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AIM: To analyze the chemical ingredients of Qingxin-zishen prescription decoction (QZPD) and predict its main pharmacodynamic substances and mechanism in the prevention and treatment of menopause syndrome (MPS) with the help of high performance liquid chromatography-quadrupole-time of flight mass spectrometry (HPLC-Q-TOF/MS) combined with network pharmacology. METHODS: The chemical ingredients of QZPD were identified after analyzing the retention time, exact mass, secondary mass spectrometry fragmentation and other information obtained from HPLC-Q-TOF/MS and comparing them with the established chemical ingredients database and the literatures. The targets of ingredients in QZPD were predicted by Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) and SwissTargetPrediction database. The disease targets of MPS were obtained through Online Mendelian Inheritance in Man (OMIM) and GeneCards Database. Gene ontology (GO) function enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of potential targets were analyzed with the Metascape database. Cytoscape 3.7.2 software was used to construct the network of active components-key targets-pathways. AutoDockTools 4.2.5 software was applied in the molecular docking verification between the key active components and key targets. RESULTS: A total of 83 components were identified in QZPD and 847 drug targets were predicted. After intersection them with 3 050 disease targets, 395 common targets were obtained. After network topology analysis, 74 key targets were obtained, involving mitogen-activated protein kinase (MAPK), phosphatidylinositol-3 kinase/protein kinase B (PI3K/Akt), transforming growth factor-β (TGF-β) and other signaling pathways. Molecular docking analysis results indicated that 23 key active components, such as berberine, epiberberine, coptisine, geissoschizine methyl ether, liensinine, norcoclaurine, palmatine, quercetin, and luteolin, had good binding activity with several of the key targets. CONCLUSION: This study preliminarily identifies the potential effective chemical ingredients of QZPD, predicts its targets in the prevention and treatment of MPS, which provides supporting information for the further study of the pharmacodynamic substances and mechanisms of QZPD.
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OBJECTIVE@#To explore the pharmacologically active ingredients in granules (TJQW) for treatment of coronavirus disease 2019 (COVID-19) in light of systemic pharmacology.@*METHODS@#We performed database search, literature mining and drug-like index screening to identify the bioactive components in TJQW, the positive drugs for disease treatment and their therapeutic targets. The core disease target was investigated based on the cross-linking interaction of the bioactive components, positive drug and potential disease target, and the target proteins at the key nodes were analyzed by GO and KEGG analyses. Based on the therapeutic targets for COVID-19, virtual screening was conducted to screen the compounds in TJQW and construct the network cross-linking the key bioactive molecules in TJQW, key node targets of the disease, and the related biological pathways.@*RESULTS@#We identified 159 compounds in TJQW and obtained 18 core proteins based on the cross-linking of the bioactive components, positive drugs and disease targets. The key node targets consisted of 22 targets including the latest 4 COVID-19 proteins. Virtual screening results showed that at least 14 compounds could bind with the core disease target proteins. The material basis of TJQW for COVID-19 treatment was explained in multi-pathway, multi-component and multi-target perspectives. In terms of the structural characteristics of the compounds, we screened the top 30 molecules with strong binding with the target proteins, among which flavonoids were the predominant components.@*CONCLUSIONS@#This investigation reveals the therapeutic mechanism of TJQW for COVID-19 involving multiple components, targets and pathways from the perspective of key bioactive molecules, disease key node targets and related biological pathways. We screened 30 active precursors from TJQW, which provides reference for the clinical application and further development of TJQW.
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Humans , Betacoronavirus , Coronavirus Infections , Drug Therapy , Drugs, Chinese Herbal , Medicine, Chinese Traditional , Pandemics , Pneumonia, Viral , Drug TherapyABSTRACT
OBJECTIVE:To study the effects of different processing methods and extraction solvents on the contents of major components in Polygonum multiflorum. METHODS:Decoction of black soybean and water were used to steam the raw P.multiflorum. Water,50％ ethanol,70％ ethanol and 90％ ethanol were used to extract the raw,black soybean steamed,water steamed and commercial processed P. multiflorum respectively. HPLC method was used to detect the contents of gallic acid,2,3,5, 4′-tetrahydroxystilbene-2-O-β-D-glucoside (THSG),emodin and physcion. RESULTS:The contents of 4 major components in 4 kinds of extracts from 3 kinds of processed P. multiflorum were higher than raw sample;the content of gallic acid extracted with water was the highest;the content of THSG extracted with 90％ ethanol was the lowest;the contents of emodin and physcione extracted with 50％ and 70％ ethanol were the higher. CONCLUSIONS:Different processing methods and different extraction solvents had effect on the contents of the main compounds of P. multiflorum. The contents of each components in the processing products didn't show certain regularity.
