The half-maximal inhibitory concentration (IC50) values for maltase were as follows: B2-3’-O-gallate (1.73 ± 1.37 μM), ECG (3.64 ± 2.99 μM), and EC (6.25 ± 1.84 μM). Inhibition kinetic assays revealed the inhibition constants (Ki) of the mixed-competitive inhibitors of maltase, as follows: B2-3’-O-gallate (1.99 ± 0.02 μM), ECG (3.14 ± 0.04 μM), and EC (7.02 ± 0.26 μM). These compounds also showed a strong inhibitory activity toward sucrase, and the IC50 values of B2-3’-O-gallate, ECG, and EC were 6.91 ± 3.41, 18.27 ± 3.99, and 18.91 ± 3.66 μM, respectively. Inhibition kinetic assays revealed the inhibition constants (Ki) of the mixed-competitive inhibitors of sucrase as follows: B2-3’-O-gallate (6.05 ± 0.04 μM), ECG (8.58 ± 0.08 μM), and EC (13.72 ± 0.15 μM). Overall, these results suggest that B2-3’-O-gallate, ECG, and EC are potent maltase and sucrase inhibitors.The inhibitory properties of epicatechin-(4 Over the past three decades, the number of people with diabetes mellitus has quadrupled, with the result being that diabetes mellitus is now the ninth leading cause of death worldwide. Approximately 1 in 11 adults have diabetes mellitus, and roughly 90% of those cases are type 2. The most pronounced symptom of diabetes mellitus is an abnormal postprandial increase in blood glucose levels . It is wα-Glucosidases are divided into four hydrolase types, i.e., maltase (Enzyme Commission (EC) 3.2.0.20), sucrase EC 3.2.1.48), glucoamylase (EC 3.2.1.3), and isomaltase EC 3.2.1.10). These four enzymes form two complexes with different substrate specificities, maltase–glucoamylase and sucrase–isomaltase complexes . Maltase. These f8, glucoaRhodiola crenulata (R. crenulata) were shown to significantly reduce the inhibitory activity of α-glucosidase. The genus Rhodiola L. (Crassulaceae) comprises approximately 90 species found throughout the world, and more than 70 species are found in the western and northern regions of Asia. Among these, R. crenulata, is an important member of the Crassulaceae family. For centuries, the roots of R. crenulata have been used as a health supplement. It is possible that these extracts could be used as a supplement for postprandial hyperglycemia associated with diabetes mellitus. In our previously study, we isolated and characterized α-glucosidase inhibitory constituents, including epicatechin--epicatechingallate , epicatechin gallate (ECG), and epicatechin (EC) from R. crenulata [O-gallate, ECG, and EC against α-glucosidase activity.In a study by Kwon et al. , water erenulata . These rO-gallate, ECG, and EC has previously been reported, the inhibitory properties of B2-3’-O-gallate, ECG, and EC toward maltase and sucrase have not been examined. Thus, this paper describes the inhibition kinetics of B2-3’-O-gallate, ECG, and EC toward maltase and sucrase. Our primary objective in this study was to elucidate the inhibitory effects of 2-3’-O-gallate, ECG, and EC on maltase and sucrase.The above potent maltase and sucrase inhibitors were considered for use in treating diabetes mellitus. Although the inhibition of α-glucosidases by B2-3’-R. crenulata were obtained from a traditional Chinese medicine pharmacy in Chiayi in south Taiwan. The authenticity was confirmed by Dr. Hsiang-Wen Tseng  using DNA sequencing technology and an internal transcribed spacer sequence database. Isolates of B2-3’-O-gallate, ECG, and EC were prepared from a crude extract of R. crenulata in accordance with the methods reported by Chu et al. [R. crenulata roots was milled and extracted using distilled water (900 mL). After centrifugation at 12,000 × g for 15 min, the supernatant was collected and freeze-dried to yield 16 g of water extract. The extract that displayed IC50 value for maltase was 5.23 ± 0.18 μg/mL, whereas the extract that displayed IC50 value for sucrase was 21.42 ± 0.45 μg/mL. The water extract underwent column chromatography on a vacuum manifold using solid-phase extraction (SPE) cartridges. Each sample (50 mg) was chromatographed on a SPE cartridge. The samples were then eluted stepwise using 0%, 10%, 20%, 30%, and 40% methanol in distilled water. A total of 5 fractions, one for each methanol elution (20 mL), were collected. The samples were concentrated using a rotary evaporator and then freeze-dried. The total yield of the fractions was 92.4%, whereas the yields of the individual fractions were as follows: 0% elution (67.6%), 10% elution (16.5%), 20% elution (5.1%), 30% (2.3%), and 40% (0.9%). The 30% fraction was redissolved in distilled water and subjected to column chromatography on a HPLC high-performance liquid chromatography (HPLC) system comprising a pump  and a detector  with a C18 packed column . A gradient elution from H2O/acetonitrile (86:14) to H2O/acetonitrile (72:28) was used to isolate B2-3’-O-gallate, ECG, and EC. Elution began with a solvent flow rate of 1 mL/min for the collection of B2-3’-O-gallate, ECG, and EC. The total yields were as follows: B2-3’-O-gallate (8.7 mg), ECG (6.1 mg), and EC (7.6 mg).Dried roots of u et al. . BrieflySaccharomyces cerevisiae (S. cerevisiae) were obtained from Sigma Chemical Co. . A reaction mixture was formulated from 30 μL of maltose (86.3 mM) with 20 μL of maltase (0.33 units/mL) and 50 μL of phosphate buffer . The mixture was incubated at 37 °C for 10 min and then at 100 °C for a further 10 min to stop the reaction. The glucose concentrations released by the reaction mixture were determined via the glucose oxidase method  using a VersaMax microplate reader  at an absorbance wavelength of 540 nm.Maltase activity was characterized using the methods outlined by Adisakwattana et al.  with sliO-gallate, ECG, EC, and quercetin, with quercetin  as positive controls. All of the samples were assessed in terms of maltase inhibitory activity under a range of concentrations (0–30 μM). Maltase inhibitory activity was evaluated by dissolving the samples directly in phosphate buffer solution . Note that each sample was assayed in triplicate. Inhibitory activity was estimated in terms of IC50 values based on percent inhibition, as follows:In this study, we focused on B2-3’-Leuconostoc mesenteroides were obtained from Sigma Chemical Co. . The reaction mixture comprised 40 μL of sucrose (480 mM) with 10 μL of sucrase (0.33 units/mL) in 50 μL of phosphate buffer solution . The mixtures were incubated at 37 °C for 60 min and then at 100 °C for a further 10 min to stop the reaction. The glucose concentrations released from the reaction mixtures were estimated via the glucose oxidase method  using a VersaMax microplate reader  at an absorbance wavelength of 540 nm.Estimates of sucrase activity were obtained using the method outlined by Akkarachiyasit et al.  with sliO-gallate, ECG, EC, and quercetin, with quercetin  as positive controls. All samples were assessed in terms of sucrase inhibitory activity under a range of concentrations (0–50 μM). Inhibitory activity was assessed by dissolving samples directly in phosphate buffer solution . Inhibitory activity was estimated in terms of IC50 values based on percent inhibition, as follows:In this study, we focused on B2-3’-O-gallate, ECG, EC, and quercetin on maltase and sucrase. Kinetics assays were conducted using substrates of various concentrations with and without inhibitors. Maltose was used as a substrate for maltase, where the initial velocity was expressed as the rate of absorbance at 540 nm per min. Sucrose was used as a substrate for sucrase, where the initial velocity was expressed as the rate of absorbance at 540 nm per 18 min. Dixon plot analysis was used to determine the competitive inhibition constant (Ki) and uncompetitive inhibition constant (Ki’). Ki was used as the equilibrium constant for the inhibition of binding to maltase and sucrase, whereas Ki’ was used as the equilibrium constant for the inhibition of binding to maltase–maltose and sucrase–sucrose complexes. The Ki and Ki’ values of the maltase inhibitors were derived from Dixon plots in accordance with the methods described by Cornish-Bowden [Lineweaver–Burk plot analysis was used to identify the mode of inhibition of B2-3’-p < 0.05.Data are expressed as the mean ± standard deviation. All data analysis was performed using Statistical Analysis System . Statistically significant differences between treatment groups were determined using one-way ANOVA followed by Duncan’s multiple range test. Three replicates of each sample were assayed, and the level of significance was set at O-gallate, ECG, and EC derived from R. crenulata. O-gallate, ECG, and EC with that of quercetin, which is a leading candidate for the treatment of diabetes mellitus [50 value of 4.8 ± 0.4 mM against rat maltase. We assessed the inhibitory effects of B2-3’-O-gallate, ECG, EC, and quercetin on maltase at various concentrations. The addition of B2-3’-O-gallate, ECG, EC, and quercetin at a concentration of 10 µM was shown to significantly enhance maltase inhibition, as follows: B2-3’-O-gallate (90.7%), ECG (87.7%), EC (66.3%), and quercetin (56.3%). Potent maltase and sucrase inhibitors have been considered for use for treating diabetes mellitus . Thus, wmellitus . Quercetmellitus  reportedO-gallate, ECG, EC, and quercetin. The IC50 values for maltase were as follows: B2-3’-O-gallate (1.73 ± 1.37 µM), ECG (3.64 ± 2.99 µM), EC (6.25 ± 1.84 µM), and quercetin (8.33 ± 3.91 µM). The strength of the IC50 values was in the following order: B2-3’-O-gallate < ECG < EC < quercetin. We also compared the