“藏茵陈”类植物化学成分的结构鉴定及高效液相色谱/质谱分析表征

NWIPB OpenIR

	“藏茵陈”类植物化学成分的结构鉴定及高效液相色谱/质谱分析表征; Structure Identification and HPLC/MS Analysis of Chemical Constituents in Tibetan Medicinal Plants “Zangyinchen”
	李玉林
	2007-05-30
摘要	“藏茵陈”是西藏、青海、四川、云南、甘肃等地区最具特色的治疗肝胆疾病的一大类著名藏药，其原植物有20多种，属于龙胆科的獐牙菜属、龙胆属、扁蕾属、花锚属及肋柱花属和虎耳草科的虎耳草属。本学位论文工作重点对辐状肋柱花和红直獐牙菜化学成分进行提取、分离、结构鉴定，以及对植物川西獐牙菜、抱茎獐牙菜、印度獐牙菜、四数獐牙菜、橢圆叶花锚和辐状肋柱花等6种“藏茵陈”植物的主要化学成分进行了HPLC/MS、HPLC、HPCE分析表征。对青藏高原特色生物资源“藏茵陈”进行了较为全面、系统和深入的研究，取得了以下研究成果和结论。 1、从辐状肋柱花中经提取、分离、纯化和结构鉴定得到8个水溶性成分，它们是： 2-(3′-O-β-D-glucopyranosyl)benzoyloxygentisic acid(1)、异荭草苷（2）、芒果苷(3)、 Swertipunicoside(4)、当药醇苷（5）、异牡荆苷（6）、当药黄素（7）和7-O-[α-L-吡喃鼠李糖-（1→2）-β-D-吡喃木糖]-1，8-二羟基-3-甲氧基口山酮(8)。除异荭草苷外，其余化合物均首次从该种植物中得到，其中1为新的天然化合物。 2、从红直獐牙菜的水溶性部分得到7个化合物，它们分别为8-O-β-D-吡喃葡萄糖-1,5-二羟基-3-甲氧基口山酮(1)、8-O-β-D-吡喃葡萄糖-1,3,5-三羟基口山酮(2)、1-O-β－D-吡喃葡萄糖-3,7,8-三羟基口山酮(3)、异荭草苷(4)、落干酸(5)、龙胆苦苷(6)和β-龙胆二糖(7)。化合物3、4、5和7首次从该植物中得到。 3、采用高效液相色谱/质谱联用技术对6种典型“藏茵陈”药用植物川西獐牙菜、抱茎獐牙菜、印度獐牙菜、四数獐牙菜、橢圆叶花锚和辐状肋柱花提取物中的化学成分进行了全面的分析表征。它们的化学成分主要为环烯醚萜苷、黄酮苷、口山酮苷及口山酮苷元，其中口山酮类化合物为“藏茵陈”主要的特征性成分。 4、采用反相高效液相色谱法同时分离和测定了抱茎獐牙菜中9种主要活性成分：獐牙菜苦苷、芒果苷、龙胆苦苷、獐牙菜苷、异荭草苷、当药黄素、当药醇苷、7-O-[α-L-吡喃鼠李糖-（1→2）-β-D-吡喃木糖]-1，8-二羟基-3-甲氧基口山酮和雏菊叶龙胆酮。在Eclipse XDB-C8柱上，以乙腈-水-甲酸（10:90:0.1）和乙腈-水（90:10）为流动相梯度洗脱，流速1 mL/min，DAD检测波长240， 254 和 265 nm，实现了9种化合物及其它未知物的基线分离。9种化合物的线性回归系数大于0.9980, 检测限为0.0061～0.0026 mg/mL，峰面积和保留时间的RSDs小于2.07 % 和2.86 %，具有良好的重现性。此方法用于抱茎獐牙菜的测定，结果满意。 5、采用高效毛细管电泳法同时分离和测定了川西獐牙菜和抱茎獐牙菜中5种苷类成分：当药醇苷、当药黄素、异荭草苷、芒果苷和7-O-[α-L-吡喃鼠李糖-（1→2）-β-D-吡喃木糖]-1，8-二羟基-3-甲氧基口山酮。毛细管规格为48.5cm x 50μm，二极管阵列紫外检测器(DAD)检测波长254nm，最佳分离条件：电压24kV，分离温度25℃，背景电解质为含有28mmol/L 十二烷基硫酸钠(SDS)，1.0％乙腈的30mmol/L硼酸溶液，pH =9.0。5种化合物的线性回归系数在0.9980～0.9995之间。标准品迁移时间和峰面积的RSDs分别小于 0.91 % 和7.85 %，具有较好的重现性。 6、利用荧光衍生试剂吖啶酮-9-乙基对甲苯磺酸酯，将其作为柱前衍生化试剂, 在Eclipse XDB-C8色谱柱上，采用梯度洗脱对川西獐牙菜、抱茎獐牙菜和辐状肋柱花中C1～C19游离脂肪酸衍生物进行基线分离。利用柱后在线的串联质谱以大气压化学电离源正离子模式实现了各组分的质谱定性。荧光检测的激发和发射波长分别为ex =404nm，em = 440nm。19种游离脂肪酸衍生物的线性回归系数大于0.9989，检测限为12.3～43.7fmol，峰面积和保留时间RSDs小于2.24 % 和0.45 %，具有良好的重现性。此方法用于“藏茵陈”药材样品的测定，结果满意。; “Zangyinchen” are the traditional Tibetan folk medicinal plants and used to cure febrile diseases in liver and gall bladder at the regions such as Xizang, Qinghai, Sichuan, Gansu, and Yunnan Province. The medicinal plants used as “Zangyinchen” have about 20 species remaining with the families of Swertia, Gentiana, Gentianopsis, Halenia, and Lomatogonium in Gentianaceae and Saxifraga in Saxifragaceae. In this research, the structure identification of chemical constituents in two plants Lomatogonium rotatum and Swertia erythrosticta, and HPLC/MS, HPLC, and HPCE anlysis of chemical constituents in Swertia chirayita, Swertia mussotii, Swertia franchetiana, Swertia tetraptera, Halenia elliptica and Lomatogonium rotatum have been studied widely、systematically and deeply. And we get some results and conclusions as followed. 1. A new phenyl glycoside, 2-(3′-O-β-D-glucopyranosyl)benzoyloxygentisic acid, along with seven known glycosides, namely isoorientin, mangiferin, swertipunicoside, swertianolin, isovitexin, swertisin, and 7-O-[α-L-rhamnopyran osyl-（1→2）-β-D-xylopyranosyl]-1,8-dihydroxy-3-methoxyxanthone, was isolated from Tibetan herbal medicine Lomatogonium rotatum. Their structures were elucidated by spectroscopic methods including 1D and 2D NMR techniques and MS data. Except isoorientin, others were all firstly isolated from this plant. 2. Seven glycosides were isolated from Swertia erythrosticta and structurally identified as swertianolin, norswertianolin, norswertiaglucoside, isoorientin, loganic acid, gentiopicroside, and β-gentiobiose by spectral analysis and chemical evidence. Compounds norswertiaglucoside, isoorientin, loganic acid, andβ-gentiobiose were obtained firstly from this plant. 3. The chemical constituents were analyzed and characterized roundly by HPLC/UV/APCI/MS in the extracts of six typical “Zangyinchen” medicinal plants, namely Swertia chirayita, Swertia mussotii, Swertia franchetiana, Swertia tetraptera, Halenia elliptica and Lomatogonium rotatum. The main components in these plants are iridoid glycolsides, flavone glycolsides, xanthones and xanthone glycolsides. Xanthones and xanthone glycolsides are the characteristic components in “Zangyinchen”. 