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Aim To establish a LC-MS/MS method for the determination of pirfenidone(BT)and its major metabolite 5-carboxy-pirfenidone(SBT)in human plasma.Methods Human plasma samples containing BT and SBT,as well as their corresponding deuterium-labeled internal standards pirfenidone-d5(dBT)and 5-carboxy-pirfenidone-d5(dSBT),were precipitated using methanol.Chromatographic separation was carried out on an Agilent ZORBAX SB C18(3.0 mm×100 mm,3.5 μm)column with the mobile phase of water(0.5％formic acid)and acetonitrile(50/50).The detection of analytes was performed on a tandem mass system equipped with an electrospray ionization source in positive ion mode using multiple-reaction monitoring.The MS/MS ion transitions monitored were m/z 185.958→77.1 for BT,m/z 215.944→77.0 for SBT,m/z 190.965→81.1 for dBT and m/z 220.948→99.1 for dSBT.Results There was no remarkable interference in blank solvent,plasma,and there was no mutual interference between analytes or internal standards.The proposed method showed good linearity over the concentration range of 0.020 59～25.14 mg·L-1 for BT and 0.016 73～20.42 mg·L-1 for SBT.The intra-batch and inter-batch precision and accuracy were proved to be acceptable.Human samples kept stable after 4 h at room temperature,the three freeze-thaw cycles and 10,29 and 52 days at-70 ℃,and the processed samples remained stable after 24 h in the autosampler.The average extraction recovery and matrix effect were precise,reproducible and acceptable.Conclusion Our current LC-MS/MS method is proved to be sensitive,accurate and convenient,and could be suitable for the clinical pharmacokinetic studies of BT-related preparations.
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A liquid chromatography-quadrupole-time-of-flight mass spectrometer(LC-Q/TOF-MS) based urinary metabolomic approach was employed to assess the toxicity-alleviation effect of Huangqi oral solution(HOs) on cisplatin-exposed rats and explore its possible mechanisms. Rat toxicity model was developed by multiple intraperitoneal injection of low-dose cisplatin, while HOs was orally administrated to rats simultaneously for 16 consecutive days to attenuate or reduce the cisplatin-induced toxicity. 24-hour urine samples on day 18 were collected and analyzed using LC-Q/TOF-MS to obtain the dataset of urinary metabolites. Principal component analysis(PCA) and orthogonal partial least squares-discriminant analysis(OPLS-DA) were employed to assess the quality of the dataset and screen the potential toxicity-alleviation biomarkers. The serum level of rat creatinine and urea nitrogen on day 20 was determined, and the results showed that successive administration of HOs significantly reduced the cisplatin-induced increase of creatinine and urea nitrogen. PCA cluster analysis clearly demonstrated that HOs could partly improve the CDDP-induced abnormality of metabolic profiling. 35 urinary metabolites were finally screened as the potential biomarkers associated with the toxicity-attenuation effect of HOs, according to the combination of the analysis results of OPLS-DA, t-test and fold change analysis. Further metabolic pathway analysis revealed that HOs could restore the metabolic disorders of amino acid, energy and nucleotide, thereby exerted its toxicity-alleviation effect.
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Aim To establish depression rat model with kidney-yang deficiency and evaluate the related parameters.Methods A total of 24 adult SD rats were divided into control group,mod-el group and self-healing group by body weight.Model group and self-healing group were injected hydrocortisone for 21 days, meanwhile the control group was injected sodium chloride.After modeling,the related indexs detection were taken out:open-field test,sucrose preference test and forced swim test.Then the orbital blood was taken to detect 5-HT,DA and NE by LC-MS/MS.And liver,kidney,spleen,thymus,adrenal gland,testis and epididymis were taken to calculate organ index.Results Compared with the control group,model rats displayed depres-sion and kidney yang deficiency syndrome such as dry hairs, chills,back arched and so on. Correspondingly, the body weight increased slowly.Immobility in FST was significantly in-creased. Sucrose preference, horizontal scores and vertical scores was significantly decreased.Organ index decreased in liv-er,kidney,spleen,adrenal,testis and epididymis.The level of 5-HT and NE was significantly high.Compared with the model group,5-HT,NE and DA were significantly reduced,5-HT and DA were even lower than those of the control group.Conclusion Depression rat model with kidney-yang deficiency could be in-duced by continuous injection of hydrocortisone.
