Ent-11α-hydroxy-15-oxo-kaur-16-en-19-oic-acid inhibits hepatocellular carcinoma in vitro and in vivo via stabilizing IkBα.

Ent-11-hydroxy-15-oxo-kaur-16-en-19-oic-acid (5F) isolated from Pteris Semipinnata L is known to inhibit certain tumor cells in vitro. The information on the in vivo effect of 5F is limited and its effect on hepatocellular carcinoma (HCC) is unknown. In this study, the anti-tumor effect of 5F was investigated in a diethylnitrosamine (DEN)-induced mouse HCC model. In addition to therapeutic effect, the potential side effect was monitored. A panel of cultured HCC cells was used to confirm the in vivo data and explore the responsible molecular pathway. The result showed that 5F significantly inhibited the DEN-induced HCC tumors by reducing the number of tumor foci and the volume of tumors. Furthermore, 5F induced the death of cultured HCC cells in dose- and time-dependent manners. The cell death was confirmed to be apoptotic by in vivo and in vitro TUNEL assays. 5F inhibited NF-kB by stabilizing its inhibitor IkBα, reducing the nuclear p65 and inhibiting NF-kB activity. Subsequently it affected the NF-kB downstream molecules with a decrease in anti-apoptotic Bcl-2 and increase in pro-apoptotic Bax and Bak. During the whole period of the experiment, mice receiving 5F appeared to be healthy, though they suffered from a mild degree of hair loss. 5F did not damage liver and renal functions. In conclusion, 5F is effective against HCC with minimal side effects. It induces apoptosis in HCC cells via inhibiting NF-kB, leading to the decrease of Bcl-2 but the increase of Bax and Bak.

N-acetyl-cysteine protects chicken growth plate chondrocytes from T-2 toxin-induced oxidative stress.

T-2 toxin is now considered to be related to bone malformation such as incomplete ossification, absence of bones and fused bones. In this study, primary cultures of chicken tibial growth plate chondrocytes (GPCs) were treated with various concentrations of T-2 toxin (5, 50, and 500 n m) in the absence and presence of N-acetyl-cysteine (NAC) to investigate the effects of the antioxidant NAC on T-2 toxin-induced toxicity. Our results showed that T-2 toxin markedly decreased cell viability, alkaline phosphatase activity and glutathione content (P < 0.05). In addition, T-2 toxin significantly increased reactive oxygen species levels and malondialdehyde in a dose-dependent manner. However, the T-2 toxin-induced cytotoxicity was reversed, in part, by the antioxidant NAC (P < 0.05). These results suggest that T-2 toxin inhibits the proliferation and differentiation of GPCs in vitro by altering cellular homeostasis and NAC can protect GPCs against T-2 toxin cytotoxicity by reducing the T-2 toxin-induced oxidative stress.

[Study on HPLC fingerprint of Alpinia officinarum].

OBJECTIVE: To establish the chromatography fingerprint of Alpinia officinarum by HPLC. METHODS: An optimum HPLC conditions which were obtained under the assessment of LC-MS were as follows: Shim-pack VP-ODS column (2.0 mm x 250 mm, 5 microm), 0.1% HAc aqueous solution as phase A, 15% Acetonitrile: 40% Methanol: 45% Tetrafuran as phase B, the flow rate was 0.20 mL/min, column temperature was 35 degrees C and UV detector was set at 280 nm. RESULTS: The HPLC fingerprint of Alpinia officinarum was established, the consensus 10 peaks and their relative retention times along with the ranges of relative area were determined. CONCLUSION: The method is reliable and stable and can be used for the quality control and identification of Alpinia officinarum.

[Improvement of synthesis of quercetin and genistein sulfates and identification by HPLC-MS].

OBJECTIVE: To improve synthesis of quercetin and genistein sulfates. METHODS: Quercetin and genistein were esterified with concentrated sulfuric acid for 3 hours in ice and esterfication was terminated by neutralization with 6N NaOH. Derivatives were identified by HPLC-APCI-MS. RESULTS: Quercetin and genistein derivatives were only one compound of monosulfate. CONCLUSION: The improvement of synthesis of quercetin and genistein sulfates is a convenient method for obtaining soluble derivatives of the flavonoids.

Chitosan hydrogel in combination with marine peptides from tilapia for burns healing.

The purpose of this study was to develop a promising burns dressing. Chiosan (CS) has been widely used as biomaterials, in combination with marine peptides (MPs) extracted from seawater cultured Tilapia, the newly developed material Chitosan-Marine Peptides hydrogels (CSMP) in this study showed antibacterial activity, pro-cell proliferation and migration, well burning healing. Pathological examinations by HE staining demonstrated that CSMP had pronounced wound healing efficiencies. In burn wounds treated with CSMP, reepithelialization and collagen fiber deposition were observed on day 7, the epithelium was completely regenerated by day 14, and the wounds were completely healed by day 21. Furthermore, CSMP can up-regulate the expression of FGF2 and VEGF. Collectively, these results suggest that CSMP may enhance cell migration and promote the skin regeneration, which demonstrates the potential application of CSMP in burning healing.

[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.

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.

[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.

