Preliminary Construction Of The Grading Method Of Huajuhong Decoction Pieces Based On "network Prediction→grading Quantification→pharmaceutical Effect Verification"

Objective:By integrating multi-disciplinary technology and methodology,this paper intends to carry out the classification and evaluation of Exocarpium Citri Grandis(Huajuhong)decoction pieces,and to establish a new method for the sequential evaluation of TCM decoction pieces with strong operability and controllable quality,so as to provide a basis for further guiding the grading circulation in market,clinical grading management and application for Huajuhong decoction pieces.Methods:(1)Theoretical analysis and network prediction:By consulting ancient books and searching databases such as "PubMed","ScienceDirect" and "CNKI" related to Huajuhong,the historical origin and modern research of Huajuhong were sorted out,and the traditional identification and the frontier of modern research experience of Huajuhong were summarized.The main chemical constituents of Huajuhong were screened by searching the target databases of traditional Chinese medicine(TCMSP),TCMID and BATMAN-TCM,and combining with the published literatures in various journals.TCMSP,Stitch,SwissTargetPrediction and other platforms were used to predict the potential targets and potential diseases of the above chemical constituents,and the key targets and pathways of Huajuhong for the treatment of specific diseases were screened again,and the potential pharmacodynamic constituents and key pharmacodynamic targets were summarized(2)Grade quantification and grade standard:45 batches of Huajuhong decoction pieces from different producing areas were collected.The thickness of Huajuhong decoction pieces and the chroma of Huajuhong powder were quantitatively analyzed by analytical instrument;the thin hair density and texture of Huajuhong decoction pieces were qualitatively analyzed by traditional character identification method;the methods of Chinese pharmacopoeia(2015 edition)were used to conduct qualitative and quantitative analysis on the microscopic characteristics,thin-layer characteristics,moisture content,ash content,extract content(water soluble and alcohol soluble),fingerprint,and content determination of each batch of Huajuhong decoction pieces.Through data analysis,the grading scale of Huajuhong decoction pieces was formed.Based on the grading scale and the current national and local standards of Huajuhong,the Grade standard draft of Huajuhong decoction pieces was formulated.(3)Experiment research of efficacy validation:Rat model of pulmonary fibrosis was established by tracheal infusion of bleomycin.Taking the chronic inflammatory response of pulmonary fibrosis as the research point,the pharmacodynamic efficacy of different grades of Huajuhong decoction pieces were evaluated.To evaluate the improvement of different grades of Huajuhong decoction pieces on the body level of rats with pulmonary fibrosis from the aspects of body weight,body temperature and respiratory frequency.The therapeutic effects of different grades of Huajuhong decoction pieces on pulmonary fibrosis were evaluated at the tissue level of HE staining pathological sections,Sirius red staining pathological sections,collagen ratio,HYP content.At the level of cytokines,the biological efficacy of different grades of Huajuhong decoction pieces on the predicted key inflammatory cytokines IL-6 and IL-8,key chemokines MCP-1 and MIP-lα,key pro-inflammatory TNF-α and pro-fibrotic factor TGF-β1 were evaluated.Results:(1)Theoretical analysis and network prediction:Through ancient books and modern research literature,it can be seen that Huajuhong has distinct advantages of authentic producing area,variety and young fruit,and its modern research mainly focuses on anti-inflammatory and immune regulation.29 main chemical constituents were collected and 316 targets were predicted with the network pharmacology study of Huajuhong.Further optimization of network topological parameters screened out 22 potential targets of Huajuhong,correlated with 20 chemical constituents,and predicted 17 categories of potential diseases.Naringin and rhoifolin were selected as the pharmacodynamic quality index constituents of Huajuhong.The network pharmacology study on the treatment of pulmonary fibrosis with Huajuhong showed,there are 21 key therapeutic targets,24 direct therapeutic targets and 6 key direct therapeutic targets for the treatment of pulmonary