Study On Antitumor Effects Of The Juglans Mandshurica Maxim Stem-bark Extract In Vitro

Malignant tumor is a clinical common disease,threating the person's physical and mental health severely.In the world ,the death rate of the tumor is the first or the second in all of the death causes and that has been the second in China.Tumor is a refractoriness disease because of the multiplicity of etiopathogenisis.Drug treatment,operation cure and radiation therapy are the important methods of cancer treatment.Chemotherapy is circumscribed in clinical application because the chem drug has severe side effect.Traditional Chinese drug is a traditional method of cancer therapy and in the advantage of its less side effect, it is becoming the considerable treatment in tumor combined therapy .Juglans mandshurica maxim belongs to Juglandaceae.It is distributed over north-east ,north of China,HeNan province and ShanXi province in China.Its root,stem-bark,leaves and exocarp(qinglongyi) have pharmacologic action including antibiosis,anti-virus,analgesia,anti-oxydation,anti-tumor and et al.Especially anti-tumor effect is paied closed attention by much researcher,who concentrate more investigation on anti-tumor effects of Leaves and qinglongyi .The results show that the extracts of leaves and qinglongyi can inhibit multitude kinds of tumor cells proliferation, induce the apoptosis of tumor cells,make telomerase activity down, block cell cycle, increase immune function of mice carrying tumor,influence the tumor cell membrane fluidity and erythrocyte membrane fluidity, promote the expression of TNFα-mRNA in peritoneal macrophage of mice and so on.Qinglongyi has been used for cancer treatment in clinic,which gain satisfied effects.The experiments in antitumoreffects of the Juglans mandshurica maxim stem-bark are performed by few people.We have used S180 cells to identify the functions of Juglans mandshurica maxim stem-bark extracts(JMME).The results show that the extracts of Juglans mandshurica maxim stem-bark have remarkable cytolytic and cytostatic action on S180 cells, increase weight of thymus and thymus index, increase transformation of lymphocyte,promote activities of NK cells and increase both TNF and IL-2 level.On the basement of the science conclusions,this study aimed at anti huaman tumor of SMMC-7721,MCF-7, A549 effects and mechanism of Juglans mandshurica maxim stem-bark extracts by the methods of growth curve,MTT,morphologic observation, agarose gel electrophoresis,telomerase PCR-ELISA .1.Separated Juglans mandshurica maxim stem-barkThe extracts were prepared from Juglans mandshurica maxim stem-bark by the way of ethanol soaking, low pressure distill and organic reagent abstraction. Finally, the desired part of Juglans mandshurica maxim is gotten by means of chromatography.2.Juglans mandshurica maxim stem-bark extracts influenced cells growth curveExtracts from Juglans mandshurica maxim with various doses were added to experimental groups of SMMC-7721,MCF-7 and A549. 40μl cell suspension was sampled respectively after treated 0,1,2,3,4,5,6,7 d adding 0.4% trypan blue 40μl in order to count living cells by blood counting ehamber. Drawed coordinate Diag with logarithm of living cells per milliliter and culture time.The result showed that the extracts had cytostatic action on the three kinds of tumor cells. Extended the cell growth curve's linearity to axis ofordinates,that illustrated that the number of the extracts treated own generate cells decrease at the time of 0. The cell doubling time of SMMC-7721,MCF-7,A549 was 20.96h,19.28h,20.67h respectively but the extracts treated was 34.23h,32.89h,35.69h. The cell doubling time of the experiment groups was longer than the control's.At about the fourth or fifth day ,the control cells growth reach plateau phase while extracts treated cann't. 3.Cytolytic action of Juglans mandshurica maxim stem-bark extracts Inoculated the cells during the logarithm growth phase into 96-well cell culture plate,then added extracts with various doses. MTT method was used to detected A . The result showed that the extracts had cytolytic action on tumor cells. IC50 of SMMC-7721,MCF-7,A549 was 211.21μg/ml,110.37μg/ml, 176.20μg/ml respectively. It also demonstrated that this function was dose-dependence.By q test,P<0.05,so disparation was significant.4.Juglans mandshurica maxim stem-bark extracts enhanced the LAK cell's cytolytic action on tumorInoculated the cells during the logarithm growth phase into 96-well cell culture plate,then added LAK,JMME,LAK+JMME respectively.A was detected by MTT after treatment for 72h.The result revealed that the effect of LAK 's cytolytic action on tumor was enhanced by extracts.5.Juglans mandshurica maxim stem-bark extracts induced apoptosis on tumor cells the cellular morphology of tumor cells(SMMC-7721,MCF-7,A549) was observed by inverted microscope,the DNA gragmentation of tumor cells was detected by agarose gel electrophoresis. The typical apoptosis morphology was identified under inverted microscope.The typical ladder bar can beobserved.These demonstrated that the tumor cells were induced apoptosis by Juglans mandshurica maxim stem-bark extracts.6.Juglans mandshurica maxim stem-bark extracts influenced telomerase activity of tumor cellsTelomerase activity was measured by TRAP PCR-ELISA assay.The result shows that the telomerase activities of SMMC-7721,MCF-7,A549 were stronger than negative control but the telomerase activities of the tumor cells treated with extracts were much weaker than the tumor cells untreated.The inhibition rate was 42.86%,73.30%,58.78% respectively.The results of this study have shown that Juglans mandshurica maxim stem-bark extracts can influence the grown character of tumor cells through cytostatic action, induction of apoptosis and weakening telomerase activity. Meanwhile the cytostatic action of LAK was enhanced by extracts.