Study On Quality Conformity And Chronic Toxicity Test Of Zhenhuang Yangyan Capsules

Objective: The Zhenhuang Yangyan Capsules(ZYC) is newly researched and developmented compound preparation product. It has the effects of tonifying Qi and enriching blood、nourishing skin、improving facial chloasma and so on. This research intends to study the quality control method of this product and establish an effective、stable and controllable methods of its qualitative and quantitative analysis and to provide a reference basis for the establishment of the product quality standard. In addition, this research also studys on the chronic toxic reaction of ZYC in rats and defines the safe dose for its clinical usage, provided the safety evidence for clinical use of ZYC.Method:(1) The microscopic identification of Poria、Artemisiae Argyi Folium and other nine kinds of herbs of ZYC was carried out;(2) Identificated the Rehmanniae Radix Praeparata、Chuanxiong Rhizoma、Angelicae Sinensis Radix、Salviae Miltiorrhizae Radix Et Rhizoma and other three heras herbs of ZYC by TLC;(3) Used the HPLC to determine the content of paeoniflorin and paeonol of ZYC, and the chromatographic conditions are such as: the chromatographic column is Luna C18 100A(250mm×4.6mm,5um); the mobile phase is acetonitrile-0.05% phosphoric acid, gradient elution; the wavelength detection is230nm; flow speed is 1.00mL/min; the column temperature is 35℃ and the sample size is10 ul. The methodological investigations are also carried out.(4) According to the technical demand of fingerprint about injection of traditional Chinese medicine, the chromatographic conditions are such as: the chromatographic column is Luna C18 100A(250mm×4.6mm,5um); the mobile phase is acetonitrile-0.05% phosphoric acid, gradient elution; the wavelength detection is 228nm; flow speed is 0.8mL/min; the column temperature is 35℃ and the sample size is 10 ul. The methodological investigations are also carried out.The correlation 、 cosine of vectors and similarity evaluation system for chromatographic fingerprint of TCM was used to ten batch samples.(5) Rats were given this Capsules for successive 180 days in the doses of 2.93、1.47、0.74 g(crude drugs).kg-1three expermental groups and the control group was established. The toxic reactions of after being administrated 90 days、180 days and the recovery condition within 30 days of stopping medication were observed.Result:(1) The microscopic identification methods of Poria 、 Artemisiae Argyi Folium and other nine kinds of herbs of ZYC with strong microscopic characteristic were meaningful to identify the ZYC.(2) The TLC identification methods of Rehmanniae Radix Praeparata、Chuanxiong Rhizoma、Angelicae Sinensis Radix、Salviae Miltiorrhizae Radix Et Rhizoma and other three herbs of ZYC were established in this study. The spots of the TLC chromatogram were quite clear, same color sports of the sample were found in the same loction of the corresponding reference or the reference drug and the blank test showed no interference.(3) The determination of paeoniflorin and paeonol of ZYC by HPLC: the method had a good linearity in the ranges of 0.144ug~1.44ug(r=0.9997) of paeoniflorin and 0.0316~0.316ug(r=0.9998) for paeonol. The RSD of precision、stablity、repeatability and recovery were less than 3%. We finally determined that paeoniflorin(C23H28O11) should not be less than 1.0965 mg and the paeonol(C9H10O3)should not be less than 0.2665 mg each Capsule of ZYC.(4) HPLC was used to establish the reference peak,35 peaks were identified as the general peaks, and 5 general peaks’ composition were confirmed by contrasting with reference substance. The RSD of relative retention time and relative peak area of the total peaks was less than 3%, excepted some total peaks’ RSD of relative peak area in repeated test is less than 3.5%. Compared with standard fingerprint,each sample’s similarity was more than 0.9.(5) Compared with the normal control group,Dosing after 90 days, The food intake of high dose decreased and the rats’ body weights in high、medium dose grown slowly. The kidney index of rats in high dose increased and thelung index、thymus index decreased. 180 days later,the values of platelets、aspartate aminotransferase 、 alkaline phosphates increased and the mean corpuscular volume decreased of high、medium dose. Drug withdrawal 30 days after, the values of platelets increased of high dose and the mean corpuscular volume of high、medium dose decreased.Some of the index mentioned above has no difference.Conclusion:(1) The method of microscopic identification、TLC identification、high performance liquid chromatography and HPLC fingprint in this study have a strong specificity and reproducible, and can use to provide the scientific basis for quality control of ZYC.(2) the dose of 0.74 g( crude drugs).kg-1of ZYC was found no obvious toxic effects in all the check.This test indicates that the mdication safety dose of ZYC should be less than 0.74 g( crude drugs).kg-1.