inhibitory activities of B2-3’-O-gallate, ECG, EC, and quercetin toward sucrase. As shown in O-gallate, ECG, EC, and quercetin at a concentration of 20 µM was shown to significantly enhance sucrase inhibition, as follows: B2-3’-O-gallate (84.6%), ECG (56.1%), EC (51.4%), and quercetin (51.7%).50 values for sucrase were as follows: B2-3’-O-gallate (6.91 ± 3.41 µM), ECG (18.27 ± 3.99 µM), EC (18.91 ± 3.66 µM), and quercetin (18.98 ± 2.53 µM). The strength of the IC50 values was in the following order: B2-3’-O-gallate < ECG < EC < quercetin. We can see that the inhibitory effects of quercetin on sucrase were far less pronounced than those of B2-3’-O-gallate, ECG, and EC (p < 0.05). There has already been considerable research into the use of these polyphenols as maltase and sucrase inhibitors as an alternative to pharmaceutical treatments such as acarbose for the management of type 2 diabetes. Pyner et al. [50 values of polyphenol-rich green tea extract, acarbose, and epigallocatechin gallate for rat maltase were 0.035 ± 0.005 mg/mL, 0.42 ± 0.02 μM, and 14.0 ± 2.0 μM, respectively. Furthermore, the IC50 values of polyphenol-rich green tea extract, acarbose, and epigallocatechin gallate for rat sucrase were 1.8 ± 0.3 mg/mL, 12.3 ± 0.6 μM, and 950 ± 86 μM, respectively. In addition, quercetin showed inhibitory activities with IC50 values of 3.5 ± 0.3 mM against rat sucrose [50 = 12 mM), siamenoside I (IC50 = 10 mM), and mogroside III (IC50 = 1.6 mM) isolated from Siraitia grosvenori also have maltase inhibitory activity [Polyopes lancifolia. That compound had an IC50 value of 1.00 ± 0.03 mM against sucrose in rats. According to the above results, B2-3’-O-gallate, ECG, and EC have the potential to be maltase and sucrase inhibitors.As shown in r et al.  reported sucrose . Furtheractivity . Kim et activity  isolatedO-gallate, ECG, EC, and quercetin toward maltase. In this study, the Michaelis constant (Km) of maltase was 1.01 mM, whereas in the study by Pyner et al. [Km value of maltase was 7.5 mM. The Lineweaver–Burk plots of B2-3’-O-gallate , ECG , and quercetin  were mixed-competitive inhibitors of α-glucosidase [Ki calculated from a Dixon plot was 5.93 ± 0.26 µM [We also evaluated the inhibition kinetics of B2-3’-r et al. , the Km -gallate A, ECG , ECG (6.38 ± 0.04 µM), EC (18.88 ± 0.22 µM), and quercetin (14.97 ± 0.75 µM). Note that all of the Ki values were lower than the Ki’ values. As mentioned previously, Ki is the equilibrium constant for the inhibition of binding to maltase, whereas Ki’ is the equilibrium constant for the inhibition of binding to the maltase–maltose complex. In a previous report by Cortés et al. [Ki values are smaller than Ki’ values in cases of reversible mixed-competitive inhibition. This is an indication that inhibitor–enzyme binding affinity exceeds inhibitor–enzyme–substrate binding affinity, resulting in the mixed-competitive inhibition of maltase. These findings suggest that B2-3’-O-gallate, ECG, EC, and quercetin may bind to either maltase or the maltase–maltose complex. Nonetheless, the binding sites and underlying mechanisms of inhibition have yet to be elucidated.The Dixon plots in s et al. , Ki valuO-gallate, ECG, EC, and quercetin toward sucrose, the Michaelis constant (Km) was 100.4 mM. In a previous report by Pyner et al. [Km value of sucrase activity was 9.5 mM. The Lineweaver–Burk plots of B2-3’-O-gallate  was found in litchi and sweet cherry. Kinetics analysis revealed that sucrase was inhibited by cyanidin-3-rutinoside in a mixed-type manner [Ki calculated from a Dixon plot was 0.77 µM [In our analysis of the inhibition kinetics of B2-3’-r et al. , the Km -gallate A, ECG , ECG (15.74 ± 0.32 µM), EC (21.36 ± 0.30 µM), and quercetin (18.54 ± 0.07 µM). Note that the Ki values of B2-3’-O-gallate, ECG, EC, and quercetin were lower than the Ki’ values, indicating that these compounds were mixed-competitive inhibitors of sucrase. These findings suggest that B2-3’-O-gallate, ECG, EC, and quercetin may bind to either sucrase or the sucrase–sucrose complex. Nonetheless, the binding sites and underlying mechanisms of inhibition have yet to be elucidated.The Dixon plots in O-gallate, ECG, EC, and quercetin). Ki and Ki’ are the dissociation constants of the maltase–inhibitor complex and the maltase–maltose–inhibitor complex, respectively. According to our results, B2-3’-O-gallate , ECG , EC , and quercetin  are mixed-competitive inhibitors of maltase. Note that mixed-competitive inhibitors bind at sites that are distinct from maltose active sites; however, they bind to either maltase or the maltase–maltose complex. Moreover, a similar inhibition scheme was also observed in the results of B2-3’-O-gallate, ECG, EC, and quercetin against sucrase , ECG , EC , and quercetin  are mixed-competitive inhibitors of sucrase. Mixed-competitive inhibitors bind at sites that are distinct from sucrose active sites; however, they bind to either sucrase or the sucrase–sucrose complex. Note that B2-3’-O-gallate, ECG, and EC all had significant inhibitory effects on maltase as well as sucrase. sucrase B. In theO-gallate, ECG, and EC isolated from R. crenulata displayed maltase and sucrase inhibitory properties in a mixed-competitive mode. The IC50 values of these compounds for both maltase and sucrose were in the following order: EC > ECG > B2-3’-O-gallate. Among these compounds, B2-3’-O-gallate had the highest maltase and sucrose inhibitory activities. We observed that the IC50 values of B2-3’-O-gallate, ECG, and EC were lower than those of quercetin, which means that they have greater potential for the prevention of hyperglycemia associated with maltase or sucrase. Our findings clearly identify B2-3’-O-gallate, ECG, and EC as likely candidates for the treatment of diabetes mellitus, warranting further in vivo studies.B2-3’-


Insr) and insulin receptor substrate 1 and 2 (Irs1 and Irs2), as well as those involved in lipid metabolism, including Forkhead box protein O1 (FOXO1) and carnitine palmitoyl transferase 1 (CPT1) following GRE treatment. GRE showed a strong potential to ameliorate hepatic insulin resistance by improving insulin sensitivity through the regulation of PI3K/AKT, FOXO1 and AMPK-mediated pathways.We previously demonstrated that an aspalathin-enriched green rooibos extract (GRE) reversed palmitate-induced insulin resistance in C2C12 skeletal muscle and 3T3-L1 fat cells by modulating key effectors of insulin signalling such as phosphatidylinositol-4,5-bisphosphate 3-kinase/protein kinase B (PI3K/AKT) and AMP-activated protein kinase (AMPK). However, the effect of GRE on hepatic insulin resistance is unknown. The effects of GRE on lipid-induced hepatic insulin resistance using palmitate-exposed C3A liver cells and obese insulin resistant (OBIR) rats were explored. GRE attenuated the palmitate-induced impairment of glucose and lipid metabolism in treated C3A cells and improved insulin sensitivity in OBIR rats. Mechanistically, GRE treatment significantly increased PI3K/AKT and AMPK phosphorylation while concurrently enhancing glucose transporter 2 expression. These findings were further supported by marked stimulation of genes involved in glucose metabolism, such as insulin receptor ( Increased consumption of diets high in saturated fats such as palmitate, commonly found in processed and fast foods, contributes to the development of insulin resistance and related conditions, including obesity and type 2 diabetes . In the Recently, studies reported that dietary polyphenols can reduce or prevent insulin resistance and enhance insulin sensitivity. Thus, sources of such polyphenols are of interest as potential therapeutic modulators of glucose and lipid metabolic dysfunction ,10,11,12p < 0.05) in the vehicle control. Palmitate exposure resulted in impaired glucose metabolism by reducing both basal (without insulin) and insulin-stimulated glucose uptake, respectively, by ~30 ± 4.6%, (p < 0.001) and ~43 ± 4.7% (p < 0.001) (p < 0.01) in insulin-stimulated C3A liver cells (p < 0.001). Moreover, GRE enhanced palmitate fatty acid uptake both in basal  and insulin-stimulated palmitate treated cells . Furthermore, GRE increased insulin-stimulated ATP content in palmitate-treated cells from 87.8 ± 7.0% to 139.2 ± 7.3% (p < 0.001), respectively, compared to the palmitate control  A. Similaer cells B, confir control C. Howeve control .p < 0.001) in basal conditions and by ~40 ± 5.0% (p < 0.001) in insulin-stimulated cells compared to vehicle control (p < 0.001) and 153.3 to 89.2 ± 5.2% (p < 0.001) compared to the palmitate control (p < 0.01). This reduction was reversed (p < 0.05) after culturing with GRE with or without insulin compared to the palmitate control , while palmitate exposure significantly reduced insulin-stimulated AKT (Ser 473) activation from 296.0 ± 55.1% to 136.3 ± 13.6% (p < 0.001) compared to the vehicle control with insulin (p < 0.05)  (p < 0.05) under basal conditions when compared to vehicle control (p < 0.05) (p < 0.05) and 83.46 ± 6.7% to 106.0 ± 2.3% (p < 0.05), respectively  B. Palmit< 0.001) C. Intere control C. In add < 0.05) D. GRE reectively D.p < 0.05) (p < 0.05) (p < 0.05) for basal cells and from 123.0 ± 5.5% to 85.3 ± 9.9% (p < 0.001) for insulin-stimulated cells (p < 0.01), when compared to the palmitate control (p < 0.01) for basal cells and from 73.6 ± 3.4% to 116.9 ± 8.2% for insulin-stimulated cells  A, and th < 0.05) B. Inhibied cells C. Palmit control D. The exed cells E.After establishing