Study On Pharmacodynamics And Related Substances Of The Metabolites Of Endophytes Isolated From Glycyrrhiza Uralensis

Objectives: To research the bacteriostasis,anti-inflammatory,antitussive and expectorant effects of metabolites of endophytes isolated from wild and cultivated Glycyrrhiza uralensis in Gansu,as well as its related substances of effective metabolites of endophytes,to reveal the correlations of metabolites of endophytes with host plant Glycyrrhiza uralensis in terms of efficacy and chemical components,and provide evidence for the application of metabolites of endophytes from Glycyrrhiza uralensis as microbial resources.Methods:1.Isolation and identification of endophytes.Endophytes were isolated from wild and cultivated Glycyrrhiza uralensis,and molecular biological identification was carried out for 20 strains of endophytes that was filtrated by pre-experiment.Endophytic bacteria were subject to 16 Sr DNA identification,and endophytic fungi were subject to internal transcript spacer(ITS)identification.2.Study on bacteriostasis.With Streptococcus pneumoniae,Staphylococcus aureus and Escherichia coli as the tested bacteria,bacteriostatic test in vitro was performed by quantitative bacteriostasis to determine the inhibition zone diameter and the minimum inhibitory concentration(MIC)of the metabolites of endophytes from Glycyrrhiza uralensis.Bacteriostatic test in vivo.Bacterium suspensions of the tested bacteria(streptococcus pneumoniae,staphylococcus aureus and escherichia coli)at 6 gradient concentrations were prepared respectively.There are 3 batches of Kunming mice,60 Kunming mice of each batch were randomly divided into 6 groups.Each group was intraperitoneally injected a suspension at a gradient concentration,0.5ml for each mouse.The deaths of each group were observed within 4 days,and the minimum lethal dose was determined for the tested bacteria.500 Kunming mice were randomly divided into 25 groups,these are blank group,model group,positive control group,wild Glycyrrhiza water decoction group,cultivated Glycyrrhiza water decoction group,and 20 groups of endophyte fermentation product.Gavage administration with a specified dose was maintained for each group once per day for four consecutive days.0.5h after the last gavage,each mouse in each group was injected with 0.5ml of the tested bacterium solution based on MLD for modeling.The death of infected mice in each group on day 4 to 7 to calculate the mortality,rate of death protection and mean survival days.3.Anti-inflammation research in vitro.RAW264.7 cells in the logarithm growth period were added with lipopolysaccharide(LPS)for MTT assay in order to calculate the cell survival rate.LPS in vitro was used to stimulate RAW264.7 macrophagocyte to establish an inflammation model.These cells were inoculated onto a 96-well plate and divided into blank group,LPS model group,wild and cultivated Glycyrrhiza water extract groups,and 20 groups of endophyte fermentation product.The model group was added with 1mg/ml LPS for culture.Each test drug group was added with Glycyrrhiza water extracts or endophyte fermentation products(10mg/L,20mg/L and 40mg/L)for two hours of treatment,then LPS(1mg/ml)was added for stimulation.The nitric oxide(NO)content was detected by Griess method,while the contents of Interleukin-1?(IL-1?)and Prostaglandin E2(PGE2)were measured by enzyme-linked immunosorbent(ELISA)method.Anti-inflammation test in vivo.160 Kunming mice were randomly divided into 16 groups: model group,positive control group,wild Glycyrrhiza water decoction group,cultivated Glycyrrhiza water decoction group,and 12 groups of endophyte fermentation products(effective groups for anti-inflammation in vitro).Different drugs were given by gavage administration once per day for seven consecutive days.1 hour after the last administration,50?l xylene was evenly applied on the two sides of the right ear of each mouse,with the left ear as the control.These mice were sacrificed 30 min later,and auricles were taken by punching before weighting them and calculating the degree of swelling and the inhibition rate of swelling.Glacial acetic acid-induced mouse abdominal capillary permeability increase test,the grouping and drug adminstration of mice were the same as above,once per day for five days.30 minutes after the last adminstration,0.5% Evans blue solution(10m L/kg)was injected