Prebiotic effects of resistant starch from purple yam (Dioscorea alata L.) on the tolerance and proliferation ability of Bifidobacterium adolescentis in vitro.

The in vitro prebiotic effects of resistant starch (RS), prepared by different treatments from purple yam, on Bifidobacterium adolescentis (bifidobacteria for short), were investigated. Tolerance tests indicated that bifidobacteria in PDS (prepared by debranching combined with autoclaving) and PDS.H (PDS further treated by double enzyme hydrolysis) media adapted better to simulated upper gastrointestinal conditions (at pH 1.5-3.0 and 0.3% and 1.0% bile acid) than those in GLU (glucose) and DAS (prepared by autoclaving) media. PDS.H, which had the highest digestion resistibility, exhibited significant effects on the OD600 nm value (1.544) and the pH value (4.21) when the carbohydrate concentration was 20 g L-1. Additionally, the exponential growth phase of bifidobacteria was 2 h in the PDS or PDS.H media, whereas it was 4 h in the GLU or DAS media. A higher content of short-chain fatty acids (SCFAs) was obtained in the PDS.H medium. Analysis of the structural features of RS and fermented RS indicated that PDS, especially PDS.H, had a rougher surface and higher crystallinity than DAS. Fermented RS in a simulated large bowel environment showed an eroded surface and decreased crystallinity. All of these findings suggest that RS with a rough surface and perfect crystalline structure could protect bifidobacteria from gastrointestinal conditions and enhance the proliferation of bifidobacteria.

New bioactive compounds from aquatic endophyte Chaetomium globosum.

Two new oxidation products-related aureonitol and cytochalasan were isolated from Chaetomium globosum fermented in Chinese yam (Dioscorea opposita) and determined as 10,11-dihydroxyl- aureonitol (1) and yamchaetoglobosin A (2). Compound 2 indicated significant inhibitory effect on nitric oxide production in LPS-activated macrophages, anti-acetylcholinesterase activity with the inhibition ratios of 92.5, 38.2% at 50 µM, and cytotoxicity to HL-60, A-549, SMMC-7721, MCF-7 and SW480 with the range of inhibition ratio at 51-96% for a concentration of 40 µM. Compounds 1, 2 showed weak anticoagulant activity with PT at 16.8 s. Few work was reported on the anti-acetylcholinesterase, and anticoagulant activities of aureonitol, and cytochalasan derivatives. The preliminary structure-activity relationship stated that the oxidation ring-opening in yamchaetoglobosin A can retain the inhibitory effect against NO production and tumor cell.

Larvicidal spirobisnaphthalenes from the endophytic fungus Berkleasmium sp. against Aedes albopictus.

BACKGROUND: In our screening programme for new agrochemicals from endophytic fungi, the ethyl acetate extract of an endophytic Berkleasmium sp. isolated from the medicinal plant Dioscorea zingiberensis was found to possess strong larvicidal activity against the Asian tiger mosquito, Aedes albopictus. RESULTS: Bioassay-guided fractionation of the fungal extract has led to the isolation of seven spirobisnaphthalenes, including palmarumycins C8, C12, C15 and B6 and diepoxins γ, δ and ζ. Among them, palmarumycins C8 and B6 showed strong larvicidal activity against the fourth-instar larvae of A. albopictus, with LC50 values of 8.83 and 11.51 µg mL(-1) respectively. Interestingly, only spirobisnaphthalenes with a chlorine substituent possessed strong larvicidal activity. CONCLUSION: The results indicated that the spirobisnaphthalenes derived from the endophytic fungus Berkleasmium sp. could be promising leads for the development of new larvicides against A. albopictus.

Screening for differentially expressed genes in endophytic fungus strain 39 during co-culture with herbal extract of its host Dioscorea nipponica Makino.

