Peiqin Li

Diterpenoid Tanshinones and Phenolic Acids from Cultured Hairy Roots of Salvia miltiorrhiza Bunge and Their Antimicrobial Activities

Four diterpenoid tanshinones and three phenolic acids were isolated from the crude ethanol extract of the cultured hairy roots of Salvia miltiorrhiza Bunge by bioassay-guided fractionation. By means of physicochemical and spectrometric analysis, they were identified as tanshinone ΙΙA (1), tanshinone Ι (2), cryptotanshinone (3), dihydrotanshinone Ι (4), rosmarinic acid (5), caffeic acid (6), and danshensu (7). These compounds were evaluated to show a broad antimicrobial spectrum of activity on test microorganisms including eight bacterial and one fungal species. Among the four tanshinones, cryptotanshinone (3) and dihydrotanshinone Ι (4) exhibited stronger antimicrobial activity than tanshinone ΙΙA (1) and tanshinone Ι (2). The results indicated that the major portion of the antimicrobial activity was due to the presence of tanshinones and phenolic acids in S. miltiorrhiza hairy roots, which could be used as the materials for producing antimicrobial agents for use in agricultural practice in the future.

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.

Enhancement of diosgenin production in Dioscorea zingiberensis cell cultures by oligosaccharides from its endophytic fungus Fusarium oxysporum Dzf17.

The effects of the oligosaccharides from the endophytic fungus Fusarium oxysporum Dzf17 as elicitors on diosgenin production in cell suspension cultures of its host Dioscorea zingiberensis were investigated. Three oligosaccharides, DP4, DP7 and DP10, were purified from the oligosaccharide fractions DP2-5, DP5-8 and DP8-12, respectively, which were prepared from the water-extracted mycelial polysaccharide of the endophytic fungus F. oxysporum Dzf17. When the cell cultures were treated with fraction DP5-8 at 20 mg/L on day 26 and harvested on day 32, the maximum diosgenin yield (2.187 mg/L) was achieved, which was 5.65-fold of control (0.387 mg/L). When oligosaccharides DP4, DP7 and DP10 were individually added to 26-day-old D. zingiberensis cell cultures at concentrations of 2, 4, 6, 8 and 10 mg/L in medium, DP7 at 6 mg/L was found to significantly enhance diosgenin production, with a yield of 3.202 mg/L, which was 8.27-fold of control. When the cell cultures were treated with DP7 twice on days 24 and 26, and harvested on day 30, both diosgenin content and yield were significantly increased and reached the maximums of 1.159 mg/g dw and 4.843 mg/L, both of which were higher than those of single elicitation, and were 9.19- and 12.38-fold of control, respectively.

Enhancement of Diepoxin ζ Production by Yeast Extract and Its Fractions in Liquid Culture of Berkleasmium-Like Endophytic Fungus Dzf12 from Dioscorea zingiberensis

This study was to examine the effects of yeast extract (YE) and its fractions (YE1 and YE2) on the growth and diepoxin ζ (a spirobisnaphthalene with a diversity of bioactivities) production in liquid culture of Berkleasmium-like endophytic fungus Dzf12 from Dioscorea zingiberensis. When YE was applied to the liquid medium at 10 g/L on day 3 of culture, the diepoxin ζ production was most effectively enhanced 3.2-fold (378.70 mg/L versus 120.09 mg/L in control) after another 10 days culture. Feeding with 15 g/L of YE on day 9, the mycelia biomass reached 16.44 g/L, about 2.3-fold in comparison with the control (7.15 g/L). The polysaccharide fraction (YE1) was mainly responsible for stimulating diepoxin ζ accumulation, and the non-polysaccharide fraction (YE2) was mainly responsible for promoting mycelia growth. The results showed that the diepoxin ζ production in liquid culture of endophyte Dzf12 could be effectively enhanced by YE and its fractions.

