Extraction Of Effective Compounds From Vaccaria Segetalis And Its Activity Evaluation

AbstractBackground and Objective:Tumour angiogenesis has become an intensely investigated area over the last decade because it is an essential component of the growth and spread of cancer and is therefore potentially an important therapeutic target.Vascular endothelium cells play an important role in angiogenesis,by inhibiting vascular endothelial cells proliferation can restrain tumor angiogenesis,to inhibit tumor growth and metastasis.HMEC-1 cell model was used in this study to compare with angiogenesis effect of different extracts from Vaccaria, select a good efficacy component to study acute toxicology and pharmacodynamics in vivo.Methods:Vaccaria water extract and ethanol extract were isolated separately by system solvent extraction and objective extraction,keeping the organic extraction phase then removing organic solvent under reduced pressure,the product was concentrated by freeze drying.Determination human micro vascular endothelial cell(HMEC-1)proliferation and IC50 by SRB method;Study the chemical constituents of active fractions, to determine saponin content by UV spectrophotomrtry,to determine residual n-butanol content in active fractions by GC method, scraping line method was used to observe cell migration,to monitor and compare the cytotoxicity of different extracts by measuring the release of LDH in extracellular fluid.The minimum toxicity dose and the minimum lethal dose were determined according to conventional acute toxicology methods,take high and low doses between the minimum toxicity dose and the minimum lethal dose,observe acute toxicity among different components and doses, blood pressure, autonomic activity and blood coagulation time of mice in each group were measured before administration and 24 hours,7 days and 14 days after administration.14 days after administration,the mice were killed and weighed the organs,calculate organ index;observed pathological changes in vital organs by HE staining;biochemical method determined tissue SOD activity,MDA content,serum BUN, GOT and GPT content.The solid carcinoma model of mouse was made by H22 cells subcutaneous injection and they were divided into five groups including blank control group, model control group,1mg/kg treatment dose,2.5 mg/kg treatment dose and 5mg/kg treatment dose groups of Vaccaria segetalis(A4).After oral administration treatment for 13 days,the mice were killed,and the inhibited rate of tumor growth was calculated.The pathological morhpologic change of tumor was observed by HE staining;the cell apoptosis in tumor tissues was observed by TUNEL method.The expression of CD31 of tumor endothelial cells was measured by immunohistochemical method.Results:IC50 of A4,A7,B4,B7 are corresponding to 6.44±0.2,12.78±0.4,7.25±0.07 and 10.35±0.21,which means efficacy order was A4> B4> B7> A7; four active fractions all contain saponin,saponin content are A4> B4> B7> A7,there may be flavonoids in active fractions,residual n-butanol content were under 0.5%.24h after drug intervention cell migration distance of A4,A7,B4,B7 were 19.78±3.14μm,30.56±6.99μm,20.99±1.69μm and 36.88±0.42μm respectively;cell extracellular fluid LDH activity(U/L) were 758.1±19.73,1006.54±129.41,1111.11±36.97 and 617.45±75.93 corresponding to A4,A7,B4,B7.The minimum lethal dose of A4 and B4 were 1500 mg/kg,A7,B7 were 1000 mg/kg;the minimum toxicity dose of A4 was 100 mg/kg,others were 80 mg/kg. After one time administration,Compared with control group,200 mg/kg dose groups blood coagulation time got shorter,blood pressure decreased,14 days later,blood coagulation time and blood pressure returned to normal except blood pressure of A7 group remained lower than normal group,tissue SOD activity,MDA content,serum GOT, GPT, BUN, content did not change significantly;After one time administration,compared with control group,1000 mg/kg dose groups blood coagulation time got shorter,blood pressure decreased,14 days later,blood coagulation time and blood pressure returned to normal except blood pressure of B4,B7 groups increased,tissue SOD activity decreased,MDA content increased,serum GOT, GPT content did not change significantly,serum BUN content increased,there are pathological changes in heart liver,brain,lung and other major organs.Vaccaria segetalis can improve the quality of life on mice invaded by tumor, inhibit tumor growth.There were necrosis, tumor cell apoptosis,and expression of CD31 decreased in tumor tissues.Conclusion:N-butanol extract of Vaccaria alcohol extract isolated by system solvent extraction(A4) was the relative high activity part with low toxicity,this component has strong cell proliferation and migration inhibitory activity in vitro. LDH release show low cytotoxicity in vitro,weaker acute toxicity was also showed in vivo.It can improve the the quality of life on mice invaded by tumor,and inhibit the growth of tumor.The mechanisms were likely due to inhibiting the angiogenesis,and inducing apoptosis in tumor tissues.In summary this component has the good prospect of development for five categories of antitumor drugs.