Interactions of Taxol-producing endophytic fungus with its host (Taxus spp.) during Taxol accumulation.

Endophytic fungi (Fusarium mairei) culture broth (EFCB) was added to cell suspension cultures of Taxus cuspidata. After 5 days, cultures of T. cuspidata given 4 ml of EFCB produced a maximal yield of 6.11 mg/l paclitaxel, with a release ratio of 75%, 2- and 6.8-fold, respectively, greater than the controls. The active element in EFCB is an exopolysaccharide of approximately 79 kD. Endophytic fungi produced 0.19 mg/l of paclitaxel in its producing medium. However, when the supernatant of Taxus cell suspension cultures from day 20 was added to the paclitaxel-producing medium, the biomass of fungi decreased by 24% and the yield of paclitaxel by 45%. In a co-culture system of plant and fungus, the yield of paclitaxel (12.8 mg/l) was >2-fold higher than that in the EFCB-treatment system.

Paclitaxel production using co-culture of Taxus suspension cells and paclitaxel-producing endophytic fungi in a co-bioreactor.

The co-culture of the suspension cells of Taxus chinensis var. mairei and its endophytic fungi, Fusarium mairei, in a 20-L co-bioreactor was successfully established for paclitaxel production. The co-bioreactor consists of two-unit tanks (10 L each) with a repairable separate membrane in the center, culturing Taxus suspension cells in one tank and growing fungi in another. By optimizing the co-culture conditions, there was a desirable yield of paclitaxel in Taxus cell cultures. The Taxus cell cultures by co-culture produced 25.63 mg/L of paclitaxel within 15 days; it was equivalent to a productivity of 1.71 mg/L per day and 38-fold higher than that by uncoupled culture (0.68 mg/L within 15 days). The optimum conditions for co-culture in the co-bioreactor were: B5 medium, inoculating fungi when Taxus cells had grown for 5 days in the co-bioreactor, hydrophilic separate membrane in the center of the co-bioreactor, and air flow rate of 1:0.85 v/v/m in fungus cultures.

Induction of apoptosis in SGC-7901 cells by polysaccharide-peptide GFPS1b from the cultured mycelia of Grifola frondosa GF9801.

The biological function of GFPPS1b, a novel polysaccharide-peptide isolated from cultured mycelia of Grifola frondosa GF9801, was well investigated. GFPS1b has anti-tumor activity and can significantly inhibit the proliferation of SGC-7901 cells, whereas slightly influences the growth of human normal liver cell line L-02. When treated with GFPS1b, SGC-7901 cells showed typical apoptotic morphological features such as the loss of villus and appearance of apoptotic bodies on the cell surface, volume reduction, and chromatin condensation, by scanning electron microscopy (SEM) and fluorescent microscopy (Hoechst 33342). The results of flow cytometry analysis and annexin V-PI assay showed that the SGC-7901 cell cycle was arrested in the G(2)/M phase, the subdiploid peak of DNA characteristic of apoptotic was also observed, and the apoptosis ratio was about 15.08%. DNA isolated from SGC-7901 cells cultured with GFPS1b showed a typical DNA 'ladders' of apoptosis in agarose gel electrophoresis. Further investigation results showed that the apoptotic machinery of SGC-7901 induced by GFPS1b was associated with drop in mitochondrial trans-membrane potential, upregulation of Bax, downregulation of Bcl-2, and activation of caspase-3. Our finding suggests that GFPS1b could suppress SGC-7901 cell growth and reduce cell survival via arresting cell cycle and inducing apoptosis of tumor cells.

Protective effects of mycelia of Antrodia camphorata and Armillariella tabescens in submerged culture against ethanol-induced hepatic toxicity in rats.

