Preliminary safety assessment of oridonin in zebrafish.

Context: Oridonin, isolated from the leaves of Isodon rubescens (Hemsl.) H.Hara (Lamiaceae), has good antitumor activity. However, its safety in vivo is still unclear. Objective: To investigate the preliminary safety of oridonin in zebrafish. Materials and methods: Embryo, larvae and adult zebrafish (n = 40) were used. Low, medium and high oridonin concentrations (100, 200 and 400 mg/L for embryo; 150, 300 and 600 mg/L for larvae; 200, 400 and 800 mg/L for adult zebrafish) and blank samples were administered. At specific stages of zebrafish development, spontaneous movement, heartbeat, hatching rate, etc., were recorded to assess the developmental effects of oridonin. VEGFA, VEGFR2 and VEGFR3 gene expression were also examined. Results: Low-dose oridonin increased spontaneous movement and hatching rate with median effective doses (ED50) of 115.17 mg/L at 24 h post-fertilization (hpf) and 188.59 mg/L at 54 hpf, but these values decreased at high doses with half maximal inhibitory concentrations (IC50) of 209.11 and 607.84 mg/L. Oridonin decreased heartbeat with IC50 of 285.76 mg/L at 48 hpf, and induced malformation at 120 hpf with half maximal effective concentration (EC50) of 411.94 mg/L. Oridonin also decreased body length with IC50 of 324.78 mg/L at 144 hpf, and increased swimming speed with ED50 of 190.98 mg/L at 120 hpf. The effects of oridonin on zebrafish embryo development may be attributed to the downregulation of VEGFR3 gene expression. Discussions and conclusions: Oridonin showed adverse effects at early stages of zebrafish development. We will perform additional studies on mechanism of oridonin based on VEGFR3.

Antiangiogenic effects of oridonin.

BACKGROUND: Oridonin, the major terpene found in Rabdosia rubescens (Henmsl.) Hara, is widely used as a dietary supplement and therapeutic drug. Oridonin has been proven to possess good anti-tumour activity, but little is known about its effect on angiogenesis. The aim of this study was to investigate the antiangiogenic effects of oridonin in vivo and in vitro and prove that oridonin anti-tumour activity is based on suppressing angiogenesis. METHODS: In vitro, the antiangiogenesis effect was studied by proliferation, apoptosis, migration, invasion, and tube formation experiments on human umbilical vascular endothelial cells (HUVECs). In vivo, using the Tg (fli1: GFP) zebrafish model, the embryonic vasculogenesis and postnatal regeneration were evaluated. The vascular endothelial growth factor (VEGF) signalling pathway gene expressions were assessed by reverse transcription-polymerase chain reaction (RT-PCR). Furthermore, the inhibition effects on tumour growth and metastasis were observed using a xenograft zebrafish tumour model and xenograft nude mouse tumour model. Angiogenesis was assayed by immunostaining with cluster of differentiation 31. Importantly, the proteins were identified as being differentially expressed in an in vivo model by two-dimensional electrophoresis-mass spectrometry (2D-MS) and western blot (WB). RESULTS: The results indicated that oridonin inhibited HUVEC proliferation, migration, invasion, and tube formation and induced cell apoptosis. Oridonin inhibited zebrafish angiogenesis during embryonic development and tail fin regeneration. RT-PCR showed that oridonin decreased the VEGFA, VEGFR2, and VEGFR3 expressions in zebrafish, while the TP53 expression increased. Moreover, oridonin had strong effects on tumour growth and metastasis in vivo. 2D-MS identified a total of 50 proteins differentially expressed (17 up-expressed, 28 down-expressed). Lastly, WB showed that Claudin 1, Claudin 4, and Claudin 7 were closely related to tumour growth and metastasis. CONCLUSION: This study demonstrated that oridonin could inhibit tumour growth and metastasis, which mainly based on oridonin antiangiogenic effects. Claudin 1, Claudin 4, and Claudin 7 were the main contributors to the mechanism.

In Vitro and In Vivo Antitumor Effects of n-Butanol Extracts of Pterocephalus hookeri on Hep3B Cancer Cell.

