| Objective: To explore the seperation and purification method of arctiin from fructus arctii and study its protective mechanism on the vascular endothelial cell of experimental diabetic rats.Methods: (1) Silica column chromatography and LH-20 were used in phytochemistry study. (2) Based on the extraction ratio of arctiin, the best extraction procedure was acheived under the direction of L9(34) orthogonal experimental design. From 10 types of resins, the one with the maximal binding ability for Arctii was selected and silica-column choramatograpy was used in its seperation and purification procedure. (3) The animal diabetic experimental model in Wistar rats was established with streptozotocin (STZ) and many biochemical factors were used to indicate the pharmacological effects of arctiin, including glycosylated hemoglobin, serum creatinine, seralbumin, monoxide nitrogen, glycerin trilaurate, cholesterol, HDL-ch, LDL-ch, Blood urea nitrogen, creatinine, SOD, ET, MDA, total Urine protein and albuminuria. (4) The aorta Vascular endothelial cell was purified from Wistar rats by differential digestion, subcultured and then investigated the high sugar environment's effects on its activities, which include the abilities of releasing LDH, MDA and NO and expressing eNOS.Results: (1) 12 compounds was separated and purified from Fructus Arctii, their structures have been certified by MS, 1H-NMR, 13C-NMR, DEPT and they were listed in the following: arctigenin (1), mat-airesinol (2), lappaol B (3), lappaol A (4), lappaol F (5), arctiin (6), 8-hydroxypinoresinol (7), (+)-Fraxiresinol (8), acanthoside B (9), kaempferol (10), quercetin (11) and isoquercitrin (12). (2) Based on the extraction ratio of arctiin, the best extraction procedure, which included 70 % ethanol , 1 hour of reflux , 8-fold solvent and 3 times of extraction, was acheived under the direction of L9(34) orthogonal experimental design. The optimum purification conditions were as follows: the concentration of sample extract was 5.5mg/mL, the adsorbtion velocity was 2BV/h, the ratio of diameter to height of resin column was 1: 5, 50% alcohol was chosen as elution agent, the flow speed of eluting was 2BV/h, 4 times as the volume of the resin, the content of arctiin could reach to more than 50%. At last, the purifying technology was made by silica-column chramatograpy, CHCl3-CH3OH (9:1) as elution solvent, detected by HPLC, and the arctiin in the total solid could amount to more than 90%. Study on the quality standard of arctiin has been carried out. The detecting ways of the related substance and the content of arctiin by HPLC in effective parts have been carried on quality of 3 batches. The physics and chemistry nature, pH value, clarity and color, moisture, residue on ignition, heavy mentals ect were studied at the same time. (3) Arctiin could affect the blood-fat metabolism of TC, TG, LDL-ch and HDL-ch; lower the quality of 24-h Urine total protein and Urine albuminuria; adjust the content of MDA, NO, ET and SOD in rat serum. (4) Arctiin had no effect on Vascular endothelial cell in low concentration while could restrain RAECs, according to the concentration of arctiin. In low concentration (1μg·ml-1, 10μg·ml-1 and 100μg·ml-1), arctiin could greatly reduce the release of LDH,ET and MDA induced by high sugar; greatly increase the ability of excrete NO by RAECs; increase the expression of RAECs eNOS in high sugar. It indicated that in low concentration.Conclusion: (1) The content of arctiin was rather high in the alcohol extract of Fructus arctii. (2) The seperation and purification method of arctiin was simple, stable and was suitable to be used in the manufacture。The quality control standard of the arctiin was established. (3) Arctiin may have therapeutical effects on diabetes mellitus vascular complications, prevent the injure of Vascular endothelial cell, and protect Vascular endothelial cell by interfering nitricoxide synthase.
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