The hepatoprotective effects of the mycelia of Antrodia camphorata and Armillariella tabescens were evaluated in vivo using acute ethanol-intoxicated rats as an experimental model. Animals were orally treated with Antrodia camphorata (0.5 or 1.0 g/kg b.w.) or Armillariella tabescens (0.5 or 1.0 g/kg b.w.) for 10 days whereas controls received vehicle only. At the end of the experimental 10-day period, the animals were administered by gavage with an acute ethanol dose of 5.0 g/kg b.w. diluted in deionized water (6:4, v/v) and sacrificed at 18 h after ethanol administration. The degree of protection was measured by using biochemical parameters like serum transaminases (AST and ALT), alkaline phosphatase (ALP), bilirubin. Meanwhile, the histopathological studies were carried out to support the above parameters. Administration of Antrodia camphorata or Armillariella tabescens markedly prevented ethanol-induced elevation of levels of serum AST, ALT, ALP, and bilirubin comparable with standard drug silymarin.

Study on the structure-activity relationships of phoxalone and induction of apoptosis in H446 tumor cells.

A novel macrolide with dioxa and double ring pentadecanone, named as phoxalone, isolated from the culture broth of a myxobacterium Sorangium cellulosum WXNXJ-C, was well investigated. The chemical structure of Phoxalone was elucidated by spectroscopic analysis including UV, IR, (1)H NMR, MALDI-TOF-MS, and LC/MS spectra. The results of cytotoxic bioactivities on human non-small lung cancer H446, in vitro, showed that not only did phoxalone express higher antitumor bioactivity than many antitumor drugs, but it also displayed less cytotoxicity to normal human liver L02 cell lines (IC(50), 286 microg mL(-1)). Further cytotoxic bioactivity study indicated that phoxalone could induce H446 cell line apoptosis in vitro. More investigation results of flow-cytometric analysis suggested that phoxalone arrested the mitosis of H446 cell line at G2/M phase. Hence, phoxalone and its derivatives or analogs would reveal huge research value and fascinating foreground.

A myxobacterium strain Sorangium cellulosum AHB125 producing epothilone B and other anticancer substances.

A myxobacterium strain AHB125 belonging to genus Sorangium cellulosum was isolated from Anhui area in China and identified with morphological analysis by electron microscopy and phase contrast microscope according to Bergey's manual of systematic bacteriology (8th Ed.). Its high-antitumor bioactivity metabolites was evaluated by bioassay-directed screening technique with B16 tumor cell line etc. Research results showed that it exhibited not only strong antitumor ability bioactivities and broad-spectrum antitumor abilities to B16, Bel7402, H446, SGC7901 cell lines, but also has selectivity and pertinence to B16 and SGC7901 cell lines. The compound was confirmed as epothilone B by HPLC and LC/MS analysis, compared to the epothilone B standard sample. Bioassay indicated that there were other high-bioactive substances in the metabolites.

Preparation of a chemically sulfated polysaccharide derived from Grifola frondosa and its potential biological activities.

This report describes the preparation, characterization and potential biological activities of a chemically sulfated polysaccharide (S-GAP-P), which was derived from water-insoluble polysaccharide of Grifola frondosa mycelia. S-GAP-P was determined to be a glucan sulfate with the average molecular weight of 28 kDa and the sulfur content of 16.4%. The antitumor and immunomodulating activities of the sulfated derivative were estimated in vitro and in vivo. S-GAP-P inhibited the proliferation of SGC-7901 cells and induced apoptosis, in a dose-dependent manner. And the results from in vivo experiments demonstrated that S-GAP-P significantly inhibited the tumor growth and enhanced the peritoneal macrophages phagocytosis in S180-bearing mice. It is noteworthy that S-GAP-P could accelerate the antitumor activity of CTX and improve the immunocompetence damaged by CTX, suggesting the combination might increase cytotoxic efficacy and decrease toxicity of some chemotherapeutic agents in cancer treatment.

Antitumor and immunomodulatory effects of weikangfu granule compound in tumor-bearing mice.

