Synergistic effect of combining paeonol and cisplatin on apoptotic induction of human hepatoma cell lines

Aim： To investigate whether paeonol （Pae） has synergistic effects with cisplatin （CDDP） on the growth-inhibition and apoptosis-induction of human hepatoma cell lines HepG2 and SMMC-7721. Methods： The cytotoxic effect of drugs was measured by 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyl-tetrazolium bromide assay. The coefficient of drug interaction was used to analyze the nature of drug interactions. Morphological changes were observed by acridine orange fluorescence staining. Cell cycle and the apoptosis rate were detected by flow cytometry. Bcl-2 and Bax expression were assayed by immunohistochemical staining. Results： Pae or CDDP had antiproliferative effect on the 2 cell lines in a dose-dependent manner, with different sensitivities to drugs. More interestingly, a synergistic inhibitory effect on the viability of the 2 cell lines was observed after treatment with a combination of Pae （15.63, 31.25, and 62.5 mg/L） with various concentrations of CDDP. Further study showed typical morphological changes of apoptosis if the cells were exposed to the two agents for 24 h. The apoptotic rate of the cells with combination treatment was significantly higher than that of cells treated with Pae or CDDP alone. The expression of Bcl-2 decreased and that of Bax increased in the treated groups, especially in the combination group, with the ratio of Bcl-2/Bax decreasing correspondingly. Additionally, a combination of Pae with CDDP resulted in a stronger S phase arrest, compared with Pae or CDDP alone. Conclusion： Pae, in combination with CDDP, had a significantly synergistic growth-inhibitory and apoptosis-inducing effect on the 2 human hepatoma cell lines, which may be useful in hepatoma treatment.     

Anticancer effect of aloe-emodin on cervical cancer cells involves G2/M arrest and induction of differentiation

Aim： The aim of this study was to investigate the effects of aloe-emodin, a natural compound from the root and rhizome of Rheum palmatum, on the growth of hu- man cervical cancer cells, HeLa. Methods： HeLa cells were treated with various concentrations of aloe-emodin for 1-5 d, and cell growth was measured by 3-（4, 5- dimethylthiazol-2-yl）-2, 5-diphenyl tetrazolium bromide assay. The long-term growth effect was investigated by crystal violet assay. The distributions of the cell cycle and apoptosis were analyzed by flow cytometry. The alkaline phos- phatase （ALP） activity was analyzed by a chemical analyzer. Finally, Western blotting was used to indicate the abundant changes of protein kinase C （PKC）, c- myc, cyclins, cyclin-dependent kinases （CDK）, and proliferating cell nuclear anti- gen （PCNA）. Results： Aloe-emodin inhibited the growth of HeLa cells in a dose- dependent manner at concentrations ranging between 2.5 and 40 0rnol/L. The flow cytometric analysis showed that HeLa cells were arrested at the G2/M phase. This effect was associated with the decrease in cyclin A and CDK2, and the increase in cyclin B 1 and CDK1. More importantly, the ALP activity was found to be increased by aloe-emodin treatment, and accompanied by the inhibition of PCNA expression. In addition, aloe-emodin suppressed the expression of PKCα and c-myc. Conclusion： These findings provide a possible mechanistic explana- tion for the growth inhibitory effect of aloe-emodin on HeLa, which includes cell cycle arrest and inducing differentiation.     

Ginsenoside Rgl promotes endothelial progenitor cell migration and proliferation

Aim： To investigate the effect of ginsenoside Rgl on the migration, adhesion, proliferation, and VEGF expression of endothelial progenitor cells （EPCs）.
Methods： EPCs were isolated from human peripheral blood and incubated with different concentrations of ginsenoside Rgl （0.1, 0.5, 1.0, and 5.0 μmol/L） and vehicle controls. EPC migration was detected with a modified Boyden chamber assay. EPC adhesion was determined by counting adherent cells on fibronectin-coated culture dishes. EPC proliferation was analyzed with the 3.（4,5.dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） assay. In vitro vasculogenesis was assayed using an in vitro vasculogenesis detection kit. A VEGF-ELISA kit was used to measure the amount of VEGF protein in the cell culture medium.
Results： Ginsenoside Rgl promoted EPC adhesion, proliferation, migration and in vitro vasculogenesis in a dose- and timedependent manner. Cell cycle analysis showed that 5.0 μmol/L ofginsenoside Rgl significantly increased the EPC proliferative phase （S phase） and decreased the resting phase （G0/G1 phase）. Ginsenoside Rgl increased vascular endothelial growth factor production. Conclusion： The results indicate that ginsenoside Rgl promotes proliferation, migration, adhesion and in vitro vasculogenesis.     

