Identification and Quantification of Monosaccharides from Fungal Cell Walls and Exopolysaccharides by Gas Chromatography Coupled to Mass Spectrometry.

The fungal cell wall and secreted exopolysaccharides play an important role in the interactions between fungi and their environment. Despite their central role in fungal biology, ecology, and host-pathogen interactions, the composition of these polymers and their synthetic pathways are not well understood. The protocols presented in this article describe an approach to isolate fungal cell wall polysaccharides and to identify and quantify the monosaccharide composition of these polymers by gas chromatography-mass spectrometry (GC-MS). © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: O-methyl trimethylsilyl monosaccharide derivatives composition analysis by GC-MS Support Protocol: Fungal cell wall extraction.

Purification, Structural Characterization, and Anti-Inflammatory Effects of a Novel Polysaccharide Isolated from Orostachys fimbriata.

The present study elucidated the structural characteristics and anti-inflammatory activity of a novel polysaccharide isolated from Orostachys fimbriata, which is a traditional Chinese medicinal plant. O. fimbriata polysaccharide (OFP) was extracted and subsequently purified by chromatography using a DEAE cellulose-52 and Sephadex G-75 column. The molecular weight was determined as 6.2 kDa. HPGPC and monosaccharide composition analysis revealed a homogeneous polysaccharide containing only Glc. Chromatography and spectral analysis showed that the possible chemical structure consisted of →4)-α-Glcp-(1→ and a small quantity of →4,6)-ß-Glcp-(1→ in the main chain and →6)-ß-Glcp-(1→, α-Glcp-(1→, and ß-Glcp-(1→ in the side chain. Morphological analysis using scanning electron microscopy (SEM) and atomic force microscopy (AFM) indicated that OFP had a multi-branched structure, and the sugar chain molecules of polysaccharide appeared aggregated. OFP was found to exhibit anti-inflammatory activity by reducing the secretion of inflammatory factors in RAW264.7 cells and by decreasing the extent of xylene-induced ear swelling in mice.

Separation of three chromones from Saposhnikovia divaricata using macroporous resins followed by preparative high-performance liquid chromatography.

Prim-O-glucosylcimifugin, cimifugin, and 5-O-methylvisamminoside are three major chromone derivatives of Saposhnikovia divaricata that have many pharmacological activities, such as anti-inflammatory and antitumor activities. In the present work, an effective method for the simultaneous separation of prim-O-glucosylcimifugin, cimifugin, and 5-O-methylvisamminoside with high purities was established using HPD-300 resin coupled with preparative high-performance liquid chromatography. The adsorption kinetics curves of the three compounds on the HPD-300 resin were studied and found to fit well according to the pseudo-second-order equation. The adsorption isotherm results indicated that the adsorption process of the three compounds was exothermic. After a one-run treatment with the resin, the contents of prim-O-glucosylcimifugin, cimifugin, and 5-O-methylvisamminoside increased from 0.29, 0.06, and 0.37% to 13.07, 2.83, and 16.91% with recovery yields of 76.38, 78.25, and 76.73%, respectively. Finally, the purities of the three compounds were found to reach more than 95% after further separation using preparative high-performance liquid chromatography. The method developed in this study was effective and could simultaneously separate three chromones from Saposhnikovia divaricate. The experimental results also showed that the HPD-300 resin is suitable for the separation of chromone derivatives.

Toxicological assessment of Opuntia dillenii (Ker Gawl.) Haw. cladode methanol extract, fractions and its alpha pyrones: Opuntiol and opuntioside.

