Screening And Activity Analysis Of Some Astragalus Plant Germplasm Resources In Shanxi Province Based On Flavonoids

The problems of food safety and environmental pollution caused by the large-scale abuse of feed antibiotics have received more and more attention.Prohibiting the use of feed antibiotics in animal production has become the consensus and inevitable trend of all countries in the world.Therefore,finding a safe and effective substitute for feed antibiotics has become a hot topic in animal husbandry research at home and abroad.At present,the commonly used feed antibiotic substitutes include oligosaccharides,antimicrobial peptides,enzymes,acidifiers,probiotics and feed plant extracts.Among them,feed plant extracts have become one of the ideal substitutes for feed antibiotics in animal production due to their rich resources,natural sources,comprehensive functions,high safety,low toxic and side effects,difficulty in producing drug resistance and no(low)residues.Flavonoids,as a kind of secondary metabolites widely existing in plants,have immunomodulatory,antioxidant,and antibacterial activities.However,there are few reports on the application of flavonoids in germplasm evaluation and screening.Therefore,this study screened the germplasm resources based on the biological activity of flavonoids in some Astragalus plants in Shanxi province.The main experimental results are as follows:1.The total flavonoids content,antibacterial and antioxidant activities of the aboveground parts of 9Astragalus plants(Astragalus capillipes Fisch.ex Bunge,Astragalus arkalycensis Bunge,Astragalus melilotoides Pall.,Astragalus discolor Bunge ex Maxim.,Astragalus complanatus Bunge,Astragalus dahuricus(Pall.)DC.,Astragalus membranaceus(Fisch.)Bunge,Astragalus limprichtii Ulbr.,and Astragalus laxmannii Jacq.)collected in Shanxi Province were determined.The results showed that the content of flavonoids in the A.membranaceus stems and leaves was the highest,which was 22.66 mg/g(powder dry weight).The results of antibacterial activity showed that A.membranaceus stem and leaf extracts had the strongest inhibitory activity against Bacillus cereus,and the diameter of inhibition zone was 6.25 mm at 5 mg/m L.2.The antibacterial and antioxidant activities of total flavonoids,total saponins and total polysaccharides in the A.membranaceus stems and leaves were evaluated.The results showed that the biological activity of total flavonoids in the A.membranaceus stems and leaves was the strongest.The antibacterial activity showed that the inhibition zone diameter of flavonoids against B.cereus was 10.05 mm at 5 mg/m L.The antifungal activity showed that the EC50 of total flavonoids against Aspergillus flavus,Penicillium citrinum,Aspergillus niger,Fusarium moniliforme,Rhizopus oryzae,and Trichoderma harzianum were 0.90 mg/m L,1.11 mg/m L,1.10 mg/m L,1.18 mg/m L,1.26 mg/m L,and 0.97 mg/m L,respectively.The antioxidant activity showed that the EC50 of total flavonoids on DPPH,ABTS free radical scavenging and iron ion reduction/antioxidant capacity were 0.15 mg/m L,0.03 mg/m L,and 3.19 mg/m L,respectively.3.Ultrasonic-assisted ethanol extraction was used to extract total flavonoids from A.membranaceus stems and leaves,and the extraction process was optimized by response surface methodology.The results showed that the optimum extraction conditions were as follows: solid-liquid ratio 40 m L/g,ethanol concentration 75%,extraction time 35 min,and extraction temperature 58 °C.Under these conditions,the yield of flavonoids from A.membranaceus stems and leaves was 22.03 ± 2.57 mg/g(powder dry weight).4.B.cereus is one of the three major pathogens that cause mastitis in dairy cows.In this study,the inhibitory activity of total flavonoids from A.membranaceus stems and leaves against B.cereus was evaluated.The results showed that total flavonoids from A.membranaceus stems and leaves could inhibit the growth of B.cereus in many ways,including growth curve,disrupting the integrity of cell wall and cell membrane,inhibiting biofilm formation,and inhibiting bacterial protein synthesis.In addition,the total flavonoids of A.membranaceus stems and leaves could also inhibit the expression of virulence factors.After 24 h of 1/2 MIC treatment,the relative expression levels of cyt K,nhe C,hbl C and hbl D were 28.66%,0.57%,36.70%,and 15.41% of the control group,respectively.5.The antibacterial and antioxidant activity-guided separation and purification of total flavonoids from A.membranaceus stems and leaves were carried out by Sephadex LH-20 gel column chromatography and thin-layer chromatography method.Two target components were obtained,namely Frac.ccd component with strong antioxidant activity and Frac.50-2c component with strong antibacterial activity,and the main active components were isoquercitrin and isoliquiritigenin,respectively.Molecular docking showed that isoquercitrin form 5 hydrogen bonds with superoxide dismutase(SOD),and isoliquiritigenin form 2 hydrogen bonds with filamentous temperature sensitive mutant Z(Fts Z).6.The A.membranaceus stems and leaves were collected for transcriptome and metabolomics sequencing at 6 stages(Flowerless period-FL,Flowerless bud period-FB,Flowering period-F,Green fruit period-GF,Mature fruit period-MF,and Withering period-W)during the growth and development of A.membranaceus.A total of 184121 Unigene genes were obtained by assembling the transcriptome sequencing results.Among them,38 differentially expressed genes were annotated in the flavone and flavonol biosynthesis pathway,and 107 differentially expressed genes were annotated in the isoflavone biosynthesis pathway.A total of 944 metabolites were identified by metabolomics sequencing,of which flavonoids were the most abundant,accounting for 39.51 %.Among them,13 differential metabolites were annotated in the flavone and flavonol biosynthesis pathways,and 15 differential metabolites were annotated in the isoflavone biosynthesis pathway.Isoquercitrin only detected differences between FL vs W and GF vs W,and isoliquiritigenin did not detect differential metabolism during the 6 periods.And 4 genes,UGT79B30,PMAT1,5MAT,and F3’H,which may be related to the biosynthesis of isoquercitrin,were identified by combined transcriptome and metabolomics analysis.In this study,9 species of Astragalus in Shanxi Province were screened based on the biological activity of flavonoids.The results showed that the content of flavonoids in A.membranaceus stems and leaves was the highest,and it had good antibacterial and antioxidant activities.The research results not only lay a foundation for the development of feed antibiotic substitutes for A.membranaceus,but also promote the healthy and green development of A.membranaceus industry by comprehensive development and utilization of A.membranaceus resources.