Initial study on apoptosis in HepG-2 Human heptocarcinoma cell line by CSS

Objective To discuss on mechanism of the killing and apoptosis inducing effect induced by total alkaloid in the CSS(Capparis spinosa L.saponin,CSS)on human hepatocarcinoma cell Line HepG-2.Methods The killing effect of the CSS on human hepatocarcinoma cell Line HepG-2 was observed by MTT method.Morphological observation of the HepG-2 cells was completed by fluorescence microscope.This test was signed to observe the changes of the cell cycle of HepG-2 cells affected by the CSS by PI single-staining,and to observe if there were typical apoptosis peaks.The apoptosis inducing effect and changing of mitochondria membrane potential of the CSS on the HepG-2 cells were studied by flow cytometry.The effect of intracellular Ca2+ level of CSS on the HepG-2 cells was measured by laser confocal microscope.Results CSS has growth inhibiting on the HepG-2 and seems to be enhanced with the increasing concentration of CSS,and its IC50 value was 46.16 μg·mL-1.The HepG-2 cells are characteristic apoptosis morphologic changed,and the apoptosis percentage is increased to 66.652% in the 50 μg·mL-1 dosage group.The cells cycle has been changed obviously that the progresses of cells cycle of G1 period and G2 period in high dosage group have been blocked,and the cellular proportion in G2 period is decreased by the function of CSS for 24 h.The mitochondria membrane potential of HepG-2 cells induced by CSS is decreased in various degrees.In addition,the intracellular Ca2+ level is increased by the function of CSS in the middle and high dose groups.Conclusions The CSS has obviously killing and apoptosis inducing effect on human hepatocarcinoma cell Line HepG-2 by the mechanism of decreasing the mitochondria membrane potential and increasing the intracellular Ca2+ level.     

Blockade of sonic hedgehog signal pathway enhances antiproliferative effect of EGFR inhibitor in pancreatic cancer cells

Aim： To investigate the expression of sonic hedgehog （SHH） and epidermal growth factor receptor （EGFR） signal molecules in pancreatic cancer cells, and to assess the inhibitory effects through the blockade of the SHH and EGFR signaling pathways by cyclopamine and Iressa, respectively. Methods： The expression of SHH and EGFR in pancreatic cancer cell lines （PANC- 1, SUIT-2, and ASPC- 1） was detected by RT-PCR and Western blot analysis. After treatment with different concentrations of cyclopamine, alone or in combination with Iressa, the antiproliferative effect on pancreatic cancer cells was analyzed by methyl thiazolyl tetrazolium assays. A flow cytometry analysis was used to detect the cellular cycle distribution and apoptosis of pancreatic cancer cells. Results： All of the 3 pancreatic cancer cell lines expressed SHH, Smoothened （SMO）, and EGFR. Cyclopamine could downregulate the expression of EGFR in all cell lines. Cyclopamine or Iressa could induce a growth inhibitory effect in a dose-dependent manner. Moreover, the combined use of 2.5 μmol/L cyclopamine and 1 μmol/L Iressa induced an enhanced inhibitory effect and a greater apoptosis rate than any agent alone. The percentage of the cell population of the G0/G1 and sub-G1 phases was significantly increased along with the increasing dose of cyclopamine and/or Iressa. Conclusion： The blockade of the sonic hedgehog signal pathway enhances the antiproliferative effect of the EGFR inhibitor through the downregulation of its expression in pancreatic cancer cells. The simultaneous blockade of SHH and EGFR signaling represents possible targets of new treatment strategies for pancreatic carcinoma.     