BACKGROUND: Weikangfu granule compound (WKC) is a drug preparation based on a clinical prescription drug, Weikangfu-tang, which has been found to have therapeutic effects on gastric cancer. WKC comprises 7 components, including polysaccharides, saponin, flavonoids, and essential oil. OBJECTIVE: The purpose of this study was to assess the antitumor and immunomodulatory effects of WKC in a tumor-bearing rodent model. METHODS: Male and female Kuming mice weighing ∼20 g were subcutaneously implanted with sarcoma 180 (S180) tumor cells and randomly assigned to 1 of 5 treatment groups: oral WKC 175, 350, or 525 mg/kg·d, isotonic saline (negative control), or intraperitoneal cyclophosphamide 25 mg/kg·d (positive control). All treatments were administered daily for 10 days. After euthanization on day 11, the mice, tumors, and spleens were weighed. Lymphocyte proliferation and cytotoxic T lymphocyte (CTL) activity were determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cellular viability assay method. Macrophage phagocytosis was identified using a yeast test. RESULTS: Fifty mice were included in the study (10 mice were assigned to each group). The tumors of the mice administered WKC 175, 350, and 525 mg/kg·d were significantly regressed, as determined using MICs, compared with those in the negative-control group (P<0.05, P<0.01, and P<0.01, respectively), and the inhibitory rates were 30.43%, 46.72%, and 54.35%, respectively. Compared with those in the negative-control group, CTL activities and lymphocyte proliferations in the presence of concanavalin A were significantly greater in the WKC-treated groups at all doses (CTL activities: P<0.05, P<0.01, and P<0.01, respectively; lymphocyte proliferations: P<0.05, P<0.01, and P<0.01, respectively). In the groups receiving WKC 175, 350, and 525 mg/kg·d, the phagocytic rates were 1.5- to 2.0-fold those in the negative-control group (P<0.05, P<0.01, and P<0.01, respectively). In the groups receiving WKC 175, 350, and 525 mg/kg·d, the phagocytic indexes were 3.7- to 5.0-fold those in the negative-control group (all, P<0.01). In contrast, lymphocyte proliferation in the positive-control group was significantly less compared with that in the negative-control group (P<0.01), but no significant differences were found in CTL activities or macrophage phagocytosis between these 2 groups. CONCLUSION: The results of this study in a rodent model suggest that WKC exhibited antitumor and immunomodulatory activities in S180-bearing mice, and that WKC improved nonspecific and specific immune functions in mice, such as lymphocyte proliferation, CTL activity, and macrophage phagocytosis.

Effects of cerium on growth and physiological mechanism in plants under enhanced ultraviolet-B radiation.

Effect of cerium (Ce3+) on the growth, photosynthesis and antioxidant enzyme system in rape seedlings (Brassica juncea L.) exposed to two levels of UV-B radiation (T1: 0.15 W/m2 and T2: 0.35 W/m2) was studied by hydroponics under laboratory conditions. After 5 d of UV-B treatment, the aboveground growth indices were obviously decreased by 13.2%-44.1% (T1) and 21.4%-49.3% (T2), compared to CK, and except active absorption area of roots, the belowground indices by 14.1%-35.6% (T1) and 20.3%-42.6% (T2). For Ce+UV-B treatments, the aboveground and belowground growth indices were decreased respectively by 4.1%-23.6%, 5.2% -23.3% (Ce+T1) and 10.8%-28.4%, 7.0%-27.8% (Ce+T2), lower than those of UV-B treatments. The decrease of growth indices appeared to be the result of changes of physiological processes. Two levels of UV-B radiation induced the decrease in chlorophyll content, net photosynthesis rate, transpiration rate, stomatal conductance and water use efficiency by 11.2%-25.9% (T1) and 20.9%-56.9% (T2), whereas increase in membrane permeability and activities of antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) by 6.9%, 22.8%, 21.5%, 9.5% (T1) and 36.6%, 122.3%, 103.5%, 208.9% (T2), respectively. The reduction of the photosynthetic parameters in Ce+UV-B treatments was lessened to 3.2%-13.8% (Ce+T1) and 4.9%-27.6% (Ce+T2), and the increase of membrane permeability and activities of antioxidant enzymes except POD in the same treatments were lessened to 2.4%, 8.4%, 6.6% (Ce+T1) and 30.1%, 116.7%, 75.4% (Ce+T2). These results indicate that the regulative effect of Ce on photosynthesis and antioxidant enzymatic function is the ecophysiological basis of alleviating the suppression of UV-B radiation on growth of seedlings. Furthermore, the protective effect of Ce on seedlings exposed to T1 level of UV-B radiation is superior to T2 level.

