| Objective:The team’s research confirmed that the total flavonoids of the litchi core have significant anti-liver fibrosis effects in vivo and in vitro.Guangxi is the second largest litchi producing area in the country,litchi kernel resources are very rich,the total flavonoids obtained from litchi seeds have the potential to be developed into anti-liver fibrosis drugs.This topic is in accordance with the requirements of the Chinese Pharmacopoeia2020 edition,carry out and complete the research on the preparation process,quality standard and stability of Lizhihe jiaonang,provide research data for the development of new drugs for the treatment of liver fibrosis.Methods:1.Investigation and quality control of litchi nuclear resources.（1）According to the statistical yearbook of relevant departments,complete the resource survey of litchi annual yield and annual planting area in recent years,and predict the litchi nuclear yield in 2019.（2）Collection of litchi seeds.Fresh litchi fruits of different varieties,different origins and different batches were collected and dried to obtain raw materials.UV spectrophotometry was used to determine the content of raw materials,and the determination conditions were optimized.（3）Study on fingerprint of Litchi Seed.HPLC method was used to study the fingerprint of raw materials.The chromatographic conditions were optimized and the methodology was investigated.The HPLC fingerprint of raw materials was established.The similarity was calculated by using the similarity evaluation system of chromatographic fingerprint of traditional Chinese medicine（version A,2012）.The correlation analysis and evaluation of the fingerprint of traditional Chinese medicine were carried out by using SPSS,Stata and SIMCA software.2.Extraction,purification and component analysis of TFL.（1）TFL was extracted and purified.The extraction and purification of TFL were optimized by UV Spectrophotometry with proanthocyanidin A2 as the index,and the pilot scale-up and content determination were completed.（2）TFL component analysis.The chemical components of TFL were collected,and the targets of chemical components were predicted by Swiss database and Dra-cpi（drug repositioning and adverse reaction via chemical-protein interactome）.The disease targets of liver fibrosis were screened by genecards database,the protein-protein interaction was analyzed by string database,and go（gene ontology）and KEGG（Kyoto Encyclopedia of genes and genes）were analyzed.The chemical composition,target,pathway network and potential q-marker of TFL were established and visualized by Cytoscape software.HPLC method was used to study the fingerprint of raw materials.The chromatographic conditions were optimized and the methodology was investigated.The TFL HPLC fingerprint was established.The similarity was calculated by using the similarity evaluation system of traditional Chinese medicine chromatographic fingerprint（version A,2012）.The TFL fingerprint was analyzed and evaluated by SPSS,Stata and SIMCA software.3.Study on the preparation technology of Lizhihe jiaonang:Taking angle of repose,hygroscopicity,molding rate,critical relative humidity and bulk density as indexes,the varieties and dosage of excipients,wetting agent and drying time were selected to determine the optimal preparation process.4.Study on the quality standard of Lizhihe capsule:The quality standard of the preparation was studied from two aspects of content determination and related inspection of Jiaonang.The contents of proanthocyanidins A2 and rutin were determined by HPLC,and the Jiaonang were checked according to the Chinese Pharmacopoeia（2015edition and 2020 Edition）（general rule 0103）.5.Preliminary stability test of Lizhihe capsule:According to the guiding principles for stability test of APIs and preparations（general rule9001）in Chinese Pharmacopoeia（2020 Edition）,accelerated test and long-term test were carried out for the determination of properties and contents of three batches of products（batch number:20200901,20200902 and20200903）.Results:1.Resource investigation shows that litchi resources in Guangxi are very rich,which provides a stable source for the full development of Lizhihe Jiaonang.Using proanthocyanidin A2 as the reference substance,the method of concentrated hydrochloric acid vanillin was stable and reliable.The results showed that the content of total flavonoids in Litchi seeds ranged from 0.0009 to 0.0047 g·g-1,and Guangxi Heiye,Feizixiao and Heli,Guangdong Heiye,Feizixiao and Guiwei,Hainan Feizixiao and Hainan Feizixiao were the best varieties for extraction.The fingerprints of litchi seeds were established,and 15common peaks were calibrated.Among them,No.6 peak was identified as epicatechin,No.9 peak was identified as proanthocyanidin A2,and 10peaks were selected as potential markers.The content of total flavonoids in Litchi seeds was significantly correlated with 6 peaks.2.A stable and reliable method for the determination of TFL was established.The purity of TFL was 28.41%～69.26%.Four batches of TFL raw materials（batch number:20190801,20191201,20200101,20200201）were obtained through pilot scale-up.The fingerprint of TFL was established and 20 common peaks were demarcated,among which 4components were identified,which were proanthocyanidin B3（peak 3）,proanthocyanidin B2（peak 6）,epicatechin（peak 10）and proanthocyanidin A2（peak 16）.Ten chromatographic peaks were selected as potential markers,and there was significant relationship between TFL purity and six chromatographic peaks.Network pharmacology analysis showed that TFL could inhibit liver fibrosis and protect liver by interfering with the expression of ALB,PLG,HSP90AA1,EGFR and MAP2k1,which may be related to regulating PI3K Akt pathway and other signal pathways related to liver fibrosis.The results showed that the quality of TFL could be controlled by the potential q-marker,such as jopinene,quercetin,epicatechin,proanthocyanidin A2,naringenin,chuanpeioside,phloridzin and rutin.3.The best preparation process of Lizhihe capsule is as follows:TFL powder is extracted and purified,after screening,microcrystalline cellulose is added according to drug adjuvant ratio of 3:2 to mix well,10%starch slurry is used as wetting agent to make granules,dried at 60℃,whole granules are packed separately to obtain capsule.4.A method for the determination of proanthocyanidin A2 and rutin in Lizhihe Jiaonang was established,and the method has strong stability,which can be used to control the quality of Lizhihe Jiaonang.The properties,moisture,loading difference,disintegration time limit,heavy metals and pesticide residues of Lizhihe Jiaonang meet the relevant requirements of Chinese Pharmacopoeia（2015 edition and 2020 Edition）.5.3 batches of Lizhihe Jiaonang were stored under the conditions of accelerated test and long-term test,and all indexes met the requirements within 1 month,which preliminarily showed that the product was stable.Conclusion:A large number of high-purity TFL raw materials were obtained through the extraction and purification of litchi seed,and the fingerprints and TFL fingerprints of litchi seed from multiple varieties and origins were established.Meanwhile,the chemical components were analyzed by network pharmacology and multivariate statistics,which laid a foundation for the establishment of quality standards of Litchi seed and provided a reference for the selection of raw materials.A stable and feasible extraction and molding process was established,and qualified capsule products were prepared,which provided reliable parameter basis for industrial production,and established a simple,feasible and effective quality standard,which laid a foundation for ensuring the safety and effectiveness of clinical medication. |
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