Structural elucidation a complex galactosyl and glucosyl-rich pectin from the pericarp of immature fruits of Juglans mandshurica Maxim.

A glucosyl-rich pectin, JMMP-3 (Mw, 2.572 × 104 g/mol, O-methyl % = 3.62%), was isolated and purified from the pericarp of the immature fruit of Juglans mandshurica Maxim. (QingLongYi). The structure of JMMP-3 was studied systematically by infrared spectroscopy, monosaccharide compositions, methylation analysis, partial acid hydrolysis, and 1/2D-NMR. The backbone of JMMP-3 possessed a smooth region (→ 4GalA1 →) and a hairy region (→ 4GalA1 → 2Rha1 →) with a molar ratio of 2: 5. The substitution of four characteristic side chains (R1-R4) occurs at C-4 of → 2,4)-α-Rhap-(1→, where R1 is composed of → 5)-α-Araf-(1→, R2 is composed of → 4)-ß-Galp-(1 → and ß-Galp-(1→, R3 is composed of α-Glcp-(1→, →4)-α-Glcp-(1 → and → 4,6)-α-Glcp-(1→, and R4 is composed of → 5)-α-Araf-(1→, ß-Galp-(1→, → 4)-ß-Galp-(1→, → 3,4)-ß-Galp-(1→, → 4,6)-ß-Galp-(1 → and → 2,4)-ß-Galp-(1 → . In addition, the antitumor activity of JMMP-3 on HepG2 cells was preliminarily investigated.

Complete composition analysis of polysaccharides based on HPAEC-PAD coupled with quantitative analysis of multi-components by single marker.

INTRODUCTION: Monosaccharide compositions analysis (MCA) is indispensable for structural characterisations and structure-activity relationships of plant polysaccharides. OBJECTIVES: To develop a concise and direct MCA method, we established a quantitative analysis of the multi-monosaccharaides by single marker (QAMS) by high-performance anion-exchange chromatography with pulsed-amperometric detection (HPAEC-PAD) method. METHODOLOGY: A stable and reproducible HPAEC-PAD method for simultaneous determination of aldoses, ketoses and uronic acids (i.e., l-arabinose, d-xylose, d-ribose, l-rhamnose, d-fucose, d-mannose, d-glucose, d-galactose, d-fructose, d-glucuronic acid and d-galacturonic acid) was established by systematic optimisation of stationary phases, column temperatures and elution programmes. On this basis, the QAMS method was proposed through comprehensive investigations of relative correction factor (RCF) variations under different influencing factors, for example, sample concentrations, flow rates, and column temperatures. RESULTS: Using rhamnose as an internal reference standard, the contents of the other monosaccharide components in polysaccharides from Panax quinquefolium L. and Achyranthes bidentata Bl. samples were simultaneously determined by QAMS, and there was no significant difference between the results from the QAMS and external standard method (t test, P > 0.520). In addition, a MCA fingerprinting of 30 batches of P. quinquefolium polysaccharide was established by HPAEC-PAD, and six common peaks were assigned and determined. CONCLUSIONS: The established HPAEC-PAD-QAMS method was successfully applied to the MCA of polysaccharides from P. quinquefolium and A. bidentata after optimisation of hydrolysis conditions. HPAEC-PAD-QAMS was proposed and established for MCA of plant polysaccharides for the first time.

An alternative strategy based on ultra-high-performance supercritical fluid chromatography for full monosaccharide compositional analysis of polysaccharides in Schisandra chinensis fruits.

Due to green and environment-friendly characteristics, ultra-high-performance supercritical fluid chromatography has been widely used in analytical fields in recent years, but until now few reports are available for monosaccharide compositional analysis of macromolecule polysaccharides. In this study, an ultra-high-performance supercritical fluid chromatography technology with an unusual binary modifier is used to determine the monosaccharide compositions of natural polysaccharides. Each carbohydrate herein is simultaneously labeled as 1-pheny-3-methyl-5-pyrazolone and acetyl-derivative via pre-column derivatizations aiming to increase UV absorption sensitivity and decrease water solubility. Ten common monosaccharides are fully separated and detected on ultra-high-performance supercritical fluid chromatography combined with a photo-diode array detector by systematic optimization of multiple relevant parameters, for example, column stationary phases, organic modifiers, additives, flow rates, and so on. Compared with carbon dioxide as a mobile phase, the addition of a binary modifier increases the resolution of analytes. Additionally, this method has the advantages of small consumption of organic solvent, safety, and being environmental-friendly. It has been successfully applied for full monosaccharide compositional analysis of heteropolysaccharides from Schisandra chinensis fruits. To sum up, a new alternative approach is provided for monosaccharide compositional analysis of natural polysaccharides.