the model , we examInsr mRNA expression from 100 ± 9.8% to 170.4 ± 7.8% (p <0.05) when compared to the normal control (Irs1 and Irs2 in the liver tissue from 100.0 ± 18.3% to 400.7 ± 68.9% (p < 0.05) and from 100.0 ± 6.9% to 190.4 ± 36.9% (p < 0.05), respectively (Pi3k gene expression (120% ± 28.6% (p < 0.01) for GRE) (Ampk gene expression affected by GRE when compared to the control (p < 0.01) and vildagliptin (p < 0.01)  D. This w < 0.01) E. Moreov in vivo F.Excessive intake of saturated fatty acids is a major causal factor of hepatic lipid accumulation, insulin resistance, and the subsequent development of type 2 diabetes . TherefoThe use of 0.75 mM palmitate was previously shown to effectively reduce insulin sensitivity in muscle cells . This waCaenorhabditis elegans -d-glucose (DOG) and 14C palmitate were obtained from American Radiolabelled Chemicals . Bradford and reducing agent compatible and detergent compatible (RC DC) protein assay kits were obtained from Bio-Rad Laboratories , while Lonza  supplied Eagle’s Minimum Essential Medium (EMEM), Dulbecco Modified Eagle’s Medium (DMEM) and the MycoAlert™ mycoplasma detection and ViaLight plus ATP kits. Protease and phosphatase inhibitor tablets were purchased from Roche . Palmitic acid (C18:0), Vildagliptin, glycerol release assay kit, Oil Red O powder, crystal violet, and other cell culture reagents were obtained from Sigma-Aldrich . The 50% Dextrose-Fresenius 50% was from Intramed . Mini-PROTEAN TGX Stain-Free™ Precast Gels and Midi Nitrocellulose Transfer Packs were from Biorad Laboratories  while, chemiluminescence kits and nitrocellulose membrane were from Amersham Bioscience . TRIzol reagent , RNeasy Mini kit, RNAlater , and all other chemicals , unless specified, were also obtained from Sigma-Aldrich .Human C3A liver cells  were obtained from the American Type Culture Collection . GRE, previously designated SB1 which is a solvent-based extract and characterized in terms of its flavonoid profile, was used . AspalatGlut2 (Hs01086390_m1), Irs1/2 , Pi3k (Hs00979691_m1) and Ampk (Hs00272166_m1) were supplied by Applied Biosystems .Cell Signalling Technology  supplied primary antibodies raised against AKT (cat. # 9272), p-AKT (Ser473) (cat. # 9271), AMPK (cat. # 2532), p-AMPK (Thr172) (cat. # 2531), ACC (cat. # 3662), p-ACC (Ser79) (cat. # 3661), FOXO1 (C29H4) (cat. # 2880), p-FOXO1 (Ser256) (E1F7T) (cat. # 84192) and INSR (β (4B8) (cat. # 3025). Antibodies for GLUT2 (cat. # ab54460), CPT1 (cat. # ab53532) and MCD (cat. # ab95945) were from Abcam . The reference control, β-actin (C4): (sc-47778), and the secondary antibodies, donkey-anti mouse (IgG-HRP: sc-2314) and donkey-anti rabbit (IgG-TR: sc-2784)—horseradish peroxidase, were purchased from Santa Cruz Biotechnology . Taqman gene expression assays for 3H]-d-glucose and palmitate uptake, 96-well plates  for ATP analysis, and 6-well plates  for protein analysis. The medium was refreshed after two days. C3A cells were confirmed to be free from mycoplasma following the manufacturer’s instructions. We used cells at passages 5–20 for all experiments. A dose study was performed using glucose uptake on normal C3A liver cells to determine effective concentration and insulin (1 µM) was used as a positive control (results presented in w/v) at a stock concentration of 0.1 mg/µL and further diluted in the DMEM culture media without phenol red to yield a final concentration of 10 µg/mL. Final DMSO concentration in the media for GRE was < 0.001% and did not affect cell viability. On the day of the assay, palmitate-induced insulin-resistant C3A liver cells were serum and glucose starved for 30 min before treating with GRE (10 µg/mL) and insulin (1 µM) in DMEM without phenol red containing 8 mM glucose, 2% BSA, and 0.75 mM palmitate for 3 h at 37 °C in 5% CO2 and humidified air. Insulin was used to stimulate insulin signaling.C3A liver cells were cultured and sub-cultured in EMEM with essential amino acids, sodium pyruvate, and L-glutamine. Cells were seeded into 24-well plates  for 2-deoxy-[3H-2-DOG and 14C palmitate uptake assays were performed separately by following previously published protocols [3H-2-DOG or 1 µCi/mL 14C palmitate in medium supplemented with GRE for 3 h at 37 °C in 5% CO2 and humidified air for determination of glucose and palmitate uptake, respectively. Insulin (1 µM), the positive control, was added during the last 15 min in each experiment. Thereafter, cells were lysed with 0.1 M NaOH before 3H-2-DOG or 14C activity in the lysate was assessed by liquid scintillation . Intracellular ATP was measured as previously described [rotocols . Brieflyescribed  using thThe Oil Red O assay was performed as previously described  with sliLipolysis was evaluated by measuring the amount of glycerol released into the medium after 3 h culture with relevant treatment as previously described in . Brieflyw/v) low-fat milk powder in Tris-buffered saline with Tween 20 at room temperature for 2 h. Membranes were then labelled overnight at 4°C with the relevant primary antibodies (p-AKT (Ser 473), AMPK, p-AMPK (Thr172), GLUT2, CPT1, FOXO1, MCD, INSR) and horseradish peroxidase conjugated secondary antibody (β-actin) applied for 1.5 h the following day. Proteins were detected and quantified by chemiluminescence using a Chemidoc-XRS imager and Quantity One 1-D software. Molecular weight band detection was confirmed using image J software , whereas β-actin was used as the reference control.Western blot analysis was performed by modifying a previous protocol . Brieflyhttp://www.mrc.ac.za/ethics/ethicsbook3.pdf). Three-week-old weaning male Wistar rats were obtained and housed at the Primate Unit of the SAMRC . The rats were housed individually in wired top and bottom polycarbonate cages, fitted with PerspexTM houses and maintained in a temperature-controlled room of 24–26 °C, humidity of 45–55% with 15–20 air changes per hour and on a 12 h light/dark cycle.This study was approved by the Ethics Committee for Research on Animals (ECRA) of the South African Medical Research Council (SAMRC) . The study was executed in accordance with the principles and guidelines of the SAMRC as outlined in Guidelines on Ethics for Medical Research: Use of Animals in Research and Training, 2004 (n = 60), 12 rats per group were fed a high-fat, high-sugar diet (40% fat as energy of which 18.3% were saturated fats) and 44% carbohydrates . In addition, 30% sucrose and fructose (1:1 w/w) was added in drinking water ad libitum for 12 weeks to induce obesity and insulin resistance (OBIR), according to an already published model [Wistar rats . OGTTs were performed after 12 weeks of treatment. Briefly, after 16 h of fast, rats were gavaged with GRE treatments 1 h (t = −60 min) before administration of a glucose bolus (t = 0 min) at 2 g/kg glucose (50% Dextrose-Fresenius 50%). Plasma glucose concentrations were determined at −60 and at 0, 15, 30, 60, 120, and 240 min, respectively, relative to the glucose bolus (t = 0). Twenty-four-hour water and food intake were determined, in metabolic cages, prior to commencement of treatment with GRE, as well as 12 weeks after treatment with GRE . Upon coIrs1 (Hs00178563_m1), Irs2 (Hs00275842_s1), Pi3k (Hs00979691_m1), Ampk (Hs00178903_m1) and glucose transporter 2  were investigated. mRNA was normalised to mouse β-actin (β-actin Hs02742610_g1) and glyceraldehyde-3-phosphate dehydrogenase . Data were analysed using standard curve and 2−delta Ct ABI Standard Quantification (AQ) software .Total RNA was extracted from liver tissue using TRIzol reagent. Briefly, 80–100 mg of liver tissue was homogenized twice using a TissueLyser  at 25 Hz for 2 min. Gene expression was measured by quantitative real-time PCR (qPCR) using the fluorescent TaqMan 5′nuclease assay on an Applied Biosystems 7500 sequence detection system . qPCR was performed using cDNA with 2× TaqMan Master Mix and the 20× premade TaqMan gene expression assays (Applied Biosystems) and qPCR conditions were 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Gene expression of insulin receptor , t-test for analysis of data obtained with the animal experiment. A p < 0.05 was considered significantly different. All statistical analyses were performed using GraphPad Prism 5 software .In vitro data were expressed as the mean ± SEM of three independent experiments. Significant differences between treatments were calculated using one-way or two-way analysis of variance (ANOVA), followed by a Tukey post hoc test or Student The results presented in this study, of an aspalathin-enriched green rooibos extract, provides evidence of its partial beneficial effect in ameliorating metabolic complications associated with insulin resistance and type 2 diabetes. Modulation of glucose and lipid metabolism by targeting the PI3K/AKT and AMPK signaling cascade may possibly provide a strategy for therapeutic intervention in the reversal of insulin resistance and potential treatment of type 2 diabetes and its complications. However, future research directions, which are also important in addressing limitations of the current studywill include testing the therapeutic effects of GRE on an established model of OBIR. Furthermore, investigating phosphorylation/activation status of major proteins will involve insulin independent pathways such as AMPK. This will include inhibitory or gene targeted overexpression experiments to further elaborate on the protective effect of GRE against the metabolic syndrome.


