中藥成分之定性與定量分析--訶子、黃柏、黃芩及微量元素

莊麗貞(Lih-Jeng Jan)

指導教授 ： 許順吉

國立臺灣師範大學/理學院/化學系/博士(2003年)

摘要

高效液相層析(HPLC)及毛細管電泳(CE)同屬液相分離技術，它們遵循不同的分離機制，各有其優缺點，可適用於多種類型的化合物分析，是目前最常用於定量中藥材指標成分的分析方法。本研究利用此兩分析方法分析訶子、黃柏以及黃芩藥材，整理歸納，以建立化學指紋圖譜辨識基原的方法；應用感應耦合電漿質譜儀(ICP-MS)分析防己、丹皮、厚朴之微量元素含量，以探討其比值與品種之間的關係。另外，以多變項統計分析，驗證前項辨別法則；以藥物動力學，探討不同組合三黃瀉心湯製劑中黃芩成分的生體轉化及可用率。
訶子為著名治喉痛中藥，常用作收斂劑，其主要藥效成分為多種水解型單寧。本研究以開發磷酸與氰甲烷為沖提液的高效液相層析方法，可在八十分鐘內成功分析十四個單寧指標成分；以硼酸鈉、磷酸鹽及界面活性劑配製緩衝溶液，利用MEKC毛細管電泳模式，可在四十分鐘內得到近似的分析結果。總體而言，HPLC條件在分離極性較高的小分子單寧效果較佳；CE條件則對中性溶液中能解離的化合物較理想。應用高效能液相層析法分析臺灣的市售訶子藥材，計收集長圓形的訶子(Terminalia chebula Retz.)22件及卵圓形的小花訶子(T. chebula Retz. var. parviflora Thwaites) 6件。實驗結果顯示，成分1,2,3,4,6-penta-O-galloyl-b-D-glucose與punicalagin的含量比值可用於分辨該兩品種；chebulinic acid、chebulagic acid及1,2,3,4,6-penta-O-galloyl-b-D-glucose三成分含量可用於評鑑訶子品質；而chebulagic acid與1,2,3,4,6-penta-O-galloyl-b-D-glucose的比值，則可同時用於品種辨別及品質評價。
以毛細管區帶電泳(CZE)定量黃柏的五個生物鹼，分析市售黃柏樣品28批，發現大都來自禿葉黃皮樹(Phellodendron chinense var glabrius-culum Schneid.)，與1993年的調查結果不同，本文綜合所有資料，建立臺灣黃柏(P. wilsonii Hayata et Kanehira)、日本黃柏(P. amurense Rupr. var. sachalinense Fr. Schm.)、關黃柏(P. amurense Rupr.)、川黃柏(P. chinense Schneid.)及禿葉黃皮樹等五品種的辨識方式。市售黃芩經組織鏡檢，共有黃芩(Scutellaria baicalensis Georgi)、粘毛黃芩(S. viscidula Bge.)、西南黃芩(S. amoena C.H. Wright)、連翹葉黃芩(S. hypericifolia Levl.)等四品種；本研究綜合32批黃芩樣品的六個黃酮成分分析資料，發現粘毛黃芩的oroxylin A 7-O-glucuronide含量最高為主要分辨點；家種黃芩成分與黃芩相當接近，但是baicalein 7-O-glucuronide含量較多；西南黃芩與連翹葉黃芩則明顯含有兩未知吸收峰，可有效作為辨別依據，經LC-MS檢測與文獻查考，推斷該等成分結構為5,7,2‘-trihydroxy-6- methoxy-flavone及其glucuronide。
川丹皮與西丹皮，廣防己、粉防己與日防己，川厚朴、凹葉厚朴及混淆品武當玉蘭等三類藥材共八品種，可用其藥效或特殊成分的有無或比值，明確分辨。我們用ICP-MS測定各不同品種間的水溶性微量元素含量比例，發現植物因彼此蓄積效果不同，各藥材間與各品種間確實存在不同含量的特徵元素。以十四個稀土元素(La~Lu)及Li, Be, Al, Ga, Ge, Sr, Mo, Ag, Cd, Cs, Ba, Tl等特徵元素，可分辨川丹皮與西丹皮；以稀土元素及Mn、Rb、Sr等特徵元素，可鑑別廣防己、粉防己與日防己。但同樣方法用於川厚朴、凹葉厚朴及混淆品的分辨，其效果則不盡理想。
多變項統計分析是生物資訊的重要工具之一，適用於多樣品多變數的辨識應用。我們利用群聚分析對化學成分的含量變異做解析，可協助人工比對，客觀而高效率的檢視以往的藥材分類成果；應用主成分分析，由丹皮的68個無機元素中篩選出16個特徵元素，可有效降低變數數目；另外，經由區別分析瞭解變數的特徵性，建立黃芩五品種的區別函數，其中以正品及家種黃芩的效果最好，分辨效率將近100 %；試驗結果也發現，少數關鍵性成分含量或其比值的加入，可有效幫助厚朴的基原辨識。
不同萃取方法的三黃浸膏在動物體內的吸收代謝有明顯差異。本研究以共煎、50%乙醇萃取、混合等三種三黃浸膏樣品作比較，發現餵食共煎三黃浸膏之大鼠，血清中的藥效成分濃度最高(Cmax of BG = 6.03 mg/mL, Cmax of WG = 4.38 mg/mL)，相對生體可用率最好(共煎的Frel為50%乙醇萃的三倍，為混合製劑的五倍)，且濃度時間曲線為單峰形式(Tmax =3小時)。50%乙醇萃取與共煎較類似；混合樣品則使排尿量明顯增多，尿藥回收率(WG=1.1%, B=3.2%)明顯高於共煎者(WG=0.3%, B=1.5%)，而且各成分的血藥濃度時間曲線呈雙峰現象(Tmax =1和12小時)，生體轉化機制與共煎顯著不同，推測可能是大黃的rhein或chrysphanol與黃芩藥效成分發生拮抗作用所致。本實驗數據顯示，方劑適當配伍有其意義與重要性。

