第一部分：苦藤葉部抑制甲型葡萄糖水解酶活性成分之研究 第二部分：柳葉石櫟葉部成分之研究

Part 1 α-Glucosidase inhibitors from Tinospora crispa leaves Part 2 Chemical investigation of Pasania dodoniifolia leaves

張吉智(Chi-Chih Chang)

指導教授 ： 李水盛

國立臺灣大學/藥學專業學院/藥學研究所/博士(2015年)

https://doi.org/10.6342/NTU.2015.10236

摘要

第一部分 苦藤葉部抑制甲型葡萄糖水解酶活性成分之研究
苦藤Tinospora crispa (Linn.) Hook. f. et Thoms.為防己科青牛膽屬植物，帶有瘤狀莖的藤蔓，廣泛分布於亞洲熱帶與亞熱帶區域，包含印度、泰國、越南、菲律賓、印尼與馬來西亞等國家，其乾燥莖作為民俗療法應用於許多方面，並被證實具有抗糖尿病的活性。我們實驗室最近的研究發現，從莖部分離出來的活性成分，borapetosides A 與C具有降血糖之功效，其作用機轉與活化胰島素之訊息傳遞相關。延續這部分的工作至葉部，其成分還未被報導，同時本研究發現葉部的乙醇萃取物，具有抗甲型葡萄糖水解酶的活性，由於此標的抑制劑可用於治療第二型糖尿病，本研究擬揭示苦藤葉部這類活性成分。
苦藤葉部之乙醇萃取物具有抗甲型葡萄糖水解酶的活性，以生物活性為導向對此活性萃取物進行成分分離純化，藉助於HPLC-SPE-NMR連結技術，最後得到十七個黃酮類成分，包含了八個黃酮醯化苷(6-11, 13, 15)，其中isoorientin-2'-(E)-sinapate (6)、isovitexin-2'-(E)-p-coumarate (8)、cosmosiin- 6'-(E)-ferulate (9)與cosmosiin-6'-(E)-cinnamate (15)為新化合物，它們的結構解析乃依據核磁共振及質譜光譜分析。酵素活性抑制試驗顯示，isovitexin- 2'-(E)-p-coumarate (8)具最強的抑制效果，其IC50值為4.3 ± 1.4 μM；然而isoorientin- 2'-(E)-p-coumarate (7)，為3′位置羥基化，活性大幅降低(IC50: 35.7 μM)。其他cosmosiin之醯化衍生物，包含6'-(E)-ferulate (9)、6'-(E)-p-coumarate (10)、6'-(Z)-p-coumarate (11)與 6'-(E)-cinnamate (15)，皆具有抑制活性，其IC50值分別8.8、14.6、10.1與 11.3 μM。

第二部分 柳葉石櫟葉部成分之研究
柳葉石櫟Pasania dodoniifolia Hayata為殼斗科柯屬植物，常綠中型喬木，高度可達9公尺，直徑約10公分，屬於台灣特有植物，分布於台灣南部中低海拔，在我們研究之前，還未有相關的成分報導，因此本研究擬研究其化學組成。
柳葉石櫟(Pasania dodoniifolia Hayata)葉部之乙醇萃取物，被劃分成二氯甲烷、乙酸乙酯、正丁醇與水可溶等部分，乙酸乙酯可溶部分進一步使用離心分配層析(CPC)、Sephadex LH-20管柱層析與RP-18管柱層析，等等一系列的分離純化後，得到 kaempferol-3-O-peracylated glucosides (18-21)、(2S)-6-C-β-glucopyranosyl-naringenin (22)、(2R)-6-C-β-glucopyranosyl naringenin (23)、(2S)-8-C-β-glucopyranosyl naringenin (24)、(2R)-8-C-β-glucopyranosyl naringenin (25)、(–)-epicatechin (26) 與 (7S,7'S,8R,8'R)-icariol A2 (27)。化合物22~27絕對立體構型的確認，係由所測得的CD圖譜與參考文獻進行比對。在這些化合物中，kaempferol-3-O-(3',4'-di-O-acetyl-2'-O-(Z)-p-coumaroyl-6'-O-(E)-p-coumaroyl)-β-glucopyranoside (20) 與 kaempferol-3-O-(3',4'-di-O-acetyl-2',6'-di-O-(Z)-p-coumaroyl)-β-glucopyranoside (21)為新化合物，其結構係由二維核磁共振光譜分析與質譜解析而確定。

