[Apoptosis effect and mechanism of 5F from Pteris semipinnata on HepG2 cells].

OBJECTIVE: To investigate the effect of Pteris semipinnata L. (PsL) extract Ent-11alpha-hydroxy-15-oxo-kaur-16-en-19-oic-acid (5F)-on HepG2 cells and explore its potential mechanism. METHODS: Cytotoxicity of 5F was studied in HepG2 cells treated with different doses of 5F (0 - 80 mg/L) for 24 h and cell viability was determined by MTT assay. To analyze apoptosis qualitatively, the Hoechst/PI assay was used. The level of Bax in mitochondria fraction of 5F-treated HepG2 cells was determined by western blotting. The levels of cyto-c and AIF in the cytosol were analyzed by western blotting. RESULTS: The cytotoxicity of 5F on HepG2 cells was elevated with the increasing of 5F concentrations, as evidenced by the cell viability assay. The apoptotic cells characterized by condensed neclei were observed after the exposure of HepG2 cells to 5F. The level of Bax in mitochondria fraction of 5F-treated HepG2 cells increased. The levels of cyto-c and AIF in the cytosol of 5F-treated HepG2 cells increased. CONCLUSION: 5F mediated apoptosis involves mitochondria-dependent pathway and 5F might have a therapeutic value against human cancer cell lines and especially on hepatocellular carcinoma (HCC) cells.

[Studies on quality standard of PsL 5F injections].

OBJECTIVE: To establish the quality standard of PsL injections containing mainly 5F (ent-11alpha-hydroxy-15-oxo-kaur-16-en-19-oic-acid). METHOD: The identification of PsL was performed by thin-layer chromatography, and the content was determined by HPLC. The column was Hypersil C18 (4.6 mm x 250 mm, 5 microm), the mobile phase was the mixture of methane-water-acitic acid (55:45: 0.045) with a flow rate of 1.0 mL x min(-1), the detective wavelength was 254 nm, and the column temperature was maintained at 35 degrees C. The pH value and K+ content of the three batchs injection were determined with pH meter and flame photometric meter, and the contents of tannin, protein, oxalic acid salt and heavy metals were detected by deferent methods. RESULT: The TLC method was suitable for the identification of PsL5F. The linearity for 5F was obtained over the range of 30-240 microg x mL(-1) (r = 0.999 8), the average recovery of 5F was 99.8%. The injections were of pH value range from 7.80 to 8.20, K+ contents less than 10 mmol x L(-1), and the contents of tannin, protein, oxalic acid salt and heavy metals were qualified with the Chinese pharmacopoeia, respectively. CONCLUSION: It's sensitive and reliable that can be used as quality control methods of PsL5F injections.

[Advances in study on chemical constituents and pharmacological activities of plants of genus Pteris].

The main chemical constituents in plants of genus pteris include diterpenoids, diterpenoid glycosides, flavonoids, flavonoid glycosides, sesquiterpenoids and volatile oils, etc. Some of extracts show the following activities, such as antitumor, antifungi and antibacteria. Some of compounds have inhibitory effect on platelet aggregation and antiinflamatory action. The latest progress on chemical constituents and pharmacological activity were reviewed in the paper. Main problems and study directions in future of pteris were indicated.

[Study on the effect of resveratrol on metastasis-associated ability of ovarian carcinoma HO-8910PM cells in vitro].

OBJECTIVE: To investigate the inhibitory effect of resveratrol on the metastasis-associated ability of human highly metastatic ovarian carcinoma HO-8910PM cells in vitro. METHODS: MTf assay was used to examine the cytotoxicity of resveratrol in HO-8910PM cells; Transwell Chamber assay was performed to determine the effect on invasion and migratory capacity of the cells by resveratrol; Effect on adhesion potential of HO-8910PM cells was tested by cell-Matrigel adhesion assay. RESULTS: Resveratrol showed no cytotoxicity on HO-8910PM cells after 6 h treatment. Resveratrol significantly inhibited migration and adhesion capacity of HO-8910PM cells in vitro. Their inhibitory rates after treated with the chemical of 100 micromol/L for 6 h were (30. 1 +/- 10. 8) % ,(34. 27 +/- 1. 28)% , respectively. However, Resveratrol had no effect on invasion capacity of HO-8910PM cells. CONCLUSION: Resveratrol can inhibit the migration and adhesion of HO-8910PM cells in vitro. Resveratrol might be a potential drug to inhibit tumor metastasis.

[Determination of 5F in Pteris semipinnata L. from various origins by TLC-scanning].

OBJECTIVE: To determine the content of 5F in Pteris semipinnata L. from various origins. METHODS: 5F was determined by TLC-Scanning. RESULTS: The linear relationship was in range of 0. 504 - 2. 520 microg. The mean recovery was 96. 68% and RSD = 1.24% (n = 5). CONCLUSION: The method is available with a good reproducibility, and pretreatment is simple and easy to operate.

[Effect of three flavones on enzyme activity of recombinant human phosphoinositide 3-kinase p110beta catalytic subunit].

