Isolation, purification, and structural characterization of polysaccharides from Codonopsis pilosula and its therapeutic effects on non-alcoholic fatty liver disease in vitro and in vivo.

Codonopsis pilosula is a famous edible and medicinal plants, in which polysaccharides are recognized as one of the important active ingredients. A neutral polysaccharide (CPP-1) was purified from C. pilosula. The structure was characterized by HPSEC-MALLS-RID, UV, FT-IR, GC-MS, methylation analysis, and NMR. The results showed that CPP-1 was a homogeneous pure polysaccharide, mainly containing fructose and glucose, and a small amount of arabinose. Methylation analysis showed that CPP-1 composed of →1)-Fruf-(2→, Fruf-(1→ and Glcp-(1→ residues. Combined the NMR results the structure of CPP-1 was confirmed as α-D-Glcp-(1 â†’ [2)-ß-D-Fruf-(1 â†’ 2)-ß-D-Fruf-(1]26 â†’ 2)-ß-D-Fruf with the molecular weight of 4.890 × 103 Da. The model of AML12 hepatocyte fat damage was established in vitro. The results showed that CPP-1 could increase the activity of SOD and CAT antioxidant enzymes and reduce the content of MDA, thus protecting cells from oxidative damage. Subsequently, the liver protective effect of CPP-1 was studied in the mouse model of nonalcoholic fatty liver disease (NAFLD) induced by the high-fat diet. The results showed that CPP-1 significantly reduced the body weight, liver index, and body fat index of NAFLD mice, and significantly improved liver function. Therefore, CPP-1 should be a potential candidate for the treatment of NAFLD.

Comparison of two commercial methods with a UHPLC-MS/MS method for the determination of multiple mycotoxins in cereals.

Immunoassay-based techniques are important on-site screening tools for the detection of mycotoxins in cereals. This study aims to evaluate the trueness, precision, repeatability and cross-reactivity of commercially available test strips, ELISA kits and UHPLC-MS/MS on analyzing zearalenone, ochratoxin A, deoxynivalenol, T-2 toxin and fumonisin B1. The results showed that false negative rate (25.7 %-37.4 %) of all tested mycotoxins by test strips was higher than the false positive rate (0 %-31.0 %). The repeatability of ELISA kits at the declared LOD dispersed from -85.7 % to +98.4 %. ELISA kits were more accurate at 50 % of the maximum residue limit (MRL) of mycotoxins than 150 % and 200 %. All the tested deoxynivalenol/zearalenone derivatives had cross-reactivity with different level, and sample matrix could reinforce this overestimation of target mycotoxin. This study emphasized that higher-quality antibody screening and more analytical performance investigations are need to address for on-site detection of mycotoxins in the future.

Preparation, characterization and immunoregulatory activity of derivatives of polysaccharide from Atractylodes lancea (Thunb.) DC.

A polysaccharide (ALP-1) extracted from Atractylodes lancea (Thunb.) DC. was carboxymethylated (C-ALP-1), phosphorylated (P-ALP-1) and acetylated (A-ALP-1) to improve its physicochemical properties and bioactivities. The solubility of all derivatives was increased, and the solubility of A-ALP-1 increased to 137.5 mg/mL, which was much higher than the solubility of ALP-1 (15.0 mg/mL). The results of HPSEC-MALLS-RID showed that the molecular weight of polysaccharides was slightly increased after the modification, and the root mean square radius of rotation (Rz) and morphology of polysaccharides in solution were also changed. The scanning electron microscopy (SEM) and X-ray diffraction (XRD) results confirmed that the surface morphology of ALP-1 changed dramatically and the crystallinity decreased after structural modification. From thermal analysis results, the T50 of ALP-1, C-ALP-1, P-ALP-1 and A-ALP-1 were 281.34, 292.14, 333.75 and 298.70 °C, which showed that derivatives had stronger thermal stability than ALP-1. The immunomodulatory activity studies displayed that P-ALP-1 showed the best ability to stimulate RAW264.7 macrophages to release NO, and A-ALP-1 showed the best capacity to stimulate TNF-α and IL-6 releasing. These results indicated that chemical modification could enhance the solubility, the thermal stability and immunomodulatory activity of polysaccharides, which is beneficial for the development and utilization of natural polysaccharides.

