Study On The Mechanism Of Ginger Processing And Compatibility Based On GC-MS Metabolomics

Processing and compatibility are two major characteristics of Chinese Medicine(CM).Chinese herbral medicine must be processed into Chinese herbal slices before they are used in clinic.But Chinese herbal compound is composed of Chinese herbal slices under the theory of CM compatibility.Therefore,it is in accord with practical clinic to study the processing of CM in CM prescription.Ginger is one of the most commonly used in CM clinic,and its commonly processd products include fresh ginger,dried ginger and stir-frying ginger,which have different efficacy.The predecessors said that “ fresh ginger ‘go’ but not ‘hold’;dried ginger not only ‘go’ but also ‘hold’;stir-frying ginger ‘hold’ but not ‘go’ ”.It can be seen that different processed products of ginger have different effects.The preliminary study of the research group showed that there were significant differences in the contents and pharmacodynamics of gingerols in different processed ginger products.The changes of component caused by processing may be one of the main reasons for the differences in clinical efficacy.Chinese doctors often use CM prescription in clinic.The results of processing changes of single herb can only partly reflect the actual situation.It is more in line with clinical practice to study processing in CM prescription.Linggan Wuwei Jiangxin(LGWWJX)prescription originated from the “Jingui Yaolüe”(“Synopsis of Golden Chamber”),which has been used to treat cold asthma over a thousand year in clinic.In this prescription,dried ginger is monarch drug,by its characteristics of “warming the stomach to dispel cold,warming the lung to resolve fluid”.From the perspective of the efficacy of processed ginger products,fresh ginger can reduce phlegm and stir-frying ginger can warm spleen and stomach.It is worth discussing the effect and mechanism of different processed ginger products on this prescription.Objective Based on the previous research of our group,the model of cold asthma rats were established by using “ovalbumin(OVA)+ice bath”.We compare the effects of pharmacodynamics and metabolomics of ginger processing and compatibility against cold asthma rats.The scientificity and rationality of ginger processing and compatibility was discussed from the perspective of pharmacodynamics and metabolomics.Which provided a theoretical basis for the research of the mechanism of ginger processing and compatibility,and provided a basis for clinical rational selection of ginger processed products.Methods 1.The rat model of cold asthma was established by “OVA+ice bath”.Rats were randomly divided into control group,model group,Linggan Wuwei(fresh/dried/ stir-frying)Jiangxin decoction group and positive control Guilong Kechuanning group,8 of each group.General morphology observation,body weight,visceral index,HE staining of rats’ s lung tissue,the counting of eosinophils,mononuclear,neutrophils in bronchoalveolar lavage fluid(BALF)and the determination of IgE,IL-4 and IFN-γ in serum were used to evaluate the effects of the model of cold asthma rats and the efficacy of Linggan Wuwei(fresh/dried/stir-frying)Jiangxin decoction against cold asthma rats.2.Gas chromatography-mass spectrometry(GC-MS)was used to determine metabolites of serum and urine in each group.Principal component analysis(PCA),partial least squares discriminant analysis(PLS-DA)and orthogonal partial least squares discriminant analysis(OPLS-DA)were established.OPLS-DA was used to screen differential metabolites of serum and urine between control and cold asthma model group by variable importance projection(VIP,VIP>1)and T(p<0.05),finally comparing with NIST library(Match>700).To structure metabolic pathways for differential metabolites using Metaboanalysis database.Pearson correlation analysis was used between pharmacodynamic indicators and differential metabolites,presented as a heat map.3.The serum and urine metabolites of each group were determined by GC-MS.The variable importance projection(VIP)was obtained by orthogonal partial least squares discriminant analysis(OPLS-DA),VIP<1 and T(p<0.05)is a criterion for screening differential metabolites of serum and urine between cold asthma model group and Linggan Wuwei(fresh/dried/stir-frying)Jiangxin decoction group,then comparing with the NIST library(Match>700).To explore the mechanism of ginger processing