Preparation And Evaluation Of Qianjin Huanglian Ganjiang Decoction Enteric Coated Pellets

Objective:Huanglian ganjiang decoction(HD),which is a traditional classic prescription in China,contains Coptidis Rhizoma,Phellodendri Chinensis Cortex,Zingiberis Rhizoma,Angelicae Sinensis Radix,Sanguisorbae Radix,Granati Pericarpium,and Asini Corii Colla.This prescription was first recorded in the Tang Dynasty "Thousand-Golden-Prescriptions(Beiji Qianjin Yao Fang)" volume 14,which also had been recorded in "Yixin Fang" and"ShengJiZongLu".Clinically,it was mainly used for the treatment of small intestine distension and pain,hematochezia or loose stools,and intestinal peristalsis,which are clinical manifestations similar to those of ulcerative colitis(UC).Based on the preparation method of HD determined in the preliminary experiment,this paper used network pharmacology to screen of active ingredients and predict the targets of this prescription,and then used modern drug delivery technology to prepare it as an oral colon-targeting preparation-enteric-coated pellets.Pharmacy in vitro and pharmacodynamic evaluation in vivo of enteric-coated pellets were conducted to improve the efficacy of HD and achieve the goal of targeted colonic administration.Methods:1.Network pharmacology technology was used to predict the potential targets of main components of HD.The active ingredients of HD were screened out by using the TCMSP database(traditional Chinese medicine system pharmacology technology platform)with OB>30%and DL>0.18.The potential targets of active ingredients in HD were predicted by using TCMSP and BATMAN-TCM.The String site converted the target name to get the kgene name.The software Cytoscape 3.5.1 was used to construct the relationship network between active components and target points of HD.Network Analyzer was used to analyze the topology parameters of network nodes to obtain the corresponding topological characteristic values(degrees).The median of the two degrees as the screening condition was used to obtain the potential key targets for the action of active components of HD.HPO database,Disgenet database and Drugbank database were used to collect target information of ulcerative colitis.String software was used to analyze and predict interactions between proteins,and Cytoscape 3.5.1 software was used to construct a drug-target-disease network and extract the junction part of the network,which was the key target of HD for the treatment of ulcerative colitis.KEGG pathway enrichment analysis of molecular functions and biological processes under Gene Ontology of key targets/genes of HD active ingredients were performed using DAVID 6.8 database tools.2.Preparation of pH-dependent colon-targeting preparation--HD enteric-coated pellets.By measuring density,fluidity and water content,the powder properties of HD extract were determined,which provided reference for preparation process.According to the active ingredients screened by network pharmacology,the determination of dissolution and content determination indicators was guided.A HPLC method for the determination of berberine hydrochloride,palmatine,ferulic acid and ellagic acid was established.Through single factor investigation,the technological parameters of preparation of drug-containing pellets by extrusion spherification were determined.The types and dosage of excipients,wetting agents and disintegrating agents for drug-containing pellets were investigated.Determine the type and dosage of plasticizer and adhesive required by coating agent.The technological parameters of preparing HD enteric-coated pellets by coating machine were determined.The type of coating agent and coating weight gain were investigated with dissolution as index.3.Pharmaceutical evaluation of HD enteric-coated pellets in vitro.The HPLC method for the determination of gallic acid and the GC method for the determination of zingerone and ligustilide was established.The characters,contents,dissolution and stability of HD enteric-coated pellets were evaluated in vitro.The influence of factors(high temperature,high humidity,light,5 days and 10 days)on pellets character,content and dissolution in vitro were investigated.4.Pharmacodynamic evaluation of HD enteric-coated pellets in vivo.The model of ulcerative colitis was established by 5%TNBS combined with anhydrous ethanol(v/v=3/2)on SD rats.The model animals were divided into 5 groups,and each group was given distilled water,mesalazine,HD and HD enteric-coated pellets low dose and HD enteric-coated pellets high dose respectively.The drugs were administered once a day for 7 consecutive days to investigate the protective effect of enteric pellets on UC rats.During the administration period,the rats were weighed and recorded daily.Meanwhile,fecal characteristics and blood in the stool were observed,and DAI of each rat was calculated.Blood was taken from the abdominal aorta 24 hours after the last administration.The spleen,thymus and colon from anus to ileocecal region were taken.H&E staining was performed on the colon tissues to observe the histopathology.The potential targets of network pharmacology were predicted and the relevant phenotypes were selected.The serum levels of SOD,MDA,GSH-Px,TNF-α,IL-1 β,IL-6,and IL-10 were detected.To analyze whether the phenotype of HD enteric-soluble pellets protected UC rats was consistent with the network pharmacologic prediction pathway,and to evaluate the colonic targeting of HD enteric-soluble pellets from the perspective of efficacy.Results:1.Network pharmacology technology was used to predict the potential targets of main components of