TMT-Based Proteomics Reveal the Mechanism of Action of Amygdalin against Rheumatoid Arthritis in a Rat Model through Regulation of Complement and Coagulation Cascades.

The limitations of current medications for treating rheumatoid arthritis (RA) emphasize the urgent need for the development of new drugs. This study aimed to investigate the potential anti-RA mechanism of amygdalin using tandem mass tag (TMT)-based quantitative proteomics technology. First, the anti-RA activity of amygdalin was evaluated in a Complete Freund's adjuvant (CFA)-induced rat model. Then, the roles and importance of proteins in the extracted rat joint tissue were evaluated using TMT-based quantitative proteomics technology. A bioinformatics analysis was used to analyze differentially abundant proteins (DAPs). A proteomics analysis identified 297 DAPs in the amygdalin group compared with the model group, of which 53 upregulated proteins and 51 downregulated proteins showed opposite regulatory trends to the DAPs produced after modeling. According to enrichment analyses of the DAPs, the signaling pathways with a high correlation degree were determined to be the complement and coagulation cascades. Furthermore, western blotting and molecular docking were used to further validate the key node proteins, e.g., complement C1s subcomponent (C1s), component C3 (C3) and kininogen 1 (Kng1). These results suggest that amygdalin may be a promising agent for treating RA by regulating the complement and coagulation cascades.

A Modified GC-MS Analytical Procedure for Separation and Detection of Multiple Classes of Carbohydrates.

A modified GC-MS analytical procedure based on trimethylsilyl-dithioacetal (TMSD) derivatization has been established for a simultaneous determination of thirteen carbohydrates. Different from previous approaches, the current GC-MS method was featured by a powerful practicability for simultaneous detection of aldoses, uronic acids, ketoses, and amino sugars; simplifying GC-MS chromatograms and producing a single peak for each derivatized sugar, as well as high resolution, sensitivity, and repeatability. An additional liquid-liquid extraction from derivatization mixtures was performed not only to increase the detection sensitivity of amino sugars but also to decrease the by-products of derivatization. Contrarily, three amino sugars were detected at a very low intensity or not detected at all. The effect of time on monosaccharide- mercaptalated reaction was systematically investigated. The effect of trimethylsilylation on the formation of TMSD was also optimized. The established GC-MS based on TMSD derivatization was suitable for complex carbohydrate analysis and has been successfully applied for the detection of free carbohydrates in water extracts of Anemarrhena asphodeloides roots and determination of monosaccharides in Glossy ganoderma polysaccharides.

Quality Analysis of American Ginseng Cultivated in Heilongjiang Using UPLC-ESI--MRM-MS with Chemometric Methods.

American ginseng (Panax quinquefolium) has long been cultivated in China for the function food and medicine. Here, ultra-high performance liquid chromatography was coupled with electrospray ionization and triple quadrupole mass spectrometry (UPLC-ESI--TQ-MS) for simultaneous detection of 22 ginsenosides in American ginseng cultivated in Mudanjiang district of Heilongjiang. The extraction conditions also were optimized by a Box Behnken design experiment. The optimized result was 31.8 mL/g as ratio of liquid to raw materials, 20.3 min of extraction time, and 235.0 W of extraction powers. The quantitative MS parameters for these 22 compounds were rapidly optimized by single factor experiments employing UPLC-ESI--multiple reaction monitoring or multiple ion monitoring (MRM/MIM) scans. Furthermore, the established UPLC-ESI--MRM-MS method showed good linear relationships (R² > 0.99), repeatability (RSD < 3.86%), precision (RSD < 2.74%), and recovery (94⁻104%). This method determined 22 bioactive ginsenosides in different parts of the plant (main roots, hairy roots, rhizomes, leaves, and stems) and growth years (one year to four years) of P. quinquefolium. The highest total content of the 22 analytes was in the hairy roots (1.3 × 105 µg/g) followed by rhizomes (7.1 × 104 µg/g), main roots (6.5 × 104 µg/g), leaves (4.2 × 104 µg/g), and stems (2.4 × 104 µg/g). Finally, chemometric methods, hierarchical clustering analysis (HCA) and partial least squares discrimination analysis (PLS-DA), were successfully used to classify and differentiate American ginseng attributed to different growth years. The proposed UPLC-ESI--MRM-MS coupled with HCA and PLS-DA methods was elucidated to be a simple and reliable method for quality evaluation of American ginseng.

