ABSTRACT
OBJECTIVE： To establish the method for the content determination of astragaloside Ⅳ， emodin and chrysophanol in Jianpi yishen pills （JYP） and to investigate the effects of JYP on calcium， phosphorus metabolism and inflammatory factors in chronic renal failure （CRF） model rats. METHODS： HPLC method was adopted. The determination of astragaloside Ⅳ， emodin and chrysophanol was perform on Agilent Zorbax SB-C18， Agilent TC C18 column， respectively； mobile phase consisted of acetonitrile-water （36 ∶ 64， V/V） and methanol-0.1％ phosphoric acid solution （75 ∶ 25， V/V）； the detectors were evaporative light-scattering detector and diode-array detector （detection wavelength of 254 nm）； the column temperatures were set at 30 ℃and 25 ℃ at the flow rate of 1.0 mL/min； the sample sizes were 20 and 10 μL. SD rats were randomly divided into normal group， model group， Niaoduqing group （1.80 g/kg） and JYP low-dose， medium-dose and high-dose groups （1.71， 3.43， 6.85 g/kg）， with 10 rats in each group. Except for normal group， CRF model of other groups were established by 5/6 nephrectomy in other groups. Four months after modeling， normal group and model group were given constant volume of water intragastrically； admi- nistration groups were given relevant medicine intragastrically， once a day， for consecutive 12 weeks. The levels of serum creatinine （Scr）， urea nitrogen （BUN）， parathyroid hormone （PTH） and inflammatory factors （IL-6， TNF-α） were measured by ELISA. Methyl thymol blue colorimetric method and phosphomolybdic acid method were used to detect the contents of blood calcium and phosphorus. Correlation of inflammatory factors with related calcium and phosphorus metabolism indexes （blood calcium， blood phosphorus， PTH） were investigated with Pearson assay. RESULTS： The linear range of astragaloside Ⅳ， emodin and chrysophanol were 54.537-381.759， 2.960-20.720， 6.318-44.223 μg/mL， respectively. The limits of quantitation were 0.010， 0.288， 0.216 μg/mL； the limits of detection were 0.003， 0.096， 0.072 μg/mL. RSDs of precision， reproducibility and stability tests were all lower than 3.0％. The recoveries were 97.18％-102.33％（RSD＜3％，n＝9）. After modeling （before medication）， serum contents of Scr and BUN in model group and administration group were increased significantly， compared with normal group （P＜0.01）. After medication， above indexes of administration group were decreased significantly， compared with model group and the same group before medication （P＜0.01）. Compared with normal group， the content of blood calcium were decreased significantly， while the contents of IL-6 and TNF-α were increased significantly （P＜0.01）. Compared with model group， the content of blood calcium were increased significantly in JYP medium-dose and high-dose groups， while serum content of PTH in Niaoduqing group， serum contents of PTH and IL-6 in JYP medium-dose and high-dose groups as well as serum content of TNF-α in administration group were decreased significantly （P＜0.05 or P＜0.01）. JYP had no significant effect on blood phosphorus in rats， and there was no correlation of inflammatory factors with related calcium and phosphorus metabolism indexes （P＞0.05）. CONCLUSIONS： The established content determination method is simple， specific and sensitive， and can be used for content determination of astragaloside Ⅳ， emodin and chrysophanol in JYP. JYP can improve renal function of CRF model rats， relieve calcium metabolism disorder and inhibit the expression of inflammatory factors.
ABSTRACT
Objective Former standards for Yishen Pills only identify angelica root , astragalus root and ligustrum lucidum by thin layer chromatography ( TLC) and there is no test for the content of effective components .The study was to improve the quality of Yishen Pills by perfecting the quality standards . Methods We determined the content of specnuezhenide in Yishen Pills by high-per-formance liquid chromatography(HPLC), with a mobile phase of MeOH-Water, a chromatography column of Agilent TC-C18(2) (4.6 × 250 mm, 5μm), a flow rate of 1.0 mL/min, a column temperature of 25℃, a detection wavelength of 224 nm, and a sample loop volume of 10μL. Results The linear relationship of specnuezhenide content was good in the range of 15.375μg/mL~246.000μg/mL.The relative standard deviation of precision experiment , stablity experiment and repeatablity experiment was 0.44%, 0.95%and 2.65%re-spectively.The average recovery was 99.60%.The qualified standard for specnuezhenide in Yishen Pills was ≥0.3 mg/g. Conclusion The method is simple , accurate and reliable , with good reproducibility , and it is applicable for the quality control of Yishen Pills .