Study On Chemical Constituents Of Aconitum Soongaricum Stapf And Its Effects On Endogenous Metabolites In Rats

Objective: To detect chemical composition of ethanol extracts in A. soongaricum Stapf. To study the effects of its ethanol extracts on the rat’s plasma biochemical indicators and endogenous metabolites,and to provide information for the further study of its toxicity、 clinical diagnosis of poisoning and drug development.Method: The qualitative and quantitative analysis of chemical composition of the ethanol extracts in A. soongaricum Stapf., was performed with HPLC. The serum of rats were collected at 0.0 h, 0.5, 1.0, 2.0, 6.0, 12.0 and 24.0 h after fed with the ethanol extracts(7.8mg/kg). The biochemical Analysis of AST, ALT, TP, ALB, TC, TG, TBIL, Crea, GLU, LDH was performed on automatic biochemical analyzer. The serum metabolomics study was performed on NMR spectrometer. Multivalent static analysis of the biochemical and NMR data to identify the endogenous metabolites biomarkers was performed on SIMCA software with methods of Principal component analysis(PCA), partial least squares(PLS-DA) and orthogonal partial least squares- discriminant analysis(OPLS-DA).Result:(1) The HPLC method: the mobile phase, Acetonitrile-0.3% phosphoric acid water(B: A); gradient elution: 10~16%B(0~12min), 16~35%B(12~30min), 35~45%B(30~40min), 45%B(40~45min); detection wavelength: 235 nm; column temperature: 35?C; flow rate : 1.0 mL/min; injection volume:20μL. This HPLC condition is feasible in concern with the separation, precision, stability, and recoveries.(2) Under above HPLC condition, 5 main alkaloids were identified: songorine(54.9±4.5 mg), benzoylaconine(7.3±1.6 mg), aconitine(3.7±0.8 mg), bulleyaconitine A(1.2±0.3 mg), and 3-deoxy-aconitine(3.6±0.5 mg) in 100 g raw herbs.(3) The animal experiment showed that the serum biochemical indexes change significantly over time after single oral administration(7.8 mg/kg) of the ethanol extract. Among the observed biochemical indexes, ALB,TP,TC,TBIL and Crea reduced(p<0.05) and AST, TG, GLU, LDH increased(p<0.05); no significant effect on ALT and Crea(p>0.05).(4) The 1H-NMR metabolomics study showed that the serum of rats had a significant difference before and after administration of the extracts. With the multivalent static analysis, toxicity markers such as α-glucose, β-glucose, creatine were raised; amino acid(alanine, glutamate, glycine, tyrosine, leucine, threonine, lysine, phenylalanine), lipids were decreased first and then increased; pyruvate and lactate were decreased.Conclusion: The ethanol extracts of A. soongaricum Stapf. contains 5 alkaloids of songorine, benzoylaconine, aconitine, bulleyaconitine A, and 3-deoxy-aconitine. By analysis of the blood biochemical markers and its endogenous metabolites, we concluded that the ethanol extracts may cause damage on liver and heart cells with different degrees which maybe relate to the impact on glucose metabolism, lipid metabolism, and amino acid metabolism. The rat serum 1H-NMR metabolomics study showed that the metabolic changes were significant and the identified biomarkers may be further used as medicine toxicity testing indicators.