A New UPLC-MS/MS Method for the Characterization and Discrimination of Polysaccharides from Genus Ephedra Based on Enzymatic Digestions.

Ephedra sinica polysaccharides have been reported to possess important activities, so quality evaluation of polysaccharides from the genus Ephedra is urgent. In this study, enzymatic digestions were performed to establish multiple saccharide fingerprints by ultra-performance liquid chromatography with electrospray ionization triple quadrupole linear ion trap mass spectrometry (UPLC-ESI-TQ-MS/MS) based on a multiple-reaction monitoring in negative mode. Under optimum UPLC-ESI⁻-TQ-MS/MS conditions, excellent separation and quantification of 21 constituents were achieved within 20 min on a solid core column with a 1.6 µm particle using pre-column derivatization with a PMP reagent. This method, coupled with enzymatic digestions and principal component analysis, has been successfully applied to characterize and discriminate Ephedra polysaccharides attributed to different species and plant parts. The results suggest that the proposed analytical strategy could achieve a quality evaluation of plant polysaccharides from traditional Chinese medicines.

[Therapeutic Material Basis of Chemical Subdivisions of Anemarrhenae Rhizoma on Anti-inflammatory and Immunomudulatory Effects].

OBJECTIVE: To study the relationship between chemical subdivision and immune suppressive activity in order to find out the anti-inflammatory and immunomudulatory pharmacological activity material basic of Anemarrhenae Rhizoma. METHOD: C57 mice was induced by 2,4-Dinitrofluorobenzene to build immune inflammation which was also called contact allergic dermatitis. The influence of Anemarrhenae Rhizoma decoction and chemical subdivisions on immune organ, ear edema and the Th1/Th2 immune balance was measured by analytical balance and Elisa method. The inflammatory factor TNF-α and NO level excreted by macrophage RAW264. 7 induced by LPS were also investigated. RESULT: AA decoction, timosaponin and polysaccharides significantly reduced the immune organ index and ear edema degree (P < 0.05), protein expression of IFN-γ was inhibited by AA timosaponin fraction and polysaccharides fraction. In vitro experiments showed that the proliferation of spleen cells was inhibited by timosaponin and polysaccharides after induced by ConA (P < 0.05). The release of NO and TNF-α induced by LPS significantly decreased by Anemarrhenae Rhizoma decoction and timosaponin (P < 0.05). CONCLUSION: The significant anti-inflammatory and immunomudu latory effects of AA are related to timosaponin and polysaccharides.

[Exploring effective components of laxative effect of Anemarrhenae Rhizoma based on Chinese herbal processing theory].

Anemarrhena asphodeloides processed by salt and raw product was compared including both chemical composition and laxative function in order to find the possible active substance to cure constipation. Processed and raw Anemarrhenae laxative effect on experimental constipation models was observed as well as chemical composition using UPLC-MS technology and the total sugar content was determined by phenol sulfuric acid method. Processed Anemarrhenae water extract improved excrement more than raw which has significant difference compared with the blank group (P < 0.05). On the other hand, the total ion flow spectrum showed no significant difference in most substance, but the total sugar content was significantly higher than raw product. Anemarrhenae ancient be recognized benefitting for draining body water in traditional Chinese medicine which has been lost in modern books because it is manifested as excellent laxative effect not diuretic effect. Saccharides carbohydrate may have closely relationship with this magically effect.

Quantitative analysis and fingerprint profiles for quality control of Fructus Schisandrae by gas chromatography: mass spectrometry.