ETHNOPHARMACOLOGICAL RELEVANCE: The edible plant Opuntia dillenii (Ker Gawl.) Haw. commonly known as Nagphana, belongs to the Cactaceae family. It is traditionally used to treat various ailments including inflammation, gastric ulcers, diabetes, hepatitis, asthma, whooping cough and intestinal spasm. AIM OF THE STUDY: Despite its traditional use in various countries, detailed toxicological studies of O. dillenii cladode are few. Thus in the current study, toxicity of O. dillenii cladode derived methanol extract, fractions and its α-pyrones: opuntiol and opuntioside have been addressed. METHODS: The test agents were assessed using both in vitro and in vivo toxicity assays. MTT on human embryonic kidney cell line (HEK-293), tryphan blue exclusion in rat neutrophils, Cytokinesis-B block micronucleus (CBMN) in human lymphocytes and genomic DNA fragmentation using agarose gel electrophoresis were performed. In acute toxicity test, mice orally received extract (5 g/kg) for 7 days followed by measurements of relative organ weight, biochemical (blood profile, liver and kidney function test) and histological studies (liver and kidney) were carried out. Rat bone marrow micronucleus genotoxicity assay was also conducted. RESULTS: O. dillenii derived test agents were non-cytotoxic and had no effect on the integrity of DNA. Methanol extract (5 g/kg) orally administered in mice did not cause any significant change in relative organ weights, biochemical parameters and liver and kidney histology as compared to vehicle control. In parallel, extract did not stimulate micronuclei formation in rat bone marrow polychromatic erythrocytes. CONCLUSION: These results led to conclude that edible O. dillenii extract is non-toxic via the oral route and appears to be non-cyto-, hepato-, nephro- or genotoxic, thereby supporting its safe traditional use against various ailments. Therefore, opuntiol and opuntioside may serve as lead compounds in designing new drug(s) derived from edible plants.

Recovery of Anthocyanins and Monosaccharides from Grape Marc Extract by Nanofiltration Membranes.

Wastewaters and by-products generated in the winemaking process are important and inexpensive sources of value-added compounds that can be potentially reused for the development of new products of commercial interest (i.e., functional foods). This research was undertaken in order to evaluate the potential of nanofiltration (NF) membranes in the recovery of anthocyanins and monosaccharides from a clarified Carménère grape marc obtained through a combination of ultrasound-assisted extraction and microfiltration. Three different flat-sheet nanofiltration (NF) membranes, covering the range of molecular weight cut-off (MWCO) from 150 to 800 Da, were evaluated for their productivity as well as for their rejection towards anthocyanins (malvidin-3-O-glucoside, malvidin 3-(acetyl)-glucoside, and malvidin 3-(coumaroyl)-glucoside) and sugars (glucose and fructose) in selected operating conditions. The selected membranes showed differences in their performance in terms of permeate flux and rejection of target compounds. The NFX membrane, with the lowest MWCO (150-300 Da), showed a lower flux decay in comparison to the other investigated membranes. All the membranes showed rejection higher than 99.42% for the quantified anthocyanins. Regarding sugars rejection, the NFX membrane showed the highest rejection for glucose and fructose (100 and 92.60%, respectively), whereas the NFW membrane (MWCO 300-500 Da) was the one with the lowest rejection for these compounds (80.57 and 71.62%, respectively). As a general trend, the tested membranes did not show a preferential rejection of anthocyanins over sugars. Therefore, all tested membranes were suitable for concentration purposes.

Physicochemical and macromolecule properties of RG-I enriched pectin from citrus wastes by manosonication extraction.

The properties of pectin extracted from mandarin citrus peels by manosonication extraction (MSp) were systematically studied and compared with pectin obtained by the conventional maceration method (CMp). The yield of MSp (25.5%) was significantly higher than that of CMp (18.3%), while MSp exhibited two Mw fraction distributions. Monosaccharide analysis demonstrated that MSp had more branched RG-I regions (78.3 mol%) than CMp (36.6 mol%) with a high content of arabinose and galactose. The branched-chain morphological characteristics of samples were directly imaged by atomic force microscopy. MSp exhibited a significantly lower degree of methoxylation than CMp by FT-IR and NMR analysis, but X-ray diffraction analysis showed little difference in the level of crystallinity. Moreover, MSp and CMp showed non-Newtonian behaviour, and the increasing order of apparent viscosities was 1.0 w/v% MSp < 1.0 w/v% CMp < 2.0 w/v% CMp < 2.0 w/v% MSp. Thermal analysis and weight loss measurements indicated MSp exhibited greater thermal stability. The results also indicated that both MSp and CMp significantly enhanced the emulsion activity at high concentrations; the emulsions containing 1.5 w/v% pectin showed no phase separation over 21 days, suggesting that MSp could be a potential effective stabiliser in the food and beverage industry.

A New, Quick, and Simple Protocol to Evaluate Microalgae Polysaccharide Composition.

