香苦草抑制黃嘌呤氧化酶活性成分之研究及花生衣原花青素之成分研究

Abstract

第一部份 香苦草抑制黃嘌呤氧化酶活性成分之研究 痛風是一種由尿酸結晶堆積於關節而造成的代謝性疾病；其中，黃嘌呤氧化酶在尿酸的形成中扮演很重要的角色，故臨床上常使用其抑制劑allopurinol來做為長期治療的用藥。然而，此藥物可能引發嚴重的過敏反應Stevens-Johnson syndrome，所以找尋治療痛風的替代藥物仍屬必須。根據本實驗室過去的研究，頭花香苦草(Hyptis rhomboids)中具有抑制黃嘌呤氧化酶的成分。因此，本研究旨在探索同屬物種的香苦草(H. suaveolens)中是否同樣具有抑制黃嘌呤氧化酶的成分。 香苦草莖部的乙醇萃取物經極性切割後劃分為二氯甲烷、乙酸乙酯、正丁醇和水可溶部分，再進一步對活性較佳的乙酸乙酯和正丁醇可溶部分進行分離。過程中使用Sephadex LH-20、silica gel和逆相層析管柱、離心式分配層析，以及半製備HPLC等，共計得到17個化合物，經由核磁共振光譜及質譜技術分析確認dimethyl melitrate A (9)和9'-O-methyl melitrate A (10)為新的化合物。其餘15個化合物為ursolic acid (1)、caffeic acid (2)、rosmarinic acid (3)、methyl rosmarinate (4)、kaempferol 3-O-(4-O-acetyl-alpha-L-rhamnopyranoside) (5)、oresbiusin A (6)、danshensu (7)、methyl melitrate A (8)、netpetoidin B (11)、melitric acid A (12)、sagecoumarin (13)、netpetoidin A (14)、apigenin-6,8-di-C-glucoside (15)、adenosine (16)和rutin (17)。最後，經由黃嘌呤氧化酶抑制活性測試，發現netpetoidin B (11)具有最佳抑制活性，與本實驗室過去結果一致(IC50 11.7 ± 2.5 uM vs. allopurinol 5.3 ± 0.6 uM)。 第二部分 花生衣原花青素之成分研究 糖尿病是一種代謝性疾病，其中，又以第二型糖尿病為主，佔患者總數的90%，主要致病原因可能為胰島素感受性下降。在臨床治療上，常使用以metformin為基礎的處方，再依照病情加入其他用藥，甲型葡萄糖抑制劑便是其中一項。根據電腦分子模擬發現，部分A-type dimeric proanthocyanidins對甲型葡萄糖水解酶具有良好的結合能力，而該化合物曾被報導富含於花生衣中。因此，本研究計畫以花生衣(Arachis hypogaea L.)作為A-type proanthocyanidins的來源，進行化合物結構與甲型葡萄糖水解酶抑制活性間的探討，並期望藉此賦予低經濟價值的花生衣新的再利用途徑。 花生衣的乙醇萃取物經二氯甲烷、乙酸乙酯、正丁醇和水進行極性切割，共劃分為四個可溶部分。取乙酸乙酯可溶部分經Sephadex LH-20做初步分離，利用proanthocyanidins在anisaldehyde顯色後呈現深紅色的特性，並透過ESI-MS尋找對應的分子量作為進一步分離的依據。過程中使用離心式分配層析(centrifugal partition chromatography, CPC)、逆相層析管柱及半製備HPLC等，共計得到13個化合物，經核磁共振光譜及質譜技術分析確認化合物結構為(+)-catechin (18)、(-)-epicatechin (19)、cinnamtannin D1 (20)、proanthocyanidin A1 (21)、proanthocyanidin A4 (22)、proanthocyanidin A2 (23)、epicatechin-(2beta→O→7,4beta→6)-catechin (24)、procyanidin B3 (25)、procyanidin B4 (26)、epicatechin-(2beta→O→7,4beta→8)-ent-epicatechin (27)、epicatechin-(2beta→O→7,4beta→6)-ent-catechin (28)、proanthocyanidin A6 (29)、epicatechin-(2beta→O→7,4beta→6)-ent-epicatechin (30)。另外，經甲型葡萄糖水解酶抑制活性測試發現，化合物24具有最佳抑制活性(IC50 9.7 ± 0.2 uM vs. acarbose 0.023 uM)。

