Small Molecular Weight Fraction Research Articles

Enzymatic Conversion of Hydrolysis Lignin—A Potential Biorefinery Approach

Lignin is an abundant and renewable source capable of replacing different raw materials in the chemical industry. It can be obtained from lignocellulosic biomass (LCB) via different pretreatment methods. In the present study, hydrolysis lignin (HL) from the SunburstTM pretreatment technology was utilized to investigate its enzymatic conversion. At first, soluble HL fractions were obtained via alkali solubilization followed by acid precipitation, referred to as acid precipitated lignin (APL). Furthermore, the APL was tested with three different bacterial laccases to identify the optimal conditions for its conversion into small molecular weight fractions. Among the tested laccases, Streptomyces coelicolor A3(2) (ScLac) displayed the highest rate of APL conversion with a high lignin dosage and under extremely alkaline conditions, i.e., 50 g/L in 0.25 M NaOH solution, resulting in higher molecular weight fractions. The increase in the molecular weight and quantitative linkages before and after the enzymatic oxidation of the APL were characterized by size exclusion chromatography (SEC), Fourier-transform infrared spectroscopy (FT-IR), and two-dimensional heteronuclear single quantum correlation nuclear magnetic resonance (2D HSQC NMR) methods.

Dynamic Change in Starch Biosynthetic Enzymes Complexes during Grain-Filling Stages in BEIIb Active and Deficient Rice.

Starch is the predominant reserve in rice (Oryza sativa L.) endosperm, which is synthesized by the coordinated efforts of a series of starch biosynthetic-related enzymes in the form of a multiple enzyme complex. Whether the enzyme complex changes during seed development is not fully understood. Here, we investigated the dynamic change in multi-protein complexes in an indica rice variety IR36 (wild type, WT) and its BEIIb-deficient mutant (be2b) at different developmental stages. Gel permeation chromatography (GPC) and Western blotting analysis of soluble protein fractions revealed most of the enzymes except for SSIVb were eluted in smaller molecular weight fractions at the early developing stage and were transferred to higher molecular weight fractions at the later stage in both WT and be2b. Accordingly, protein interactions were enhanced during seed development as demonstrated by co-immunoprecipitation analysis, suggesting that the enzymes were recruited to form larger protein complexes during starch biosynthesis. The converse elution pattern from GPC of SSIVb may be attributed to its vital role in the initiation step of starch synthesis. The number of protein complexes was markedly decreased in be2b at all development stages. Although SSIVb could partially compensate for the role of BEIIb in protein complex formation, it was hard to form a larger protein complex containing over five proteins in be2b. In addition, other proteins such as PPDKA and PPDKB were possibly present in the multi-enzyme complexes by proteomic analyses of high molecular weight fractions separated from GPC. Two putative protein kinases were found to be potentially associated with starch biosynthetic enzymes. Collectively, our findings unraveled a dynamic change in the protein complex during seed development, and potential roles of BEIIb in starch biosynthesis via various protein complex formations, which enables a deeper understanding of the complex mechanism of starch biosynthesis in rice.

Looking for peptides from rice starch processing by-product: Bioreactor production, anti-tyrosinase and anti-inflammatory activity, and in silico putative taste assessment.

One of the major challenges for the modern society, is the development of a sustainable economy also aiming at the valorization of agro-industrial by-products in conjunction with at a significant reduction of generated residues from farm to retail. In this context, the present study demonstrates a biotechnological approach to yield bioactive peptides from a protein fraction obtained as a by-product of the rice starch production. Enzymatic hydrolysis, with the commercial proteases Alcalase and Protamex, were optimized in bioreactor up to 2 L of volume. The two best digestates, selected with respect to peptide release and extract antioxidant capacity, were further fractionated (cut-offs of 10, 5, and 1 kDa) via cross-flow filtration. Amino acid composition indicated that most of the fractions showed positive nutritional characteristics, but a putative bitter taste. A fraction obtained with Alcalase enzyme (retentate 8 kDa) exerted anti-inflammatory potential, while the smaller molecular weight fractions (retentate 1–5 kDa and permeate < 1 kDa) were more active in tyrosinase inhibition. The latter were further sub-fractionated by size-exclusion chromatography. From the 15 most anti-tyrosinase sub-fractions, 365 peptide sequences were identified via liquid chromatography coupled with high resolution mass spectrometry. The present data support the possible exploitation of bioactive peptide from rice starch by-product as ingredients into food, nutraceutical, pharmaceutical, and cosmetic formulations.

