Metabonomic Study Of Traditional Chinese Medicine Of Yi Guan Jian Decoction

As a part of Chinese excellent cultural achievements,traditional Chinese Medicine(TCM)is a reflection of Chinese culture and philosophy.In the course of TCM's 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.Metabonomics with its impressive and ever-increasing coverage of endogenous compounds has been successfully employed in many areas.What's more,metabonomics analysis can offer an unbiased view of changes in metabolism,covering entire metabolic pathways to characterize pathological states and providing diagnostic information.Therefore,metabonomics has attracted a widely attention in recent decades years and developed a lot in life science.Yi Guan Jian comes from ?Xu Ming Yi Lei'An?which belongs to Wei Zhixiu in Qing dynasty.Its major functions recorded in volume 18 said: “Hypochondriac pain,acid regurgitation,spit acid,abdominal hernia,all liver diseases”.Discourse on prescription that selected from ?Zhong Feng Jiao Quan? in volume 3 said: “Any lateral thorax pain or abdominal pain is mostly because of disturbance of the liver-qi,rigid and wood unrestraining.A temporary solution of relieving stagnant Qi and clearing disease often effective.…… to Regulat unreasonableness of liver Qi,obey harmonious in our body,which would be the best choice to conserve “yin” of the liver……”.Yi Guan Jian consists of six kinds of traditional Chinese medicine: Radix Rehmanniae,Radix Glehniae,Ophiopogon root,Angelica,Lycium chinensis,Fructus meliae toosendan,which could be used to smooth the liver and discharge hot,activate blood and relieve pain in clinical.In the formula of Yi Guan Jian,Radix Rehmanniae was put in an important position which serves as monarch drug and could nourish yin and blood,soft liver and drainage depression,nourish liver and kidney,its means are nourishing water and wood.Angelica and Lycium chinensis nourish blood and liver;Radix Glehniae and Radix Ophiopogonis nourish lung and stomach,nourish “Yin” and promoting production of body fluid,intend to keep smooth in our body,and these four drugs are corporated as the ministerial drug.The prescription is accompanied by a small amount of fructus meliae toosendan,and has the ability to smooth the liver and discharge heat,regulate qi further to alleviate pain and recovery harmony.Although these medicines are bitter-cold herbs,the compatibility with large amount of drugs with the features of sweet and nourishing yin and blood has set against it.In essence,the combination of the six herbs is a systematic and complex interaction system which is composed of multiple components of Chinese herbal medicine.These components may have a direct effect on the body,or may enter the body after interaction with the body's metabolic system to produce pharmacological effects.The research on Yi Guan Jian decoction just about compound prescription,the drug ratio and the preparation of the decoction in domestic,however,the exploration of the treatment mechanism is not profound.This experiment was to explore the role of the known or unknown biological pathways in the treatment of Yi Guan Jian decoction,by using the combination of gas chromatography-mass spectrometry(GC-MS)or liquid chromatography-mass spectrometry(HPLC-MS/MS)to combine with the methods of chemical analysis and find the significant difference in concentration or abundance.By identifying the structure of metabolites,we established metabolic networks in the relationship between metabolic profile and metabolic phenotype in vivo and further revealed the mechanisms of Yi Guan Jian on anti-inflammation,anti-fatigue and anti-hepatic fibrosis.Specific research results are as follows:One,study on the mechanism of the anti-inflammatory effect of Yi Guan Jian based on metabonomics.In order to explore the regulation of the inflammatory mechanism of the organism in the treatment of Yi Guan Jian decoction,we used a combination of liquid and gas chromatography-mass spectrometry technology coupled with metabonomics method to observe the anti-inflammatory effect on carrageenan induced rat's paw edema and xylene induced ear swelling in mice.Then the content of interleukin-6(IL-6),interleukin-1(IL-1)and tumor necrosis factor alpha(TNF-?)were measured by enzyme linked immunosorbent assay(ELISA)in rats and mice,respectively.The spectrums obtained in MS2Scan(HPLC-MS/MS)and Scan(GC-MS)mode were imported to the XCMSonline software to have the spectral peak for baseline correction,peak identification and peak alignment,retention time correction,then exported related data matrix to SIMCA-P 11.5.PCA and PLS-DA analysis were carried out with it.According to the value of VIP>1,we screened out differential metabolites related to inflammation.In carrageenan induced rats' s paw edema model,we screened out 25 differential metabolites: Spermine,citric