[Inhibitory effect of weikangfu recipe on growth of mouse S180 tumor and its apoptotic induction].

OBJECTIVE: To study the growth inhibition of Weikangfu recipe on S180 tumor and its apoptotic induction. METHOD: S180-bearing mice were orally administrated with different dosages of Weikangfu recipe, and the growth inhibition was evaluated; apoptotic cells induced were detected by flow cytometry and DNA agarose gel electrophoresis. RESULT: Weikangfu recipe showed significant inhibition on the growth of S180 tumor in a dose-dependent manner, compared with the control group. From apoptotic analyses, Weikangfu recipe induced a dose-dependent apoptosis of S180 tumor cells and arrested the cell cycle distribution at G0-G1 phase. At the same time, the up-regulation of p53 and bax and down-regulation of bcl-2 were observed in S180 tumor cells of the treated groups. CONCLUSION: Our findings demonstrate that Weikangfu recipe can significantly inhibit the growth of S180 tumor and induce apoptosis through expression alteration of p53, bax and bcl-2.

[Studies on active substance of anticancer effect in Polygonum cuspidatum].

OBJECTIVE: To supply the scientific basis of research and development of the medicinal value of Polygonum cuspidatum. METHODS: One composition was isolated from the roots of Polygonum cuspidatum by cytotoxicity based fractionation and identified by HPLC-MS, UV scanning and IR. The inhibition and morphology of L-02, Hep G2, SHZ-888, MCF-7, MCF-7/ADM cells growth caused by this composition was determined by MTT assay and HE dyeing. RESULTS: This composition was identified as trans-and cis-resveratrol. It could specifically inhibit proliferation of many cancer cells but not human normal liver cell. We investigated the cytotoxicity of resveratrol to adriamycin-resistant MCF-7 cell in virtro. CONCLUSION: Resveratrol is a new anticancer composition which is less toxicity and higher efficiency in Polygonum cuspidatum.

Responses of antioxidant enzyme and photosynthesis in rape seedling to the combined stresses of acid rain and ultraviolet-B radiation.

Effects of the simulated acid rain (AR) and ultraviolet-B (UV-B, 280-320 nm) radiation with a single or two ways simultaneously (AR + UV-B) on the antioxidant enzyme and photosynthesis of the rape seedlings were investigated by the hydroponic culture. The results of static experiment indicated that the tolerance of rape seedling to single stress (AR or UV-B) is stronger than that to dual stresses (AR + UV-B). Furthermore, the dual stresses had additive effect on catalase activity, and a synergistic effect on MDA content, net photosynthesis rate, water use efficiency as well as intercellular CO2 concentration. Meanwhile, it has an independent effect on chlorophyll content, stomatal conductance, and transpiration rate as well as membrane permeability. During 64 h restoration course, the dynamic change in the curves of physiological and biochemical indices were not identical, and none of them show a simple linear variation. According to the static and dynamic experiments, it was found that a responsive sequence of catalase activity, membrane permeability, MDA content and photosynthetic characteristics to the above-mentioned stresses was as follows: AR + UV-B > UV-B > AR.

[Identification of psychrotrophs SYP-A2-3 producing cold-adapted protease from the No. 1 Glacier of China and study on its fermentation conditions].

The psychrotrophs SYP-A2-3 producing the cold-adapted protease has been isolated from the bacterial samples collected from the No. 1 Glacier of China and identified as Bacillus cereus according to its morphological and physiochemical characteristics and 16s rDNA gene sequence analysis. It could grow between 0 degree C and 38 degrees C while its optimal growth temperature was 25 degrees C and the optimal temperature for its protease production was 15 degrees C. The cold-adapted protease was identified as neutral metallo-protease, the molecular weight was 34.2 kD shown by SDS-PAGE, the optimal pH and temperature for activity was 7.0-8.5 and 42 degrees C, respectively. Various fermentation conditions of its protease production were also investigated. The results showed that casein was the best nitrogen source while glucose and starch were suitable carbon source for its protease production. The initial pH of fermentation broth ranged from 6.5 to 7.0 was optimal. Under optimized conditions, the protease activity produced by SYP-A2-3 could reach 3800 U/mL and 4800 U/mL conducted in shaking flask and 5 L stirred jar experiment, respectively.