Strain 39 is an endophytic fungus which was isolated from Dioscorea nipponica Makino (DNM). After Strain 39 co-cultured with ethanol extract of DNM rhizomes for several days, the content of saponins in this culture mixture would be obviously increased. To analyze the mechanism of this microbial transformation, we used the differential display reverse transcription polymerase chain reaction (DDRT-PCR) method to compare the transcriptomes between Strain 39 cultured in normal PD medium and in PD medium which added ethanol extract of DNM rhizomes. We amplified 29 DDRT-PCR bands using 12 primer combinations of three anchored primers and five random primers, and six bands were re-amplified. Analysis of real-time PCR and sequence alignment showed that three clones were up-regulated in sample group: squalene epoxidase, squalene synthase, and catalase, one clone was expressed only in sample group. The possible roles and origins of the above genes were discussed, and the molecular mechanism of Strain 39 biotransformation was speculated. This study is the first report of the molecular biotransformation mechanism of saponins production by endophytic fungus of DNM.

Use of Lactococcus lactis to enrich sourdough bread with γ-aminobutyric acid.

Fried sourdough bread (bhatura) with an elevated amount of γ-aminobutyric acid (GABA) was produced using lactic acid bacteria (LAB). The LAB starter was screened and isolated from pickled yam showing highest GABA content and was identified as Lactococcus lactis subsp. lactis. The maximum GABA production in de Man Rogosa Sharpe (MRS) media supplemented with monosodium glutamate (MSG) was 110 mg/100 ml at pH 5, and 1-3% NaCl did not change the production of GABA significantly (p>0.05). When MSG was replaced with Vigna mungo in sourdough, the amount of GABA for bhatura was 226.22 mg/100 g representing about 10-fold increase. A sensory evaluation resulted as the overall general acceptability of bhatura to be 4.91 ± 0.03 on a five-point hedonic scale. Thus, the results indicated the potential of L. lactis as a LAB starter for the production of GABA-enriched bhatura. Although other physiological effects can be expected in the product, animal and clinical studies are mandatory prior to application of this food.

Red mold dioscorea: a potentially safe traditional function food for the treatment of hyperlipidemia.

A study was undertaken to evaluate whether the interaction between Monascus-fermented products and lovastatin contributes to increased risk of rhabdomyolysis. Rhabdomyolysis is a potentially dangerous side effect of statin drugs. In this study with hyperlipidemic hamsters fed lovastatin only, lovastatin with 1-fold red mold dioscorea (RMD), and lovastatin, the functional components of red mold fermented products, HMG-CoA reductase inhibitors, did not exacerbate pre-existing diseases, and actually helped in improving existing disease conditions, respectively, as compared with the control. Administration of RMD, alone or in combination with lovastatin did not cause significant rhabdomyolysis as assessed by measuring the levels of creatinine phosphokinase. Further, we did not find any study that clearly implicates the involvement of RMD, which have long been considered a food product, in liver and kidney toxicity. RMD alone or in combination with lovastatin, does not increase the risk of rhabdomyolysis, even when administered at a high dosage (including HMG-CoA reductase inhibitors >75 mg/day/adult).

Effects of polysaccharide elicitors from endophytic Fusarium oxysporium Dzf17 on growth and diosgenin production in cell suspension culture of Dioscorea zingiberensis.

Three polysaccharides, namely exopolysaccharide (EPS), water-extracted mycelial polysaccharide (WPS) and sodium hydroxide-extracted mycelial polysaccharide (SPS), were prepared from the endophytic fungus Fusarium oxysporium Dzf17 isolated from the rhizomes of Dioscorea zingiberensis. The effects of the time of addition and polysaccharide concentration on the growth and diosgenin accumulation in cell suspension culture of D. zingiberensis were studied. Among them, WPS was found to be the most effective polysaccharide. When WPS was added to the medium at 20 mg/L on the 25th day of culture, the cell dry weight was increased 1.34-fold, diosgenin content 2.85-fold, and diosgenin yield 3.83-fold in comparison to those of control. EPS and SPS showed moderate and relatively weak enhancement effects on cell growth and diosgenin accumulation, respectively. The dynamics of cell growth and diosgenin accumulation when WPS was added to the medium at 20 mg/L on the 25th day of culture were investigated, and results showed that dry weight of cells reached a maximum value on day 30 but the maximum diosgenin content was achieved on day 31.

