| The Chinese folk medicine Potentilla chinensis Ser. Bge (Rosaceae) is a perennial herb. It is also called "Fan-bai-cai", "Bai-tou-weng", "Ha-ma-cao" and "Tian-qing-di-bai", listed in Chinese Pharmacopoeia 2000 and 2005. As a folk medicine, Potentilla chinensis has anti-inflammatory and detoxification properties. In addition, P. Chinensis revealed the therapeutic effects for controlling blood glucose and improving glucose tolerance of body according to clinical researches from the Jixian Chinese Medicine Hospital of Tianjin and hospitals of Tianjin Traditional Chinese Medicine Universty. Our preliminary study showed that the main constituent compound of its hypoglycemic effect is potentilla flavone (trans-tiliroside), and its hypoglycemic effect is better than phenformin hydrochloride. And its structure is specially different from biguanides, sulfonylureas, a-glycosidase inhibitors, thiazolidinedione,GLP-1R peptide agonist drug, which indicates potentilla flavone has a different anti-diabetic mechanism and is an active leading compound worth studying.For furder quality control of herbs P. Chinensis and pharmacokinetic study provide a practical analysis methods, we have done the preparation of reference substance and established analysis methods for potentilla flavone.Firstly, we have established the separating conditions for reference substance of potentilla flavone with high-speed countercurrent chromatography, combined with HPLC to select solvent systems and adjust the solvents' ratios. The extracting crude of potentilla flavone was for the HSCCC purification. The results showed that the methylene chloride-methanol-water (5:4:2,v/v) was the best solvent system. The separating conditions as follow:stationary phase:upper phase; mobile phase:lower phase; flow late of the mobile phase:2.0mL/min; rotation speed:800r/min. The purity of potentilla flavone was over 98%, which was separated by HSCCC.And then determination methods in herb P. chinesis and biological samples for potentilla flavone were studied. Firstly, we have estabilshed a content method of potentilla flavone from herb P. chinesis by HPLC, with investigating different proportions of methanol-acetic acid, acetonitrile-acetic acid solutions in order to separate potentilla flavone and disturb chemicals. It was showed that methanol-3%o acetic acid(49:51) was the best mobile phase.By measuring the potentilla flavone from Potentilla chinesis in different growth periods and parts, the determination results showed that the highest content of potentilla flavone are in July and its leaves of P. chinesis, with the contents are 1.018%o and 1.477%o, respectively.A rapid, precise and sensitive HPLC method for the determination of potentilla flavone in rat plasma has been developed. Acetonitrile-3‰acetic acid solution(35:65) on potentilla flavone and endogenous substances in plasma was better. It could be used for potentilla flavone and plasma protein-binding study. By investigating equilibrium dialysis and ultrafiltration, we have found that potentilla flavone has a low solubility and poor stability in water. Otherwise, its equilibrium dialysis time was longer than one or two days. But free potentilla flavone could be separated fastly within 30 minutes by ultrafiltration method. So we have chosen the ultrafiltration method for the binding rate of potentilla flavone to rat plasma proteins. The plasma protein binding rate of potentilla flavone was meanly 87.95%.Lastly, a rapid, sesitive and specific method was established for determing potentilla flavone in rat tissue samples by HPLC. Acetonitrile-3‰acetic acid solution(28:72) could be achieved to separate potentilla flavone and endogenous substances in tissue samples, which the resolution was greater than 1.5. It was a good method for the further tissue distribution research of potentilla flavone in rats with oral administration. The initial pretest of tissue distribution was carried out and found that the content in the pancreas was the highest one, except the small intestine. It was useful for providing basic datas of the further tissue distribution study. |
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