4. A sensitive and specific reversed-phase high performance liquid chromatography method with diode array detection was established for the quantitative determination of the nine active components, namely swertiamarin, mangiferin, gentipicroside, sweroside, isoorientin, swertisin, swertianolin, 7-O-[α-L-rhamnopyranosyl-(1→2)-β-D-xylopyranosyl]-1,8-dihydroxy-3-methoxyx anthone and bellidifolin used as the external standard, in Tibetan folk medicinal species Swertia franchetiana. Based on the baseline chromatographic separation of most components from methanolic extract of Swertia franchetiana on a reversed-phase Eclipse XDB-C8 column with water-acetonitrile-formic acid as mobile phase, the nine components were identified by comparing with standard samples and qualified by using external standard method with DAD at 254 nm. The correlation coefficients of all the calibration curves were founded to be higher than 0.9980. The relative standard deviations of the peak areas and retention times for the nine standards were less than 2.07 % and 2.86 %, respectively. 5. The contents of five pharmacologically active flavone and xanthone glycosides, namely swertianolin, swertisin, isoorientin, mangiferin, and 7-O-[α-L- rhamnopyranosyl-(1→2)-β-D-xylopyranosyl]-1,8-dihydroxy-3–methoxyxanthone, extracted from Tibetan folk medicinal species Swertia mussotii and S. franchetiana were determined by capillary electrophoresis with diode array detection. Separation of five components has been optimized with a capillary column of 48.5 cm total length, effective length 40 cm (50 m i.d). Influence of several important parameters such as running buffer, SDS concentration, and organic modifier. Etc. on resolution were evaluated. The background electrolyte contained 30 mmol/L borate buffer, 28 mmol/L SDS, 1.0 % (v/v) acetonitrile, and adjusted to pH 9.0 with 0.1 mol/L NaOH. A good baseline resolution was obtained for the separation of five components within 5 min with working voltage at 24 kV and column temperature at 25 ℃. The relative standard deviations of the peak areas and retention times for five standards are < 7.85 % and 0.91 %, respectively. The established method was rapid and reproducible for the separation and determination of five flavone and xanthone glycosides from the extracts of S. mussotii and S. franchetiana plant samples with satisfactory repeatability and recovery. 6. A simple and sensitive method for the determination of free fatty acids (FFAs) using acridone-9-ethyl-p-toluenesulfonate (AETS) as fluorescence derivatization reagent by HPLC has been developed. Free fatty acid derivatives were separated on Eclipse XDB-C8 column with a good baseline resolution and detected with the fluorescence which excitation and emission wavelengths of derivatives were set at lex =404 and lem=440nm, respectively. Identification of 19 fatty acid derivatives was carried out by online post-column mass spectrometry with atmospheric pressure chemical ionization source under positive-ion detection mode. 19 FFAs from the extracts of Swertia mussotii, S. franchetiana, and Lomatogonium rotatum are sensitively determined. The validation of the method including linearity, repeatability, and detection limits was examined. Most linear correlation coefficients for fatty acid derivatives are >0.9989, and detection limits (at signal-to-noise of 3:1) are 12.3-43.7 fmol. The relative standard deviations of the peak areas and retention times for 19 FFAs standards are < 2.24 % and 0.45 %, respectively. The established method is rapid and reproducible for the separation and determination of FFAs from these medicinal plants “Zangyinchen” with satisfactory results.
文献类型	学位论文
条目标识符	http://210.75.249.4/handle/363003/31412
专题	中国科学院西北高原生物研究所
推荐引用方式 GB/T 7714	李玉林. “藏茵陈”类植物化学成分的结构鉴定及高效液相色谱/质谱分析表征, Structure Identification and HPLC/MS Analysis of Chemical Constituents in Tibetan Medicinal Plants “Zangyinchen”[D],2007.