OBJECTIVE: To study the effect of three flavones-luteolin, apigenin and genistein on activity of recombinant human phosphoinositide 3-kinase (PI3-K) p110beta catalytic subunit. METHODS: Recombinant human P13-K p110beta catalytic subunit was expressed by gene engineering. PI3-K activity was assayed by incubation recombinant PI3-K p110beta with phosphatidylinostiol-4,5-bisphosphate and [gamma-32P] ATP; the 32P-radiolabeled lipids were extracted with cholroform and methanol, and assessed by scintillation counter. RESULTS: Luteolin and apigenin showed inhibition on the recombinant p110beta catalytic subunit with IC50 8. 65 micromol/L and 11.56 micromol/L, but genistein had no inhibition. CONCLUSION: Luteolin and apigenin are inhibitors of P13-K. The recombinant P13-K p1100 catalytic subunit may be used as a molecular target for simpler screening and development of more effective inhibitors of P13-K.

Cell death induced by Pteris semipinnata L. is associated with p53 and oxidant stress in gastric cancer cells.

In this study, we demonstrated that Ent-11alpha-hydroxy-15-oxo-kaur-16-en-19-oic-acid (5F) had stronger cytotoxicity against MKN-45, a gastric cancer cell line bearing wild-type p53 than MKN-28, another gastric cancer cell line containing missense mutation in p53. The rapid increase of ROS level was involved in the mechanism of cytotoxicity. Classical features of apoptosis induced by 5F were observed in MKN-45 cells only or more significant in MKN-45 cells than MKN-28 cells. Translocation of Bax from cytosol to mitochondria, reduction of delta psi m and DNA fragmentation were induced by 5F in the p53-dependent manner. We conclude that the expression of Bax and its downstream molecules requires the presentation of a wild-type p53 in the cells treated by 5F.

[Inhibitory effect and its kinetic analysis of baicalein on recombinant human protein kinase CK2 holoenzyme].

BACKGROUND & OBJECTIVE: Protein kinase CK2 is a ubiquitous and pleiotropic Ser/Thr protein kinase in eukaryotic cells. CK2 activity has been shown to be markedly elevated in solid tumors and leukemia cells. Its alpha or alpha' gene is a protooncogene. CK2 is attracting increasing attention as a potential target for anti-neoplastic. This study was to search specific CK2 inhibitors in tumor cells through observation of the inhibitory effects of baicalein on recombinant human protein kinase CK2 holoenzyme and its kinetics in vitro. METHODS: Recombinant human protein kinase CK2 alpha' and beta subunits were cloned and expressed by gene engineering, and purified to homogeneous. These 2 subunits were mixed at equal molar ratio to reconstitute CK2 holoenzyme. The CK2 activity was evaluated by detecting radioactivity of 32P of [gamma-32P]ATP which was incorporated into the substrate in various conditions. RESULTS: Baicalein was shown to strongly inhibit the holoenzyme activity of CK2 with IC50 of 2.54 micromol/L. Kinetic studies of baicalein on CK2 showed that baicalein acted as an inhibitor of noncompetitive with ATP(KI=7.73 micromol/L) and mixed types with casein(KI=3.07 micromol/L). CONCLUSION: Baicalein is an effective inhibitor of protein kinase CK2 in vitro.

[Analysis of the diterpenoids in the extract of Pteris semipinnata L by HPLC-APCI-MS].

AIM: To establish an accurate and reliable method for quantitative analysis of the diterpenoids in Pteris semipinnata L. METHODS: A quadruple mass spectrometer coupled with atmospheric pressure chemical ionization interface was employed as a detector for HPLC. As to MS detector, selective ion monitoring (SIM) scan mode was used. For ent-11 alpha-hydroxy-15-oxo-kaur-16-en-19-olic acid (5F) and ent-11 alpha-hydroxy-15-oxo-kaur-16(R) methyl-19-olic acid (4F), the majority of the diterpenoids in Pteris semipinnata L, the [M-H]-1 ion were observed, and the [M-H2O-H]-1 ion could be observed from the collision-induced dissociation spectua. [M-H]-1 was selected as the SIM ion in quantification, the mobile phase and the MS conditions were optimized. The mobile phase of HPLC was 30% CH3CN-70% 2 mmol.L-1 NH4Ac, analytical column was Diamonsil ODS (4.6 mm x 150 mm), flow rate 1.0 mL.min-1, inject volume 5 microL. The area of ion flow peak were used for quantitative determination. As an example of its application, this method was used to determine the content of 5F as an antitumor diterpenoid in Pteris semipinnata L. RESULTS: The content of 5F accounted 1.18 mg.g-1 in Pteris semipinnata L sample. For 5F, RT is about 4.3 min, the standard curve showed good linearity over the range of 0.05-2.5 micrograms, gamma = 0.9998 (n = 5); the recovery was 97.8% (n = 5); the limit of detection was 0.4 ng (inject 5 microL). CONCLUSION: This method is highly sensitive, accurate and fast, which can be applied to study the antitumor drug of diterpenoids in Pteris semipinnata L and to establish the raw herb standard.