[Effect of liuwei dihuang decoction and its additives on boosting spermatogenesis in spermatogenesis obstruction rat].

OBJECTIVE: To study the effect of Liuwei Dihuang Decoction (LDD) and its additives on boosting the spermatogenesis in spermatogenesis obstruction rat. METHOD: The model of spermatogenesis obstruction of rat was made with gossypol acetate. Boosting spermatogenesis of LDD and its additives was studied by observing the number and quality of sperm. RESULT: The sperm quantity of LDD and its additives in treated group is significantly different from model group, and higher than that of positive group. The sperm quality of treated group is better than that of model group. CONCLUSION: LDD and its additives could rise the quantity and quality of spermatogenesis obstruction rat.

Identification of a Novel Antibiotic from Myxobacterium Stigmatella Eracta WXNXJ-B and Evaluation of its Antitumor Effects I n - vitro.

This work was to isolate and identify the bioactive secondary metabolite which was produced by myxobacterium Stigmatella eracta WXNXJ-B, and to evaluate its antitumor and apoptosis-inducing effects. The results showed that one novel compound (molecular formula C29H25NO3) was isolated, purified by Sephadex LH-20 column chromatography and preparative RP-HPLC, and identified as 5-(6-benzyl-quinolin-3-ylmethyl)-6- phenyl-3,7-dioxa- bicycle [4.1.0] heptan-3-one (named as quinoxalone) according to its UV, IR, HRMS and NMR spectra. The compound showed strong antitumor activity on B16, HepG2, MCF-7, SGC-7901, MDA-MB231 and CT-26 six tumor cell lines in-vitro. Nevertheless, it showed less cytotoxic to the mouse normal spleen cells (IC50 was 836.27 ± 13.02 µg mL(-1)). The cytotoxic study on HepG2 cells in-vitro showed that quinoxalone could induce the change of cell nuclear and arrested the cell division in the S and G2/M phase. Our results suggest that quinoxalone could be a potential anti-cancer agent.

Further studies on the hepatoprotective effect of Antrodia camphorata in submerged culture on ethanol-induced acute liver injury in rats.

To further understand the hepatoprotective activity of Antrodia camphorata in living systems and the possible mechanisms of this protection, the effects of fractions from A. camphorata in submerged culture on the liver and its antioxidative system in acute ethanol intoxicated rats were investigated. The results showed that the ethanolic extract (Fr-I) of A. camphorata was the most effective in the prevention of ethanol-induced acute liver injury and free radical generation in rats. The ethanolic extract administrated prior to ethanol significantly prevented the increase in serum levels of hepatic enzyme markers such as aspartate aminotransferase and alanine aminotransferase. It also normalised the increase of hepatic malondialdehyde concentration and the decrease of glutathione levels in the liver. Moreover, Fr-I improved the ethanol-induced decrease of hepatic glutathione peroxidase and reductase activities. On the basis of these results, the ethanolic extract of A. camphorata may exert its hepatoprotective activity by up-regulating GSH-dependent enzymes and inhibiting free radical formation in the liver.

Analysis of volatile compounds of Antrodia camphorata in submerged culture using headspace solid-phase microextraction.

In this work a headspace solid-phase microextraction (HS-SPME) coupled with gas chromatography-mass spectrometry (GC-MS) and GC-olfactometry (GC-O) was developed to evaluate the profile of the volatile compounds that contribute to the aroma of Antrodia camphorata in submerged culture. For this purpose, the HS-SPME sampling method for the volatile compounds of A. camphorata in submerged culture was optimised by a D-optimal design. A HS extraction of the culture broth of A. camphorata followed by incubation on a carboxen/polydimethylsiloxane (CAR/PDMS) fibre during 31.8min at 54.6°C gave the most effective and accurate extraction of the volatile compounds. By the optimised method, a total of 49 volatile compounds were identified in culture broth of A. camphorata, while a total of 55 volatile compounds were identified in the mycelia. A series of C(8) aliphatic compounds (mushroom-like odour), several lactones (fruity odour) and l-linalool (citrus-like odour) were the most potent key odourant in both the mycelia and culture broth. This combined technique is fast, simple, sensitive, inexpensive and useful to monitor volatile compounds associated to A. camphorata.