This paper describes a simple, rapid, and effective quality assessment method for Fructus Schisandrae by gas chromatography-mass spectrum (GC-MS). The method was established by using specific lignan fingerprint profiles and quantitation of characteristic compounds in this herbal medicine. The GC-MS fingerprints of 15 batches of Schisandra samples from different regions of China showed similar lignan profiles. Five peaks were selected as characteristic peaks, and all of these were identified by using GC-MS techniques. The relative retention times of these characteristic peaks in the GC-MS fingerprint were established as an important parameter for identification of Schisandra samples. Meanwhile, relative peak areas may be a feasible approach to discriminate the S. chinensis and S. sphenanthera. Finally, these pharmacologically active constituents in the titled plant, schisandrins A-C and schizandrols A and B, were quantitatively determined using a validated GC-MS method.

Five withanolides from the leaves of Datura metel L. and their inhibitory effects on nitric oxide production.

Four new withanolides named dmetelins A-D (compounds 1-4), along with the known compound 7α,27-dihydroxy-1-oxo-witha-2,5,24-trienolide (5) were isolated from the leaves of Datura metel L. (Solanaceae). Their structures were elucidated on the basis of detailed analysis of 1D and 2D NMR and mass spectrometry data. All the compounds were evaluated for their inhibitory effects on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW264.7 cells. Compounds 1, 4 and 5 showed significant inhibitory activities, and compounds 2 and 3 showed moderate inhibitory activities with IC50 values of 17.8, 11.6, 14.9, 33.3 and 28.6 µM, respectively.

A Reverse Thinking Based on Partially Methylated Aldononitrile Acetates to Analyze Glycoside Linkages of Polysaccharides Using Liquid Chromatography-Multiple Reaction Monitoring Mass Spectrometry.

Glycoside linkage analyses of medicine and food homologous plant polysaccharides have always been a key point and a difficulty of structural characterization. The gas chromatography-mass spectrometry (GC-MS) method is one of the commonly used traditional techniques to determine glycoside linkages via partially methylated alditol acetates and aldononitrile acetates (PMAAs and PMANs). Due to the simplicity of derivatization and the highly structural asymmetry of PMANs, reverse thinking is proposed using liquid chromatography-electrospray ionization-multiple reaction monitoring mass spectrometry (LC-ESI-MRM-MS) for the first time to directly determine the neutral and acidic glycosyl linkages of polysaccharides. The complete characterization of glycoside linkages deduced from PMANs was achieved using a combination of tR values, characteristic MRM ion pairs, diagnostic ESI+-MS/MS fragmentation ions (DFIs), and optimal collision energy (OCE). The DFI and OCE parameters were confirmed to be effective for the auxiliary discrimination of some isomers of the PMANs. The practicality of LC-ESI+-MRM-MS was further verified by analyzing the glycoside linkages of polysaccharides in five medicine and food homologous plants. This method can serve as an alternative to GC-MS for the simultaneous determination of neutral and acidic glycosyl linkages in polysaccharides.

Distinction and quantification of Panax polysaccharide extracts via attenuated total reflectance-Fourier transform infrared spectroscopy with first-order derivative processing.

Derivative spectroscopy is used to separate the small absorption peaks superimposed on the main absorption band, which is widely adopted in modern spectral analysis to increase both the valid spectral information and the identification accuracy. In this study, a method based on attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) with first-order derivative (FD) processing combined with chemometrics is proposed for rapid qualitative and quantitative analysis of Panax ginseng polysaccharides (PGP), Panax notoginseng polysaccharides (PNP), and Panax quinquefolius polysaccharides (PQP). First, ATR-FTIR with FD processing was used to establish the discriminant model combined with principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA) and linear discriminant analysis (LDA). After that, two-dimensional ATR-FTIR based on single-characteristic temperature as external interference (2D-sATR-FTIR) was established using ATR-FTIR with FD processing. Then, ATR-FTIR with FD processing was combined with PLS to establish and optimize the quantitative regression model. Finally, the established discriminant model and 2D-sATR-FTIR successfully distinguished PGP, PNP and PQP, and the optimal PLS regression model had a good prediction ability for the Panax polysaccharide extracts content. This strategy provides an efficient, economical and nondestructive method for the distinction and quantification of PGP, PNP and PQP in a short detection time.

Structural identification, rheological properties and immunological receptor of a complex galacturonoglucan from fruits of Schisandra chinensis (Turcz.) Baill.

