| Objective:Determine the optimum dissolution medium for cassia seed extract,analysis the degree of contribution of the main components of cassia seed extract to the equilibrium solubility in the optimum dissolution medium and define the markers of equilibrium solubility of cassia seed extract.Prepare cassia seed granules and establish their quality standards.The effect and mechanism of granules in the treatment of STC was investigated using a replication of the slow transit constipation（STC）model in rats using compound difenoxil.To screen the relevant targets and pathways of cassia seeds for the treatment of STC using network pharmacology.Methods:1)The equilibrium concentrations of the cassia seed extract in water,0.1mol·L-1hydrochloric acid and p H=6.8 phosphate buffer at 37℃were determined by differential spectrophotometry,and the optimal solubilization medium was determined based on the previous research of the group.The effects of bound anthraquinone and free anthraquinone on the equilibrium solubility of the cassia seed extract were analyzed by partial least squares regression analysis;the fingerprints of the total anthraquinone of the cassia seed extract were established by HPLC with reference to the fingerprints of the total anthraquinone of the cassia seed extract at different equilibrium time points in the optimal dissolution medium.The fingerprints of the equilibrium concentrations of the main constituents of Cassia extract in the optimum dissolution medium were analyzed to determine the influence of the equilibrium solubility of Cassia extract in the optimum dissolution medium.2)The granules were prepared using the melt extrusion method,and the optimum ratio of polyethylene glycol 4000（PEG4000）and polyethylene glycol 6000（PEG6000）,the amount of microcrystalline cellulose（MCC）added and the amount of drug loading were investigated on the basis of single-factor examination,and the overall score（R）was used as the index.The prescription process was optimized by the Box-Behnken optimization method in Design-Expert,and its quality standard was established.3)Rats were randomly divided into normal groups(saline,10 m L·kg-1),model group(saline,10 m L·kg-1),positive group(hemp seed pill 1.16 g·kg-1),and cassia granules low,medium and high groups(1.5,3,6 g·kg-1),The STC model was prepared using compound difenoxanthin(10 m L·kg-1,once daily for 14d)in all groups except for the normal group.The effect and mechanism of granules in the treatment of STC were investigated by measuring the levels of nitric oxide（NO）,total nitric oxide synthase（T-NOS）and 5-hydroxytryptamine（5-HT）in serum and NO,NOS and acetylcho-linesterase（ACh E）in colonic tissues of rats in each group.4)To investigate the mechanism of action of anthraquinone components in cassia seed granules for the treatment of STC through network pharmacology and molecular docking techniques.The target proteins of the five anthraquinone components in cassia seed granules,including orange cassia and the target proteins of STC,were screened and collated from the database to construct a biological functional network map of anthraquinone-component-slow transport constipation-signaling pathway.The target proteins and corresponding anthraquinone components were processed using SYBYL-X2.1.1 software,and molecular docking was performed to predict the anthraquinone components in cassia granules for STC using Total Score≥5 as the criterion.Results:1)The equilibrium solubilities of cassia extract measured at 37℃in p H=6.8phosphate buffer,0.1mol·L-1hydrochloric acid and water were 11.099,10.91and10.344μg·m L-1（in terms of total anthraquinone content）,respectively,and the equilibrium time points were reached at 24h,3h and 7h,respectively.In the optimum dissolution medium,the contribution of the main components of the cassia seed extract to the equilibrium solubility of the cassia seed extract at different time points varied,with 22 chemical components having a VIP value greater than 1,of which the retention time was 33.104min The chemical components with retention times of 33.104min,50.839 min,77.774min,96.237 min and 109.358min were the known components orange cassia,rhubarb acid,rhubarbin,rhubarbol and rhubarbin methyl ether,with VIP values of 1.13987,1.15565,1.12806,1.14825 and 1.1488,respectively.values were 1,2,5,9 and13.2)The optimum prescription for cassia granules is PEG4000:PEG6000=3:2(g·g-1);the MCC addition is 1.5g;the drug loading is 23%.The quality standards are in accordance with the provisions of the Chinese Pharmacopoeia,2020 Edition,Part IV.3)Before modelling,the daily faecal water content data of each group of rats were counted and found to be not significantly different and the data were not statistically significant（P>0.05）;during the first week of modeling,the fecal water content of rats in all five groups decreased,except for the normal group,and there was a significant difference in the fecal water content of rats in the normal group compared with the model group.（P<0.01）;in the second week of Modeling,the overall water content in the faeces of the rats was similar to that of the first week of modeling,and there was also a significant difference（P<0.01）.In the first week of administration,the fecal water content of rats in the positive and cassia high dose groups was significantly higher compared to the model group,with significant differences（P<0.01）,the water content of cassia seeds in the low and medium dose groups was statistically significant（P<0.05）;in the second week of administration,the water content in the faeces of the rats was significantly different from that of the model group.（P<0.01）.There was a significant difference between the model group and the normal group in terms of reduced values of ink propulsion rate in the model group rats（P<0.01）;the model group had significantly higher values of ink advancement in rats in each group after administration of the drug compared to each administration group（P<0.01）.Compared to the normal group,the NO and NOS contents in serum and colonic tissues of rats in the model group were increased;compared to the model group,the NO and NOS contents in serum and colonic tissues of rats in each administration group were decreased.Compared to the normal group,the ACh E content in the colonic tissues of rats in the model group decreased;compared with the model group,the ACh E content in the colonic tissues of rats in each administration group increased.4)The total number of targets for the five anthraquinone components of cassia seeds,including orange-yellow cassia,was 138,the total number of STC targets was 3894,and the total number of common targets for both was 103.The results of molecular docking indicate that the Total Score of Colorectal cancer and PI3K-Akt signaling pathway to the involved target proteins GSK3B and HSP90AA1 is≥5,indicating that stable conformation can be formed and binding activity is high.Conclusion:p H=6.8 phosphate buffer was the best solubility medium for cassia seed extract,and the equilibrium solubility of cassia seed extract could be investigated with orange cassia,rhubarb acid,rhubarbin,rhubarbol and rhubarbin methyl ether as solubility markers.The prescription and preparation process of cassia seed granules are reasonable and the quality standard is in line with the provisions and requirements of granules in Part IV of the Chinese Pharmacopoeia（2020 edition）.The anthraquinones in cassia seed granules,such as MOL000472 and MOL001729,may play a role in the treatment of chronic transport constipation by acting on colorectal cancer,GSK3B and HSP90AA1 target proteins in the PI3K-Akt signaling pathway... |
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