Novel flavan-3-ol-dithiothreitol conjugates derived from the degradation of grape seed proanthocyanidins and their neuroprotective potential.

Dithiothreitol (DTT) was adopted as a nucleophile to develop a new acid-catalyzed degradation method for grape seed proanthocyanidin extraction (GSPE). Backpropagation neural network and Box-Behnken design were employed and compared to establish the optimized degradation conditions. GSPE was reacted with DTT at a ratio of 1:1 under mild conditions with 0.14 M HCl at 40.8 °C for 60 min. Three monomeric proanthocyanidins and six novel flavan-3-ol-DTT conjugates consisting of three pairs of diastereomers were simultaneously obtained with a high yield (929 mg/g). All the degradation products showed protective effects against Aß25-35-induced neurotoxicity in PC-12 cells and prevented Aß25-35 aggregation based on the results from MTT and thioflavin T fluorescence assays, respectively. Detailed intermolecular interactions leading to the prevention of Aß25-35 aggregation were elucidated using molecular docking. This work would provide new compounds from functional foods that can be explored for their neuroprotective potential.

A saponin-polybromophenol antibiotic (CU1) from Cassia fistula Bark Against Multi-Drug Resistant Bacteria Targeting RNA polymerase.

BACKGROUND: Gradual increase of multidrug resistant infections is a threat to the human race as MDR plasmids have acquired.>10 mdr and drug efflux genes to inactivate antibiotics. Plants secret anti-metabolites to retard growth of soil and water bacteria and are ideal source of antibiotics. PURPOSE: Purpose of the study is to discover an alternate phyto-drug from medicinal plants of India that selectively kills MDR bacteria. METHODS: MDR bacteria isolated from Ganga river water, milk, chicken meat and human hair for testing phyto-extracts. Eighty medicinal plants were searched and six phyto-extracts were selected having good antibacterial activities as demonstrated by agar-hole assays giving 15 â€‹mm or greater lysis zone. Phyto-extracts were made in ethanol or methanol (1:5 w/v) for overnight and were concentrated. Preparative TLC and HPLC were performed to purify phytochemical. MASS, NMR, FTIR methods were used for chemical analysis of CU1. In vitro RNA polymerase and DNA polymerase assays were performed for target identification. RESULTS: CU1 belongs to a saponin bromo-polyphenol compound with a large structure that purified on HPLC C18 column at 3min. CU1 is bacteriocidal but three times less active than rifampicin in Agar-hole assay. While in LB medium it shows greater than fifteen times poor inhibitor due to solubility problem. CU1 inhibited transcription from Escherichia coli as well as Mycobacterium tuberculosis RNA Polymerases. Gel shift assays demonstrated that CU1 interferes at the open promoter complex formation step. On the other hand CU1 did not inhibit DNA polymerase. CONCLUSION: Phyto-chemicals from Cassia fistula bark are abundant, less toxic, target specific and may be a safer low cost drug against MDR bacterial diseases.

Remote loading paclitaxel-doxorubicin prodrug into liposomes for cancer combination therapy.

The combination of paclitaxel (PTX) and doxorubicin (DOX) has been widely used in the clinic. However, it remains unsatisfied due to the generation of severe toxicity. Previously, we have successfully synthesized a prodrug PTX-S-DOX (PSD). The prodrug displayed comparable in vitro cytotoxicity compared with the mixture of free PTX and DOX. Thus, we speculated that it could be promising to improve the anti-cancer effect and reduce adverse effects by improving the pharmacokinetics behavior of PSD and enhancing tumor accumulation. Due to the fact that copper ions (Cu2+) could coordinate with the anthracene nucleus of DOX, we speculate that the prodrug PSD could be actively loaded into liposomes by Cu2+ gradient. Hence, we designed a remote loading liposomal formulation of PSD (PSD LPs) for combination chemotherapy. The prepared PSD LPs displayed extended blood circulation, improved tumor accumulation, and more significant anti-tumor efficacy compared with PSD NPs. Furthermore, PSD LPs exhibited reduced cardiotoxicity and kidney damage compared with the physical mixture of Taxol and Doxil, indicating better safety. Therefore, this novel nano-platform provides a strategy to deliver doxorubicin with other poorly soluble antineoplastic drugs for combination therapy with high efficacy and low toxicity.