into tail vein,while 0.8% glacial acetic acid normal saline solution(10ml/kg)was injected.These mice were sacrificed 20 minutes later.Peritoneal washing absorbance was determined for each group.For the intervention test of LPS-induced acute pneumonia rat,170 SD rats were randomly divided into 17 groups,and each group was given the corresponding drug by gavage administration once per day for five days.Except the blank group,the other groups were intravenously injected with LPS(1mg/kg)to establish an acute pneumonia model,while the blank group was injected with the same volume of normal saline.After 6 hours,blood was collected from rat femoral artery to detect peripheral white blood cell counts,and the contents of serum TNF-?,IL-6 and IL-10 were detected.4.192 Kunming mice were randomly divided into 16 groups in two batches,these are model group,positive control group,cultivated and wild Glycyrrhiza water decoction groups,and 12 groups of endophyte fermentaiton product(effective groups for anti-bacterial and anti-inflmmatory).Each group was orally administrated corresponding drugs once per day for seven days.1 hour after the last administration,ammonia water and SO2 were used to induce cough.EDT50,R value,cough incubation period,time of coughs within 2min,elongation rate and cough relieving rate were taken as the detection indexes.5.The model of phlegm blocking in lung was induced by inhaleing SO2 and clod wind.10 days after modeling,each group was given the corresponding drug by gavage administration for seven days.The expectorant effects were studied by observing the changes of rats' general activities and pathological of lung tissues.In addition,the contents of NO,ICAM-1 and COX-2 in lung tissue were measured by ELISA method,and Na+-K+-ATP enzyme activity were measured.The expressions and distributions of AQP1 and AQP5 in lung tissue were measured by immunohistochemical method.6.Detection of chemical component.With liquiritin and ammonium glycyrrhetate as the controls,the contents of total flavonoids and total saponins in the fermentation products of endophyte by ultraviolet spectrophotometry with wavelengths of 250 nm and 334 nm.Hanbon Sci.&Tech.Phecda C18(5?m,4.6×250 mm)chromatographic column was adopted,acetonitrile-0.5% phosphoric acid water was used as moving phase,and gradient elution was performed.With a wavelength of 250 nm,a velocity of 1.0m L/min and an injection amount of 10?L,the contents of ammonium glycyrrhetate,liquiritin and liquiritigenin in the fermentation products of endophytes in licorice were determined at a column temperature of 35?.Results:1.140 strains of endophytes were isolated from wild and cultivated Glycyrrhiza uralensis,including 94 strains of endophytic bacteria and 46 strains of endophytic fungi.Molecular biological identification showed that there are 6 strains of Bacillus atrophaeus(JTZB002,JTZB006,JTZB014,JTZB018,JTZB020 and JTYB006),5 strains of Bacillus subtilis(JTZB005 ?JTZB016?JTZB058?JTZB059?JTZB060),2 strains of Bacillus mojavensis(JTZB043 and JTYB029),2 strains of Bacillus sonorensis(JTZB054 and JTZB063),1 strain of Bacillus vallismortis(JTYB012),1 strain of Firmicutes Lactobacillales Aerococcus viridans(JTZB062),and 3 strains of Aspergillus ochraceus(JTZF011,JTZF014 and JTYF023).2.18 strains of endophytes fermentation products(JTZB002,JTZB005,JTZB006,JTZB014,JTZB016,JTZB018,JTZB020,JTZB043,JTZB054,JTZB058,JTZB059,JTZB060,JTZB062,JTZB063,JTZF011,JTYB006,JTYB029 and JTYF023)showed significant bacteriostatic activity in vitro against staphylococcus aureus,and JTZB059 and JTYB029 showed strong bacteriostatic activity.3 strains of endophytes fermentation products(JTZB005,JTZB043,JTZF014)showed significant bacteriostatic activity against streptococcus pneumonia.3 strains of endophytes fermentation products(JTYB012,JTZB018 and JTZB020)showed significant bacteriostatic activity against escherichia coli.Bacteriostatic test in vivo indicated that compared with the model group,10 groups of endophytes fermentation products(JTZB002,JTZB005,JTZB006,JTZB014,JTZB020,JTZB043,JTZB058,JTZB060,JTZB063 and JTYB029)could significantly increase the death protection rate and survival days of mouse infected with staphylococcus aureus(P<0.05 or P<0.01),and 2 groups of endophytes fermentation products(JTZB005 and JTZB043)could significantly increase the death protection rate and survival days of mouse