Preparation Of Gracilaria Lemaneiforms Polysaccharides And Its Hyperglycemic Activities In STZ-induced Diabetes Mice

Gracilaria lemaneiformis, which belongs to the family of Gracilaria, is widely consumed as a healthy food and traditional Chinese medicine(TCM) in China. Phytochemical studies have demonstrated that Gracilaria lemaneiformis contains various active ingredients,of which polysaccharide is one of the main active ingredients. It has been reported that Gracilaria lemaneiformis polysaccharide has many effective bioactivities, such as antioxidant,antitumor and immunomodulatory activities et al. In the present study, systematic studies about the extraction, isolation and purification, physicochemical property, hyperglycemic effects in diabetes mice and the hyperglycemic mechanism of Gracilaria lemaneiformis polysaccharide were carried out. Main results are listed as follows:(1) Four polysaccharides(GWP, GUP, GUWP and GCP) were obtained from Gracilaria lemaneiformis by hot water, ultrasonic wave, ultrasonic-hot water and citric acid,respectively. And their properties were compared in this paper. Citric acid extraction method was selected for further research for GCP has a better biological activity. The optimal conditions of citric acid extraction(p H: 2.0; extraction temperature: 100 °C; extraction time:2 h;solvent-material ratio: 50:1 m L/g) were determined. Under this condition, the polysaccharide yield was 17.81±0.34% with a molecular weight of 21.17 KDa, and the sulfated group content was 19.64±1.0%. GCP exhibited the strongest antioxidant ability. Its oxygen radical absorbance capacity was 298.735±6.57 μmol Trolox/g. At 2.0mg/m L, the DPPH free radical scavenging activity and the reducing power were 65.79±0.3% and0.765±0.01, respectively. In addition, the result of α-amylase inhibition assay revealed that GCP can act as potent α-amylase inhibitor.(2) The anti-diabetic activity of GCP in streptozotocin-induced diabetic mice was investigated. The results showed that GCP could decrease blood glucose level, enhance glucose tolerance, improve polydipsia 、 polyphagia 、 polyuria and slim down. Furthermore,GCP could antagonize enlargement of liver and kidney and atrophy of thymus and spleen in diabetes mice. It could be concluded that GCP has hyperglycemic effect and immune-enhancing activity in diabetes mice to some extent.(3) Hyperglycemic mechanism of GCP from the glucolipid metabolism was investigated. The results showed that GCP could significantly improve the lipid metabolism in diabetes mice. The mice serum TC、TG and LDL-c contents were decreased dramatically and the HDL-c content has a great promotion after administration of GCP for 6 weeks. GCP(225mg/kg/d) could increase the insulin secretion and hepatic glycogen content in diabetic mice,and enhance the enzyme activities of GCK and G-6-PD which play critical roles in glucose metabolism. GCP could also decrease the activity of G-6-Pase, thus inhibit gluconeogenic and glycogenolytic pathways.(4)Hyperglycemic mechanism of GCP from the antioxidant effect was investigated.The results showed that GCP could significantly dectease MDA level, while increase enzyme activities of SOD, CAT and GSH-Px evidently. GCP could remarkably suppress the increase of the activities of ALT 、 AST and content of BUN simultaneously. It was reasonable to suggest that GCP has a positive effect on oxidative stress in diabetes mice.(5)GCP was purified by DEAE-Sepharose Fast Flow chromatogram to obtain GLP-1、GLP-2、GLP-3、GLP-4 and GLP-5. The physical and chemical properties, antioxidant activity,hypoglycemic activity and structural differences were systematically compared. GLP-3 has comparatively higher antioxidant activity, both GLP-3 and GLP-5 have significant effect on glucose consumption in IR-Hep G2. No sulfated group was detected in GLP-1 and GLP-2,GLP-3 has a relative higher content of uronic acid(galacturonic acid content is 9.57%) and a small quantity of sulfated group(2.55%), GLP-4(22.84%) and GLP-5(24.31%) are riched in sulfated group. The monosaccharide composition of GLP-1 was quite different from those of others. GLP-1 was totally composed of glucose(90.76%) almostly; GLP-3 was composed of galactose, glucose, rhamnose, arabinose, xylose and mannose with a molar ratio of3.93:1.84:1:0.03:1.61:0.14. No arabinose and mannose was detected in GLP-4 and GLP-5.Periodate oxidation analysis showed that the five fractions were contained a large amount of(1→3) linkages except for GLP-5, while GLP-2 has a relatively higher content of(1→) or(1→6) linkages(42.8%). The content of(1→2)or(1→4) and(1→3) linkages was 78.65%,12.51%,respectively. The IR spectrum analysis showed that typical polysaccharide absorption peaks were exhibited clearly in the five polysaccharide fractions. Wherein,absorption peak at 858 cm-1 was detected in GLP-3,GLP-4 and GLP-5, which representing a symmetrical C-O-S vibration associated with the sulfated group in C4 site substitution; Both GLP-3 and GLP-5 have absorption peak at 818 cm-1 which represent the C6 site substitution of sulfated group. The difference of uronic acid content and the site of sulfated group substitution may contribute to the biological activities of GLPs.