Synthesis, Characterization And Antitumor Activity Evaluation Of Baicalin-rare Earthmetal (Ce, La, Y) Complex

Seeking new and efficient antitumor drugs focus has been paid attention in pharmacy and biology fields,becasue tumor serious threat to human lives and livelihoods. Recent studies show that Baicalin belongs toflavonoids, itself has a special structure and anti-tumor structure. However, Antitumor activity of baicalin-rareearth metal complexes has been rarely reported, and its mechanism of action is unclear for further study.interdisciplinary means, including medicinal chemistry, cell biology, molecular biology, electrochemistry,animal toxicological tests and drug safety evaluation experiments were used this paper, Three weresynthesized BMC(Ce3+, La3+, Y3+) and Their effects on human hepatoma SMMC-7721cells and its interactionwith DNA of liver cancer were studied to explore the BMC and hepatocellular DNA binding capacity of thestrength associated with anti-tumor activity. Discussion on the strength of BMC and liver DNA bindingcapacity of its anti-tumor activity of the association. In addition, We carried out animal pathology andsynthetic drugs safety evaluation experiments to seek further toxicological and safety. The main work is asfollows:(1) Method baicalin-rare earth Complexes BMC (Ce3+, La3+, Y3+) was synthesised by complexingligand. The composition and structure of BMC were characterized with liquid chromatography-massspectrometry, elemental analysis and a variety of technical TGA, IR, UV spectra. The results show that threenew BMC Successfully prepared, Molar ratio of BC-Ce was3:1, Both molar ratio of BC-La and BC-Y was2:1. Complex formula was Na3Ce(C21H16O11)3·10H2O, Na2La(C21H16O11)2·8H2O, Na2Y(C21H16O11)2·6H2Orespectively.(2) MTT was used to analyze the effects of BMC on SMMC-7721cell proliferation, as shown that BMCcan inhibit cell proliferation. The result of Fluorescence polarization was indicated that BMC action bySMMC-7721significantly reduced membrane fluidity. Cell cycle analysis showed that BMC caused cell cyclearrest in G2/M phase; The results of immunofluorescence show BMC have some damaging effects on actinand nuclear of SMMC-7721hepatoma cells to inhibit cell proliferation; Bax, Bcl-2mRNA and protein testresults show: BMC up-regulated the expression of Bax mRNA and protein in SMMC-7721cells, meanwhiledown-regulated that Bcl-2mRNA and protein; The results of Western Bloting(WB) further validate it. Theresults demonstrate that BMC inhibited cell proliferationby by disrupting the cell membrane fluidity, cellcycle arresting, destructing nuclei and actin to described that three kinds of complexes have good anti-tumoractivity: Baicalin-Cerium(BC-Ce)> Baicalin-LanthanumL(BC-La)>Baicalin-Yttrium(BC-Y)>Baicalin(BC),and showed dose-response relationship.(3) In order to determine the security of three kinds of complexes, Stable transfection method to obtainthe HEK293cells loading of various ion channels. Whole-cell patch-clamp technique was used to recordchanges on each ion channel currents in HEK293cells to investigate the mechanism of cellular pharmacologyof baicalin metal complexes drugs (BMC). The results showed that, BMC have impact on various ionchannels, for Kv1.4, Cav3.2have obvious impact, and BMC, the same ligands, the different metal ions, havedifferent effects on ion channels. Among the ion channels, the percentage inhibition for BC-La, BC-Y, BC-Ceon Kv1.4rates are91%,76%,-10%respectively; for Cav3.2inhibition rate are43%,57%,-14%respectively.Manifested as class-effect relationship of metal ions, that is, pharmacological effects show a strong correlation (4) with the type of metal ions in class-effect complexes. Three baicalin metal complexes, BC-La, BC-Ywith respect to BC-Ce strong efficacy, can act on ion channels and related diseases, but represents the strongerthe greater efficacy toxicity, so Conversely safety. BC-La, BC-Y with respect to BC-Ce was more strongefficacy, witch acted on ion channels and related diseases, but represents the stronger the efficacy, the greaterefficacy toxicity, so Conversely safety.(5) The total DNA were extracted SMMC-7721cells as its targeting, to further study the interactionmechanism between the three complexes with DNA, using an electrochemical method on the DNA level. Theresults show that the action mode of BMC and DNA of liver cancer is the two mixed-mode, includingelectrostatic interactions and inserting mode, to forming electrically inactive supramolecular compound.Binding number m=1, Binding constant as follow: βBC=1.27×105L mol-1, βBC-Y=3.46×105L mol-1,βBC-La=6.24×105L mol-1, βBC-Ce=7.29×106L mol-1, namely the the strength binding of BMC and DNA:BC-Ce> BC-La> BC-Y> BC.(6) Toxicological experiments was carried out in vivo to further study the pharmacological value of threecomplexes. First, immunodeficient mice were combined to Co tumor, The best concentration of cellsSMMC-7721was1×107cells/ml and the best combined time of tumor was12days by pre-experiment. Insubsequent experiments, the cell concentration as targeting was injected into immune deficiency nude mice bysubcutaneously to co tumors. Intraperitoneal injection was after a successful co-tumor, with taking the tumorand pathology experiments, It showed that the capacity of inhibit tumor growth of these complexes as follow:Baicalin-Cerium (BC-Ce)>Baicalin-LanthanumL(BC-La)>Baicalin-Yttrium (BC-Y)> Baicalin(BC).(7) The above results show that anti-tumor activity of three kinds of BMC: BC-Ce> BC-La> BC-Y> BC.From the cellular level, its mechanism is that nucleus may be destroyed, cell membrane fluidity andmitochondrial membrane potential were changed after drugs on cellular to inhibit cell proliferation. From thepoint of view gene and protein levels, Up-regulated bax mRNA and protein or down-regulated bcl-2mRNAand protein to achieve apoptosis. Pathology experimental and toxicological experimental in animals showedthat three kinds of complexes by inhibiting tumor growth to extend the life of sick animals. Moreover, Ionchannel HEK293cell mode was builded from the ion channel level for electrophysiological experiments anddrugs safety evaluation of BMC. Related work provide to a new mechanism of reference for pharmacologicalbaicalin Metal Complexes. We can draw that three-drug anti-tumor activity is closely related to the DNAbinding capacity by electrochemical means, indicating that the replication of cellular DNA was Prevented tothus inhibit cell proliferation after the drug into the cells.This thesis successfully synthesized three new metal complexes, the mechanism of anti-tumor activityand toxicity of the three drugs were studied from all levels. Ion channel HEK293cell model antitumor activityof BMC binding to DNA was established. Related results provide new ideas for the design of new anticancerdrugs and efficacy evaluations, but anti-tumor mechanism in vivo needs further study.