Shan-lü-cha, the leaf of Ilex hainanensis Merr., is a well-known traditional Chinese medicine in Guangxi Province. It is mainly distributed in the south of China. The tablet made from its leaves is a traditional Chinese medicine used as antihyperlipidemic, antihypertensive, anti-inflammatory on clinic. Pharmacology research showed that Ilex hainanensis has the potential of protecting cardiovascular, anti-inflammatory and antihypertensive activities. However, the quality evaluation of Ilex hainanensis were limitedly focus on total flavonoids and triterpenoids, respectively. Method for comprehensively evaluating this herb was still lack. Thus, the objective of present study was to investigate the chemical components in Ilex hainanensis and develop a valid method for quality control of Ilex hainanensis. The dissertation consists of three chapters. Chapter I reviewed the research progresses on chemical, pharmacological, clinical and quality control of Ilex hainanensis. Chapter Ⅱ showed the chemical study in Ilex hainanensis. In order to investigate the bioactive compounds in Ilex hainanensis, macroporous resin column, silica gel column chromatography and pre-HPLC were employed in the systematical separation. As the results, ten compounds were isolated and purified. Based on the physiochemical and spectroscopic evidence, their structures were elucidated as follows: Ilexgenin A (1), Ilexhainanoside D (2), Ilexsaponin A1 (3), 2-(Caffeoyloxy)-methyl-3-hydroxy-1-butene-4-O-β-D-glucopyranoside (4), Rutin (5), Eriodictyol-7-O-β-D-glucopyranoside (6), Dibutyl Phthalate (7), Syringaresinol-4-O-β-D-glucopyranoside (8), (2E)-2-methyl-2-butene-1,4-diol-4-O-β-D-(6′′-caffeoyl)-glucopyranoside (9), 1-O-caffeoyl-(2E)-2-methyl-2-butene-1,4-diol-4-O-β-D-glucopyranoside (10), respectively. Among them, (8) was isolated and identified in Ilex hainanensis for the first time. In Chapter III, an HPLC-DAD-ELSD method was developed for simultaneous quantification of eight major bioactive components including two flavonoids, two isochlorogenic acids and four triterpenoids in Ilex hainanensis. The analysis was performed on an Agilent ZORBAX SB-C18 column (4.6 mm×250 mm, 5µm), with a mixture solution of acetonitrile and 0.2% methanic acid as mobile phase using a gradient elution. The flow rate was 1.0 mL/min, the column temperature was 30 ℃ and the detection wavelength was 283 nm. The method was validated in terms of linearity, sensitivity, precision, stability and accuracy. It was found that the calibration curves for all analytes showed good linearity (R 2 >0.9991) within the test ranges. The limit of detection (LOD) and limit of quantification (LOQ) were less than 0.21 and 0.78 µg/mL, respectively. The relative standard deviations (RSD) for intra- and inter-day repeatability were no more than 1.46 % and 1.78 %, respectively. The sample was stable for at least 48 h. The recoveries of eight components were between 94.6 %-103.3 %. In this study, ten compounds were isolated from Ilex hainanensis, Compound (8) was separated and identified in this herb for the first time. An effective HPLC method was developed for simultaneous determination of eight major compounds of Ilex hainanensis, and was successfully applied in four samples. The results showed that the developed HPLC-DAD-ELSD method was linear, sensitive, precise and accurate. It could be applied for quality control of Ilex hainanensis.