Assessment of the PM2.5 oxidative potential in a coastal industrial city in Northern France: Relationships with chemical composition, local emissions and long range sources.

The objective of this work was to relate PM2.5 Oxidative Potential (OP) data to PM composition and PM local and distant source contributions. PM2.5 collected in Dunkerque, a coastal industrial city in North of France, was extensively characterized for major and minor chemical species. PM2.5 filters were extracted using a synthetic pulmonary fluid to achieve OP estimation based on Ascorbic Acid (AA) and dithiothreitol (DTT) depletion assays. In order to evidence relationships between OP values, chemical composition and local and distant source contributions, correlation coefficient, Principal Component Analysis (PCA), concentration roses, polar plots and concentration weighted trajectories were used. Heterogeneous conclusions were drawn using the three first methods as the bivariate polar plots lead to dismiss some of the correlations evidenced using correlation coefficient and PCA. Both AA and DTT tests appeared complementary as they were not sensitive to the same species/source contribution. The bivariate polar plot representation of OP values versus wind direction and wind speed revealed that PM2.5 concentration and combustion sources were linked to OP-AA, whereas emissions from integrated steelworks, electric steelworks, heavy fuel oil combustion and traffic non-exhaust significantly contribute to OP-DTT. Sea-salts, aged sea-salts, crustal, secondary sulfates and secondary nitrates sources were not found to contribute to OP values. Constant weighted trajectories evidenced several source regions responsible for high OP values with Belgium, Germany, Netherlands and France at the leader position. Contribution of inland regions appeared possibly related to the biomass and traffic related combustion while heavy fuel oil combustion could also be involved in the contribution of marine and coastal areas.

Monoacylglycerol lipase inhibitors: modulators for lipid metabolism in cancer malignancy, neurological and metabolic disorders.

Monoacylglycerol lipase (MAGL) is a serine hydrolase that plays a crucial role catalysing the hydrolysis of monoglycerides into glycerol and fatty acids. It links the endocannabinoid and eicosanoid systems together by degradation of the abundant endocannabinoid 2-arachidaoylglycerol into arachidonic acid, the precursor of prostaglandins and other inflammatory mediators. MAGL inhibitors have been considered as important agents in many therapeutic fields, including anti-nociceptive, anxiolytic, anti-inflammatory, and even anti-cancer. Currently, ABX-1431, a first-in-class inhibitor of MAGL, is entering clinical phase 2 studies for neurological disorders and other diseases. This review summarizes the diverse (patho)physiological roles of MAGL and will provide an overview on the development of MAGL inhibitors. Although a large number of MAGL inhibitors have been reported, novel inhibitors are still required, particularly reversible ones.

Noni (Morinda citrifolia L.) fruit as a new source of milk-clotting cysteine proteases.

This work reports the characterisation of caseinolytic and milk-clotting activities of proteases extracted from ripe fruits of Morinda citrifolia L., as a potential of their use in cheese production. Noni puree extract (NPE) was obtained by homogenising the fresh puree in 150 mM NaCl/50 mM sodium phosphate buffer (pH 7.0). The resulting protein concentration was of 0.367 ±â€¯0.006 mg/mL, and an electrophoretic profile of the extract revealed protein bands ranging from 14 to 55 kDa. The proteolytic activity of NPE was higher when the extract had been previously incubated at pH 6.0 (8.859 ±â€¯0.216 U/mg), whereas the optimum caseinolytic activity was observed at 50 °C. Noni puree proteases were strongly (98%) inhibited by iodoacetamide and E-64, suggesting the presence of only cysteine proteases in the crude extract. NPE proteases showed a milk-clotting activity (MCA) of 238.80 ±â€¯5.29 U/mL, a specific milk-clotting activity (SMCA) of 9950.17 ±â€¯220.74 U/mg, and an SMCA/PA ratio of 1124.31 ±â€¯24.94, this last being comparable to those of commercial calf rennet. The cheese manufactured using NPE presented brittle and soft texture, high humidity, and showed sanitary conditions compatible with current Brazilian regulations. The product showed a slightly bitter taste, but still good acceptability, rating between 6 and 7 in the hedonic scale for flavour, texture, and overall acceptance. Lastly, there was 60% of positive purchase intent, demonstrating that noni fruit is a promising source of milk-clotting enzymes for the dairy industry.

