Synthesis and evaluation of ursolic acid derivatives as potent cytotoxic agents.

Structural modification was performed at the C-3 and C-28 positions of ursolic acid (UA). Ten UA derivatives with distinct electrical property were synthesized. They could be divided into two groups according to their charge under physiological conditions: (1) Group I negatively charged and (2) Group II positively charged. The anti-proliferative capability of the derivatives was evaluated against HepG2, AGS, HT-29 and PC-3 cells by the MTT assay. Flow cytometry and Annexin V/PI dual staining assay were carried out to explore the antitumor mechanism. The results showed the cytotoxic capacity of the compounds was: Group I

Synthesis of [3ß-acetoxy-urs-12-en-28-oyl]-1-monoglyceride and investigation on its anti tumor effects against BGC-823.

Ursolic acid (UA) as the leader compound was designed to prepare a series of derivatives (three novel compounds UA-1a, UA-1b and UA-2) by modification at the C3 and C28 positions. Their chemical structures were confirmed by IR, (1)H NMR and MS. The cytotoxic activity of the derivatives was evaluated against HepG2, BGC-823 and HT-29 by the MTT assay. The novel derivative UA-1a, [3ß-acetoxy-urs-12-en-28-oyl]-1-monoglyceride showed significant anti-growth ability against the assayed cancer cell lines, particularly against BGC-823, while low cytotoxicity to human normal gastric cell line GES-1. Further investigation revealed that UA-1a could induce apoptotic events of the treated BGC-823 cells, such as comet-like DNA bend, sub-G0/G1 phase accumulation and phosphatidylserine externalization. The activity of Caspase-3 was found to be up-regulated, while the expression of Bcl-2 and Survivin were down-regulated in UA-1a treated cells. UA-1a might trigger the death of BGC-823 cells by inducing apoptosis via the mitochondria pathway. UA-1a exerted stronger ability than Taxol to retard tumor growth in nude mice without leaving apparent toxicity to the hosts. The experimental data suggested that UA-1a would have a therapeutic potential in the treatment of gastric cancer.

[Transcriptional regulation of cytochrome P450 3A4 by four kinds of traditional Chinese medicines].

OBJECTIVE: To screen a group of traditional Chinese medicines with effect on pregnane X receptor (PXR)-mediated transcription regulation of P450 3A4 (CYP3A4); and to study whether they can induce the expression of CYP3A4 with a dose, time-dependent manner. METHOD: Transient cotransfection reporter gene assays were performed with pCI-hPXR-neo, pGL3-CYP3A4-Luc and beta-galactosidase expression plasmid in HepG2 cells. RESULT: Rhizoma Curcumae, Atractylodes lancea, A. macrocaphala and Poria cocos could induce transcriptional expression of CYP3A4. In the dose-effect study, 24 h after induction, 500 mg x L(-1) Rhizoma Curcumae, A. lancea, A. macrocaphala and Poria cocos, respectively, could induce the CYP3A4 gene expression with (6.82 +/- 0.09), (6.76 +/- 0.20), (5.49 +/- 0.13) and (4.97 +/- 0.07) folds, as compared with 0.1% DMSO treated cells. In the time-effect study, 500 mg x L(-1) Rhizoma curcumae, A. lancea, A. macrocaphala and Poria cocos for 48 h could induce the CYP3A4 gene expression with (7.74 +/- 0.54), (7.34 +/- 0.10), (5.54 +/- 0.11) and (5.32 +/- 0.18) folds, compared with 0.1% DMSO treated cells. CONCLUSION: Rhizoma Curcumae, A. lancea, A. macrocaphala and Poria cocos could induce the expression of CYP3A4 gene transcription through activating PXR.

[Effects on the activities and mRNA expression of CYP3A in rat's liver by four kinds of extracts from anti-cancer Traditional Chinese Medicines].

OBJECTIVE: To study the effects of four kinds of extracts from anti-cancer Taditional Chinese Medicines on the activity and mRNA expression of CYP3A in rat's liver. METHODS: Rat's liver microsomal cytochrome P450 and CYP3A isoemzymes--erythromycin N-demethylase(ERD) activities were determined by UV chromatography, the mRNA expression levels of CYP3Al and CYP3A2 were detected by reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: Extracts of Rhizoma Curcumae, Rhizoma Atractylodes and Rhizoma Atractylodes Macrocaphalae markedly increased the P450 content of liver microsomes and induced the enzyme activity of CYP3A, but extract of Poria cocos did not. At the mRNA level, extracts of Rhizoma Curcumae, Rhizoma Atractylodes Lanceae and Rhizoma Atractylodes Macraphalae induced the expression of CYP3A1, but extract of Poria cocos did not. The expression of CYP3A2 were induced by extracts of Rhizoma Curcumae and Rhizoma Atractylodes Lanceae, but extracts of Rhizoma Atractylodes Macrocaphalae and Poria cocos were found of remarkable inhibition of the mRNA expression of CYP3A2. CONCLUSION: Extracts of Rhizoma Curcumae, Rhizoma Atractylodes Lanceae and Rhizoma Atractylodes Macrocaphalae can regulate CYP3A on the levels of enzyme activity and mRNA expression, but Poria cocos extract only regulated CYP3A on the level of mRNA expression, possibly by affecting the metabolism of other drugs in the body.