Phoxalone, a novel macrolide from Sorangium cellulosum: structure identification and its anti-tumor bioactivity in vitro.

A novel macrolide, isolated from the myxobacteria Sorangium cellulosum WXNXJ-C, was identified as 1,7,12,13-tetrahydroxy-14-methoxy-8,10-dimethyl-6-phenyl-5,15-dioxa-bicyclo[9.3.1]pentadecan-4-one, "Phoxalone". It had minimum IC(50) values of 0.24, 6.9, 10.3, 0.98 and 4 microg/ml, respectively, against tumour cell lines: B16, Bel7402, H446, MCF-7, and SGC7901. In addition, it had less cytotoxicity to normal human liver L02 cell lines (286 microg/ml, 24 h). A cytotoxic bioactivity study on H446 cell line in vitro suggested that Phoxalone arrested the mitosis in the G2/M phase.

[Ozonation of odorous organic compounds in eutrophic water].

Ozonation was applied to investigate the removal efficiency of 6 odorous organic compounds in eutrophic water from Taihu Lake (Wuxi). The results showed that the ozonation process could be described by first order kinetic model and the overall kinetic rates of the odorous compounds were determined by their chemical characteristic and structure, which could be ranked: beta-Ionone (3.3 x 10(-3) s(-1)) > beta-Cyclocitral (2.8 x 10(-3) s(-1)) > 2,4-Decadienal (2.7 x 10(-3) s(-1)) > Neryl Acetone (2.2 x 10(-3) s(-1)) > Geosmin (1.4 x 10(-3) s(-1)) > 2-Methylisoborneol (5.6 x 10(-4) s(-1)). Ozonation of eutrophic water led to the formation of a series of aldehydes, ketones, alcohols and esters. The influence of bicarbonate and natural organic carbon on odorous compounds ozonation were further investigated, results showed that the bicarbonate could restrain the effect of ozonation by cutting down the radical pathway and natural organic carbon in the water by competition with target compounds.

[Rapid determination of volatile flavor compounds in soy sauce using head space solid-phase microextraction and gas chromatography-mass spectrometry].

A rapid and simple method was developed for the determination of volatile flavor compounds (VFCs) in soy sauce by head space solid-phase microextraction (HS-SPME) coupled to capillary gas chromatography-mass spectrometry (GC-MS). Five types of SPME fibers, including 85 microm PA, 100 microm PDMS, 75 microm CAR/PDMS, 65 microm PDMS/DVB, 50 microm DVB/CAR/PDMS were investigated. Three parameters for HS-SPME in terms of adsorption time, salt concentration, and extraction temperature were optimized. Adsorption time tested in this study were 20, 40 and 60 minutes; the salt concentrations were 180, 210, 250, 270 and 300 g/L; and extraction temperatures were 25, 35, 45, 55 and 65 degrees C. The concentrations of the compounds were calculated based on their relative peak areas to the internal standard of 2-octanol. An 85 microm PA fiber, adsorption time of 40 min, a temperature of 45 degrees C and NaCl concentration of 250 g/L were selected as th optimum conditions. This optimized method was applied to evaluate a real sample. As a result, 97 compounds in a soy sauce sample were isolated and identified successfully. The results showed that alcohols, carboxylic acids, esters and phenols were the major VFCs of soy sauce. The most important groups of volatile compounds in the soy sauce sample were ethanol, hexadecanoic acid, phenylethyl alcohol and 2,3-butanediol. In addition, some oxo-compounds and heterocyclic compounds were also found. The average relative standard deviation of the relative peak area was 12.1%, and the recoveries were 79.9% - 109.6%. The method is simple, fast and accurate with high reproducibility, high sensitivity and low cost.