| In the course of Traditional Chinese Medicine(TCM)development, there was no doubt of its validity, practicability and science in traditional culture of China. Metabonomics as a useful technology, which provides a good development direction for TCM. Therefore, metabonomics have increase a widely attention in recent years.Jin Ling-zi powder was a basic TCM prescription to cure anti-inflammation and analgesic, which was originally described from the old chinese book of“Su Wen”. The prescription of Jin Ling-zi powder was composed of corydalis rhizoma and toosendan fructus(Jin Ling-zi), which could promote blood circulation and relieve pain. Earlier studies in anti-inflammation and analgesic effect about Jin Ling-zi powder have detected from pharmacology aspect, but there have not studied about it from metabolomics.In order to explore Jin Ling-zi powder in the process of treatment of biological pathways, we have used 1H NMR and LC-MS analysis methods and chemometrics methods to found out significantly expressed metabolites with the identification of metabolite structure between metabolic phenotype of metabolic profile and disease metabolic network. In this study, we were used metabolomics study to observe Jin Ling-zi powder in the mechanism of anti-inflammatory, sedative, analgesia, hepatotoxicity and nephrotoxicity.1. Anti-inflammatory To explore Jin Ling-zi powder inflammatory mechanisms in the treatment of inflammatory adjustment. This study had adopted the 1H NMR and LC-MS technology. From Jin Ling-zi powder pharmacology experiment research on mice ear swelling induced by xylene for anti-inflammatory effect and use Elisa measurement detected inflammatory cytokines in the rat serum. Finally, through the analysis of carrageen induce rat foot swelling model of inflammation to finding out biomarkers related to inflammation models of Jin Ling zi powder in SD rat. Through the analysis of detection spectrum, we had found 18 biomarkers of metabolites(citrate, pyruvate, malic acid, succinate, glutamate, lysine, tartrate, 2-oxobutyric acid, glycine, guanosine, 9-cis-retinoic acid, triphosphate, inosine 5’-diphosphate, inosine diphosphate, tripolyphosphate, inorganic triphosphate, glycerophosphocholine, 21-deoxycortisol)and the pathway analysis results were the TCA cycle, pyruvate metabolism, glycine, serine and threonine metabolism, and dicarboxylic acid metabolism. From the metabolic network we can see that the anti-inflammatory effect of Jin Ling zi powder could regulate citric acid, succinic acid and glycine content to resisting oxygen free radical and reduce body damage by ROS.2. Analgesia In order to explore Jin Ling-zi powder effects in the process of treatment in metabolic pathways of analgesic. This study using the metabolomics research methods of LC-MS technology, with the formalin model, hot plate model and acetic acid model to find out the changes of biomarkers associated with pain in the mice serum. Speculation pains related role model of formalin, which about 34 differences metabolic material and four related metabolic pathways which were ketone synthesis, glutathione metabolic pathway, primary bile acid synthesis and degradation, arginine and proline metabolism. Hot plate also have four related metabolic pathways includes penascorbate and aldarate metabolism, pentose and glucuronate interconversions, taurine metabolism and arginine and proline metabolism. The arginine and proline metabolism was the common pathway in formalin model and the hot plate model. Pentose and glucuronate interconversions pathway have significantly effects to release mice pain. From three model results suggested that Jin Ling-zi powder have both peripheral and central analgesic effect.3. Sedative And Hypnotic Based on 1H NMR technique, Jin Ling-zi powder extract was observed metabolites changes after given sodium pentobarbital substance-induced sleep in rat metabolites and explained its mechanism. We have compared urine and serum metabolic profiles from sprague-dawley rat using 1H NMR-based metabonomics. The related metabolites data were transported to Simca-p and Chenomx Software to identification and classification. Consequently, we concluded that 16 different metabolites(glycine, glucose, fumaric acid, glucose-6-phosphate, 2-oxoglutate, betaine, creatine phosphate, phosphoric anhydride, taurine, choline, succinate, creatine, lactate, glutamine, succinylacetone, acetate) related to sedative and hypnotic effect of Jin Ling-zi powder. Compared with the control group, Jin Ling zi powder groups have found that lactic acid and taurine were increased and glutamine was decreased in rat serum. The content of 2-oxoglutate and phosphoric anhydride were decreased, betaine and taurine were increased in rat urine. Alanine, aspartate and glutamate metabolism, glycine, serine and threonine metabolism, arginine and proline metabolism and citrate cycle(TCA cycle) were four related metabolic pathways. Jin Ling-zi powder group and indiplon group can prolong pentobarbital induced rats sleeping time, which indicated Jin Ling-zi powder has a synergistic effect with pentobarbital sodium. Endogenous compounds such as glutamine and 2-oxoglutate had relieved the exciting of cranial nerve excitability and play a role in regulating sleep.4. Toxicity To further understand the metabolic characteristics of Jin Ling-zi powder toxicity effect in rats and explore the role of Jin Ling-zi powder biological pathways in the treatment process. After administration of Jin Ling-zi powder, the rest of rats in each group have 7 days to recovery. With three different dose for administration, urine and serum of rats samples were use LC-MS for analysis. Under the XCMS online analysis, we have found 44 metabolomics about Jin Ling-zi powder toxicity. Finally, using Met-PA for metabolic pathways enrichment and analysis, the results obtained: steroid hormone biosynthesis, tryptophan metabolism, pentose and glucuronate interconversions, ascorbate and aldarate metabolism, glutathione metabolism for five related metabolic pathways. The main metabolic pathways were related to toxicity energy conversion, cholesterol metabolism and amino acid metabolism. High and middle dose groups of Jin Ling-zi powder have significantly deviated with control group, suggesting that high and middle dose groups of Jin Ling-zi powder have interfered with endogenous metabolites in 14 days and this group had already have potential toxicity. Through the metabolites pathway we can see that five metabolic pathways, which unbalance metabolism have caused toxicity of liver and kidney in rat.Overall, Traditional Chinese Medicine of Jin Ling-zi powder was involved multiple system organ for strengthening the mechanism in drug treatment. With the use of high-throughput metabolomics analysis to identified significant changes in metabolic, Jin Ling-zi powder intervention mechanism of related diseases for metabolic pathways had provided the usefulness of 1H-NMR and LC-MS that based metabonomics approach in the field of the modernization of Chinese Medicine. |
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