infected with streptococcus pneumonia(P<0.05 or P<0.01).3.MTT showed that compared with the blank group,the cell survival rates of different drug administration group at mass concentrations of 10mg/L,20mg/L and 40mg/L had no significant differences(P>0.05).When the mass concentration was maintained at 40mg/L,the contents of NO,PGE2 and IL-1? in 12 groups of endophytes fermentation products(JTZB005,JTZB006,JTZB018,JTZB020,JTZB059,JTZB060,JTZB063,JTZF011,JTYB006,JTYB012,JTYB029 and JTYF023)were significantly lower than those of the model group(P<0.05 or P<0.01).Anti-inflammatory test in vivo indicated that 7 groups of endophytes fermentation products(JTYB006,JTYB029,JTYF023,JTZB006,JTZB005,JTZB060 and JTZB063)could significantly inhibit xylene-induced mice ear swelling and inhibit acetic acid-induced abdominal capillary permeability of mice(P<0.05 or P<0.01).8 groups of endophytes fermentation products(JTYB006,JTYB012,JTYB029,JTYF023,JTZB005,JTZB006,JTZB060 and JTZB063)could significantly reduce blood WBC of the pneumonia model rats,7 groups of endophytes fermentation products(JTYB006,JTYB029,JTYF023,JTZB005,JTZB006,JTZB060 and JTZB06)could significantly decrease the contents of TNF-? and IL-6 and increase the content of IL-10 in rat serum(P<0.05 or P<0.01).4.After inducing cough by ammonia,8 groups of endophytes fermentation products(JTYB029,JTZB005,JTZB006,JTZB014,JTZB020,JTZB043,JTZB060 and JTZB063)had significant antitussive effect(R>150),2 groups of endophytes fermentation products(JTYB006 and JTZB058)had antitussive effect(130<R<150).After inducing cough by SO2,the cough incubation period of rats in 6 groups of endophyte fermentation product(JTYB029,JTZB005,JTZB006,JTZB043,JTZB060 and JTZB063)was prolonged,the number of coughs within 2 min was significantly reduced(P<0.05 or P<0.01),the cough incubation period of JTZB060 was the highest,and the antitussive rate of JTYB029 was the highest.5.Compared with the model group,the rat syndrome manifestations and pathomorphism of the fermentation products of 5 group endophytes(JTYB029?JTZB005?JTZB006?JTZB058?JTZB063)were milder,the distributions and expressions of aquaporin-1 and aquaporin-5 increased,the contents of NO,ICAM-1 and COX-2 in lung tissue remarkably decreased,and Na+-K+-ATP activity in lung tissue significantly increased(P<0.05 or P<0.01).6.There were differences in total flavonoids and total saponins between the fermentation products of 20 group endophytes from Glycyrrhiza uralensis.The content of total flavonoids was 1.13~2.24mg·g-1 and reached the maximum in JTZB002,while the content of total saponins was 50.01~71.93 mg·g-1 and reached the maximum in JTYB029.In the fermentation product powders of endophytes,the content of ammonium glycyrrhetate was 0.012-0.074 mg·g-1 and reached the maximum in JTYB006,the content of liquiritin was 0.01-0.06 mg·g-1 and reached the maximum in JTZB020,and the content of liquiritigenin was 0.003-0.027 mg·g-1 and reached the maximum in JTZB043.Conclusions:1.Among 20 strains of endophyte from Glycyrrhiza uralensis,16 strains of endophytic bacterium are Bacillus,belonging to five species,1 strain of endophytic bacterium is Aerococcus viridans,and 3 strains of endophytic fungi are Aspergillus ochraceus.2.Both in vitro and in vivo,the metabolites of endophytes in 10 strains(JTZB002,JTZB005,JTZB006,JTZB014,JTZB020,JTZB043,JTZB058,JTZB060,JTZB063 and JTYB029)have inhibitory effects on staphylococcus aureus and streptococcus pneumonia.3.The metabolites of endophytes in 7 strains(JTZB005,JTZB006,JTZB060,JTZB063,JTYB006,JTYB029 and JTYF023)have anti-inflammatory effects both in vitro and in vivo.4.The metabolites of endophytes in 6 strains(JTZB005,JTZB006,JTZB043,JTZB060,JTZB063 and JTYB029)have antitussive effects on ammonia water and SO2 induced rats.5.The metabolites of endophytes in 5 strains(JTZB005,JTZB006,JTZB058,JTZB063 and JTYB029)have expectorant effects on the rats in the model of phlegm blocking in lung.6.The metabolites of endophytes in 20 strains and host plant Glycyrrhiza uralensis have similar effective parts(total flavonoids and total saponins),the metabolites of endophytes in 12 strains and host plant Glycyrrhiza uralensis have similar active ingredients(glycyrrhizic acid,liquiritin and liquiritigenin).7.The metabolites of endophytes and host plant Glycyrrhiza uralensis have similar pharmacologic action and active ingredients.