A complex heteropolysaccharide SCP-2 named schisanan B (Mw = 1.005 × 105 g/mol) was obtained from water extracts of Schisandra chinensis fruits, and its planar structure was finally deduced as a galacturonoglucan by a combination of monosaccharide compositions, methylation analysis, partial acid hydrolysis, enzymatic hydrolysis and 1D/2D-nuclear magnetic resonance spectroscopy. The conformation of SCP-2 exhibited a globular shape with branching in ammonium formate aqueous solutions. The rheological properties of SCP-2 were investigated on concentrations, temperature, pH and salts. The in vitro immunomodulatory activity assay demonstrated that SCP-2 significantly enhanced the production of nitric oxide (NO) and stimulated the secretion of tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) in macrophages. Through a combination of high-resolution live-cell imaging, surface plasmon resonance, and molecular docking techniques, SCP-2 exhibited a strong binding affinity with the Toll-like receptor 4 (TLR4). Moreover, western blot analysis revealed that SCP-2 effectively induced downstream signaling proteins associated with TLR4 activation, thereby promoting macrophage activation. The evidence strongly indicates that TLR4 functions as a membrane protein target in the activation of macrophages and immune regulation induced by SCP-2.

Oligo/polysaccharides from Cyathula officinalis and Achyranthes bidentata: a review of structures and bioactivities.

OBJECTIVES: Oligo-/polysaccharides from Cyathula officinalis Kuan (COPs) and Achyranthes bidentata Blume (ABPs) have attracted researchers' attention in the fields of healthy food supplements and traditional Chinese medicine (Niúxi) due to their multiple bioactivities combined with their nontoxic and highly biocompatible nature. The purpose of this paper was to provide a systematic and comprehensive overview of the extraction, purification, and structural analysis methods, chemical characteristics, biological activities, and structure bioactivity relationship. Furthermore, the possible development trends and perspectives for future research, and traditional uses of Niúxi are also summarized. METHODS: All the information was gathered from a library search and scientific databases. KEY FINDINGS: Although COPs and ABPs are derived from different plants, they have similar structural features in type, structure, and glycosidic linkage patterns and biological activities in vivo and in vitro. However, there are differences in monosaccharide compositions, which can be used as an identification mark. CONCLUSIONS: As traditional Chinese herbal medicine, C. officinalis and A. bidentata have similar pharmacological activities. The COPs and ABP possess wide pharmacological effects such as antitumor, antioxidant, anti-osteoporosis, and anti-inflammatory. Meanwhile, the biological activity and structure-activity relationship of purified COPs and ABPs are less studied, future research should focus on them.

Reversible ON- and OFF-switch receptors Clec4G and Rab1A reveal the hormetic effects of a pectin polysaccharide in Aralia elata (Miq.) Seem.

BACKGROUND: Numerous studies indicate that natural polysaccharides have immune-enhancing effects as a host defense potentiator. Few reports are available on hormetic effects of natural polysaccharides, and the underlying mechanisms remain unclear. PURPOSE: AELP-B6 (arabinose- and galactose-rich pectin polysaccharide) from Aralia elata (Miq.) Seem was taken as a case study to clarify the potential mechanism of hormetic effects of natural polysaccharides. METHODS: The pharmacodynamic effect of AELP-B6 was verified by constructing the CTX-immunosuppressive mouse model. The hormetic effects were explored by TMT-labeled proteomics, energy metabolism analysis, flow cytometry and western blot. The core-affinity target of AELP-B6 was determined by pull down, nanoLC-nanoESI+-MS, CETSA, immunoblot and SPR assay. The RAW264.7Clec4G-RFP and RAW264.7Rab1A-RFP cell lines were simultaneously constructed to determine the affinity difference between AELP-B6 and targets by confocal laser scanning live-cell imaging. Antibody blocking assays were further used to verify the mechanism of hormetic effects. RESULTS: AELP-B6 at low and medium doses may maintain the structural integrity of thymus and spleen, increase the concentrations of TNF-α, IFN-γ, IL-3 and IL-8, and alleviate CTX-induced reduction of immune cell viability in vivo. Proteomics and energy metabolism analysis revealed that AELP-B6 regulate HIF-1α-mediated metabolic programming, causing Warburg effects in macrophages. AELP-B6 at low and medium doses promoted the release of intracellular immune factors, and driving M1-like polarization of macrophages. As a contrast, AELP-B6 at high dose enhanced the expression levels of apoptosis related proteins, indicating activation of the intrinsic apoptotic cascade. Two highly expressed transmembrane proteins in macrophages, Clec4G and Rab1A, were identified as the primary binding targets of AELP-B6 which co-localized with the cell membrane and directly impacted with immune cell activation and apoptosis. AELP-B6 exhibits affinity differences with Clec4G and Rab1A, which is the key to the hormetic effects. CONCLUSION: We observed hormesis of natural polysaccharide (AELP-B6) for the first time, and AELP-B6 mediates the hormetic effects through two dose-related targets. Low dose of AELP-B6 targets Clec4G, thereby driving the M1-like polarization via regulating NF-κB signaling pathway and HIF-1α-mediated metabolic programming, whereas high dose of AELP-B6 targets Rab1A, leading to mitochondria-dependent apoptosis.