In this work, a new methodological approach, relying on the high specificity of enzymes in a complex mixture, was developed to estimate the composition of bioactive polysaccharides produced by microalgae, directly in algal cultures. The objective was to set up a protocol to target oligomers commonly known to be associated with exopolysaccharides' (EPS) nutraceutical and pharmaceutical activities (i.e., rhamnose, fucose, acidic sugars, etc.) without the constraints classically associated with chromatographic methods, while maintaining a resolution sufficiently high to enable their monitoring in the culture system. Determination of the monosaccharide content required the application of acid hydrolysis (2 M trifluoroacetic acid) followed by NaOH (2 M) neutralization. Quantification was then carried out directly on the fresh hydrolysate using enzyme kits corresponding to the main monosaccharides in a pre-determined composition of the polysaccharides under analysis. Initial results showed that the enzymes were not sensitive to the presence of TFA and NaOH, so the methodology could be carried out on fresh hydrolysate. The limits of quantification of the method were estimated as being in the order of the log of nanograms of monosaccharides per well, thus positioning it among the chromatographic methods in terms of analytical performance. A comparative analysis of the results obtained by the enzymatic method with a reference method (high-performance anion-exchange chromatography) confirmed good recovery rates, thus validating the closeness of the protocol. Finally, analyses of raw culture media were carried out and compared to the results obtained in miliQ water; no differences were observed. The new approach is a quick, functional analysis method allowing routine monitoring of the quality of bioactive polysaccharides in algal cultures grown in photobioreactors.

Purification, characterization and immunostimulatory effects of polysaccharides from Anemarrhena asphodeloides rhizomes.

The crude polysaccharide was extracted from A. asphodeloides rhizomes and further purified to produce two fractions F1 (50.0%) and F2 (19.6%). The chemical constitutions of the polysaccharides were neutral sugars (51.4%-89.7%), uronic acids (1.0%-30.2%) and sulfate esters (3.4%-8.1%), with various ratios of monosaccharides including rhamnose (1.4%-6.1%), arabinose (7.1%-21.2%), xylose (0.2%-4.8%), mannose (39.9%-79.0%), glucose (6.0%-11.1%) and galactose (2.6%-22.0%). The molecular properties of the polysaccharides were investigated by the HPSEC-UV-MALLS-RI system, revealing the Mw 130.0 × 103-576.5 × 103 g/moL, Rg 87.6-382.6 nm and SVg 0.3-54.3 cm3/g. The polysaccharides stimulated RAW264.7 cells to produce considerable amounts of NO and up-regulate the expression of TNF-α, IL-1 and COX-2 genes. Polysaccharides exhibited the growth inhibitory effects on cancer cells lines of AGS, MKN-28 and MKN-45, in which F2 fraction exhibited prominent bioactivities. The AGS cells treated with F2 experienced condensed cytoplasm, shrinkage of nucleus and chromatin marginalization with the highest number of cells at early-stage apoptosis reaching 54.6%. The inhibitory effect of F2 polysaccharide on AGS cells was through MAPKs and STAT3 signaling pathways. The backbone of the F2 was mainly linked by (1 â†’ 4)-linked mannopyranosyl and (1 â†’ 3)-linked galactopyranosyl. Taken together, the polysaccharide from A. asphodeloides rhizomes could be utilized as medicinal, pharmacological and functional food ingredients.

Isolation of an acidic polysaccharide from the flowers of Leucosceptrum canum Smith and its immunomodulatory activity evaluation.

A water-soluble polysaccharide (LCP-05) was isolated from the flowers of Leucosceptrum canum Smith. LCP-05 was an acidic polysaccharide with a molecular weight of approximately 8.9 kDa. Monosaccharide composition analysis indicated that LCP-05 was composed of Man, Rha, GlcA, GalA, Glc, Gal and Ara in a molar ratio of 0.83:1.68:0.33:2.15:1.00:1.45:1.22. The framework of LCP-05 was speculated to be a branched rhamnogalacturonan with the backbone consisting of α-1,2,4-linked Rhap and α-1,4-linked GalAp, and bearing branches at the O-4 position of the Rha residues. The side chains are terminated primarily with the Araf and Glcp residues. LCP-05 was found to be able to significantly induce the production of NO, IL-6, and TNF-α in RAW 264.7 cells, and to induce RAW 264.7 cell's suppressive effect on both cell growth and cell migration of 4 T1 mammary breast cancer cells.

Comprehensive evaluation of alkali-extracted polysaccharides from Agrocybe cylindracea: Comparison on structural characterization.

