赤芍之高極性化學成分及降血糖多酚之研究

Chemical Investigation of Polar Constituents and Hypoglycemic Polyphenols from Paeoniae Rubra Radix

王智恆(Chi-Heng Wang)

指導教授 ： 張嘉銓

國立臺灣大學/醫學院/藥學研究所/碩士(2016年)

https://doi.org/10.6342/NTU201603104

摘要

赤芍(Paeoniae Rubra Radix)為芍藥科 (Paeoniaceae) 芍藥屬 (Paeonia) 之植物的帶皮乾燥根，是傳統中藥中的一種重要草藥，常用於治療糖尿病之方劑中。赤芍的乙醇萃取物經液─液萃取以極性劃分成正己烷、二氯甲烷和水可溶部分，水可溶部分進一步利用Sephadex LH-20、silica gel、Lobar RP-18和半製備HPLC等管柱分離純化，化合物結構透過NMR與質譜技術判定。共計得到10個化合物，包括5個單萜類 (monoterpenoids) 成分：paeoniflorin (1) 、 oxypaeoniflorin (2)、benzylpaeoniflorin (3)、4-O-methyl paeoniflorin (4)、paeonidanin (5)、 1個黃酮類 (flavonoid) 成分：ent-catechin (6); 1個苷類 (glycoside) 成分：1,2,3,4,6-penta-O-galloyl-β-D-glucopyranose (7); 以及3個苯甲酸類 (benzoic acids) 成分：benzoic acid (8)、4-hydroxybenzoic acid (9)、vanillic acid (10) 。其中化合物7屬於可水解單寧類 (hydrolyzable tannin)，具有擬胰島素 (insulin－mimetic) 的活性1，能使胰島素受器磷酸化而活化胰島素訊號傳遞路徑，達到降低血糖的效果，因此具有極高的藥物發展潛力。此篇研究中化合物7由赤芍之分離方法，較其他種類或分離方法4, 66, 67提升了赤芍中取得化合物7的量高1.6~4.1倍。本研究使用之分離方法可進一步研究以對可水解單寧之分離純化有更多進展。
多酚類成分常見於天然植物中，屬於次級代謝物，在抵抗外部病菌侵入發揮了重要功用，因此具有極高的藥物研發潛力。本研究探討赤芍乙醇萃取物經高多孔性樹脂Diaion HP20吸附後，以極性切割劃分為乙酸乙酯可溶部分、水可溶部分以及沉澱物NPF (non－PGG fraction)。NPF經過三氟乙酸進行酸水解反應後，以液─液萃取法將水解產物為乙酸乙酯及水可溶部分。將兩部分與縮合單寧、可水解單寧分別進行顯色反應以比較其特性，在使用三氯化鐵 (FeCl3, 1%)、溴水 (Br2, 3%)、四乙酸鉛 [Pb(C2H3O2)4, 1%] 及硫酸亞鐵胺 [FeNH4 (SO4)2, 1%] 等試劑反應後，得到此NPF之化學性質與縮合單寧有類似之實驗結果，以香草醛─鹽酸法檢驗不同批次生產之NPF，判斷含縮合單寧比例分別為25.8%與32.1%。其後以兩種硫化物試劑反應後，確立了以苄硫醇(benzyl mercaptan) 試劑進行聚合性多酚之硫解反應，可得到具再現性之液相層析圖譜，並以Lobar B type製備管柱純化，得到T−1、T−2、T−3、T−4、T−5五個化合物，其中T-2之化學結構以NMR與質譜技術判定，推測為(−)-epicatechin 4β-benzylthioether，可得知NPF之部分縮合單寧結構。

關鍵字

赤芍 ； 多酚 ； 降血糖 ； 五?食子醯葡萄糖 ； 單寧

並列摘要

Paeoniae Rubra Radix (PRR), the dried root with bark of Paeoniaceae, is an important traditional Chinese medicine often used in anti-diabetic drugs. Ethanol extract of PRR was divided into fractions soluble in n-hexane, dichloromethane (DCM) and water via liquid−liquid partitioning process, The water-soluble fractions was separated by chromatographic methods including Sephadex LH-20, Silica gel, Lobar RP-18 column and semi-preparative HPLC to afford 10 compounds (1-10).
These compounds were characterized as five monoterpenoids: paeoniflorin (1), oxypaeoniflorin (2), benzoylpaeoniflorin (3), 4-O-methylpaeoniflorin (4), paeonidanin (5); one flavonoids: ent-catechin (6); one glycoside: 1,2,3,4,6-penta-O-galloyl-β-D-glucopyranose (7); and three benzoic acid derivates: benzoic acid (8), 4-hydroxybenzoic acid (9), vanillic acid (10).
In terms of its anti-diabetic activity, Compound 7 have insulin-mimetic activity1, which enables the phosphorylation of insulin receptors, and activate the insulin signal transmission path, thereby achieve the function of lowering blood sugar, thus, Compound 7 has high potential for drugs development. The method of extraction and isolation in this study4, 66, 67 enhance the yield of 7 about 1.6~4.1 times from peony plants, this method is expected to further develop for hydrolyzed tannin purification.
Polyphenols are common second metabolites found in a number of plants, These compounds play important roles against herbivory and infections of bacteria, and therefore being high potential for the development of anti-diabetic drug. This study was to evaluate the dried Paeoniae Rubra Radix ethanol extracts, after adsorbed with high porous Diaion HP20, the Diaion HP20 absorbate were suspended in water and partitioned with ethyl acetate, then divided into fractions soluble in EtOAc, water and the precipitate via liquid−liquid partitioning process. The precipitate was hydrolyzed by trifluoroacetic acid, and divided hydrolysates into water-soluble fraction and ethyl acetate fration, then the two fractions and condensed tannins, hydrolyzable tannins were subjected to colormetric analysis to compare its chemical characteristic, the reagents include: ferric chloride (FeCl3, 1%), bromine (Br2, 3%), lead(IV) acetate [Pb(C2H3O2)4, 1%] and iron(II) ammonium sulfate, the results show that the precipitate has similar chemical characteristics to condensed tannins in this experiment, and the results of Vanillin−HCl assay, examing two different batch of NPF, show 25.8%, 32.1% of the content of condensed tannin respectively. Followed by acid hydrolysis with different concentration and two thiolysis reagent, the method was established by using benzyl mercaptan to thiolyze the polymeric polyphenols, and the liquid chromatography spectrum of the thiolysate shows high reproducibility, after that, use Lobar RP-18 B type to separate thiolysate of the polyphenols to afford 5 compounds: T−1、T−2、T−3、T−4、T−5. Wherein T-2 was analysed by NMR and mass spectrometry, the chemical structure is presumed to be (−)-epicatechin 4β -benzylthioether, that can be part of the NPF condensed tannin structure.

並列關鍵字

Paeoniae Rubra Radix ； polyphenols ； Hypoglycemic ； 1,2,3,4,6-O-petna-galloyl-β-D-glucopyranose ； tannin

參考文獻

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