| Brucea javanica is the dehydrated ripe fruit of Brucea javanica(L.)Merr(Simaroubaceae).It is a bitter,cold natured traditional chinese herb medicine with the functions of clearing away heat and detoxification,killing insects and stopping phlegm uptaken by large intestine and liver.Cultivation areas in China are tropical and subtropical zones,including Guangdong,Guangxi,Yunnan,Fujian,Taiwan and Hainan provinces.In recent years,the incidence of melanoma has rapidly increased.As a skin cancer drug commonly used in traditional Chinese medicine,drug efficacy substances of Brucea javanica are mainly concentrated in Brucea oil and bitter lignin compounds.Since our previous study found that Brucea javanica proteolysis has a significant inhibitory effect on tumor cell proliferation,we believed that the Brucea javanica polypeptide component should have anti-melanoma activity.In this study,Brucea globulin was used as the research object in order to investigate the effects of the content of globulin and the activity of biopeptide anti-melanoma of Brucea javanica in different areas and to optimize the extracting method of globulin.After pepsin hydrolyzed globulin,the polypeptide was separated and purified by ultrafiltration and gel chromatography so that we could obtain a high activity group of biopeptide anti-melanoma and then identify the amino acid sequence by mass spectrometr.Principal components analysis and globulin components research of Brucea javanica from different producing areas.By collecting Brucea javanica from four producing areas including Guangdong,Guangxi,Yunnan,and Fujian and then determining the total protein,moisture,ash,crude fat,and starch content respectively by Kjeldahl method,oven method,muffle furnace method,optical rotation method and cable extraction method,the results showed that the total protein content of Brucea javanica in the Guangxi origin was 17.23%which was significantly higher than that in other three producing areas(P<0.05).The protein content of Brucea javanica in Yunnan,Fujian and Guangdong provinces were 14.20%,13.20%and 12.61%,respectively.The Brucine globulin was extracted with 5%NaCl for qualitative and quantitative analysis and the result showed that there was no significant difference in the results of SDS-PAGE electrophoresis of Brucea globulin from different regions.The results were mainly distributed in 33 kDa,32 kDa,19 kDa and 11 and the vicinity of kDa showed there was a significant difference(P<0.05)among producing areas.The Brucea javanica content from Guangxi was highest,accounting for 6.52%of the total protein,followed by the globulin content from Yunnan and Fujian regions 4.07±0.06%and 4.06,respectively.±0.04%(no significant difference,P>0.05),but the lowest globulin in Guangdong,only 2.63±0.04%of total protein.Pepsin hydrolyzed Brucella globulin of different origins respectively.The reaction conditions were as follows:substrate concentration 1%,ratio of enzyme to substrate 1:10(w/w),pH=2.0,temperature 37℃,time 48 h.The hydrolysates were obtained by ultrafiltration(MWCO=3 kDa)to obtain small molecule polypeptide fractions less than 3 kDa.MTT assay was used to compare the cytotoxicity of Brucella gut proteins derived from different origins on melanoma cell A375.The results showed that the anti-melanoma activity of Brucea protein derived from Fujian origin was the best(P<0.05),IC50 value was 2.78±0.03μg·mL-1 at 72 h,followed by the Guangxi origin sample,IC50 value was 5.54±0.03 μg·mL-1。Guangdongensis and Yunnan isolates on A375 cells were 8.16±0.01μg·mL-1 and 8.34±0.04 ug·mL-1,respectively.Study on the optimization of preparation method of Brucea javanica globulin.Precipitation method was used to determine the isoelectric point of Brucea javanica globulin and it’s 2.8.Then the globulin was primarily purified by isoelectric precipitation.The extraction efficiency and purity of Brucea globulin were studied.The results showed that the isoelectric precipitation did not cause the loss of the globin protein band by SDS-PAGE electrophoresis.The protein purity was 83.77%,which was 4.49%higher than the globulin without precipitation treatment.And the extraction rate was 21.61%,The hydrolysates(≤3 kDa)of B.javanica globulin produced by pepsin showed significantly high growth inhibitory activity on A375 cell with the IC50 value of 2.09 μg·mL-1.The purification and structure identification of anti-tumor peptides.Globulin was hydrolyzed by pepsin,separated by ultrafiltration(molecular weight cut-off=3 kDa)and Sephadex G-10 chromatography.At the same time,MTT assay was used to screen highly active anti-tumor peptides.After separation by Sephadex chromatography,9 fractions,(F1-F9),were obtained.There was a significant difference in the inhibitory effect of each component on the proliferation activity of A375 cells,the inhibitory rate of component F3 was-18.85%,while the inhibition rates of components F7,F8 and F9 were 90.69%,91.83%and 90.58%respectively(the drug concentration was 2.2 μg·mL-1).The IC50 values for further calculation of the cytotoxicity of F7,F8 and F9 were 0.47μg·mL-1,0.52 μg·mL-1,and 0.2μg·mL-1,respectively,and the F9 activity was highest.F9 reversed high performance liquid phase separation,a total of 7 components were obtained.The amino acid sequence of component F9 was analyzed by liquid chromatography and mass spectrometry.Four 8-10 amino acid polypeptides were obtained from the Uniprot Sapindales database,and their molecular weights ranged from 851.97 to 1036.21. |
