Extraction, Purification, Prebiotic Properties And Immunoregulation Activity Of Verbascose From Mung Bean

Verbascose, which has three a-D-galactosyl residue linked(1â†'6) to the D-glucosyl residue of sucrose, is a member of raffinose family and distribute in legume seeds. There are two ways to prepare verbascose, one method is enzymatic synthesis, another is extracted from natural plants. As a kinds of a-galactooligosaccharide, recent report about verbascose were mainly concentrated on its composition, separation, structure analysis, and there is hardly any report about active substances. In this thesis, verbascose was studied systemically, including isolation and purification from mung bean, probiotic properties to intestine and immunomodulatory activity. Main results are listed as follows:1Isolation and purification of verbascose from mung beanSoluble sugar was extracted from mung bean, then the extracting solution was isolated and purificated through activated carbon-diatomate (1:1, w/w) medium pressure chromatography column, sugar content was analyzed by HPLC. Calculation according to per gram of mung bean, composition of soluble sugar are fructose2.53mg/g, glucose1.82mg/g, sucrose17.12mg/g, melibiose4.32mg/g, raffinose2.58mg/g, stachyose13.72mg/g, verbascose25.25mg/g, Totle67.34mg/g; the percentage is fructose3.76%, glucose2.70%, sucrose25.42%, melibiose6.42%, raffinose3.84%, stachyose20.37%, verbascose37.50%. verbascose was purificated through activated carbon-diatomate (1:1, w/w) column and analyzed by HPLC, its purity is93.85%.2Study on Probiotic properties to intestine of verbascoseAnaerobic fermentation cultivation was carried out for verbascose and α-galactooligosaccharide of polymerization degree prepared by anaerobe faecal fermentation in vitro, treatment without verbascose or α-galactooligosaccharide was used as the control, and fructooligosaccharide was used as the positive control. The change of the amount of beneficial bacterium(bifidobacterium, lactobacillus), harmful bacterium (clotridium, bacteroides), total bacterial were detected by fluorescent in situ hybridization. Results show that verbascose has a significant influence on the growth of bifidobacterium and lactobacillus and a negtive effect on the growth of clotridium, bacteroides, the result is best at24h. In addition, the variation of different short chain fatty acid in the fermentation process was detected by HPLC. After verbascose was used by intestinal microorganism, the most is Acetic acid, then Lactic acid and Formic acid, last were Proponic acid and Butyric acid. Contrast with control, Acetic acid, Lactic acid, Formic acid and Butyric acid increase obsservably, on the contrary, Proponic acid decrease. This trend exerted dose and time-dependent effect on acid, and the effect is the most obvious at24h..3Study on Immunomodulatory activity of verbascoseImmunomodulating activities in vitro of verbascose were evaluated by using the macrophage cell line of RAW264.7. Results showed that verbascose could promote proliferation of RAW264.7cells, could enhance the ability of devouring neutral red of peritoneal macrophages, could promote the release of NO and the immune reactive molecules such as IL-6, IL-1β, IFN-a, IFN-y. Verbascose at a dose200μg/mL showed the best function.Immunomodulating activities in vivo of verbascose were measured by using the animal model experiments. Results showed that verbascose could significantly increase the index of spleen and thymus, activity of LZM in spleen and serum, hemolysin levels in serum and the swelling rate of ear in DTH of immune suppress mice. These results suggested that verbascose could improve specific immune ability and nonspecific immunity of Cy-immunosuppressed mice.