山綠茶為冬青科冬青屬植物海南冬青(Ilex hainanensis Merr.)的葉，在我國廣東、 廣西、雲南等地多有分佈，生長於海拔 500-1000 米的山坡密林或疏林裡，四季皆可 採收，曬乾儲藏，作為廣西少數民族草藥有著悠久的使用歷史。在《廣西中藥材標 準》（1990 年版）中已有收錄，山綠茶降壓片在 2010 版《中國藥典》和《中藥部頒 標準》亦有收載。山綠茶性寒，味辛；歸肝、脾、肺、腎經，有平肝息風、清熱解 毒、消腫止痛等功效，臨床主用於降血脂、降血壓、治療風熱咳嗽及痢疾等疾病。 作為一味傳統藥材，目前對於山綠茶的品質控制研究卻十分缺乏，僅局限於總黃酮 或三萜類的分析。因此本課題以山綠茶為研究對象，應用現代分離技術對山綠茶進 行化學成分研究，並在此基礎上運用高效液相色譜技術對山綠茶主要活性成分進行 了定量分析。 本論文包括三個章節。第一章介紹本課題的研究背景，從化學研究、藥理研究、 臨床療效、分析方法等方面對山綠茶研究現狀進行了總結，提出本課題的立題基礎 和研究可行性。 第二章為山綠茶的化學成份研究。為探索山綠茶藥材的活性物質基礎，本課題 對山綠茶的化學成分進行了分離和結構鑒定。利用矽膠色譜、凝膠色譜和製備高效 液相色譜技術對山綠茶進行了系統分離，從中分離得到 10 個化合物，並通過紫外光 譜、質譜、核磁共振氫譜和碳譜的方法進行了結構鑒定，分別為冬青素 A (Ilexgenin A, 1)，海南冬青苷 D (Ilexhainanoside D, 2)，毛冬青皂苷 A1 (Ilexsaponin A1, 3），咖 啡 醯 -2- 甲 基 -3- 羥 基 -1- 丁 烯 -4-O-β-D- 吡 喃 葡 萄 糖 苷 [2-(Caffeoyloxy)-methyl-3-hydroxy-1-butene- 4-O-β-D-glucopyranoside, 4] ， 蘆 丁 (Rutin, 5)，北美聖草素-7-O-β-D-吡喃葡萄糖苷 (Eriodictyol-7-O-β-D-glucopyranoside, 6)，鄰苯二甲酸二丁酯 (Dibutyl Phthalate, 7)，丁香脂素-4-O-吡喃葡萄糖苷 (Syringaresinol-4-O-β-D-glucopyranoside, 8) ， (2E)-2- 甲 基 -2- 丁 烯 -1,4- 二 醇 -4-O-β-D-(6′′-O- 咖啡醯基 )- 吡 喃 葡 萄 糖 苷 [(2E)-2-methyl-2-butene-1,4-diol-4-O-β-D-(6′′- caffeoyl)-glucopyranoside, 9]，1-O-咖啡 醯 基 -(2E)-2- 甲 基 -2- 丁 烯 -1,4- 二 醇 -4-O-β-D- 吡 喃 葡 萄 糖 苷 [1-O-caffeoyl-(2E)-2-methyl-2-butene-1,4-diol-4-O-β-D-glucopyranoside, 10]。其中化合 物 8 為從本植物首次分離與鑒定。 第三章為含量測定研究。我們採用 HPLC-DAD-ELSD 聯用分析技術，對山綠茶 藥材中的 8 個主要活性成分進行了含量測定，建立了穩定可靠的品質控制方法。這 8 個成分包括了 2 種黃酮（蘆丁、北美聖草素-7-O-β-D-吡喃葡萄糖苷），2 種綠原酸 （異綠原酸 A、異綠原酸 C）和 4 種三萜（冬青素 A、海南冬青苷 D、毛冬青皂苷 A1、熊果酸）。其中北美聖草素-7-O-β-D-吡喃葡萄糖苷、異綠原酸 A 和異綠原酸 C 首次作為山綠茶的質量控制指標。色譜柱採用 Agilent ZORBAX-C18 (5µm, 4.6mm×250mm)；流動相：0.2%甲酸溶液 (A)-乙腈 (B)，梯度洗脫程式為（B）：0-15 min, 15-18%; 15-25 min, 18-23% ; 25-35 min 23-30% ; 35-40 min 30-40% ; 40-50 min 40-50% ; 50-55 min 50-80% ; 55-60 min 80-100% ; 60-70 min 100-100%；流速：1.0 mL·min-1；檢測波長：283nm；柱溫為 30℃。在相應的線性範圍內，8 種化合物的 標準曲線方程均 R 2 >0.9991，檢測限 (LOD)與定量限 (LOQ)分別小於 0.21 與 0.78 µg/mL，日內精密度與日間精密度 RSD 值分別小於 1.46 % 與 1.78 %，平均加樣回 收率為 94.6 %-103.3 %，樣品在 48 h 內穩定。 本課題從山綠茶中分離得到10個化合物，其中1個為首次從該植物中分離鑒定。 在此基礎上建立了同時實現對山綠茶中主要活性成分黃酮、異綠原酸、三萜類的分 離檢測的色譜方法，並測定了來自 3 個批次樣品及山绿茶提取物中 8 種化合物的含 量。該方法具有線性良好，快速準確，重現性好等特點，為山綠茶質量評價及質量 控制提供了科學依據。