| AimTo develop the quality control method of WZYZW using UPLC-PDA,and establish a reliable and sensitive method for analyzing chemical compounds and elucidating direcet material base in rat serum after oral administration of Wuzi Yanzong Wan using Ultra-Performance LC coupled with high resolution mass spectrometry.The metabonomics approach was adopted to assess the rat model with kidney deficiency and essence damage,explain the therapeutic effects and mechanism of tonifying Kidney and strengthening essence of WZYZW.MethodsBased on UPLC coupled with high resolution MS technique,the chemical constituents of WZYZW were systematic screened and identified in vitro and in vivo,the quality stability of WZYZW was evaluated.An estabilished UPLC-MS/MS based metabolomic method with pattern recognition analysis(PCA,OPLS-DA and VIP)was applied to investigate the rat model with kidney deficiency and essence damage for exploring and identifying biomarkers,meanwhile,the Pathway Topological Analysis tool was employed to measure the most correlation pathways.The model was evaluated based on clinical and metabolomic method.The developed UPLC-ESI MS based metabolomic method coupled with clinical data was also introduced to elucidate the therapeutic effects and the mechanism of tonifying Kidney and strengthening essence of WZYZW.Results1.Chemical constituents analysis and and quality evaluation of WZYZWThe Chromatographic Fingerprint of methanol extract of WZYZW was established using UPLC-PD A technique and 20 common peaks were determined,then a professional similarity evaluation software was employed to conduct similarity investigation,as a result,the similarity among 10 batches of WZYZW was favorable,It provide a quality basis for subsequent studies.In this work,a rapid and sensitive method for systematic screening and identifying the chemical constituents of WZYZW in both positive and negative ion modes have been developed using Ultra-Performance LC coupled with ESI-linearion trap-Orbitraptandem mass spectrometry(UPLC-ESI-LTQ-Orbitrap-MS)for the first tme.Based on the chromatographic and spectrometric data,referring to literatures,we could tentatively identify 109 compounds,including organic acids,flavonoids,phenylpropanoids,alkaloids and terpenoids.35 ingredients were identified from flavonoids,34 ingredients were from phenylpropanoids,17 ingredients were from organic acids,11 ingredients were from terpenoids,and 8 ingredients were from alkaloids.It revealed that six categories of compounds all validated contributed to chemical composition in vitro of WZYZW.2 Analysis of the constituents absorbed in serumThe preparation method of plasma sample was developed.A validated UPLC-ESI-LTQOrbitrap-MS method was developed to simultaneously analyze the chemical constituents of WZYZW in vitro and in vivo,and the results from pattern recognition analyses have proved that the UPLC-ESI-LTQ-Orbitrap-MS method illustrated well reproducibility and—discrimination which can be apply to analyse the constituents absorbed in serum.The profiles of serum sample from control and post dosing WZYZW were achieved by UPLC-ESI-LTQ-Orbitrap-MS analyses,then subjected to MetQuest analyses.Based on the chromatographic and spectrometric data,databases,referring to literatures,we could tentatively identify 43 compounds,20 ingredients were from prototypes,and 23 metabolites.Prototypes were Gallic acid,Chlorogenic acid,Quinic acid,Caffeic acid,Hyperoside,Quercetin,Abromine,Atropine,Geniposidic acid,Schisandrin,Schisandrol B,Schisantherin A,Deoxyschizandrin,Schizandrin B,Schisanhenol,Astragalin,Isoquercitrin,Isorhamnetin,Iuteolin and Apigenin.Metabolites were Demethyl-schisandrol B,Hydroxyl-schisantherin A,Dihydroxyl-deoxyschizandrin,Hydroxyl-schizandrin B,Demethyl-schisanhenol,Gallic acid-O-glucuronide,Chlorogenic acid-O-glucuronide,Caffeic acid-O-glucuronide,Methyl-caffeic acid,Methyl-hyperoside,Quercetin-O-glucuronide,Methyl-quercetin,Ferulic acid-O-glucuronide,Kaempferol-O-glucuronide,Kaempferol-O-sulfate,Methyl-kaempferol,Isoquercitrin-O-glucuronide,Isorhamnetin-O-sulfate,Iuteolin-O-glucuronide,Methyl-baicalein-O-glucuronide,Methylbaicalein,Homoplantaginin-O-glucuronide,Hydroxyl-salicylicacid-O-glucuronide.The result provide a basic data for the further studies on effective constituents and the effecting mechanism of WZYZW.3.The duplication and evaluation of the rat model with kidney deficiency and essence damageThe rats with unilateral testicular were used to duplicate the model with kidney deficiency and essence damage.The testis index,prostate index,seminal vesicle index,the activity of SOD andMDA,the level of FSH,T and LH in plasma,the activity rate and quantity of sperm were changed significantly in kidney deficiency and essence damage rat model.Besides,for the