槲皮苷含量的方法,并比较不同产地黄蜀葵茎叶中黄酮类成分的含量差异。3.结果：(1)采用比色法建立了黄蜀葵花和茎叶中总黄酮的含量测定方法,线性范围为12～72μg/ml,平均回收率为97.96%,相对标准偏差(RSD)为2.63%。方法学考察表明,所建立的含量测定方法测定结果准确,且操作简便,可用于黄蜀葵花和茎叶中总黄酮的含量测定。(2)采用HPLC法建立了同时测定黄蜀葵花中4种指标性成分(金丝桃苷、异槲皮苷、杨梅素、槲皮素)含量的方法,金丝桃苷、异槲皮苷、杨梅素、槲皮素的回归方程分别为：Y=1489889X一14982(r=0.9999)；Y=2183245X-23801(r=1)；Y=2830492X-44686 (r-0.9990);Y=3135211X-25706(r-0.9994)。方法学考察表明,所建立的含量测定方法操作简便,结果可靠,可用于黄蜀葵花药材的质量控制。(3)测定了10个不同产地黄蜀葵花的HPLC指纹图谱,相似度评价均大于0.9,整体相似度较好,说明各产地黄蜀葵花药材质量有较好的相似性。(4)采用气相色谱-质谱(GC-MS)联用技术,从黄蜀葵花中鉴定出47个挥发性成分,其中,相对含量>2%的化合物有八个,分别为：植烷(37.83%)、二十五烷(13.49%)、9-辛基十七烷(9.78%)、柏木脑(3.07%)、(Z,Z,Z)-9,12,15-十八碳三烯酸甲酯(2.79%)、四十四烷(2.78%)、环二十四烷(2.61%)和十六烷酸甲酯(2.32%)。结果表明,黄蜀葵花中挥发性成分主要是由非环状烷烃类化合物、羟基化合物和芳香化合物组成,且植烷的含量最高。(5)采用高效液相色谱串联离子阱质谱(HPLC-ITMS),快速从黄蜀葵茎叶中鉴定出5个黄酮类化合物,分别为槲皮素-3-O-葡萄糖基阿拉伯糖苷、槲皮素-3-O-半乳糖基鼠李糖苷、芦丁、金丝桃苷和异槲皮苷,均为黄酮醇苷,这5个黄酮类化合物也存在于黄蜀葵花药材中。(6)采用多种分离纯化方法和结构鉴定手段(如1H-NMR、13C-NMR等波谱学技术),对黄蜀葵茎叶中的黄酮类成分进行研究,共分离得到9个化合物,鉴定出6个化合物,其中黄酮类成分3个,其他类成分3个,分别为金丝桃苷、异槲皮苷、芦丁、二十六碳酸、二十碳烯酸和十七烷酸乙酯,所鉴定出的3个黄酮类成分也存在于黄蜀葵花药材中。(7)采用HPLC法建立了同时测定黄蜀葵茎叶中3种指标性成分(芦丁、金丝桃苷、异槲皮苷)含量的方法,芦丁、金丝桃苷、异槲皮苷的回归方程分别为：Y=1522361X +4585(r=0.9999)；Y=1511417X-273(r=1)；Y=2286106X-434(r=0.9999)。方法学考察表明,所建立的含量测定方法操作简便,结果可靠,可用于黄蜀葵茎叶药材的质量控制。4.结论：所建立的质量评价方法专属性强、重复性良好,可用于黄蜀葵花的质量控制；对黄蜀葵花中挥发性成分的研究,为黄蜀葵花药材资源的进一步开发利用奠定了基础；揭示了黄蜀葵茎叶中黄酮类有效成分的物质基础,所建立的含量测定方法操作简便,结果准确,可用于黄蜀葵茎叶的质量控制。更多还原

【Abstract】 Objectives:To establish a method for the determination of main constituents and HPLC fingerprint of Abelmoschus corolla in order to optimize the quality evaluation system; To analyze the volatile constituents from Flos Abelmoschus manihot in order to provide reference for the development and utilization of Flos Abelmoschus manihot; To study the chemical constituents of stems and leaves of Abelmoschus manihot and establish a method for the determination of main constituents of stems and leaves of Abelmoschus manihot, and reveal the effective constituents of stems and leaves of Abelmoschus manihot, which was greatly contributed to the study of quality control and pharmacodynamics.Methods:(1) To establish a method for the determination of total flavonoids of flowers, stems and leaves of Abelmoschus manihot by UV-VIS spectrophotometry.(2) To establish a method for simultaneous determination of hyperin, isoquercitrin, myricetin and quercetin in the flowers of Abelmoschus corolla from different areas by HPLC, and compare their content differences.(3) To establish HPLC fingerprint of Abelmoschus corolla, and the data was processed on the similarity of fingerprint software and the SPSS analysis software for cluster analysis.(4) To analyze the volatile constituents from Flos Abelmoschus manihot by GC-MS method.(5) To analyze the flavonoids in stems and leaves of Abelmoschus manihot by HPLC-ITMS.(6) To study the chemical constituents of stems and leaves of Abelmoschus manihot by using several separation and purification methods and determination technology (such as 1H-NMR,13C-NMR).