Agrocybe cylindracea is a common source of active polysaccharides, but their fine structures are not clearly elucidated. In the present study, four fractions were purified from the alkaline extract of A. cylindracea (JACP), and their chemical components and structures were compared by HPAEC-PAD, methylation combined with GC-MS, and 1D/2D NMR analysis. Results showed the purified fractions' physicochemical properties, including monosaccharide compositions, molecular weights, viscosities and surface morphology considerably varied. JACP-30 was identified as a fucoglucogalactan with a α-(1 → 6)-galactopyranosyl as main chain. JACP-50p and JACP-80r were characterized as ß-(1 → 6)-glucans with side chains composed of terminal and 3-substituted ß-glucopyranosyl residues attached at O-3 for every three residues. Similarly, the backbone of JACP-80 was ß-(1 → 6)-linked glucopyranosyl and ß-(1 → 3,6)-linked glucopyranosyl residues at a ratio of 4:1. This work provides more information to the understanding of polysaccharides from A. cylindracea, further guiding its biological researches and developing the application in food and biomedicine industries.

Determination of terminal glycan and total monosaccharide profiles of reelin glycoprotein in SH-SY5Y neuroblastoma cell line by lectin blotting and capillary liquid chromatography electrospray ionization-ion trap tandem mass spectrometry system.

Reelin (400 kDa) is an extracellular matrix glycoprotein that is a key regulator of the many significant biological processes including the brain formation, cell aggregation, and dendrite formation. The glycosylation contributes to the nature of the protein through folding, localization and trafficking, solubility, antigenicity, biological activity, and half-life. Although reelin is to be known as a glycoprotein, the knowledge of its glycosylation is very limited. In this study, we aimed to characterize the terminal glycan profile of reelin by lectin blotting and monosaccharide analysis of glycan chains by capillary liquid chromatography electrospray ionization ion trap tandem mass spectrometry (CapLC-ESI-MS/MS) in SH-SY5Y neuroblastoma cell line. According to our results, reelin was detected in different protein fragments (310, 250, and 85 kDa) in addition to full-length form (400 kDa) in the cell line. The reelin glycoprotein was found to carry the ß-N-Acetylglucosamine, α-Mannose, ß-Galactose, and α-2,3 and α2,6 linked sialic acids by lectin blotting. Nevertheless, these terminal monosaccharides were found in different intensity according to reelin fragments. Besides, we purified a reelin fragment (250 kDa), and we analyzed it for their monosaccharide by CapLC-ESI-MS/MS. We found that reelin contained five types of monosaccharides, which were consisted of N-Acetylgalactosamine, N-Acetylglucosamine, Galactose, Glucose, Mannose and Sialic acid, from high to low abundance respectively. The present results provide a valuable guide for biochemical, genetic, and glycobiology based further experiments about reelin glycosylation in cancer perspective.

Screening active fractions from Pinus massoniana pollen for inhibiting ALV-J replication and their structure activity relationship investigation.

The objective was to identify the active fractions of polysaccharide against replication of ALV-J and elucidate their structure activity relationship. The optimal extraction conditions were extracting temperature 90℃, pH 9 and the ratio of liquid to solid 30:1. Under these conditions, extraction yield of total polysaccharide was 6.5 % ± 0.19 %. Total polysaccharide was then purified by DEAE-52 cellulose and Sephadex G-200 gel. Three fractions, PPP-1, PPP-2, and PPP-3, were identified with molecular weight of 463.70, 99.41, and 26.97 kDa, respectively. Three polysaccharide fractions were all composed of 10 monosaccharides in different proportions. Compared with PPP-1, which was mainly composed of glucose, PPP-2 and PPP-3 contained a higher proportion of galactose, glucuronic acid and galacturonic acid. The Congo red assay indicated that the PPP-2 may have a triple helical structure, while PPP-1 and PPP-3 were absent. In vitro assay showed that there was no significant cytotoxicity among the polysaccharide fractions under the concentration of 800 µg mL-1 (P > 0.05). The antiviral test showed that PPP-2 had the strongest activity, indicating PPP-2 was the major antiviral component. The structure-activity relationship showed that the antiviral activities of polysaccharide fractions were affected by their monosaccharide composition, molecular weight, and triple helical structure, which was a result of a combination of multiple molecular structural factors. These results showed that the PPP-2 could be exploited as a valued product for replacing synthetic antiviral drugs, and provided support for future applications of polysaccharide from Pinus massoniana pollen as a useful source for antiviral agent.

Composition, physicochemical properties, and anti-fatigue activity of water-soluble okra (Abelmoschus esculentus) stem pectins.

