Preparation Of Cassia Seeds Extract And Its Protective Effect And Related Mechanism On Liver Injury Induced By Fluorouracil In Mice

Objective:To optimize the extraction process of total anthraquinones from cassia seeds with Box-Behnken response surface methodology combined with fingerprint technology.To establish a quantitative analysis of multi components by single marker（QAMS）method for the simultaneous determination of the contents of 5 anthraquinones in cassia seeds,including aurantio-obtusin,rhein,emodin,chrysophanol and physcion by HPLC with 1,8-dihydroxyanthraquinone as the internal standard.A study on the material basis and mechanism of antioxidant activity of cassia seeds based on spectroscopic integration and molecular docking technique.To investigate the effect and mechanism of total anthraquinone extract of cassia seeds（hereinafter referred to as the cassia seeds extract,the same as the whole text）on liver injury induced by fluorouracil（5-Fu）,to screen the effective components group in cassia seeds extract on liver injury by using spectrum-effect relationship integrated molecular docking,and to predict and verify the relationship between the anti liver injury effect of cassia seeds extract and oxidative stress based on network pharmacology.Methods:1)Response surface test of 3 factors and 3 levels was conducted on the basis of single factor test with comprehensive evaluation as the measurement index.The comprehensive evaluation indexes include the extraction rate of total anthraquinones in cassia seeds or the amount of total anthraquinones equivalent to the medicinal materials per gram of cassia seeds（the weight is 0.2）,the normalized value of the peak areas of 5indicator components including aurantio-obtusin in the fingerprint of each sample accounting for 16 common peaks（with a weight of 0.5）and the fingerprint similarity（with a weight of 0.3）（the reference spectrum was established with the extraction solvent for the determination of cassia seeds content in Volume I,Chinese Pharmacopoeia,2020）.2)The chromatography was carried out by HPLC using a YMC-Pack ODS-A C18（4.6 mm×250 mm,5μm）column with gradient elution at a flow rate of 1.0 m L﹒min^-1and a column temperature of 30℃.The mobile phase was acetonitrile-0.1%aqueous phosphoric acid solution with a detection wavelength of 254 nm.With 1,8-dihydroxyanthraquinone as the internal standard,the relative correction factors of the internal standard with aurantio-obtusin,rhein,chrysophanol,emodin and physcion were calculated,and the contents of the 5 anthraquinones in cassia seeds were calculated by using the relative correction factors.At the same time,the contents of the 5 components were determined by Working Curve Method（WCM）,and then the differences between the results of QAMS and WCM are compared.3)HPLC fingerprint of cassia seeds extract was established.The scavenging ability and total reducing abilities of cassia seeds on 1,1-diphenylpicrylphenylhydrazine radical（DPPH·）,superoxide anion radical（O₂^-·）,ABTS⁺radical,hydroxyl radical（·OH）were studied.The spectrum-effect relationship and material basis of the antioxidation of cassia seeds extract were analyzed by using the Deng’s correlation degree calculation method with Matlab language,and the effective components group of the antioxidation of cassia seeds extract were obtained.The common target proteins between the antioxidant components of cassia seeds extract and the disease"anti oxidant"were obtained by referring to relevant literature and using TCMSP,Swiss Target Prediction,Gen CLIP3,Gene Cards,Venny.2.1,STRING,DAVID and other databases,and their GO function enrichment analysis and KEGG pathway enrichment analysis were carried out.The main molecular mechanism of antioxidant effective components in cassia seeds extract was verified by SYBYL-X 2.1.1 software.4)Fingerprint of 10 batches of cassia seeds extract was established by HPLC.The model of liver injury in mice was established by intraperitoneal injection of 5-Fu,with abifidonates as the positive group,and the protective effect of cassia seeds extract(0.4,0.8 and 1.6 g·kg^-1)on the liver injury induced by 5-Fu was studyed.The body weight of each group of mice was recorded and the organ index was calculated.The activities of ALT and AST in serum and MPO,SOD and T-AOC in liver tissue of mice in each group were determined.After hematoxylin-eosin（HE）staining,the pathological changes of liver tissue in mice were observed.The spectrum-effect relationship of cassia seeds extract on liver injury induced by 5-Fu was studied based on Grey Correlation Degree method,and its pharmacodynamic components were screened out.5)The pharmacodynamic components group and predicted targets of cassia seeds extract were collected through TCMSP,Swiss Target Prediction,Gencards,Gen CLIP,STRING,DAVID and other databases,the common target proteins of diseases and components were screened,and the biological functions and pathways were analyzed.The PPI network diagram of protein interaction was constructed.The network diagram of pharmacophore-targets-pathways of cassia seeds extract was constructed through Cytoscape 3.8.2 