The effect of α-tocopherol and dithiothreitol in ameliorating emamectin benzoate cytotoxicity in human K562 cells involving the modulation of ROS accumulation and NF-κB signaling.

Emamectin benzoate (EMB) toxicity contributes a potential risk to environment and human health. To investigate the effect of α-tocopherol (VitE) and dithiothreitol (DTT) in ameliorating EMB-induced cytotoxicity in human K562 cells, in vitro cultured human K562 cells were incubated with different concentrations of EMB in supplement with VitE and DTT when the cells were in the logarithmic phase. Next, the cell growth inhibition was evaluated using the MTT assay and cellular morphology observation. Reactive oxygen species (ROS) production was monitored using DCFH-DA probe and NF-κB signaling was determined using Western blotting. The results demonstrated that treatment with EMB (time- and concentration-dependent) showed significantly greater inhibition on K562 cell viability, heavier chromatin condensation and DNA fragmentation, and stronger suppression of NF-κB/p105 and p65/RelA expression of K562 cells than the control group (p < 0.01). The supplementation of VitE or DTT could help protect K562 cells against EMB-induced cytotoxicity by improving cell viability, preventing ROS accumulation and up-regulating NF-κB signaling through their ameliorating effects against oxidative stress induced by EMB. VitE had a stronger synergistic effect in limiting EMB cytotoxicity than DTT. Our findings indicate that VitE and DTT are potent antioxidants for human K562 cells, offering a promising means of ameliorating EMB cytotoxicity.

Improved analysis of anthocyanins and vitamin C in blue-purple potato cultivars.

Methods were optimized for extraction and quantification of anthocyanins (ACY) and vitamin C in potatoes. Acidified aqueous methanol (70%) was the optimal extraction solvent and freeze-drying significantly improved the extraction yield of ACY. The content of ACY varied widely in five potato cultivars from 0.42 to 3.18mg/g dry weight, with the latter being the highest value found in the Finnish cultivar 'Synkeä Sakari'. Compared with dithiothreitol (DTT), tris(2-carboxyethyl) phosphine hydrochloride (TCEP) was more efficient in reducing dehydroascorbic acid (DHA) to ascorbic acid (AA) and for quantifying the content of total ascorbic acid (TAA). For raw potatoes, quantification of TAA after treatment with TCEP was more reliable than a direct analysis of AA, whereas AA can be analyzed directly in steam-cooked samples. The TAA contents in the three potato cultivars were around 0.30-0.35mg/g dry weight. The loss of AA in steam cooking was 24%.

Tissue specific effects of feeds supplemented with grape pomace or olive oil mill wastewater on detoxification enzymes in sheep.

The aim of the present study was to investigate the effects of livestock feed supplemented with grape pomace (GP) or olive oil mill wastewater (OMW) byproducts on the enzymatic activity and protein expression of antioxidants enzymes, in liver and spleen tissue of sheep. Thus, 36 male sheep of Chios breed were divided into 3 homogeneous groups, control group (n = 12), GP group (n = 12) and OMW group (n = 12), receiving standard or experimental feed. Liver and spleen tissues were collected at 42 and 70 days post-birth. The enzymatic activity of superoxide dismutase (SOD) and glutathione-s-transferase (GST) and also the protein expression of γ-synthase glutamyl custeine (γ-GCS) were determined in these tissues. The results showed GP group exhibited increased enzymatic activity of GST and protein expression of γ-GCS in liver compared to control group. In GP group's spleen, GST activity was increased compared to control but γ-GCS expression was not affected. In OMW group's liver, GST activity was increased and γ-GCS expression was reduced compared to control. In OMW group's spleen, GST activity was increased but GCS expression was not affected. SOD activity was not affected in both tissues either in GP or OMW group.

Epiberberine, a natural protoberberine alkaloid, inhibits urease of Helicobacter pylori and jack bean: Susceptibility and mechanism.