acid,succinic acid,uric acid,creatine,acetoacetate,2-ketoglutaric acid,hippuric acid,anserine,guanidine acetic acid in the urine;and Histamine,carnosine,proline,taurine,phenylpyruvic acid,cysteine,tryptophan,hippuric acid,choline,acetylcholine,four arachidonic acid in the serum;we screened out 14 differential metabolites in xylene induced ear edema: Anserine,phosphoserine,succinic acid,linoleic acid,citric acid,glycine,pyruvic acid,oxaloacetic acid,glutaric acid,2-oxidation of uracil,spermidine,acetic acid,lactic acid in the serum.Then we had an enrichment of related metabolic pathways with Met PA,found out the related metabolic pathways of impact>0.1,aimed to have a construction of metabolic network and further explain the mechanism of inflammation in the whole.Two,study on the mechanism of the anti-fatigue effect of Yi Guan Jian based on metabonomics.In order to further understand the changes of the metabolism of anti-fatigue in mice and explore the metabolic pathway of the organism in the treatment process,we adopted GC-MS and multivariate statistical analysis techniques based on metabonomics to measure the changes of metabolites in fatigue mice,found and screened out biological markers associated with anti-fatigue,surmised the mechanisms related to anti-fatigue.The spectrums obtained in Scan mode were imported to the XCMSonline software to have the spectral peak for baseline correction,peak identification and peak alignment,retention time correction,then exported related data matrix to SIMCA-P 11.5 to have an analysis of PCA and PLS-DA,screened out the metabolites related to inflammation.Differential metabolites were L-2-amino-acetoacetate,taurine,fumaric acid,malic acid,aspartic acid and hippuric acid,acetic acid,glutamine,glycine,serine,urea et al.We had an enrichment of metabolic pathway with the Met PA/MBrole method,and finally built the network map,further carried out the anti-fatigue mechanism of the explanation.We found that Yi Guan Jian decoction in high dose group can significantly improve the swimming time of mice,and observed differences in the content of metabolites such as amino acids,some organic acids and sugars in the disorder in the body were reversed due to the intervention of Yi Guan Jian.At the same time,we also observed the levels of BLA,BUN and SOD in disorder gradually reversed to the normal state by Elisa experiment.To sum up,we concluded that Yi Guan Jian showed strong anti-fatigue effect.Three,study on the mechanism of the Anti-hepatic fibrosis effect of Yi Guan Jian based on metabonomics.In this study,we used HPLC-MS/MS technology based on metabonomics to determine the effect of yin deficiency syndrome of liver fibrosis rats and observed the effect of Yi Guan Jian decoction of nourishing liver and kidney on “Yin” deficiency syndrome.60 male Sprague-Dawlay rats were randomly divided into 6 groups: normal group,model group,positive control group,Yi Guan Jian high-dose group(21.56g/kg),Yi Guan Jian medium-dose group(10.78g/kg)and Yi Guan Jian low-dose group(5.39g/kg).Each group had 10 rats.Except the normal group,hepatic fibrosis model induced by CCl4 was established.After 3 days when the CCl4 injection was stopped,each rat in Yi Guan Jian group was given Yi Guan Jian(2m L/200 g)by intragastric administration,1 time/d.Positive control group was given Colchicine(0.25mg/kg)by intragastric administration,1 time/d.The normal group and model group were fed with same volume of saline daily.After 4 weeks,serum levels of aspartate aminotransferase(AST)and alanine aminotrans-ferase(ALT)were measured by routine methods;the HE staining was used to evaluate the semi-quantitative scoring of liver inflammation(G)and liver fibrosis(S);the features of Yin deficiency syndrome like body weight,tongue humidity,temperature of tongue,drinking,stool and urine were detected.At the same time,PCA and PLS-DA were used to screen out the differential metabolites for an analysis of metabolic profiles.Result: Through pharmacological observation,the serum levels of ALT and AST were significantly tended to the normal group.The vitro indicators such as humidity and temperature has gradually turned to the normal state with the increasing time of administration and in a dose-dependent.Conclusion: the Yi Guan Jian decoction has obvious anti-hepatic fibrosis effect.In conclusion,compound traditional Chinese medicine of YGJ participated in the regulation mechanism of diseases covers the interaction of multiple body organs and systems,it is important to study the mechanism of strengthening the treatment of related diseases.In this study,we used the high throughput analysis method to find the difference in the metabolic spectrum and identified and analyzed the treatment strategy and the intervention mechanism of the diseases.A new experimental basis for the experimental study of Yi Guan Jian decoction was provided.