We were gratified to note that, like us, the authors obtained a strong correlation between EPS (derived from ref. 3) and the association rates they have measured using a distinct method. We are also pleased that the new odds ratio they calculated for remoxipride fits our rebinding hypothesis, strengthening the overall correlation between EPS and kr  as a surrogate of agonist binding at the D2R. This approach assumes that there is a direct, linear relationship between agonist occupancy at the receptor and GIRK channel activation. This is not, however, commonly observed for receptor signaling events, even for measurements of proximal events such as G protein activation. Instead, operational models of receptor activation describe a hyperbolic relationship between agonist binding and downstream effector activation5.Rather than directly measuring binding at the Drs) of 0.90 (P < 0.0001) between the two data sets  for koff and kon is 0.77 (P = 0.0033) and 0.59 (P = 0.0428) for the whole data set, but rises to 0.82 (P = 0.0021) and 0.84 (P = 0.0015) when remoxipride is removed from the analysis. It therefore appears that this discrepancy in kinetics for remoxipride may be related to the compound itself, rather than due to differences in assay methodology. In Sahlholm’s study4, remoxipride was tested at a 10-fold higher concentration (100 μM) than any of the other ligands, despite having an apparent Kd similar to that of clozapine. It is possible that at this higher concentration remoxipride could display off-target effects that contribute to the much faster apparent association rate. For example, several other antipsychotics have previously been shown to directly block GIRK channels expressed in xenopus oocytes6. If the high concentrations of remoxipride used in the study of Sahlholm and colleagues4 were to directly block GIRK channels, it would manifest in the appearance of a more rapid association to the D2R. This potential off-target action is supported by a previous study7 that directly measured the dissociation of [3H]-remoxipride from the D2R. They reported an off rate of 3.2 min−1, similar to our own value of 1.9 min−1, but almost 4-fold different from the value of 12.2 min−1 obtained by Sahlholm and colleagues4.However, despite these differences in assay methodology, the affinity measurements reported for a selection of antipsychotics are surprisingly similar to ours, with a Pearson’s correlation  is much more rapid than the dissociation rate of our fluorescent tracer (F-PPHT), and is therefore more appropriate for assessing rapid binding kinetics. Indeed, they require a rapid tracer because they make the assumption that the rate-limiting step is binding and unbinding of the antagonist, rather than the combined summation of the steps between dopamine binding, receptor-mediated activation of G proteins and subsequent GIRK channel activation. This, they have acknowledged, creates an upper limit of sensitivity (or ceiling effect) that limits their analysis to decay rates slower than ~1 s and antagonist concentrations below 10 μM2R and use a mathematical approach that explicitly incorporates the association and dissociation rates of the tracer molecule when calculating the kinetics of competing antagonists. In this way we avoid the necessity for the tracer kinetics to be much more rapid than the test compound. As mentioned by Zeberg and Sahlholm, we have previously found that using this assay, rapid competitor dissociation rates cannot be accurately determined using a tracer ligand with a much slower rate of dissociation. This observation was made using the slowly dissociating antagonist spiperone as a tracer (koff = 0.026 min−1)9. For this reason we selected PPHT-red as the tracer ligand for our most recent studies as it has a dissociation rate 10-fold higher than spiperone of 0.52 min−1. We acknowledge that there will still be a theoretical limit of detection with this tracer, but believe 1.9 min−1 (our koff for remoxipride) is within our levels of detection as we have measured ligands with more rapid dissociation rates 9. We have also independently verified the binding kinetics of remoxipride and clozapine using another rapidly dissociating tracer, clozapine-red. The association and dissociation values calculated for remoxipride using this tracer were 1.00 × 107 M−1 min−1 and 1.63 min−1, respectively, demonstrating tracer independence with regard to kinetic determinations. Data from these experiments are shown in Fig. To avoid the above assumptions, we chose to measure binding directly to the DZeberg and Sahlholm also suggest that the inclusion of GppNHp in our assay might influence the observed kinetics for remoxipride, and that their assay is more physiologically relevant because it measures interactions with the higher affinity “functional” state of the receptor. This high affinity (G protein-coupled) state is, however, extremely transient in a whole cell due the high intracellular concentrations of GTP. We include GppNHp with our membrane preparations to mimic this high concentration. All current evidence suggests that once the agonist dissociates, then in a whole cell, the receptor rapidly reverts to the inactive uncoupled state before an antagonist binds. This, as previously discussed, is supported by the good agreement of the kinetic values between our membrane assay and their whole cell oocyte system, with the only clear exception being remoxipride Fig. b–d. In s1 receptor10. Our study revealed a compelling correlation between EPS and association rate, and we propose a mechanism of drug-rebinding that can explain this correlation2. Given the complex action of antipsychotic drugs it would, however, be naive to expect that all drugs would fall within this correlation. Those that do not may act via distinct mechanisms. Indeed, in our study we observed that aripiprazole, a drug that acts as a D2R partial agonist rather than an antagonist, was an outlier. We proposed that this was because of its distinct action at the D2R. As such, we feel that one should not discount the potential importance of drug rebinding at the D2R due to a small number of drugs that do not fit this correlation. Instead, just as the work of Seeman and colleagues11 stimulated our study, we hope that our work will inspire future studies aimed at carefully testing our hypothesis and unraveling the complex mechanisms that determine antipsychotic drug efficacy.The final comment raised by Zeberg and Sahlholm relates to the antipsychotic thioridazine, which they state has a favourable EPS profile despite its rapid association rate. Note that this drug was not included in the meta-analysis of Leucht et al. so could not be included in our correlation. As discussed in our paper, it was not our intension to present the rebinding hypothesis as the only mechanism that might predict EPS. Rather, many antipsychotics display complex poly-pharmacology that may also contribute to their relative efficacy and side-effect profiles. For example the superior EPS profile of thioridazine may relate to off-target activity at the muscarinic MClozapine-red was synthesized in house as follows Fig. .Fig. 3SyN-Desmethylclozapine (2): Clozapine  was dissolved in 1,2-dichloroethane (20 mL) under N2 and cooled to 0 °C. 1-Chloroethyl chloroformate  was added dropwise to the reaction mixture. After 10 min, the reaction mixture was warmed up to r.t and subsequently heated at reflux for 24 h. The brown reaction mixture was concentrated in vacuo, and the residue was dissolved in methanol (30 mL) and heated at 50 °C for 2 h, cooled, and again concentrated in vacuo. The resulting residue was partitioned between ethyl acetate (50 mL) and 1 M aqueous hydrochloric acid (50 mL). The aqueous layer was collected and the pH adjusted to ~10 using concentrated sodium hydroxide then extracted with ethyl acetate (3 × 50 mL). The combined organic layers were washed with water (50 mL) and saturated brine (50 mL), dried over anhydrous Na2SO4, filtered and evaporated to dryness. Purification was achieved via column chromatography  to give a yellow foam . 1H NMR (CDCl3) δ 2.57 , 3.01 , 3.48 , 4.90 , 6.61 , 6.81–6.84 , 7.02 , 7.06 , 7.25–7.32 . 13C NMR (CDCl3) δ 45.7 (CH2), 48.2 (CH2), 120.1 (CH), 120.2 (CH), 123.2 (CH), 123.2 (CH), 123.5 (C), 126.9 (CH), 129.2 (C), 130.4 (CH), 132.0 (CH), 140.5 (C), 141.9 (C), 152.8 (C), 163.1 (C).tert-Butyl piperazin-1-yl)propyl)carbamate (3): N-Desmethylclozapine , sodium iodide  and N,N-diisopropylethylamine  were added to acetonitrile (30 mL) under N2. tert-Butyl (3-bromopropyl)carbamate  was added and the reaction mixture was heated at reflux for 24 h. After cooling to room temperature the solvent was removed and the residue was dissolved in ethyl acetate (30 mL), washed with water (2 × 30 mL) followed by brine (50 mL), dried over anhydrous Na2SO4, filtered and concentrated to give the crude product. Further purification via column chromatography  gave the title compound as a yellow foam . 