| Objective:Based on the research method of spectrum-effect relationship,the spectrum-effect-dryness relationship of Fructus aurantii(FA)was investigated so as to screen its bioactive components including regulating-qi and dryness effects.To sort out the efficacy(regulating-qi)and dryness quality markers(Q-marker)of FA,and provide scientific basis for the clinical application and quality evaluation of FA.Methods:1.HPLC fingerprints of five fractions of FA were established by high performance liquid chromatography(HPLC)to evaluate and ensure the homogeneity,stability and comparability of the samples.UHPLC-Q-TOF/MS(ultra-performance liquid chromatography quadrupole time of flight mass spectrometry)fingerprints of different fractions of FA were established by UHPLC-Q-TOF/MS.The chemical components were identified to provide the basis for the subsequent screening of active and dryness substances.2.The effect of different fractions of FA on gastric motility in normal KM mice were investigated including the whole gastrointestinal propulsion time test,intestinal propulsion and gastric emptying test.3.The model rats of gastrointestinal motility disorder(GMD)were established by intraperitoneal injection of L-arginine hydrochloride.The biochemical indexes such as 5-HT,SP,MLT,GAS and VIP in rat plasma were used to evaluate the gastric motility effects of different fractions of FA on GMD rats.4.The dryness of FA was characterized by the amount of drinking water,AQP2 content in kidney,AQP3 content in colon and AQP5 content in submandibular gland of normal mice and GMD rats.5.Principal component analysis(PCA),Pearson correlation analysis and orthogonal partial least squares analysis(OPLS)were used to study the spectrum-effect-dryness relationship of different fractions of FA.The data of fingerprint,pharmacology experiment and dryness effect experiment were imported into SPSS 21.0 software for PCA analysis,and the Pearson correlation coefficients of chromatographic peak areas with pharmacodynamics and dryness were also calculated.The data of fingerprint,pharmacology experiment and dryness effect experiment were imported into SIMCA-P 14.1 software,the parameters such as score,load distribution,the variable weight value(vip)and regression coefficient(coefficent)of common peaks in the fingerprint were obtained after model fitting.According to the vip value and regression coefficient order,the components that contributed greatly to the regulating qi and dryness of FA were screened out.6.Combined with our previous studies on serum pharmacochemistry and pharmacokinetics,the quality marker of regulating qi and dryness of FA were preliminarily established by the ternary correlation "components-efficacy-pharmacokinetics".7.Using the network pharmacology research method,the key regulating-qi and dryness targets of the Q-markers were explored by target prediction,gene ontology enrichment and signal pathway analysis,and the main action pathways and biological processes were analyzed.Then,the network relationship of components,targets and pathways of FA was explored,and molecular docking technology was used to verify the results.Finally,the regulating-qi and dryness mechanism of the Q-markers was elucidated.8.The effectiveness of quality markers was verified by smooth muscle test in vitro and gastric emptying and intestinal propulsion test in vivo.9.The measurability of Q-markers in FA was evaluated with HPLC fingerprint qualitative method and multi-index content determination method,and the content determination method was established for the Q-markers.Results:1.There were 74 common peaks in petroleum ether fraction,71 common peaks in chloroform fraction,72 common peaks in ethyl acetate fraction,72 common peaks in n-butanol fraction and 58 common peaks in water fraction.2.The results of gastric motility test in normal mice showed that different fractions had a certain degree of promoting gastrointestinal