| With the unreasonable and large-scale use of antibiotics,the resistance of bacteria is increasing,which makes the treatment of bacterial diseases once again difficult.Finding new antibacterial drugs has become a very important topic.The development and utilization of natural products is the current research hotspot.Angelica dahurica(Fish.ex Hoffm.)Benth.et Hook.f.var.formosana(Boiss.)Shan et Yuan is a kind of medicinal material of Sichuan origin,and its root is the drug-injecting part,which has good pharmacodynamic value and various pharmacological activities.However,the antibacterial effect of Angelica dahurica has not been widely explored,and the metabolic pathway of the active ingredient is rarely studied.In this study,the Angelica dahurica materials selected in the previous research were tested for their antibacterial activity and mechanism by in vitro bacteriostasis test,quorum induction inhibition test and traditional Chinese medicine-antibiotic combination for extracts and extracts.The new bacteriostatic drugs provide a theoretical basis.In addition,through the molecular mining of the transcriptome data of Angelica dahurica,the key enzyme genes regulating the metabolic pathways of bacteriostatic substances in Angelica dahurica are sought,in order to further develop and utilize the antibacterial components of Angelica dahurica.Breeding and metabolic engineering lay the foundation and provide scientific evidences.Below are key research findings:(1)The crude extract of Angelica dahurica was obtained by alcohol extraction from the root of Angelica dahurica,and the in vitro antibacterial test was carried out on Escherichia coli,Staphylococcus aureus,Klebsiella pneumoniae and Pseudomonas aeruginosa.The results showed that the crude extract of Angelica dahurica carp had a certain degree of inhibition on the four pathogenic strains,and the crude extract had better inhibition effect against S.aureus and E.coli than the other two strains.Using the continuous dilution method,the MICs of the extracts of Angelica dahurica var.formosana against the four bacteria were: S.aureus=E.coli=P.aeruginosa=250 mg/mL,K.pneumoniae=500 mg/mL.Five extraction phases were obtained by organic solvent extraction,namely,petroleum ether phase(PE),trichloromethanechloroform phase(CE),ethyl acetate phase(EE),n-butanol phase(NE)and residual phase(RE),antibacterial test.The growth of the four tested bacteria was inhibited by all the extraction phases.Among them,EAE has the broadest antibacterial spectrum and the strongest antibacterial activity,and it exhibits antibacterial activity against all four tested bacteria.Combined with the inhibition spectrum and antibacterial activity,the order of antibacterial strength of each test substance was EE > NE > CE > PE > RE.Using the continuous double dilution method,the results showed that the best antibacterial effect against S.aureus was EE phase,the MIC was 1.56mg/mL;the best antibacterial effect against E.coli was EE,CE,NE phase,The MIC was1.56 mg/mL;the best antibacterial effect against K.pneumoniae was EE and NE,and the MIC was 75 mg/mL;the best antibacterial effect against P.aeruginosa was EE and NE,MIC It is 100 mg/mL.The qualitative analysis of the extracts showed that the lactones contained coumarin,flavonoids and phenols.The coumarins were mainly concentrated in ethyl acetate extract and N-butanol extract.In the middle,it is indicated that the best component of the bacteriostatic action of Angelica dahurica is coumarin.(2)P.aeruginosa,a model strain of quorum sensing research,was tested for its virulence factors,pyocyanin,extracellular protease,biofilm formation,and mobility by ethyl acetate extract of Angelica dahurica extract.The results showed that when the concentration of extract was 25 mg/mL,it could significantly inhibit the four virulence phenotypes of P.aeruginosa,which proved that the components of Angelica dahurica could inhibit bacterial quorum induction.When the concentration of extract is lower than25 mg/mL,the composition of Angelica dahurica has the effect of promoting quorum induction.It is presumed that the chemical structure of some coumarins or other compounds in Angelica dahurica extract is similar to that of quorum sensing system.At high concentrations,it may compete with the signal molecule receptors in the quorum sensing system to block the quorum-sensing network,eventually leading to a decrease in the expression of bacterial virulence factors;at lower concentrations,it will act as asignaling molecule.Combine more receptors.The results of the qualitative analysis of the chemical components of the different extraction phases show that the active component of the extract of the Angelicae Dahuricae is mainly the coumarin,the flavone and the flavone which are larger in polarity.(3)The main reason for the formation of bacterial drug resistance is that bacteria form biofilm,which can block the destruction of bacteria by antibiotics.We tested the drug-resistant S.aureus and P.aeruginosa with better membrane-producing ability.The results showed that the coumarins in Angelica dahurica could inhibit the production of S.aureus and P.aeruginosa biofilm and also destroy mature Biofilm;When coumarins were combined with antibiotics,the combination of coumarins and antibiotics played a positive synergistic role,and a better combination scheme was obtained.In the process of bacteriostatic and bactericidal,both the use of antibiotics and the production of drug-resistant strains could be reduced.Generally speaking,the inhibitory activity of simple coumarin combination on its biofilm is higher than that of other coumarin derivatives and falcarindiol(for Staphylococcus aureus),while the inhibitory effect of xanthotoxol and ampicillin combination on Pseudomonas aeruginosa biofilm is better.(4)Two coumarin synthesis-related genes were successfully cloned from Angelica dahurica based on the sequence information obtained by sequence analysis of transcripts and analyzed by bioinformatics.The results showed that the open reading frame of the cloned 12956 gene was 1104 bp in length and encoded 367 amino acid residues.The theoretical molecular weight(MW)of the protein was 40.69 kDa,and the isoelectric point(pI)was 6.06.The cloned 2646 gene cDNA was open for reading.The frame is 1080 bp in length and encodes 359 amino acid residues.The theoretical molecular weight(MW)of the protein is 39.33 kDa and the isoelectric point(pI)is 5.82.The two genes obtained are all hydrophilic proteins,which are stable protein structures and may not be secreted proteins,which are basically outside the membrane.The spatial structure predicts that the secondary structure of the protein mainly contains an alpha helix,an extended chain,a beta sheet,and a random coil.Structural analysis indicated that 12956 belongs to the2-oxoglutaric acid(2OG)and Fe(II)-dependent oxygenase superfamily,and 2646 belongs to the O-methyltransferase family.Through the current research on the biosynthetic metabolic pathway of coumarins,it is predicted that both 12956 and 2646 enzymes are involved in coumarin anabolism and are downstream of the phenylpropanoid metabolic pathway,and 12956 is the key enzyme in the production of 6’-Hydroxyferuloyl-CoA from Feruloy-CoA,2646 is the key enzyme in the production of Bergaptol from Bergapten,its specific function needs to be further studied. |
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