Effects of DOM characteristics from real wastewater on the degradation of pharmaceutically active compounds by the UV/H2O2 process

The characteristics of dissolved organic matter (DOM) can significantly affect the degradation of target compounds by the advanced oxidation processes. In this study, the effects of the different hydrophobicity/hydrophilicity fractions, molecular weight (MW) fractions, fluorescence components and molecular components of DOM extracted from municipal wastewater on the degradation of 4 pharmaceutically active compounds (PhACs), including carbamazepine, clofibric acid, atenolol and erythromycin by the UV/H2O2 process were investigated. The results showed that the degradation rate constants of 4 PhACs decreased dramatically in the presence of DOM. The linear regressions of 4 PhACs degradation as a function of specific fluorescence intensity (SFI) are exhibited during the degradation of 4 PhACs and the SFI may be used to evaluate effect of DOM on target compounds in wastewater. The hydrophobic acid (HPO-A) exhibited the strongest inhibitory effect on degradation of 4 PhACs during oxidation process. The small MW fractions of DOM significantly inhibited the degradation of 4 PhACs during oxidation process. Among three fluorescence components, hydrophobic humic-like substances may significantly inhibit the degradation of 4 PhACs during oxidation process. At the molecular level, the formulas may be derived from terrestrial sources. CHO compound may significantly inhibit the degradation of 4 PhACs during oxidation process on formula classes. The unsaturated hydrocarbons, carbohydrates and tannins compounds may significantly inhibit the effectiveness of the UV/H2O2 process on compound classes.

Gamma-irradiation-induced molecular-weight distribution and complexation affinity of humic acid with Cs+, Sr2+, and Eu3+

Solutions of humic acid (HA) were irradiated with 0, 1, 5, 10, 50, and 100 kGy of gamma irradiation using a 60Co source. The non-irradiated and irradiated HA molecules were fractionated by ultrafiltration into four categories: > 100, 50–100, 10–50, and < 10 kDa. Total organic carbon measurements and potentiometric titration analysis suggested that (1) some gamma-irradiated HA molecules were degraded into smaller molecules and (2) radiolytic degradation caused phenolic –OH became the predominant functional group in the small molecular-weight fractions of HA. The effect of absorbed dose of gamma rays on the distributions of Cs+, Sr2+, and Eu3+ ions in the molecular-weight fractions of the metal–HA systems was examined to discuss the complexation affinity. The metal ions were distributed in the smaller molecular-weight fractions at different doses, which corresponded to the degradation of HA molecules. For a predetermined absorbed dose, Cs+ ions did not change the molecular-weight distribution of the total organic carbon content of the degraded HA molecules. Conversely, the Sr2+ and Eu3+ ions redistributed organic carbon toward the larger molecular-weight fractions.

Water and heat stresses during grain formation affect the physicochemical properties of waxy maize starch

Maize is frequently subjected to simultaneous water (drought or waterlogging) and heat (HS) stresses during grain formation in southern China. This work examined the effect of high temperature combined with drought (HD) or waterlogging (HW) during grain formation on the starch physicochemical properties of two waxy maize hybrids, namely Suyunnuo5 (SYN5) and Yunuo7 (YN7). Heat stress enlarged the starch granule size, and water stresses aggravated this effect. Heat stress reduced the ratio of small molecular weight fractions for both hybrids, and HD aggravated this reduction only in SYN5. Relative crystallinity in SYN5 was increased by stresses but in YN7 it was unaffected by HD, reduced by HS, and increased by HW. Fourier-transform infrared (FTIR) spectrometry results showed that the 1045/1022 cm-1 ratio in SYN5 was not influenced by HW but was increased by other stresses, and that in YN7 it was increased by all stresses, with the highest value induced by HW. Peak viscosity was decreased, whereas gelatinization temperatures and retrogradation percentage were increased by all of these stresses. These effects were exacerbated by combined heat and water stresses. The maximum decomposition rate was severely increased by HW. Drought or waterlogging at grain formation stage aggravated the detrimental effects of HS on the starch physicochemical properties of waxy maize. © 2020 Society of Chemical Industry.