關鍵字

苦藤 ； 甲型葡萄糖水解酶抑制劑 ； 黃酮醯化苷 ； 柳葉石櫟 ； 葉部 ； 黃酮類

並列摘要

PartⅠ: α-Glucosidase inhibitors from Tinospora crispa leaves
Tinospora crispa (Linn.) Hook. f. et Thoms. (Menispermaceae) is a vine with warty stem and is widely distributed over tropical and subtropical Asia, including India, Thailand, Vietnam, Philippines, Indonesia, and Malaysia. The dried vines of this plant have been used as a folk medicine for many purposes and demonstrated to possess inhibitory activity against diabetes. Recent studies in our lab found borapetosides A and C, isolated from this vine, to be the active constituents possessing hypoglycemic activity with the mechanism of action related to activation of the insulin signaling pathway. Extension of such work on the leaf part, whose constituents have not been reported yet, found the ethanolic extract to be active against α-glucosidase, one of the therapeutic targets for type-2 diabetes mellitus. Thus, this study was aimed to disclose the active constituents from the leaf part of this folk plant.
The ethanolic extract of Tinospora crispa leaves had shown inhibitory activity toward α-glucosidase. Bioassay guided fractionation and separation of this active extract with the aid of HPLC-SPE-NMR led to the isolation of 17 flavonoids, including 8 acylated glycosylflavonoids (6-11, 13, 15). Among them, isoorientin-2'-(E)-sinapate (6), isovitexin -2'-(E)-p-coumarate (8), cosmosiin-6'-(E)-ferulate (9), and cosmosiin-6'-(E)-cinnamate (15) are new compounds. Their structures were elucidated on the basis of NMR and mass spectroscopic analyse. From the enzyme inhibitory assay, isovitexin-2'-(E)-p-coumarate (8) showed the best activity against α-glucosidase with an IC50 value of 4.3 ± 1.4 μM. Isoorientin-2'-(E)-p-coumarate (7), the 3′-hydroxylated 8, however is much less active (IC50: 35.7 μM). Other acylated derivatives of cosmosiin, including
6'-(E)-ferulate (9), 6'-(E)-p-coumarate (10), 6'-(Z)-p-coumarate (11), and 6'-(E)-cinnamate (15), also exhibited inhibitory activities with the IC50 value of 8.8, 14.6, 10.1, and 11.3 μM, respectively.

PartⅡ: Chemical investigation of Pasania dodoniifolia leaves
Pasania dodoniifolia Hayata (Fagaceae), a medium-sized evergreen tree up to 9 m high and 10 cm in diameter, is an endemic species to southern Taiwan from medium to low attitude. Before our study, no chemical information about this plant had been reported. Thus, this study was aimed to investigate its chemical constituents.
The ethanolic extract of its leaf was divided into fractions soluble in CH2Cl2, ethyl acetate, n-BuOH, and H2O. The EtOAc-soluble fraction was further chromatographed on a centrifugal partition chromatograph (CPC), Sephadex LH-20, and RP-18 columns to give four kaempferol 3-O-peracylated glucosides (18−21) in addition to (2S)-6-C-β-glucopyranosylnaringenin (22), (2R)-6-C-β-glucopyranosyl naringenin (23), (2S)-8-C-β-glucopyranosyl naringenin (24), (2R)-8-C-β-glucopyranosyl naringenin (25), l-epicatechin (26), and (7S,7'S,8R,8'R)-icariol A2 (27). The absolute configurations of compound 22~27 were determined by comparing their CD data with those reported. Among the isolated compounds, kaempferol-3-O-(3',4'-di-O-acetyl-2'-O-(Z)-p-coumaroyl-6'-O-(E)-p-coumaroyl)-β-glucopyranoside (20) and kaempferol-3-O-(3',4'-di-O-acetyl-2',6'-di-O-(Z)-p-coumaroyl)-β-glucopyranoside (21) are new compounds and their structures were elucidated by 2D NMR spectroscopic analyses and MS data.

並列關鍵字

Tinospora crispa ； α-Glucosidase inhibitors ； Acylated glucosylflavones ； Pasania dodoniifolia ； leaves ； flavonoids

參考文獻

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