關鍵字

中藥

並列摘要

Both high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) belong to the liquid-chromatography technology. They follow different separation mechanisms and have their respective advantages and drawbacks. The techniques are applicable to the analysis of various compounds and are currently the most commonly used analytical methods for assaying the marker substances of Chinese herbal medicines. In this study we applied these two methods to analyze the following Chinese herbal drugs: Chebulae Fructus, Phellodendri Cortex and Scutellariae Radix. Systematic rules for the chemical fingerprints of individual origins were established and proper flowcharts were devised to aid the identification of their botanical origins. Inductively coupled plasma-mass spectrometry (ICP-MS) was applied in assaying the contents of trace elements in water extracts of Stephaniae Cortex, Moutan Cortex and Magnoliae Cortex in order to investigate the relationship between the elemental contents and plant species. In addition, the statistical concept of multivariate analysis was employed and processing algorithms were developed to verifying the reliability of the pattern recognition rules previously deployed by human. Finally, the pharmacokinetic parameters were attempted to investigate the bio-transformation and bioavailability of the constituents of Scutellariae Cortex in the Chinese herbal formula San-Huang-Hsieh-Hsin-Tang (Coptis and Rhubarb Combination).
Chebulae Fructus, a renowned Chinese herbal remedy, is commonly used as astringents for treating sore throat and contains multifarious hydrolysable tannins. In this study we developed an HPLC method by using phosphate aqueous solution and acetonitrile as mobile phases, which could separate 14 tannin marker substances within 80 minutes. With a buffer solution comprised of sodium borate, sodium dihydrogen phosphate and surfactants, the MEKC mode of capillary electrophoresis could achieve an similar separation result within 40 minutes. Generally speaking, HPLC is better for analyzing small and polar tannins while CE is ideal for separating compounds that tend to dissociate in neutral solutions. In analyzing Chebulae Fructus samples sold in Taiwan by the HPLC method, totally 22 samples of oblong-shaped Terminalia chebula Retz and 6 samples of ovoid-shaped T. chebula Retz var. parviflora Thwaites were collected. Experimental results showed that the ratio of the contents of 1,2,3,4,6-penta-O-galloyl-β-D-glucose and punicalagin could be utilized in differentiating the two species; the contents of the three constituents, chebulinic acid, chebulagic acid and 1,2,3,4,6-penta-O-galloyl-β-D-glucose could be used to assess the quality of the herbal drug; whereas the content ratio of chebulagic acid and 1,2,3,4,6-penta-O-galloyl-β-D-glucose could be used both in identifying the species and in assessing the quality of the herb drug.
As the five alkaloids of Phellodendri Cortex were quantified by capillary zone electrophoresis, the 28 commercial samples collected in Taiwan were found to originate mostly from Phellodendron chinense var. glabrius-culum Schneid and the result is different from the previous investigation made in 1993. Therefore, in this study we combined data of two investigations and established the discernment of the five Phellodendron plants: P. wilsonii Hayata et Kanehira, P. amurense Rupr. var. sachalinense Fr. Schm, P. amurense Rupr, P. chinense Schneid and P. chinense var. Glabrius-culum Schneid. Microscopic histological examination on commercial samples of Scutellariae Radix found that most of them originated from four species, namely Scutellaria baicalensis Georgi, S. viscialula Bge, S. amoena C.H. Wright and S. hypericifolia Levl. In this study we sorted the contents of six flavones from 32 scutellaria samples and found that S. viscialula had the highest contents of oroxylin A 7-O-glucuronide which could serve as its main identifier. The cultivars of S. baicalensis had close semblance to the non-cultivated species but with higher content of baicalein 7-O-glucuronide. S. amoena and S hypericifolia exhibited two unidentified peaks which were useful for their identification and were postulated to be 5,7,2’-trihydroxy-6-methoxy- flavone and its glucuronide after LC-MS determination and thorough literature investigation.