Chemistry, pharmacology and analysis of Pseudostellaria heterophylla: a mini-review.

Pseudostellaria heterophylla is one of the well-known traditional Chinese medicines and has been used in clinics for 100 years in China. The chemistry and pharmacology of P. heterophylla were reviewed to understand its active compounds. Then analysis of these compounds related to quality control of this herb was discussed. For the analysis of chemicals, three aspects have been discussed in this review. The first two aspects focused on the methodologies for analysis of cyclic peptides and carbohydrates in P. heterophylla, respectively. The last one dealt with the other methods used for identification of P. heterophylla. Some rich chemicals such as oligosaccharides in this plant were rarely evaluated. Many analyses were performed on this plant, however, few of them were accepted as quality control method.

Comparison of Antioxidant Activity and Main Active Compounds Among Different Parts of Alpinia officinarum Hance Using High-Performance Thin Layer Chromatography-Bioautography.

Background: Alpinia officinarum Hance (ginger family) is an important Chinese medicine, especially in Southern China. Objective: A simple and effective high-performance thin-layer chromatography coupled with 2, 2-diphenyl-1-picrylhydrazyl bioautography (HPTLC-DPPH) and electrospray ionization quadrupole time-of-flight tandem mass spectrometry (ESI-Q-TOF-MS/MS) method was developed for the bioactivity-based quality control of A. officinarum. Methods: The HPTLC-DPPH and ESI-Q-TOF-MS/MS were applied for the analysis of different parts of A. officinarum after using methanol extraction for 23 batches of taproot, four batches of aerial, and three batches of fibril parts. Results: The systematic evaluation showed that similar components in taproot and aerial parts make the major antioxidant activity. However, based on our evaluation, the antioxidant ability of the aerial parts is lower than the taproot parts. There is also a significant difference (P < 0.05) between taproot and fibril parts of the root. The chemical structures of compounds with the antioxidant capacity were tentatively identified as 5R-hydroxy-7-(4-hydroxy-3-methoxyphenyl)-1-phenyl-3-heptanone (band 1), kaempferide (band 2), and galangin (band 3) based on ESI-Q-TOF-MS/MS analytical results and further confirmed by standards. Conclusions: This identification indicated that two flavonoid compounds and one diarylheptanoid compound possessed high potentials to be used as the antioxidant biomarkers for the quality control of A. officinarum. Highlights: The comparison of different parts could be considered as guidelines for the usage of A. officinarum.

Qualitation and quantification of water soluble non-starch polysaccharides from Pseudostellaria heterophylla in China using saccharide mapping and multiple chromatographic methods.

Qualitative and quantitative analysis of non-starch polysaccharide (NSP) from Pseudostellaria heterophylla in China (Guizhou, Anhui and Fujian) were performed using high performance size exclusion chromatography coupled with multi-angle laser light scattering and refractive index detector (HPSEC-MALLS-RID), saccharide mapping based on polysaccharide analysis by using carbohydrate gel electrophoresis (PACE) and high performance thin layer chromatography (HPTLC). Results showed that PACE and HPTLC fingerprints of enzymatic hydrolysates of NSP were similar. Moreover, the results indicated that 1,4-ß-d-Galp, 1,5-α-Araf, 1,4-α-d-GalAp and 1,4-ß-d-Glcp linkages existed in NSP. Based on the results of HPSEC-MALLS/RI, the Mw, Rg, and contents of NSP in P. heterophylla were ranging from 4.37 × 104 to 1.70 × 105 Da, 46.4 to 65.9 nm, and 0.43% to 1.41%, respectively. There were no significant difference (p>0.05) observed among the three main producing areas, which suggested that polysaccharides could be used as marker for quality control of P. heterophylla.

Preparation and Application of Standardized Typical Volatile Components Fraction from Turmeric (Curcuma longa L.) by Supercritical Fluid Extraction and Step Molecular Distillation.