Revealing the "Yin-Jing" mystery veil of Platycodon grandiflorum by potentiating therapeutic effects and lung-oriented guidance property.

ETHNIC PHARMACOLOGICAL RELEVANCE: "Yin-Jing" medicine (YJM) has been widely used by both ancient and modern Chinese medicine practitioners during long-term clinical practice. However, it remains unclear how to best guide other medicines to the targeted organs in a traditional Chinese medicine (TCM) prescription. Here, in an attempt to explain the scientific connotation of the YJM property (YJMP) attributed to a basic TCM theory, Platycodon grandiflorum (PG) was chosen as a case study to reveal the mystery of YJMP theory. AIM OF THE STUDY: The main purpose of this study is to employ modern chemical and molecular biology methods to confirm the "Yin-Jing" effect of PG, and further clarify its material basis and related possible mechanism. MATERIALS AND METHODS: The ammonia-induced lung injury rat model was utilized to determine the optimal dosage of traditional prescription Hui Yan Zhu Yu decoction (HYZYD) using Wright Giemsa staining, HE staining, Masson staining, and TUNEL analysis. With the same way, PG was confirmed to have potentiating therapeutic effect (PTE) by comparison with HYZYD and [HYZYD-PG]. TMT proteomics was used to reveal the "Yin-Jing" mechanism of action. Western blot assay (WB) was employed for verification of differentially expressed proteins. Additionally, four non-crossing fragmentations (Fr. A-D) were characterized by RPLC/SEC-ELSD and HILIC-ESI--Q-OT-IT-MS techniques. The PTE and guidance property assays were utilized to evaluate "Yin-Jing" functions by a compatible combination of hydroxysafflor yellow A (HYA) using qPCR, FCM, WB, HPLC, high content cell imaging (HCI) and high-resolution live-cell imaging (HRLCI) techniques. RESULTS: The HYZYD-M (medium dose group) significantly improved the lung injury level in a pneumonia model of rats. PG enhanced the therapeutic effect of HYZYD ascribed to Yin-Jing PTE functions. TMT proteomics revealed a category of differentially expressed proteins ascribed to Golgi-ER between HYZYD and [HYZYD-PG]. Fr. C (i.e., saponins) and Fr. D (i.e., lipids) were determined as therapeutic fragmentations via the LPS-induced A549 cell injury model; however, Fr. B (fructooligosaccharides and small Mw fructans) had no therapeutic effect. Further compatibility PTE assays confirmed Fr. B significantly improved efficiency by a combination of HYA. The guidance assays showed Fr. B could significantly increase the uptake and distribution of HYA into lung cells and tissues. HCI assays showed that Fr. B increased uptake of HYA accompanied by significant activation of Golgi-ER. Unlike Fr. B, HRLCI showed that Fr. A, C and D were not only unobvious activations of Golgi-ER but also insignificant facilitation of colocalizations between HYA and Golgi-ER. CONCLUSIONS: Fr. B is believed to be a key YJMP material basis of PG attributed to Yin-Jing PTE with characteristic of lung-oriented guidance property, whereas another abound Fr. C was determined to have synergistic effects rather than Yin-Jing material basis.

Structure of an unprecedent glucuronoxylogalactoglucomannan from fruit bodies of Auricularia auricula-judae (black woody ear).