Cloning and expression of hydroxynitrile lyase gene from Eriobotrya japonica in Pichia pastoris.

Hydroxynitrile lyase gene (hnl) from Eriobotrya japonica was successfully amplified using the method of SEFA PCR (Self-Formed Adaptor PCR). The complete sequence was 5.5 kbp in length, including 3100 bp of the upstream promoter region, 1659 bp of the coding sequence, three introns and 315 bp of the downstream transcription terminator. The phylogenetic analysis illustrated that the obtained hnl exhibited 66-70% identity to the reported isozymes from almond, black cherry and Japanese apricot. The EjHNL had 552 amino acids including a 25 amino acid-long signal peptide. The conserved characteristic structures of HNLs, such as FAD-binding motif, N-glycosylation sites and active sites were observed. The coding sequence of the hnl was inserted into pPIC9K vector for heterologous expression in Pichia pastoris. The HNL activity of the culture supernatant reached 15 U/ml after 96 h of induction by methanol. The specific activity of the recombinant HNL was about 197 U/mg. The enantiomeric excess value of the product R-mandelonitrile attained 98.6% and the value of K(m) of the recombinant HNL was determined to be 0.47 mM based on the kinetic data. The optimum temperature and pH of the recombinant HNL were 40°C and 6.0 respectively. The experimental data indicated that the obtained recombinant HNL showed similar catalytic characteristics with the natural EjHNL. The expression of the recombinant HNL in P. pastoris could present another available biocatalyst for the synthesis of R-selective cyanohydrins.

In vitro and in vivo anticancer activity evaluation of ursolic acid derivatives.

Twenty-three ursolic acid (1) derivatives 2-24 (ten novel compounds 8-10, 14-17 and 22-24) modified at the C-3 and the C-28 positions were synthesized, and their structures were confirmed by IR, (1)H NMR, MS, and elemental analysis. The single crystals of compounds 15 and 17 were obtained. The cytotoxic activity of the derivatives was evaluated against HepG2, BGC-823, SH-SY5Y, HeLa and HELF cells by the MTT assay. The induction of apoptosis and affects on the cell cycle distribution with compound 14 were assessed by fluorescence microscopy, flow cytometry and the activity of caspase-3 in HepG2 cells. Compounds 14-17 had more significant antiproliferative ability against the four cancer cell lines and low cytotoxicity to human embryonic lung fibroblast cells (HELF). Compounds 11, 14-16, 21 and 23 were particularly active against HepG2 cell growth. Compound 14 was selected to investigate cell apoptosis and cell cycle distribution. Flow cytometric analysis and morphologic changes of the cell exhibited that treatment of HepG2 cells with compound 14 led to cell apoptosis accompanied by cell cycle arrest at the S phase in a dose-dependent manner. Furthermore, the activity of the caspase-3 enzyme was increased in the treated cells. In vivo studies using H22 xenografts in Kunming mice were conducted with compound 14 at doses of 50, 100 and 150 mg/kg body weight. The results revealed that the medium dosage group (100 mg/kg) showed significant anticancer activity (45.6 ± 4.3%) compared to the control group.

[Preparation and identification of OmpW monoclonal antibodies].

AIM: To prepare and characterize the mouse monoclonal antibodies against Vibrio parahaemolyticus OmpW. METHODS: The OmpW amino acid sequence from three diseased Vibrio was analyzed by Bioinformatics. Mice were immunized with r-OmpW which was highly expressed and purified in E.coli. Five Vibrio(Va, Vp, Vh, Vv, Van) were chosen as antigen for mAb selection.The characters of the anti-OmpW monoclonal antibodies were studied by Western blot, Flow Cytometry, indirect immunofluorescence. RESULTS: OmpW was testified a highly conservative membrane protein.Three clones of anti-OmpW mAb was obtained. The Ig subclass of the mAb secreted from fused cell S5C10 was IgG3, which of the titer was 4.6×10(4);. The mAb could specifically recognize Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio harveyi, Vibrio anguillarum, Vibrio vulnificus, which could not react with Pseudomonas flurosecens, Aeromonas hydrophila, Aeromonas sobria, Aeromonas hydrophila, Escherichia coli. CONCLUSION: The mAb could specially recognize five diseased Vibrio, which is a useful tool for the further study of the diagnosis of Vibrio.