A green and reliable method using supercritical fluid extraction (SFE) and molecular distillation (MD) was optimized for the separation and purification of standardized typical volatile components fraction (STVCF) from turmeric to solve the shortage of reference compounds in quality control (QC) of volatile components. A high quality essential oil with 76.0% typical components of turmeric was extracted by SFE. A sequential distillation strategy was performed by MD. The total recovery and purity of prepared STVCF were 97.3% and 90.3%, respectively. Additionally, a strategy, i.e., STVCF-based qualification and quantitative evaluation of major bioactive analytes by multiple calibrated components, was proposed to easily and effectively control the quality of turmeric. Compared with the individual calibration curve method, the STVCF-based quantification method was demonstrated to be credible and was effectively adapted for solving the shortage of reference volatile compounds and improving the QC of typical volatile components in turmeric, especially its functional products.

Potential molecular mechanisms for fruiting body formation of Cordyceps illustrated in the case of Cordyceps sinensis.

The fruiting body formation mechanisms of Cordyceps sinensis are still unclear. To explore the mechanisms, proteins potentially related to the fruiting body formation, proteins from fruiting bodies, and mycelia of Cordyceps species were assessed by using two-dimensional fluorescence difference gel electrophoresis, and the differential expression proteins were identified by matrix-assisted laser desorption/ionisation tandem time of flight mass spectrometry. The results showed that 198 differential expression proteins (252 protein spots) were identified during the fruiting body formation of Cordyceps species, and 24 of them involved in fruiting body development in both C. sinensis and other microorganisms. Especially, enolase and malate dehydrogenase were first found to play an important role in fruiting body development in macro-fungus. The results implied that cAMP signal pathway involved in fruiting body development of C. sinensis, meanwhile glycometabolism, protein metabolism, energy metabolism, and cell reconstruction were more active during fruiting body development. It has become evident that fruiting body formation of C. sinensis is a highly complex differentiation process and requires precise integration of a number of fundamental biological processes. Although the fruiting body formation mechanisms for all these activities remain to be further elucidated, the possible mechanism provides insights into the culture of C. sinensis.

Microwave-Assisted Extraction, Chemical Structures, and Chain Conformation of Polysaccharides from a Novel Cordyceps Sinensis Fungus UM01.

Cordyceps sinensis is a well-known tonic food with broad medicinal properties. The aim of the present study was to investigate the optimization of microwave-assisted extraction (MAE) and characterize chemical structures and chain conformation of polysaccharides from a novel C. sinensis fungus UM01. Ion-exchange and gel filtration chromatography were used to purify the polysaccharides. The chemical structure of purified polysaccharide was determined through gas chromatography-mass spectrometry. Moreover, high performance size exclusion chromatography combined with refractive index detector and multiangle laser light scattering were conducted to analyze the molecular weight (Mw ) and chain conformation of purified polysaccharide. Based on the orthogonal design L9 , optimal MAE conditions could be obtained through 1300 W of microwave power, with a 5-min irradiation time at a solid to water ratio of 1:60, generating the highest extraction yield of 6.20%. Subsequently, the polysaccharide UM01-S1 was purified. The UM01-S1 is a glucan-type polysaccharide with a (1→4)-ß-d-glucosyl backbone and branching points located at O-3 of Glcp with a terminal-d-Glcp. The Mw , radius of gyration (Rg ) and hydrodynamic radius (Rh ) of UM01-S1 were determined as 5.442 × 10(6)  Da, 21.8 and 20.2 nm, respectively. Using the polymer solution theory, the exponent (ν) value of the power law function was calculated as 0.38, and the shape factor (ρ = Rg /Rh ) was 1.079, indicating that UM01-S1 has a sphere-like conformation with a branched structure in an aqueous solution. These results provide fundamental information for the future application of polysaccharides from cultured C. sinensis in health and functional food area.

Chemical characteristics of different parts of Coreopsis tinctoria in China using microwave-assisted extraction and high-performance liquid chromatography followed by chemometric analysis.

