Study On Flavonoids,Quality Evaluation And Anti-glycation Activity Of Abrus Precatorius Leaves

ObjectivesAbrus precatorius,a plant in Abrus Adans.of the family of Leguminosae,has the functions of clearing away heat and detoxifying,diuretic and anti-inflammatory,moisturizing the lungs and protecting the liver.And it has certain advantages in hypoglycemic.However,there is a lack of research on component characteristics,quality and hypoglycemic material basis of it.In this paper,flavonoids were extracted and isolated from Abrus precatorius leaves（APL）.The methods of determination and fingerprinting of APL were established to analyze the component distribution characteristics and quality of APL from different sources.The antioxidant capacity and anti-glycation activity in vitro were determined to screen out the most active compound.The key target and pathway of APL to improve the diabetes and diabetes syndrome were predicted by network pharmacology.The results would lay a good foundation for the source selection and quality evaluation of APL,as well as further research and development of flavonoids of APL.MethodsThe solvent method was used to separate the flavonoids of APL combined with different column chromatography.The structures of the components were analyzed and identified by means of rationalization property determination,UV,HPLC,MS and NMR.The optimal extraction technology of total flavonoids from APL was screened out by response surface methodology.The specific content determination method of flavonoids in APL was established by UV technology combined with different color development methods.The multi-index content determination method of flavonoids in APL was established by HPLC external standard method.The ingredient characterization analysis and fingerprint evaluation system of APL were established to evaluate the quality of different batches of APL by UPLC-MS/MS,as well as the relationship between quality and components were analyzed by chemical pattern recognition method.The in vitro anti-glycation activity of APL and flavonoids was evaluated by inhibiting the formation of AGEs test.The correlation between the content of flavonoids and its activity in different batches of APL was explored.The in vitro antioxidant activity of APL and flavonoids was investigated and evaluated based on total antioxidant capacity test,ABTS free radical scavenging test and DPPH free radical scavenging test.Meanwhile,the key components,related pathways and target proteins of APL to improve the diabetes and diabetes syndrome were predicted through network pharmacology.Molecular docking technique was used to verify the molecular docking of chemical components and key targets in the APL.ResultsSeven compounds were obtained from the ethanol extract of APL whose structures were identified as hemiphloin（1）,（2R）-naringenin6-C-β-D-glucopyranoside（2）,isohemiphloin（3）,hispidulin4’-O-β-D-glucopyranoside（4）,cirsimarin（5）,hispidulin（6）,cirsimaritin（7）.The extraction process results of flavonoids from APL showed that the effects of various factors on the content of total flavonoids were extraction times>ethanol concentration>liquid-material ratio>extraction time.The response analysis results showed that ethanol concentration and extraction times had a great impact on the content of total flavonoids in the extraction solution.Combined with the actual situation,the best extraction process was determined as follows:The heating and reflow method was used.The extraction solvent is dilute ethanol,the liquid-material ratio was 50:1,the extraction time was 60 min and the extraction times was twice.With compound 5（cirsimarin）as reference,the results showed that Al Cl₃ method was more reasonable to determine the content of total flavonoids in APL by comparing three different color rendering methods.When the concentration of APL was 4 mg/m L,and the Al Cl₃chromogenic solution at the concentration of 0.1 mol/L was added into 2m L.The measurement effect was more accurate.The methodological inspection results showed that the recovery rate was in the range of95.16%～100.20%and RSD value was 2.1%.The content of total flavonoids in 16 batches of samples from different sources was in the range of 2.6%～4.8%.An HPLC method was established for the simultaneous determination of seven flavonoids in APL.The recoveries of seven components were in the range of 95.16%～104.61%with the RSD values of less than 3%.The sum of the seven flavonoids in 16 batches of samples was in the range of 2.3%～11%.By comparing the content of total flavonoids measured by HPLC and UV methods,it was found that the total content measured by HPLC method was significantly different from that measured by UV method,which had a multiple relationship,except for individual samples.An UPLC-MS/MS analysis method was established,and the ethanol extract of APL was analyzed