A Water-Soluble Polysaccharide from the Fruit Bodies of Bulgaria inquinans (Fries) and Its Anti-Malarial Activity

A water-soluble polysaccharide (BIWS-4b) was purified from the fruit bodies of Bulgaria inquinans (Fries). It is composed of mannose (27.2%), glucose (15.5%) and galactose (57.3%). Its molecular weight was estimated to be 7.4 kDa (polydispersity index, Mw/Mn: 1.35). Structural analyses indicated that BIWS-4b mainly contains (1 → 6)-linked, (1 → 5)-linked and (1 → 5,6)-linked β-Galf units; (1 → 4)-linked and non-reducing terminal β-Glcp units; and (1 → 2)-linked, (1 → 6)-linked, (1 → 2,6)-linked and non-reducing terminal α-Manp units. When examined by the 4-day method and in a prophylactic assay in mice, BIWS-4b exhibited markedly suppressive activity against malaria while enhancing the activity of artesunate. Immunological tests indicated that BIWS-4b significantly enhanced macrophage phagocytosis and splenic lymphocyte proliferation in malaria-bearing mice and normal mice. The anti-malarial activity of BIWS-4b might be intermediated by enhancing immune competence and restoring artesunate-suppressed immune function. Thus, BIWS-4b is a potential adjuvant of anti-malaria drugs

Simultaneous Determination of 25 Ginsenosides by UPLC-HRMS via Quantitative Analysis of Multicomponents by Single Marker

A method using UPLC-HRMS has been developed for a rapid, simultaneous qualitative and quantitative analysis of twenty-five ginsenosides. Chromatographic separation was achieved on a C18 analytical column with an elution gradient comprising 0.1% aqueous formate/acetonitrile as the mobile phase. HRMS detection acquired full mass data for quantification and fullms-ddms2 (i.e., data-dependent scan mode) yielded product ion spectra for identification. Furthermore, quantitative analysis of multiginsenosides by single marker (QAMS) was developed and validated using a relative correction factor. Under optimal conditions, we could simultaneously separate eight groups of isomers of the 25 ginsenosides. Good linearity was observed over the validated concentration range for each analyte (r2 > 0.9924), showing excellent sensitivity (LODs, 0.003–0.349 ng/mL) and lower limit quantification (LOQs, 0.015–1.163 ng/mL). The LC-MS external standard method (ESM) and QAMS were compared and successfully applied to analyze the ginsenoside content from Panax ginseng roots. Overall, our UPLC-HRMS/QAMS approach provides high precision, stability, and reproducibility and can be used for high-throughput analysis of complex ginsenosides and quantitative analysis of multiple components and quality control of traditional Chinese medicines (TCM)

Pectic Bee Pollen Polysaccharide from Rosa rugosa Alleviates Diet-Induced Hepatic Steatosis and Insulin Resistance via Induction of AMPK/mTOR-Mediated Autophagy

Despite it is used as a nutraceutical against diabetes and obesity, the mechanism of action of bee pollen is still unclear. Pectic bee pollen polysaccharide (RBPP-P) was isolated from Rosa rugosa, and its structure was characterized by 13C-NMR and Fourier transform-infrared spectroscopy (FT-IR). Using high glucose and fatty acids-treated HepG2 cells and high fat diet (HFD)-induced obesity mice, we detected its effect on insulin function and lipid metabolism based on autophagy. RBPP-P contained arabinogalactan, rhamnogalacturonan I, and homogalacturonan domains. In vivo studies demonstrated that RBPP-P markedly ameliorated insulin resistance, glucose intolerance, and liver steatosis in obese mice. The suppressive effects of RBPP-P on liver steatosis and triglyceride content were mediated by increased autophagy and lipase expression in liver. In AMPK knockdown cells (prkaa 1/2−/− MEF) and HFD-fed mice tissues (liver, gonadal white adipose, and inguinal white adipose), RBPP-P enhanced autophagy in AMPK/mTOR-dependent way in liver, but not in adipose tissue. These findings demonstrated that bee pollen polysaccharide alleviated liver steatosis and insulin resistance by promoting autophagy via an AMPK/mTOR-mediated signaling pathway, suggesting that RBPP-P could be a novel therapeutic agent used for the treatment of obesity and diabetes