software,finally,the core target was verified by molecular docking.Results:1)The optimal extraction process of cassia seeds extract:The ratio of liquid to material is 20:1(m L·g^-1),the extraction solvent is 60%ethanol-ethyl acetate（2:1）mixed solution,and the extraction time is 15 minutes.The results of 5 validation experiments showed that the comprehensive evaluation of cassia seeds extract with the optimal technology was 0.528（RSD=0.45%）,the model prediction result of response surface method was 0.531,and the relative error with the prediction result was 0.56%.The optimal extraction technology was consistent with the model prediction result,and the optimal technology could be used for the extraction of cassia seeds extract.2)The relative correction factors of 1,8-dihydroxyanthraquinone with aurantio-obtusin,rhein,emodin,chrysophanol and physcion were 2.978,1.790,1.680,1.652 and1.333 within a certain linear range,respectively.There was no statistical significance between the contents of 5 total anthraquinones determined by QAMS and WCM.3)The results of antioxidant activity in vitro showed that scavenging rate and reducing ability of free radicals increased with the increase of the concentration of cassia seeds extract.,and it had good antioxidant activity.The spectrum-effect correlation degree of cassia seeds extract is positively correlated with the sample concentration,and the contribution of each component to the antioxidant activity was not the same,but there were effective components group,such as aurantio-obtusin（peak 6）,rhein（peak 11）,emodin（peak 22）,chrysophanol（peak 29）,and physcion（peak 30）.The network pharmacological study found that the components such as aurantio-obtusin and rhein acted on 80 protein targets such as CASP3,EGFR,HSP90AA1,MTOR,and played an antioxidant role through 96 signal pathways such as cancer in cancer,Erb B signaling path,HIF-1 signaling path,and PI3K-Akt signaling path,which involves 249 biological processes,including response to hypoxia,cellular response to oxidative stress,and positive regulation of apoptotic process.4)Compared with the normal group,the activities of ALT and AST in serum were significantly increased（P<0.05）,the activities of SOD and T-AOC in liver were significantly decreased（P<0.05）,and the activity of MPO in liver was significantly increased in 5-Fu model group（P<0.05）,and the liver cells appeared swelling,inflammatory infiltration and death.Compared with 5-Fu model group,the activities of ALT and AST in serum of mice treated with cassia seeds extract decreased significantly（P<0.05）.The activities of SOD and T-AOC increased significantly（P<0.05）.The level of MPO decreased significantly（P<0.05）.And the liver histopathological damage was reduced in the mice of each dose group of cassia seeds extract.5)There were 67 prediction targets for the anti-liver injury of the effective components group in cassia seeds extract,such as aurantio-obtusin.The enrichment analysis of the pathway obtained 105 related signal pathways.The analysis found that the anti-liver injury effect of aurantio-obtusin and other components in cassia seeds extract may be mainly exerted through the core targets such as CASP3,HSP90AA1,EGFR,ESR1,ERBB2,SRC,MTOR,and these targets are mainly enriched in many important pathways,such as Pathways in cancer,Lipid and atherosclerosis,Prostate cancer,Endocrine resistance,Apoptosis,EGFR tyrosine kinase inhibitor resistance,Micro RNAs in cancer,Gastric cancer,Erb B signaling pathway,PI3K-Akt signaling pathway,and other important pathways,and aurantio-obtusin and other components have good combination effects with core targets.Conclusion:The optimal extraction technology of cassia seeds extract can be obtained by Box-Behnken response surface method combined with fingerprint technology.The QAMS method can simultaneously determine the contents of aurantio-obtusin,rhein,emodin,chrysophanol and physcion in cassia seeds extract.The determination results are accurate and reliable,and can be used for the content determination and quality evaluation of multiple index components in cassia seeds.Cassia seeds extract has good antioxidant activity in vitro,aurantio-obtusin,rhein,emodin,chrysophanol,physcion are its antioxidant effective components group.Cassia seeds extract has the effect of anti liver damage in mice induced by 5-Fu,and aurantio-obtusin,rhein,emodin,chrysophanol,and physcion are the main active ingredients that play the role of anti liver damage in mice induced by 5-Fu.The mechanism of cassia seeds extract against 5-Fu induced liver injury in mice may be related to oxidative stress.The results of network pharmacology and molecular docking showed that the effective components of cassia seeds extract,such as aurantio-obtusin,rhein,emodin,chrysophanol,physcion,could play the role of anti drug liver injury through multiple targets and multiple pathways based on antioxidant activity.Among them,HSP90AA1,EGFR,PIK3CA,ERBB2 and MTOR target proteins of PI3K-Akt signaling pathway are one of the core pathways and key targets of drug active components to play a role in anti-drug-induced liver injury based on antioxidant activity.