| Objective:To supplement and improve quality standard of Lanhong Capsules and to conduct a comprehensive quality control and evaluation. To establish the method for quality control and fingerprint analysis of E. latifolius, and to determine the origin and source of E. Latifolius, which is used by Lanhong Capsules. Study E. latifolius’s acute toxicity and anti-inflammatory effects, which can lay a foundation for clinical applications and develpoment of E. Latifolius. Methods:Carthamus tinctorius and E. latifolius in Lanhong Capsules were identified by TLC, using silica gel as stationary phase, using the mixture of trichloromethane-ethyl acetate-methanol-methanoic acid(4:1:1:0.5) as developing solvent, using white light and AICl3solution as coloring reagent. Eucommia ulmoides Oliv and Notoginseng Radix were. identified by TLC, using silica gel as stationary phase, using trichloromethane-methanol-water(3:1:0.1) and upper layer of the mixture of butanol-ethyl acetate-water(4:1:5) as developing solvent, and sulphuric acid ethanol solution (1→10) as coloring reagent. Margarita in Lanhong Capsules was identified by physicochemical method. The test sample preparation method of Lanhong Capsules was selected by using single factor method. The content of apigenin-7-O-β-D-glucoside and hydroxysafflor yellow A (HYSA) in Lanhong capsules could be determined by HPLC. The separation was performed on C18column with acetonitrile-ammonium acetate solution (21:79) and (9:91) as mobile phase, the column temperature were maintained at25℃and30℃,the flow rate was1ml·min-1and the detection wavelength were set at340nm and403nm. Pinoresinol diglucoside and notoginsenoside R1, ginsenosideRg1, Re, Rb1, Rd in Lanhong capsules could be determined by HPLC. The separation was performed on C18column with methanol-water and acetonitrile-water as mobile phase under flow rate of1ml·min-1by gradient elution, the column temperature were maintained at30℃and25℃, use the wavelength of277nm and ELSD as detection methods. The content of apigenin-7-O-β-D-glucoside in E. latifolius could be determined by the quantitative method of Lanhong Capsules. A HPLC fingerprint analysis method of E. latifolius was developed. The chromatographic separation was performed on C18column with acetonitrile-ammonium acetate solution as mobile phase under flow rate of lml·min-1by gradient elution, the column temperature was maintained at30℃and the detection wavelength was340nm. The extracts from E. Latifolius were separated and purified by D101macroporous resins. Finally, the maximum dose method was used to study acute toxicity on mice which caused by extracts from E. Latifolius. The anti-inflammatory effect of extracts from E. Latifolius in mice was observed in test of ear swelling caused by xylene. Results:The thin-layer chromatogram of Lanhong Capsules showed clear spots with no interference from negative control. Margarita in Lanhong Capsules could be identified by physicochemical method effectively. Determined the test sample preparation method of Lanhong Capsules. The determination of index components in Lanhong Capsules could be used as quality control method. Apigenin-7-O-β-D-glucoside, HSYA, notoginsenosideR1, ginsenosideRg1, Re, Rb1, Rd showed good linearity in the range of the standard curve, with correlation coefficients all being than0.9993. The results of precisions, reproducibility, tolerability and stability were good. The mass fraction of apigenin-7-O-β-D-glucoside, HSYA, notoginsenosideR1, ginsenosideRg1, Re, Rb1,Rd in Lanhong Capsules were within the ranges of0.038%～0.050%,0.35%～0.91%,0.25%～0.51%,1.13%～2.02%,0.13%～0.24%,0.87%～1.59%,0.19%～0.49%, respectively. The results of pinoresinol diglucoside determined from Lanhong Capsules within the range of0.023%～0.083%(within the range of0.10mg～0.34mg). The result of apigenin-7-O-β-D-glucoside determined from E. latifolius indicated that a large fluctuation exists within the range of0.004%～0.081%. In the fingerprint of E. latifolhis, there were12common peaks, the relative retention time of the common peaks compare with S peak were1(0.423),2(0.535),3(0.639),4(0.858),5(S,1.000),6(1.042),7(1.072),8(1.307),9(1.751),10(1.790),11(1.891),12(1.932). The peaks of1,3,8,11were chlorogenic acid, caffeic acid, apigenin-7-O-β-D-glucoside and apigenin. The extracts from E. Latifolhis were separated into D101-water+30%ethanol part, D101-50%ethanol part and the D101-70%+95%ethanol part. In the acute toxicity research, the mice did not appear dead, the organ was normal. The maximum dose value of D101-water+30%ethanol part, D101-50%ethanol part and the D101-70%+95%ethanol part were equal to854,415,383times of daily dosage in clinic. The anti-inflammatory experiment showed that aspirin, the medium-dose and high-dose of mixture of D101-water+30%ethanol, D101-50%ethanol, and mixture of D101-70%+95%ethanol could apparently inhibit auricle swelling induced by xylene. Comparing between groups, the high-dose of the parts of D101-50%and mixture of D101-70%+95%ethanol has the similar anti-inflammatory effect with asprine. The anti-inflammatory effect of the mixture of D101-water+30%ethanol was relatively small. Conclusions:The qualitative and quantitative methods are simple, rapid, available with high specificity and good reproducibility, which can be applied for the quality control of Lanhong Capsules and E. latifolius effectively, made the clinical safety and effectiveness of Lanhong Capsules has been fully improved. Fingerprint of E. Latifolius provided more comprehensive quality information, which could control the quality of Lanhong Capsules better, ensure the stability of clinical use. The studies of preliminary toxicology and anti-inflammatory effect in extracts from E. Latifolius laid a solid foundation of clinical application and development. |
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