Comparative Study On Antimicrobial Activity Beween The Gansu Wild And Cultivated Licoce Endophyte Fermentation Product And The Host Plant And Its Medicinal Ingredient

OBJECTIVE: Study on the antibacterial activity of effective fermentation, host decoction, total flavonoids, total saponins on Staphylococcus aureus, Streptococcus pneumoniae and E.coli, compare the differences on the antibacterial activity between the effective strains and the host. Provide a theoretical basis for solving the wild licorice resources shortage and seeking the active ingredients of licorice substitute.METHODS: 1. The wild and cultivated licorice endophytic bacteria and fungi were isolated by the method of lines and five points by NA and PDA respectively, using the method of bacteria cake to screen the effective strains of endophytic bacteria with inhibitory effect that to Staphylococcus aureus, Streptococcus pneumoniae, E. Coli.2. Using ultrasonic extraction method, extraction temperature, extraction time, ethanol concentration, solid-liquid ratio as factors, the total flavonoids and total saponin content as evaluation index, apply L9(34) orthogonal test design, optimization the optimum extraction process of total flavonoids and total saponins in wild and cultivated licorice.3. Use the method of quantitative antibacterial to observed 20 Licorice entophytes fermentation Product, decoction, total flavonioids, total saponins in wild and cultivation Licorice.4. By intraperitoneal in of Staphylococcus aureus, Streptococcus pneumoniae, E. coli,was observed by oral administration to mice infected with endophytes 20 licorice fermentation product, the host decoction, total flavonoids and total saponins gavage for protection rate of infection in mice.RESULTS 1. Isolated of licorice endophytic and screening of effective strains: 63 strains of endophytic bacteria were isolated from the wild licorice, including 31 strains of endophytic bacteria, numbered JTYB001, JTYB002 and JTYB003... JTYB031, 32 strains of Endophytic fungi, numbered JTYF001, JTYF002 and JTYF003... JTYF032. 77 strains of endophytic bacteria were isolated from the cultivated licorice, including 63 strains of endophytic bacteria,numbered JTYB001, JTYB002 and JTYB003... JTYB031, 14 strains of Endophytic fungi,numbered JTYF001, JTYF002 and JTYF003... JTYF032. 18 strains with antibacterial activity were screened out from the above 140 strains. There are 18 strains of endophytic which have inhibitory effect on Staphylococcus aureus: JTYB006、029,JTYF023,JTZB002、005、006、014、016、018、020、043、054、058、059、060、062、063,JTZF011;There are 3 strains of endophytic which have inhibitory effect on Streptococcus pneumoniae: JTZB005 、JTZB043、JTZF014;There are 3 strains of endophytic fungi which have inhibitory effect onE.coli: JTYB012、JTZB018、JTZB020。2.Ultrasonic extraction technology of licorice total flavonoids and total saponins:(1)total flavonoids: B extraction time> A extraction temperature> C ethanol concentration> D solid-liquid ratio. Four factors are significant, optimum extraction is B3A3C2D1;(2) Total saponin: A extraction temperature> C ethanol concentration> D solid-liquid ratio> B extraction time, A extraction temperature, C ethanol concentration and solid-liquid ratio have significant, optimum extraction is A2C3D1B2.3. In vitro antibacterial test: to Staphylococcus aureus, every group has antibacterial activity(P <0.01), each entophyte group were weaker than the host group; The antibacterial activity of endophytes numbered JTZB059, JTYB029 is stronger, the minimum inhibitory concentrations were 0.1250 g /m L, 0.0625 g/m L; The antibacterial activity of total flaonoids is best in wild and cultivated in Licorice. To Streptococcus pneumoniae, every group has antibacterial activity(P <0.01); To compared with decoction and total saponins in cultivated Licorice JTZB005 has a stronger bacteriostatic effect, the minimum inhibitory concentrations were 0.0625 g/m L; The antibacterial activity of total flaonoids is best in cultivated in Licorice.To E. coli, every group has antibacterial activity(P <0.01), To compared with decoction and total saponins in cultivated Licorice JTZB018 has a stronger antibacterial effect, the minimum inhibitory concentrations were 0.1250g/m L; The antibacterial activity of total flaonoids is best in wild and cultivated in Licorice.4. In vivo antibacterial test: influence of Staphylococcus aureus infection in mice:Endophyte JTYB029, JTZB002,005,006,014,020,043,058,060,063, Licorice root, total flavonoids and total saponins against Staphylococcus aureus infection in mice has a protective effect(P<0.05,P<0.01); JTYB029 and JTZB002, 005, 006, 058 and the water decoction, the total flavonoids, the total saponins made the mice average survival time up to 3 days(P<0.01);JTZB014,020,043,060,063 reached the mice average time to 2 days(P<0.05 or P<0.01).Influence of Streptococcus pneumoniae infection in mice: JTZB005,043 and the water decoction, the total flavonoids, the total saponins against Staphylococcus aureus infection in mice has a protective effect(P<0.05 or P<0.01); TZB005, the wild licorice decoction, the total flavonoids, the total saponins made the mice average survival time up to 3 days(P<0.01);JTZB043 and the cultivated decoction made the mice average survival time up to 2 days(P<0.01). No protective effect of endophytic bacteria on the mice infected with E.coli.CONCLUSION 1. Isolated the 63 endophytes from the wild licorice,isolated the 77 endophytes from the cultivate licorice, Licorice has its own endophyte species diversity.2.Ultrasonic extraction and orthogonal experimental design used to extract total flavonoids and total saponins, the process is simple, convenient and fast.3.Part licorice endophytes have the same or similar inhibitory activity of the host.4.Prompt, JTYB029 alternative wild licorice inhibit Staphylococcus aureus, JTZB005 alternative cultivated Glycyrrhiza total flavones inhibit Streptococcus pneumoniae.