Okra, Abelmoschus esculentus (L.) Moench, an annual herbaceous plant, is widely distributed in tropical and subtropical regions. Water-soluble pectic hydrocolloids from okra stems (HOS) were extracted and purified using polydivinylbenzene HP-20 resins. The sugar composition of the purified HOS with an weight-average molecular weight of 178.4 ± 2.1 kDa and a polydispersity index of 1.02 ± 0.02 contained galacturonic acid (34%), galactose (31%), rhamnose (21%), arabinose (4.2%), glucuronic acid (2.5%), xylose (1.2%), and other monosaccharides (6.1%) by weight. Its favorable rheological behaviors were evident on relatively higher concentrations (20, 25, and 30 mg/mL) and moderately lower pH levels (3 and 5) of HOS. The anti-fatigue experiments in vivo demonstrated that a high dose of HOS (450 mg/kg feed) prolonged the exhaustive swimming time of mice, significantly induced an increase in blood glucose and glycogen, and decreased lactic acid and serum urea nitrogen levels. HOS digestion in vivo was fairly conducive to the improvement of energy storage capacity and renal function for physically induced fatigue, compared with the conventional herbal supplement Panax quinquefolium. Accordingly, HOS exhibits potential for reutilization of okra stem waste.

Enhanced separation and analysis of low abundant soy proteins by dual washing extraction process.

Soybean seeds provide a rich source of proteins, fats, carbohydrates, and micronutrients. Extraction and analysis of low abundant soybean seed proteins are challenging because of its complex seed composition. For characterizing various proteins, it is paramount to remove the other interfering components, primarily oils, and carbohydrates. In the present study, we used a sequential dual washing process initially with hexane to remove oil and non-polar interferences, followed by 80% ethanol washing to remove about 60% of the total soluble sugars. The extracted soluble sugars were quantified using a newly developed and validated high-performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD). This newly developed combined washings process significantly enhanced the separation of both low molecular weight and low abundant proteins using 1D (one dimensional)- and 2D (two dimensional) gel electrophoresis. The separated proteins were trypsinized and analyzed by using Bruker amazon speed ion trap mass spectrometer equipped with an ESI source. This combined washing process allowed the identification of 18 additional low abundant soy proteins as compared to the simple hexane washed samples. This purification process will allow researchers to identify and investigate the role of low molecular weight and low abundant proteins as it relates to plant functions, nutrition, and health.

Studies on chemical composition, rheological and antioxidant properties of pectin isolated from Riang (Parkia timoriana (DC.) Merr.) pod.

Although synthetic antioxidant food additives are widely used in a variety of food products, some of them are suspected of having a noxious effect on human health. As a consequence, much research attention has been focused on developing natural antioxidant compounds from plants. Riang (Parkia timoriana (DC.) Merr.) is known as a traditional medicinal plant in which its various parts have been reported to exhibit antioxidant and numerous biological activities. In this study, pectins from Riang pod husk and pod powder were extracted, and their physico-chemical, rheological, and antioxidant properties were characterized. The extracted pectins showed high uronic acid content (> 65%) and high molecular weight (200-250 kDa) and the yields were approximately 15 and 36%w/w (dry basis), for Riang husk pectin (RHP) and Riang pod powder pectin (RPP), respectively. Furthermore, both pectins were classified as a high methoxyl with their DE of ~66%. Rheological measurements revealed a pseudoplastic behavior above 2% w/v. RHP contained higher content of total phenolics, flavonoids and tannin, compared with RPP. Antioxidant activities of RHP were consequently higher than RPP in all studied assays. The highest antioxidant activities of RHP and RPP, obtained from ABTS assay, were 0.95 and 0.24 mmol Trolox equivalents/g, respectively.

The heteropolysaccharide of Mangifera indica fruit: Isolation, chemical profile, complexation with ß-lactoglobulin and antioxidant activity.