clinical HE diagnosis,the teste and the adrenal gland were found pathological change.These results indicate that the model was successfully reproduced.The procedure of sample preparation and the operation parameters of UPLC/Q-TOF-MS were optimized,and the experimental method was validated.Then,the developed metabonomics method was adopted to investigate the urine and plasma samples of the model rats.The comparison among profiles was performed with Pattern recognition analyses(PCA,OPLS-DA and VIP).The results were as follow:in scores plot,it is indicated that control urine and plasma samples were obviously separated from the model rats,and the obviouse discrimination was visually observed.43 biomarkers were identified as 8-isoprostanes,sphingosine,cystathionine,dehydroepiandrosterone sulfate,two methyl arginine,3-hydroxy decanonic acid,adenosine,succinyl prolyl phenylalanine,2,8-two hydroxy beta-Dglucuronide,4 ene valproic acid,coenzyme Q1,3-methyl histidine,5-ketocapric acid,3a,6a 7b-,three hydroxy-5b-formic acid and 3-to bile duct hydrogen glucose and two hydroxy mandelic acid,n-octanoyl glucuronide,4-hydroxy cyclohexyl acetate,a-ketoacid octanoic acid,3-hydroxy decanoic acid,azelaic acid,N-acetylcysteine,ketoglutarate,epinephrine,pyridoxamine,sebacic acid,adipic acid,two methyl Xin Ersuan 2-,hydroxyl octanoic acid,methoxyepinephrine,4,6-two hydroxyquinoline,5'-methylthioadenosine,3-methoxy-4hydroxy phenylglycol sulfate,N-acetyl-7-oxygen-acetylneuraminic acid and pyridoxal;blood potential biomarkers in 8,package Including cholic acid,glycocholic acid,3-hydroxy alkyl acid,twelve double norepinephrine,sixteen acyl carnitine,testosterone,twenty-four carbon six acid and linoleic acid.Their related 15 metabolic pathways were aslo founded as Tyrosine metabolism,Vitamin B6 metabolism,Sphingolipid metabolism,Cysteine and methionine metabolism,Primary bile acid biosynthesis,Steroid hormone biosynthesis,Citrate cycle(TCA cycle),Alanine,aspartate and glutamate metabolism,D-Glutamine and D-glutamate metabolism,Nitrogen metabolism,Pentose and glucuronate interconversions,Butanoate metabolism,Glycine,serine and threonine metabolism,Fatty acid metabolism and Tryptophan metabolism.4.Study on the mechanisms of tonifying Kidney and strengthening essenceThe model rats were treated with WZYZW,GWS and WZYZW+GWS,respectively.The testis index,prostate index,seminal vesicle index,the activity of SOD andMDA,the level of FSH,T and LH in plasma,the activity rate and quantity of sperm in model were obviously regulated by WZYZW,besides,the pathological change of teste and adrenal gland were also alleviated.WZYZW showed the strongest effects,compared with positive medicine,there was no significant difference.WZYZW had little synergism with GWS.WZYZW corrected 18 biomarkers and 15 metabolic pathways to normal,GWS corrected 6 biomarkers and 7 metabolic pathways to normal,WZYZW+GWS corrected 7 biomarkers and 8 metabolic pathways to normal,WZYZW showed the strongest effects,compared with positive medicine,there was no significant difference.WZYZW had little synergism with GWS.WZYZW corrected 18 biomarkers were Dihydroceramide,sphingosine,cysteine sulfur ether,dehydroepiandrosterone sulfate,3-to hydrogen-L gulonic acid,alpha ketoglutarate,epinephrine,pyridoxamine,methoxy to norepinephrine,6-bis(8-hydroxyquinoline),5'-a sulfur adenosine,pyridoxal,cholic acid,sweet ammonia acid,palmitoyl meat poisonous alkali and testosterone.the related metabolic pathways were demonstrated as Tyrosine metabolism,Vitamin B6 metabolism,Sphingolipid metabolism,Cysteine and methionine metabolism,Primary bile acid biosynthesis,Steroid hormone biosynthesis,Citrate cycle(TCA cycle),Alanine,aspartate and glutamate metabolism,D-Glutamine and D-glutamate metabolism,Nitrogen metabolism,Pentose and glucuronate interconversions,Butanoate metabolism,Glycine,serine and threonine metabolism,Fatty acid metabolism and Tryptophan metabolism.which were systematically contributable to elucidate the therapeutic effects and the mechanism of tonifying Kidney and strengthening essence of WZYZW.ConclusionThis is the first report on systematic analysis of WZYZW.The quality control method was established which is validated used to quality evaluation of WZYZW.This study has developed a UPLC-MS/MS method for systematic screening and identifying the chemical constituents of WZYZW in vitro and in vivo,the direcet material base was tentatively identified.Besides,the developed UPLC-ESI MS based metabolomic method has been apply to assess the model and elucidate the therapeutic effects and the mechanism of tonifying Kidney and strengthening essence of WZYZW.This study has may provide essential data for further research and clinical evaluation of WZYZW... |
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