反义寡脱氧核苷酸抑制U937泡沫细胞血管内皮生长因子的表达(英文)

AIM:To study the expression of vascular endothelial growth factor (VEGF) induced by oxidized low density liprotein (ox-LDL) and the inhibitory effects of antisense oligodeoxynucleotide (asODN) on the levels of VEGF protein and mRNA in the U937 foam cells. METHODS: U937 cells were incubated with ox-LDL 80 mg/L for 48h, then ,the foam cells were treated with asODN (0,5,10, and 20μmol/L). The VEGF concentration in the media was determined by ELISA. The VEGF protein expression level in cells was measured by immuohistochemistry; the positive ratio detected by a morphometrical analysis system was used as the amount of the VEGF expression level. The VEGF mRNA level was examined by Northern blotting. RESULTS: After U937 cells were incubated with ox-LDL, VEGF expression level increased greatly both in the cells and in the media. asODN markeldy inhibited the increase of VEGF. After treatment with asODN 20μmol/L, the VEGF protein concentration in the media decreased by 45.0%, the VEGF positive ratio detected by immuohistochemistry in cells decreased by 64.9%, and the VEGF mRNA level decreased by 47.1%. CONCLUSION: The expression of VEGF in U937 foam cells was strong. asODN inhibited VEGF expression significantly in U937 foam cells in vitro.     

Biphasic effects of haloperidol on sodium currents in guinea pig ventricular myocytes

Aim： To study the effects of haloperidol on sodium currents （INa） in guinea pig ventricular myocytes. Method： Whole-cell patch clamp technique was employed to evaluate the effects of haloperidol on INa in individual ventricular myocytes. Results： Haloperidol （0.1-3 wnol/L） inhibited INa in a concentration-dependent manner with an IC50 of 0.253±0.015 larnol/L. The inhibition rate of haloperidol （0.3 μmol/L） on INa was 22.14%±0.02%, and the maximum conductance was reduced. Haloperidol significantly reduced the midpoints for the activation and inactivation of INa by 2.09 and 4.09 mV, respectively. The time constant of recovery was increased. The increase in time intervals could only recover by 90.14%±1.4% （n=6）; however, haloperidol at 0.03 μmol/L enhanced INa conductance. The midpoints for the activation and inactivation Of INa were shifted by 1.38 and 5.69 mV, respectively, at this concentration of haloperidol. Conclusion： Haloperidol displayed a biphasic effect on INa in guinea pig cardiac myocytes. High concentrations of haloperidol inhibited INa, while lower concentrations of haloperidol shifted the activation and inactivation curve to the left. Full recovery of recovery curve was not achieved after 0.3 μmol/L haloperidol administration, indicating that the drug affects the inactivated state of sodium channels.     

The effects and mechanisms of cytoplasmic Macrophage colony-stimulating factor (M-CSF) on the proliferation, migration and invasion of HeLa cells

Objective To explore the effects and mechanisms of cytoplasmic M-CSF on the proliferation,migration and invasion of HeLa cells.Methods Both pCMV/cyto/myc vector and pCMV/cyto/myc-M-CSF vector was transfected into HeLa-cell by transfectaimine.After screening by G418,the positive clones were amplified and confirmed by RT-PCR,Western blot and immunocytochemistry.The effect of cytoplasmic M-CSF on the proliferation of HeLa cells were analyzed by cell conuting and antisense oligonucleotides.The migration and invasion of cell was measured by in vitro Transwell assay and Matrigel-coated polycarbonate filters.The expression of cyclinE,cyclinD1/2/3,CDK2/4/6,Rac1,and matrix metalloproteinase 2 and 9(MMP2/9)were assayed by semiquantitative RT-PCR.And expression of both α-tubulin and cdc42 were displayed by immunofluorescence.The activity of MMP2 was detected by gelatin zymography.Results Results A cell line(referred as to HeLa-M cell)that highly expresses cytoplasmic M-CSF was successfully established in the test.Our result indicated that HeLa-M cell had a larger volume,faster growth rate and shorter doubling time than either pCMV/cyto/myc transfected HeLa cells(referred as to HeLa-C cell)or untransfected HeLa cells(referred as to HeLa cell).M-CSF-specific antisense oligonucleoside significantly inhibited HeLa-M cell proliferation and had little effect on either HeLa-C cell or HeLa-C cell growth.Cytoplasmic M-CSF up-regulated both the expression of cyclinE,cyclinD1 and cyclinD3,CDK2,CDK 4 and CDK6,a Rho GTPase ralative protein(Rac1),cdc42 and MMP2,but had little effect on expression of MMP9 and cyclin D2.Furthermore,cytoplasmic M-CSF induced the rearrangement of the α-tubulin in HeLa cells and significantly promoted the migration and invasion of HeLa cells in vitro.Conclusions Cytoplasmic M-CSFs up-regulate the expression of cyclinE,cyclinD1 and cyclinD3,CDK2,CDK 4 and CDK6 and induces the proliferation of HeLa cells.Cytoplasmic M-CSFs up-regulate the expression of Rac1 and cdc42 and cause the rearrangement of the α-tubulin in HeLa cells.Furthermore Cytoplasmic M-CSFs increase both the expression and activity of MMP2 and promote the migration and invasion of HeLa cell in vitro.But cytoplasmic M-CSFs have little effect on expression of cyclin D2 and MMP9.     