Digestive solubilization of Cd in highly-contaminated sediment by marine deposit feeders: The roles of intestinal surfactants in Cd mobilization and Re-Adsorption processes

Marine deposit feeders are of ecological significance in transferring sedimentary Cd along aquatic food chains. A key process for this transfer is these organisms’ dietary uptake of Cd via solubilization of Cd present in ingested contaminated sediment. To better understand the bioavailability of sedimentary Cd to deposit feeders, the present study used in vitro extraction experiments to explore the contribution of different digestive agents (proteins, amino acids and surfactants) to the solubilization of Cd from sediment collected in a highly-contaminated Chinese bay. This was done for various commercially-available mimetic digestive agents (the protein BSA, a mixture of amino acids, and the surfactants rhamnolipid and SDS), and for proteins and surfactants collected from the gut juice of a sipunculan worm. The Cd mobilization capacity of BSA was significantly higher than that of the amino acids and the commercial surfactants. In the presence of BSA, > 70% of the released Cd became associated with this protein. In contrast, the digestive proteins from the sipunculan had a lower Cd mobilization capacity than was the case for the other digestive agents and the majority of the released Cd (∼80%) was associated with small molecular weight fractions. The differences in Cd mobilization between the BSA and the digestive proteins were attributed to differences in their sediment-adsorption tendencies and their Cd-complexing capacities. While the digestive surfactants had minor effects on the release of sedimentary Cd, they significantly enhanced Cd mobilization by the digestive proteins when both were present simultaneously. Our results suggest that the characteristics of proteins should be considered when using commercially-available mimetic digestive agents to explore Cd bioavailability in sediments. Furthermore, digestive surfactants seem to have important effects on the solubilization of Cd during gut passage by reducing the adsorption of the digestive proteins to the sediments.

Treatment of organosilicon wastewater by UV-based advanced oxidation processes: Performance comparison and fluorescence parallel factor analysis

Organosilicon wastewater, generally contains plentiful silicone polymers, high salinity and high chemical oxygen demand (COD), and therefore, has great impact on the environment. This study explored the degradation of organosilicon wastewater by UV-based advanced oxidation processes: UV/H2O2, UV/peroxydisulfate (PDS) and UV/peroxymonosulfate (PMS). At natural pH of wastewater and 100 mM of oxidant concentration, UV/PDS achieved a higher COD removal (90.6%) than UV/PMS (80.8%) after a 240 min reaction time, whereas only half time was required for UV/H2O2 to reach nearly the same COD removal (89.7%) as UV/PDS. Electron paramagnetic resonance (EPR) analysis indicated hydroxyl and sulfate radicals were the main reactive species for COD removal. Parallel factor analysis (PARAFAC) of excitation emission matrix (EEM) fluorescence spectroscopy was employed to monitor the transformation of dissolved organic matter (DOM) present in the organosilicon wastewater. The results illustrated that the tyrosine-, tryptophan- and humic-like substances were three components of DOM and all of them were significantly degraded into smaller molecular weight fractions.

Aerobic denitrification performance of strain Acinetobacter johnsonii WGX-9 using different natural organic matter as carbon source: Effect of molecular weight