An unprecedent glucuronoxylogalactoglucomannan (GXG'G″M), ME-2 (Mw, 2.60 × 105 g/mol; O-acetyl % = 16.7 %), was isolated and purified from water extracts of Auricularia auricula-judae (black woody ear). Firstly, due to much higher O-acetyl contents, we prepared its fully deacetylated products (dME-2; Mw, 2.13 × 105 g/mol) for convenient structure survey. The repeating structure-unit of dME-2 was readily proposed based on Mw determination, monosaccharide compositions, methylation analysis, free-radical degradation and 1/2D NMR spectroscopy. The dME-2 was identified as a highly branched polysaccharide with an average of 10 branches per 10 sugar backbone units. The backbone was only repeating →3)-α-Manp-(1→ residues, substituted at the C-2, C-6 and C-2,6 positions. The side chains included ß-GlcAp-(1→, ß-Xylp-(1→, α-Manp-(1→, α-Galp-(1→ and ß-Glcp-(1→. Secondly, the complex substituted positions of O-acetyl groups in ME-2 were determined to be at C-2, C-4, C-6 and C-4,6 in the backbone and at C-2 and C-2,3 in some side chains. Finally, the anti-inflammatory activity of ME-2 was preliminarily explored on LPS-stimulated THP-1 cells. The above date not only provided the first example for structural studies of GXG'G″M type polysaccharides, but also facilitated development and application of black woody ear polysaccharides as medicinal agents or functional dietary supplements.

Direct acetylation for full analysis of polysaccharides in edible plants and fungi using reverse phase liquid chromatography-multiple reaction monitoring mass spectrometry.

It is vitally important to characterize polysaccharides by monosaccharide composition method. In this study, a direct acetylation strategy combined with reversed-phase liquid chromatography electrospray tandem multiple reaction monitoring mass spectrometry (RPLC-ESI-MRM-MS) was developed for simultaneous determination of 8 aldoses (Glc, Gal, Man, Ara, Xyl, Rib, Rha and Fuc), a ketose (Fru), 2 alditols (Glc-ol and Man-ol) and 2 uronic acids (GlcA and GalA) on a high-pressure resistant reversed-phase column. Employing 1-MeIm as catalyst for direct acetylation, even though no DMSO was used to inhibit the transformation of configurations, each carbohydrate still produced a single chromatographic peak in RPLC conditions due to the ɑ- and ß- isomers merged together. Except for Fru and Man, all the other 11 carbohydrates were base-line separated in a 1.7 µm CYANO column. Therefore, correction factor method is further proposed to perfectly solve co-elution problem of Fru and Man because of occurrence of a specific Q3 ion for aldoses rather than ketose. The result was verified on a 1.7 µm Fluoro-Phenyl column with a full separation of Fru and Man. Herein, the established direct acetylation as followed RPLC-ESI-MRM-MS method was successfully applied for compositional analysis of complex polysaccharides from edible plants and fungi.

Structure of a highly branched galacturonoglucan from fruits of Schisandra chinensis (Turcz.) Baill.

A novel galacturonoglucan, named SCP-1, is isolated and purified from Schisandra chinensis fruits. The structure of SCP-1 is systematically investigated by a combination of monosaccharide compositions, absolute Mw, methylation analysis, partial acid hydrolysis, isoamylase degradations, and nuclear magnetic resonance spectroscopy. The structure of SCP-1 is theoretically described as follows: (i) Glc and GalA in a molar ratio of 17:3; (ii) â†’ 4)-α-Glcp-(1→, →4,6)-α-Glcp-(1→, →3,4,6)-α-Glcp-(1→, α-Glcp-(1→, →4)-α-GalAp-6-OMe-(1→, α-GalAp-6-OMe-(1→, ß-Glcp-(1→, →6-)-ß-Glcp-(1 â†’ and →3,4)-ß-Glcp-(1 â†’ in a molar ratio of 48:5:3:3:10:5:12:5:9; (iii) a repeating unit of →4)-α-Glcp-(1 â†’ as a backbone with branched points at C-3 and C-6, substituted by different types of acidic and neutral side chains to form multiple branches; and (iv) a rigid rod configuration deduced from α value of 1.26 in Mark-Houwink equation ([η] = kMα). Anti-tumor assay investigated the effects of SCP-1 on human HepG2 cancer cell lines in vitro. This is for the first time to report a galacturonoglucan in S. chinensis fruits.