| Objective:Primary liver carcinoma （PLC） is a frequent malignant tumor doing great harm tohuman’s health. TCM treatment with its particular effect is still an important part ofintegrate therapy of PLC. It is a hot research topic to find effective anticancer componentand extract from natural products by molecular biologic method. This topic should be basedon the theoty of TCM, to study the application of the therapy of eliminating pathogenicfactors on PLC and the the study evolution on the anticancer function of Annona squamosaL. To select the most active anticancer lactones compound from the seeds of Annonasquamosa L. and evaluate its anticancer action through experiments in vitro. The study is toestablish a fundamental base for putting annonaceous acetogenins into a new anticancerdrug by disclosing its anticancer mechanism in molecular biologic method.Methods:Both of the literature and experimental research were studied.1 The literature study included summarizing the understanding of ancient and modernliterature of TCM, to explain the concept of toxin and application of the therapy ofeliminating pathogenic factors on PLC, to analyze the study evolution and applicationoutlook on the anticancer function ofAnnona squamosa L. and annonaceous acetogenins.2 Experimental study2.1 Abstraction of annonaceous acetogeninsTo study and research the chemical composition of the Annona squamosa L. seeds to getoverall lactones extractive at the dichlormethane site and monomer by alcohol steeping,dichlormethane extracting, silica gel and column chromatography repeatedly, highperformance liquid chromatography, etc.2.2 Depressant effect of serum with medicine of overall lactones extractive at thedichlormethane site on the human hepatocarcinoma cell line HepG2 in vitro.By serum pharmacological method to PrePare the serum with medicine of overall lactones extractive on the dichlormethane site, to observe the inhibitory action of the serum withmedicine on the human hepatocarcinoma cell line HepG2 by MTT assay and find out thecorrelation of dosage-effect and time-effect.2.3 Selection of monomerTo observe the inhibitory action of 3 lactone compounds on the human hepatocarcinomacell line HepG2 by MTT assay and find out the correlation of dosage-effect and time-effect.2.4 Anticancer mechanism①Inverted phase-contrast microscope was used to observe the morphological changes ofcells.②Transmission electron microscope was used to observe the change of theultramicrostructure of the cells, e.g., apoptotic bodies, nuclear condensation, collapse, etc.③FCM（flow cytometer） was used to observe the apoptotic peak and analyze the cellularcycle and calculate the apoptotic rate.④Inverted biological microscop was used to observe the change of theultramicrostructure of the cells, N/C ratio of cells was processed by Image-Pro Plus andSPSS software.⑤Western blot was used to test the ERK and p-ERK, scanner was used to scan the straps,to analyze the coloration areas and integral optical density value （IOD） by software.Results:1 The stagnation of phlegm-turbid, blood-stasis and heat accumulation could change totoxin and lead to PLC. There are heat-clearing and toxin-resolving, phlegm-resolving andtoxin-resolving, blood-stasis-removing and toxin-resolving, synergia and toxin-resolving,which are important healing methods on PLC.2 From the alcohol extract of Annona squamosa L., overall lactones extractive at thedichlormethane site was separated, from the ethanolic extract of Annona squamosa L.,seven compounds were separated by column chromatography on silica gel, SephadexLH-20, recrystallization and PREP HPLC, which were identified by the means of IR,1H-NMR, 13C-NMR, MS. Among the total cherimolin-1 （AS-3）, desacetyluvaricin （AS-4）and cherimolin-2 （AS-5） are annonaceous acetogenins monomers.3 Different treatment of serum with medicine of overall lactones extractive at thedichlormethane site had the depressant effect on the human hepatocarcinoma cell lineHepG2. Among 3 treatments, the depressant effect was concentration and time dependent essentially. The highest inhibitory rate of high dose was 54.16％.4 Among 3 lactone compounds, AS-4 had the best depressant effect, and IC50 was23.79μg/ml of 48h drug action on HepG2 cell line. After treated by AS-4, HepG2 cellswere found typical morphological changes through inverted phase-contrast andtransmission electron microscope: cellular volume decreases, membrane crinkles, nucleuscondenses, collapses, apoptotic bodies came, etc. Typical sub-diploid peaks were found byFCM analysis at different dosage groups. Apoptotic rate was 24.22％after treated by50μg/ml AS-4 for 36h, and it made cells of synthesis phase to decrease 15.22％.5 High dose serum with medicine of annonaceous acetogenins and AS-4 had thesignificant effect on N/C ratio of cells, and had the time dependence. To follow theextension of action time, the N/C ratio was gradually degraded.6 Western blot showed that ERK and p-ERK were inhibited after treated by AS-4 for 36h,and the luminous intensity of straps was concentration-dependent.Conclusion:1 Toxin is the key to induce the occurrence and development of PLC, the therapy ofeliminating pathogenic factors is an important healing theorem on PLC. The therapy ofremoving toxic substance can increase the clinical curative effect.2 The overall extractive at the dichlormethane site and lactone compound AS-4 from theseeds of Annona squamosa L. have anticancer activity on HepG2 cell line. AS-4 have thebest depressant effect on the human hepatocarcinoma cell line HepG2, and it can induce theapoptosis and differentiation on HepG2 cell line.3 The mechanism of AS-4’s inhibiting proliferation on HepG2 is related todownregulating the proteinum expression of ERK and p-ERK, blocking the Ras-MAPKsignal transduction pathway. |
