Study On Anti-infective Active Components Of Houttuynia Cordata Extract

Objective To screen anti-infective activity extracts of Houttuynia cordata by antiviral experiment in vitro,bacteriostatic experiment in vitro and anti-Staphylococcus aureus experiment in vivo;to optimize its extraction process and macroporous adsorption resin purification process,to prepare the anti-infective active components of Houttuynia cordata;to evaluate the efficacy and the process optimization effect of the components by anti-inflammatory experiments in vivo.Methods 1.Houttuynia cordata was used as a raw material.Four different solvent extracts of water,30% ethanol,50% ethanol and 75% ethanol were prepared by reflux method.Two extracts of the supernatant and the precipitated part were prepared by the water extraction and alcohol precipitation method.A method was established for determining the total flavonoids content of Houttuynia cordata.The total flavonoid content of the six extracts was determined.The anti-infective activities of six extracts were evaluated by antiviral experiment in vitro,bacteriostatic experim-ent in vitro and anti-S.aureus experiment in vivo.Among them,the test viruses for in vitro antiviral experiments were EV71(Enterovirus type 71),HSV-1(Herpes simplex virus type 1),RSV(Respiratory syncytial virus),and CV-B3(Coxsackie virus type B3),CV-B5(Coxsackie virus type B5);the test bacteria in the in vitro bacteriostatic test is S.aureus standard strain CMCC(B)26003 and its clinical isolates of methicillin-resistant strain,Proteus standard strain ATCC49027 and its clinical isolates,Klebsiella pneumoniae standard strain ATCC13883 and its clinical isolates.2.Using the total flavonoids as an indicator,the extraction process of 50% ethanol extract of Houttuynia cordata was optimized by single factor experiment and orthogonal experiment design.The anti-infective activity extract of Houttuynia cordata was prepared by the optimal extraction process.3.The anti-infective activity extract of Houttuynia cordata was purified by macroporous adsorption resin technology.Taking the total flavonoids as the index,the adsorption and elution conditions of the samples were investigated by single factor experiment,and the purification process of the macroporous adsorption resin was optimized.The anti-infective active component of Houttuynia cordata was prepared by the optimal purification process.4.The acute inflammation model of mice was established by intraperitoneal injection of lipopolysaccharide.The anti-inflammatory activity of anti-infective compo-nents of Houttuynia cordata was evaluated by using the levels of pro-inflammatory factors TNF-α,IL-1β and IL-6 in serum.Results 1.The content of total flavonoids in six extracts of Houttuynia cordata showed that the content of total flavonoids in 50% ethanol extract was the highest.The results of antiviral and antibacterial experiments in vitro showed that: all the six different extracts had certain antiviral and antibacterial activities.The TI value of 30% ethanol extract for EV71 and HSV-1 viruses was 18.566 and 83.725.The MIC and MBC for S.aureus was 3.125 mg/mL and 6.25 mg/mL.The TI value of 50 % ethanol extract for EV71 and HSV-1 viruses was 20.716 and 64.677.The MIC and MBC for S.aureus was 1.5625 mg/mL and 3.125 mg/mL.According to the results of in vitro experiments,50% and 30% ethanol extracts showed significant anti-infective activity.The results of anti-S.aureus test in vivo showed that: at the same dose,the 50% ethanol extract administration group was higher than the 30% ethanol extract administration group,and the mouse signs recovered better.Comprehensive in vitro and in vivo experimental results,the anti-infective activity of the 50% ethanol extract was higher.2.The results of the orthogonal test used to optimize the extraction process show that: the best extraction process of 50% ethanol extract of Houttuynia cordata was the extraction temperature was 75 ℃,and the extraction time was 1.5 h,Material to liquid ratio is 1 to 20.The content of total flavonoids in the anti-infective extract of Houttuynia cordata was 22.58%.3.The single-factor experimental optimization of macroporous adsorption resin purification process showed that: the sample adsorption conditions were sample concentration of 9.032 mg/mL(based on total flavonoid content),adsorption flow rate of 1 mL/min;elution conditions were eluted with 70% ethanol.The elution flow rate is 2 BV/h and the elution volume is 3 BV.The optimal flavonoid content of Houttuynia cordata anti-infective active ingredient was 60.96%.4.The results of anti-inflammatory experiment showed that: the levels of pro-inflammatory factors TNF-α,IL-1β and IL-6 in the serum of the anti-infective component of Houttuynia cordata were significantly lower than those in the model group.The inflammatory response was treated and controlled.At the same time,compared with the anti-infective extract of Houttuynia cordata,the anti-infective active component of Houttuynia cordata prepared by the purification process is significantly improved.Conclusion This study confirmed that the anti-infective activity of 50% ethanol extract of Houttuynia cordata was the most significant in terms of antiviral,bacteriostatic and anti-S.aureus experiments in vitro.According to the literature,this component is mainly composed of flavonoids.Optimize the total flavonoid content of the components by extraction and purification processes,in vivo anti-inflammatory experiments further confirmed that process optimization can increase the anti-infective activity of the components.