The aim of this study was to investigate the effect of natural organic matter (NOM) including humic acid (HA) and fulvic acid (FA), intracellular organic matter (IOM) extracted from Microcystis aeruginosa (MA) and Chlorella sp. (CH), and their different molecular weight (MW) fractions on the aerobic denitrification performance of bacterial strain WGX-9 by monitoring nitrogen removal efficiency and testing changes in organic matter with HA, FA, MA-IOM and CH-IOM as the sole carbon source. Strain WGX-9 was identified as Acinetobacter johnsonii and exhibited excellent aerobic denitrification capability. The nitrate removal efficiency with IOM as the sole carbon source was relatively higher than that with NOM as the sole carbon source. The prepared NOM and extracted IOM samples were separated into six fractions with MW cut-offs of 100, 30, 10, 5 and 1 kDa. The fraction of MW > 100 kDa contributed the largest amount to the MW distribution, accounting for 77.11%, 29.00%, 44.97% and 24.81% of HA, FA, MA-IOM, and CH-IOM, respectively. Nitrate removal efficiency was improved with decreasing MW of organic matter. For example, nitrate removal efficiency was 26.50%, 32.41%, 27.88% and 43.89% using HA, FA, MA-IOM, and CH-IOM fractions of MW > 100 kDa as the carbon source, whereas with MW < 1 kDa, it increased to 36.67%, 37.88%, 60.90%, and 68.90%, respectively. This is probably because the smaller MW fraction is more suitable for bacterial growth. These results demonstrate that the strain WGX-9 can utilize lower MW organic matter, which lays the foundations for nitrogen removal in actual drinking water reservoirs.

Novel natural rubber-based epoxy coating

The use of renewable resources in development of polymeric products is becoming more relevant in the current times, braced by the scarcity of fossil resources and threat from global warming. The manuscript describes the preparation of epoxy resin from epoxidized natural rubber (ENR) via ultraviolet (UV) treatment, followed by investigation on the film properties of the coating produced by blending the natural rubber-based epoxy resin with pentaerythritol tetrakis(3-mercaptopropionate), PETMP as the hardener. The UV treatment involved is responsible to break down the natural rubber into smaller molecular weight fractions so that it becomes more compatible with common solvents and chemicals, and at the same time improves the solubility and viscosity of the resin. The results obtained are very encouraging, with significant reduction in the molecular weight of the rubber observed after 6 hours of UV treatment, and spectroscopic characterization suggests that substantial amount of epoxide group in the ENR was effectively preserved during the treatment for subsequent reaction with PETMP. The epoxide group in the UV-degraded ENR was found to react with the –SH group of PETMP after the two were blended, applied into film, and heated at 80 °C. The properties of the coatings produced were investigated via pencil hardness test, crosshatch adhesion tape test, thermal stability test, water, and chemical resistance test. Most of the coating formulations produced film with excellent hardness, adhesion and chemical resistance. Coating B and C are among the best coating formulations, with both recorded pencil hardness grade ≥ 4H, zero film removal in the adhesion test, no film defect observed during saltwater, acid and alkaline resistance test, and reasonably good thermal stability with onset of degradation above 200 °C.

Fluorescent and molecular weight dependence of THM and HAA formation from intracellular algogenic organic matter (IOM)

This study (i) examined the formation of two major carbonaceous disinfection by-products (C-DBPs), trihalomethanes (THMs) and haloacetic acids (HAAs), during the chlorination of intracellular algogenic organic matter (IOM) extracted from two commonly blooming algae M. aeruginosa (MA) and Chlorella sp. (CH), and (ii) investigated the roles and relationships of fluorescent and molecular weight (MW) properties on/with IOM-derived THMs and HAAs. The extracted IOM samples were separated into different MW fractions by centrifugal devices with membrane support with MW cut-offs of 100, 30, 10, 3, and 1 kDa. We observed an overall reduction of C-DBPs with a decrease of IOM-MW from >100 kDa to <1 kDa. Of six fractionated IOM, a large fraction (>100 kDa) contributed the largest amount to the MW distribution of IOM, accounting for 33 and 42% of the total dissolved organic carbon (DOC) of MA and CH, respectively. It also had the highest-yielding potential to produce significant levels of THMs and HAAs, and total C-DBPs over other small MW fractions. Although small MW fractions (>10 kDa) contributed around 50% of the total DOC, they made an insignificant contribution (>20%) to the THMs, HAAs, and overall C-DBPs. Furthermore, the decrease of IOM MW caused a shift from the domination of HAA formation to THM formation, especially when MW was <10 kDa. By canonical correspondent analysis, the relationship of IOM-derived THMs and HAAs with IOM properties was examined. In particular, large fractions of IOM, exhibiting aromatic protein- (AP) and soluble microbial product- (SMP) like fluorescence, are favorable for the formation of HAAs, whereas small MW fractions of IOM with HA- and FA-like fluorescence preferentially tends to form THMs. Our findings evidently show the strong dependence of IOM-derived THMs and HAAs on the fluorescent and MW properties. Therefore, the characterization of MW and fluorescent properties can provide the advantages in the control of algae-derived DBPs upon the chlorination of eutrophic water.

