| Due to the unhealthy eating habits and lifestyles,the global prevalence of diabetes has increased dramatically.Diabetes has become the second major type of disease after high blood pressure,which brought heavy living and economic burdens to patients.Diabetic nephropathy(DN),one of the most common microvascular complication of diabetes,is the leading cause of end-stage renal disease.It’s characterized by proteinuria,thickening of the renal basement membrane,expansion of mesangial area,continuous renal fibrosis and progressive renal hypofunction.The diminished renal function caused by DN will not only shorten the life expectancy but also increase the risk of cardiovascular disease of patients,which will seriously affect the quality of life.At present,there is no specific effective drug for the treatment of DN.The progress of DN is mainly delayed by improving lifestyles,controlling blood glucose and blood pressure as well as correcting lipid metabolism disorders.Therefore,research on drugs for the prevention and treatment of DN has important social and economic significance.Traditional Chinese medicine(TCM)has a long history in China.There have been many studies on the therapeutic effects of TCM and their extracts on DN.The exploration of material basis and molecular mechanism of TCM for their therapeutic effects has become a focus for current research.Cordyceps is one of the TCM with beneficial effects on lungs and kidneys.The artificial Cordyceps sinensis prepared through fermentation process makes up for the serious shortage of wild Cordyceps.These wild and artificial Cordyceps have been widely used in the treatment of various kidney diseases,including DN,and have achieved good clinical results.At present,the research on active components,mechanism and medicinal preparations of Cordyceps sinensis in DN treatment is still in the initial stage.Most studies have only focused on research of Cordyceps sinensis powder and its crude extracts.Besides,artificial Cordyceps sinensis preparations on the market are only simple capsules filled with Cordyceps sinensis powder.Although few studies on artificial Cordyceps sinensis polysaccharides have been reported,there is still a lack of systematic research on active components,mechanism and high-end preparations of artificial Cordyceps sinensis for DN.To explore the mystery of artificial Cordyceps sinensis as a therapeutic drug for DN,we chose a kind of artificial Cordyceps sinensis commonly used in the pharmaceutical market as raw material and conducted component separation,activity screening,mechanism research and formulation improvement.The research content can be divided into four parts:(1)isolation and purification of various components in artificial Cordyceps sinensis and preliminary screening of anti-DN activity;(2)therapeutic effects and mechanisms of active components on experimental DN;(3)preparation of artificial Cordyceps sinensis improved formulation and its pharmacokinetics;(4)preparation,characterization and evaluation of ergosterol nano-formulation.(1)Isolation and purification of various components in artificial Cordyceps sinensis and preliminary screening of anti-DN activityThe work in this section focuses on separation and purification of polysaccharides,nucleosides and sterols(active fractions)that are rich in Cordyceps sinensis and have good biological activities,as well as preliminary screening of anti-DN activity.The polysaccharides were separated from artificial Cordyceps sinensis by water extraction and alcohol precipitation,and purified by deproteinization with Sevage reagent,decolorization with hydrogen peroxide,and removal of small molecules by dialysis.The quality of the prepared polysaccharides was evaluated.It was found that the polysaccharide content in the sample was 49.73%(based on glucose).Besides,the polysaccharides sample contained saccharides and had no reductive monosaccharides and proteins.The nucleosides component extracted from artificial Cordyceps sinensis(CS-N)was purified by macroporous resin column and sephadex column.Qualitative and quantitative analysis of nucleoside compounds in CS-N was performed by HPLC-DAD and HPLC-MS.The 10 nucleoside compounds identified were uracil,adenine,guanine,hypoxanthine,Cytidine,uridine,adenosine,2’-deoxyadenosine,guanosine and thymidine,among which the content of guanosine was the highest.Sterols component was extracted from artificial Cordyceps sinensis by ethanol extraction and ethyl acetate extraction,then separated and purified