Coreopsis tinctoria, also called "snow chrysanthemum" in China, is a flower tea material that has been reported to possess excellent pharmacological properties such as antioxidant and antidiabetic activities. The chemical characteristics of different parts (flowers, buds, seeds, stems, and leaves) of C. tinctoria were investigated based on microwave-assisted extraction and the simultaneous determination of 13 major active compounds by high-performance liquid chromatography, including taxifolin-7-O-glucoside, chlorogenic acid, (R/S)-flavanomarein, isocoreopsin, quercetagetin-7-O-glucoside, isookanin, 5,7,3',5'-tetrahydroxyflavanone-7-O-glucoside, marein, 3,5-dicaffeoylquinic acid, coreopsin, okanin, 5,7,3',5'-tetrahydroxyflavanone, and N(1) ,N(5) ,N(10) ,N(14) -tetra-p-coumaroylspermine. Chemometric analysis based on the contents of investigated compounds from 13 samples showed that C. tinctoria and the related flower tea materials, Chrysanthemum morifolium cv "Hangju" and "Gongju," were in different clusters, and different parts (flowers, buds, seeds, stems, and leaves) of C. tinctoria were obviously different. This study is helpful for the quality control and pharmacological evaluation of different parts from C. tinctoria and its related products.

Comparison and Characterization of Compounds with Antioxidant Activity in Lycium barbarum Using High-Performance Thin Layer Chromatography Coupled with DPPH Bioautography and Tandem Mass Spectrometry.

Methanol extracts from 50 batches of Lycium barbarum (L. barbarum, wolfberry) in China were compared and characterized using high-performance thin-layer chromatography coupled with 2,2-diphenyl-1-picrylhydrazyl (DPPH) bioautography (HPTLC-DPPH) and electrospray ionization quadrupole time-of-flight tandem mass spectrometry (ESI-Q-TOF-MS/MS), respectively. Results showed that similar components occupying the major antioxidant activity existed in L. barbarum collected from different origins. However, the average antioxidant capacities of methanol extracts of L. barbarum collected in Ningxia were significantly higher than those of Qinghai, Xinjiang, Inner Mongolia, and Gansu, which may contribute to rational use of L. barbarum in China. Furthermore, the chemical structure of compound with the highest antioxidant capacity was tentatively identified as 2-O-ß-d-glucopyranosyl-l-ascorbic acid using ESI-Q-TOF-MS/MS analysis, which possessed high potentials to be used as an antioxidant biomarker for the quality control of L. barbarum. Results are helpful for the bioactivity-based quality control of L. barbarum, and beneficial for the improvement of their performance in functional/health foods area, suggesting that HPTLC-DPPH bioautography with ESI-Q-TOF-MS/MS could be used as a routine approach for quality control of antioxidant components in L. barbarum.

A comparative study on immunomodulatory activity of polysaccharides from two official species of Ganoderma (Lingzhi).

Two Ganoderma species, G. lucidum and G. sinense, are listed as Lingzhi in Chinese Pharmacopoeia and they are considered to have the same therapeutic effects. Polysaccharides were the main immunomodulatory and anticancer components in Ganoderma. In this study, the chemical characters and the effects of polysaccharides from G. lucidum (GLPS) and G. sinense (GSPS) on macrophage functions were investigated and compared. Chemical studies showed that GLPS and GSPS were different, displaying various molecular weight distribution and ratio of monosaccharide components. In vitro pharmacological studies showed that both GLPS and GSPS had potent effects on macrophage functions, such as promoting macrophage phagocytosis, increasing their release of nitric oxide and cytokines interleukin (IL)-1α, IL-6, IL-10, and tumor necrosis factor-α. Generally, GLPS was more powerful than GSPS. This study is helpful to elucidate the active components and pharmacological variation between the 2 Ganoderma species. The structure-activity relationship of polysaccharides from Ganoderma needs further study.

Chain conformation and immunomodulatory activity of a hyperbranched polysaccharide from Cordyceps sinensis.