1H NMR (CDCl3) δ 1.44 , 1.69 , 2.46 , 2.52 , 3.21 , 3.47 , 4.90 , 5.26 , 6.61 , 6.80–6.83 , 7.01 , 7.06 , 7.24–7.32 . 13C NMR (CDCl3) δ 26.6 (CH2), 28.6 (CH3), 39.9 (CH2), 47.3 (CH2), 53.3 (CH2), 57.0 (CH2), 79.0 (C), 120.1 (CH), 120.2 (CH), 123.2 (CH), 123.2 (CH), 123.5 (C), 126.9 (CH), 129.2 (C), 130.4 (CH), 132.0 (CH), 140.5 (C), 141.9 (C), 152.8 (C), 156.2 (C), 162.8 (C).4): Compound 3  was dissolved in DCM (10 mL) and TFA (2 mL) was added dropwise. Stirring at room temperature occurred for 1–2 h before the reaction mixture was diluted with a further 20 mL of DCM. Saturated K2CO3 (20 mL) was added slowly and the mixture further extracted with 3 × 20 mL portions of DCM. The combined organic layers were further washed with water (50 mL), brine (50 mL), dried over anhydrous Na2SO4, filtered and concentrated to give the product as a yellow foam which was used in the subsequent reaction without further purification . 1H NMR (CDCl3) δ 1.30 , 1.67 , 2.46 , 2.53 , 2.77  3.47 , 4.90 , 6.61 , 6.80–6.83 , 7.01 , 7.06 , 7.24–7.32 . 13C NMR (CDCl3) δ 30.7 (CH2), 40.9 (CH2), 47.4 (CH2), 53.4 (CH2), 56.6 (CH2), 120.1 (CH), 120.2 (CH), 123.2 (CH), 123.2 (CH), 123.6 (C), 126.9 (CH), 129.2 (C), 130.4 (CH), 132.0 (CH), 140.5 (C), 141.9 (C), 152.8 (C), 162.9 (C).3-piperazin-1-yl)propan-1-amine (5): The free amine  followed the sulfo-Cy5 NHS ester (1 equiv.) was added to DMF (1 mL) under a N2 atmosphere. The reaction was stirred at room temperature for 12 h in the absence of light, then purified immediately via preparative-HPLC. The clean fractions were collected, pooled and the solvent removed via lyophillization to obtain the product as the TFA salt. Blue solid . 1H NMR (d6-DMSO) δ 0.83 , 1.15–1.29 , 1.51 , 1.69 , 2.02 , 2.97 , 2.97–3.40 , 3.60 , 3.94 , 4.12 , 6.29 , 6.56 , 6.88–6.94 , 7.01–7.09 , 7.29–7.41 , 7.64–7.66 , 7.83–7.84 , 8.36 , 9.58 . HPLC purity (λ = 214 nm): 95%, tR = 4.89 min. HRMS (ESI)-TOF (m/z): [M + H]+ 994.3762 calcd for C52H60ClN7O7S2; found [M + H]+ 994.3765.1-piperazin-1-ium-1-yl)propyl)amino)-6-oxohexyl)-3,3-dimethyl-2--5-penta-1,3-dien-1-yl)-3H-indol-1-ium-5-sulfonate 2,2,2-trifluoroacetate (2).All other reagents and materials were obtained as previously described  was used to define the level of nonspecific binding.kon) and dissociation rate (koff) values of clozapine-red was determined using multiple different concentrations of clozapine-red. Clozapine-red (50–6.25 nM) was incubated with human D2L CHO cell membranes (2 μg/well) in assay binding buffer . Specific binding of clozapine-red bound to the D2L receptor was measured at 20 s intervals by HTRF detection allowing construction of clozapine-red association curves. Data were globally fitted to the association kinetic model to derive a single best-fit estimate for kon and koff.Determination of clozapine-red binding kinetics: The association rate  and unlabelled competitor to the receptor preparation of interest in this case the human dopamine D2LR, so that at t = 0 all receptors are unoccupied. 50 nM clozapine-red (a concentration which avoids ligand depletion in this assay volume), was added simultaneously with the unlabelled compound (at t = 0) to CHO cell membranes containing the human dopamine D2LR (2 μg per well) in 40 μl of assay buffer. The degree of clozapine-red bound to the receptor was measured at 20 s intervals by HTRF detection.Competition binding kinetics: To determine the association and dissociation rates of Dkon and koff as previously described.As described previously nonspecific binding was determined in the presence of haloperidol (10 μM) and was subtracted from each time point. Time points were performed on the same 384 well Optiplate plate maintained at constant temperature, 37 °C with orbital mixing (1 s of 100 RPM/cycle). For determination of rate parameters multiple concentrations of unlabelled competitor were employed and data were globally fitted to simultaneously calculate 2).Signal detection and data analysis: Signal detection was performed on a Pherastar FS  using standard HTRF settings and experiments were analyzed by non-regression using Prism 6.0  all as previously described (Sykes et al.


Baupain belongs to the α+β class of proteins with a secondary structure-content of 44% α-helix, 16% β-sheet and 12% β-turn. The structural transition induced by pH was found to be noncooperative, with no important differences observed in the pH range from 3.0 to 10.5. At pH 2.0 the protein presented substantial non-native structure with strong ANS binding. Guanidine hydrochloride (GdnHCl)-induced unfolding did not change the protein structure significantly until 4.0 M, indicating the high rigidity of the molecule. The unfolding was cooperative, as seen by the sigmoidal transition curves with midpoints at 4.7 ± 0.2 M and 5.0 ± 0.2 M GdnHCl, as measured by CD and fluorescence spectroscopy. A red shift of 7 nm in intrinsic fluorescence was observed with 6.0 M GdnHCl. Temperature-induced unfolding of baupain was incomplete, and at least 35% of the native structure of the protein was retained, even at high temperature (90 °C). Baupain showed characteristics of a molten globule state, due to preferential ANS binding at pH 2.0 in comparison to the native form (pH 7.0) and completely unfolded (6.0 M GdnHCl) state. Combined with information about N-terminal sequence similarity, these results allow us to include baupain in the papain superfamily. Bauhinia forficata leaves, named baupain , defined as [θ] = θobs/(10.C.l.n), where θobs is the CD in millidegrees, C is the protein concentration (M), l is the path length of the cuvette (cm), and n is the number of amino acid residues (261). The CDPro software was used to estimate the fractions of secondary structures . The reaction was followed for 10–30 min and stopped by the addition of 50 µL acetic acid 30% (v/v). The fluorescence release was measured on a FluoroCount PackardPrior to the addition of the substrate Z–Phe–Arg–MCA (0.4 mM), 40 µL of baupain (0.23 µM) was pre-incubated at a series of temperatures of 0, 37, 40, 60, 80, 100 °C for 30 min with 100 µL of the 0.1 M sodium phosphate buffer, pH 6.3 containing 0.4 M NaCl, 10 mM EDTA in a final volume of 250 µL by adding 90 µL of distillated water.In this study we found that baupain, a papain-like enzyme that hinders thrombin-induced human platelet aggregation and shows kinogenase activity ,2, fullyi.e., with no detectable intermediates) from native to unfolded form, with a red shift of 7 nm of intrinsic fluorescence in 6.0 M GdnHCl. Finally, a weak cooperative unfolding was observed when varying the pH. At pH 2.0, baupain still retained a significant content of secondary structure, but without its tertiary packaging, as indicated by ANS binding assays reflecting the exposure of its hydrophobic regions. These results at pH 2.0 demonstrate the presence of a transient intermediate state during unfolding of baupain, possibly a molten globule state, similar to papain and ficin [In addition, at 60 °C baupain was unable to cleave the substrate suggesting that changes in tertiary structure lead to a modification of the enzyme active site, thus affecting baupain activity. Exposure of baupain to GdnHCl led to a single step unfolding process (nd ficin ,9. The snd ficin .These fi


Vasconcellea cundinamarcensis latex, obtained by chromatographic separation on Sephadex-G10 and ultrafiltration. This fraction enhances healing in different models of skin lesions, and displays a protective/healing effect against gastric ulcers, where it was suggested an antioxidant role. Methods: We evaluated here the effect of topical treatment with P1G10, in mice lesions induced by UVB. Results: After single exposure to 2.4 J cm−2 UVB, P1G10 reduced erythema, increased cellularity of hypodermis, enhanced MPO activity and IL1β, and inhibited COX2 levels. These results point to an anti-inflammatory effect by P1G10. This fraction displayed antioxidant activity by reversing the depletion of glutathione (GSH), glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and reducing the catalase activity increased by UVB. These changes may be related to a reduction in MDA observed in groups treated with P1G10. P1G10 also inhibited MMP-9, caspase-3 and pkat while increasing p53 levels.Background: P1G10 is a cysteine proteolytic fraction from  The skin is an organ acting as a selective barrier between the body and the external environment. It is constantly exposed to a pro-oxidant environment, including ultraviolet light . Solar UThe mechanism underlying UV lesions involves damage through photons targeting DNA, proteins, and smaller molecules. The activated derivatives alter their biological properties or act as inducers of chemical reactions involving reactive free radicals (ROS). 