Anticancer efficacy of 5F in NNK-induced lung cancer development of A/J mice and human lung cancer cells.

The mechanism responsible for the apoptotic effect induced by ent-11α-hydroxy-15-oxo-kaur-16-en-19-oic-acid (5F) is not fully understood and its in vivo effect has not been tested. In this study, the effect and mechanism of 5F was investigated in cigarette smoking carcinogen 4-methylnitrosamino-1-3-pyridyl-butanone (NNK)-induced mouse lung tumor model and in cultured lung cancer cells NCI-H23 and CRL-2066. 5F were given to mice after they were treated with NNK for 18 weeks. The effect of 5F on the lung tumor formation was examined, and its side effect was monitored. Cell proliferation and apoptosis were determined through expression of PCNA, Bcl-2, Bax, and TUNEL assay in in vivo animal model. 5F significantly inhibited the NNK-induced lung tumors by inducing apoptosis and suppressing cell proliferation in vivo with minimal side effects. Cell culture experiments showed that 5F translocated Bax into the mitochondria, downregulated Bcl-2, activated caspase-9 and caspase-3, released cytochrome c into the cytosol, and translocated AIF from the mitochondria to the nucleus, which leading to G2-M cell cycle arrest and cell apoptosis. 5F also activated ERK1/2 and the inhibition of ERK1/2 suppressed 5F-mediated changes in apoptotic molecules. In addition to ERK1/2, 5F activated Akt. The inhibition of Akt further facilitated the apoptosis induced, suggesting that Akt activation was anti-apoptotic rather than pro-apoptotic. Collectively, 5F is effective against lung cancer in vivo with minimal side effects. It induces apoptosis in lung cancer through the mitochondrial-mediated pathway, in which the activation of ERK is critical.

Induction of laryngeal cancer cell death by Ent-11-hydroxy-15-oxo-kaur-16-en-19-oic acid.

BACKGROUND: Ent-11-hydroxy-15-oxo-kaur-16-en-19-oic acid (5F) is known to exhibit antitumor activity, but its mechanism is not completely understood. 5F has not been tested in laryngeal cancer. METHODS: Two laryngeal cancer cell lines were treated with 5F. Cell death was analyzed by MTT [3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyltetrazolium bromide] and Annexin V assay. Nuclear factor kappa beta (NF-κB)- and apoptosis-related molecules were examined. RESULTS: 5F induced laryngeal cancer cell death in a dose-dependent manner. The Annexin V assay and the measurement of cleavage of procaspase-3 and poly(ADP-ribose) polymerase demonstrated that the 5F-induced cell death was mainly apoptotic. 5F slightly reduced the basal level of NF-κB, but significantly suppressed the inducible NF-κB by reducing its transcriptional activity, protecting its inhibitory subunit IκBα from degradation, and suppressing its level in the nucleus. 5F also inhibited pro-proliferative and anti-apoptotic molecules but promoted pro-apoptotic Bax. CONCLUSIONS: 5F induces apoptosis of laryngeal cancer cells by inhibiting NF-κB activation/induction, suppressing pro-proliferative and anti-apoptotic molecules, and promoting pro-apoptotic Bax.

Tanshinone prevents cancellous bone loss induced by ovariectomy in rats.

AIM: To investigate the skeletal effects of total tanshinone in ovariectomized rats by analyzing cancellous bone histomorphometry of fourth lumbar vertebrae (LV4) and proximal tibial metaphyses (PTM). METHODS: Four-month-old Sprague-Dawley female rats were sham-operated and treated with vehicle or ovariectomized and treated with either vehicle, total tanshinone (200 mg x kg(-1) x d(-1), equivalent to 35 microg x kg(-1) x d(-1) of tanshinone II A and 16 microg x kg(-1) x d(-1) of cryptotanshinone), or 17alpha-ethynylestradiol (30 microg x kg(-1) x d(-1) as positive treatment group) starting one day post-surgery for 10 weeks. Double in vivo fluorochrome labeling was administered to all rats. The undecalcified longitudinal LV4 and PTM sections were cut and stained with Goldner's Trichrome (4-microm thickness) or unstained (8-microm thickness) for the bone histomorphometric analysis. RESULTS: A significant decrease in trabecular bone volume (BV/TV) and trabecular number (Tb.N) and a significant increase in osteoclast surface (OCS/BS) and mineralizing surface (MS/BS) were found in both LV and PTM of vehicle-treated OVX rats compared with sham controls. Tanshinone completely prevented the decreases in BV/TV and Tb.N and the increase in OCS/BS in the LV4, and partially prevented the decreases in BV/TV and Tb.N in the PTM of OVX rats. In addition, tanshinone increased trabecular thickness (Tb.Th) whereas it did not alter MS/BS. Moreover, tanshinone had no effect on uterine weight and body weight of OVX rats. Estrogen treatment increased BV/TV and Tb.N and decreased OCS/BS, but, also markedly decreased MS/BS and increased uterine weight in OVX rats. CONCLUSION: The current study demonstrated that the adequate supply of tanshinone prevented OVX-induced cancellous bone loss in rats through inhibition of elevated bone resorption.