A polysaccharide, named as cordysinan, extracted from natural Cordyceps sinensis, was identified as a hyperbranched heteropolysaccharide from the results of FT-IR, GC-MS, and carbohydrate analysis by carbohydrate gel electrophoresis analysis, as well as the degree of branching of cordysinan was 43.3%. The solution properties of cordysinan were investigated by using size exclusion chromatography coupled with multi-angle laser light scattering and triple detector array, respectively. The molecular weights, the radius of gyration and the intrinsic viscosity of cordysinan were determined as 22.45±0.26 kDa and 22.37 kDa, 15.4±2.4 nm and 1.41 mL/g, respectively. By applying the polymer solution theory, the exponent (ν and α) values of g1/2=kMwv and [η]=kMwα were calculated as 0.28 and 0.42, respectively, which firstly revealed that cordysinan existed as a globular shape in 0.9% NaCl aqueous solution. Moreover, the results showed that cordysinan could obviously stimulate macrophages functions.

Advance of CE and CEC in phytochemical analysis (2012­2013).

This article presents an overview of the advance of CE and CEC in phytochemical analysis, based on the literature not mentioned in our previous review papers [Chen, X. J., Zhao, J., Wang, Y. T., Huang, L. Q., Li, S. P., Electrophoresis 2012, 33, 168­179], mainly covering the years 2012­2013. In this article, attention is paid to online preconcentration, rapid separation, and sensitive detection. Selected examples illustrate the applicability of CE and CEC in biomedical, pharmaceutical, environmental, and food analysis. Finally, some general conclusions and future perspectives are given.

Characterization of polysaccharides from Ganoderma spp. using saccharide mapping.

Polysaccharides from Ganoderma spp. and their adulterants were firstly investigated and compared using saccharide mapping, enzymatic (endo-1,3-ß-D-glucanase and pectinase) digestion followed by polysaccharide analysis using carbohydrate gel electrophoresis analysis. The results showed that both 1,3-ß-D-glucosidic and 1,4-α-D-galactosiduronic linkages were existed in Lingzhi (Ganoderma lucidum and Ganoderma sinense), and the similarity of polysaccharides from G. lucidum and G. sinense was high, which may contribute to rational use of Lingzhi. Different species of Ganoderma and their adulterants can be differentiated based on the saccharide mapping, which is helpful to well understand the structural characters of polysaccharides from different species of Ganoderma and to improve the quality control of polysaccharides in Lingzhi.

Mycelia extracts of fungal strains isolated from Cordyceps sinensis differently enhance the function of RAW 264.7 macrophages.

ETHNOPHARMACOLOGICAL RELEVANCE: Cordyceps sinensis, an entomogenous fungus used in traditional Chinese medicine with multiple pharmacological activities. However, its usage has been limited due to the high price and short supply. Isolate of fungi strains from natural Cordyceps sinensis to achieve a large-scale production by fermentation is an alternative choice. The aim of this study was to investigate and compare the effects of mycelia extracts of different fungal stains isolated from natural Cordyceps sinensis on macrophage functions in vitro. MATERIALS AND METHODS: Macrophages' proliferation, phagocytosis, nitric oxide (NO) production, cytokines secretion, iNOS, NF-κB p65 activation and translocation were investigated by the MTT assay, flow cytometry assay, Griess reagent method, ELISA, western blot and immunostaining assay, respectively. RESULTS: The results showed that the effects of cultured Cordyceps mycelia of different fungal strains isolated from natural Cordyceps sinensis on macrophages greatly variant. Among 17 Cordyceps aqueous extracts, only five extracts (UM01, QH11, BNQM, GNCC and DCXC) could significantly increase cell proliferation and NO production of RAW 264.7 mouse macrophages. Moreover, the five extracts, especially UM01 and QH11, significantly enhanced phagocytosis and promoted cytokines release of macrophages. Polysaccharides in cultured UM01 mycelia were found to be the main immune stimulating compounds. CONCLUSIONS: The variation of biological effects of fermented mycelia of different fungal strains from natural Cordyceps sinensis may be derived from their chemical diversity, especially polysaccharides, which need further study in future.

