A novel polypeptide from Cervus nippon Temminck proliferation of epidermal cells and NIH3T3 cell line.

A novel polypeptide, velvet antler polypeptide (VAPPs), having a stimulary effect on proliferation of some cell was isolated from the velvet antler of sika deer (Cervus nippon Temminck). This polypeptide consists of a single chain of 32 amino-acid residues VLSAT DKTNV LAAWG KVGGN APAFG AEALE RM. VAPPs showed marked stimulary effect on rat epidermal cells and NIH3T3 cell line (dose range from 10-40 mg x L(-1) and 5-80 mg x L(-1), respectively).

[Studies on evodiamine induced HeLa cell apoptosis].

AIM: To study the mechanism of evodiamine-induced growth inhibition of HeLa cells. METHODS: HeLa cells viability and the effect of caspase inhibitors on evodiamine-induced apoptosis were measured by crystal violet assay. Changes in cellular morphology were observed by phase-contrast microscopy. Apoptosis-specific nucleosomal DNA fragmentation was assayed by agarose gel electrophoresis. RESULTS: Evodiamine was found to inhibit HeLa cell growth in dose- and time-dependent manners. Caspase-3 inhibitor, z-Asp-Glu-Val-Asp-fmk (z-DEVD-fmk) was shown to partially inhibit evodiamine-induced apoptosis. However, caspase-1 inhibitor, Ac-Tyr-Val-Ala-Asp-chloromethyl-ketone (Ac-YVAD-cmk), did not antagonize evodiamine induced cell death. CONCLUSION: Evodiamine suppresses the growth of HeLa cells in vitro by apoptosis. Evodiamine-induced apoptosis is partially dependent on caspase-3 pathway in HeLa cells. Other apoptotic pathways might be also related to the induction of apoptosis by evodiamine.

[The antitumor activity of Diosgenin in vivo and in vitro].

OBJECTIVE: To investigate the antitumor activity of Diosgenin in vivo and in vitro. METHOD: S-180, HepA, U14 and EAC transplant mice were given Diosgenin ig or i.p. everyday for 10 days, from the next day when they were inoculated in axilla. Tumor growth inhibit rates were calculated. Four kinds of cells, MCF, L929, A375-S2 and HeLa, were incubated respectively with Diosgenin in vitro. Tumor growth inhibit rates were also calculated. RESULT: In vivo, both ig and i.p., Diosgenin inhibited S-180, HepA, U 14 mice transplant tumor, the inhibit rates being 30%-50%, but it did not inhibit the EAC mice transplant tumor. In vitro, Diosgenin inhibited L929, HeLa, MCF cell growth, and IC50 were 1.2, 18.2, 19.8 micrograms.mL-1 respectively, but it did not significantly affect A375-S2 cells. CONCLUSION: Diosgenin has an obvious antitumor activity on S-180, HepA, U14 transplant mice in vivo and L929, HeLa, MCF cells in vitro.

[Intestinal bacteria metabolism of TSDP and characterization of metabolites in rats].

OBJECTIVE: To study the rat intestinal bacteria metabolism of total saponins of Dioscorea pathaica (TSDP) in vitro, and characterize the metabolites in serum and urine of rats after oral administration of TSDP 900 mg.kg-1. METHOD: TSDP metabolites were detected with thin-layer chromatography (TLC) and combination of electrospray ionization mass spectrometry (ESI-MS) and sequential tandem mass spectrometry (MSn). RESULT: In vitro, TSDP was decomposed easily by rat intestinal bacteria, and metabolites DP-1, DP-2, DP-4, DP-5 and diosgenin (Dio) were observed with prolongation of incubation time by ESI-MS2. In vivo, in the full-scan positive mass spectrum of the rat urine sample, the ion peak at m/z 415 (M-H) and its characteristic fragmentations at m/z 397 and m/z 271 in the MS/MS spectrum were identified with that of metabolite Dio, therefore metabolite Dio was deduced to exist in the rat urine, and metablite Dio was allso detected in the rat serum sample. CONCLUSION: TSDP is decomposed easily by rat intestinal bacteria and metabolite diosgenin is absorbed into blood after oral administration of TSDP.

[Comparative study on anti-hypercholesterolemia activity of diosgenin and total saponin of Dioscorea panthaica].