Pterocephalus hookeri is a widely applied Tibetan medicinal prescription for treatment of diseases such as flu, rheumatoid arthritis, and enteritis in China. It has been reported that Pterocephalus hookeri has anti-inflammatory and analgesic actions. However, the antitumor activity of Pterocephalus hookeri remains unknown. In the present study, we demonstrate that n-butanol extracts of Pterocephalus hookeri (YSC-ZDC) has a strong antitumor activity against hepatoma carcinoma cell in vitro and in vivo. YSC-ZDC inhibited proliferation of all cancer cell lines and significantly inhibited Hep3B cells proliferation in a dose- and time-dependant manner. Transmission electron microscopy, hoechst 33258 staining, and flow cytometry analysis revealed that YSC-ZDC induced apoptosis in Hep3B cells. YSC-ZDC treatment dramatically inhibited PDK1 and Akt phosphorylation in Hep3B cells. Moreover, YSC-ZDC increased Bax expression and inhibited Bcl-2 expression. In addition, YSC-ZDC inhibited growth hepatoma xenografts in vivo with no effect on body weight and spleen index. Consistent with results in vitro, YSC-ZDC increased Bax expression and inhibited Bcl-2 expression in tumor tissue. Taken together, this study shows YSC-ZDC with an antitumor activity both in vitro and in vivo. Its mechanism underlying is related to blocking of the Akt pathway and regulation of Bcl-2 family proteins expression.

[Application of zebra fishes in studies on traditional Chinese medicines].

The zebra fish model, as an integral animal model, features small volume, high throughput, low cost, short cycle and reliable experimental results, thus has been widely used in medical studies. Traditional Chinese medicines (TCM) constitute a complex system, their active ingredients and action mechanisms are among study hotspots in during the development of modern TCMs. Along with the constant improvement of advanced technologies and methods, zebra fishes have been increasingly applied in studies on TCMs and shown advantages in active screening, and toxicity and metabolism studies. In this paper, TCM studies by using zebra fishes in recent years are summarized to provide new ideas and methods for basic studies on TCMs.

Four new bis-iridoids isolated from the traditional Tibetan herb Pterocephalus hookeri.

Pterocenoids A-E (1-4), which Pterocenoids A(1) is one novel dimer containing a pyridine monoterpene alkaloid; and Pterocenoids B-E (2-4) are rare arranged non-glycosidic bis-iridoids were isolated from Pterocephlus hookeri. The structures of the compounds were established by 1D and 2D NMR spectroscopy and mass spectrometry. All bis-iridoids isolated from P. hookeri were found to possess secoiridoid/iridoid subtype skeletons. Hence, bis-iridoids can be regarded as the chemotaxonomic markers of P. hookeri. The origins of the new bis-iridoids (1-4) were postulated and their activities of inhibition of the NF-κB pathway were assayed and compounds 1-3 showed moderate activity in inhibiting NF-κB.

New isoprenylated flavonoids and cytotoxic constituents from Artocarpus tonkinensis.

Two new isoprenylated flavonoids, artotonins A and B (1 and 2), along with 13 known compounds (3-15), were isolated from the roots of Artocarpus tonkinensis A. Chev. ex Gagnep. The structures were elucidated by spectroscopic methods. Cyclocommunol (6), isocyclomulberrin (7), cudraflavone C (11), and morusin (13) exhibited cytotoxicity against hepatocellular carcinoma (SMMC-7721) and gastric carcinoma (BGC-823 and SGC-7901) cell lines.

[Effects of cycloartocarpin A and artocarpin extracted from Fructus Artocarpin Heterophylli on apoptosis of SMMC-7721 and SGC-7901 cells].

