大黃葛根黃芩之定量分析研究

吳權益

指導教授 ： 許順吉教授

國立臺灣師範大學/理學院/化學系/碩士(2000年)

摘要

高效液相層析（HPLC）及毛細管電泳（CE）是目前最常用來測定中藥成分含量的分析方法。綜合兩者的優點，合併運用，可以拓展中藥化學評價的範疇。
本研究分三個部分，第一部分為開發大黃藥材的分析方法。大黃(Rhei Rhizoma)為蓼科植物的乾燥根莖，是重要的瀉下藥材。本研究利用高效液相層析儀(HPLC)與毛細管電泳(CE)分析其anthraquinones，dianthraquinones，stilbenes，galloylglucoses等十九個組成成分。實驗結果顯示利用醋酸與甲醇混合液為沖提液，HPLC可在75分鐘內成功地分析這十九個成分；CE無法一次完成分離，但以CZE模式，用硼酸鹽及磷酸二氫鈉配製成兩種不同pH值緩衝溶液，則可達到分離二十二個成分的目標。利用所開發的HPLC條件，分析含大黃製劑之小承氣湯，可分析其中大黃十九個成分、厚朴兩個成分和枳實四個成分，共二十五個組成成分的含量。運用該分析方法於大黃之安定性及炮製研究，發現加熱對各成分的含量有很大影響，而且加熱溫度越高，對藥材的保存越不利，此變化於加熱七天後漸趨平穩；酒浸後陰乾或烘乾的成分含量變化不大，酒炒則使各成分大為減少，酒蒸炮製品之anthraquinones和dianthraquinones類化合物普遍減少，stilbenes和galloylglucoses類化合物則增加。
第二部分為開發葛根藥材之HPLC分析方法。葛根是豆科植物（Leguminosae）甘葛藤(Pueraria lobata Ohwi.)的乾燥根，為重要的解表藥材，主要含黃酮類化合物。本研究利用醋酸、氰甲烷和甲醇混合液為沖提液，在65分鐘內分析出十二個成分。市售葛根藥材有野葛與粉葛兩種，各成分總含量以野葛較多，其中3’-methoxypuruerarin與puerarin兩成分的比值是它們的主要分辨點，該值野葛大於0.1，粉葛小於0.03；同時，本分析方法可作為葛根單方及葛根湯製劑之原料藥材基原鑑別。
第三部分為開發黃芩藥材HPLC之分析方法。黃芩為唇形科(Labiatae)植物黃芩(Scutellariae baicalensis Georgi)的乾燥根部，具有清熱燥濕、瀉火解毒之效。本研究利用醋酸和氰甲烷混合液為沖提液，可在50分鐘內分析baicalin(1, BG)，oroxylin A 7-O-glucuronide(2, OG)，wogonin 7-O-glucuronide(3, WG)，baicalein(4, B)，wogonin(5, W)與oroxylin A(6, O)等六個黃酮類成分，並可應用於分析三黃瀉心湯及探討三黃瀉心湯製劑在人工腸液中的成分變化。黃芩成分在人工胃液中幾不發生變化，但在人工腸液中，配醣體成分則有明顯水解現象，該水解現象可為大黃藥材所抑制。另外，以三黃瀉心湯餵予小白鼠，發現三小時後的尿液量明顯增多，而以六小時最多，黃芩各成分含量分別是BG 102.20 mg/mL，OG n.d.，WG 57.37 mg/mL，B 5.70 mg/mL，W 1.39 mg/mL，O 1.74 mg/mL；分析血清中黃芩成分含量，則以餵食三小時後有最高黃芩成分含量(BG 1.82 mg/mL，OG n.d.，WG 1.26 mg/mL，B 1.14 mg/mL，W 0.18 mg/mL，O 0.47 mg/mL)。