In our previous study, Rhizoma Coptidis extract was found to exert more potent inhibitory effect than its major component berberine towards urease from Helicobacter pylori (HPU) and jack bean (JBU). In continuation of our work, the present study was designed to further comparatively investigate the urease inhibitory activities of five major protoberberine alkaloids in Rhizoma Coptidis, namely berberine, palmatine, coptisine, epiberberine, jateorhizine to identify the bioactive constituent, and illuminate the potential mechanism of action. Results indicated that the five protoberberine alkaloids acted as concentration-dependent inactivators of urease with IC50 values ranging between 3.0 and 5087µM for HPU and 2.3->10,000µM for JBU, respectively. Notably, epiberberine (EB) was found to be the most potent inhibitor against both ureases with IC50 values of 3.0±0.01µM for HPU and 2.3±0.01µM for JBU, which was more effective than the standard urease inhibitor, acetohydroxamic acid (83±0.01µM for HPU and 22±0.01µM for JBU, respectively). Further kinetic analysis revealed that the type of EB inhibition against HPU was slow-binding and uncompetitive, with Ki of 10.6±0.01µM, while slow-binding and competitive against JBU with Ki of 4.6±0.01µM. Addition of thiol reagents, such as l-cysteine, glutathione and dithiothreitol, significantly abolished the inhibition, while Ni2+ competitive inhibitors, boric acid and sodium fluoride, synergetically inhibited urease with EB, indicating the obligatory role of the active site sulfhydryl group for the inhibition. In addition, binding of EB with the urease proved to be reversible, as about 65% and 90% enzymatic activity of HPU and JBU, respectively, could be restored by dithiothreitol application. These findings highlighted the potential role of Rhizoma Coptidis protoberberine alkaloids, especially EB, as a lead urease inhibitor in the treatment of diseases associated with ureolytic bacteria. Thus, EB had good potential for further development into a promising therapeutic approach for the treatment of urease-related diseases.

EDTA is superior to DTT treatment for overcoming the prozone effect in HLA antibody testing.

A limitation of solid-phase human leukocyte antigen (HLA) antibody assays is the falsely low/negative result of samples with high-titer antibodies, a phenomenon known as the prozone effect. Here we compared the efficacy of ethylenediaminetetraacetic acid (EDTA) and dithiothreitol (DTT) treatment of serum samples in overcoming the prozone effect. A total of 21 serum samples were treated with either EDTA or DTT before HLA single antigen bead assay. The efficacy of prozone effect reversal, compared with untreated samples, was examined on fourfold, serially diluted samples, from neat to 1:256, using PBS as diluent. EDTA reversed the prozone effect in all tested samples, with an efficiency of greater than 84%, estimated by the ratio of undiluted sample mean fluorescence intensity (MFI) to peak MFI, for any given dilution. In contrast, the efficiency of DTT treatment was as low as 47%. These results show superior prozone effect reversal with EDTA treatment, compared with DTT.

Dithiothreitol enhanced arsenic-trioxide-induced cell apoptosis in cultured oral cancer cells via mitochondrial dysfunction and endoplasmic reticulum stress.

Arsenic is naturally occurring toxic metalloid and drinking As2 O3 containing water are recognized to be related to increased risk of neurotoxicity, liver injury, blackfoot disease, hypertension, and cancer. On the contrary, As2 O3 has been an ancient drug used in traditional Chinese medicine with substantial anticancer activities, especially in the treatment of acute promyelocytic leukemia as well as chronic wound healing. However, the cytotoxicity and detail mechanisms of As2 O3 action in solid cancer cells, such as oral cancer cells, are largely unknown. In this study, we have primarily cultured four pairs of tumor and nontumor cells from the oral cancer patients and treated the cells with As2 O3 alone or combined with dithiothreitol (DTT). The results showed that 0.5 µM As2 O3 plus 20 µM DTT caused a significant cell death of oral cancer cells but not the nontumor cells. Also As2 O3 plus DTT upregulated Bax and Bak, downregulated Bcl-2 and p53, caused a loss of mitochondria membrane potential in oral cancer cells. On the other way, As2 O3 also triggered endoplasmic reticulum stress and increased the levels of glucose-regulated protein 78, calpain 1 and 2. Our results suggest that DTT could synergistically enhance the effects of As2 O3 on killing oral cancer cells while nontoxic to the nontumor cells. The combination is promising for clinical practice in oral cancer therapy and worth further investigations. © 2015 Wiley Periodicals, Inc. Environ Toxicol 32: 17-27, 2017.

