| Objective:The preparation intermediate "Black Ginseng Instant Release Pellets" was prepared from the processed product "Black Ginseng" of traditional Chinese medicine ginseng,determine the optimal formulation and preparation process;formulate the quality standard of black ginseng quick-release pellets;investigate the pharmacodynamic effects and pharmacological mechanism of intervening renal interstitial fibrosis(RIF)through in vivo and in vitro experiments.Methods:1.Extraction process.The ultrasonic extraction method was used,ethanol was used as the solvent,and the total content of ginsenosides Rg3,Rg5,and Rk1 in the extract was used as an index.The extraction process of black ginseng was optimized by response surface analysis(RSM).2.Formulation preparation.Using black ginseng extract(BGE)as raw material,the black ginseng immediate-release pellets were prepared by extrusion-spheronization technology,and the comprehensive score of the pellet yield and disintegration time was used as indicators to screen the formulation accessories and optimize the formulation.3.Quality standard research.Check the pharmaceutic preparation indicators such as the appearance and sophericity of the pellets;determine the contents of ginsenosides Rg3,Rg5,and Rk1 by HPLC;examine the dissolution characteristics of the main effective components in the pellets.4.In vivo experiment.SD rats were randomly divided into 7 groups.Except for the blank group(Control),the other groups were injected with adriamycin(ADR)6.2 mg·kg-1 in the tail vein at one time to establish a chronic kidney disease model.The model was divided into model control group(ADR),BGE high-dose group(ADR+BGE 1200),BGE medium-dose group(ADR+BGE 600),BGE low-dose group(ADR+BGE 300),and Haikun Shenxi group(ADR+HKSX 70),Losartan group(ADR+LOS 5.25),from the 1st day after the modeling,the corresponding dose of the drug was administered intragastrically daily.The Control and ADR groups were given the same volume of normal saline,24 h urine protein detection once a week during dosing;9 weeks after dosing,serum was collected to determine changes in serum creatinine(SCr)and urea nitrogen(BUN)levels;the left and right kidneys were weighed,and the left kidney histopathological examination(HE,Masson staining)was performed.Immunohistochemistry(IHC)was used to determine α-smooth muscle actin(α-SMA)and type Ⅰ collagen(COL-Ⅰ)expression;the expression of TGF-β1,p-Smad3,Smad3 and Smad7 was measured by Western blotting(WB).5.In vitro experiments.HK-2 cells were divided into blank group,TGF-β1 model group,model administration group,and positive control group,TGF-β1 5 ng·mL-1 was stimulated for 24,48,and 72 h,respectively-Cell proliferation of different groups was measured by thiazole blue method(MTT).Cell morphological changes of different groups were observed under microscope;WB assessed the expression of E-cadherin,α-SMA,COL-Ⅰ,TGF-β1,p-Smad3,Smad3 and Smad7 in HK-2 cells.Results:1.The best extraction process for black ginseng is to use 65%ethanol as the solvent,the material-liquid ratio is 1:20,the ultrasonic frequency is 80 KHZ,and the ultrasonic extraction is performed 3 times for 48 min each time.2.The optimal formulation was determined to be black ginseng extract 25%,microcrystalline cellulose(MCC)36%,lactose 25%,Sodium carboxymethyl starch(CMS-Na)7%,and low-substituted hydroxypropyl cellulose(L-HPC)7%and 50%ethanol.3.In the quality standard,the black ginseng immediate-release pellets are brownish-yellow pills with a round and uniform appearance and a particle size of 0.09-0.13 mm.The disintegration time is 30 s,and the cumulative dissolution rate is greater than 90%for 10 min.HPLC determination of ginsenosides Rg3,Rg5,Rk1 showed a good linear relationship in the range of 10-100 μg·mL-1;the methodological investigation results showed that the precision,repeatability,and stability RSD%were all less than 2%,and the sample recovery rates were 98.79%,98.99%,and 99.01%;the pellets contained ginsenoside Rg3,Rg5,and Rk1 not less than 0.295 mg·g-1,0.294 mg·g-1,0.529 mg·g-1.4.in vivo experiments,(1)There was no significant difference in 24 h urine protein(UP)excretion between all groups at 0-1 weeks(P>0.05).The 24 h UP of ADR group was significantly higher than that of Control group at week 2(P<0.05),the 24 h UP in the drug-administered group at week 9 was lower than that in the ADR group(P<0,05).Compared with the control group,the levels of BUN and Scr in the ADR group were significantly increased(P<0.05).Compared with the ADR group,the levels of BUN and Scr in each administration group were significantly decreased(P<0.05).(2)HE results,pathological changes such as apoptosis and necrosis of epithelial cells,loss of renal tubular morphology,excessive deposition of glomerular stroma,inflammatory cell infiltration,and interstitial fibrosis in the renal tissues of the ADR group,the degree of change in the BGE administration group was significantly weaker than that in the ADR group.Masson results showed that the collagen deposition and fibrosis area in the BGE administration group was significantly lower than that in the ADR group.(3)IHC results,compared with the ADR group,BGE inhibited the increase of a-SMA and COL-I protein in a dose-dependent manner,and the BGE high-dose group showed a significant difference(P<0.05);WB results,compared with the ADR group,the BGE group down-regulated the expression of TGF-β1 and p-Smad3 and up-regulated the expression of Smad7 in a dose-dependent manner(P<0.05).5.In vitro experiments,(1)MTT experimental results show,TGF-β1 can stimulate the growth of HK-2 cells,and it has the strongest induction effect at 72 h at 5 ng·mL-1;compared with the model group,each group of BGE could improve the induction of HK-2 by TGF-β1,and the inhibitory effect was the strongest in the 800 μg·mL-1 group(P<0.05).(2)WB results,BGE group significantly inhibited TGF-β1-induced E-cadherin,Smad7 down-regulation(P<0.05),α-SMA,COL-Ⅰ,TGF-β1,and p-Smad3 up-regulated in a dose-dependent manner(P<0.05).Conclusion:The extraction process of black ginseng is reasonable and feasible;the formulation of black ginseng immediate-release pellets is reasonable;the established quality standard has good reproducibility and strong specificity,and can be used for this quality control;in vitro and in vivo experiments have confirmed that BGE can improve the process of adriamycin-induced renal interstitial fibrosis and protect renal function,which may be related to its regulation of the TGF-β1/Smad3 signaling pathway. |
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