| Objective Pueraria montana var.thomsonii（Bentham）M.R.Almeida,a medicinal and edible homologous plant,has many health functions such as antiwine,hypoglycemia,antioxidation,antitumor and antibacterial.Plant tissue culture is an asexual propagation method,which can be used for large-scale production of active secondary metabolites,has many advantages,such as not restricted by region,season,insect pests and short growth cycle.However,at present,the research on callus culture of P.thomsonii Benth is not systematic and imperfect,and the related research on its active secondary metabolites is not in-depth and unclear.Based on the good antibacterial activity of callus of P.thomsonii Benth in previous experiments,this paper intends to systematically explore the culture system of its antibacterial callus,the biological activity and chemical composition of secondary metabolites,and the preparation process and application of active components,in order to provide scientific basis for the development and utilization of the secondary metabolites of callus of P.thomsonii Benth.Methods Firstly,the different disinfectants were used to optimize the disinfection conditions of different explants,different plant hormones were used to optimize the induction conditions of callus,and then the proliferation conditions of callus types with strong antibacterial ability were optimized and a large number of cultures were conducted.The secondary metabolites of different polarity of the callus were prepared by grades,and their biological activities were evaluated using 8 strains of Gram-positive and Gram-negative bacteria,antioxidant indexes such as DPPH,ABTS and total antioxidant capacity,and cell lines such as human gastric cancer cell SGC-7091 and human lung cancer cell A549.Further,the antibacterial secondary metabolites in callus were tracked by chromatography,activity test and spectral technique.Finally,the preparation technology of active ingredient was optimized and its antiseptic effect on fresh pork was studied.Results 1.The greenhouse seedling explants were determined as suitable culture materials.The optimal disinfection conditions for leaves,petioles and stems were 75%alcohol 30 s+0.1%Hg Cl2 10 min+2%Na Cl O 10 min,75%alcohol 30 s+0.1%Hg Cl2 5 min+2%Na Cl O 15 min,75%alcohol30 s+0.1%Hg Cl2 10 min+2%Na Cl O 5 min,and the optimum conditions for callus induction were 2.0 mg·L-1 6-BA+0.5 mg·L-1 NAA,2.0 mg·L-1 6-BA+0.1 mg·L-1 NAA,2.0 mg·L-1 6-BA+0.1 mg·L-1 NAA.The highest induction rate of stem segment was96.3%.2.The type of stem callus with strong antibacterial activity was determined.The optimal conditions of proliferation were 1.10 mg·L-1 6-BA+0.25 mg·L-1 NAA+0.95mg·L-1 2,4-D,MS base medium,2%sucrose,0.8%AGAR,pH 5.8,25℃,illumination18 h/dark 6 h,5～8 pieces/bottle were inoculated,and the transfer time was 15 d.Under these conditions,the callus was mainly yellow-green or yellow,the tissue was relatively loose,and the proliferation coefficient could reach 9～10.3.Among the four different polar extracts,ethyl acetate extract showed strong activity on the whole,and showed good inhibitory effect on Gram-positive bacteria such as Enterococcus faecalis,Bacillus thuringiensis,Staphylococcus aureus,and Micrococcus luteus,its MIC were0.195～1.536 mg·m L-1.Its IC50 on DPPH and ABTS were 0.2795 and 0.0640 mg·m L-1,respectively,and when mass concentration at 1.5 mg·m L-1,it was equivalent to the reducibility of 1.3420μmol·m L-1Fe SO4.And it also had a good inhibitory effect on human lung cancer cell A549 which EC50 was 260.90μg·m L-1.However,the four different polar extracts had no significant inhibitory effect on human gastric cancer cells SGC-7091.4.The concentration of total flavonoids and total phenols was higher in ethyl acetate extract,and the concentration of total polysaccharides was higher in water extract.The correlation analysis indicated that total flavonoids might be an important antibacterial component.Through activity tracking,daidzein（1）and genistein（2）were isolated from the antibacterial component Fr.2.4 of ethyl acetate extract,daidzein（3）and genistein（4）were isolated from the crude component Fr.3,both of which were isoflavone components.The antibacterial results showed that genistein was the main component to inhibit Enterococcus faecalis and Bacillus thuringiensis,and its MIC were 19.53μg·m L-1 and 4.88μg·m L-1,respectively,while daidzin,genistin and daidzein had no obvious inhibitory effects on the two strains.Three potential antibacterial components,5-hydroxymethylfurfural,β-sitosterol and lupinol,were identified from the volatile components of ethyl acetate extract by GC-MS analysis,these volatile components may be involved in the inhibition of some components on the tested bacteria.5.The optimal enzyme-ultrasonic method for the preparation of total flavonoids from stem callus was cellulase dosage 1%,enzymatic hydrolysis temperature 57℃,enzymatic hydrolysis time 100 min,ethanol volume fraction 66%,solid-liquid ratio 1:50 g·m L-1,ultrasonic power 200 W,ultrasonic time 50 min,extraction for 3 times.Under these conditions,the total rate of flavonoids was 3.055%.The total flavonoids prepared by this method can effectively delay the rise of p H and TVB-N values of fresh pork within 6-9 days,inhibit the growth of microorganisms on pork surface and maintain the brightness of pork surface to a certain extent.Conclusion A system of disinfection,induction and proliferation of the callus of P.thomsonii Benth stem with antibacterial ability was established.The secondary metabolites of the callus showed strong antibacterial,antioxidant and weak cytotoxic activities,and genistein was the main component to show the antibacterial activity.It was also found that the total flavonoids of the callus had a certain antiseptic effect on fresh pork.The results provided some reference for the development and utilization of secondary metabolites from callus of P.thomsonii Benth in food and pharmaceutical fields. |
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