Na Guo

Simultaneous Determination of Salidroside and Its Aglycone Metabolite p-Tyrosol in Rat Plasma by Liquid Chromatography-Tandem Mass Spectrometry

Salidroside and its aglycone p-tyrosol are two major phenols in the genus Rhodiola and have been confirmed to possess various pharmacological properties. In our present study, p-tyrosol was identified as the deglycosylation metabolite of salidroside after intravenous (i.v.) administration to rats at a dose of 50 mg/kg, but was not detectable after intragastric gavage (i.g.) administration through HPLC-photodiode array detection (PDA) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Next, an accurate and precise LC-MS/MS method was developed to quantitatively determine salidroside and p-tyrosol in rat plasma samples. Samples were analyzed by LC-MS/MS on a reverse-phase xTerra MS C18 column which was equilibrated and eluted with an isocratic mixture of acetonitrile-water (1:9, v/v) at a flow rate of 0.3 mL/min. The analytes were monitored by multiple reaction monitoring (MRM) under the negative electrospray ionization mode. The precursor/product transitions (m/z) were 299.0→118.8 for salidroside, 137.0→118.9 for p-tyrosol and 150.1→106.9 for the internal standard (IS), paracetamol, respectively. The calibration curve was linear over the concentration ranges of 50–2,000 ng/mL for salidroside and 20–200 ng/mL for p-tyrosol. The inter- and intra-day accuracy and precision were within ±15%. The method has been successfully applied to the pharmacokinetic study and the oral bioavailability was calculated.

Iron/dextran sulfate multilayered microcapsules for controlled release of 10-hydroxycamptothecin.

Stable 10-hydroxycamptothecin (HCPT) microcrystals with a length of about 5-10μm and a ζ-potential of -38.5mV were produced by pH-induced reprecipitation in presence of a stabilizer hydroxypropylmethylcellulose. Sequential layer growth was achieved by the layer-by-layer (LbL) assembly of Fe(3+) and dextran sulfate (DS) on the surface of HCPT microcrystals via both electrostatic interaction and chemical complexation process. The satisfactory drug loading content (67.2±0.82%) as well as high encapsulation efficiency (60.56±0.82%) for four bilayers of Fe(3+)/DS coating was achieved. Both in vitro and in vivo release study revealed that the release time increased as the number of deposited Fe(3+)/DS bilayers increased. These results indicated that such iron-polysaccharide multilayered microcapsules can be a promising approach for the construction of an effective controlled release delivery system of HCPT as well as other drugs with potential cytotoxicity or short half-life time.

A two-step pH-dependent liquid–liquid extraction combined with HPLC-fluorescence method for the determination of 10-hydroxycamptothecin in mouse liver tissue

Context: Liquid–liquid extraction (LLE) shows high efficiency in the plasma sample preparation. However, this extraction method is not optimal for the biological samples containing complex organic interferences, such as liver and brain tissues. Some plant secondary metabolites can be converted between water-insoluble and water-soluble forms by pH adjustment. Objective: A two-step pH-dependent LLE method was introduced in this study to eliminate both water-soluble and lipidic interferences using the properties of pH-dependent interconvertible forms of analytes during sample preparation. A sensitive and reliable method using a reverse-phase HPLC coupled with a fluorescence detector was developed and validated. Materials and methods: 10-Hydroxycamptothecin (HCPT) with internal standard camptothecin and liver tissues were used as model compounds and biological samples. The lactone form of HCPT was converted to the water-soluble carboxylate form under moderate alkaline conditions, and the water-insoluble interferences were extracted with a nonpolar solvent. Afterward, the water-insoluble lactone form of HCPT was regenerated by acidification and then extracted using an organic solvent in a second LLE step. Results: The calibration curve was linear (r2 > 0.999) for HCPT concentrations ranging from 2.5 to 160 ng/mL. The mean recoveries of HCPT were 114.94 ± 3.98, 104.30 ± 2.44 and 95.90 ± 1.40% (n = 6) at concentrations of 2.5, 10 and 80 ng/mL, respectively. The stability determination data showed that no significant degradation occurred under the experimental conditions. This method was successfully applied to liver tissue distribution study of HCPT in mice. Discussion and conclusion: This two-step LLE can be applied to distribution studies of compounds with pH-dependent interconvertible forms in other biological matrices.