2 and iNOS, triggering further cellular and tissue damage [An initial inflammatory response involves cutaneous vasodilation resulting in erythema and includes epidermal hyperplasia, enlargement of stratum corneum, increased vascularization, and melanogenesis ,9. Mediae damage ,12.Changes in interleukins IL-10, IL-4 and PGE2 affecting the immune response are also observed . The impNo significant decrease on earth UVB radiation resulting from recovery of ozone depletion is yet observed . Thus, o−2 UVB, were observed in mice at 6, 12, 24, and 48 h following exposure. Erythema peaked 12 h after irradiation (The evolution of cutaneous macro and microscopic lesions induced with 2.4 J cmadiation A.p < 0.01) compared to the basal group (445.0 ± 18.21 µm), followed by a reduction at 48 h , followed by a decline to basal level at 12 h and ensued by a second peak 220% at 24 h (p < 0.01) that remained elevated (210%) at 48 h (p < 0.05) (p < 0.001), followed by gradual rise without attaining normal level , as shown on The profile of redox markers at different intervals after UVB-radiation was investigated. The SOD activity peaked (3-fold) at 12 h, then declined to basal at 24 and 48 h. Interestingly, CAT displayed two maxima, one, 6 h post-irradiation reaching 180% ( < 0.05) A. In conal level B. The efp < 0.01) decrease in skin thickness following 0.1% P1G10 at 24 h, and 35% at 1% P1G10, (p < 0.05) compared to the (control) vehicle group  on UVB-irradiated skin at 24 h was chosen for analysis, as many of the changes induced by UV were maximized during this period. In these conditions A, in untle group B.To investigate the ability of P1G10 to quench the surge of ROS induced by UV, we evaluated the activities of SOD, CAT, GSH-Px and GSH content after 24 h UVB-exposure. The depletion of SOD activity induced by UVB after 24 h observed in the saline and control group was reversed by 0.1% P1G10, but the effect vanishes at 1% P1G10 A. MeanwhAnalysis of GSH and GSH-Px B,C, showp < 0.01) (p < 0.05) compared to the vehicle group.A morphometric analysis showed a 30% reduction in inflammatory infiltrate in the group treated with 1.0% P1G10 relative to the vehicle, ( < 0.01) A. Also,  < 0.01) B, in the2 .We measured the level of inflammatory mediators after 24 h A, to est2 B, an infp < 0.05), at both concentrations (trations B.p < 0.001) compared to basal level. The presence of 0.1% or 1.0% P1G10 reversed the MMP-9 increase (p < 0.05), becoming like the basal group.We also analyzed by zymography the activity of MMP-9 in skin samples, 24 h after UVB irradiation in the presence or absence of P1G10 B. Gel de−1) was evaluated by DPPH radical scavenging assay, using ascorbic acid as positive control (100% inhibition). The concentration of P1G10 that was able to sequester 50% of free radicals (EC50) in solution was estimated by linear regression to be 20.35 ± 0.66 μg mL−1 (The antioxidant capacity of P1G10 (6.2–100.0 μg mL μg mL−1 A.p < 0.01). Application of 0.1% P1G10 post-irradiation moderately depleted MDA, but, in the 1.0% P1G10 group, MDA decreased by 48% (p < 0.01), compared to the vehicle control. We also analyzed ROS, by via oxidized dichlorofluorescein (DCF) (p < 0.01). Meanwhile, in UVB-induced groups treated with 0.1% or 1.0 % P1G10, ROS production was inhibited by 50% and 60%, respectively, both compared to the irradiated control. These ROS values in P1G10 treated samples became like the Basal level  and dichlorofluorescin diacetate (DCFH-DA) used as markers for photo-oxidative damage. After UVB-exposure, we observed an 80% increase in MDA level B in the in (DCF) C. In UVBal level C.p < 0.05) and 8-fold increase of caspase-3 (p < 0.001), relative to the basal non irradiated group. The increase in pAkt was fully reversed at 0.1% and became lower than basal at 1% P1G10. A similar trend is observed for caspase-3, but the decline caused by P1G10, although significant, did not attain basal level (p < 0.01)  A. Histoc < 0.01) B.To validate the effect of P1G10 on UV irradiated skin of hairless mice, we carried initial experiments to assess between 6 and 48 h, changes in selected agents associated to redox status and the macro- and microscopic effects induced by radiation . Erythem−2 UVB treatment and continued to be depressed up to 72 h post-UVB. In their study the authors report strong fluctuations in basal levels which they attributed to circadian variations of these enzymes. On the other hand, Iizawa et al. [−2). We find that GSH, crucial for removal of ROS species O2− and derivatives [The status of antioxidant enzymes after irradiation showed a single SOD peak at 12 h and a bimodal response of CAT with two maxima at 6 h and 24 h post UV A. Pence a et al.  reportedivatives , was depivatives  reportedivatives   species, was supported by the decrease in MPO activity at 24 h B, whereaIn this model, the antioxidant activity in SOD and CAT showed opposite changes after UV, and P1G10 drove these two enzyme levels back to basal, implying a reduction of the oxidative stress. Depletion of GSH induced by UV was also abolished in P1G10 groups, beyond basal level, creating a surplus of the reducing agent. These changes provide additional support for the UV antioxidant/anti-inflammatory action of P1G10.2O2 by oxidation of GSH. We investigated the effect of UV on the activity of glutathione peroxidase (GSH-Px), the enzyme that removes HC. papaya which is equivalent to P1G10 in V. cundinamarcensis (https://www.drugbank.ca/drugs/DB11193), displays strong antioxidant activity and inhibits lipid peroxidation [As described so far, the anti-inflammatory role of P1G10 was caused at least in part by a reduction of ROS species, thus, we investigated the possible antioxidant power in P1G10. The ROS scavenging activity using the DPPH assay supported the ROS scavenging ability of P1G10 in a concentration dependent manner A. In a sxidation . We remixidation ,31.2, the limiting enzyme during synthesis of PGs, induced after UV-irradiation [2 was stimulated at 24 h post-UV, in P1G10 treated groups the enzyme was downregulated to Basal, preventing the formation of inflammatory mediators. In previous experiments, our group showed that induced gastric lesions treated with P1G10 had reduced expression of COX2 as well [2 is targeted to treat diverse inflammatory symptoms, and several synthetic drugs [2 [Ananas comosus, which downregulates the expression of COX2 and PGE-2 in murine and human cells [V. cundinamarcensis share the same allergenic feature.We also analyzed changes in COXadiation . While C as well . COX2 isic drugs  and plandrugs [2 . A similan cells . On the an cells . WhetherTo verify a role of cytokines in connection with the anti-inflammatory response, we analyzed TGF-β, IL1β, IL8, and IL10 after treatment with P1G10. While P1G10 had no effect on the depletion in TGFβ and IL8 induced by UV, a drop of the proinflammatory IL1β was observed with 1% P1G10, supporting the anti-inflammatory action assigned to P1G10. Meanwhile, IL10, an immunoregulator that plays a key role reducing immune responses and preventing excessive inflammation , was indTo gain insight into possible pathways affected in groups treated with P1G10, we assessed the levels of akt, pakt, p53, and capase-3. A strong decrease in pakt and increase in p53 were observed in the group receiving 1% P1G10, inducing a cell-cycle arrest. An intense crosstalk between p53 and pakt/mTOR was reported following UV stress to control the cellular fate through; cell-cycle arrest, cell death, autophagy, and senescence . Howeverapelin human peptide that inhibits vasodilation of blood vessels induced by UVB exposure [Many antioxidant/anti-inflammatory small molecules from plants have been reported in the literature, but only a few of them are proteins, except for endogenous proteins directly involved in detoxification of ROS. An exception is the exposure . In thisThe possibility of modulating the anti-inflammatory action by incorporating an active agent into liposomes is being established ,44 and tEDTA, sodium acetate, sodium hydroxide, dimethylsulfoxide, absolute ethanol, potassium monophosphate, and Triton X-100 were from Merck, Darmstadt, Germany. Milli-Q-water was purified through Millipore, System Bedford, MA, USA. All the reagents used in formulations were for analysis Galena  or Clariant . Sephadex G-10 was from GE Healthcare; Coomassie G-250 was from Bio-Rad Laboratories, Hercules, CA, USA; TdT-Fragel DNA-Fragmentation Detection Kit was from Calbiochem, San Diego, CA, USA).hairless (HRS/J) mice (8–10 weeks old) whose matrices were provided by Universidade de São Paulo-Campus Ribeirão Preto were maintained in the animal house facility from the Laboratório de Substâncias Antitumorais (LSAT), UFMG. The experiments were carried out in accordance to internationally accepted guidelines on animal handling and were approved by the institutional Ethics Committee on Animal