Screening And Mechanism Of The Antineoplastic Effective Components From Semen Litchi

1Purpose and significanceThe study aims to screen of the anti-tumor active components of Semen litchi, thanpreliminary discusses its possible anti-tumor mechanism and analyzes the chemicalcompositions of the active parts on the base of components extraction and isolation in thenext place. The achievement would provide the fundamental basis for the further researchof the pharmacological effect on the anti-tumor effective components from Semen litchi.The significance of the research is to select out the component of Semen litchi with thebest anti-tumor effect, and confirm its way of inhibition on tumor cell is induction of cellapoptosis, and analyse the probably effective components which would be its flavonoidsand tetranuclear diterpenoids components.2Methods2.1Selection of Tumor Cell Lines and Study on Their CharacterizationsThe research had a study on the growth characteristics on human lung cancer cell-A549、liver cancer cell-Hep G2、breast cancer cell-MCF-7、cervical cancer cells-SiHaand Hela those had a high rates of cancer deaths, that provided prerequisite for druganti-tumor experiment. The growth curves of tumor cells with different concentrations weredrawed by continuous cultivation method and the MTT assay. Thus we could find the bestexperimental cell number and time for detection. What’s more, this experiment also studiedthe harmless concentration of DMSO as a drug co-solvent by MTT assay.2.2Extrcation and Sepration of Semen Litchi ComponentsTo discuss the anti-tumor effects of different components from Semen litchi, we needto extract and isolate the components of Semen litchi firstly. The Semen litchi extractwas acquired by70%ethanol and ultrasonic extraction method, and petroleumether phase component、ethyl acetate phase component、adsorbed component of D101macroporous resin and aqueous phase component from Semen litchi are acquired byliquid-liquid extraction combined with D101macroreticular resin.These fourisolates would be the components for the experiment on screening of theantineoplastic effective drugs.2.3Screening of Anti-tumor Effective Components from Semen LitchiTo define the anti-tumor effect of different components from Semen litchi, thisstudy detected the effects of the petroleum ether phase component、ethyl acetate phase componen、adsorbed component of D101macroporous resin and aqueous phase componentfrom Semen litchi on proliferation of A549、HepG2、MCF-7、SiHa and Hela cells, andfiltered out the common sensitive cell line of the four phase separations, than with afurther comparison of the inhibitory effects of each separation components on it, we finallyselected out the components with the best anti-tumor ability and the corresponding sensitivetumor cell line.2.4The Study on the Mechanism of Anti-MCF-7cell Ability of Ethyl AcetatePhase Components from Semen LitchiThrough in vitro anti-tumor experiment, we selected out the ethyl acetate phasecomponent from Semen litchi has the best inhition effect on MCF-7cell. To further exploreits mechanism, firstly, this study used Annexin V-FITC/PI double staining method withobservation of cell morphology under the fluorescence microscope, in order to explorewhether the drug has induction of cell apoptosis. Then we combined Rhodamine-123staining method with flow cytometry, to explore the effects of drug on mitochondrial ΔΨm.Furthermore, the chemical compositions of ethyl acetate phase component of Semen litchiwere analyzed by infrared spectrum and ultraviolet spectrum scanning methods.3Results3.1Characterizations of Tumor Cell LinesBy drawing the cell growth curve,we found that A549、Hep G2、MCF-7and SiHa cellshave the best proliferation activity with the inoculation concentration of3.5×104cell/ml,Hela cell has the best proliferation activity with the inoculation concentration of0.5×104cell/ml. These five cell lines proliferated rapidly in24h～48h, and had the best cell viabilityat48h, then reached a plateau, so we could determine48h as the time node for their drugexperiments.Moreover, the experiment of testing the toxicity of DMSO in tumor cellsshowed that DMSO(≤0.1%)had no differences of survival rates between the experimentalgroup and negative group，but decreased the survival rates of Hep G2、MCF-7and SiHacells(p<0.05),when the concentrantion≥0.2%. Therefore we were assured≤0.1%is thesafe concentration range for DMSO used in cells.3.2Extrcation and Sepration of Semen Litchi ComponentsFour different polar components were separated from Semen litchi in this experiment：the petroleum ether phase component is non polar, the ethyl acetate phase component isweak polar, the adsorbed component of D101macroporous resin is polar, and the aqueousphase component is strong polar. Drying each component to constant weight, finally wegot：6.0404g of the petroleum ether phase component,13.5507g of the ethyl acetate phasecomponent,15.5811g of the adsorbed component of D101macroporous resin and8.0623gof the aqueous phase component.3.3Screening of Anti-tumor Active Components from Semen LitchiThe experiment of anti tumor in vitro showed that the petroleum ether phasecomponent、 the ethyl acetate phase component、 the adsorbed component of D101macroporous resin and the aqueous phase component from Semen litchi all have a goodgrowth inhibition on MCF-7cell, p<0.01.The comparison of the inhibition action of each separation component on MCF-7cell showed that the proliferation inhibition level of thepetroleum ether phase component was the lowest, the maximum inhibition rate is less than50%; The IC50of ethyl acetate phase component、the adsorbed component of D101macroporous resin and the aqueous phase component on MCF-7cell respectively were53.80mg/L、260.89mg/L and239.80mg/L. Thus we could found the ethyl acetate phasecomponent from Semen litchi has the best inhibition effect on MCF-7cell.3.4The Priliminary Study on the Mechanism of Anti-MCF-7cell Effect of EthylAcetate Extraction Phase Components from Semen LitchiThe cell apoptosis detection by Annexin V-FITC/PI double staining method found thatthe nucleus became solid and contractile when MCF-7cell had been treated with the ethylacetate phase component (100mg/L) from Semen litchi with24h, apoptotic bodies formedwith48h. The study of mitochondrial activity detected by Rh123staining method showedthe Rh123accumulation in mitochondrion of MCF-7cell treated with the ethyl acetatephase component(25～100mg/L) from Semen litchi with24h had different degrees ofdecline, and the increasing drug doses enhanced the effect(p<0.01). The further results ofinfrared and ultraviolet scanning found the characteristic absorption peak of flavonoids andtetranuclear diterpenoids were showed in the ethyl acetate phase components from Semenlitchi.4Conclusions4.1By discussing the growth regularity of these five tumor cell lines in vitro，we masteredthe best concentration for cell planting and their growth duration. We clarified that3.5×104cell/ml is the best plantingconcentration for the anti-tumor experiments of A549、Hep G2、MCF-7and SiHa cells in vitro, while0.5×104cell/ml is for Hela cell; their correspondingbest detection time of the efficacy are al48h. This result provided the premise for thefollow-up study on the antineoplastic effective components of Semen litchi.4.2Through the screening of the antineoplastic effective components of rough isolationsfrom Semen litchi in vitro, we found the sensitivities of different tumor cell lines onTraditional Chinese Medicine composition are different. The ethyl acetate phase componentof Semen litchi had the best anti-tumor efficacy, and the corresponding sensitive tumor cellline was MCF-7cell (IC50was53.80mg/L).4.3The study on the mechanism of anti-tumor cells effect of Semen litchi confirmed thatthe ethyl acetate phase component from Semen litchi could control the pathway ofmitochondrion to mediate the PCD of MCF-7cell. The further results of infrared andultraviolet scanning showed flavonoids and tetranuclear diterpenoids were the mainconstitutes of this antineoplastic effective component.