OBJECTIVE: To compare the anti-hypercholesterolemic and cholesterol absorption inhibitory activities between total saponin of Dioscorea panthaica (TSDP) and diosgenin (Dio). METHOD: TSDP and Dio were given ig or i.p. to mice or rats treated with cholesterol feed to evaluate their preventive and therapeutic effect on hypercholesterolemia. TSDP or Dio and cholesterol were mixed with pig bile to form the micelle, then the freeing cholesterol was detected to evaluate inhibitory effect of the both compounds on cholesterol absorption. RESULT: Dio (80 and 160 mg.kg-1) showed significantly therapeutic and preventive effect on hypercholesterolemia in mice, while TSDP showed a certain preventive activity only at a big dose (400 mg.kg-1). The intraperitoneal injection of Dio (20 and 40 mg.kg-1) to mice suffered from hypercholesterolemia was effective, but TSDP showed no effective. The serum total cholesterol level was decreased when rats were pre-treated with TSDP (200 and 400 mg.kg-1, ig) and Dio (200 and 100 mg.kg-1, ig). However, the hypercholesterolemia-preventing activity of Dio was stronger than that of TSDP. In addition, inhibitory effect of Dio on cholesterol micelle formation was still stronger than that of TSDP. CONCLUSION: The preventive and therapeutic activity of Dio against hypercholesterolemia indused by cholesterol in mice or rats is stronger than that of TSDP. The anti-hypercholesterolemia mechanism of Dio is probably related with its cholesterol absorption inhibitory activity.

Microbial metabolism of pseudoprotodioscin.

Microbial transformation of the furostanol saponin pseudoprotodioscin ( 1) using Aspergillus fumigatus resulted in the isolation of two new steroidal metabolites, 3- O-[bis- alpha- L-rhamnopyranosyl-(1-->2 and 1-->4)- beta- D-glucopyranosyl]-22 R,25 R-spirost-5-ene-3 beta,20 alpha-diol ( 2) and 3- O-[bis- alpha- L-rhamnopyranosyl-(1-->2 and 1-->4)- beta- D-glucopyranosyl]-25 R-furost-5-ene-3 beta,22 alpha,26-triol ( 3), in addition to the previously reported steroidal saponins: dioscin ( 4) and progenin II ( 5). The structure elucidation of these metabolites was based primarily on 1D and 2D NMR analyses. Metabolites 2 - 5 showed significant cytotoxicity against cancer cell lines A375, L929, and HeLa with IC (50) values ranging from 1.18 microM to 17.88 microM.

Apoptotic effects of ginsenoside Rh2 on human malignant melanoma A375-S2 cells.

AIM: To study the mechanism of ginsenoside-Rh2 (G-Rh2)-induced growth inhibition of A375-S2 cells. METHODS: A375-S2 cell viability and the effect of caspase inhibitors on G-Rh2-induced apoptosis were measured by crystal violet assay. Changes in cellular morphology were observed by phase-contrast microscopy. Apoptosis-specific nucleosomal DNA fragmentation was assayed by agarose gel electrophoresis. Cell cycle distribution was measured by flow cytometry. RESULTS: G-Rh2 inhibited the A375-S2 cell growth in concentration- and time-dependent manners. Caspase family inhibitor, z-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk), caspase-3 inhibitor, z-Asp-Glu-Val-Asp-fluoromethylketone (z-DEVD-fmk), and caspase-8 inhibitor, z-Ile-Glu-Asp-fluoromethylketone (z-IETD-fmk), partially inhibited G-Rh2-induced apoptosis. But caspase-1 inhibitor, Ac-Tyr-Val-Ala-Asp-chloromethyl-ketone (Ac-YVAD-cmk), did not antagonize G-Rh2 induced-cell death. CONCLUSION: G-Rh2 suppresses the growth of A375-S2 cells in vitro by inducing apoptosis. G-Rh2-induced apoptosis is partially dependent on caspase-8 and caspase-3 pathway in A375-S2 cells. Other apoptotic pathways might be also related to the induction of apoptosis by G-Rh2.

Hypoglycemic activity of ginseng glycopeptide.

AIM: To study the hypoglycemic activity of ginseng glycopeptide (GGP). METHODS: Normal mice or rabbits and alloxan or streptozotocin-induced hyperglycemic rats or mice were used in the study. Blood glucose and liver glycogen levels of the experimental animals during the trial period were analyzed by spectrophotometry with O-toluidine and iodine reagents, respectively. RESULTS: Significant decreases in blood glucose and liver glycogen levels were induced in a dose-dependent manner after administration of GGP 50, 100, or 200 mg/kg injected ip or sc to normal mice and injected im 30 or 60 mg/kg to normal rabbits. The hypoglycemic activity of GGP lasted for about 16 h, and were examined in both normal animals and hyperglycemic animals. CONCLUSION: GGP injection induced the pronounced decreases in blood glucose and liver glycogen levels in both normal and hyperglycemic animals.