for its components.Total 29 compounds,including 3 alkaloids,16 flavonoids,9 triterpenoid saponins and 1 fatty acid were rapidly identified.Among them,flavonoids mainly existed in the form of flavonoid glycosides,dihydro-flavonoid glycosides and their aglycones.By analyzing the UPLC-MS fingerprint of 16 batches of APL samples,18 common peaks were matched.The RSD values of the relative retention time and relative peak area of common peaks were 0.09%～1.5%and 16%～184%,respectively.It indicated that component distributions of APL from different sources were similar,but there were differences in content.16 batches of samples could be divided into 4 categories through cluster analysis and OPLS-DA analysis.The 5 principal components were extracted by principal component analysis,and their cumulative contribution rate reached 92.29%.The prediction results of VIP value showed that components 1-7 were the key components to identify and distinguish the quality of APL.The anti-glycation ability of APL extraction was positively correlated with the concentration.The order of the inhibition of seven flavonoids was 4>6>3>1>7>2>5.Particularly hispidulin4’-O-β-D-glucopyranoside（4）and hispidulin（6）showed significant anti-AGEs activity,which IC₅₀ value were about 1/40 of aminoguanidine in the positive control.The total antioxidant capacity,ABTS radical scavenging capacity and DPPH radical scavenging clearance of APL had a positive linear relationship with concentration.The order of DPPH radical scavenging capacity of seven flavonoids was 4>6>1>2>3>7>5.The results of network pharmacology showed that there were 96intersecting targets of diabetes and flavonoids in APL.The GO enrichment analysis of the target of diabetes drug interaction showed that there were 281 biological processes,97 molecular functions and 120 cell components.The results of KEGG enrichment analysis showed that the relative pathways of flavonoids of APL to improve the diabetes and diabetes syndrome could be predicted through lipid and atherosclerosis,AGE-RAGE signaling pathway in diabetic complications,NF-kappa B signaling pathway and others.Top 4 targets of degree,named TNF,AKT1,EGFR,SRC,were screened out through the topological analysis of intersecting targets between diabetes and drug.And they were used as the receptor protein,the results of molecular docking verification showed that hispidulin 4’-O-β-D-glucopyranoside（4）had strong binding effect to key targets.ConclusionsThe seven flavonoids isolated from APL were flavonoids,dihydro-flavonoids and their glycosides.Among them,compounds 1-4and 6 were isolated for the first time from APL.The extraction method of flavonoids from APL was optimized,which laid a good foundation for subsequent quality analysis and purification and preparation of parts.An UV method suitable for the content of total flavonoids in APL was established.The method had good stability,specificity and simple operation.It could be used for the determination of total flavonoids in APL and preliminary quality evaluation.An HPLC method suitable for the simultaneous determination of multi-index content of flavonoids in APL was established.The method had good precision,stability and reproducibility.It could be used for the quality evaluation of flavonoids from APL.The total flavonoid content obtained by the two determination methods was different,but this difference had a conversion ratio of about1:2（UV:HPLC）.An UPLC-MS/MS method suitable for the analysis of composition characteristics and fingerprints of APL was constructed.The fingerprint combined with chemical pattern recognition evaluation was used to completely characterize the chemical components characteristics and quality relationship in medicinal materials,and revealed that the quality of APL from different producing areas had regional classification.Flavonoids of APL had significant anti-glycation potential and good antioxidant capacity,and they have a certain correlation,especially hispidulin 4’-O-β-D-glucopyranoside（4）and its aglycone component hispidulin（6）.Network pharmacology studies suggested that TNF,AKT1,EGFR and SRC were key targets of flavonoids in APL to improve the diabetes and diabetes syndrome,and molecular docking validation results showed that the hispidulin 4’-O-β-D-glucopyranoside（4）had the closest docking with the key target.This was consistent with the screening results of anti-glycation potential.In this paper,the isolation,identification,composition characteristics,quality evaluation,anti-glycation potential and mechanism of flavonoids in APL were preliminary studied.The structure characteristics and quality evaluation basis of the medicinal material were further clarified,which laid the foundation and basis for the establishment of quality standards,clinical application,and research and development to improve the diabetes and diabetes syndrome.