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Clinically, kidney?yang deficiency is one of the main TCM syndromes in patients with major depressive disorder. Warming yang and reinforcing kidney as a therapeutic measure for the treatment of depressive disorder has a?chieved good clinical curative effect. However, there are some problems on the study of kidney?yang deficiency depression. One of the reasons is that the establishment methods of depression animal model with kidney?yang deficiency are not uni?fied. In order to provide reference and further improve the replication method of depression animal models with kidney?yang deficiency, we systematically collected and analyzed the experiment results published in the past 15 years about the depres?sion animal model with kidney?yang deficiency induced by glucocorticoid hormone drugs and find out their similarities and differences.
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Objective To develop an UPLC-MS/MS method for simultaneously determination of five components (adenosine,cytidine,guanosine,mannitol and adenine) in Qiangshenpaidu capsules. Methods The UPLC separation was performed on an Agilent ZOBAX SB-C18(2.1 mm×150 mm,5 μm) column.Isocratic elution was carried out with mobile phase consisting of methanol-0.1%formic acid (5∶95) at a flow rate of 0.2 mL.min-1.The mass spectrometer was operated in the positive ionization electrospray ( ESI) mode using multiple monitoring ( MRM) for analysis of five components. Results Adenosine,cytidine,guanosine,mannitol and adenine were all analyzed with good precision and accuracy. The linear ranges were 35-1 120 ng.mL-1( r=0.998 1) ,10-320 ng.mL-1( r=0.996 4) , 30-980 ng.mL-1( r=0.999 3) , 40-1 280 ng.mL-1( r=0.993 4), 25-800 ng.mL-1(r=0.996 5),respectively.The recoveries of six analytes ranged from 97.4% to 103.6% and the relative standard deviations were all below 4.7%. Conclusion A sensitive,accurate and suitable UPLC-MS/MS method has been developed,and the method could be applied for the determination of adenosine,cytidine,guanosine,mannitol,and adenine in Qiangshenpaidu Capsules.
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Aim To establish a LC-MS/MS method for determination of 5-HT, NE, DA and observe the con-centration of 5-HT, NE, DA in rat plasma of CUMS. Methods Twenty-two male SD rats were divided into control group and model group. Model group was given 9 kinds chronic unpredictable mild stimulating factors every day. 21 days later, behavior and orbital blood were measured before and after modeling. Using benzo-yl chloride as a pre-column derivatization reagent, three analytes and IS were derivatized before LC-MS/MS detection. Change in three kinds of neurotransmit-ter concentration was measured in rat plasma before and after modeling. Results After modeling, com-pared with control group, the weight of rats in model group was declined significantly ( P<0. 05 ) . Horizon-tal scores, vertical scores and sugar consumption were declined significantly ( P <0. 01 ) . Calibration curves of 5-HT, NE, DA were linear between 1. 47 ~752, 1. 75 ~898 , 2. 05 ~1 053 μg · L-1 and LOQ were 1. 47, 1. 75, 2. 046μg·L-1 ,respectively. The recov-ery of 5-HT, NE, DA from plasma was over than 70%, and RSD of inter-day and intra-day assay was limited in 15%. Compared with control group, the con-centration of 5-HT, NE, DA in rat plasma of model group was declined to ( 3. 99 ± 1. 21 ) , ( 6. 24 ± 1. 94), (6. 07 ± 1. 98) μg·L-1(P <0. 01). Con-clusion After making CUMS model of depression, three kinds of neurotransmitters in rat plasma are de-creased.