Hypoglycemic mechanism of ginseng glycopeptide.

AIM: To study the hypoglycemic mechanism of ginseng glycopeptide (GGP). METHODS: After administration of GGP, the levels of insulin, lactate dehydrogenase (LDH), lactic acid (LC), and oxygen consumption, as well as blood glucose (BG) and liver glycogen (LG) were measured. Based on these measurement results, the effects of GGP on insulin secretion and anaerobic/aerobic glycolysis were evaluated. Adenylate cyclase (AC) activity and cAMP level were measured to study the effects of GGP on BG and LG metabolism and to determine whether the effects were through second transmitting message system. Propranolol (beta-receptor antagonist) and phentolamine (alpha-receptor antagonist) were used to investigate whether hypoglycemic activity of GGP was through beta- or alpha-adrenoceptor. [3H]DHA (antagonist of beta-adrenoceptor) was used to determine GGP binding affinity to beta-adrenoceptor. Citrate synthetase (CTS), succinate dehydrogenase (SDH), malate dehydrogenase (MDH), and cytochrome oxidase (CCO) activities were measured to explore GGP effects on aerobic glycolysis in liver mitochondria. Phosphorylase (PP) activity was measured to study GGP effects on liver glycogen metabolism. RESULTS: cAMP content and AC activity were increased when BG and LG contents in liver of mice decreased. The decrease in liver glycogen induced by GGP was inhibited by pretreatment with propranolol. Radioligand receptor assay showed that GGP was competing in vitro with [3H]DHA to bind to beta-adrenoceptor of duck erythrocyte membrane, and IC50 of GGP was 63 nmol/L. GGP inhibited LDH activity at an appropriate dosage, at which contents of BG and LG could be effectively lowered. GGP also stimulated activities of SDH, MDH, CCO, CTS, and PP. CONCLUSION: The hypoglycemic activity of GGP may be attributed to the enhancement of aerobic glycolysis through stimulation of beta-adrenoceptor and increase of various rate-limiting enzyme activities related to tricarboxylic acid cycle.

Diosgenin induces apoptosis in HeLa cells via activation of caspase pathway.

AIM: To investigate the mechanism of diosgenin-induced HeLa cell apoptosis. METHODS: HeLa cell growth was measured by MTT method. Apoptosis was detected by electron microscopy and agarose gel electrophoresis. Ratio of apoptotic cells was measured by APO-BRDU kit. Cell cycle distribution and changes of mitochondrial membrane potential were monitored by flow cytometry. Caspase activities were assayed by caspase apoptosis detection kit. Western blot analysis was used to evaluate the level of mitochondrial Bcl-2 expression. RESULTS: Diosgenin inhibited HeLa cell growth. HeLa cells treated with diosgenin showed typical characteristics of apoptosis including the morphological changes and DNA fragmentation. Caspase family inhibitor (z-VAD-fmk), caspase-9 inhibitor (Ac-AAVALPAVLLALLAPLEHD-CHO), and caspase-3 inhibitor (z-DEVD-fmk) partially prevented diosgenin-induced apoptosis, but not caspase-8 inhibitor (z-IETD-fmk) and caspase-10 inhibitor (z-AEVD-fmk). Diosgenin caused reduction of mitochondrial membrane potential and down-regulated Bcl-2 expression. CONCLUSION: Diosgenin induced HeLa cell apoptosis through caspase pathway.

Evodiamine, a constituent of Evodiae Fructus, induces anti-proliferating effects in tumor cells.

We found that evodiamine, a major alkaloidal component of Evodiae Fructus (Goshuyu in Japan), inhibited proliferation of several tumor cell lines, but had less effect on human peripheral blood mononuclear cells (PBMC). We used human cervical cancer cells, HeLa, as a model to elucidate the molecular mechanisms of evodiamine-induced tumor cell death. The results showed that evodiamine induced oligonucleosomal fragmentation of DNA in HeLa cells and increased the activity of caspase-3, but not that of caspase-1, in vitro. Both evodiamine-induced DNA fragmentation and caspase-3 activity were effectively inhibited by a caspase-3 inhibitor, z-DEVD-fmk (z-Asp-Glu-Val-Asp-fmk). In addition, evodiamine increased the expression of the apoptosis inducer Bax, but decreased the expression of the apoptosis suppressor Bcl-2 in mitochondria. Taken together, our data indicated that evodiamine alters the balance of Bcl-2 and Bax gene expression and induces apoptosis through the caspase pathway in HeLa cells.