OBJECTIVE: To investigate the effects of cycloartocarpin A (ACR-2) and artocarpin (ACR-3), monomeric compounds isolated from Fructus Artocarpi Heterophylli, on apoptosis of SMMC-7721 and SGC-7901 cell lines. METHODS: SMMC-7721 and SGC-7901 cells were routinely cultured, and divided into experiment group and control group. The SMMC-7721 cells were treated with different concentrations of ACR-2 (3.46 x 10(-3), 13.82 x (-3), 55.30 x 10(-3) mmol/L) and ACR-3 (6.88 x 10(-3), 27.52 x 10(-3), 110.09 x 10(-3) mmol/L), and the SGC-7901 cells were also treated with different concentrations of ACR-2 (8.06 x 10(-3), 32.26 x 10(-3), 129.03 x 10(-3) mmol/L) and ACR-3 (2.87 x 10(-3), 11.47 x 10(-3), 45.87 x 10(-3) mmol/L), with PBS (DMSO<0.1%) as control treatment. Cell apoptosis was measured by double labeled staining with Hoechst33342/propidium iodide (PI) and TdT-mediated dUTP-biotin nick end labeling (TUNEL) and flow cytometry. RESULTS: ACR-2 and ACR-3 could induce apoptosis of SMMC-7721 and SGC-7901 cells. Some of SMMC-7721 and SGC-7901 cells demonstrated typical apoptosis after being treated with ACR-2 and ACR-3. Hoechst33342/PI staining showed that cells were fraught with overlapping nuclei and nuclear debris or lobule, and the nuclear appeared light blue. TUNEL showed that cells permeated with overlapping nuclei and nuclear debris or lobule, and the nuclear appeared brown. Less apoptotic cells were observed in negative control group, and the nuclear appeared light blue. The apoptosis rates of SMMC-7721 and SGC-7901 cells in the ACR-2 and ACR-3 treated groups were significant higher than those in the control group (P<0.05, P<0.01). CONCLUSION: ACR-2 and ACR-3 can induce apoptosis of SMMC-7721 and SGC-7901 cells.

[Effects of three compounds extracted from Tripterygium wilfordii Hook on angiogenesis in chick chorioallantoic membrane].

OBJECTIVE: To investigate the effects of three compounds extracted from Tripterygium wilfordii Hook (TW) on angiogenesis in the chick chorioallantoic membrane (CAM). METHODS: Fifty fresh Hongkong Mahua chicken eggs were divided into five groups: PBS-treated group, TW1-, TW2- and TW3-treated groups and Rg3-treated group. After disinfection, the eggs were incubated for six days in a constant temperature box with the temperature being controlled within 37.8 degrees C, then exposed CAM, laid the filter papers with specimen on the CAM, and the eggs were incubated for another two days. CAM was fixed with the mixture of methyl alcohol and acetone at room temperature for about 15 min, and then cutting the CAM, taking photos and observing the angiogenesis in the CAM. RESULTS: There were many CAM vessels in the PBS-treated group and the blood vessel net could be seen clearly. The number of CAM vessels in the TW1-, TW2- and TW3-treated groups (10 microg/egg) was much less than that in the PBS-treated group. Furthermore, the frame of the vessels was not clear, and the color was obscure. Inhibition rates of angiogenesis in the TW1-, TW2- and TW3-treated groups were 80%, 60% and 100% respectively, while the inhibition rate of angiogenesis in the Rg3-treated group (10 microg/egg) was only 10%. CONCLUSION: TW1, TW2 and TW3 can obviously restrain the angiogenesis in CAM and still need further study.

Cytotoxic and antifungal isoprenylated xanthones and flavonoids from Cudrania fruticosa.

A new isoprenylated xanthone, cudrafrutixanthone A, was isolated from the roots of Cudrania fruticosa, together with 18 known compounds. Their structures were elucidated by spectroscopic methods. Most isoprenylated xanthones exhibited cytotoxicity against HCT-116, SMMC-7721, SGC-7901, and BGC-823 cell lines with IC (50) values of 1 - 5 microg/mL. Toxyloxanthone C and wighteone showed antifungal activity against Candida albicans with MICs of 25 and 12.5 microg/mL, respectively.

Cytotoxic isoprenylated xanthones from Cudrania tricuspidata.

Eight new isoprenylated xanthones, cudratricusxanthones A-H (1-8), were isolated from the roots of Cudrania tricuspidata, together with ten known compounds, cudraxanthones H (9) and M (10), xanthone V(1a) (11), toxyloxanthone C (12), macluraxanthone B (13), 1-hydroxy-3, 6, 7-trimethoxyxanthone (14), cycloartocarpesin (15), artocarpesin (16), cudraflavone B (17), and kaempferol (18). Their structures were characterized by spectroscopic methods. Xanthones 5, 7, 10, and 12 showed inhibitory effects on four kinds of human digestive apparatus tumor cell lines (HCT-116, SMMC-7721, SGC-7901, and BGC-823) with IC(50) values of 1.6-11.8 microg/mL. Xanthones 2, 4, and 11 displayed significant cytotoxicity against HCT-116, SMMC-7721, and SGC-7901 (IC(50)=1.3-9.8 microg/mL). Flavonoids 15-17 were almost inactive.