台灣特有種植物林氏茜草暨市售茜草藥材之HPLC指紋圖譜分析及品質評價

HPLC Fingerprint Analysis and Quality Evaluation of Rubia linii Chao, an Endemic Plant in Taiwan, and Commercial Rubiae Radix

葉瓊文(Chiung-Wen Yen)

指導教授 ： 顏銘宏

高雄醫學大學/藥學院/藥學系碩士在職專班/碩士(2016年)

摘要

目的：本研究應用高效液相層析法（High Performance Liquid Chromatography; HPLC）建立台灣市售茜草藥材（從中國輸入）的化學指紋圖譜（Chemical Profile Fingerprint）。收集台灣北、中、南部市售茜草藥材共15個藥材，進行HPLC化學指紋圖譜分析，及指標成分大葉茜草素（Mollugin）含量測定，分析市售茜草藥材品質差異，並建立HPLC化學指紋圖譜科學參數，做為茜草藥材基原確立之依據。另外再採集台灣同屬植物並且是台灣特有種的植物林氏茜草(Rubia linii Chao)的根同時做比較，以評估代用茜草藥材之可行性。
方法：HPLC分析條件，Mightysil RP-18 GP column(250 x 4.6 mm i.d.；5 µm; Kanto Chemical Co. Inc., Tokyo, Japan)；流動相A為0.1%磷酸，B為乙腈，使用梯度沖提方法(0∼25 min, 80∼65% A; 25∼55 min, 65∼45% A; 55∼60 min, 45∼30% A; 60∼76 min, 30%∼60% A; 76∼90 min, 60∼80% A)。體積流量1. 0 mL/min；柱溫室溫；檢測波長272 nm；進樣量20 μL。檢測材料有15個台灣市售茜草藥材及台灣特有種植物林氏茜草(R. linii)的根。使用的對照指標成分（Standard Reference）為大葉茜草素（Mollugin）。
結果：本研究結果顯示Mollugin線性範圍在22.22∼177.78 µg/mL 有良好的線性關係（R²= 0.9995）。本研究將HPLC圖譜之吸收峰積分面積值(Peak Area)小於25,000者視為極微弱吸收峰或雜訊，並將其排除不列入分析；積分面積值在25,000以上之吸收峰則依據吸收峰面積比值(Peak Area Ratio; PAR)進一步分類。PAR的分析結果測得15個台灣市售茜草藥材的HPLC化學指紋圖譜介於17〜29支明顯吸收峰（Peak Level Limit:25,000）。林氏茜草的HPLC指紋圖譜具有17支明顯吸收峰（Peak Level Limit:25,000）。依據各吸收峰的相關位置，本研究建立台灣市售茜草藥材的HPLC指紋圖譜的相對吸收峰面積（Relative Peak Area Ratio; PAR）、α-值（α-value）、滯留因子（Retention Factor k）及分離因子（Separation Factor），可作為茜草飲片品質控制及基原鑑定的科學依據。在檢品品質分析方面，收集自全台15家中藥房市售茜草藥材，其大葉茜草素(Mollugin)含量，從最小(0.53 mg/g檢品乾重)至最大(5.02 mg/g檢品乾重)，差距達10倍，顯見台灣市售茜草藥材品質極不穩定。而台灣特有種林氏茜草（R. linii）的根，其指紋圖譜與台灣市售茜草藥材相比較相去甚遠，顯示林氏茜草（R. linii）無法代用台灣市售茜草藥材。

關鍵字

高效液相層析法（HPLC） ； 指紋圖譜 ； 茜草藥材 ； 林氏茜草

並列摘要

Purpose: In this study, we utilized high performance liquid chromatography (HPLC) to establish the chemical fingerprint of commercial Rubiae Radix, which was origined from the roots of Rubia cordifolia and mainly imported from mainland China. The quality control of standard reference was mollugin. In addition, we compared the roots of Rubia linii Chao, an endemic plant in Taiwan, with the commercially available Rubiae Radix to investigate if it was a good alternatives.
Method: HPLC analysis condition, Mightysil RP-18 GP column( 250 x 4.6 mm i.d.；5 µm; Kanto Chemical Co. Inc., Tokyo, Japan);mobile phase A was 0.1% phosphoric acid, B was acetonitrile, and gradient method was applied. (0∼25 min, 80∼65% A; 25∼55 min, 65∼45% A; 55∼60 min, 45∼30% A; 60∼76 min, 30%∼60% A; 76∼90 min, 60∼80% A). Volume flow was 1.0 mL/min; column temperature was room temperature; detection wavelength was set at 272 nm; volume injected was 20 μL. Target samples included commercially Rubiae Radix, and the endemic species Rubia linii Chao. The standard reference in comparison was mollugin.
Results: The results of this study showed a positive linear relationship, with its range of 22.22 ~ 177.78 μg/mL, and R² = 0.9995 respectively. As the HPLC fingerprints result, 17〜29 absorption peaks (peak level limit:25,000) were found in 15 commercial Rubiae Radix samples. In addition, there were 17 obvious absorption peaks (peak level limit:25,000) existed in the HPLC fingerprint of R. linii. We also established the relative peak area ratio (PAR),α-value, retention factor(k) and separation factor in the HPLC fingerprint to help offer a method for quality validation of Rubiae Radix. As the results indicated, there was a 10-fold difference in regard to mollugin content from the minimum of 0.53 mg/g?Odried material to the maximum of 5.02 mg/g?Odried material. HPLC fingerprints of R. linii, an endemic species of Taiwan, and that of commercially Rubiae Radix were compared, and the results suggested a significant difference between these two medicine.

並列關鍵字

High Performance Liquid Chromatography (HPLC) ； Fingerprints ； Rubiae Radix ； Rubia linii

參考文獻

參考文獻

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