Enhanced hypolipidemic effect and safety of red mold dioscorea cultured in deep ocean water.

Red mold dioscorea (RMD) produced by Monascus sp. was proven to be a hypolipidemic functional food. Deep ocean water (DOW), that is, water obtained from over 200 m deep in the ocean, was found to promote the growth of fungus via its mineral richness. On the basis of the advantages, this study used 650 m DOW as the culture water to culture Monascus purpuresus NTU 568 and produce the DOW-RMD. The goal of this study is to compare the difference between DOW-RMD and reverse osmosis water-cultured RMD (ROW-RMD) on the hypolipidemic effect. Hyperlipidemic hamsters were fed a high-cholesterol diet and administered various doses of DOW-RMD or ROW-RMD for 8 weeks. After sacrifice, biochemical analyses in serum, liver, and feces were carried out. The results showed that DOW-RMD had a greater effect on lowering cholesterol levels and lipid peroxidation in serum and lipid plaque in heart aorta than ROW-RMD. However, DOW was likely to modulate the Monascus metabolite biosynthesis pathway toward the formation of hypolipidemic yellow pigments (such as monascin and ankaflavin) rather than red pigments and the mycotoxin citrinin. In addition, the DOW with higher Mg(2+) ion was proven to absorb into DOW-RMD; however, the accumulation of Mg(2+) ions should contribute a greater hypolipidemic effect to DOW-RMD. Comprehensively, the DOW-induced metabolism modulation and the ions of DOW were a benefit to the development of safe DOW-RMD with low citrinin levels and high hypolipidemic, antiatherosclerosis, and anti-fatty liver effects.

Effects of red mold dioscorea on oral carcinogenesis in DMBA-induced hamster animal model.

Monascus-fermented products offer valuable therapeutic benefits and have been extensively used for centuries in East Asia. Dioscorea has been proved to have anti-cancer effect. The aim of this study is to investigate the anti-tumor ability of the ethanol extract of red mold dioscorea (RMDE) on 7,12-dimethyl-1,2-benz[a]anthracene (DMBA)-induced hamster buccal pouch carcinogenesis. We induced oral squamous cell carcinoma (OSCC) in the buccal pouch of male Syrian golden hamsters by painting with 0.5% DMBA three times a week for 14 weeks. From 9 to 14 weeks, a dose of 50, 100, and 200 mg RMDE per kg body weight were painting with the hamsters for 6 weeks on days alternate to the DMBA application. The results demonstrated that RMDE decreased nitric oxide (NO), reactive oxygen species (ROS), and prostaglandin E(2) (PGE(2)) overexpression in hamster buccal pouches in the DMBA treatment group and increased p53, serum tumor necrosis factor-α (TNF-α), and interleukin-1ß (IL-1ß) to significantly stimulate caspase-8 and -3 activities, indicating that RMDE reduced oxidative damage causing by DMBA and induced apoptosis in oral cancer cells. Therefore, RMDE may have therapeutic potentials against OSCC.

Red mold dioscorea-induced G2/M arrest and apoptosis in human oral cancer cells.

BACKGROUND: Monascus-fermented products are among the most commonly used traditional food supplements. Dioscorea is known to exhibit anticancer properties. In this study the effects of the ethanol extract of red mold dioscorea (RMDE) on cell proliferation, cell cycle and apoptosis in human oral cancer cells were investigated. RESULTS: RMDE exercised growth inhibition on squamous cell carcinoma-25 (SCC-25) cells. RMDE-mediated G2/M phase arrest was associated with the down-regulation of NF-κB, resulting in the inhibition of cyclin B1 and CDK1 expression; this may be the mechanism by which RMDE inhibits cancer cells. Furthermore, the proapoptotic activity of RMDE was revealed by the Annexin V-FITC/PI double-staining assay. In addition, the proapoptotic effect of RMDE was evident by the inhibition of Bax expression in the mitochondria, resulting in the activation of caspase-9 and caspase-3 and subsequent triggering of the mitochondrial apoptotic pathway. RMDE also enhanced caspase-8 activity, indicating the involvement of the death receptor pathway in RMDE-mediated SCC-25 cell apoptosis. CONCLUSION: RMDE treatment inhibited the growth of SCC-25 cells by arresting cell cycle at the G2/M phase and induced apoptosis in a time- and dose-dependent manner. Therefore RMDE may be a good candidate for development as a dietary supplement against oral cancer.

