采用超滤 HPLC-MS 筛选，溶剂和流速梯度高速逆流色谱法从翻白草中分离 α-葡萄糖苷酶抑制剂。

Separation of α-glucosidase inhibitors from Potentilla kleiniana Wight et Arn using solvent and flow-rate gradient high-speed counter-current chromatography target-guided by ultrafiltration HPLC-MS screening.

机构信息

College of Chemistry and Chemical Engineering, Central South University, Changsha, China.

Key Laboratory of Hunan Province for Water Environment and Agriculture Product Safety, Central South University, Changsha, China.

出版信息

Phytochem Anal. 2019 Nov;30(6):661-668. doi: 10.1002/pca.2839. Epub 2019 May 6.

DOI:10.1002/pca.2839

PMID:31059189

Abstract

INTRODUCTION

Potentilla kleiniana Wight et Arn is widely used as a herbal medicine to treat type 2 diabetes. However, detailed information about its active compounds is lacking.

OBJECTIVE

To develop an efficient method for the rapid screening and separation of α-glucosidase inhibitors from Potentilla kleiniana Wight et Arn.

METHODOLOGY

Potential α-glucosidase inhibitors from Potentilla kleiniana Wight et Arn were rapidly screened out through ultrafiltration high-performance liquid chromatography mass spectrometry (HPLC-MS), and then followed by a target-guided high-speed counter-current chromatography (HSCCC) separation using two-phase solvent systems composed of n-hexane/ethyl acetate/methanol/water (1:10:1:10, v/v/v/v and 1:10:5:6, v/v/v/v), and adopting increasing flow-rate from 1.5 to 3.0 mL/min after 200 min. Their structures were identified by ultraviolet (UV), MS, proton nuclear magnetic resonance ( H-NMR) and carbon-13 ( C)-NMR, and their α-glucosidase inhibitory activities were assessed by in vitro assay.

RESULTS

Five α-glucosidase inhibitors including gallic acid (25.7 mg, 98.2%, 1), brevifolincarboxylic acid (9.86 mg, 95.3%, 2), ethyl evifolincarboxylate (13.26 mg, 97.6%, 3), 3,3'-di-O-methylellagic acid-4'-O-β-d-glucopyranoside (16.26 mg, 95.1%, 4), and 3,3'-di-O-methylellagic acid (10.54 mg, 96.8%, 5) were successfully purified from 250 mg n-butanol extract in a single run. Compounds 1, 2, 4 and 5 exhibited stronger α-glucosidase inhibitory activities[half maximal inhibition concentration (IC ) values at 173.41 ± 6.35, 323.46 ± 8.08, 44.63 ± 2.50, and 20.73 ± 2.56 μM, respectively] than acarbose (IC value at 332.12 ± 5.52 μM, reference compound).

CONCLUSIONS

Notably, compounds 2-5 were reported in the Potentilla kleiniana Wight et Arn for the first time. The results indicated that the proposed method could be applied for the rapid screening and preparative separation of α-glucosidase inhibitors from a complex matrix.

摘要

简介

翻白草广泛应用于治疗 2 型糖尿病的草药中。然而，关于其活性化合物的详细信息还很缺乏。

目的

开发一种从翻白草中快速筛选和分离α-葡萄糖苷酶抑制剂的有效方法。

方法

采用超滤高效液相色谱-质谱联用（HPLC-MS）快速筛选出翻白草中的潜在α-葡萄糖苷酶抑制剂，然后采用正相高速逆流色谱（HSCCC）分离，两相溶剂系统由正己烷/乙酸乙酯/甲醇/水（1:10:1:10，v/v/v/v 和 1:10:5:6，v/v/v/v）组成，200 min 后采用 1.5-3.0 mL/min 的流速递增。通过紫外（UV）、MS、质子核磁共振（H-NMR）和碳-13（C）-NMR 鉴定其结构，并通过体外测定评估其α-葡萄糖苷酶抑制活性。

结果

从 250mg 正丁醇提取物中单次运行成功分离出 5 种α-葡萄糖苷酶抑制剂，包括没食子酸（25.7mg，98.2%，1）、短叶苏木酚羧酸（9.86mg，95.3%，2）、乙基埃夫醇羧酸酯（13.26mg，97.6%，3）、3,3'-二-O-甲基鞣花酸-4'-O-β-D-吡喃葡萄糖苷（16.26mg，95.1%，4）和 3,3'-二-O-甲基鞣花酸（10.54mg，96.8%，5）。化合物 1、2、4 和 5 对α-葡萄糖苷酶的抑制活性更强[半最大抑制浓度（IC ）值分别为 173.41±6.35、323.46±8.08、44.63±2.50 和 20.73±2.56μM]，低于阿卡波糖（IC 值为 332.12±5.52μM，对照化合物）。