Extraction Optimization of Water-Extracted Mycelial Polysaccharide from Endophytic Fungus Fusarium oxysporum Dzf17 by Response Surface Methodology

Water-extracted mycelial polysaccharide (WPS) from the endophytic fungus Fusarium oxysporum Dzf17 isolated from Dioscorea zingiberensis was found to be an efficient elicitor to enhance diosgenin accumulation in D. zingigerensis cultures, and also demonstrated antioxidant activity. In this study, response surface methodology (RSM) was employed to optimize the extraction process of WPS from F. oxysporum Dzf17 using Box-Behnken design (BBD). The ranges of the factors investigated were 1–3 h for extraction time (X1), 80–100 °C for extraction temperature (X2), and 20–40 (v/w) for ratio of water volume (mL) to raw material weight (g) (X3). The experimental data obtained were fitted to a second-order polynomial equation using multiple regression analysis. Statistical analysis showed that the polynomial regression model was in good agreement with the experimental results with the determination coefficient (R2) of 0.9978. By solving the regression equation and analyzing the response surface contour plots, the extraction parameters were optimized as 1.7 h for extraction time, 95 °C for extraction temperature, 39 (v/w) for ratio of water volume (mL) to raw material weight (g), and with 2 extractions. The maximum value (10.862%) of WPS yield was obtained when the WPS extraction process was conducted under the optimal conditions.

Extraction optimization of polysaccharide from Zanthoxylum bungeanum using RSM and its antioxidant activity.

Polysaccharide prepared from pericarp of Zanthoxylum bungeanum was proved to possess excellent antioxidant activities in vitro by using reducing ferric iron power, DPPH radical scavenging activity, chelating ferrous iron capacity, and hydroxyl radical scavenging activity assays in the present study. In those four antioxidant assay models, Z. bungeanum polysaccharide (ZBP) displayed prominent antioxidant activities with low EC50 values of 0.011, 0.021, 0.056 and 0.008 mg/mL, respectively. Moreover, the extraction process of ZBP was further optimized by response surface methodology combined with Box-Behnken design. The highest polysaccharide yield 13.96%, which agreed closely with the predicted yield 13.20%, was obtained under the optimal extraction conditions as follows: extraction temperature 89 °C, extraction time 3h, ratio of water volume (mL) to raw material weight (g) 29 (v/w), and extraction number two times. The present research not only provide theoretical basis for exploitation of natural polysaccharide antioxidants, but also establish the foundation of large-scale production and further system utilization of ZBP.

In vitro antioxidant activities of polysaccharides from endophytic fungus Fusarium oxysporum Dzf17

Three polysaccharides, namely exopolysaccharide (EPS), water-extracted mycelial polysaccharide (WPS) and sodium hydroxide-extracted mycelial polysaccharide (SPS), from the endophytic fungus Fusarium oxysporum Dzf17 were investigated for their in vitro antioxidant activities. Among them, SPS was the most active antioxidant component, and WPS exhibited moderate antioxidant activity. The median effective concentration (EC 50 ) values of the polysaccharides were 162.38 µg/ml (for WPS), 63.37 µg/ml (for SPS) by DPPH radical scavenging activity assay, and 54.54 µg/ml (for WPS) and 44.91 µg/ml (for SPS) by using ferrous ions chelating activity assay. The polysaccharides from F. oxysporum Dzf17 could be an alternative source as the antioxidant components.