關鍵字

高效液相層析儀 ； 毛細管電泳儀 ； 大黃 ； 葛根 ； 黃芩

並列摘要

High-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) are currently the most widely used analysis methods for assaying Chinese herb drugs. By combining the advantages of both methods, we can expand the scope of chemical evaluation for Chinese herb drugs.
This study is divided into three parts. The first part deals with the analysis method for the drug Rhei Rhizoma, which is available as the dried rhizome of a polygonaceaous plant and has been used as a laxative. This study employs HPLC and CE to analyze the drug’s 19 constituents that belong to anthraquinones, dianthraquinones, stilbenes and galloylglucoses. Experiment results show that by using a combination of acetic acid and methanol as the eluent, HPLC can successfully analyze the 19 constituents within 75 minutes, whereas CE cannot accomplish this all at a time. However, with the CZE mode using a buffer of borate and sodium dihydrophosphate in different ratios for different pH values, it is possible to achieve the goal of analyzing 22 constituents at a time. Using the HPLC method developed the herbal formula Hsiao-cheng-chi-tang (Minor Rhubarb Combination) can also be analyzed with a total of 25 constituents including 19 constituents from the formula’s rhubarb ingredient, 2 constituents from the formula’s magnolia ingredient and 4 constituents from the formula’s chih-shih (Aurantii Fructus Immaturus) ingredient. By applying this method to the study on rhubarb’s stability and processing, we have found that heating has great influences on the herb’s constituents and that the higher the temperature of heating the herb is subjected to, the more disadvantageous it will be to the herb’s preservation. Such influences gradually become stabilized seven days after heating. Processing by immersing the herb in wine and then drying it in the shade dose not cause much change in the contents of the constituents, but it is found to have greatly reduced after processing by stir-frying the herb with wine. Processing by steaming the herb with wine commonly reduces the contents of anthraquinones and dianthraquinones, but increases the contents of stilbenes and galloylglucoses.
The second part is about the development of the HPLC method for analyzing the herb drug Puerariae Radix, which is available as the dried root of the legume plant Pueraria lobata Ohwi and has been used as an important diaphoretic drug. The drug contains flavones. In this study we used an eluent composed of acetic acid, acetonitrile and methanol whereby we are able to separate 12 constituents within 65 minutes. Commercial articles of this drug are of two kinds: the wild pueraria (derived from P. lobata Ohwi) and the powdery pueraria (derived from P. thomsonii Denth.). The wild pueraria has high contents of constituents. The ratio between 3’-methoxypuruerarin and puerarin differentiates the two articles. For the wild pueraria, the ratio is larger than 0.1, while the powdery pueraria has a ratio smaller than 0.03. Meantime, this analysis method can be used for identification and discrimination between a pueraria simple and a pueraria-containing formula.
The third part contains the HPLC method developed for analyzing the herb drug Scutellariae Radix, which is derived from the dried root of the labiate plant (Scutellaria baicalensis Georgi). In traditional Chinese medicine, the drug is claimed to be capable of clearing heat, purging fire and detoxifying toxicosis. This study employs the combination of acetic acid and acetonitrile as an eluent, whereby 6 flavones including baicalin (1, BG), oroxylin.A 7-O-glucuronide (2, OG), wogonin 7-O-glucuronide (3, WG), baicalein (4, B), wogonin (5, W) and oroxylin A (6, O) can be separated within 50 minutes. The method can also be applied to the analysis of the baicalin-containing formulas San-huang-hsieh-hsin-tang (Coptis and Rhubarb Combination) and its changes in the artificial intestinal fluid. The constituents of scutellaria do not undergo changes in artificial gastric fluid, but the glucosides hydrolyze markedly in artificial intestinal fluid. Such hydrolysis phenomenon can be inhibited by rhubarb. Besides, as Coptis and Rhubarb Combination was fed to mice, the mice’s urine output was found to increase markedly three hours later, reaching the maximum in six hours. The contents of the constituents of scutellaria are BG 102.20 μg/ml, OG n.d., WG 57.37 μg/ml, B 5.70 μg/ml, W 1.39μg/ml, and O 1.74μg/ml. Concentrations of the constituents of scutellaria in serum were found to reach the peak values three hours after feeding of the drug to the mice. (BG 1.82 μg/ml, OG n.d., WG 1.26μg/mlμg/ml, B 1.14μg/mlμg/ml, W 0.18μg/ml and O 0.47μg/ml)

並列關鍵字

HPLC ； CE ； Rhei Rhizoma ； Puerariae Radix ； Scutellariae Radix