Beauvericin from the endophytic fungus, Fusarium redolens, isolated from Dioscorea zingiberensis and its antibacterial activity.

From the fungal endophyte, Fusarium redolens Dzf2, isolated from rhizomes of the Chinese medicinal plant Dioscorea zingiberensis, beauvericin was obtained by TLC in combination with bioautographic antibacterial assay. The compound was identified by spectroscopic and physicochemical means. The median effective inhibitory concentration (IC50) values of beauvericin against six test bacteria (Bacillus subtilis, Staphylococcus haemolyticus, Pseudomonas lachrymans, Agrobacterium tumefaciens, Escherichia coli and Xanthomonas vesicatoria) were between 18.4 and 70.7 microg/mL. The beauvericin content of F. redolens Dzf2 mycelia was 9.60 mg/g dw, and beauvericin yield 62.4 mg/L. The obtained results show the potential use of the endophytic fungus for its biological role in providing its host plant with protection, as well as the possible development of beauvericin as an antibacterial.

Protection of Monascus-fermented dioscorea against DMBA-induced oral injury in hamster by anti-inflammatory and antioxidative potentials.

Monascus -fermented products offer valuable therapeutic benefits and have been extensively used in East Asia. This study investigated the prevention of oral tumor formation and antioxidative ability of the ethanol extracts from red mold dioscorea (RMDE) on 7,12-dimethyl-1,2-benz[a]anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinogenesis. The HBP was painted with DMBA three times per week for 14 weeks, and animals were painted with celecoxib, RMDE (50, 100, and 200 mg/kg of bw), and ethanol extracts from dioscorea (200 mg/kg of bw) on days alternate to the DMBA application. The results demonstrated that RMDE attenuated tumor formation by elevating the antioxidase activity and suppressing the overproduction of reactive oxygen species, nitric oxide, prostaglandin E(2), and pro-inflammatory cytokines in the HBP caused by DMBA induction. These results indicated that RMDE exerted anti-inflammatory and antioxidative activity to prevent oral cancer. Therefore, the metabolite from Monascus fermentation may serve as a possible functional edible substance for the prevention of oral cancer.

Enhancement of diosgenin production in Dioscorea zingiberensis cell culture by oligosaccharide elicitor from its endophytic fungus Fusarium oxysporum Dzf17.

Diosgenin accumulation in cell suspension cultures of Dioscorea zingiberensis C. H. Wright was enhanced by treatment with saccharide elicitors from its endophytic fungus Fusarium oxysporum isolate Dzf17. The crude oligosaccharide was prepared by partial acid hydrolysis of the isolated Dzf17 fungal cell wall fragments. Optimal elicitation of diosgenin production by the isolated Dzf17 oligosaccharide in cell suspension culture was achieved when the oligosaccharide was added to the medium at a concentration of 30 mg/L after 16-day's continuous cell suspension culture, and the cells were cultured for another 8 days before harvesting. By using these optimal conditions, the diosgenin yield of the cultured cells reached its maximum of 5.25 mg/L, which was over a three-fold increase.

Spirobisnaphthalenes from the endophytic fungus Dzf12 of Dioscorea zingiberensis and their antimicrobial activities.

Five spirobisnaphthalenes, namely palmarumycin CP17 (1), diepoxin kappa (2), diepoxin eta (3), diepoxin xi (4), and diepoxin gamma (5), were isolated from the endophytic fungus Dzf12 associated with the medicinal plant Dioscorea zingiberensis C. H. Wright. Their structures were identified by physicochemical and spectrometric analysis. Among these spirobisnaphthalenes, 2 was found to have antibacterial activity, and the mixture of 3 and 4 was detected to have both antibacterial and antifungal activities.