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Aim To establish a combined method ofβ-glucuronidase hydrolysis and LC-MS-MS analysis for the determination of scutellarein in human plasma, and investigate the pharmacokinetics of scutellarin prepara-tion in healthy male volunteers. Methods Plasma samples were prepared by enzymolysis with β-glucu-ronidase and protein precipitation with methanol. The analytes scutellarein and quercetin ( IS ) were separa-ted on an Agilent ZORBAX SB C18 column ( 2. 1 mm × 150 mm, 5 μm) with the mobile phases consisting of acetonitrile, methanol and water. Multiple reaction monitoring ( MRM) on MS was used to monitor precur-sor to produce ion transitions of m/z 285. 0→136. 8 for scutellarein and m/z 301. 1→120. 8 for IS. After method validation, this method was applied to deter-mine the plasma concentration of scutellarein in 12 male volunteers following single oral administration of 120 mg scutellarin preparation. Drug And Statistic soft-ware (1. 0) was used to process data and the pharma-cokinetic parameters were calculated. Results The assay was validated with linear range of 4 . 01-513. 38μg · L-1 for scutellarein. The intra- and inter-batch precisions ( RSD%) were within 7. 22%. The absolute recoveries were more than 84. 23%. The pharmacoki-netic parameters after a single dose were as follows:Cmax (μg · L-1 ): 159. 97 ± 58. 14; AUC(0-19) (μg · L-1·h):1151. 37 ±279. 80; AUC(0-∞)(μg·L-1· h):1194. 13 ± 264. 51; Tmax ( h):6. 33 ± 1. 67; T1/2 (h):2. 83 ± 0. 60. Conclusion The assay method is proved to be sensitive, accurate and convenient. It can be successfully applied to a pharmacokinetic study of scutellarin in healthy male volunteers.
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This study aimed to explore the impact of depression caused by chronic unpredictable mild stress (CUMS) on in vivo activity of six kinds of CYP450 isoforms in rats. According to 'Katz' method, the model of CUMS was established. Tolbutamide, chlorzoxazone, theophylline, midazolam, omeprazole and dextromethorphan were chosen as probe substrates of CYP2C6, CYP2E1, CYP1A2, CYP3A2, CYP2D1 and CYP2D2 of rats. Plasma concentration of six kinds of CYP450 in control group and model group were determined by LC-MS/MS and computed pharmacokinetic parameters. Consequently, metabolism of theophylline and chlorzoxazone accelerated significantly (P < 0.01), but tolbutamide, dextromethorphan, omeprazole and midazolam had no significant difference. The present study proved that depression caused by CUMS had strong induction to CYP1A2 and medium induction to CYP2E1.
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Aim To investigate the pharmacokinetic effect of aspirin on caffeic acid in dengzhanxixin injec-tion( DI) . Methods Concentration of caffeic acid in rat plasma was detected by LC-MS/MS after rats were given intravenous administration of DI or DI combined with aspirin by gavage. Pharmacokinetic parameters were calculated by DAS 1. 0 pharmacokinetic software. Results In vivo pharmacokinetic models of caffeic acid were two-compartment open models in both the caffeic acid group and the caffeic acid combined with aspirin group. After compatibility, caffeic acid showed a significant increase in T 12β, with a slight decrease in CL. Conclusions Aspirin can reduce metabolic process of caffeic acid in vivo.
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Aim To investigate the influence of sarpog-relate hydrochloride (SH)on the pharmacokinetic pro-file of dextromethorphan (DM),the typical substrate of CYP2D1 /2,in rats when they were administered co-instantaneously.Methods A total of 1 2 SD rats were randomly divided into two groups:the control group (DM,1 0 mg·kg-1 )and the sarpogrelate group (SH, 1 0 mg·kg-1 ;DM,1 0 mg·kg-1 ),which received in-tragastric administration.Plasma samples were collected immediately before and at different time points after drug administration.A LC-MS /MS method was used to determine the concentrations of DM in rat plasma. Pharmacokinetic parameters were analyzed using Drug and Statistics (DAS 2.0).Results There were signif-icant differences in the pharmacokinetic parameters of DM,including T1 2 (2.49 h ±0.93 h vs 1 .47 h ±0.20 h,P <0.05 ),Cmax (325.7 μg·L -1 ±1 33.2 μg· L -1 vs 1 04.5μg·L -1 ±52.4 μg·L -1 ,P <0.05), AUC0 -t(785.5 μg·L -1 ·h ±451 .9 μg·L -1 ·h vs 244.8 μg·L -1 ·h ±1 68.3μg·L -1 ·h,P <0.05) and AUC0 -∞(804.7 μg·L -1 ·h ±445.6 μg·L -1 ·h vs 251 .4 μg·L -1 ·h ±1 73.4 μg·L -1 ·h,P<0.05 )between the two groups.Conclusion SH could significantly inhibit the elimination of DM,the substrate of CYP2D1 /2 in rats.