Part 1. Chemical investigation of Hyptis suaveolens guided by xanthine oxidase inhibitory activity Gout is a kind of metabolic diseases, caused by accumulation of uric acid crystals in the joints. Xanthine oxidase plays an important role in the production of uric acid. Thus, its inhibitor, allopurinol, is often used clinically for long-term gout therapy. This drug, however, might cause Stevens-Johnson syndrome. Therefore, the search for alternative drugs for treating gout is still in need. Our recent studies indicated that some constituents from Hyptis rhomboides are potent xanthine oxidase inhibitors. Thus the aim of this research was to investigate whether the chemical constituents of the related native species, H. suaveolens, possess similar activity. The ethanol extract of H. suaveolens stem was divided into fractions soluble in CH2Cl2, EtOAc, n-BuOH, and water via liquid-liquid partitioning. Further separation was conducted on the EtOAc and n-BuOH-soluble fractions, which possess better anti-xanthine oxidase activity among four fractions. This effort led to the isolation of 17 compounds by combination of Sephadex LH-20, silica gel and reverse-phase column chromatography, centrifugal partition chromatography, and semi-preparative RP-HPLC. Of these, two are new, i.e., dimethyl melitrate A (9) and 9'-O-methyl melitrate A (10). The 15 known ones were identified as ursolic acid (1), caffeic acid (2), rosmarinic acid (3), methyl rosmarinate (4), kaempferol 3-O-(4-O-acetyl--L-rhamnopyranoside) (5), oresbiusin A (6), danshensu (7), methyl melitrate A (8), netpetoidin B (11), melitric acid A (12), sagecoumarin (13), netpetoidin A (14), apigenin-6,8-di-C-glucoside (15), adenosine (16), rutin (17). Their structures were elucidated by MS data and NMR (1D and 2D) spectroscopic analysis. Through xanthine oxidase bioassay, netpetoidin B (11) with IC50 11.7 ± 2.5 uM was found to possess the best inhibitory activity. Part 2. Chemical investigation of proanthocyanidins from peanut skin Diabetes mellitus (DM) is a kind of metabolic disorder. Type 2 DM, which accounts for 90% DM patients, might cause by the decrease of insulin sentivity. Metformin-based regimen was usually used to treat this disorder and the other drugs, such as -glucosidase inhibitor, were added depending on the personal condition. According to the molecular docking, some A-type proanthocyanidins, which are rich in peanut skin, show good docking score toward alpha-glucosidase. As a result, such type compounds were isolated from peanut skin and assayed against alpha-glucosidase to confirm the result of this virtual screening. The ethanol extract of peanut skin was divided into fractions soluble in CH2Cl2, EtOAc, n-BuOH, and water via liquid-liquid partitioning. The EtOAc-soluble fraction was chromatographed over Sephadex LH-20, centrifugal partition chromatography, reverse-phase columns, and semi-preparative RP-HPLC, guided by ESI-MS and TLC, showing red color for dimeric proanthocyanidins while spraying with anisaldehyde reagent. This effort led to the isolation of 13 compounds. Based on 1H and 13C NMR analyses and MS data, they were identified as (+)-catechin (18), (-)-epicatechin (19)、cinnamtannin D1 (20), proanthocyanidin A1 (21), proanthocyanidin A4 (22), proanthocyanidin A2 (23), epicatechin-(2beta→O→7,4beta→6)-catechin (24), procyanidin B3 (25), procyanidin B4 (26), epicatechin-(2beta→O→7,4beta→8)-ent-epicatechin (27), epicatechin-(2beta→O→7,4beta→6)-ent-catechin (28), proanthocyanidin A6 (29), epicatechin-(2beta→O→7,4beta→6)-ent-epicatechin (30). Of these, epicatechin-(2beta→O→7,4beta→6)-catechin (24) with IC50 9.7 ± 0.2 uM was found to possess the best inhibitory activity against alpha-glucosidase.