The Film-Forming Characterization and Structural Analysis of Pectin from Sunflower Heads

A natural low-methoxyl pectin (termed AHP, DM=25.9%) was extracted from dried heads of sunflower and showed better film-forming performance blended with hydroxypropyl methylcellulose (HPMC). The solutions and films of different HPMC/AHP blends were characterized by viscosity, transparency, mechanical properties, loss on drying, water drop penetration time (WDPT), disintegration and SEM. In order to analyze the structure-property relationship of film forming, AHP was separated by ion-exchange chromatography and characterized. The results showed that the blends were immiscible, but the formation of AHP gel would give the blended film better mechanical properties. AHP was fractionated into one neutral fraction and two acidic fractions (AHPA-1 and AHPA-2). The analytical results showed that AHPA-1 and AHPA-2 were identified to be homogalacturonan- (HG-) rich pectins with low DM, and the molecular weights of them were estimated to be 106 kDa and 226 kDa, respectively. Due to the high content of the HG domain, low DM and high molecular weights, AHP had excellent gelling properties induced by Ca2+ and was added to improve the film-forming properties of HPMC and to develop plant hollow capsules

Screening of a Novel Polysaccharide Lyase Family 10 Pectate Lyase from <i>Paenibacillus polymyxa</i> KF-1: Cloning, Expression and Characterization

Pectate lyase (EC 4.2.2.2) catalyzes the cleavage of &#945;-1,4-glycosidic bonds of pectin polymers, and it has potential uses in the textile industry. In this study, a novel pectate lyase belonging to polysaccharide lyase family 10 was screened from the secreted enzyme extract of Paenibacillus polymyxa KF-1 and identified by liquid chromatography-MS/MS. The gene was cloned from P. polymyxa KF-1 genomic DNA and expressed in Escherichia coli. The recombinant enzyme PpPel10a had a predicted Mr of 45.2 kDa and pI of 9.41. Using polygalacturonic acid (PGA) as substrate, the optimal conditions for PpPel10a reaction were determined to be 50 &#176;C and pH 9.0, respectively. The Km, vmax and kcat values of PpPel10a with PGA as substrate were 0.12 g/L, 289 &#956;mol/min/mg, and 202.3 s&#8722;1, respectively. Recombinant PpPel10a degraded citrus pectin, producing unsaturated mono- and oligogalacturonic acids. PpPel10a reduced the viscosity of PGA, and weight loss of ramie (Boehmeria nivea) fibers was observed after treatment with the enzyme alone (22.5%) or the enzyme in combination with alkali (26.3%). This enzyme has potential for use in plant fiber processing

Structural Characterization of a Rhamnogalacturonan I Domain from Ginseng and Its Inhibitory Effect on Galectin-3

A rhamnogalacturonan I domain, named RG-I-3A, was prepared from ginseng pectin by pectinase digestion and chromatography separation. Monosaccharide composition analysis revealed that it was mainly composed of galacturonic acid, rhamnose, galactose, and arabinose in a molar ratio of 32.5:11.2:31.9:16.5, with a molecular weight of 50 kDa. Partial acid hydrolysis, monoclonal antibody detection, and NMR spectra analysis suggested RG-I-3A was composed of →4)-α-GalpA-(1→2)-α-Rhap-(1→disaccharide repeating units as backbone, with β-1,4-galactan, α-1,5-arabinan, AG-I, and AG-II side chains substituted via the O-4 of Rhap. Galectin-3-mediated hemagglutination and biolayer interferometry assay indicated that RG-I-3A had inhibitory activity on galectin-3. These findings suggest the potential use of this ginseng RG-I domain as a galectin-3 inhibitor in drug development applications

