具有免疫調節活性的黑豆物質之研究---萃取、純化及結構分析

Abstract

長久以來，黑大豆一直廣泛地使用於傳統醫藥中。黑大豆多醣具有免疫調節活性、抑制人類血癌細胞U937細胞生長，並誘導血癌細胞分化成正常細胞及具有活化造血系統的功能。 為了進一步的了解黑大豆多醣的免疫調節功能，本研究首先先將黑大豆磨成粉狀、再利用二氯甲烷及乙醇去除脂溶性及小分子物質，最後使用70 ℃的二次水來萃取黑大豆多醣。經過二次水的萃取、冷凍乾燥、Sephadex LH 20管柱層析及TSK HW-55F管柱層析後，再利用proteinase K將蛋白質水解，即可得到黑大豆多醣。醣的檢測部分利用酚-硫酸法，最後利用小鼠J774A.1巨噬細胞株檢測多醣的免疫調節活性。 本實驗利用氣相層析質譜儀來分析黑大豆多醣的單糖組成分，G1、G1P1及G2P1的主要單糖成分是甘露糖與半乳糖、只有含少量的葡萄糖及其他單糖，但是G2P2則以葡萄糖、半乳糖為主要單糖成分，只含少量的甘露糖。免疫調節活性方面顯示，甘露糖含量越多的多醣則免疫調節活性越佳。 經由1H-核磁共振分析G2P2之結構發現，G2P2就是水蘇醣，水蘇醣是由一個果糖、半乳糖及葡萄糖組成，經由α-1,6的鍵結將半乳糖及葡萄糖鍵結在一起，在氣相層析質譜儀也有相同的結果。比較水蘇醣及G2P2的免疫調節活性發現，G2P2有些微的免疫調節活性，但是在水蘇醣中並沒有免疫調節活性。探究其原因可能是因為G2P2含有一些可以刺激巨噬細胞分泌細胞激素的甘露糖殘留。

Black-soybean [Glycine max (L.) Merr.] has been used extensively as a healthy food and a traditional Chinese medicine for promoting good health. The related therapeutically qualities including immunomodulative activity, inhibition of human leukemic U937 cell proliferation, inducement of cell differentiation and promotion of hematopoiesis were reported from it’s polysaccharide. To further investigate the immunomodulatory property of black-soybean polysaccharides, black-soybean powder was first dissolved in dichloromethane and ethanol in order to remove the lipid-soluble and low-molecule weight components, and then extracted by water at 70 oC. The water soluble crude extracts were collected, lyophilized, further fractionated by Sephadex LH 20, TSK HW-55F chromatographies and deproteinized by proteinase K. The fractions containing polysaccharides were detected by phenol-sulfuric acid and the immuno-stimulatory activities were conducted in vitro by mouse J774A. 1 macrophage cell line. The carbohydrate composition analysis of crude black soybean extract was analyzed by Gas Chromatography-Mass Spectroscopy (GC-MS). Our result suggests that the monosaccharide composition of G1, G1P1, and G2P1 all pinpointed mannose and galactose as the major components together with smaller components of other sugars; G2P2 are composed of glucose and galactose as major and minor mannose. With regard to imuno-modulative activities, the more mannoses the better the activity. Through NMR, the structure of G2P2 is determinated as stachyose that contains one fructose, one glucose and two of galactose. The linkage of G2P2 was terminal- fructose, terminal-hexose and two of α-1,6-hexose that determinated by Gas Chromatohraphy-Mass Spectroscopy (GC-MS), and the data was similar to NMR. Comparing G2P2 and stachyose immuno-modulative activity we found that G2P2 had immuno-modulative activities depending on dose-dependent, but stachyose were bio-inactive. The reason for immuno-modulative activity was that G2P2 was contaminated by mannose residue that can interact with macrophage and induce the cytokines secreted.