| The common pathogenic bacteria in the environment can be classified:gram-positive bacteria,gram-negative bacteria and fungi,which seriously endanger human health.With the unreasonable use of antibiotics,the tolerance of pathogens are increasing.Natural antimicrobial agents have different chemical structures and categories,such as polyphenols,terpenoids and flavonoids,which presents the complexity and diversity,so the strains are not susceptible to drug resistance.So many researchers turned to the goal from the plant to find efficient and natural compounds which have the antibacterial activity.Morus alba L.?mulberry?twig has a high medicinal value and healthcare function in traditional Chinese medicine.It has been recorded as a treatment for athlete's foot in many medical books from past dynasties,such as the"Bencao Tujing"written in Song Dynasty from AD 960–1279,which states that"Morus twig may be used to treat body dryness and itchiness,athlete's foot and difficulty in extension of limbs".But whether it has the effect of treating beriberi,how well does it work,it is worth for us to use modern research methods to research and verification.The main cause of tinea pedis is Trichophyton rubrum?T.rubrum?,which accounts for over 60%of tinea pedis symptoms,However,the anti-microbial effects of its extract on T.rubrum have not been reported to date.At the same time,some studies have shown that plant in adversity?acid alkali,induced,microbial induced drought stress,physical and chemical induction,etc.?,it can synthesize some new compounds or significantly increase the content of some existing compounds to resist adversity.Therefore,this paper studied the antimicrobial activity of Morus alba L.?mulberry?twig extract which induced by microorganisms on T.rubrum.Then,we applied column chromatography of polyamide and silicon gel to isolate and identify the major inhibiting substance of T.rubrum from Morus alba L.twig under activity tracking,and get higher content of antibacterial active ingredient oxyresveratrol?OXY?.We also investigated whether the compound also has antibacterial activity of other pathogens,andproved its antibacterial mechanism.Based on this,we determined the inhibitory effect of single active substance,with the combined use of antibiotics.This provided a theoretical basis for the development of natural antimicrobial agents from M.alba twigs.The specific research contents and results are as follows:1 Study on the inhibitory activity on T.rubrum of mulberry extract and main active substanceThe experimental results showed that the extracts of M.alba twig extracts had a certain inhibitory effect on T.rubrum.Among them,ethyl acetate extract?TEE,it was taken up about 50%of the total extract?and the minimal inhibitory concentration?MIC?of TEE on T.rubrum was 1.000mg/mL.The second was TPE?it was taken up about 2%of the total extract?and the total extract of M.alba twig?TAE?,when the concentration was 1.00 mg/mL,the inhibition rate was 84.31%and83.01%respectively.Finally,it was TNE?it was taken up about 20%of the total extract?and TWE?it was taken up about 30%of the total extract?,when the concentration was 1.00 mg/mL,the inhibition rate was 50.98%and 44.44%respectively.Under the restrain activity of T.rubrum,we purified a main active substance monomer compound—OXY from TEE with various purification and identification methods.OXY had a good inhibitory activity on T.rubrum,and the MIC and IC500 value were 0.500 mg/mL and 0.065 mg/mL respectively.At the same time,HPLC and SPSS were used to calculate the contribution of OXY in the activity of TEE inhibiting T.rubrum,which was about 80.52%.Therefore,it was concluded that OXY played a major role in the inhibition of TEE on T.rubrum,and it was one of the main antibacterial active substance in M.alba twig.Synergistic inhibition test was obtained that the MIC value of OXY combined with MN was reduced to 1/2 MIC.Through the FICI index of two drug combinations,it was concluded that the combined use of OXY and MN had a superimposed effect on the inhibitory effect of T.rubrum.2 The antibacterial activity of OXY on E.coli,S.aureus and C.albicansIt was concluded that OXY could change the growth rate of the strains and prolong the logarithmic period and stability of the strains.Moreover,it had a killing effect on three bacterias.The MIC of OXY on E.coli,S.aureus and C.albicans were 1.00 mg/mL,0.50 mg/mL and 1.00mg/mL respectively,and the MBC were 4.00 mg/mL,0.50 mg/mL and 4.00 mg/mL,respectively.At the same time,in the synergistic effect of OXY and antibiotics,the MIC jointoint value of OXY and gentamicin?GS?combined on E.coli were their respective 1/2 MIC,MBC joint>1/2 MIC respectively,the MIC jointoint value of OXY and GS combined on S.aureus were their respective 1/4MIC,MBC joint=1/2 MIC;the MIC jointoint value of OXY and proconazole?MN?combined on C.albicans were 1/4 MIC,MBC joint>1/2 MIC.Through the FICI index of the combination of two drugs,it was concluded that the combination of OXY and GS had a superposition effect on the inhibitory effect of E.coli,and had synergistic effect on the inhibitory effect of S.aureus.The combination of OXY and MN had synergistic effect on the inhibitory effect of C.albicans.3 Study on the antibacterial mechanism of OXYTransmission electron microscope observation indicated that OXY changed the cell morphology,caused the cytoplasm to become light and empty,it also caused the separation of E.coli and C.albicans,and damaged cell walls of S.aureus and C.albicans.The cell membrane integrity test showed that OXY changed the cell membrane conformation of the strains,but there was no obvious damage to the cell membrane.At the same time,it inhibited the biofilm formation of three strains,which affected the growth and reproduction of the strains.This study provides a theoretical basis for the development of natural antibacterial agents from M.alba twigs,and also has important practical significance.OXY can be widely used in skin care and healthcare products as a safe constituent.Meanwhile,OXY has the potential to reduce the use of antibiotics as an adjuvant therapy to fungal infection. |
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