by silica gel column and recrystallization.The sterol compounds in sterol sample were analyzed by HPLC-DAD and GC-MS.It was found that ergosterol was the main component of the sample.The content of ergosterol in the sample could reach 92%.Therefore,commercially available ergosterol was used in subsequent research.Preliminary screening of the anti-diabetic nephropathy activity of these artificial Cordyceps sinensis components includes in vitro modeling and activity screening.High glucose-induced rat mesangial cells were used as cell model.Fibronectin and type 1 collagen(extracellular matrix components)were used as screening indicators.The preliminary activity screening results showed that the nucleoside extract and sterol extract(ergosterol)inhibited the expression of extracellular matrix more effectively in the three main components.(2)Therapeutic effects and mechanisms of active components on experimental DNThere have been many studies on polysaccharides in Cordyceps sinensis.Thus,this project focuses on the therapeutic effects and mechanisms of nucleoside extracts(CS-N)and sterol extracts(ergosterol)on experimental DN.The study of CS-N against DN includes modeling in vivo and in vitro as well as exploration of action efficacy and signaling pathways.HK-2 cells stimulated by high glucose were used as in vitro model.The results of efficacy studies showed that CS-N treatment effectively inhibited high glucose-induced epithelial-mesenchymal transition and extracellular matrix accumulation.The in vivo model was a C57BL/6 mouse model induced by STZ.CS-N was administered by gavage at 40 and 80 mg/kg/day for 8 consecutive weeks.In vivo study showed that CS-N treatment could effectively restore the body weight and improve renal function of DN mice.Hyperglycemia induced kidney cell damage,glomerular hypertrophy,mesangial expansion,glycogen and collagen deposition were also ameliorated by CS-N administration.Signaling pathway study showed that CS-N administration reduced epithelial-mesenchymal transition and extracellular matrix deposition by inhibiting the activation of p38 and ERK signaling pathways.The in vivo and in vitro modeling and action efficacy in the study of sterol extract(ergosterol)against DN are similar to that of nucleoside extract(CS-N),the main difference is in the mechanism of action.The in vitro study has established a DN cell model by stimulating mesangial cells with high glucose.The results of in vitro efficacy study showed that ergosterol inhibited high glucose-induced abnormal proliferation of mesangial cells and overexpression of extracellular matrix components.Mechanism study found that ergosterol could down-regulate the expression of TGF-β1,inhibit the phosphorylation of Smad2 level and regulate its downstream signals to exert its anti-DN effect.In animal experiments,STZ-induced C57BL/6 diabetic mice were orally administered with ergosterol(10,20,40 mg/kg/day)for 8 weeks.In vivo study found that ergosterol administration increased body weight,improved islet β-cell function and restored renal function of DN mice.In addition,ergosterol significantly ameliorated the glomerular volume increase,mesangial matrix expansion,tubulointerstitial damage,glycogen and collagen deposition in the kidney tissue of diabetic mice.Mechanism study found that ergosterol played a role in the treatment of DN in vivo through the same signal pathway as in vitro.(3)Preparation of artificial Cordyceps sinensis improved formulation and its pharmacokinetics in ratsErgosterol is an important active ingredient in artificial Cordyceps sinensis for the treatment of DN.However,the water solubility of ergosterol is extremely poor,which will affect the dissolution and release of ergosterol from fungus powder and the absorption in gastrointestinal tract,leading to low oral bioavailability.Therefore,it’s necessary to prepare an improved preparation of artificial Cordyceps sinensis to increase the solubility of ergosterol.The main work is the screening of solubilizers.The solubilization of ergosterol by different solubilization techniques were investigated.We determined the solubility of ergosterol in several common solubilizers and latent solvents.The results showed that SDS had the best solubilizing effect on ergosterol.Therefore,SDS was used in the preparation of artificial Cordyceps sinensis improved formulation.The study of pharmacokinetics of improved formulation includes the establishment of methodology and the investigation of pharmacokinetic parameters.We established