Study On The Preparation Of Trichosanthes Pellets And Its Anti-acute Gastric Ulcer Effect And Mechanism In Rats

Objective:To investigate the material basis of antioxidant in Trichosanthes extract based on the homogenized Deng’s correlation calculation method,and to screen and verify the antioxidant effective components group,and then to determine the content of total flavonoids in Trichosanthes extract based on the antioxidant effective components group.To prepare Trichosanthes pellets and establish its quality standard.To establish a rat model of acute gastric ulcer caused by anhydrous ethanol,to study the anti-acute gastric ulcer effect of Trichosanthes pellets and to explore its mechanism of action,and to construct the spectrum-effect relationship between HPLC fingerprint of Trichosanthes pellets and anti-acute gastric ulcer effect in rats by Deng’s correlation calculation method.To analyze the anti-gastric ulcer mechanism of 5 flavonoids in Trichosanthes pellets based on the network pharmacology-molecular docking technique.Method:1)The fingerprints of Trichosanthes extract were established by HPLC,the scavenging rate of Trichosanthes extract(27,9,3 mg·m L^-1)on DPPH·and superoxide anion radical（O₂^-·）was determined.The antioxidant spectrum-effect relationship of Trichosanthes extract was investigated and the antioxidant effective components group was screened by the homogenized Deng’s correlation calculation method.The mixed control solution was prepared according to the contents of known components in the antioxidant effective components group of Trichosanthes extract and the scavenging rates of DPPH·and O₂^-·were determined.2)The antioxidant effective components group of Trichosanthes extract was prepared by mixing 5 monomeric compounds,namely rutin,luteolin,apigenin,isoquercitrin and kaempferol,in the ratio of 1:2:1:2:3（mass ratio）.The standard curve was established by UV-vis spectrophotometry,and the content of total flavonoids in Trichosanthes extract was determined.3)Trichosanthes pellets were prepared by extrusion-rolling method.On the basis of single factor test,the preparation process was optimized by Box-Behnken response surface with yield,roundness,Hausner ratio（HR）and frability as indexes,drug loading,rolling time and rolling frequency as factors.Taking the cumulative release of total flavonoids as an index,the in vitro dissolution of Trichosanthes pellets was investigated,and its quality standard was established.4)The rat model of acute gastric ulcer was established by intragastric administration of anhydrous ethanol.The protective effect of Trichosanthes pellets on acute gastric ulcer was evaluated by taking the proportion of ulcer area as an index,the pathological changes of gastric tissue were observed,the p H and volume of gastric acid were determined,and the pharmacodynamic indexes in rat serum and gastric tissue were detected,and the spectrum-effect relationship of Trichosanthes pellets against gastric ulcer was studied by grey correlation degree method.5)The mechanism of 5 flavonoids in Trichosanthes pellets against acute gastric ulcer in rats was studied based on network pharmacology and molecular docking technique.The target proteins of 5 flavonoids（rutin,luteolin,apigenin,isoquercitrin and kaempferol）,gastric ulcer and antioxidation target proteins were screened by consulting the related literature and using the database,and the protein interaction network（PPI）was obtained based on the common target proteins of the three.Molecular docking was conducted between the top 20 core targets in PPI network with their corresponding active components by using SYMBOL software,and the mechanism of 5 flavonoids in Trichosanthes pellet against acute gastric ulcer was analyzed with Total Score≥5 as the index.Results:1)There were 36 common peaks in the HPLC fingerprint of Trichosanthes extract with different correlation degrees with the scavenging rates of DPPH·and O₂^-·The common peaks whose correlation degree was greater than the median value were:peak 21,36,8,31,14,5,27,2,24,15,18,33,22,34,35,19,28 and 25.The 5 components of kaempferol（peak 36）,isoquercitrin（peak 8）,luteolin（peak 31）,rutin（peak 5）and apigenin（peak 35）were preliminarily identified as antioxidant effective components group（referred to as antioxidant effective components group for short）of Trichosanthes extract,with kaempferol,isoquercitrin,luteolin,rutin and apigenin=3:2:2:1:1（mass ratio）.The scavenging rates of antioxidant effective components group 6.12,2.04,0.68μg·m L^-1)for DPPH·were 65.4%,64.0%and 61.0%,respectively,and the scavenging rates for O₂^-·were 12.9%,9.5%and 8.3%,respectively.2)The linear equation of antioxidant effective components group was:ΔA=0.2324c-0.0107,r=0.9995,the linearity of antioxidant effective components group was good in the range of 0.18～1.45μg·m L^-1,and the methodological validation results met the requirements.The content of total flavonoids in Trichosanthes extract based on the determination of antioxidant effective components group was 0.0022%（RSD=0.05%）,which was significantly different from that determined by the standard curve method with rutin and other single reference substance（P<0.01）.3)The prescription and main preparation process parameters of Trichosanthes pellets were as follows:Trichosanthes e extract:40%,MCC:60%,wetting agent:70%ethanol,rolling frequency:25Hz,rolling time:4 min.The cumulative dissolution of the prepared Trichosanthes pellets in 30min was more than 80%,and that in 45min was more than90%,which met the dissolution requirements of quick-release pellets.The RSD values of precision,repeatability,stability and sample adding recovery were all less than 5%.4)Trichosanthes pellets could reduce the pathological changes of rats with acute gastric ulcer induced by anhydrous ethanol.Compared with the model group,it could reduce the secretion of gastric acid and ulcer area（P<0.05 or P<0.01）;It could increase the level of SOD in serum and gastric tissue,decrease the level of MDA and NO in serum and gastric tissue,as well as the activity of pepsin（P<0.01）.5)500 target proteins of 5 flavonoids active components,5094 target proteins of gastric ulcer,4334 target proteins of antioxidation and 72 common target proteins of the three components were screened and obtained.The research results of network pharmacology showed that the 5 flavonoids active components in Trichosanthes may play a role on gastric ulcer treatment by regulating metabolic pathway,chemical carcinogenesis-reactive oxygen species,PI3K-Akt and other signal pathways.The score results of molecular docking（Total Score≥5 as the index）showed that the 5 flavonoids active components in Trichosanthes such as luteolin,apigenin and kaempferol had good docking ability with AKR1C1,SRC,PTK2,EGFR and AKT1,and apigenin also had good binding ability with CBR1.Conclusion:Kaempferol:isoquercitrin:luteolin:rutin:apigenin=3:2:2:1:1（mass ratio）is the antioxidant effective components group of Trichosanthes extract.The content of total flavonoids in Trichosanthes extract can be determined based on the antioxidant active components group,and the determination method is reliable and stable,which can more truly reflect the content of total flavonoids in Trichosanthes extract.The prescription and preparation technology of Trichosanthes pellets are reasonable,and the dissolution in vitro of pellets conforms to the provisions of general rule 0931 of Part IV of Chinese Pharmacopoeia 2020.Trichosanthes pellets have a good preventive effect on acute gastric ulcer in rats,which comes from the combined action of multiple effective components related to the dosage.The results of network pharmacology and molecular docking showed that the 5 flavonoids effective components in Trichosanthes such as luteolin,apigenin and kaempferol could exert anti-gastric ulcer effect through multi-targets and multi-pathways.