motility.In the whole gastrointestinal propulsive movement time test of mice,the defecation promoting effects of five different fractions were as follows: ethyl acetate > petroleum ether >n-butanol > chloroform > water.In the experiment of gastric emptying and intestinal propulsion in mice,the effects of five fractions on gastric emptying and intestinal propulsion in mice were as follows: ethyl acetate > petroleum ether >chloroform > n-butanol > water.The ethyl acetate fraction of FA was the main effective fraction for promoting defecation,gastric emptying and intestinal propulsion in mice.3.The results of pharmacodynamic experiment in GMD rats showed that different extracts could increase 5-HT,SP,MLT and gas,and decrease the expression of VIP.The order of effect on 5-HT expression was ethyl acetate > petroleum ether >n-butanol > chloroform > water;the order of effect on SP expression was n-butanol > ethyl acetate > petroleum ether > chloroform > water;the order of effect on MLT and GAS expression was ethyl acetate > petroleum ether >chloroform > n-butanol > water;the order of effect on VIP expression was: water >n-butanol > chloroform > petroleum ether > ethyl acetate.Ethyl acetate fraction of FA was the main effective fraction to promote 5-HT,MLT and GAS and inhibit VIP expression,while n-butanol fraction was the main effective fraction to promote SP expression.4.The results showed that the expression of AQP2 and AQP5 was significantly decreased and the expression of AQP3 and the amount of drinking water were significantly increased in the partial fractions groups,which was significantly different from the control group.The order of effect on AQP2 expression was water > chloroform > n-butanol > petroleum ether > ethyl acetate;the order of effect on AQP3 expression was ethyl acetate > petroleum ether > n-butanol >chloroform > water;the order of effect on AQP5 expression was water >n-butanol > chloroform > petroleum ether > ethyl acetate.Ethyl acetate fraction of FA was the main source of dryness in mice.5.The results also showed that the expression of AQP2 and AQP5 was significantly decreased and the expression of AQP3 and the amount of drinking water were significantly increased in the partial fractions groups,which was significantly different from the control group.The order of effect on AQP2 expression was water > n-butanol > chloroform > petroleum ether > ethyl acetate;the order of effect on AQP3 expression was ethyl acetate > petroleum ether > n-butanol >water > chloroform;the order of effect on AQP5 expression was water >n-butanol > chloroform > petroleum ether > ethyl acetate.Ethyl acetate fraction of FA was also the main source of dryness in GMD rats.6."Spectrum-effect" relationship study showed that the regularing qi chemical components of FA were synephrine,N-methyltyramine,isovitexin,naringenin-7-O-glucoside,naringin,narirutin,naringenin,hesperidin,neohesperidin,hesperitin-7-O-β-D-glucoside,hesperetin,obacunoic acid-17-β-D-glucoside,(2S)-6’’-O-acetylprunin,nxypeucedanin,poncirin,isovitexin-7-O-xylocoside-2’’-O-arabinoside,apigenin,obacunoic acid,eriodictyol-7-O-glucoside and nomilin;"spectrum-dryness" relationship study showed that the dryness chemical components of FA were brutieridin,neohesperidin,hesperidin,7-O-6’’-malonylnaringenin,rhoifolin,nomilin,hesperitin-7-O-β-D-glucoside,byakangelicol,isovitexin,3’-Methoxyl isovitexin,hesperetin,naringin,narirutin,(2S)-6’’-O-acetylprunin,naringenin,naringenin-7-O-glucoside,obacunoic acid-17-β-D-glucoside,eriodictyol-7-O-glucoside,poncirin,oxypeucedanin and ioquatoside;"spectrum-effect-dryness" relationship study showed that isovitexin,naringin,narirutin,naringenin,naringenin-7-O-glucoside,neohesperidin,hesperidin,hesperitin-7-O-β-D-glucoside,hesperetin,(2S)-6’’-O-acetylprunin,poncirin,oxypeucedanin,obacunone-17-O-β-D-glucoside,eriodictyol-7-O-glucoside and nomilin were the dual components of regulating qi