(7) To establish a method for simultaneous determination of rutin, hyperin and isoquercitrin in the stems and leaves of Abelmoschus manihot from different areas by HPLC, and compare their differences.Results:(1) Established the content determination method of total flavonoids, the linearity of flavonoids was fine in 12～72μg/ml, average recovery rate 97.96%, RSD 2.63%(n=6). The method has satisfactory accuracy, stability and reproducibility.(2) Established the simultaneous determination method of hyperin, isoquercitrin, myricetin and quercetin, the regression equation:Y= 1489889X-14982(r=0.9999); Y=2183245X-23801(r=1); Y=2830492X-44686(r=0.9990); Y=3135211X-25706(r=0.9994). The method has satisfactory accuracy, stability and reproducibility.(3) Seventeen common peaks in HPLC fingerprint of Abelmoschus corolla were separated from the 10 batches of samples, the common pattern was established, and the similarity of Abelmoschus corolla from different areas was above 90%.(4) A total of 47 volatile components from Flos Abelmoschus manihot were successfully identified by GC-MS, which were mainly composed of hydroxy compounds, acyclic alkane compounds and other aromatic compounds. And the eight components with the highest relative content fraction (>2%) are hexadecane,2,6,10,14-tetramethyl-(37.83%), pentacosane (13.49%), heptadecane,9-octyl-(9.78%), cedrol (3.07%), 9,12,15-octadecatrienoic acid,methyl ester, (Z,Z,Z)-(2.79%), tetratetracontane (2.78%), cyclotetracosane (2.61%) and hexadecanoic acid, methyl ester (2.32%).(5) 5 flavonol glycosides from stems and leaves of Abelmoschus manihot were successfully identified by HPLC-ITMS, including:quercetin-3-O-glucosyl-arabinoside, quercetin-3-O-galactorhamnsoide, rutin, hyperin and isoquercitrin, which are also riched in Flos Abelmoschus manihot.(6) 9 compounds were isolated from stems and leaves of Abelmoschus manihot, and 6 compounds were identified, including 3 flavonoids (hyperin, isoquercitrin and rutin) which are also riched in Flos Abelmoschus manihot and 3 other compounds (n-Hexacosanoic acid,10-Eicosenoic acid and ethyl heptadecanoate).(7) Established the simultaneous determination method of rutin, hyperin and isoquercitrin, the regression equation:Y= 1522361X+4585(r= 0.9999); Y= 1511417X-273 (r= 1); Y= 2286106X-434 (r=0.9999). The method has satisfactory accuracy, stability, reproducibility and has laid the foundation for the control of the inherent quality in the stems and leaves of Abelmoschus manihot.Conclusions:The method established for quality evaluation has excellent specificity and reproducibility for the quality control of Abelmoschus corolla. The qualitative analysis of volatile constituents from Flos Abelmoschus manihot laid the foundation for the development and utilization of Flos Abelmoschus manihot. This study revealed the effective constituents of stems and leaves of Abelmoschus manihot, and the method established for content determination has satisfactory accuracy, stability, reproducibility and has laid the foundation for the control of the inherent quality in the stems and leaves of Abelmoschus manihot.更多还原