Ultrafiltration for the Determination of Cu Complexed with Dissolved Organic Matters of Different Molecular Weight from a Eutrophic River, China.

The molecular weight of dissolved organic matter (DOM) is one of the essential factors controlling the properties of metal complexes. A continuous ultrafiltration experiment was designed to study the properties of Cu complexes with different molecular weights in a river before and after eutrophication. The results showed that the concentration of DOM increased from 26.47 to 38.20mg/L during the eutrophication process, however, DOM was still dominated by the small molecular weight fraction before and after eutrophication. The amount of Cu-DOM complexes increased with the increasing of molecular weight, however, the amounts of DOM-Cu complexes before eutrophication were higher than those after eutrophication. This is because DOM contained more -COOH and -OH before eutrophication and these functional groups are the active sites complexed with Cu.

Adsorption of Dissolved Organic Matter with Different Relative Molecular Masses on Inorganic Minerals and Its Influence on Carbamazepine Adsorption Behavior

In this work, sorption experiments were conducted to understand the adsorption characteristics of dissolved organic matter (DOM) of different relative molecular masses on minerals and the effects of their interaction with PPCPs on the adsorption process. This study chose carbamazepine (CBZ) as the target pollutant, quartz sand as the inorganic mineral, and commercial humic acid (HA) as the DOM. We studied the structural characteristics of HA with different relative molecular masses and the impact of their interaction with CBZ on their adsorption based on dialysis experiments and infrared spectroscopy. It was found that large molecular weight fractions of HA, which contain more hydroxyl, aromatic hydrocarbons, and aliphatic hydrocarbons, were mainly hydrophobic substances, while the smaller fractions, containing more alcohols and carboxyl groups, were mainly hydrophilic substances. The combination of HA and CBZ had two major mechanisms, the combination of amino groups of CBZ and polar functional groups in small molecular weight fractions of HA and hydrophobic interactions between hydrophobic groups and aliphatic or/aromatic compounds in large molecular weight HA. The interaction between CBZ and HA resulted in obvious differences in the adsorption of HA or CBZ. When CBZ does not exist, hydrophobic combinations occurred between HA through the hydrophobic components and quartz sand, which also experienced the exchange coordination reaction with the hydroxyl or carboxyl group of HA. In addition, HA adsorbed the aliphatic and hydrophobic macromolecular components. Quartz sand was used to adsorb the large hydrophobic molecules. When CBZ existed, it adsorbed the small molecular weight fractions of HA. The maximum HA adsorption capacity of quartz sand decreased. CBZ could be adsorbed on quartz sand by hydrophobic interaction, van Edward forces, and polarity interactions in the absence of HA. When adding HA, the adsorption increment of carbamazepine on quartz sand was due to the co-adsorption or accumulation of HA and CBZ after their combination.

The molecular weight distribution of dissolved organic carbon after application off different sludge disintegration techniques

Abstract The effect of sludge disintegration on molecular weight distribution of dissolved organic carbon (DOC) is demonstrated by size exclusion chromatography with online organic carbon detection (SEC-OCD). Ozone, ultrasound and sodium hydroxide were used to disintegrate excess sludge. Significant differences on the molecular weight distribution of the released DOC were found for the different treatments. Ozonation did not change the DOC distribution based on the molecular size, compared to untreated sludge. Small molecular weight compounds were the dominant constituents solubilized by sodium hydroxide treatment. Ultrasound disintegrated the sludge flocs and dissolved large molecules into the sludge liquid phase. Especially for sodium hydroxide and ultrasound treatment the analysis with SEC-OCD clearly shows the preferential fractions of organic carbon, which are more quickly transferred to methane in batch anaerobic experiments. For sodium hydroxide, it is the small molecular weight fraction as for ultrasound treatment the degradation is evenly distributed throughout all weight fractions.