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Aim To establish a LC-MS/MS method for the determination of hydroxysafflor yellow A ( QA ) in human plasma. Methods After being added into 0. 2M ammonium acetate (1∶1,V/V), QA was extrac-ted using solid-phase extraction technique, and the eluent was directly injected into LC-MS/MS systems. Agilent ZORBAX SB C18 (3. 0 × 100 mm, 3. 5 μm) column and isocratic elution system composing of meth-anol and 0. 2 mM ammonium acetate (70 ∶ 30, V/V) provided chromatographic separation of QA and internal standard isorhamnetin-3-O-neohespeidoside ( SLS) fol-lowed by detection with mass spectrometry. The mass transition ion-pair was followed as m/z 611 . 131→490. 900 for QA and m/z 623. 032→298. 800 for SLS. Results The retention time of QA and SLS was 2. 7 min and 3. 9 min respectively, with no interference in human blank plasma. The proposed method showed good linearity over the concentration range of 8. 57 ~4185 μg·L-1 for QA with a correlation coefficient≥ 0 . 9949 . The lower limit of quantitation was 8. 570 μg ·L-1 . The intra-batch and inter-batch precision and accuracy were within 7%. The average matrix effect ranged from 115. 72% to 119. 06% with RSD less than 5%. The average extraction recovery ranged from 77. 75% to 80. 76% with RSD less than 5%. Stability of human samples after 4 h at room temperature, after the three freeze-thaw cycles and after 31 days at -70℃, and post-preparative stability of the processed sam-ples after 24 h was acceptable. Plasma samples with the concentration beyond the upper quantitation limit could be accurately determined after being diluted using 6. 25 times ( V/V ) of human blank plasma. Conclusion Our current LC-MS/MS method is sensitive, accurate and convenient, and is proved to be suitable for the sys-tematic study on clinical pharmacokinetics of QA.
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<p><b>OBJECTIVE</b>To detect pharmacokinetics of Danshensu Sodium in Danshen dripping solution in Beagles.</p><p><b>METHOD</b>Danshen dripping solution was dripped intravenously into healthy Beagles at a dose of 10 mL x kg(-1). Their plasma samples were extracted with acetic ether, the blood concentrations were determined by HPLC method.</p><p><b>RESULT</b>Danshensu Sodium showed a good linear relationship within the range of 0.225-18.000 mg x L(-1), with the lowest detectable limit of 0.113 mg x L(-1). Its pharmacokinetic parameters were as follows: tmax was 30 min, Cmax was (9.5742 +/- 2.3715) mg x L(-1), t1/2 was (19.23 +/- 2.97) min, CL was (0.0127 +/- 0.0030) L x min(-1) x kg(-1), AUC(0-tn) was (474.954 +/- 95.483) mg x min x L(-1) and AUC(0-infinity) was (482.494 +/- 95.353) mg x min x L(-1).</p><p><b>CONCLUSION</b>The accurate, stable and reliable blood concentration method shows a one-compartment mode of Danshensu Sodium in Beagles.</p>
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Animals , Dogs , Female , Male , Chromatography, High Pressure Liquid , Drug Stability , Drugs, Chinese Herbal , Pharmacokinetics , Lactates , Pharmacokinetics , SolutionsABSTRACT
Aim To establish an UPLC-ESI-MS method for determination of asiaticoside and investigate its application to pharmacokinetic study in rats.Methods Eight rats were given 40 mg·kg~(-1) asiaticoside iv respectively.Drug plasma concentration was determined by UPLC-ESI-MS.Pharmacokinetic parameters were evaluated.Results Calibration curves were linear over 0.038～7.6 mg·L~(-1) and LLOQ was 38 μg·L~(-1),the recoveries of asiaticoside from plasma were larger than 95%,and RSD of inter-day and intra-day assay were below 10%.After iv administration of 40 mg·kg~(-1) asiaticoside,the pharmacokinetic parameters of AUC(0-t),T(1)/(2)β,CL,Vd were (81 443.67±57 156.81) μg·L~(-1)·min~(-1),(23.44±9.60) min,(0.19±0.07) L·min~(-1)·kg~(-1),(8.92±6.68) L·kg~(-1),respectively.Conclusion The method described in this report was sensitive and specific,and suitable for pharmacokinetic studies of asiaticoside in rats.