A 91 kDa heteropolysaccharide (F2) was isolated from Mangifera indica fruit via extraction with H2O, purification by C2H5OH, starch removal and ion exchange chromatography. This polymer was made up mostly of Ara, Gal, Glc, Rha, Xyl, and GalA in a 37: 29: 9:3:2:19 molar proportion. It inherited a small backbone containing GalpA and Rhap units substituted with very large side chains containing differently linked Ara and Gal units plus esterified gallic acid (GA) residue. Several enzymes generated oligosaccharides including (i) Ara2-10Ac6-22, (ii) Gal1-8Ac5-26 and (iii) GA1Gal1Ac7 were characterized. This polysaccharide, which showed dose dependent antioxidant activity, exhibited synergism with gallic acid, and formed a complex (K = 1.2 × 106 M-1) with ß-lactoglobulin. Accordingly, H2O treatment produces a polysaccharide with desired biochemical properties; this could be effective in designing innovative functional food with flexible makeup.

In Silico Identification of Potential Natural Product Inhibitors of Human Proteases Key to SARS-CoV-2 Infection.

Presently, there are no approved drugs or vaccines to treat COVID-19, which has spread to over 200 countries and at the time of writing was responsible for over 650,000 deaths worldwide. Recent studies have shown that two human proteases, TMPRSS2 and cathepsin L, play a key role in host cell entry of SARS-CoV-2. Importantly, inhibitors of these proteases were shown to block SARS-CoV-2 infection. Here, we perform virtual screening of 14,011 phytochemicals produced by Indian medicinal plants to identify natural product inhibitors of TMPRSS2 and cathepsin L. AutoDock Vina was used to perform molecular docking of phytochemicals against TMPRSS2 and cathepsin L. Potential phytochemical inhibitors were filtered by comparing their docked binding energies with those of known inhibitors of TMPRSS2 and cathepsin L. Further, the ligand binding site residues and non-covalent interactions between protein and ligand were used as an additional filter to identify phytochemical inhibitors that either bind to or form interactions with residues important for the specificity of the target proteases. This led to the identification of 96 inhibitors of TMPRSS2 and 9 inhibitors of cathepsin L among phytochemicals of Indian medicinal plants. Further, we have performed molecular dynamics (MD) simulations to analyze the stability of the protein-ligand complexes for the three top inhibitors of TMPRSS2 namely, qingdainone, edgeworoside C and adlumidine, and of cathepsin L namely, ararobinol, (+)-oxoturkiyenine and 3α,17α-cinchophylline. Interestingly, several herbal sources of identified phytochemical inhibitors have antiviral or anti-inflammatory use in traditional medicine. Further in vitro and in vivo testing is needed before clinical trials of the promising phytochemical inhibitors identified here.

3,4,5-Tri-O-caffeoylquinic acid methyl ester isolated from Lonicera japonica Thunb. Flower buds facilitates hepatitis B virus replication in HepG2.2.15 cells.

Caffeoylquinic acids are well known for their prominent antiviral activities. Beyond our expectations, we initially found 3,4,5-Tri-O-caffeoylquinic acid methyl ester (3,4,5-CQME) from L. japonica can facilitate HBV DNA and antigens secretion. This study aimed to investigate its underlying molecular mechanism. The results indicate that 3,4,5-CQME signally increased intracellular and secreted HBsAg levels by more than two times in HepG2.2.15 cells and HepAD38 cells. Furthermore, levels of HBeAg, HBV DNA and RNA were significantly enhanced by 3-day 3,4,5-CQME treatment; it didn't directly affect intracellular cccDNA amount, although it slightly increased cccDNA accumulation as a HBV DNA replication feedback. In addition, treatment with 3,4,5-CQME significantly induced HBx protein expression for viral replication. We utilized a phospho-antibody assay to profile the signal transduction change by 3,4,5-CQME to illuminate its molecular mechanism. The results indicate that treatment with 3,4,5-CQME activated AKT/mTOR, MAPK and NF-κB pathways verified by immunoblot. Moreover, 3,4,5-CQME upregulated the expression of nuclear transcriptional factors PGC1α and PPARα. In short, 3,4,5-CQME promotes HBV transcription and replication by upregulating HBx expression and activating HBV transcriptional regulation-related signals. As caffeoylquinic acids are widely present in traditional Chinese medicines, the risk of intaking caffeoylquinic acids-containing herbs for hepatitis B treatment requires more evaluation and further research.

[High-efficiency separation and analysis of monosaccharides in Pueraria polysaccharides by pressurized capillary electrochromatography].