and dryness of FA.7.Combined with our previous studies on serum pharmacochemistry and pharmacokinetics,fifteen regulating-qi Q-markers were preliminarily established according to the ternary correlation of "components-efficacy-pharmacokinetics",including synephrine,N-methyltyramine,umbelliferone,narirutin,naringin,naringenin,hesperidin,neohesperidin,hesperetin,meranzin hydrate,poncirin,marmin,nobiletin,tangeretin and auraptene.In the same way,eight dryness markers were determined including narirutin,naringin,naringenin,hesperidin,neohesperidin,hesperetin,poncirin and 3,5,6,7,8,3’,4’-heptamethoxyflavone.8.The results of network pharmacology experiments showed that the mechanism of15 quality markers of regulating qi of FA’s therapeutic effect may regulate the signal pathways of PI3K-Akt,serotonergic synapse,Rap1,RAS,erb B,HIF-1 and vascular endothelial growth factor through AKT1,ESR1,EGFR,GAPDH,CCND1,HRAS,SRC and VEGFA,so as to participate in the regulation of nitric oxide biosynthesis and smooth muscle cells,the positive regulation of proliferation,signal transduction,ERK1 and ERK2 cascade and the regulation of nitric oxide synthase activity;the mechanism of 8 quality markers of dryness of FA’s dryness may regulate PI3K-Akt,prolactin,Rap1,RAS,HIF-1,AMPK and other signaling pathways through 41 dryness related targets,such as HRAS,IGF1 R,KDR,ESR1,MMP9,PTGS2 and VEGFA,so as to participate in the regulation of positive regulation of nitric oxide biosynthesis,smooth muscle cell proliferation,RNA polymerase II promoter transcription and cell proliferation.9.The results of in vitro small intestinal smooth muscle test showed that 16 quality markers such as synephrine,N-methyltyramine,umbelliferone,narirutin,naringin,naringenin,hesperidin,neohesperidin,hesperetin,meranzin hydrate,poncirin,marmin,nobiletin,3,5,6,7,8,3’,4’-heptamoxyflavone,tangeretin and auraptene,had the same effect on promoting gastrointestinal motility as the water extract and alcohol extract of FA.In vivo gastric emptying and intestinal propulsion experiments showed that different extracts of FA had different degrees of promoting gastrointestinal motility.10.The HPLC method for content determination is simple,stable and reliable for the determination of 14 quality markers included umbelliferone,narirutin,naringin,naringenin,hesperidin,neohesperidin,hesperetin,meranzin hydrate,poncirin,marmin,nobiletin,3,5,6,7,8,3’,4’-heptamoxyflavone,tangeretin and auraptene.The content of 10 batches of FA from different producing areas and different harvest time was determined and the results showed that the contents of umbelliferone,narirutin,naringin,naringenin,hesperidin,neohesperidin,hesperetin,meranzin hydrate,poncirin,marmin,nobiletin,3,5,6,7,8,3’,4’-heptamoxyflavone,tangeretin and auraptene in 10 batches of FA were as follows: 0.08300～0.3874,0.8995～13.99,40.96～83.42,0.8478～4.130,31.70～52.12,0.4164～2.390,1.1390～4.328,0.1162～0.1461,0.08930～0.2639,0.07460～0.1914,0.1589～0.8841,0.04150～0.08230,0.1627～0.7031,0.05410～0.1980 mg/g,respectively.Conclusions:Different extracts of FA have significant stimulating-gastrointestinal-motility effect on normal mice and GMD model rats,and also have a certain dryness,among which the n-butanol fraction and ethyl acetate fraction have the strongest effect,and the ethyl acetate fraction have the strongest dryness.Fifteen compounds in FA including synephrine,N-methyltyramine,umbelliferone,narirutin,naringin,naringenin,hesperidin,neohesperidin,hesperetin,meranzin hydrate,poncirin,marmin,nobiletin,tangeretin and auraptene can be used for the regulating-qi quality markers.The compounds including narirutin,naringin,naringenin,hesperidin,neohesperidin,hesperetin,poncirin,and 3,5,6,7,8,3’,4’-heptamethoxyflavone may be the potential dryness Q-markers.This work can provide experimental basis for the clinical application and quality evaluation of FA. |
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