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<p><b>OBJECTIVE</b>To establish a LC-MS-MS method for quantification of cinnamic acid in human plasma and study its pharmacokinetics after administration of Mailuoning injection at a single and the multiple doses to healthy volunteers.</p><p><b>METHOD</b>Plasma samples were acidified with hydrochloric acid and extracted with ethyl acetate-ether. Cinnamic acid was determined by LC-MS-MS using a ZOBAX SB C18 column with a mobile phase of methanol-water (containing 2 mmol x L(-1) ammonium acetic) (45: 55) at a flow rate of 0.5 mL x min(-1) and detected using ESI with negative ionization mode. Ions monitored in the multiple reaction monitoring (MRM) mode were m/z 146.8 /103.1 [M - H]- for cinnamic acid and m/z 245.6 /126.0 [M - H] - for tinidazole (IS). After administration of Mailuoning injection at a single and the multiple doses via intravenous guttae (ivgtt) to 10 healthy volunteers, the concentration of cinnamic acid in plasma was determined by LC-MS-MS method. The concentration-time curves were simulated by DASver 1.0 and the pharmacokinetic parameters were calculated.</p><p><b>RESULT</b>The calibration curve was linear within the range of 0.5400 microg x L(-1). The LLOQ was 0.5 microg x L(-1) and RSDs of intra and inter day were less than 10%. The concentration-time curves of cinnamic acid were consistent with the two-compartment model. The pharmacokinetic parameters after administration of Mailuoning Injection at a single and the multiple injections were as follows: C(max) (microg x L(-1)), 115.73 +/- 44.31 and 113.79 +/- 25.61; T1/2beta (h), 0.41 +/- 0.087 and 0.52 +/- 0.132; V(L x kg(-1)), 0.519 +/- 0.134 and 0.651 +/- 0.322; CL(L x kg(-1) x h(-1)), 0.899 +/- 0.295 and 0.830 +/- 0.222; AUC(0-tn) (microg x L(-1) x h), 158.64 +/- 56.019 and 166.49 +/- 46.788.</p><p><b>CONCLUSION</b>The developed LC-MS/MS method was sensitive and selective, and there was no interference from endogenous substances. After administration of Mailuoning injection via ivgtt to healthy volunteers, the pharmacokinetic parameters of cinnamic acid between a single and the multiple doses, and between the male and female were no significant difference. There was no accumulation with multiple injections for cinnamic acid, but there were significant individual differences in the pharmacokinetics of cinnamic acid in volunteers.</p>
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Adult , Female , Humans , Male , Young Adult , Chromatography, High Pressure Liquid , Methods , Cinnamates , Blood , Pharmacokinetics , Drugs, Chinese Herbal , Injections , Tandem Mass Spectrometry , MethodsABSTRACT
0.05).Conclusion When given concomitantly as a Cocktail,theophylline,chlorzoxazone and dapsone have no pharmacokinetic interaction,which could be used together to evaluate CYP1A2,CYP2E1 and CYP3A4 activity.
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AIM: The metabolites of scutellarin were studied in gastrointestinal tract.METHODS: Scutellarin was hydrolyzed with hydrochloric acid and ?-glucuronidase.Human intestinal flora and scutellarin were incubated in vitro.The metabolites were identified by UPLC-MS/MS method.RESULTS: Scutellarin could be hydrolyzed with hydrochloric acid and ?-glucuronidase,and could be metabolized by human intestinal bacteria.A main metabolite,scutellarein,also was found in the plasma of healthy volunteers after oral administration.CONCLUSION: There were scutellarin and scutellarein in intestinal before absorption.It was suitable for scutellarin pharmacokinetic studies to determine scutellarein or total aglycone.
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0.05), meanwhile,after being treated with higher-dose Huangkui Capsule,there were no significant differences for theophylline pharmacokinetics in rats,but AUC_(0-24 h) of chlorzoxazone after treatment was 1.75 times larger than that before treatment(P