Enhancement of Palmarumycin C12 and C13 Production in Liquid Culture of the Endophytic Fungus Berkleasmium sp. Dzf12 by Oligosaccharides from Its Host Plant Dioscorea zingiberensis

Three crude oligosaccharides were respectively prepared by acid hydrolysis of three polysaccharides, which were water-extracted polysaccharide (WEP), sodium hydroxide-extracted polysaccharide (SEP) and acid-extracted polysaccharide (AEP) from the rhizomes of Dioscorea zingiberensis. Among the three oligosaccharides, the crude oligosaccharide prepared by acid hydrolysis of WEP was found to be the most efficient elicitor to enhance the production of palmarumycins C12 and C13 in liquid culture of endophytic fungus Berkleasmium sp. Dzf12. When OW was applied to the medium at 300 mg/L on day 3 of culture, the maximal yields of palmarumycin C12 (87.96 mg/L) and palmarumycin C13 (422.28 mg/L) were achieved on day 15 of culture, which were 9.83 and 3.24-fold in comparison with those (8.95 and 130.43 mg/L) of control, respectively. The results indicate that addition of the oligosaccharides from the host plant D. zingiberensis should be an effective strategy for enhancing production of palmarumycins C12 and C13 in liquid culture of endophytic fungus Berkleasmium sp. Dzf12.

Medium Optimization for Exopolysaccharide Production in Liquid Culture of Endophytic Fungus Berkleasmium sp. Dzf12

Berkleasmium sp. Dzf12, an endophytic fungus from Dioscorea zingiberensis, is a high producer of spirobisnaphthalenes with various bioactivities. The exopolysaccharide (EPS) produced by this fungus also shows excellent antioxidant activity. In this study, the experimental designs based on statistics were employed to evaluate and optimize the medium for EPS production in liquid culture of Berkleasmium sp. Dzf12. For increasing EPS yield, the concentrations of glucose, peptone, KH2PO4, MgSO4·7H2O and FeSO4·7H2O in medium were optimized using response surface methodology (RSM). Both the fractional factorial design (FFD) and central composite design (CCD) were applied to optimize the main factors which significantly affected EPS production. The concentrations of glucose, peptone and MgSO4·7H2O were found to be the main effective factors for EPS production by FFD experimental analysis. Based on the further CCD optimization and RSM analysis, a quadratic polynomial regression equation was derived from the EPS yield and three variables. Statistical analysis showed the polynomial regression model was in good agreement with the experimental results with the determination coefficient (adj-R2) as 0.9434. By solving the quadratic regression equation, the optimal concentrations of glucose, peptone and MgSO4·7H2O for EPS production were determined as 63.80, 20.76 and 2.74 g/L, respectively. Under the optimum conditions, the predicted EPS yield reached the maximum (13.22 g/L). Verification experiment confirmed the validity with the actual EPS yield as 13.97 g/L, which was 6.29-fold in comparison with that (2.22 g/L) in the original basal medium. The results provide the support data for EPS production in large scale and also speed up the application of Berkleasmium sp. Dzf12.

Quantitative determination of diosgenin in Dioscorea zingiberensis cell cultures by microplate- spectrophotometry and high-performance liquid chromatography

Quantitative determination of diosgenin was carried out by multi-well microplate-spectrophotometry and high-performance liquid chromatography (HPLC) in this study. Diosgenin detection by microplatespectrophotometry depended on the formed yellow substance developed by perchloric acid, and measured at 410 nm. The calibration graph was linear over the range of 2 to 10 μg diosgenin in each well with correlation coefficient (R) as 0.9988, detection limit as 0.6111 μg, and quantification limit as 1.8518 μg. For HPLC analysis, diosgenin detection was conducted at 203 nm on an Agilent TC-C18 column with a mobile phase of acetonitrile-water (90:10, v/v). The calibration graph was linear over the range of 0.0625 to 1.000 µg diosgenin with correlation coefficient (R) as 0.9995, detection limit as 0.0372 μg, and quantification limit as 0.1127 μg of diosgenin. Both methods were characterized by satisfactory precision and accuracy, which were then employed to detect diosgenin content in cell cultures of Dioscorea zingiberensis. No statistical significant differences were observed between the results obtained by microplate-spectrophotometry and HPLC (ANOVA, p < 0.05). Quantitative determination of diosgenin by microplate-spectrophotometry should be a rapid, accurate, simple and economic alternative method, though HPLC is more sensitive for diosgenin analysis.