Proteomic analysis of coffee grains exposed to different drying process.

Many biochemical events occur inside grains during post-harvest processes. Several methods have been developed to relate the chemical composition of the coffee grain to the beverage quality, including identification of possible molecular markers for flavor characterizing. This study was aimed at evaluating the changes in the proteomic profile of pulped and natural C. arabica grains dried in a yard or dryer at 60°C. It was observed that fruits dried in a dryer at 60°C showed an altered proteomic profile, with a reduction in the most abundant proteins compared to those yard-dried grains. Among the identified proteins, those involved in the metabolism of sugars and stress response were highlighted. Results have shown that post-harvest processes that impact coffee quality are related to changes in protein abundance, indicating that proteomic analysis may be effective in the identification of biochemical changes in coffee grains subjected to different post-harvest processes.

Uvaria rufa Blume attenuates benign prostatic hyperplasia via inhibiting 5α-reductase and enhancing antioxidant status.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional medicine has used Uvaria rufa Blume as an ethnomedicinal plant for treating fever, skin allergies, intestinal ulcers and prostate disorders including BPH. However, no scientific evidence supports the traditional use. AIM OF THE STUDY: This study aimed to evaluate the therapeutic potential of U. rufa on BPH using in vitro and in vivo models. MATERIALS AND METHODS: In vitro studies screened the efficacy of a 5α-reductase (5αR) inhibition and antioxidant activity of petroleum ether, ethyl acetate, ethanol and aqueous extracts from the stem of U. rufa. Phytochemical screening was performed to determine the active compound using high-performance liquid chromatography (HPLC). Ethyl acetate extract (UR-EtOAc) of U. rufa was used to evaluate the therapeutic efficacy in vivo models. BPH was induced by subcutaneous injection of testosterone propionate (3mg/kg) to male rats for 30 days. After 30 days of oral administration of UR-EtOAc at doses of 10 and 20mg/kg and finasteride at a dose of 1mg/kg, the prostate weight, prostate index (PI), testosterone and androgen receptor (AR) levels, and histopathological alteration of prostate gland were determined. Also, oxidative status and toxicity indices were assessed. RESULTS: UR-EtOAc exhibited the highest potency of inhibition of 5αR and possessed potent antioxidants rich in phenolics and flavonoids contents. The active compound analyzed by HPLC was ß-sitosterol. In vivo results show a significant reduction in prostate weight, PI, and AR in all treated groups when compared to the BPH model group (P<0.001). Also, the UR-EtOAc and finasteride treated groups had increased prostatic and serum testosterone levels when compared to the BPH model group. A histopathological investigation of the prostate glands supported the above results. UR-EtOAc elevated the antioxidant enzymes and reduced the malondialdehyde level in BPH-induced rats. Moreover, treatment of UR-EtOAc at all doses had no toxic effects on the vital organs and serum biochemical indices. CONCLUSIONS: UR-EtOAc from the stem of Uvaria rufa Blume appears to have the potential as a phytotherapeutic agent in the management of BPH, which provides the scientific evidence for traditional use.

Urban PM2.5 oxidative potential: Importance of chemical species and comparison of two spectrophotometric cell-free assays.

Oxidative potential (OP) of particulate matter (PM) - defined as the capacity of PM to oxidize target molecules generating reactive oxygen species (ROS) - has been proposed as a more health relevant metric than PM mass. In this study two cell-free methods were used to assess the OP of PM filters collected at an urban site and to evaluate correlation with PM mass and PM composition. Among the different assays existing, two inexpensive and user-friendly methods were used both based on spectrophotometric measurements of depletion rate of target reagents oxidized by redox-active species present in PM. One assay measures the consumption of dithiothreitol (OPDTT) and the other the ascorbate (OPAA). Although both assays respond to the same redox-active species, i.e., quinones and transition metals, no correlations were found between OPDTT and OPAA responses to compounds standard solutions as well as to ambient samples. When expressed in relation to air volume, OPDTT m-3 strongly correlates with PM2.5 mass whereas no correlation was found for OPAA m-3 with PM2.5. When expressed on mass basis, both OPDTT µg-1 and OPAA µg-1 show a strong dependence on the sample composition, with higher OP for summer samples. OPDTT m-3 were highly correlated with the determined metals (Cu, Zn, Cr, Fe, Ni, Mn) whereas OPAA m-3 showed only moderate correlation with Cu and Mn. Thus, the two assays could potentially provide complementary information on oxidative potential characteristic of PM. Consequently, the combination of the two approaches can strengthen each other in giving insight into the contribution of chemical composition to oxidative properties of PM, which can subsequently be used to study health effects.