Production of Laccase by a New Myrothecium verrucaria MD-R-16 Isolated from Pigeon Pea [Cajanus cajan (L.) Millsp.] and its Application on Dye Decolorization

The present study was conducted to screen a laccase-producing fungal endophyte, optimize fermentation conditions, and evaluate the decolorization ability of the laccase. A new fungal endophyte capable of laccase-producing was firstly isolated from pigeon pea and identified as Myrothecium verrucaria based on a ITS-rRNA sequences analysis. Meanwhile, various fermentation parameters on the laccase production were optimized via response surface methodology (RSM). The optimal fermentation conditions were a fermentation time of five days, temperature 30 °C and pH 6.22. Laccase activity reached 16.52 ± 0.18 U/mL under the above conditions. Furthermore, the laccase showed effective decolorization capability toward synthetic dyes (Congo red, Methyl orange, Methyl red, and Crystal violet) in the presence of the redox mediator ABTS, with more than 70% of dyes decolorizing after 24 h of incubation. Additionally, the activity of laccase was relatively stable with pH (4.5–6.5) and a temperature range of 35–55 °C. Therefore, the high laccase production of the strain and the new fungal laccase could provide a promising alterative approach for industrial and environmental applications.

Development and validation of an LC-ESI/MS/MS method with precolumn derivatization for the determination of betulin in rat plasma.

Neutral pentacyclic triterpenes with only one or two hydroxyl groups, such as betulin, are not easily ionized by electrospray ionization (ESI). However, because betulin is reactive and neutral, derivatization may improve ionization efficiency. In the present study, the potency of different derivatization reagents was evaluated and p-toluenesulfonyl isocyanate (PTSI) was proven to be the optimal. The derivative generated by the reaction of betulin with PTSI was ionizable and fragmentable in the negative mode by liquid chromatography-electrospray ionization/tandem mass spectrometry (LC-ESI/MS/MS). Based on this chemical derivatization, an LC-ESI/MS/MS method was developed and validated for the quantification of betulin in rat plasma. The sample was extracted with ethyl acetate, derivatized with PTSI, separated on an ACQ UPLC BEH phenyl column, and analyzed in negative multiple reaction monitoring (MRM) mode. The calibration curve was linear over the betulin concentration range 2.5-200ng/mL. The lower limit of quantification was 2.5ng/mL. The inter- and intra-day accuracy and precision were within ±15%. Betulin recoveries were 86.7% or higher at three quality control levels (5, 50, and 160ng/mL). This validated method was subsequently applied to a pharmacokinetic study of betulin in rat plasma after oral administration.

Rapid extraction of Amomum tsao-ko essential oil and determination of its chemical composition, antioxidant and antimicrobial activities

A simple, green and efficient extraction method named modified-solvent free microwave extraction (M-SFME) was employed for the extraction of essential oils (EOs) from Amomun tsao-ko. The process of M-SFME was optimized with the prominent preponderance of such higher extraction yield (1.13%) than those of solvent free microwave extraction (SFME, 0.91%) and hydrodistillation (HD, 0.84%) under the optimal parameters. Thirty-four volatile substances representing 95.4% were identified. The IC50 values of EOs determined by DPPH radical scavenging activity and β-carotene/linoleic acid bleaching assay were 5.27 and 0.63mg/ml. Furthermore, the EOs exhibited moderate to potent broad-spectrum antimicrobial activity against all tested strains including five gram-positive and two gram-negative bacteria (MIC: 2.94-5.86mg/ml). In general, M-SFME is a potential and desirable alternative for the extraction of EOs from aromatic herbs, and the EOs obtained from A. tsao-ko can be explored as a potent natural antimicrobial and antioxidant preservative ingredient in food industry from the technological and economical points of view.