Three different herbal drugs with 8 species in total which were Paeonia delavayi FRANCH, P. Suffruticosa ANDREWS; Aristolochia fangchi WU, Stephania tetrandra S. MOORE, Sinomenium acutumn REHD. et WILS; Magnolia officinalis REHD. et WILS., M. officinalis ssp. Biloba (REHD. et WILS.) Law, and their adulterant, M. sprengeri PAMP, could be clearly identified based on the presence or absence of efficacious or special constituents or their ratios within the plants. We used ICP-MS to determine the content ratios of water-soluble trace elements of each species, and found that different species of different plants contained special quantity of certain trace elements as an accumulation consequence of individual plant. With the 14 rare earth elements, La~Lu, and the special trace elements Li, Be, Al, Ga, Ge, Sr. Mo, Ag, Cd, Cs, Ba and Tl, we could distinguish P. delavayi from P. Suffruticosa; with the rare earth elements and Mn, Rb and Sr, we could identify Aristolochia fangchi, Stephania tetrandra, and Sinomenium acutumn. However, the method was not as effective in identifying Magnolia officinalis, M. officinalis ssp. Biloba and their adulterant, M. sprengeri.
Multivariate analysis is an important tool in bioinformatics, which is mostly suitable for the manipulation of multiple items with multiple variables. We employed cluster analysis in analyzing the variation of chemical components of different origins, which is an aid in artificial comparison. It can also inspect the correctness of previous pattern recognition work objectively and effectively. By principal component analysis, 16 significant trace elements were selected out of 68 inorganic elements in Moutan Cortex and the number of variables was greatly reduced. Consequently, through discriminant analysis, we could evaluate the eigenvalues of variables and established discriminant functions for the five species of Scutellariae Radix. The authentic and cultivated species were almost 100% non-mistakenly categorized. Test results also showed that the weighted inclusion of some key constituents and their ratios might assist in the origin identification of Magnoliae Cortex.
Scutellaria and Rhubarb Combination of different extraction methods produced marked differences in animal metabolism. In this study we prepared three different extracts of the formula by concomitant boiling(decoction), extracting with 50% ethanol(tincture) and mixing of component extracts(mixture) for comparison. It was found that the decoction produced the highest serum concentration (Cmax of BG = 6.03 mg/mL, Cmax of WG = 4.38 mg/mL), the best relative bioavailability (decoction was three times higher than that of tincture and five times higher than that of mixture) and the concentration curve appeared in the form of a single peak (Tmax = 3 hours). The tincture showed similar effects as the decoction, whereas the mixture caused marked increase in cumulative urine excretion and the drug recovery from urine (WG = 1.1%, B = 3.2%) was obviously higher than that of the decoction(WG=0.3%, B=1.5%). Moreover, the serum concentrations of each component assumed a double-peak shape(Tmax at 1 and 12 hours), suggesting a markedly different bio-transformation from that of the decoction. These significant differences were postulated to be resulted from the antagonistic interaction between rhein or chrysphanol of rhubarb and the active compounds in scutellaria. From the data of this study we may conclude that the correct combination of an herbal formula has significant and important meanings.

並列關鍵字

Chinese herbal medicines