Lycium barbarum polysaccharides attenuates N-methy-N-nitrosourea-induced photoreceptor cell apoptosis in rats through regulation of poly (ADP-ribose) polymerase and caspase expression.

ETHNOPHARMACOLOGICAL RELEVANCE: Lycium barbarum L., popularly known as "Goji berry", a classic of Traditional Chinese Medicine has long been used to treat ocular diseases and cardiovascular diseases. Recently, the photoreceptor cell protection of Lycium barbarum polysaccharides (LBP), a water extract from Lycium barbarum L. has received more attention. The present study was designed to investigate the effect of LBP on N-methyl-N-nitrosourea (MNU)-induced photoreceptor cell apoptosis, and the involvement of the poly (ADP-ribose) polymerase (PARP) and caspase. MATERIALS AND METHODS: Photoreceptor cell injury was induced in male Sprague-Dawley rats by an intraperitoneal injection of MNU 60mg/kg. Seven days prior to MNU injection, LBP were intragastrical administered daily, rats were sacrificed at 24h and 7 days after MNU injection. Retinal morphologies, photoreceptor cells apoptosis, and protein expression were evaluated at 24h and 7 days after MNU injection. RESULTS: Morphologically, the outer nuclear layer was well preserved in the LBP-treated rat retinas throughout the experimental period. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-digoxigenin nick-end labeling (TUNEL) assays showed that LBP could significantly suppress the loss of photoreceptor cells, as determined by the photoreceptor cell ratio at the central retina 24h and 7 days after MNU administration. Western-blot analysis demonstrated the expression levels of procaspase-9, -7, -3 and cleaved caspase-9, -7, -3 were upregulated, and PARP were downregulated both 24h and 7 days after MNU injection. LBP treatment significantly decreased protein levels of procaspase and cleaved caspase, increased the level of PARP and cleaved PARP on 24h and 7 days. CONCLUSIONS: LBP inhibits MNU-induced rat photoreceptor cell apoptosis and protects retinal structure via the regulation of the expressions of PARP and caspase.

Nitric oxide overcomes Cd and Cu toxicity in in vitro-grown tobacco plants through increasing contents and activities of rubisco and rubisco activase.

Toxic heavy metals such as cadmium (Cd) and copper (Cu) are global problems that are a growing threat to the environment. Despite some heavy metals are required for plant growth and development, others are considered toxic elements and do not play any known physiological role in plant cells. Elevated doses of Cd or Cu cause toxicity in plants and generate damages due to the stress condition and eventually cause a significant reduction in quantity and quality of crop plants. The nitric oxide (NO) donor sodium nitroprusside (SNP) is reported to alleviate the toxicity of some heavy metals like Cd and Cu. In the current study, the role of NO in alleviating stresses of Cd and Cu was investigated in in vitro-grown tobacco (Nicotiana tabacum) Based on plant growth, total chlorophyll contents, contents and activities of rubisco and rubisco activase. According to the results of this study, the growth and total chlorophyll contents of Cd/Cu stressed plants were hugely decreased in the absence of SNP, while the supplementation of SNP resulted in a significant increase of both fresh weight and total chlorophyll contents. Remarkable reductions of Rubisco and rubisco activase contents and activities were observed in Cd and Cu-induced plants. SNP supplementation showed the highest contents and activities of rubisco and rubisco activase compared to the control and Cu/Cd-stressed plants. Taken together, our findings suggest that SNP could play a protective role in regulation of plant responses to abiotic stresses such as Cd and Cu by enhancing Rubisco and Rubisco activase.

Development and implementation of an HPLC-ECD method for analysis of vitamin C in plasma using single column and automatic alternating dual column regeneration.