Pueraria polysaccharides have been proven to possess biological activities such as bacteriostasis, anti-oxidative, anti-tumor, and immunity boosting activities. The variation in the structure, composition, and amount of monosaccharides in these polysaccharides may lead to different spatial structures and biological activities. Therefore, extraction of Pueraria polysaccharides and determination of the monosaccharide composition are of great significance for activity analysis and quality control. Direct detection of saccharides is difficult because they are strongly polar and do not show absorption in the ultraviolet region. At present, the commonly used methods for saccharide detection are liquid chromatography-ultraviolet detection, gas chromatography-ultraviolet detection, and mass spectrometry. Pressurized capillary electrochromatography is a high-efficiency microseparation technology. In this study, two kinds of Pueraria polysaccharides were extracted by an ultrasonic-assisted method, and response surface methodology was performed to explore the conditions for ultrasonic-assisted extraction of polysaccharides from Pueraria. The interaction effects of four factors, the liquid-solid ratio, ultrasonic extraction time, ultrasonic extraction temperature, and ultrasonic power, on the extraction rate of the polysaccharides were analyzed. By combining the optimal conditions predicted by the software and the actual equipment conditions, the optimal extraction conditions for Pueraria polysaccharides were chosen as follows:ultrasonic extraction temperature, 90℃; liquid-solid ratio of Pueraria thomsonii Benth, 20 mL/g; liquid-solid ratio of Pueraria lobata Ohwi, 40 mL/g; ultrasonic extraction time, 30 min; ultrasonic power, 180 W. Through data fitting, the multiple quadratic regression equation of the four factors on the extraction rate of Pueraria polysaccharides was established. A novel method based on pressurized capillary electrochromatography for the separation and analysis of eight neutral monosaccharides has been established. The monosaccharides were derivatized by the 1-phenyl-3-methyl-5-pyrazolone pre-column derivatization method. The separation conditions for these monosaccharides were explored, and the buffer concentration, buffer pH, applied voltage, type of chromatographic column, and mobile phase ratio were optimized. Finally, the established pressurized capillary electrochromatography-ultraviolet detection method was applied to the detection and identification of two kinds of actual Pueraria polysaccharide samples. The results of response surface analysis showed that among the four experimental factors, ultrasonic extraction temperature had the greatest influence on the extraction rate of polysaccharides from the two kinds of Pueraria, followed by the liquid-solid ratio; the influence of the ultrasonic extraction time and ultrasonic power was relatively weak. The experimental conditions were determined as follows:the separation of eight neutral monosaccharide derivatives could be realized within 24 min on a Halo-2.7 µm core-shell C18 capillary column with acetonitrile-50 mmol/L ammonium acetate aqueous solution (18:82, v/v, pH 4.1) as the mobile phase, by detection at 250 nm under an applied voltage of-20 kV. The separation and detection speeds and the column efficiency achieved with this method were much better than those obtained with the traditional liquid chromatography method. The results show that the proposed method has a good linear relationship and good repeatability. The separation and identification results for the actual samples showed that the polysaccharides of Pueraria thomsonii Benth were mainly composed of glucose, mannose, rhamnose, and fucose in the molar ratio 1.00:0.16:0.14:0.07. The polysaccharides of Pueraria lobata Ohwi were mainly composed of glucose and mannose in the molar ratio 1.00:0.70. This study provides a novel method for the rapid and efficient separation and detection of neutral monosaccharides, and serves as a reference for analyzing the monosaccharide composition of Pueraria polysaccharides.

Arsenosugar standards extracted from algae: Isolation, characterization and use for identification and quantification purposes.

Sulfate (SO4-sug) and sulfonate (SO3-sug) arsenosugar standard solutions were obtained using preparative liquid chromatography. Several commercial algae samples were characterized (total contents and speciation) to select the most appropriate in relation to their arsenosugar contents. Water extracts from the selected sample (Fucus vesiculosus) were fractionated using a Hamilton PRP-X100 preparative column, and the presence of arsenic species in the isolated fractions was ascertained by IC-ICP-MS. Two of the fractions successfully presented only one arsenic species corresponding to sulfate and sulfonate arsenosugars at suitable concentrations. To unequivocally confirm the presence of both compounds, high-resolution mass spectrometry (ESI-TOF/MS) was used and the exact mass determined with errors lower than 0.5 ppm. The standard solutions obtained were successfully used to identify and quantify SO4-sug and SO3-sug in several edible algae samples purchased in local market. Total arsenic content for analyzed samples ranged from 34 to 57 mg kg-1, concentration values found for SO3-sug ranged from 5 to 36 mg As kg-1 and SO4-sug was only found in fucus with a concentration of 9.3 mg As kg-1.