Na Feng

Preparative isolation of cordycepin, N(6)-(2-hydroxyethyl)-adenosine and adenosine from Cordyceps militaris by macroporous resin and purification by recycling high-speed counter-current chromatography.

In this study, cordycepin, N(6)-(2-hydroxyethyl)-adenosine (HEA) and adenosine from the fruiting bodies of Cordyceps militaris were separated by using macroporous resin NKA-II adsorption. The parameters of static adsorption were tested and the optimized conditions were as follow: the total adsorption time was 12h, 50% ethanol was used for desorption and the desorption time was 9h. The crude sample that was prepared by macroporous resin NKA-II contained 3.4% cordycepin, 3.7% HEA and 4.9% adenosine. Then the crude sample was further purified by recycling high-speed counter-current chromatography (HSCCC) with ethyl acetate, n-butanol, 1.5% aqueous ammonium hydroxide (1:4:5, v/v/v) as the optimized two-phase solvent system. Three nucleosides including 15.6mg of cordycepin, 16.9mg of HEA and 23.2mg of adenosine were obtained from 500mg of crude sample in one-step separation. The purities of three compounds were 98.5, 98.3 and 98.0%, respectively, as determined by high performance liquid chromatography.

A novel Ganoderma lucidum G0119 fermentation strategy for enhanced triterpenes production by statistical process optimization and addition of oleic acid

A novel enhanced triterpenes fermentation production process by Ganoderma lucidum G0119 with the addition of oleic acid in the medium has been developed and optimized. All of the six exogenous additives tested were found to exhibit stimulatory effect on mycelial growth and triterpenes biosynthesis by G. lucidum. The results show that oleic acid addition had significant role in promoting triterpenes production. The optimal concentration and time of oleic acid addition were determined to be 30 mL/L and 0 h, respectively. Furthermore, three significant factors influencing triterpenes production were identified as glucose, magnesium sulfate and temperature using the Plackett–Burman design. The optimized conditions by central composite design were 27.83 g/L glucose, 1.32 g/L magnesium sulfate, 26.2°C temperature. The triterpenes fermentation yield with the optimized medium based on actual confirmatory experimental data in 6 L fermentor was 1.076 g/L versus the statistical model predicted value of 1.080 g/L. Our innovatively developed triterpenes fermentation production technology and process has been proven to produce high triterpenes productivity and yield conceivably useful for industrial production.

Kinetic models for the effect of temperature on flavonoid production in liquid submerged fermentation by Phellinus baumii

Kinetic models and temperature control strategy were established to reflect the effect of temperature (22 °C–30 °C) on flavonoid production of Phellinus baumii (P. baumii) in 6‐L fermentor. A modified Logistic equation, Hinshelwood model, and Luedeking Piret equation were used to describe mycelial growth and product formation. The influence of temperature on the estimated kinetic parameters was further studied by regression analysis. Based on kinetic parameters analysis, the new temperature control strategy was proposed. Briefly, at 0–43 H, decreasing temperature (30 °C–28 °C) can shorten the lag phase of mycelial growth, and at 43–90 H, fermentation temperature was reduced gradually from 28 °C to 24 °C to keep high flavonoid productivity. At the fermentation anaphase (90–161 H), temperature was controlled at 24 °C to relieve inhibition of flavonoid and maintain constant production capacity of flavonoid. As a result, the maximum flavonoid yield was reached 4.21 mg/100 mg cell dry weight by temperature control strategy, which was 70.45% higher than that at a constant temperature of 26 °C. Additionally, the establishment of kinetic models based on fermentation temperature, which presented here may provide a scientific basis for further large scales flavonoid production of P. baumii in submerged fermentation.

Preparative isolation of ganoderic acid S, ganoderic acid T and ganoderol B from Ganoderma lucidum mycelia by high-speed counter-current chromatography.

Ganoderic acid S, ganoderic acid T and ganoderal B are the main bioactive triterpenes of Ganoderma lucidum. In this study, mycelia of G. lucidum were obtained by two-stage fermentation and then extracted by ethanol and petroleum ether sequentially to obtain crude triterpenes. The crude sample was further purified by recycling high-speed counter-current chromatography with n-hexane-ethyl acetate-methanol-water (7:12:11:5, v/v/v/v) as the optimized two-phase solvent system. A 16.4 mg aliquot of ganoderol B with a purity of 90.4% was separated from 300 mg of the crude sample in a single run. After employing the recycling elution mode of HSCCC with n-hexane-ethyl acetate-methanol-water (6:10:8:4.5, v/v/v/v) for five cycles, 25.7 mg ganoderic acid T and 3.7 mg ganoderic acid S with purities of 97.8 and 83.0%, respectively, were obtained. The purities of three compounds were determined by high-performance liquid chromatography and their chemical structures were identified by NMR and MS data.

One single standard substance for the simultaneous determination of 17 triterpenes in Ganoderma lingzhi and its related species using high-performance liquid chromatography

Due to the difficulty and high cost for the preparation of triterpenes, one single standard for the simultaneous determination of multi-components (SSDMC) with high performance liquid chromatography (HPLC) is an advanced solution for multi-component analysis. Experiments were carried out to investigate the feasibility of SSDMC for the analysis of Ganoderma triterpenes, with external standard method (ESM) compared, and the samples of Ganoderma were classified by the content of Ganoderma triterpenes. The analysis was performed by using a Fortis Speed Core-C18 column (150mm×4.6mm I.D., 2.6μm) at gradient elution of 0.01% glacial acetic acid-water (V/V) and acetonitrile with diode array detection (252nm), at a flow rate of 1mL/min. The results showed that all calibration curves had good linearity (r2>0.9999) within test ranges. The LOD and LOQ were lower than 2.52ng and 6.43ng, respectively. The RSD for intra-day and inter-day of the seventeen analytes were less than 3.12% at three levels, and the recoveries were 91.4-103.0%. The contents of other 16 triterpenes were determined with ganoderic acid A by SSDMC, which showed that there were few differences compared with the results obtained by ESM. Moreover, the classification of 25 different species and strains of Ganoderma by using the content of triterpenes intuitively reflected the distinction among Ganoderma. In summary, the developed method could be readily utilized as a method of quality evaluation for Ganoderma triterpenes.

Hypoglycemic Effect of Ethanol and Ethyl Acetate Extract of Phellinus baumii Fruiting Body in Streptozotocin-Induced Diabetic Mice

We investigated hypoglycemic effect of ethanol (EtOH) and ethyl acetate extract acetate (AcOEt) extracts in streptozotocin- (STZ-) induced diabetic mice. Our data showed the maximum inhibitory effect on the fasting plasma glucose (FPG) level was detected in STZ-induced diabetic mice administered with 400 mg/kg AcOEt extract of P. baumii. A lower glycated albumin (GA) level and a higher insulin level were observed in 400 mg/kg AcOEt and EtOH extract groups. Moreover, 400 mg/kg AcOEt and EtOH extract exhibited a stronger effect on increasing size and cell number of islets. The insulin expression level of β-cells and integrated optical density (IOD) value were significantly increased by the administration of 400 mg/kg AcOEt and EtOH extracts. Taken together, AcOEt and EtOH extracts of P. baumii fruiting body exhibited considerable hypoglycemic effect on STZ-induced diabetic mice.