Optimization Of Extraction Process Of Adventitious Roots Of Hypericum Perforatum And Antibacterial And Anti-biofilm Of The Extracts

Hypericum perforatum L.is a perennial herb with a variety of pharmacological activities.However,the wild resource is decreasing year by year and the quantity and quality of artificially cultivated plant material of H.perforatum difficultly meet the demand of the product production.Therefore,the adventitious root（AR）culture is becoming an important approach for obtaining the new material of H.perforatum.At present,AR bioreactor culture system has been established,but the pharmacological effects of H.perforatum ARs are unclear yet,thereby affecting the ARs applying in the product production.Therefore,this study used bioreactor cultured ARs as the experimental material to optimize the extraction condition of flash extraction and the crude extract was futher extracted by the solvents of different polarities.Furthermore,the effects of antibacterial and anti-biofilm of the crude extract and different fractions were investigated to provide a theoretical basis for the future production of related products.The result of selecting the extraction solvent showed that the efficiency of extraction was better when methanol was as the extraction solvent compared with the solvens of ethanol and water.Therefore,methanol was used as solvent.Futhermore,the the optimal levels of extraction time,solvent concentration,and ratio of liquid to material were selected in the single factor experiments.Afterward,the extraction process was optimized in three-factor and three-level experimental design of response surface methodology（RSM）.The optimized extraction condition from the RSM was:65.44 s extraction time,77.46%methanol,and 52.13 m L g^-1 ratio of liquid to material,at which condition the flavonoid yield was upto 10.57%.In the antibacterial experiment,the crude extract（CE）and petroleum ether（PET）,dichloromethane（DCM）,ethyl acetate extract（Et OAc）,n-butanol（n-Bu OH）,and aqueous layer（AQE）fractions exerted different inhibitory effects on six different bacterial species（Bacillus subtilis,Bacillus cereus,Staphylococcus aureus,Salmonella cholerae,Pseudomonas aeruginosa）.Among extracts,the strongest comprehensive antibaterial activities was found in PET and DCM.In particular,PET and DCM had high inhibitory effects on B.subtilis,B.cereus,and S.aureus.Therefore,their effects on cell permeability and respritory metabolism of the three bacterial species were investigated.The leakage of the alkaline phosphatase enzyme,electrolyte,nucleic acid,and proteins of the three bacterial speicies were increased by the PET and DCM treatments,indicating the bacterial cell permeability increased.PET and DCM inhibited bacterial respiration,but they were involved in regulation of different respiratory metabolic pathways.Namely,PET affected the pentose phosphate（HMP）pathway of B.subtili and the tricarboxylic acid cycle pathway of B.cereus or S.aureus;DCM affected the HMP pathway of Bacillus subtilis,the hexose diphosphate（EMP）pathway of B.cereus,and both EMP and HMP pathways of S.aureus.In antibilm experiment,CE,PET,DCM,Et OAc,and n-Bu OH had the antibiofilm effect,and the highest comprehensive antibiofilm activity was determined in n-Bu OH.The five extracts can kill the bacteria inside the biofilm.The present study indicated that H.perforatum ARs possessed good antibacterial and anti-biofilm effects,thereby the ARs are feasible as a new plant material using in the production of antibacterial and anti-biofilm related products.