Study On Antibacterial And Antiviral Activities Of Three Probiotics Fermented Six Traditional Chinese Medicines And Addition And Subtraction Of Qingwenbaidu Powder Prescription

In this experiment,Bacillus subtilis K3,Bacillus subtilis E12 and Lactobacillus plantarum were used as strains to ferment single traditional Chinese medicine and traditional Chinese medicine prescription.To explore the release of active ingredients before and after fermentation of traditional Chinese medicine,the safe concentration of liquid in cells,in vitro bacteriostasis and antiviral effect and in vivo antiviral effect,so as to provide reference for in-depth study of fermentation of traditional Chinese medicine.To detect the content changes of active ingredients before and after fermentation of traditional Chinese medicine:Three different strains of Bacillus subtilis K3,Bacillus subtilis E12,Bacillus subtilis K3 and Bacillus subtilis E12(1:1)were selected to ferment Coptis chinensis,Cortex Phellodendri,Scutellaria baicalensis,Radix Isatidis,Forsythia suspense and Gardenia jasminoides at 4%inoculation dose,and the concentration of 0.1g/mL was obtained after fermentation at 37℃ for 40h.Chinese herbal decoction with the concentration of 0.1g/mL was extracted by boiling method.Finally,the peak area of the active ingredients of Chinese herbal medicine was determined by liquid chromatography,and the content changes of the active ingredients before and after fermentation were studied.The results showed that the contents of active ingredients in Coptis chinensis,Isatis indigotica,Scutellaria baicalensis and Gardenia jasminoides before fermentation were higher than those before fermentation,and the concentration of active ingredients after fermentation by Bacillus subtilis E12 and K3 was the highest.The concentration of active ingredients in Forsythia suspensa and Cortex Phellodendri after fermentation was not significantly changed compared with that before fermentation.Antibacterial test of traditional Chinese medicine:The inhibition of drugs on Staphylococcus aureus,E.coli and Salmonella was explored by plate punching method.The results showed that:(1)all the samples had no bacteriostatic effect on Salmonella;(2)only Cortex Phellodendri could inhibit the growth of E.coli on agar after fermentation by Bacillus subtilis E12,and the other samples had no inhibitory effect;(3)All samples had no inhibitory effect on Staphylococcus aureus except Gardenia decoction,Isatis decoction and Forsythia suspense fermented by Bacillus subtilis E12.Staphylococcus aureus has the effect of inhibiting,and the rest of the samples have the effect of inhibiting.Survival rate of ST cells:MTT assay was used to detect the effect of Decoction and fermentation broth of traditional Chinese medicine on ST cells at concentrations of 2mg/mL,lmg/mL,0.5mg/mL,0.25mg/mL and 0.125mg/mL.When the cell survival rate was more than 80%,it was regarded as no effect on ST cells,which was the safe concentration of drugs on ST cells.The results showed that:(1)The maximum safe concentration of Forsythia suspensa Decoction and Forsythia suspensa fermented by Bacillus subtilis K3 was 2mg/mL,the fermentation broth of Forsythia suspensa fermented by Bacillus subtilis E12 and the fermentation broth of Forsythia suspensa fermented by Bacillus subtilis K3 and Bacillus subtilis E12 mixed bacteria damaged ST cells at all dilutions,and the fermentation broth of Forsythia suspensa fermented by Lactobacillus plantarum and Forsythia suspensa fermented by three The total concentration was 1mg/mL.(2)The maximum safe concentration of Gardenia decoction is 2mg/mL,that of Gardenia fermented by Ba cillussubtilis K3 is lmg/mL,that of Gardenia fermented by Bacillus subtilis E12 is 2mg/mL,that of Gardenia fermented by Lactobacillus plantarum,that of Gardenia fermented by compound Bacillus subtilis K3 and E12,and that of Gardenia fermented by three bacteria together.The samples had no effect on ST cells at all dilutions.(3)The maximum safe concentration of Coptis chinensis decoction was 0.25mg/mL,that of Coptis chinensis fermented by Bacillus subtilis K3 was 0.5mg/mL,that of Coptis chinensis fermented by Bacillus subtilis E12 was 0.5mg/mL,that Coptis chinensis fermented by Lactobacillus plantarum was below 80%at all dilutions,and that of Coptis chinensis chinensis fermented by Bacillus subtilis E12 and K3.The maximum safe concentration of the compound bacteria fermentation broth and the fermentation broth co-fermented by the three bacteria was 0.125mg/mL.(4)The maximum safe concentration of Decoction and fermentation of Phellodendron amurense by Bacillus subtilis K3 is 0.5mg/mL,the fermentation broth of Phellodendron amurense by Bacillus subtilis E12,the fermentation broth of Phellodendron amurense by three strains of bacteria and the fermentation broth of Phellodendron amurense by Lactobacillus plantarum are all below 80%at all dilutions.Phellodendron amurense is fermented by Bacillus subtilis K3 The maximum safe concentration of the drug was 0.25mg/mL.(5)The Decoction of Radix Isatidis and the fermentation broth of Radix Isatidis fermented by three strains of bacteria had no effect on ST cells at all dilutions.The maximum safe concentration of Radix Isatidis fermented by Bacillus subtilis K3 was 1mg/mL.The fermentation broth of Radix Isatidis fermented by Bacillus subtilis E12 had a great effect on ST cells at all dilutions.The cell survival rate was below 70%,and Radix Isatidis was fermented by Lactobacillus plant.The maximum safe concentration of drugs fermented by bacteria was 1mg/mL,and that fermented by Bacillus subtilis K3 and E12 was 2mg/mL.