The Primary Study Of Antifungal Protein And Fibrinolysis Enzyme From 9 Moraceae Plant Species

The antifungal activities of nine Moraceae plant species were compared, and partial antifungal proteins were purified. At the same time, the thin polyacrylamide fibrin assay method was established. And the fibrinolysis activities of nine Moraceae plant species were studied. Antifungal proteins were found in Broussonetia papyrifera, Humulus scandens, Morus alba, Ficus carica, Cannabis sativa, and Artocarpus lakoocha. Those species were studied with a procedure involving ion exchange chromatography (Hitrp SP, 1ml) and hydrophobic interaction chromatography (RESOURCE ISO, 1ml). The results indicated that the antifungal fractions of those species were similar. Three antifungal fractions as single peak on Mono S were obtained from Ficus carica with a procedure involving ion exchange chromatography (SP-Sepharose Fast Flow, 26/30), hydrophobic interaction chromatography (Phenyl Sepharose 6 Fast Flow, 16/34 and RESOURCE ISO, 1ml) and ion exchange chromatography (Mono S, 1ml). They were named W1 andW2, respectively. They were not homogeneity identified by MALDI-TOF mass spectrometry. Four antifungal fractions as single peak on Mono S were obtained from Broussonetia papyrifera with a procedure involving ion exchange chromatography (SP-Sepharose Fast Flow, 26/30), hydrophobic interaction chromatography (Phenyl Sepharose 6 Fast Flow, 16/34 and RESOURCE ISO, 1ml) and ion exchange chromatography (Mono S, 1ml). They were named BrouinⅠ, BrouinⅡ, BrouinⅢand BrouinⅣ, respectively. Those retention volume were 4.400ml, 4.889ml, 5.437ml and 5.725ml, respectively on Mono S, when gradient elution was 0-0.12mol/LNaCl, flow was 1ml/min, and elution volume was 8ml. BrouinⅡ, BrouinⅢand BrouinⅣwere basic, with pI over 9.0. BrouinⅣhas an IC(50) of 0.1μg/μl against Trichoderma viride .BrouinⅡ, BrouinⅢand BrouinⅣhad molecular masses of 18798.703Da,31187.200Da and 31178.392Da on MALDI-TOF MS analysis,respectively.The peptide mappings lysed by trypisn were determined by MALDI-TOF MS.BrouinⅢhad 7 homologous peptides to BrouinⅣ, BrouinⅡhad no homologous peptides to BrouinⅢand BrouinⅣ. The N-terminal amino acid sequence of Brouin Ⅱwas eqcgsqvggktcpnnlccxkygwcgdtddh.The N-terminal amino acid sequence of BrouinⅢand BrouinⅣwas eqcgrqaggalcpgglccxkygwcgntpey. BrouinⅡshowed sites of high similarity with the sequences of plant chintase, the identities were 66% to 77%. BrouinⅢand BrouinⅣshowed sites of similarity with the sequences of plant chintase, the identities were 43% to 50%. Thses results indicated BrouinⅡ,BrouinⅢand BrouinⅣwere homology to plant chinitase. Polyacrlamide replaced agarose and casting gel instrument of plate electrophoresis replaced glass or plastics plate. So the controllable thickness and homogeneous thin fibrin plate was fabricated. The lysis of fibrin was indicated through protein stained. At last a method for exact detection and analysis fibrinolysis activity in vitro and high throughput screening of fibrinolysis drugs was established, the method was named the thin polyacrylamide fibrin assay method. The optimum condition was ascertained that the plate contained 0.5mg/mL fibrin and 10% acrlamide. The thin polyacrlamide plate was stained with Coomassie Brilliant Blue after incubated for 8-12 hours at 37℃in wet. It could detect fibrinolysis activity of 25ng lumbrokinase in minimum. The logarithm oflumbrokinase concentration and the logarithm of the transparent spots diameters lysed by lumbrokinase showed good linearity(R=0.9995 ) when the concentration of lumbrokinase was 1×10-2 -5mg/mL. Less variation RSD was 3.83% (n=5). The fibrinolysis activities of lumbrokinase were detected by the thin polyacrylamide fibrin plate assay and the two common methods for detection of the fibrinolysis activity in vitro, chromogenic substrate assay method and fibrin agarose plate assay method. They were compared with regard to sensitivity, reproducibility, acceptance, validity of throughput, reliability of quantitative analysis and applicability. The chromogenic substrate assay method was single and sensitive, the examination limit was low, and it could be used to detect the imperceptive change of fibrinolysis activity. The fibrin agarose plate assay method was simple operations and quantitative, it could be used to screen the fibrinolysis activity. The thin polyacrylamide fibrin plate assay have many merits, such as high sensitivity, high throughput, low cost, good reproducibility and could be used in quantitative analysis. It could be used in the high throughput screening of fibrinolysis drugs and the qualitative analysis of fibrinolysis activeity. Fibrinolysis activities of six cardiovascular medicines bought from store were detected by the thin polyacrylamide fibrin assay in vitro .The results were corresponded with some reports. This ascertained that the thin polyacrylamide fibrin plate assay could accuratly and dependably detect the fibrinolysis activity in vitro. The nine Moraceae plant species were screened by the thin polyacrylamide fibrin assay. The seeds of Cannabis sativa and the twigs of Broussonetia papyrifera indicated high fibrinolysis activity. The leaves of Broussonetia papyrifera and Ficus elastica indicated fibrinolysis activity. The fibrinolysis enzyme from the twigs of Broussonetia papyrifera was tried to purify with a procedure involving ammonium sulfate precipitation and ion exchange chromatography(SP-Serphose Fast Flow,16/16 and Mono S,1ml).But the fibrinolysis activity fractions were decentralization.