| The Traditional Chinese Medicine(TCM)of Rhizoma Coptidis has many clinical effects,and the effects are mainly for heat-clearing and damp-drying,purging fire detoxification and so on.Research shows that Rhizoma Coptidis also has extensive antitumor activity,but the major research is based on berberine and the antitumor activity of other compounds are few reported.Caenorhabditis elegans(C.elegans)is a small whole model organism which could be survival in the soil and be feed on microorganism and the decaying organism.It is easy to reproduce and has many advantages of drug screening.The C.elegans let-60(n1046)is a mutant of gain of function,which is a point mutation in codon 13 of protein LET-60(homologous of Ras),resulted in a continuous activation pathway and the Multivulva phenotype(Muv)of let-60(n1046).So the C.elegans vulval development may be a simple and effective in vivo system for evaluation of Ras protein inhibitors against Ras-activated tumors.Our research group has established a complete screening system of TCM based on C.elegans model.And we have screened out the extract of Rhizoma Coptidis could obviously inhibited the Muv phenotype of let-60(n1046).Nest we analyzed the chemical component of Rhizoma Coptidis,studied the activity of different parts and different processed products of Rhizoma Coptidis based on C.elegans,and analyzed the relationship between the content of alkaloid and the activity difference.Finally we used cell lines with a KRAS mutation to take a preliminary mechanism discussion of coptisine antitumor activity,and it may laying the foundation of the clinical application of Rhizoma Coptidis.1.The screening of effective components of extracts from TCM based on the model of let-60(n1046gf).We established a complete screening system of TCM based on let-60(n1046gf)C.elegans model and the C.elegans were cultured and administered on solid culture medium.We have screened 20 species TCM extracts,and we found that the extract of Rhizoma Coptidis could significantly inhibit the Muv phenotype of the let-60(n1046gf).Compared with the control group,when the drug concentration reached 0.5 mg/mL,the Muv%of let-60(n1046gf)could obviously reduce and the number of Vulva(similar to ovipositor)also reduce.2.Analysis of the Rhizoma Coptidis componentsWe identified the main alkaloids of Rhizoma Coptidis based on HPLC-MS,and analysis the fragmentation patterns.We have identified 12 species alkaloids including magnoflorine,greenlandrine,tetrahydropansy,tetrahydrocoptisine,jatrorrhizine,epiberberine,columbamine,berberrubine,coptisine,palmatine,berberine,worenine.We used HPLC-MS to make a rapid identification of monosaccharides that may be containing of the polysaccharide of Rhizoma Coptidis,which based on analyzing the fragmentation regularity of the monosaccharides PMP derivatization.We found that the monosaccharides of polysaccharides from Rhizoma Coptidis are Man,Rib,Rha,Gal UA,Glu,Gal,Ara/Xyl,Fuc and the Rib and Fuc were the first reported monosaccharides that obtained from polysaccharide of Rhizoma Coptidis.It suggested that monosaccharide derivatives exhibit specificity under the established mass spectrum.3.The content determination and activity evaluation of different parts and different processed products of Rhizoma Coptidis.We isolated the extract of Rhizoma Coptidis based on adsorption and desorption performances of macroporous resin.Then used water,10%ethanol,30%ethanol,50%ethanol,70%ethanol and 90%ethanol gradient elution and thus divided Rhizoma Coptidis into 6 parts,and then HPLC was used to determine the alkaloids content of different components.We processed five processed products of Rhizoma Coptidis,including crude product,processed by fried,processed by wine,processed by evodiae feuctus,processed by ginger juice.And then used HPLC to determine the alkaloids content of different processed products.We divided Rhizoma Coptidis into 3 parts,including alkaloids,non-alkaloids and polysaccharide.We test the 3 parts activity on let-60(n1046gf),and we found that when the concentration of non-alkaloids and polysaccharide reached 1mg/mL,there has no significant change on the Muv%of let-60(n1046gf).When the concentration of alkaloids reached 1mg/mL,the Muv%of let-60(n1046gf)could only slightly reduce and the inhibition ratio is far less than the extract of Rhizoma Coptidis.We test the activity of macroporous resin 6 components,and we found that when the concentration of components reached 0.25 mg/mL,the parts of 10%ethanol,30%ethanol could significantly reduce Muv%of let-60(n1046gf)and other parts have no effects.Thus we inferred the effective constituent of Rhizoma Coptidis may be highest levels in this two components.Rhizoma Coptidis has different processed products in clinical application.We test the activity of 5 kinds of Rhizoma Coptidis processed products on let-60(n1046gf),we found that they could inhibit Muv%of let-60(n1046gf)and the effects of 2 products of processed by fried and processed by evodiae feuctus are slightly weaker than extract of Rhizoma Coptidis.We analysis the relationship between the biological activity and 6 kinds of alkaloids of Rhizoma Coptidis by principal component analysis,and we found that the correlation coefficient of 6 kinds of alkaloids didn’t appear to be much different.So we guess the effect of Rhizoma Coptidis,may be the combined action of 6 kinds of alkaloids or more undetected components.4.Preliminary study on anti-tumor activity and mechanism of coptisine.We evaluate the effects of berberine alkaloids on the growth of PANC-1 and LoVo cells The anti-proliferative effect of coptisine against PANC-1 cells was most notable.It resulted in a dose-dependent inhibition of cell growth and the IC50 was 100.0 μM.Thus,we discuss the mechanism of coptisine to excavate the more effects about coptisine.The migration activity of PANC-1 cells was tested after treatment with or without coptisine by the wound healing assay,the results indicate that coptisine dose-dependently suppressed the migration of PANC-1 cells across the wound space.Hoechst 33258 staining assay was used to test the apoptosis inducing effect of coptisine against PANC-1 cells.The results showed that coptisine couldn’t affect the morphology of cell nucleus,indicating that maybe the anti-proliferative effect is irrespective of apoptosis process.Flow cytometry was performed to determine whether the anti-proliferative effect of coptisine was due to cell cycle arrest.We found that coptisine treatment exhibited arrest of PANC-1 cells at G0/G1 phase and it is in dose-dependent.We detected the phosphorylation level of Ras downstream protein kinases in PANC-1 cells by western blot.And we found that coptisine clearly inhibited p-Erkl/2 and total Erkl/2 levels,but had no significant effect on p-Mekl/2 and total Mekl/2 levels.These results indicated that maybe coptisine inhibits the cell proliferation partially by inhibition of the levels of Erk phosphorylation and total Erk,and thus inhibits Ras/MAPK sustained activation of the downstream signaling pathway. |
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