| ObjectiveDiabetic nephropathy(DN)is one of the most serious complications of diabetic,and is also a growing worldwide epidemic disease,which has become a primary cause of end-stage renal disease(ESRD).Therefore,further study of the pathogenesis and treatment of diabetic nephropathy has become a research hotspot at home and abroad.Sanziguben granule(SZGB),an experienced prescription,contain Rosa laevigata Michx,Gynostemma Pentaphyllum,Phyllanthus Emblica and Fructus Schizandrae,is widely used in clinical medication for the treatment and prevention of diabetic nephropathy.Therefore,the purpose of this paper is to study the preparation of Sanziguben Granule and its mechanism of protective effect on experimental diabetic nephropathy.Methods1.The preparation technology research of Sanziguben GranuleThrough using orthogonal experiment design method and with the total polysaccharide content and fructus schisandrae alcohol armour content as the comprehensive evaluation index to optimize the extraction process.With moisture absorption,solubility and formability as evaluation index to optimize the commonly used supplementary material such as dextrin,mannitol,lactose and soluble starch.By comparing the color of the particle uniformity and viscosity size,determine the amount of material size.With moisture absorption,solubility and formability as evaluation index to determine the dosage ratio between dextrin and mannitol.2.The fingerprint study of Sanziguben GranuleThrough optimizing the mobile phase,detection wavelength and velocity to determine the chromatographic conditions.Spectrum analysis of using traditional Chinese medicine chromatographic fingerprint similarity evaluation system was to establish the fingerprint of Sanziguben Granule.3.The serum pharmacochemistry study of Sanziguben GranuleUsing serum pharmacochemistry to track bioactive componets of Sanziguben Granule by UPLC/Q-TOF-MS/MS.Sanziguben granule was dissolved in distilled water at a concentration of 9.6 g/kg for oral and thereafter these doses were fed to the rats using gavages.Before administration and at intervals of 0.5h,1h,2h and 4h after oral administration of the extract,approximately 500μL of blood sample was drawn from the jugular vein into a vial with a fraction collector and then serum was separated.Blood sample were removed protein,sample concentration and further to analysis with UPLC/Q-TOF-MS/MS.4.Quantitative study of Sanziguben GranuleDetermining the total polysaccharides content of 15 batches of Sanziguben Granule by ultraviolet spectrophotometer method.Determining gallic acid and corilagin content of 15 batches of Sanziguben Granule by HPLC.The method validation of quantitative analysis was investigated by precision,stability,repeatability and recovery.5.The Sanziguben Granule’s mechanism study of protective effect on experimental diabetic nephropathy ratsTo accelerate the development of diabetic kidney disease,rats underwent right uninephrectomy coupled with intraperitoneal injection of streptozotocin(STZ).After treatment with Sanziguben Granule and fosinopril,observing the influences of Sanziguben Granule on experimental DN rats.Blood glucose and 24-h urinary were detected in two weeks interval.Serum was separated for detection of total cholesterol(TC),triglycerides(TG),serum creatinine(Cr),serum urea(BUN),glutathione(GSH),malondialdehyde(MDA)and catalase(CAT).The sections of kidneys tissue were stained by hematoxylin-eosin(H&E),periodic acid-Schiff(PAS)and Masson’s trichrome to evaluate for glomerulosclerosis and tubulointerstitial damage.Western blotting was used to detect 4-HNE,HO-1,Nrf2,NQO1,Bcl-2,Cleaved caspase-3,E-cadherin,Vimentin and a-SMA expression of renal tissue,which researching Sanziguben Granule’s mechanism study of protective effect on experimental diabetic nephropathy rats.Results1.The Sanziguben Granule of preparation technology:all four Chinese medicines are mixed together with twelve times the volume of water and decocted three times where each period is sustained for 1 hour.The water decoctions are mixed,filtrated and concentrated to the density of 1.30～1.35 to get extractum.2 times weight of accessories(dextrin:mannitol 1:1.5)are added and one-step-granulating is used to get the Sanziguben Granule.2.Chromatographic fingerprints from 15 batches of Sanziguben Granule was obtained and the similarity values were all upon 0.93.Each peak relative to all sample chromatograms was called the "common peak",and there by 18 common peaks were observed in all 15 batches and 4 common peaks were confirmed.3.Using serum pharmacochemistry to track bioactive components of Sanziguben Granule by UPLC/Q-TOF-MS/MS,which turned out to be gallic acid and corilagin.4.Linear relationship of total polysaccharides in the range of 1.9168μg/mL-14.3760μg/mL(R2=0.9988)was good,average recovery was 98.81%(RSD=2.7%,n=6).Linear relationship of gallic acid in the range of 4.94μg/mL-79.0μg/mL(R2=0.9996)was good,average recovery was 97.87%(RSD=2.9%,n=6).Linear relationship of corilagin in the range of 26.0μg/mL-459.0μg/mL(R2=0.9999)was good,average recovery was 98.94%(RSD=3.07%,n=6).The content of total polysaccharides,gallic acid and corilagin respectively were 112.8mg/g,7.08mg/g and 2.15mg/g of 15 batches of Sanziguben Granule.5.After 12 weeks treatment,SZGB did not have significant elevation of blood glucose and SZGB did reduce 24-h urinary protein level of rats in SZGB groups and rats in DN group had significant elevation of 24h urinary protein levels.SZGB did have improved the renal function and blood lipid of DN rats,and also promoted the antioxidant ability of DN rats through reducing levels of serum MDA,CAT and GSH activity.Meanwhile,the increase in Nrf2、HO-1、Bcl-2、E-cadherin expression levels,the decrease in NQO1、Cleaved caspase 3、Vimentin、α-SMA expression levels,it can be deduced that Sanziguben Granule’mechanism of protective effect on experimental diabetic nephropathy rats could be related to the attenuation of oxidative stress,apoptosis and EMT.ConclusionThe preparation technology of Sanziguben Granule was established and chromatographic fingerprints from 15 batches of Sanziguben granule was obtained successfully.Using serum pharmacochemistry to track bioactive components of Sanziguben Granule by UPLC/Q-TOF-MS/MS,which provided the basis for quality assessment of Sanziguben Granule.Sanziguben Granule’ mechanism of protective effect on experimental diabetic nephropathy rats could be that SZGB inhibit EMT through the Nrf2-mediated anti-oxidative effects in STZ-induced diabetic rats. |
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