OBJECTIVES: Vitamin C (l-ascorbic acid) is a water-soluble micronutrient necessary for human life. Inadequate intake can lead to the fatal disease scurvy. Measurement of vitamin C is used to assess nutritional status and to monitor supplementation. The goal of this study was to develop a chromatographic method for the quantitation of vitamin C in human plasma. DESIGN AND METHODS: Samples were prepared by protein precipitation, addition of internal standard, and reduction with dithiothreitol. Separation of ascorbic acid was accomplished by isocratic elution on a reverse-phase column; concentration was determined by coulometry. The method was validated through studies of assay linearity, sensitivity, imprecision, accuracy, analytical specificity, and carryover. RESULTS: The new assay was developed using a single pump/single analytical column HPLC system. Results correlated well with our previously used spectrophotometric method. The analytical measurement range was 1.0-2500 µmol/L. The injection-to-injection time was 13 min. Subsequently, to increase method throughput and shorten turnaround time, a dual LC pump system with a 2-position/10-port switching valve capable of performing automatic alternating column regeneration was validated and implemented. The injection-to-injection time was reduced 2-fold to 6 min. The method was linear to 5000 µmol/L; limit of quantification was 1.9 µmol/L. Total imprecision was less than 5%. CONCLUSIONS: We have developed a robust method suitable for routine clinical measurement of vitamin C in plasma specimens. The method incorporates a simplified sample preparation and a stable, non-endogenous internal standard to specifically quantify vitamin C. Faster throughput was achieved by employing an automatic alternating column regeneration system.

Celastrol blocks binding of lipopolysaccharides to a Toll-like receptor4/myeloid differentiation factor2 complex in a thiol-dependent manner.

ETHNOPHARMACOLOGICAL RELEVANCE: Tripterygium wilfordii (lei gong teng; Thunder of God Vine), which belongs to the Celastraceae family, has long been used in traditional Chinese medicine to treat inflammation and rheumatoid arthritis. Celastrol is a bioactive compound isolated from T. wilfordii. AIM OF THE STUDY: We investigated whether celastrol suppressed binding of lipopolysaccharides (LPS) to myeloid differentiation factor 2 (MD2), thereby downregulating Toll-like receptor4 (TLR4) activation in mouse primary macrophages. MATERIALS AND METHODS: Cytokine expression was determined by polymerase chain reaction analysis and enzyme-linked immunosorbent assay in bone marrow-derived primary macrophages (BMDMs). The kinase activity of tank-binding kinase 1 (TBK1) was examined by a luciferase reporter assay and an in vitro kinase assay. LPS binding to MD2 was examined by an in vitro binding assay and confocal microscopy analysis. RESULTS: Celastrol reduced LPS-induced expression of inflammatory cytokines, such as tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-12, and IL-1ß, at both the mRNA and protein levels in BMDMs. Celastrol suppressed LPS binding to MD2, as shown by the in vitro binding assay, whereas it did not inhibit TBK1. In addition, co-localization of LPS with MD2 in BMDMs was blocked by celastrol. The inhibitory effects of celastrol on LPS binding to MD2 were reversed by thiol donors (N-acetyl-L-cysteine and dithiothreitol), suggesting that the thiol reactivity of celastrol contributes to its inhibitory effects on TLR4 activation in macrophages. CONCLUSION: Our results demonstrate that celastrol suppresses TLR4 activation through the inhibition of LPS binding to the TLR4/MD2 complex. These results provide a novel mechanism of action by which celastrol contributes to the anti-inflammatory activity of T. wilfordii.

Decrease in the acrylamide content in canned coffee by heat treatment with the addition of cysteine.

Acrylamide (AA) is classified as a Group 2A carcinogen according to the International Agency for Research on Cancer. Although coffee contains a small amount of AA, it is a popular beverage worldwide. Approximately 10 billion canned coffees are consumed each year in Japan. In this study, we investigated how to decrease AA contained in canned coffee by modifying the heat treatment used for sterilization during the manufacturing process. The AA content of both types of canned coffee (black and milk) was decreased by approximately 95% by heat treatment with adding cysteine at 121 °C for 6 min. The content was also decreased by heat treatment with dithiothreitol, although that with cystine had no effect. Therefore, it is shown that thiol groups in cysteine and dithiothreitol might play an important role in decreasing the AA content.