Effects of molecular size fraction of DOM on photodegradation of aqueous methylmercury

This study investigated the photodegradation kinetics of MeHg in the presence of various size fractions of dissolved organic matter (DOM) with MW < 3.5 kDa, 3.5 < MW < 10 kDa, and MW > 10 kDa. The DOM fraction with MW < 3.5 kDa was most effective in MeHg photodegradation. Increasing UV intensity resulted in the increase of photodegradation rate of the MeHg in all size of DOM fractions. Higher rates of MeHg degradation was observed at higher pH. For the portion of MW < 3.5 kDa, the photodegradation rate of MeHg increased with increasing DOM concentration, indicating that radicals such as singlet oxygen (1O2) radicals can be effectively produced by DOM. At higher portion of MW > 3.5 kDa, the inhibition of MeHg degradation was observed due to the effect of DOM photo-attenuation. Our result indicates that radical mediated reaction is the main mechanism of photodegradation of MeHg especially in the presence of MW < 3.5 kDa. Our results imply that the smaller molecular weight fraction (MW < 3.5 kDa) of DOM mainly increased the photodegradation rate of MeHg.

Changes in the Antioxidant Potential of Nori Sheets During in vitro Digestion with Pepsin

ABSTRACTNori, the dried sheets of Porphyra, is a popular edible seaweed and a potential source of high quality protein, vitamins, and minerals. In the present study, the antioxidant potential of Nori has been investigated. For this purpose, an aqueous extract was obtained by soaking powdered Nori in KCl-HCl buffer and hydrolyzing it with pepsin. The antioxidant activity of aqueous extract of crude as well as pepsin hydrolyzate was evaluated using 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, reducing power, total antioxidant capacity and lipid peroxidation by thiobarbituric acid reactive substances (TBARS) and compared with L-ascorbic acid. After hydrolysis, the scavenging ability and inhibition of lipid per oxide was doubled, and a sevenfold increase in reducing power activity was observed. The small molecular weight fractions (F-II) of hydrolyzate obtained after gel filtration showed further increase in the activity. These results demonstrate that hydrolysis produces better antioxidants that could be used as an alternative to synthetic antioxidants.

Limited effects of land use on soil dissolved organic matter chemistry as assessed by excitation–emission fluorescence spectroscopy and molecular weight fractionation

AbstractDissolved organic matter (DOM) in soil solution represents a complex mixture of organic molecules and plays a central role in carbon and nitrogen cycling in plant–microbial–soil systems. We tested whether excitation–emission matrix (EEM) fluorescence spectroscopy can be used to characterize DOM and support previous findings that the majority of DOM is of high molecular weight (MW). EEM fluorescence spectroscopy was used in conjunction with MW fractionation to characterize DOM in soil solution from a grassland soil land management gradient in North Wales, UK. Data analysis suggested that three distinct fluorescence components could be separated and identified from the EEM data. These components were identified as being of humic‐like or fulvic‐like origin. Contrary to expectations, the majority of the fluorescence signal occurred in the small MW (&lt;1 kDa) fraction, although differences between soils from the differently managed grasslands were more apparent in larger MW fractions. We conclude that following further characterization of the chemical composition of the fluorophores, EEM has potential as a sensitive technique for characterizing the small MW phenolic fraction of DOM in soils.

