| Objectives: Arginyl-fructosyl-glucose(AFG) was considered to be formed through the early step of Maillard reaction from arginine and maltose. A new, simple, rapid and sensitive method for the simultaneous determination of two Amadori compounds(i.e., arginyl-fructose(AF) and arginyl-fructosyl-glucose(AFG)) in extracts of three types of ginseng preparations was developed. Arginyl-fructosyl-glucose(AFG) was orally administered to mice for evaluation of its effect on cyclophosphamide(CTX)-induced immunosuppression. Although antihyperglycemic activity of AFG has been reported in recent years, the amelioration effect of AFG on type 2 diabetes mellitus(T2DM) remains unclear. For this purpose, the research was performed.Methods: Ten BALB/C mice were killed, and each spleen was removed, and the spleen lymphocytes transformation was stimulated with different concentrations AFG in vitro. And there are another eighty mice, the animals were divided randomly into eight groups of 10 mice. Groups I(normal control) and II(immunosuppression model)received normal saline(NS) solution(0.9%, w/v). Groups M+AFG-H, M+AFG-M and M+AFG-L received AFG at dosages of 50, 30 and10 mg/kg bw, respectively; Groups N+AFG-H, N+AFG-M and N+AFG-L also received AFG at dosages of 50, 30 and10 mg/kg bw, respectively. Groups M+AFG-H, M+AFG-M and M+AFG-L were treated with CTX(80 mg/kg bw) on days 1,2,3,9,16,23 and 30. Determine the effect of AFG on lymphocytes transformation of BALA/C mice in vitro mice. After treatment with AFG for 30 days, the animals were killed, and various immune indices, such as thymus and spleen indices, activity of lymphocyte transformation, tumor necrosis factor-α(TNF-α)and interleukin-2(IL-2) content in serum, were measured. The separation of the two target analytes was efficiently performed on a Prevail C18 column(4.6 mm×250 mm, 5μm) at a flow rate of 0.8 m L/min in a single 15 min chromatographic run. Under the optimized conditions, the detection limits were 0.015 and 0.020 mg/ml for AF and AFG, respectively. T2 DM was induced in mice by high fat diet(HFD) and streptozotocin(STZ). Group 1(n = 10, CON) and Group 2(n = 80) were fed regular chow and HFD, respectively. After 4 weeks of HFD feeding, group 2 was injected with STZ at a dose of 100 mg/kg and tested fasting blood glucose(FBG) level for 4 weeks post-injection. Then, mice with FBG level above 7.8 mmol/L were divided into 5 groups(n=10) and treated with vehicle, metformin, 50, 30 and 10 mg/kg of AFG for 4 weeks, respectively, and CON was treated with vehicle.Results: The calibration curves of the peak area for the two analytes were linear over three orders of magnitude with correlation coefficients greater than 0.999. The relative standard deviations(RSD) of precision, recovery, reproducibility and stability were 0.43% and 0.37%, 99.5% and 100.9%, 0.46% and 0.43% and 0.41% and 0.49% for AF and AFG, respectively. The content of AF in Korean ginseng, Chinese red ginseng and sun-cured ginseng was 0.75%, 0.93% and 1.14%, and the content of AFG in Korean ginseng, Chinese red ginseng and sun-cured ginseng was 6.05%, 5.08% and 0.85%, respectively. Thymus and spleen indices were significantly higher in mice treated with every dose of AFG(P<0.01, P<0.05). The function of AFG in promoting the spleen lymphocyte transformation in vitro was enhanced with increasing concentration, when administered at a dose of 1-10μg / ml, the conversion rate was concentration-dependent. AFG-H and AFG-L can promote the spleen cells transformation significantly(P<0.01, P<0.05); T lymphocyte and B lymphocyte were transformed in mice which were oral high dose AFG-H(P<0.01, P<0.05), and mice treated with AFG-L and AFG-H were also have a significant(P<0.01, P<0.05) higher conversion rate of B lymphocyte compared with immunosuppression model. ELISA experiments showed every dose of AFG treatment enhanced production of TNF-α(P<0.01, P<0.05)and oral AFG-H and AFG-M can enhanced production of IL-2(P<0.01, P<0.05).Orally treatment with AFG resulted in the decrease of malondialdehyde, total cholesterol, total triglyceride, food and water intake and increase of body weight, superoxide dismutase and insulin. The histological examination and immunohistochemistry indicate that the hypoglycemic and insulin-sensitizing capability of AFG in diabetic mice were enhanced via improving the destruction of the pancreatic islets.Conclusions: This method was successfully applied to the quantification of AF and AFG in red ginseng, and the method is efficient, sensitive and accurate. Arginyl-fructosyl-glucose displayed strong immune enhancing activity and a protective effect against CTX-induced immunosuppression. Compared to other hypoglycemic agents and insulin, AFG treatment has many advantages, including treat diabetic radically to some extent through improve the structure of islets and increase the number of β-cell instead of just hyperglycemic or improve other symptoms of on the surface. |
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