an LC-MS method for the determination of ergosterol in rat plasma.Methodological verification results showed that the established method had good linear relationship,good specificity,high precision and accuracy,high recovery rate and good stability,thus can be used for the pharmacokinetic study of ergosterol.Pharmacokinetic study showed that the artificial Cordyceps sinensis improved formulation could significantly improve the blood concentration and oral bioavailability of ergosterol.It is speculated that compared with artificial Cordvceps sinensis powder,the artificial Cordyceps sinensis improved formulation may be able to reduce the dosage and improve the therapeutic effect in DN treatment(4)Preparation and evaluation of ergosterol-loaded nanostructured lipid carrierIn view of the good anti-DN activity of ergosterol,we prepared ergosterol-loaded nanostructured lipid carriers(ERG-NLCs)to improve the solubility and oral bioavailability of ergosterol in this part of the study.These works laid a theoretical and experimental basis for the subsequent development of ergosterol-related products.Research on ERG-NLCs includes formulation selection,process and formulation optimization,formulation characterization as well as in vitro and in vivo evaluation Nanostructured lipid carrier with good biocompatibility and high stability was selected to be the dosage form of ergosterol nano preparation.In the study of process and prescription screening,single factor screening and orthogonal design optimization were performed with encapsulation efficiency as the main evaluation index.Finally,ERG-NLCs were prepared by emulsified-ultrasonic method using glyceryl monostearate and decanoyl/caprylyl glyceride as lipid materials,Tween 80,Pluronic F68 and lecithin as emulsifiers.The prepared ERG-NLCs dispersion was a liquid with a clear blue light opalescence.The nanoparticles were smooth and nearly spherical,with high entrapment efficiency and drug loading of 92.9%and 6.51%,respectively.The particle size and zeta potential of ERG-NLCs were 81.39 nm and-30.77 mV,respectively.Stability determination results showed that ERG-NLCs had good stability.To facilitate packaging,storage and transportation,it was determined that ERG-NLCs were prepared into a lyophilized preparation with 5%mannitol as lyophilized protective agent.The prepared ERG-NLCs lyophilized products had good appearance and redispersibility.The quality evaluation results showed that the freeze-drying process had small effects on the encapsulation efficiency,drug loading,particle size,zeta potential and micromorphology of the preparation.The phase analysis results of DSC and X-ray diffraction indicated that ergosterol had been successfully entrapped into ERG-NLCs and presented as a new phase.Rats were used as pharmacokinetic research model to investigate the changes in oral pharmacokinetic parameters.The effects of ERG-NLCs on over-proliferation and ECM accumulation of RMCs induced by high glucose were studied by MTT and Western blot.These results showed that ERG-NLCs significantly increased the oral bioavailability of ergosterol,enhanced the inhibitory effects on mesangial cells over-proliferation and ECM accumulation induced by high glucose,improved the anti-DN activity in vitro.In summary,this project isolated and purified different components in artificial Cordyceps sinensis and preliminarily screened their anti-DN activity in vitro.Besides,we studied the therapeutic effects and mechanisms of nucleoside and sterol components on DN.Nucleoside extracts alleviated epithelial-mesenchymal transition and extracellular matrix deposition by inhibiting the activation of p38/ERK signaling pathway;ergosterol inhibited mesangial cell over-proliferation and extracellular matrix accumulation by regulating TGF-β1/Smad2 pathway.To overcome the poor water solubility of ergosterol,artificial Cordyceps sinensis improved formulation and ERG-NLCs were prepared and evaluated.The artificial Cordyceps sinensis improved formulation effectively improved the oral bioavailability of ergosterol;ERG-NLCs significantly increased the oral bioavailability and in vitro anti-DN activity of ergosterol.This subject provided experimental data for the research on the material basis and mechanism of Cordyceps sinensis against DN.Our work also provided theoretical and experimental basis for the improvement of Cordyceps sinensis formulations and the development of ergosterol-related products. |
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