(6)Scutellaria baicalensis Decoction and fermentation broth fermented by Bacillus subtilis K3 had no effect on ST cells at all dilutions.The maximum safe concentration of Scutellaria baicalensis fermented by Bacillus subtilis E12 was 2mg/mL,the maximum safe concentration of Scutellaria baicalensis fermented by Lactobacillus plantarum was 2mg/mL,and the maximum safe concentration of Scutellaria baicalensis fermented by Bacillus subtilis E12 and K3 was 0.125mg/mL.The maximum safe concentration of Scutellaria baicalensis fermented by three strains of bacteria was 2mg/mL.In vitro antiviral test:ST cells were incubated with PRV(pseudorabies virus)for 2 hours,then all the liquids were discarded and then added with traditional Chinese medicine solution.After 48 hours of incubation,the virus content in ST cells was detected by fluorescence quantitative PCR to explore the inhibition of PRV proliferation on ST cells at the concentration of 100mg/mL,2mg/mL,lmg/mL,0.5 mg/mL and 0.25mg/mL.The results showed that:(1)Forsythia suspensa decoction could inhibit the proliferation of PRV on ST cells.When the dilution concentration was 0.5mg/mL and 0.25mg/mL,Forsythia suspensa decoction could not inhibit the proliferation of PRV,but it still inhibited the proliferation of PRV after fermentation by Bacillus subtilis K3,Bacillus subtilisE12 or Lactobacillus plantarum.(2)Gardenia decoction can inhibit the proliferation of PRV on ST cells only at the concentration of lmg/mL,while the fermentation broth after fermentation by Lactobacillus plantarum can inhibit the proliferation of PRV at the dilution concentration of 1mg/mL and 0.5 mg/mL.In addition,Gardenia after fermentation by Bacillus subtilis E12,when the concentration of fermentation broth was 0.5mg/mL,it not only inhibited the proliferation of PRV,but also had the strongest inhibition in the same group of samples,with significant difference(P<0.001).(3)Coptis chinensis can inhibit the proliferation of PRV on ST cells,but with the decrease of the concentration of Coptis chinensis decoction,the inhibition gradually weakened.When the concentration of Coptis chinensis decoction was diluted to 0.5mg/mL,Coptis chinensis decoction could not inhibit the proliferation of PRV,while the fermentation broth fermented by Lactobacillus plantarum and Bacillus subtilis E12 could inhibit the proliferation of PRV at low concentration.(4)Scutellaria baicalensis Decoction did not inhibit the proliferation of PRV on ST cells,but after fermentation,the fermentation broth of Scutellaria baicalensis could inhibit the proliferation of PRV.(5)Phellodendron amurense decoction can inhibit the proliferation of PRV on ST cells,but it can not inhibit the proliferation of PRV after dilution,while Phellodendron amurense fermentation broth still inhibits the proliferation of PRV when dilution concentration is 0.25mg/mL.(6)Radix Isatidis Decoction and fermentation broth of Radix Isatidis could inhibit the proliferation of PRV on ST cells at all dilutions set in the experiment.Moreover,Radix Isatidis decoction,fermentation broth of Radix Isatidis by Bacillus subtilis E12,fermentation broth of Radix Isatidis by Lactobacillus plantarum and fermentation broth of Radix Isatidis by three strains of bacteria could inhibit the proliferation of PRV in the original broth of 100mg/mL.There was no significant difference between the negative control group(P>0.05),and it had anti-PRV effect.Animal experiment:In this experiment,Chinese herbal medicines including Cortex Phellodendri,Radix Isatidis,Radix Scutellariae,Coptis chinensis,Habitat,Forsythia,Gardenia jasminoides,Cortex Mouth,Gypsum,Licorice,Bamboo Leaves,Radix Scrophulariae,Rhinoceros horn,Paeonia lactiflora,Anemarrhenae and Platycodon were used to compose the prescription of QingwenBaidu Powder Addition and Reduction prescription,and fermented with Bacillus subtilis K3,Bacillus subtilis E12 and Lactobacillus plantarum.Firstly,the effect of traditional Chinese medicine prescription fermentation and non-fermentation on the survival rate of ST cells was detected by MTT assay,In vitro anti-PRV effect at concentration of 100mg/mL(stock solution),2mg/mL,1mg/mL,0.5mg/mL,0.25mg/mL,Then,the anti-PRV effect of the traditional Chinese medicine prescription in the mice non-fermentation and fermentation was studied.Test results:(1)The maximum safe concentration of the drug components non-fermentation and fermentation of Qingqi Baidu Powder Addition and Reduction prescription was 2mg/mL.(2)All treatment groups at five different concentrations had the effect of inhibiting the proliferation of PRV on ST cells,and the effect of QingwenBaidu Powder Addition and Reduction prescription on the inhibition of PRV was stronger than unfermentation.(3)In vitro antiviral test,PRV-positive mice inoculated artificially died after 4 days of exposure.All mice in the low-dose and fermentation group died.The protective rate of drugs in the low-dose and fermentation group was 16.67%,that in the high-dose and fermentation group was 66.67%,and that in the high-dose and fermentation group was 33.4%.PRV was not detected in the tissues of all surviving mice.PRV was not detected in intestinal contents of dead mice,but PRV could be detected in heart,liver,spleen,lung,kidney and brain tissues.Compared with the low-dose group,the low-dose fermentation group had less virus content in liver,lung and kidney tissues than the low-dose and non-fermentation group(P<0.05);the high-dose and fermentation group had less virus content than the high-dose and non-fermentation group(P<0.05).In liver,lung and brain tissues,PRV content in high-dose and fermentation group was lower than that in high-dose and non-fermentation group(P<0.05).