and compatibility against cold asthma.Results 1.After establishing cold asthma model according to the above method,the rats showed reduced water and food intake,weight loss,sneezing,swollen claws,dull hair,and some rats showed a series of cold asthma symptom similar to that of CM.Compared with the control group,the HE staining of model group rat’s lung tissue were significantly changed: thickening of the bronchial wall,infiltration of inflammatory cells around the bronchi,stenosis of the lumen and formation of mucus plugs;the number of eosinophils,mononuclear and neutrophils in BALF were increased significantly(P<0.01);the levels of IgE and IL-4 in serum increased significantly(P<0.01),and the levels of IFN-γ decreased significantly(P<0.01);heart,liver,spleen and lung have significant difference(P<0.05 or P<0.1).After the intervention of Linggan Wuwei(fresh/dried/stir-frying)Jiangxin decoction and Guilong Kechuanning,the pathological changes of rats’ s lung tissue were significantly improved: no obvious mucous plug was observed in each drug-administration group,the bronchial wall became thinner;the infiltration of inflammatory cells around the bronchus decreased,and lumen stenosis was improved.The counting of BALF,the level of IgE,IL-4,IFN-γ in serum,heart,liver,spleen and lung indexes all showed a trend to the control group.2.Fifteen serum metabolites related to cold asthma were screened.Compared with control group,the level of propanoic acid,heptadecane,d-(-)tagatose and hexadecanoic acid increased significantly(P<0.05).The level of glycerol,glycine,phosphate(3:1),d-pinitol,d-mannitol,d-galactose,d-glucose,glucopyranose,myo inositol,octadecanoic acid and lactose reduced significantly.Twenty two urine metabolites related to cold asthma were screened.Compared with control group,the level of benzoic acid,butanedioic acid,hexanedioic acid,β-d-glucopyranose and citric acid upregulated dramatically(P<0.05).The level of phosphate(3:1),3,4-dihydroxybutanoic acid,l-threonic acid,d-(+)arabitol,l-(-)fructose,pentanedioic acid,ribonic acid,benzeneacetic acid,d-glucose,d-galactose,glucaric acid,myo inositol,l-ascorbic acid,β-glucuronid,d-lactose,maltose and d-(+)cellobiose downregulated markedly(P<0.05).The major metabolic pathways involved include starch and sucrose metabolism,glactose metabolism,tricarboxylic acid cycle metabolism,glyoxylate and dicarboxylate,glycerolphospholipid metabolism,glycine,serine and threonine metabolism and inositol phosphate metabolism.It is mainly involved in metabolites and metabolic pathways related to energy metabolism and oxidative stress.3.After the intervention of Linggan Wuwei(fresh/dried/stir-frying)Jiangxin decoction,compared with the model group,the level of propanoic acid,glycine,phosphate,heptadecane,d-galactose,d-glucose,glucopyranose,myo inositol,hexadecanoic acid,octadecanoic acid in serum,benzoic acid,butanedioic acid,l-(-)-fuctose,pentanedioic acid,citric acid,l-ascorbic,d-lactose and maltose in urine have different degrees of callback.Linggan Wuwei(dried)Jiangxin decoction has the most powerful effect,which indicated that when ginger was processed into dried ginger,the effect of Linggan Wuwei Jiangxin decoction on the improvement of abnormal metabolic state of cold asthma rats was the best.Conclusion “OVA+ice bath” can successfully establish the model of cold asthma.Decreased basal metabolic rate and inflammatory response were observed in cold asthma rats.The disturbance of many metabolites in serum and urine also suggested that the metabolism of cold asthma rats was abnormal.Linggan Wuwei(fresh/dried/stir-frying)Jiangxin decoction can improve the pharmacodynamics index of rats and adjust potential biomarkers in serum and urine,indicating that Linggan Wuwei(fresh/dried /stir-frying)Jiangxin decoction have the function of regulating abnormal metabolism of cold asthma.Linggan Wuwei(dried)Jiangxin decoction has the most powerful effect,which indicates that it is more reasonable to process ginger into dried ginger in Linggan Wuwei Jiangxin decoction against cold asthma.We speculated that the processing mechanism may by regulating pathways of starch and sucrose metabolism,glactose metabolism,tricarboxylic acid cycle metabolism,glyoxylate and dicarboxylate,glycerolphospholipid metabolism,glycine,serine and threonine metabolism and inositol phosphate metabolism,which involved in energy metabolism and oxidative stress.