Red mold dioscorea has a greater antihypertensive effect than traditional red mold rice in spontaneously hypertensive rats.

The aim of this study is to investigate the antihypertensive effects of red mold rice (RMR) and red mold dioscorea (RMD) by low-dose oral administration to spontaneously hypertensive rats (SHRs). A single oral dose of 1-fold RMD (150 mg/kg) significantly (p < 0.05) decreased systolic blood pressure (SBP) and diastolic blood pressure (DBP) after 8 h of administration, but RMR showed no significant effect. During the chronic oral administration of 1-fold RMR (150 mg/kg), 0.5-fold RMD, 1-fold RMD, and 5-fold RMD to SHRs for 8 weeks, the increase of blood pressure was slowed significantly. The results indicated that only a 0.5-fold dose of RMD was able to significantly decrease both SBP and DBP. A 1-fold RMD showed a greater antihypertensive effect than 1-fold RMR, and both RMR and RMD can improve the vascular elastin structure remodeling. In comparison to RMR, RMD contained a higher amount of gamma-aminobutyric acid (GABA) and anti-inflammatory yellow pigments (monascin and ankaflavin). Moreover, RMD also exhibited higher angiotensin-I-converting enzyme (ACE) inhibitory activity than RMR. These results suggest that RMD has greater antihypertensive bioavailability.

Fungal endophytes from Dioscorea zingiberensis rhizomes and their antibacterial activity.

AIMS: The aim of the study was to isolate and characterize the endophytic fungi from the rhizomes of the Chinese traditional medicinal plant Dioscorea zingiberensis and to detect their antibacterial activities. METHODS AND RESULTS: After strict sterile sample preparation, nine fungal endophytes were isolated from rhizomes of the Chinese traditional medicinal plant D. zingiberensis. The endophytes were classified by morphological traits and internal transcribed spacer (ITS) rRNA gene sequence analysis. Their ITS rDNA sequences were 99-100% identical to Nectria, Fusarium, Rhizopycnis, Acremonium and Penicillium spp. respectively. Of these, the most frequent genera were Fusarium and Nectria. One isolate, Dzf7, was unclassified on the basis of its low sequence similarity. The next closest species was Alternaria longissima (c. 92.4% sequence similarity). Endophyte isolate Dzf5 showed the closest sequence similarity (c. 99.5%) to an uncultured soil fungus (DQ420800) obtained from Cedar Creek, USA. Bioassays using a modified broth dilution test were used to detect the antibacterial activity of n-butanol extracts of both mycelia and culture filtrates of D. zingiberensis showed biological activity against Bacillus subtilis, Staphylococcus haemolyticus, Escherichia coli and Xanthomonas vesicatoria. Minimal inhibitory concentration (MIC) values of the extracts were between 31 x 25 microg ml(-1) and 125 microg ml(-1). CONCLUSIONS: Endophytic fungus Dzf2 (c. 99 x 8% sequence similarity to Fusarium redolens) isolated from D. zingiberensis rhizome showed the most potent antibacterial activities. SIGNIFICANCE AND IMPACT OF THE STUDY: Endophytic fungi isolated from D. zingiberensis may be used as potential producers of antibacterial natural products.

[Selection of the microorganism for dioscin hydrolyze].

OBJECTIVE: To select the microorganism which can hydrolyze dioscin to diosgenin. METHODS: The microorganism were selected from the surface of rhizome, rhizosphere soil, the inside of the leaves and rhizome of Dioscorea zingiberensis C. H. Wright. Diosgenin was identified by thin-layer chromatography and HPLC. RESULTS: The microorganism which could hydrolyze dioscin from the experiment were identified as Aspergillus sp and Alternaria sp. Characteristics of enzymes production and fermentation technology of Aspergillus No. 1 were also studied primarily. CONCLUSION: The Aspergillus strain No. 1 can secret enzyme to hydrolyze dioscin into diosgenin effectively.