Effect on apoptosis、mitochondrial membrane potential and Ca~(2+) concentration in HepG-2 by CSEO

Objective To study on the mechanism of growth inhibiting and apoptosis inducing effect of total alkaloid in the CSEO(Capparis spinosa L.essential oil,CSEO)on human hepatocarcinoma cell Line HepG-2.Methods The growth inhibiting effect of the CSEO on human hepatocarcinoma cell Line HepG-2 was measured by MTT method.Morphological observation of the HepG-2 cells was completed by fluorescence microscope.The changing of mitochondrion membrane potential induced by CSEO was observed by staining with Rhodamine123.Effect of the CSEO on intracellular Ca2+ level of the HepG-2 cells was measured by laser confocal microscope.Results The CESO has obvious growth inhibiting effect on the HepG-2 and seems to be dose-dependent,and its IC50 is 127.5 μg·mL-1.The characteristic apoptosis morpha of HepG-2 cells has been observed,and the apoptosis percentage increase to 44.447% in the 300 μg·mL-1 dosage group.In addition,the progress of cells cycle of G1 period has been blocked,and the cellular proportion in S and G2 period is decreased in the 75 μg·mL-1 and 150 μg·mL-1 dosage groups by the function of CSEO for 48 h.The mitochondria membrane potential(Δψm)effected by CESO is decreased,while the curve moves toward left.In addition,the intracellular Ca2+ level is increased by the function of CESO in the middle and high dose groups.Conclusions The CESO has obviously growth inhibiting and apoptosis inducing effect on human hepatocarcinoma cell Line HepG-2 by the mechanism of decreasing the mitochondria membrane potential and increasing the intracellular Ca2+ level.     

Inhibition of EGFR autophosphorylation plays an important role in the anti-breast cancer efficacy of the dithiocarbamate derivative TM208

Aim： To investigate the effects of a novel dithiocarbamate derivative TM208 on human breast cancer cells as well as the pharmacoki- netic characteristics of TM208 in human breast cancer xenograft mice. Methods： Human breast cancer MCF-7 and MDA-MB-231 cells were treated with TM208 or a positive control drug tamoxifen. Cell pro- liferation was examined using SRB and colony formation assays. Cell apoptosis was analyzed with Annexin V-FITC/PI staining assay. Protein expression was examined with Western blot, ELISA and immunohistochemical analyses. MCF-7 breast cancer xenograft nude mice were orally administered TM208 （50 or 150 mg.k$1〈1-1） or tamoxifen （50 mg.kgl〈t-~） for 18 d. On d 19, the tumors were collected for analyses. Blood samples were collected from the mice treated with the high dose of TM208, and plasma concentrations of TM208 were measured using LC-MS/MS. Results： Treatment of MCF-7 and MDA-MB-231 cells with TM208 dose-dependently inhibited the cell proliferation and colony formation in vitro （the IC~o values were 36.38＋3.77 and 18.13＋0.76 pmol/L, respectively）. TM208 （20-150pmol/L） dose-dependently induced apoptosis of both the breast cancer cells in vitro. In MCF-7 breast cancer xenograft nude mice, TM208 administration dose-depend- ently reduced the tumor growth, but did not result in the accumulation of TM208 or weight loss. TM208 dose-dependently inhibited the phosphorylation of EGFR and ERK1/2 in both the breast cancer cells in vitro as well as in the MCF-7 xenograft tumor. Conclusion： Inhibition of EGFR autophosphorylation plays an important role in the anticancer effect of TM208 against human breast cancer.     

