| Objective: decodes herbal medicine on guizhi,huzhang,by high performance liquid chromatography(HPLC)method with tiger rod-huzhang,guizhi6:1 compatibility of medicinal material base,investigates methodology and discusses knotweed-huzhang,guizhi medicine of 6:1 compatibility to the acute gout arthritis of rats induced by MSU metabolomics,elaborated from the metabolic pathway its possible mechanism.Methods: 1.The giant knotweed – huzhang,guizhi medicine methodology to compatibility of medicines For giant knotweed-huzhang,guizhi the preparation of water extraction liquid,by high performance liquid chromatography(HPLC)method,the giant knotweed,huzhang,guizhiand giant knotweed-huzhang,guizhi for medicine for 2015 pharmaceutical ingredients determination of investigates methodology and the giant knotweed-huzhang,guizhi medicine study of compatibility of material base,2.huzhang,guizhi effects on foot swelling rate determination of MSU model rats 2.1 the MSU model building method in rats To adapt to the environment 1 week before experiment,free drinking water,eating,12 h,lighting,daily room temperature(25 + 2)℃.After 1 week,began to fill the stomach,in addition to the blank group was given saline,the rest of the group according to its category.Dosing volume are 10 ml/kg body weight,once a day,7 days in a row,after 7 days for 1 hour,in addition to the normal group,each group rats were acute gouty arthritis model,a one-time injection of 70 mg/ml MSU mixed suspension of 50 mu,L to summon the joint capsule on the side for the injected standard,model induced acute gouty arthritis.Blank group rats sterile saline injection in the same way.2.2 foot swelling rate determination After the success of the building,in order to observe the giant knotweed-huzhang,guizhi medicine intervention dynamic change law of gouty arthritis,respectively in the different time points after injection(0,1,2,3,4,5,6,8,12,24 h)weeks rats right hind leg joint diameter,repetitive measure 3 times,each time averaged,calculate the joint swelling index of rats(joint swelling index =(joint weeks diameter-initial weeks diameter measurement time points)/initial weeks diameter).3.The giant knotweed-determination of metabonomics of huzhang and guizhi 3.1 build model rat urine and blood collection After building each metabolism cage of rats in a urine collection through 2.1,collect urine(0-12 h),at the same time to join Na N3 0.5 m L solution as a preservative,collect urine in-20 ℃ saved for later use.namely after 12 h,build model rats after orbital venous plexus 2 m L of blood,and after solidification by EDTA(4 ℃,60 min) centrifugal(3500 r/min,15 min,4 ℃)plasma,in-20 ℃ saved for later use.Put rats back after take blood metabolic cages,namely,building 24 h,48 h,72 h after lavage,dosing after 1 h at the end of the time,take blood.3.2 sample preparation Plasma: after thawing at room temperature,in 5 mm NMR nuclear magnetic tube in serum of 400 u L tips,add 50 microliter phosphate buffer(0.2 mol/L Na HPO4 0.2 mol/L Na H2PO4,p H7.4 slightly)and 50 microliter D2O(for lock),oscillation even,under test.Urine: after thawing at room temperature,take 200 edged up phosphate buffer to join in the urine,400 < mu > l tips let stand 20 min after centrifugation,take 500 edged up that add to 5 MMNMR supernatant fluid nuclear magnetic tube,then add the 50 microliter D2 O TSP-d4(containing 0.05% W/V),shock blending,under test.3.3.the NMR experiments NMR data processing of the NMR data via Fourier transform spectrum diagram,data analysis will save the Excel format of data input to the SIMCA-P + software(Umetrics v10.04,Umea,Sweden)multivariate statistical analysis.Average data using centralized(mean centering)or Pareto scale(Pareto scaling)after pretreatment line of principal component analysis(PCA).4.The ELISA test IL-1β,TNF –α In beta,IL-1 TNF alpha MPP add 300 mu L wash wash plate twice,let stand for 15 min after removing lotion,pat dry on blotting paper,add 50 mu L immediately detect buffer,50 mu L standard solution and sample(the sample step must be completed within 15 min),50 mu L detection antibody,seal plate membrane sealing plate,with the constant temperature oscillators(100 r/min)to avoid the glare 2 h incubation,remove the liquid,the MPP 300 mu L wash washing plates with six times(every washing plate must be put on the MPP residual liquid pat dry,then add in 100 mu L mold avidin horseradish peroxide enzyme labeled chain,with the new seal plate membrane sealing plate,in the constant temperature oscillators(100 r/min)to avoid the glare incubation 45 min,take out after remove the liquid,the MPP + 300 mu L wash wash plate 6 times(must be the MPP board each wash the residual liquid on the pat dry),plus 100 m L chromogenic substrate TMB,avoid light incubation 10 ~ 30 min,after joining 100 mu L terminated liquid color have blue to yellow,if the color green or the change of the obvious uneven color,please gently taps plate and frame,so that it is thoroughly incorporated.Within 30 min,using enzyme standard meter(detection wavelength is: 570 nm,570 nm)determination of OD value,record the result of the experiment.Results 1.optimize the content of huzhang,guizhi HPLC determination results According to the experiment of giant knotweed glycosides emodin,resveratrol and emodin,the regression curve equation of methyl ether can be calculated in the giant knotweed knotweed glycosides and resveratrol,emodin,emodin content of methyl ether were 0.0082、0.0054、0.0011、0.0017 g.The giant knotweed glycosides,resveratrol,giant knotweed,emodin,the percentage content of emodin methyl ether is 1.36%、0.91%、1.1%、0.28%.According to the 2015 edition of China pharmacopoeia of giant knotweed rules: giant knotweed emodin(C15H10O5)content in the dry goods should not be less than 0.6%,giant knotweed glycosides(C20H22O8)content shall not be less than 0.15%,cinnamic aldehyde(C9H80)in Cassia twig dry product should not be less than 1.0 %.Another same knotweed medicinal materials,crushing,through 120 mesh sieve.Precision said giant knotweed powder 0.6g,28 ml,and 80% ethanol at room temperature,no glare under ultrasonic 30 min,twice extraction,the extracted liquid,use the filter head filtering,to optimize the alcohol extraction liquid.Adopting the selected chromatographic conditions,to optimize the alcohol extraction liquid of emodin in the peak area of 1512805 in 3.4 emodin in the standard curve regression equation to the content of emodin was 0.0109 g,the percentage content of emodin in the giant knotweed was1.82%.2.foot swelling determination results The experimental rats with acute gouty arthritis model success.Test shows that compared with model group,in building four hours later,the rats began to reduce foot swelling degree,a significant difference between both.3.metabonomics test results 3.1 the plasma and urine of 1 h-nmr spectrum analysis of metabolites Plasma and urine metabolites analysis,of metabolites in plasma lipoprotein,lipid,sugar,amino acid,organic acids and ketone body,etc.In the urine is mainly materials such as amino acids,organic acids and ketone bodies.3.2 rainfall distribution on building 24 hours time change of metabolic fingerprint spectrum of rats The rats induced by using MSU mixed suspension organism physiology and metabolism condition has obvious changes have taken place.To test and verify the reliability of OPLS-DA model,by using the method of cross-validation 7 times,got the OPLS-DA main parameters of the model.Plasema: R2 X = 0.836,R2 Y = 0.949,Q2= 0.832;Urine: R2 X = 0.871,R2 Y = 0.996,Q2 = 0.859.Thus,this paper adopts the model has high reliability.3.3 Associated with acute gouty arthritis of the change of potential biomarkers Potential biomarkers in the plasma has Leucine,Leucine,Lysine(Lysine)and Glutamine(Glutamine),lactic acid(Lactate)and beta Glucose(beta Glucose),urine potential biomarkers with pyruvic acid salt(Pyruvate),Succinate(Succinate),alpha ketone glutaric acid(2-Oxoglutarate),citric acid,Citrate,Trimethylamine oxide(Trimethylamine N-oxide),hippuric acid salt(Hippurate).Compared with model group,control group and giant knotweed huzhang,guizhi group of rats serum leucine,Lysine(Lysine)and Glutamine(Glutamine)and the content of lactic acid(Lactate)decreased obviously,and beta(beta Glucose)in the plasma Glucose levels increased significantly;Also through comparing with the model group,control group and giant knotweed guizhi group in the urine of rats pyruvic acid salt(Pyruvate),Succinate(Succinate),alpha ketone glutaric acid(2-Oxoglutarate)significantly lower and the content of citric acid,Citrate,and hippuric acid salt(Hippurate)in the urine content significantly increased,in the control group in the urine of rats Trimethylamine oxide(Trimethylamine N-oxide)content also increased dramatically,giant knotweed huzhang,guizhigroup content has no obvious change.3.4 Met PA analysis of metabolic pathways Affect the path through the analysis of the Met PA topology analysis and calculation of the influence of the signal path is greater than 0.1 there are five paths,Valine,leucine and isoleucine biosynthesis(Valine,leucine and isoleucine)biosynthesis,glyoxylic acid and dicarboxylic acid metabolism(Glyoxylate and dicarboxylate metabolism),citric acid cycle(Citrate cycle),Alanine,glutamic acid and aspartic acid metabolism(Alanine and aspartate and glutamate metabolism)and Pyruvate metabolism(Pyruvate metabolism).4.The results of IL – 1β and TNF-α test Compared with blank control group,IL-1 beta,TNF alpha inflammation factor content of model group have obvious difference(P < 0.05)and statistically significant;the content of IL-1 beta had obvious difference(P < 0.05)compared with blank control group,but the content of TNF alpha had no difference.Compared with model group,the blank group and giant knotweed guizhi IL-1 beta,TNF alpha inflammation factor content have obvious difference(P < 0.05),statistically significant.Conclusion: 1.Through to the giant knotweed huzhang,guizhi on the methodology of investigation,determine the giant knotweed huzhang,guizhi of emodin in medicine,giant knotweed glycosides Content are in line with the 2015 "Chinese pharmacopoeia" relevant provision,suggests that the giant knotweed huzhang,guizhi medicinal materials conform to the requirement of pharmacopoeia,can be used for drug giant knotweed guizhi decoction to water intervention efficacy studies in rats with acute gouty arthritis.2.The blank control group rats foot swelling rate is significantly lower than the model group rats foot swelling rate,show that the experiment Rat acute gouty arthritis model success.Compared with model group,the building four hours later,giant knotweed water decoction group began to reduce rats foot swelling degree,significant difference between the two appear,show that giant knotweed water decoction group for inflammatory factor stronger inhibitory effect.3.Compared with model group,the giant knotweed of huzhang,guizhi drugs can enhance metabolism of some amino acids in the blood and urine production.Protecting the intestinal microbiota metabolism had certain significance to the treatment of gouty arthritis.4.From the point of inflammatory factor test results,gouty arthritis model is very successful.Compared with model group,the giant knotweed Huzhang,guizhi group has obvious inflammatory factor inhibition. |
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