Experimentation, CETEA-Protocol 174/2010. The animals were housed in individual cages with controlled temperature, humidity and light–dark cycle, with unrestrained access to food and water.Male Vasconcellea cundinamarcensis females from Valle del Elqui, Chile. A voucher specimen of the plant was deposited at the herbarium of the Universidad de La Serena, Chile, with No 15063. Latex was collected by incisions onto the surface of unripe fruits with a steel blade. Following collection, latex was stored in the dark, at −20 °C until lyophilized. The isolation of P1G10 was described previously [Latex was obtained from unripe fruits of eviously . Brieflyeviously  and the eviously . The staw/v. The control sample contained a similar volume of saline dispersed directly in the vehicle. The formulations were stored at 4 °C.The vehicle used was a hydrosoluble base containing 5% propyleneglycol, 5% monoolein, and 0.25% hydroxyethylcellulose gel base (Natrosol) (pH 6.5). A 10% aqueous solution of P1G10 was dispersed into the hydrosoluble vehicle to a final concentration of 0.1% or 1.0 % controlAbs is the absorbance of DPPH radical + methanol; sampleAbs is the absorbance of DPPH radical + sample divided by the controlAbs.Free radical scavenging activity of P1G10 was determined using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay . Various−2 and the dose applied was 2.4 J cm−2 [The source of UVB irradiation was a Cole-Parmer (USA) ultraviolet lamp with an emission peak at 312 nm (measured in Ocean Optics spectrometer and OOI Base32 Software). The lamp was mounted at 13 cm distance from a table top where mice were irradiated. The UVB output was determined using a calibrated CMP22 radiometer Kipp and Zonen. The irradiance was set at 8 W m4 J cm−2 .n = 5/group). Before irradiation, the animals were anesthetized with an i.p. injection containing ketamine (80 mg kg−1) and xylazine (10 mg kg−1). Thereafter, each animal was placed in polyvinyl chloride chamber (15 × 25 mm2) and received a dorsal single dose of 2.4 J cm−2 of UVB. The erythema score represents the redness intensity of the irradiated area [The animals were randomly divided into five groups . The suspension was centrifuged for 10 min at 13,000× g at 4 °C. The protein content in supernatants was measured by Bradford [Skin-tissue fragments were triturated in liquid NBradford .−1 tissue.ROS levels were detected using the oxidation sensitive dye 2,7-dichlorofluorescin diacetate  assay, as previously described ,52. The −1 protein.The method , involvi2O2 as described by Shangari and O’Brien [2O2 buffered in potassium phosphate . The reduction in absorbance at 240 nm was scored after 3 min incubation at 37 °C and compared to a blank without the sample, and expressed according to the formula:This measurement is based on the decomposition of H O’Brien . Ten µL −1 of catalase (sample volume), ΔA = change in absorbance, d = sample dilution, v = volume of reaction, V = sample volume in the reaction and ε = 2.35 × 105 M−1 cm−1 . The results referred to the protein content were expressed as percentage from control.The activity is expressed as Units mL–1 glutathione reductase was incubated at 37 °C for 5 min with appropriate aliquots of tissue homogenates. The assay started with the addition of 2.2 mM H2O2. The absorbance of the samples was measured at 340 nm in a UV spectrophotometer . The data were registered at 15 s intervals during 300 s. GSH-Px activity was expressed as µM mg−1 tissue as described [The reaction mixture consisting of 100 mM potassium phosphate buffer, 1.1 mM sodium azide, 2.1 mM EDTA, 0.15 M reduced glutathione, 8.4 mM NADPH, and 100 × UmLescribed .−1 solution of o-phthalaldehyde dissolved in methanol was added to supernatants, previously diluted with 0.1 M sodium phosphate buffer, 5 mM EDTA, pH 8.0. After 15 min incubation at room temperature, the fluorescence was recorded in a plate reader  at 350 nm excitation and 420 nm emission). GSH concentration was determined using a GSH standard curve and expressed as µM mg−1 tissue [Samples were precipitated with 10% trichloroacetic acid, centrifuged, and the pellet discarded. Then, 1 mg mL1 tissue .2HPO4, pH 3 (Sigma-Aldrich), 2.5 mM butylatedhydroxytoluene  and incubated in a dry bath for 15 min, 100 °C. The reaction products were extracted with n-butanol and MDA was determined in the organic phase at 532 and 600 nm . MDA concentration was determined using a standard curve  and expressed as µM mg−1 tissue [Skin homogenates were mixed with 10 mM 2-thiobarbituric acid  solution containing 75 mM K1 tissue .g for 30 min. The supernatant was used to determine levels of IL-1β, IL8, TGF-β, and IL10 by ELISA [−1 tissue.Tissue was homogenized in PBS pH 7.4 containing 0.05% Tween-20 (Sigma-Aldrich) and centrifuged at 10,000× by ELISA . The ass4, pH 4.7 was centrifuged at 12,000× g for 10 min. Pellets were suspended in 0.05 M NaPO4 buffer (pH 5.4) containing 0.5% hexadecyltrimethylammonium bromide followed by three freeze–thaw cycles using liquid N2. After addition of 1.6 mM tetramethylbenzidine and 0.3 mM H2O2 to supernatant samples, MPO activity was measured by the change in absorbance at 450 nm. Results are expressed as percentage of basal MPO activity.The sediment recovered after centrifugation from tissue homogenates (see cytokine assay above) was suspended with buffer for myeloperoxidase assay (MPO) to monitor neutrophil, as described by . Tissue 2 and 0.15 M NaCl). Digital images and densitometric analyses of gels were obtained with ImageQuant LAS4000 , and the MMPs activity was quantified with ImageQuant software. The results are expressed as percent inhibition of the gelatinolytic activity induced by the extracts using as reference saline treated samples. Molecular mass standards (BioRad Laboratories) were electrophoresed in parallel with samples to assess protein size.Gelatinolytic activity of matrix metalloproteinase-9 (MMP-9) was assayed by zymography, as previously described by . An aliq2, and anti-p53 (Abcam). Skin samples were mechanically triturated and powdered in a mortar using liquid N2 for 3 min. The protein was extracted in 500 µL ice-cold lysis buffer . After centrifugation at 10,000× g at 4 °C for 30 min, the protein concentration was assessed by Bradford assay [Variations in pAkt, Akt, GAPDH, COX2 and p53 were evaluated by Western blot using anti-pAkt, anti-Akt, and anti-GAPDH , anti-COXrd assay ,51. The 6 mm−2, area). Skin thickness analysis was made with a 10× objective in five different random lesions and their average calculated. In Gomori trichrome stained slices, blue color intensity represents the density of collagen at 40× magnification. Mean density of each group was defined as the collagen density of the irradiated area versus the collagen density of equal area in normal tissue.Tissue sections were processed for microscopy and stained with hematoxylin and eosin (H&E)  for morpThe streptavidin-peroxidase (SP) method was used for immunohistochemical staining using the Kit Ultra vision large volume anti-polyvalent detection system, HRP-Lab Vision, DAKO, as described . The antp < 0.05; ** p < 0.01 or *** p < 0.001.The data from triplicate experiments are expressed as the mean ± standard error (SEM). Each experiment was repeated three times. Differences between three or more groups were analyzed using one-way ANOVA, followed by Newman–Keuls multiple comparisons test. Statistical significance was set at * V. cundinamarcensis exerts topic antioxidant and anti-inflammatory effects on mice dermal lesions induced by UVB radiation. These effects are mediated by increase in scavenging agents and/or the enzymes involved in their synthesis, and a reduction of pro-inflammatory agents. The changes induced by P1G10 provided a protection minimizing the symptoms triggered by UV radiation.The proteolytic fraction (P1G10) from