Diterpenoid alkaloid derivatives as potential chemotherapeutic agents in American trypanosomiasis

The use of natural products for the treatment of protozoal infections (Leishmania and Trypanosoma spp.) is well known and has been documented since ancient times. We have already established an in vitro culture system using mammalian host cells (Vero) infected with Trypanosoma cruzi in which the time course of parasite growth is determined quantitatively. This system was used to screen anti-T. cruzi agents using two experimental models: simultaneous cell infection and compound addition or preincubation of the parasite with the test compound prior to cell infection. Among 64 diterpenoid alkaloids tested, including C19 and C20 skeletons, five C20 compounds were active on T. cruzi epimastigotes: azitine, isoazitine and 15,22-O-diacetyl-19-oxodihydroatisine had moderate effects on the parasite, while atisinium chloride and 13-oxocardiopetamine were potent T. cruzi epimastigote growth inhibitors with activity levels similar to that of benznidazole, used as the reference drug. Additionally, these compounds decreased the ability of metacyclic forms to invade mammalian cells, their intracellular replications and their transformation into trypomastigotes, with no toxicity to the host cell. These results suggest that these alkaloids are structural leads of clinically active compounds against T. cruzi and probably other members of the Trypanosomatidae.     

Trypanocidal constituents in plants 5. Evaluation of some Mexican plants for their trypanocidal activity and active constituents in the seeds of Persea americana

Crude extracts of Mexican medicinal plants were screened for trypanocidal activity against Trypanosoma cruzi, which is the etiological agent for Chagas' disease, one of the most serious protozoan diseases in Latin America. There were 71 kinds of methanolic and other organic extracts from 65 plants, which were newly examined by a preliminary screening test to observe immobilization of epimastigotes and trypomastigotes of T. cruzi in vitro. The MeOH extract of seeds of Persea americana (avocado) showed moderate activity against epimastigotes. In order to identify the principal compounds for the activity, the MeOH extract was subjected to bioassay-guided fractionation. From the active fractions, six 1,2,4-trihydroxyheptadecane derivatives and two 1,2,4-trihydroxynonadecane derivatives including a new one were isolated. These compounds showed moderate activity against epimastigotes and trypomastigotes.     

Trypanocidal constituents in plants 4. Withanolides from the aerial parts of Physalis angulata

The constituents of the aerial parts of Physalis angulata (Solanaceae) were investigated based on the plant's trypanocidal activity against epimastigotes of Trypanosoma cruzi, the etiologic agent for Chagas' disease. Four new withanolides were isolated, along with six known ones, from the active fraction. Their structures were determined by spectroscopic analysis. Trypanocidal activity against trypomastigotes, an infectious form of T. cruzi, was also estimated, as well as cytotoxic activity against human uterine carcinoma (HeLa) cells in vitro. Evaluation of trypanocidal activity using the colorimetric reagent Cell Counting Kit-8 was also examined.     

Development of Trypanosoma cruzi after starvation and feeding of the vector - a review

Trypanosoma cruzi develops in the intestinal tract of reduviid bugs and may be affected by changes in the nutritional state of the vector. In regularly fed Triatoma infestans the population of T. cruzi in the rectum consists mainly of equal amounts of epi- and trypomastigotes. Starvation of the bug reduces the total number of flagellates and the number and percentage of trypomastigotes. The number and the percentage of drop-like forms and of resulting spheromastigotes, however, increases up to 30% 60 days after the last feeding (daf). Feeding of starved bugs (60 daf) reduces the original population density, which then increases again. In starved bugs 1 daf spheromastigotes (including intermediate forms) have almost disappeared and epimastigotes dominate. In addition "giant cells" (a multiple division stage) comprise about 10% of the population and in the following two days this form represents on average 30-50% of the total population, before disappearing nearly completely. Feeding the vector at 40 daf; a) induces the appearance of pure populations of trypomastigotes in immediately deposited drops of bug urine; b) induces metacyclogenesis in epimastigotes, and c) reduces metacyclogenesis in spheromastigotes. Incubating isolated recta together with the Malpighian tubules in Drosophila Ringer's solution and initiating the excretion with 5-hydroxy-tryptamine also induces metacylogenesis in epimastigotes.     