Degradation of lignin in birch sawdust treated by a novel Myrothecium verrucaria coupled with ultrasound assistance

Combined treatment of a novel fungal endophyte Myrothecium verrucaria coupled with ultrasound assistance was conducted to enhance lignin degradation in birch sawdust. The optimum treatment conditions were confirmed as the materials to liquid ratio 1:20, temperature 30°C, time 4days and pH 7, respectively. The results showed that the combined treatment led to the lignin degradation reaching 67.95±2.14%, while the lignin degradation were 45.50±2.12% and 13.75±0.66% with separate fungal treatment and ultrasound treatment, respectively. Moreover, SEM and FTIR analysis indicated that combined treatment significantly altered surface morphology and chemical structure of birch sawdust. The combined treatment greatly increased lignin removal during short time in mild environment. Therefore, these results demonstrated that the combined treatment of fungal endophyte coupled with ultrasound assistance has the high potential for the removal lignin in lignocellulose.

Application of magnetically immobilized edible fungus for the biotransformation of panax notoginseng saponin Rb1 to Rd and Rg3.

In this study, we developed a new magnetically immobilized edible fungus for the biotransformation of panax notoginseng saponins Rb1 to Rd and Rg3. The optimum biotransformation conditions were as follows: temperature 32°C, pH 6.5, time 48h and liquid-solid ratio 25:1(mL/g). The yields of Rd and Rg3 reached 41.35±0.12mg/g and 6.35±0.08mg/g which increased 6.97-fold and 3.23-fold to that of untreated control, respectively. Additionally, SEM demonstrated that vestured pits and cell walls of samples were destroyed obviously which was beneficial to the Rb1 biotransformation and target Rd and Rg3 release. Meanwhile, the reusability of magnetically immobilized microorganism was tested and the activity of the magnetically immobilized microorganism remained 83.8% after 15 runs. The recycling experiments demonstrated that magnetically immobilized fungus exhibited higher efficiency than the non-magnetical one. These results proved that this new magnetically immobilized microorganism could be applied for industrial production and pharmaceutical industry with good efficiency.

2′-O-Galloylhyperin Isolated From Pyrola incarnata Fisch. Attenuates LPS-Induced Inflammatory Response by Activation of SIRT1/Nrf2 and Inhibition of the NF-κB Pathways in Vitro and Vivo

2′-O-galloylhyperin, a major compound of Pyrola incarnata Fisch., possesses a variety of biological and pharmacological activities, including anti-oxidative and anti-inflammatory activities. Nevertheless, the underlying molecular mechanisms of 2′-O-GH in microbial infection and sepsis are not clear. In this study, we investigated the anti-inflammatory effects of 2′-O-GH. We found that 2′-O-GH significantly reduced the production of TNF-α, IL-6, and nitric oxide (NO), suppressed the expression levels of iNOS, blocked the translocation of NF-κB from the cytosol to nucleus, and decreased the MAPK activation in LPS-activated RAW 264.7 cells. 2′-O-GH also enhanced the nuclear translocation of Nrf2 and up-regulated the expression of heme oxygenase-1 (HO-1) and SIRT1. In addition, the administration of 2′-O-GH attenuated the TNF-α and IL-6 production in the serum, infiltration of inflammatory cells, liver tissue damage, and the mortality rate of LPS-challenged mice. Moreover, 2′-O-GH significantly upregulated Nrf2 and SIRT1 expression and inhibited the inflammatory responses in the liver of septic mice. The collective data indicate that 2′-O-GH could potentially be a novel functional food candidate in the treatment of sepsis.

Enhanced Production of Two Bioactive Isoflavone Aglycones in Astragalus membranaceus Hairy Root Cultures by Combining Deglycosylation and Elicitation of Immobilized Edible Aspergillus niger

A cocultivation system of Astragalus membranaceus hairy root cultures (AMHRCs) and immobilized food-grade fungi was established for the enhanced production of calycosin (CA) and formononetin (FO). The highest accumulations of CA (730.88 ± 63.72 μg/g DW) and FO (1119.42 ± 95.85 μg/g DW) were achieved in 34 day-old AMHRCs cocultured with immobilized A. niger (IAN) for 54 h, which were 7.72- and 18.78-fold higher than CA and FO in nontreated control, respectively. IAN deglycosylation could promote the formation of CA and FO by conversion of their glycoside precursors. IAN elicitation could intensify the generation of endogenous signal molecules involved in plant defense response, which contributed to the significantly up-regulated expression of genes in CA and FO biosynthetic pathway. Overall, the coupled culture of IAN and AMHRCs offered a promising and effective in vitro approach to enhance the production of two health-promoting isoflavone aglycones for possible nutraceutical and pharmaceutical uses.