High-performance anion-exchange chromatography coupled with diode array detection for the determination of dencichine in Panax notoginseng and related species.

A high-performance anion-exchange chromatography coupled with diode array detection method was developed for the determination of dencichine in Panax notoginseng and related species. The analysis was performed on an Eprogen Synchropak WAX column (4.6 × 250 mm, 6 µm) with 50 mM NaH2 PO4 aqueous solution isocratic elution. The method was validated in terms of linearity, sensitivity, precision, stability, and accuracy. It was found that the calibration curve for dencichine showed good linearity (R(2) = 0.9999) within the test range. The LOD and LOQ were 0.77 and 3.06 ng, respectively. The RSD for intra- and interday repeatability was 0.2 and 0.5%, respectively. The test solution of dencichine is stable at least for three days at room temperature and for seven days at 4 °C. The mean recovery of dencichine was 102.0%. The established method was successfully applied to determine dencichine in the raw root of P. nogoginseng, P. ginseng, and P. quinquefolium as well as the steamed root of P. notoginseng. Compared with previous reports, this method is sensitive, selective, and accurate, which is helpful to evaluate the quality of P. notoginseng and related species.

Effects of polysaccharides from different species of Dendrobium (Shihu) on macrophage function.

Dendrobium spp. are precious medicinal plants, used in China for thousands of years as health foods and nutrients. Polysaccharides are the main effective ingredients in Dendrobium plants. In this study, the chemical characteristics and the effects of crude polysaccharides (CPs) from five species of Dendrobium on macrophage function were investigated and compared in vitro for the first time. Chemical characteristic studies showed that CPs from different species of Dendrobium were diverse, displaying widely varied Mw distributions and molar ratios of monosaccharides. Their effects on macrophage functions, such as promoting phagocytosis, release of NO and cytokines IL-1α, IL-6, IL-10 and TNF-α, were also different. Moreover, CPs from D. officinale, especially collected from Yunnan Province, exerted the strongest immunomodulatory activities and could be explored as a novel potential functional food. The diverse chemical characteristics of CPs from different species of Dendrobium might contribute to their varied effects on macrophage functions, which should be further investigated.

Simultaneous determination of flavonoids, isochlorogenic acids and triterpenoids in Ilex hainanensis Using high performance liquid chromatography coupled with diode array and evaporative light scattering detection.

A high performance liquid chromatography coupled with diode array and evaporative light scattering detection (HPLC-DAD-ELSD) method for simultaneous determination of eight major bioactive compounds including two flavonoids (rutin and eriodictyol-7-O-ß-D-glucopyranoside), two isochlorogenic acids (isochlorogenic acid A and isochlorogenic acid C) and four triterpenoids (ilexhainanoside D, ilexsaponin A1, ilexgenin A and ursolic acid) in Ilex hainanensis has been developed for the first time. The 283 nm wavelength was chosen for determination of two flavonoids and two isochlorogenic acids. ELSD was applied to determine four triterpenoids. The analysis was performed on an Agilent Zorbax SB-C18 column (250 × 4.6 mm i.d., 5 µm) with gradient elution of 0.2% formic acid in water and acetonitrile. The method was validated for linearity, limit of detection, limit of quantification, precision, repeatability and accuracy. The proposed method has been successfully applied for simultaneous quantification of the analytes in four samples of Ilex hainanensis, which is helpful for quality control of this plant.

Chromatography in characterization of polysaccharides from medicinal plants and fungi.

Polysaccharides isolated from medicinal plants and fungi exhibit multiple pharmacological activities. The biological activities of polysaccharides depend on their chemical characteristics. However, characterization of polysaccahrides is a challenge because of their complicated structure and macromolecular mass. In this review, chromatography in characterization of polysaccharides, including physicochemical characterization (purity, molecular mass, and distribution), structural characterization (constituent monosaccharide composition and the ratio, the features of glycosidic linkages), and fingerprint of polysaccharides (acidic and enzymatic hydrolysates), from medicinal plants and fungi were reviewed and discussed according to the publications collected in Web of Science since 2007. The perspective for characterization of polysaccharides has also been described.