Trypanocidal constituents in plants 2.xanthones from the stem bark of Garcinia subelliptica

The constituents of the stem bark of Garcinia subelliptica (Guttiferae) were investigated based on its trypanocidal activity against epimastigotes of Trypanosoma cruzi, the etiologic agent for Chagas' disease. As the active components, nine xanthones were isolated including two new ones, 4-hydroxybrasilixanthone B and 1,3,5,6-tetrahydroxy-4,7,8-tri(3-methyl-2-butenyl)xanthone. Their structures were determined by spectroscopic analysis. Trypanocidal activity against trypomastigotes, an infectious form of T. cruzi, was also estimated as well as cytotoxic activity. Fukugetin, the major component of the bark, showed no activity.     

Psilostachyin C: a natural compound with trypanocidal activity

In this study, the antiprotozoal activity of the sesquiterpene lactone psilostachyin C was investigated. This natural compound was isolated from Ambrosia scabra by bioassay-guided fractionation and was identified by spectroscopic techniques. Psilostachyin C exerted in vitro trypanocidal activity against Trypanosoma cruzi epimastigotes, trypomastigotes and amastigotes, with 50% inhibitory concentration (IC(50)) values of 0.6, 3.5 and 0.9 μg/mL, respectively, and displayed less cytotoxicity against mammalian cells, with a 50% cytotoxic concentration (CC(50)) of 87.5 μg/mL. Interestingly, this compound induced ultrastructural alterations, as seen by transmission electron microscopy, in which vacuolisation and a structural appearance resembling multivesicular bodies were observed even at a concentration as low as 0.2 μg/mL. In an in vivo assay, a significant reduction in the number of circulating parasites was found in T. cruzi-infected mice treated with psilostachyin C for 5 days compared with untreated mice (7.4 ± 1.2 × 10(5)parasites/mL vs. 12.8 ± 2.0 × 10(5)parasites/mL) at the peak of parasitaemia. According to these results, psilostachyin C may be considered a promising template for the design of novel trypanocidal agents. In addition, psilostachyin C inhibited the growth of Leishmania mexicana and Leishmania amazonensis promastigotes (IC(50)=1.2 μg/mL and 1.5 μg/mL, respectively).     

Activities of Pt(II) and Ru(III) triazole-pyrimidine complexes against Trypanosoma cruzi and T. brucei brucei

We studied the biological activity of three newly synthesized metal complexes of triazole-pyrimidine derivatives that were previously observed to inhibit in vitro growth of epimastigotes of Trypanosoma cruzi and procyclic forms of Trypanosoma brucei brucei. We analyzed the possible inhibitory effect of these compounds on the synthesis of DNA, RNA and protein, ultrastructure and excretion of metabolites by these parasites. RNA synthesis was inhibited by all three complexes assayed. These complexes also led to anomalies of the main organelles (e.g. nucleus, kinetoplast and mitochondria). In addition, these complexes may be capable of altering the excretion of metabolites by the parasites.     

Selective activity of polyene macrolides produced by genetically modified Streptomyces on Trypanosoma cruzi

The growth inhibitory effects on Trypanosoma cruzi of several natural tetraene macrolides and their derivatives were studied and compared with that of amphotericin B. All tetraenes strongly inhibited in vitro multiplication. Proliferation of epimastigotes was arrested by all these drugs at < or =3.6 microM, which were also active on amastigotes proliferating in fibroblasts. Compared with amphotericin B, the compounds were less effective but also less toxic, showing no effect on the proliferation of J774 and NCTC 929 mammalian cells at concentrations active against the parasites. CE-108B (a polyene amide) appeared to be an especially potent trypanocidal compound, with strong in vivo trypanocidal activity and very low or no toxic side effects, and thus should be considered for further studies.     

Ursolic acid as a trypanocidal constituent in rosemary

The MeOH extract of the leaves of rosemary (Rosmarinus officinalis L.) completely inhibited the motility of cultured epimastigotes of Trypanosoma cruzi at the concentration of 2 mg/ml after 2 h of incubation. Activity-guided fractionation of the MeOH extract has resulted in the isolation of three triterpene acids, betulinic, oleanolic and ursolic acids. Ursolic acid stopped the movement of all T. cruzi epimastigotes at the minimum concentration (MC(100)) of 40 micro g/ml (88 micro M) after 48 h of incubation. Oleanolic acid was less active (MC(100): 250 micro g/ml, 550 micro M) and betulinic acid was practically inactive.