| Study on material basis for efficacy of Chinese herbal formula is the precondition and base of modernization and internationalization of Chinese materia medica, also is one of the problems demanded to be settled urgently of this course. In this course, it is the most important thing to open out material basis for efficacy of Chinese herbal formula. Material basis for efficacy of Chinese herbal formula is an aggregate of all effective components. At present, the methods to study on material basis for efficacy of Chinese herbal formula include combining Chinese medicinal chemistry and pharmacodynamics together, pharmaceutical chemistry and pharmacyology in serum, cell membrane chromatography, and also.This research investigates the substance foundation of Xiezhuo Chubi Fang (XZCBF) for hyperuricemia using the first method:contents of active elements of XZCBF were determined by modern analyzing technology (HPLC); Total flavonoids in XZCBF were isolated and purified by chromatography, its effects on renal tubular epithelial cells' (RTECs) proliferation, uric uptake and RST gene expression were investigated systematically, these experimentation results show that Total flavonoids in XZCBF were one of the effective part to treat hyperuricemia.The research include follow contents:1. Contents determination of active elements of XZCBF by HPLCContents of diosgenin, ecdysterone and astilbin in XZCBF were determined by HPLC:For diosgenin, with a agilent HC-18 column (250 mm×4.6 mm,5μm), acetonitrile:methanol (3:2) severed as the mobile phase, the speed was 1 mL-min-1 and the detection wavelength was at 206 nm, the recovery of diosgenin was 104.51%; For ecdysterone, with a agilent TC-18 (250 mm×4.6 mm,5μm) column, acetonitrile: water (17:83) severed as the mobile phase, the speed was 1 mL-min-1 and the detection wavelength was at 243 nm, the recovery of ecdysterone was 93.94%; For astilbin, with a KromasilC-18 (250 mm×4.6 mm,5μm) column, acetonitrile-phosphoric acid-water (20:0.025:80) severed as the mobile phase, the speed was 1 mL-min-1 and the detection wavelength was at 290 nm, the recovery of astilbin was 99.46%. These methods are sensitive, accurate, reproducible, specific and can be used for quality control of XZCBF efficiently.2. Separation and purification of total flavonoids in XZCBFQuality and quantity analysis methods of Smilax Glabra Roxb.(principal drug) were established, astilbin in it was identified by TLC, contents of astilbin and engelitin in Smilax Glabra Roxb. were determined by HPLC simultaneously; extraction procedure of total flavonoids was optimized by using orthogonal experiment, four factors-alcohol concentration, temperature, dosage and extraction times-were investigated, content of total flavonoid was assessed by colorimetry, and astilbin was assessed by HPLC. The best extraction procedure is the medical material should be extracted by 10 times (V/W) 60% alcohol twice in boiling water bath, the extraction efficiency is as high as 95.99%. With the contents of total flavonoids, astilbin and engelitin as indexes, the optimum process of separating and purifying flavonoid was screened by static and dynamic adsorption tests. The content of total flavonoids was 75.63% in the extractive, the transfer rate was 68.24%; the content of astilbin was 12.77% in the extractive, the transfer rate was 73.05%.3. Isolations and structure identifications of chemical constituents of XZCBFIn this research, chemical constituents of XZCBF were isolated with systematic solvent, silica gel-column chromatogram and polyamide-column chromatogram; the structures of chemical constituents were identified with dates of IR,1HNMR,13CNMR, physical-chemical characters, compared with related documents reported.7 chemical compounds were isolated in this research; among them six have been identified, they wereβ-Sitosterol (I), Oleanolic acid (II), Ursolic acid (Ⅲ), Astilbin (IV), Engelitin (V) andβ-Ecdysterone (VI).4. Study of action mechanism of total flavonoids in XZCBF in treating hyperuricemiaFirstly, conditions of RTECs primary culture were optimized and cultures underwent functional tests. The adherent rate of segments of renal proximal tubules reached 54.5% after the digestion using 400U collagenase type I for 20 minutes; the most suitable time for the first change of the culture medium was 72 hours; Log phase began from the fifth day and the cell's growth conditions became unsuitable in the tenth day. In the transport medium contained 1500μmol-L-1 uric acid, the 30-min uptake of uric acid was the highest and was taken as a measure of an initial rate. Different uric acid-uptake inhibitory effects appeared due to different concentrations of the indicate drugs probenecid and benzbromarone. The inhibition influence of benzbromarone was higher than probenecid. The mouse renal proximal tubular epithelial cells cultured by the modified method will yield rich and homogeneous harvest, and present well function of uric acid uptake.Next, effects of total flavonoids on cell proliferation and uric acid-uptake were investigated. The RTECs were isolated from the renal of mouse by the utilized method and were cultured with total flavonoids from XZCBF at different concentrations. The proliferation of RTECs was assessed by MTT assay. The uric uptake medium was prepared and the uric acid-uptake values were determined by the kit. The interest cells were isolated and it has the function of uric acid-uptake. The proliferation of the RTECs was inhibited by high concentrations (10,7.5,5 g-L-1) of the drug, while no significant effects on the proliferation of the RTECs were observed at the low concentrations (2.5,0.5 g-L-1). Moreover, uric acid-uptake tests were undertaken at this range of concentrations, and different inhibitory degrees by the drug were also determined.Effects of total flavonoids on gene RST expression were detected to reveal its molecular mechanism. The uric acid-uptake essay was carried out on the mouse renal tubular epithelia cells and the different effects of different levels of total flavonoids in XZCBF on the uric acid uptake were studied. The expression of mouse renal-specific transporter (RST) in the cells was detected by RT-PCR. Results indicated that total flavonoid in XZCBF at the concentration range from 0.5 g-L-1 to 2.5 g-L-1 was proved to inhibit the uric acid-uptake function of RTECs and the expression of RST was lower under treatment at these concentrations. The uric acid-uptake can be reduced by total flavonoids in XZCBF by inhibiting the expression of RST. |
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