Cloning, expression and biochemical characterization of a GH1 β-glucosidase from Cellulosimicrobium cellulans

β-Glucosidase plays an important role in the degradation of cellulose. In this study, a novel β-glucosidase ccbgl1b gene for a glycosyl hydrolase (GH) family 1 enzyme was cloned from the genome of Cellulosimicrobium cellulans and expressed in Escherichia coli BL21 cells. The sequence contained an open reading frame of 1494 bp, encoded a polypeptide of 497 amino acid residues. The recombinant protein CcBgl1B was purified by Ni sepharose fastflow affinity chromatography and had a molecular weight of 57 kDa, as judged by SDS-PAGE. The optimum β-glucosidase activity was observed at 55 °C and pH 6.0. Recombinant CcBgl1B was found to be most active against aryl-glycosides p-nitrophenyl-β-D-glucopyranoside (pNPβGlc), followed by p-nitrophenyl-β-D-galactopyranoside (pNPβGal). Using disaccharides as substrates, the enzyme efficiently cleaved β-linked glucosyl-disaccharides, including sophorose (β-1,2-), laminaribiose (β-1,3-) and cellobiose (β-1,4-). In addition, a range of cello-oligosaccharides including cellotriose, cellotetraose and cellopentaose were hydrolysed by CcBgl1B to produce glucose. The interaction mode between the enzyme and the substrates driving the reaction was modelled using a molecular docking approach. Understanding how the GH1 enzyme CcBgl1B from C. cellulans works, particularly its activity against cello-oligosaccharides, would be potentially useful for biotechnological applications of cellulose degradation

Biotransformation of ginsenoside Rb1 for ginsenoside Rd preparation by <i>Lysinibacillus massiliensis</i> No. 24

400-406Bacterium Lysinibacillus massiliensis No. 24 was tested for its ability to transform the major ginsenosides Rb1. The transformation product was identified by thin layer chromatography (TLC) and high performance liquid chromatography (HPLC), and its structure was assigned by 13C-NMR. The bacterium was found to specifically transform major ginsenoside Rb1 and ginsenoside Rd was the sole product. The optimal conditions for transforming Rb1 by L. massiliensis No. 24 were determined to be: substrate concentration, 3 mg/mL; temperature, 34&deg;C; pH 10; the time of substrate addition, 48 h; cell concentration, 0.30&times;109 cells/mL; and biotransformation period, 3 d. Under the optimum conditions, the maximum yield of ginsenoside Rd reached 97%. Further, a preparative scale transformation with L. massiliensis No. 24 was performed with 150 mg Rb1 to give a yield of 79%. This bacterium would be potentially applied in the preparation of Rd

Efficient Biotransformation of Polysialogangliosides for Preparation of GM1 by Cellulosimicrobium sp. 21

A new ganglioside transformed strain isolated from soil was identified as Cellulosimicrobium sp. 21. It produced a sialidase which transformed polysialo-gangliosides GD1 and GT1 into a monosialoterahexosylganglioside, i.e., ganglioside GM1. The sialidase had both NeuAc-α-2,3- and NeuAc-α-2,8-sialidase activity without producing asiolo-GM1. The optimum conditions were evaluated and it was found that the transformation was optimally performed at 30 °C and pH 7.0. The substrate should be added at the beginning of the reaction and the concentration of substrate was 3% (w/v). Under these optimum conditions, Cellulosimicrobium sp. 21 converted GD1 and GT1 into GM1 in inorganic medium in a 5 L bioreactor with the recovery rate of 69.3%. The product contained 50.3% GM1 and was purified on silica to give the product with 95% of GM1 with a recovery rate of 30.5%. Therefore, Cellulosimicrobium sp. 21 has potential to be applied in the production of GM1 in the pharmaceutical industry