HD.After searching and screening through TCMSP database,there were 67 active ingredients in the herbs of HD.After combining 10 common active ingredients,52 active ingredients were finally obtained.Through the Network Analyze Tool analysis,a total of 57 key targets were identified for the specific components of HD.The potential targets of HD were annotated by the KEGG pathway,which mainly involved tumor necrosis factor signaling pathway,inflammatory bowel disease(IBD),Toll-like receptor signaling pathway,HIF-1 signaling pathway,NF-κB signaling pathway,etc.Biological processes related to the treatment of ulcerative colitis with HD mainly involve the I-κB kinase/NF-κB signaling pathway,reactive oxygen metabolism,autophagy,mechanical stimulation response,lipid biosynthesis,Notch signaling pathway and other processes.2.Preparation of pH-dependent colon-targeting preparation-enteric coated pellets of-HD.The study of powder properties before preparation showed that the HD extract had poor fluidity and should be improved in the preparation process.A HPLC method for the determination of berberine hydrochloride,palmatine,ferulic acid and ellagic acid was established.The mobile phase system of acetonitrile-0.1%phosphoric acid water(0.2%triethylamine)was used for the determination of berberine hydrochloride and palmatine.The mobile phase system of methanol-0.1%phosphoric acid water was used for the determination of ferulic acid and ellagic acid.The precision,repeatability,stability and recovery rate of the two methods were investigated.The HD drug-containing pellets were prepared by extrusion spheronization,and the technological parameters were extrusion speed of 30 Hz,rolling speed of 20 Hz,and rolling time of 3 min.50%microcrystalline cellulose(MCC)was used as pelletizing promoter and diluent.10%ethanol was used as wetting agent.5%L-HPC+3%corn starch was used as disintegrating agent.Acrylic resin Eudragit S100 was selected as coating material,15%triethyl citrate(TEC)was selected as plasticizer,and 50%talcum powder was selected as anti-sticking agent.During the coating process,the main parameters were determined as the material temperature was 30℃,the inlet air temperature was 35℃,the inlet air speed was 1400 rpm,the rolling speed was 200 rpm,the pump speed was 5.0 mL/min,and the coating weight increased by 25%.3.Pharmaceutical evaluation of HD enteric coated pellets in vitro.HD enteric coated pellets were round gray-yellow particles.89.72%of the pellets were in the particle size ranging from 20 to 40 mesh.The resting angle of HD enteric coated pellets was 30.05° on average.The moisture was 5.75%.HPLC method for determination of gallic acid and GC method for determination of zingerone and ligustilide were established.Berberine hydrochloride,palmatine,ferulic acid,ellagic acid,gallic acid,zingerone and ligustilide were determined in the enteric coated pellets.The release of HD enteric coated pellets was investigated in vitro.The indicator components were released in a small amount in the simulated gastric fluid and simulated intestinal fluid,but they were released completely after the sudden release of the simulated colonic fluid,which basically conformed to the characteristics of colon-targeted drug release.High temperature,high humidity and strong light showed great influence on the stability of HD enteric coated pellets.4.Pharmacodynamic evaluation of HD enteric coated pellets in vivo.The model of ulcerative colitis was established by 5%TNBS combined with anhydrous ethanol(v/v=3/2)on SD rats.After 7 days of administration,the results showed that for the general observation indicators,the high dose group of HD enteric coated pellets showed the most significant weight recovery and DAI score for UC rats.HD enteric coated pellets could antagonize the shortening colon of UC rats obviously.There was a tendency to increase spleen index and thymus index in UC rats.The high dose group of HD enteric coated pellets had a significant protective effect on colon structure.HD enteric coated pellets showed the obvious effect of increasing SOD level and decreasing the level of MDA.There was a tendency to promote the vitality of GSH-Px in colitis rats.HD enteric coated pellets could reduce the levels of TNF-α,IL-1β,IL-6 and IL-10 in rats with experimental UC significantly.The results showed that HD enteric coated pellets could protect UC rats from oxidation,and regulating inflammation,and these effects were closely related to TNF pathway and NF-κB pathway.Moreover,in terms of pharmacodynamics,HD enteric coated pellets showed an advantage on colon-targeting on UC rats better than the decoction group.Conclusion:After network pharmacology screening,there were 52 active ingredients in the herbs of HD.Through the network analyze analysis,a total of 57 key targets were identified for the specific components of HD.The potential targets of HD for treating UC were mainly involved TNF signaling pathway,IBD,Toll-like receptor signaling pathway,NF-κB signaling pathway,etc.PH-dependent colonic targeting preparation--HD enteric coated pellets were prepared successfully.Pharmaceutical evaluation of enteric pellets in vitro showed that particle size,fluidity and dissolution in vitro all met the standards.High temperature,high humidity and hard light showed great influence on the content and dissolution of HD enteric coated pellets.The evaluation of pharmacodynamics of HD enteric coated pellets in vivo were carried out.HD enteric coated pellets could improve the general observation indexes of UC rats,and showed significant protective effects in antioxidant and anti-inflammatory aspects.