Role of NOM molecular size on iodo-trihalomethane formation during chlorination and chloramination

Natural organic matter (NOM) is the major precursor for the generation of disinfection byproducts (DBPs) during disinfection, but the role of the NOM molecular size on the formation of iodinated DBPs (I-DBPs) is still unclear. The objective of this study was to evaluate the function of the NOM molecular size on the formation of iodo-trihalomethane (I-THMs) during chlorination and chloramination. Humic acid was adopted as the NOM matrix and fractionated into four molecular weight (MW) groups. Various parameters, including iodide, bromide, NOM concentrations, pH, and pre-chlorination time, were investigated for each MW fraction. During chlorination, high MW fractions (i.e., MW > 100 K Da and 50 K < MW < K00 K Da) produced more I-THMs compared with small MW fractions (i.e., MW < 3 K Da and 3 K < MW < 50 K Da). With the increase in the I− or NOM concentration, the formation of I-THMs increased for small MW fractions, while a slight reduction occurred for high MW fractions during chlorination. Higher pH resulted in more I-THM formation for small MW fractions, while the opposite was true for high MW fractions during chlorination. Compared to small MW fractions, bromide was relatively more reactive with high MW fractions in the formation of I-THMs during chlorination. During chloramination, the I-THM yields decreased with the increasing NOM concentration for high MW fractions. The concentration of bromine-containing I-THMs decreased with increasing pH for all MW fractions during chloramination. Additionally, with the prolongation of pre-chlorination time, the total amount of I-THMs decreased remarkably for MWs higher than 3 K Da, while a slight change for MW lower than 3 K Da occurred during chloramination. The results from this study suggest that the molecular weight of the NOM plays an important role in the formation of I-THMs during chlorination and chloramination.

A targeted strategy for ingredients analysis and enrichment by mass-based preparative LC method: application to three isomeric C21 steroids from Marsdenia tenacissima

This paper proposed a strategy to acquire specified ingredients in small amounts from a complicated herbal extract by combination of simple Sephadex LH-20 column chromatography and a mass-based preparative liquid chromatography (LC) method. The developed method was applied to the analysis and enrichment of tenacigenin A, tenacigenin B, and 17β-tenacigenin B, which lack any significant chromophores from Marsdenia tenacissima. A chloroform extract of M. tenacissima was initially subjected to a Sephadex LH-20 column to yield two fractions. From 20 mg of the smaller molecular weight fraction, 3.0 mg tenacigenin A, 2.0 mg tenacigenin B and 3.0 mg 17β-tenacigenin B were successfully separated via preparative-LC employing mass-based fraction collection. In contrast to the active splitter system previously applied, the modified purification system via a tee was economic with a high convenience. The purities of the three obtained compounds were all above 90% as determined by LC coupled with evaporative light scattering detection (ELSD), and their structures were identified by 1D NMR spectra. The results demonstrated that the proposed strategy may well be an efficient and environmentally friendly technique for systematic isolation of bioactive components from traditional medicinal herbs.

TLR ligands of ryegrass pollen microbial contaminants enhance Th1 and Th2 responses and decrease induction of Foxp3hi regulatory T cells

Microbial contamination of grass pollens could affect sensitization, subsequent allergic response, and efficacy of allergen-specific immunotherapy. We investigated whether bacterial immunomodulatory substances can direct PBMC responses of allergic and nonatopic subjects against ryegrass pollen (RGP) toward Th1, Th2, or regulatory T (Treg) cells. Aqueous extracts of RGP with high or low LPS were fractionated into large and small molecular weight (MW) components by diafiltration. CFSE-labeled PBMCs from allergic and nonatopic subjects were stimulated with RGP extracts (RGPEs) and analyzed for cytokine secretion and T-cell responses. High LPS RGPE increased IFN-γ(+) Th1 and IL-4(+) Th2 effector cell induction and consistently decreased CD4(+) Foxp3(hi) Treg-cell induction. IL-10-producing T-cell frequency was unaltered, but IL-10 secretion was increased by high LPS RGPE. RGPE-stimulation of TLR-transfected cell lines revealed that high LPS pollen also contained a TLR2-ligand, and both batches a TLR9-ligand. Beta-1,3-glucans were detected in large and small MW fractions and were also T-cell stimulatory. In conclusion, coexposure to allergen and proinflammatory microbial stimuli does not convert an established Th2- into a Th1-response. Instead, proinflammatory responses are exacerbated and Foxp3(hi) Treg-cell induction is decreased. These findings show that adjuvants for specific immunotherapy should enhance Treg cells rather than target immune deviation from Th2 to Th1.