| Objective:To develop NMR and GC/TOF-MS based metabolomic platform for toxicological studies of Fu Zi. Investigate the toxical changed rule and toxical mechanism of Fu Zi, and explore the perspectives of metabonomics in the toxicological study of traditional Chinese medicine (TCM).Methods:The NMR and GC/TOF-MS based metabonomics methods were used to the following toxicity study of Fu Zi.1. The acute toxicity of aconitine, mesaconitine and hypaconitine on rats after a single dose; 2. The subacute toxicity of aconitine, mesaconitine and hypaconitine on rats after low doses for 15 days; 3. The effect of glycyrrhetic acid, one of the effective components of Gan Cao (Radix Glycyrrhiza), on the toxicity of mesaconitine; 4. The effects of decoction of Gan Jiang (Rhizoma Gingiberis) on the toxicity of mesaconitine. The urine samples were collected everyday from predose to the end of recovery period, while plasma and tissue samples were collected during the dissection. The NMR and GC/TOF-MS experiments were carried out for urine, plasma and tissue extract samples. The integrated data were exported for multivariable statistical analysis by using SIMCA P+, in order to find the potential biomarkers characteristic metabolites and to reveal the toxical mechanism.Results:1. In the acute toxicity study of aconitine, mesaconitine and hypaconitine, the urine NMR results showed that 6h after the administration, the urine metabolic profiles changed most significantly, these changes released after 12h, but were aggravated one more time after 24h. The concentrations of glucose, acetate and alanine were increased, and citrate, creatine, TMAO,2-oxoglutarate and huppirate were decreased in the Aconitum alkaloids treated groups after 6h. The plasma NMR results showed that the changes of metabolic profile caused by hypaconitine were different from those by aconitine or mesaconitine. The toxicity was displayed in the content changes of glucose, lactate, lipids, OAc, UFA, LDL/VLDL, PUFA and NAc. In plasma samples,38 metabolites were indentified in the TIC detected by GC/TOF-MS,included amino acids, fatty acids, sugars and organic acids. The results of t test and multivariable statistical analysis revealed that the toxicity of hypaconitine was lower than aconitine and mesaconitine, and with different mechanism.2. In the subacute toxicity study of aconitine, mesaconitine and hypaconitine, the urine NMR results showed that the metabolic profiles of Aconitum alkaloids treated groups were different from control group after the first administration. The most changed metabolic profiles by aconitine, mesaconitine and hypaconitine were observed after continuous dosing for 5d,9d and 13d respectively. And the toxicity of aconitine was higher than mesaconitine and hypaconitine. The toxicity was displayed in the content changes in acetate, succinate, glycine, creatinine, creatine, phenylalanine,2-oxoglutarate, huppirate, citrate, DMA, taurine, TMAO, alanine, DMG, glucose, valine and 3-hydroxybutyric acid. The plasma NMR results also showed that the metabolic profiles of Aconitum alkaloids treated groups were different from control group, and that the toxicity of aconitine was higher than mesaconitine and hypaconitine. The toxicity was displayed in the content changes in choline, lipids, NAc, OAc, acetate,3-hydroxybutyric acid, acetone, lactate, diacetate, glutamine, alanine, valine, leucine/isoleucine, HDL, UFA. In plasma samples,36 metabolites were indentified in the TIC detected by GC/TOF-MS, included amino acids, fatty acids, sugars and organic acids. The results of t test and multivariable statistical analysis showed that the toxicity of hypaconitine was lower than aconitine and mesaconitine, and with different mechanism.3. The urine NMR results showed that glycyrrhetic acid could release the toxicity of mesaconitine, and that the effect of multi-doses of glycyrrhetic acid was better than single-dose. This was displayed in the content changes in 2- oxoglutarate, huppirate, creatinine/creatine, lactate, alanine, acetate, succinate, citrate, DMG and glycine. The plasma NMR results showed that glycyrrhetic acid could release the toxicity of mesaconitine on the metabolites of small molecule, and the effect of multi-doses of glycyrrhetic acid was better than single-dose. This was displayed in the content changes in alanine, pyruvic acid, TMAO, glucose, lipids, lactate, NAc, diacetate. In plasma samples,34 metabolites were indentified in the TIC detected by GC/TOF-MS, included amino acids, fatty acids, sugars and organic acids. The results showed that glycyrrhetic acid could release the toxicity of mesaconitine. In addition, the metabonomic analysis of the lipid extract of heart and aqueous extract of brain also showed that glycyrrhetic acid could release the toxicity of mesaconitine.4. The urine NMR results showed that the decoction of Gan Jiang and compounded glycyrrhetic acid could display the toxicity of mesaconitine, and the effect of decoction of Gan Jiang compounded glycyrrhetic acid was better then that of Gan Jiang. These results were showed by the content of 2-oxoglutarate, huppirate, glucose, taurine, glycine. The plasma NMR results also showed that both decoction could display the toxicity of mesaconitine, and the effects of them were closer. These results were showed by alanine, lactate, acetate, glucose, lipids, NAc, OAc. In urine samples, 35 metabolites were indentified in the TIC detected by GC/TOF-MS, and in plasma samples,33 metabolites were indentified, both the results showed that the decoction of Gan Jiang compounded glycyrrhetic acid could display the toxicity of mesaconitine, and the effect of decoction of Gan Jiang compounded glycyrrhetic acid was better then that of Gan Jiang. In addition, the NMR data of hearts and brains extract samples were also indicated similar results.5. It is the first time that using the same method to detect the metabolic profiles of urine and plasma, and establishing the metabolites libraries of urine and plasma respectively. There are 35 metabolites were included in the urine library (The urine library included 35 metabolites), and 38 metabolites in the plasma library. These data can increase the speed and accuracy of the peaks’ identification in TIC. Conclusions:Aconitine, mesaconitine and hypaconitine all showed the toxicity on the rats’ metabolic profiles. The toxicity of aconitine was the highest, and that of hypaconitine was the lowest. In addition, the toxicity mechanism of hypaconitine was different from the two others. Glycyrrhetic acid could display the toxicity of mesaconitine, and effect of multi-doses of glycyrrhetic acid was better than that of single-dose. Both decoction of Gan Jiang and the decoction of Gan Jiang compounded glycyrrhetic acid could also show the toxicity of mesaconitine, and the effect of decoction of Gan Jiang compounded glycyrrhetic acid was better then that of the decoction of Gan Jiang. There will be a bright perspective for the metabonomics in the field of safety evaluation of TCM. |
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