fibrosis with Huajuhong.The treatment of pulmonary fibrosis with Huajuhong mainly regulates MAPK and Toll-like receptor signaling pathways,thereby balancing immunity,regulating inflammatory response and delaying the pathological progress of pulmonary fibrosis.The key therapeutic targets of Huajuhong on MAPK and Toll-like receptor pathways in the treatment of pulmonary fibrosis were selected as the evaluation index for the experiment research to verify the efficacy of Huajuhong decoction pieces(2)Grade quantification and grade standard:The physical and chemical indexes testing results show there were obvious grade differences in the detection data of traits such as thin hair density,thickness,texture and color of Huajuhong decoction pieces.The four grades of Huajuhong decoction pieces could be differentiated according to the experimental data of the above traits,and the experimental results were consistent with the theoretical research results And there were obvious grade differences in water content,fingerprint similarity and total flavone content,but no significant differences in ash content and extract content.Through data analysis,the gradation scale of Huajuhong decoction pieces was formed.In this paper,the Grade standard draft of Huajuhong decoction pieces was formulated by combining the grading scale with the current 14 medicinal materials and quality standards of of Huajuhong decoction pieces,and the quality control of different grades of Huajuhong decoction pieces was realized.(3)Experiment research of efficacy validation:IPF rat experiments showed there were statistically significant differences in weight of the Huajuhong decoction treatment groups and the model group on day 28(p<0.05),among which,the differences were more significant in grade Ⅰ,grade Ⅱ and grade Ⅳ groups(p<0.01).There were statistically significant differences in body temperature between the Huajuhong decoction treatment groups and the model group on day 7 and 28(p<0.05),among which,the difference on day 28 was more significant in grade Ⅱ group(p<0.01).There were statistically significant differences in respiratory frequency between the Huajuhong decoction treatment groups and the model group on day 28 only in grade I group(p<0.05).The observation results of HE staining,Sirius red staining,the analysis results of collagen type Ⅰ and Ⅲ proportions in the lung tissues of rats,and the HYP content in the lung tissues of rats all indicated that the different grades of Huajuhong decoction could delay the progress of pulmonary fibrosis.At the level of cytokines,the contents of IL-6,IL-8,MCP-1 and MIP-1a in the lung tissues of each group,as well as the contents of TNF-α and TGF-β1 in the serum were compared.The experimental results showed that the expression of the above cytokines could be inhibited to different degrees in different grades of Huajuhong decoction pieces treatment groups.In terms of the regulatory efficacy of cytokines IL-6,IL-8,TNF-α and TGF-β1,there was no significant difference between grade Ⅰ and grade Ⅱ groups,but there was significant difference between grade Ⅲ and grade Ⅳ groups.Specifically,in terms of regulating ability of IL-6,IL-8,TNF-αand TGF-β1,the efficacy of grade Ⅰ and grade Ⅱ of Huajuhong were better,the efficacy of grade Ⅲ of Huajuhong was weaker,the efficacy of grade Ⅳof Huajuhong was the weakest.In terms of regulating ability of MCP-1 and MIP-lα,the key inflammatory chemokines of pulmonary fibrosis,there was no significant difference between grade Ⅲ and grade Ⅳ groups,but there was significant difference between grade Ⅰ group and grade Ⅱ group.Specifically,in terms of regulating ability of MCP-1 and MIP-lα,the efficacy of grade Ⅰ of Huajuhong was better,the efficacy of grade Ⅱ of Huajuhong was weaker,the efficacy of grade Ⅲ and gradeⅣ of Huajuhong were the weakest.Conclusion:Combining traditional experience and modern technology,the sequential grading evaluation method of "network prediction→grading quantization→efficacy validation" can be constructed.The quality distinction,quality control and efficacy evaluation of different grades of Huajuhong decoction pieces could be realized with above method,which is helpful to guide the grading production and circulation of Huajuhong decoction pieces in the market and the grading management and application of Huajuhong decoction pieces in clinical practice.Our study can provide a reference for the grading evaluation of other Chinese herbal decoction pieces.