We previously reported that four doses or four double doses of hepatitis B vaccination regimens could not significantly increase a response rate compared with standard doses. However, the antibody levels were higher in the four doses and four double doses groups. This study followed those patients for at least 3 years and aimed to evaluate the immunogenicity of the three vaccination regimens.3, undetectable plasma HIV-1 RNA, and negative for all hepatitis B virus markers were randomly assigned to receive one of three recombinant vaccines  regimens: 20 μg IM at months 0, 1, and 6 , 20 μg IM at months 0, 1, 2, 6 , or 40 μg IM at months 0, 1, 2, and 6  between February 2011 and May 4, 2012. Of 132 participants, 126 were evaluated from August 2015 to January 2016; 42 in the standard doses, 43 in the four doses, and 41 in the four double doses groups.HIV-infected adults who had CD4+ cell counts > 200 cells/mmP = 0.067 vs. the standard doses group); and 80.5% (95% CI 67.8–93.2%) in the four double doses group (P = 0.033 vs. the standard doses group). Factors associated with a responder were the vaccination schedule (either four doses or four double doses groups) and a younger age.At a median duration of 49.7 months (range 46.7–53.7) after completion of the primary vaccination schedule, the percentages of responders with anti-HBs ≥ 10 mIU/mL were 57.1% (95% CI 41.5–72.8%) in the standard doses group; 76.7% (95% CI 63.6–89.9%) in the four doses group (3 and undetectable plasma HIV-1 RNA. The standard vaccination regimen may not be the best strategy to provide long-term immune response against hepatitis B virus among HIV-infected individuals.Despite the highly effectiveness of the standard hepatitis B vaccination regimen at 6 months after completion, the long-term immunogenicity was lower than the four double doses regimen among HIV-infected adults with CD4+ cell counts > 200 cells/mmTrial registration NCT1289106, NCT02713620 The duration of suppressed plasma HIV-1 RNA was shorter in patients in the standard doses group than the other two groups [As previously reported, demographic and clinical characteristics of participants by vaccination regimen at the time of vaccination were similar in terms of ages, body mass index, creatinine clearance, nadir CD4 cell count, time elapsed since HIV diagnosis, antiretroviral regimens, history of drug resistance, HIV risk exposure, alcohol uses and underlying diseases including hypertension, and dyslipidemia. There were more males and a longer duration of combination antiretroviral therapy in patients in the four double doses group than the standard doses group. Patients in the standard doses group had a lower median CD4 cell count than the other two groups; [400 cells/mm3 in the standard doses group, 694  cells/mm3 in the four doses group (P = 0.003 vs. the standard doses group) and 625  cells/mm3 in the four double doses group (P = 0.237 vs. the standard doses group). All patients had undetectable HIV-1 RNA.The median CD4 cell count during this follow up period was 534  cells/mmP = 0.067 vs. the standard doses group); and 80.5% (95% CI 67.8–93.2%) in the four double doses group (P = 0.033 vs. the standard doses group), respectively  were 57.1% (95% CI 41.5–72.8%) in the standard doses group; 76.7% (95% CI 63.6–89.9%) in the four doses group (P = 0.258) and 46.3% (95% CI 30.4–62.3%) in the four double doses group (P = 0.115 vs. the standard doses group)  were 28.6% (95% CI 14.3–42.8%) in the standard doses group; 41.9% (95% CI 26.5–57.2%) in the four doses group (P = 0.047 vs. the standard doses group); and 57.5 mIU/mL (95% CI 29.7–111.5) in the four double doses group (P = 0.030 vs. the standard doses group), respectively  in the standard doses group; 51.6 mIU/mL (95% CI 29.3–91.0) in the four doses group , and younger age at the time of vaccination . No factors associated with achieving a high titer level (anti-HBs ≥ 100 mIU/mL) were demonstrated.Characteristics between responders and non-responders are shown in Table 3 and undetectable HIV-1 RNA, the response rate to the standard HBV vaccination schedule ranged between 40 and 71% [Among HIV-infected individuals with CD4 cell count ≥ 200 cells/mm and 71% , 13, 16. and 71% , 17, 18. and 71% . Another3, 70% and 27% of participants retained seroprotection at 36 and 84 months after vaccination, respectively [This study followed the participants in the primary analysis with the median follow-up time of 49.6 months addressed the importance of high anti-HBs titers after primary vaccination. Studies of long-term seroprotection rates have been reported –23. The ectively . The latectively . Our stuBased on the results of this study along with other previous reports, the four double doses vaccine schedule increased long-term protective antibody levels as compared with the standard doses schedule. However, a randomised controlled trial is still needed to determine whether the four double doses schedule is superior to the four doses schedule or three-double dose schedule as the higher antibody titer after primary vaccination conveyed longer seroprotection.3 and undetectable plasma HIV-1 RNA, so the results could not be generalized to other groups of HIV-infected individuals. Second, we did not design the trial to compare the response rates between the four double doses group and the four doses group, the sample size was inadequate to detect the difference if it existed. Third, as we performed the antibody titer at a specific time point, we were unable to calculate the median time to loss of seroprotection. The appropriate time point to re-assess anti-HBs titers could not be addressed from this study.Our study had some limitations. First, the study included only HIV-infected adults with CD4+ cell counts > 200 cells/mmIn conclusion, anti-HBs titers were significantly higher with four-double doses schedule than the standard doses and therefore, conveyed a longer-term protection.Additional file 1: Fig. S1. Consort diagram of participants.


Fragments were identified with LC-hrMS and these were incorporated in the LC-MS/MS method. A method of 10 min was developed and a solid phase extraction clean-up procedure was optimised for shellfish matrix. The determined limits of detection were respectively 8 and 22 µg PlTX kg−1 for mussel and oyster matrix. Oysters gave a low recovery of approximately 50% for PlTX during extraction. The method was successfully in-house validated, repeatability had a relative standard deviation less than 20% (n = 5) at 30 µg PlTX kg−1 in mussel, cockle, and ensis, and at 60 µg PlTX kg−1 in oyster.Palytoxin (PlTX) and analogues are produced by certain dinoflagellates, sea anemones, corals and cyanobacteria. PlTX can accumulate in the food chain and when consumed it may cause intoxication with symptoms like myalgia, weakness, fever, nausea, and vomiting. The analysis of PlTXs is challenging, and because of the large molecular structure, it is difficult to develop a sensitive and selective liquid chromatography-mass spectrometry (LC-MS/MS) method. In this work, an LC-MS/MS method was developed to analyse PlTXs with use of lithium iodine and formic acid as additives in the mobile phase. For method development, initially, LC-hrMS was used to accurately determine the elemental composition of the precursor and product ions. The main adduct formed was [M + H + 2Li] Ostreopsis ovata and Ostreopsis siamensis 2+ and [M − 2H]2− ions with relatively low abundance (spectra in m/z 700 to 2800 was acquired in both positive and negative electrospray ionisation (ESI) mode. With ESI negative mode no PlTX ions were formed when the mobile phase contained Li. With positive ESI mode, PlTX ions were formed without additives, and under both acidic and alkaline conditions. However, no single charged ion species was obtained , m/z 891.8374 ([M + H + 2Li − H2O]3+), m/z 675.1345 ([M + H + 3Li]4+), and m/z 1343.2548 ([M + H + Li]2+) were observed. Under alkaline conditions two major ions were formed: m/z 897.8374 ([M + H + 2Li]3+) and m/z 902.5024 ([M + H + 2Li − 2H + O]3+). Furthermore, low abundant ions with m/z 1343.2482 ([M + H + Li]2+) and m/z 1350.2463 ([M + H + Li − 2H + O]2+) as well as some in-source fragmentation resulting in multiple losses of water were detected. No quadruple charged ions were formed under alkaline conditions. The average Δ ppm error of the measured masses compared to the theoretical masses was 2.5 ppm with a maximum of 4.1 ppm for the [M + H + Li − 2H + O]2+ ion and a minimum of 1.6 ppm for the [M + H + 2Li + O]3+ ion. Under both the acidic and alkaline conditions, an in-source oxidation product was observed, the [M + H + 2Li + O]2+ and [M + H + Li − 2H + O]2+ ion under respectively acidic and alkaline conditions. This in-source oxidation which occurred is not very often described, however, there is some literature available on the oxidation of large molecules. A possible cause might be the condition of the ESI capillary, which is described by Chen et al. who showed that corroded capillaries can cause in-source oxidation of peptides 1+, [C70H113O28N+H+Li]2+ and [C16H26O5N2+H]1+. The proposed structures of the fragments are shown in When selecting m/z, some of the OVTXs have the same m/z and are structurally similar and are therefore not separated with the fast 10 min LC gradient. To obtain an improved chromatographic separation a slightly longer LC-method with a gradient with a slower increase over time in organic strength was developed. The longer LC method is capable of separating the OVTXs a to e and putative PlTX (pPlTX) as demonstrated for an algae extract  clean-up procedure. Besides the methanol/water (50:50 v/v) extraction, a variety of extraction solvents was tried like 100% methanol or methanol/water (50:50 v/v) with additives at various pHs. Furthermore, the addition of ethylenediaminetetraacetic acid (EDTA) was explored to improve extraction efficiency. EDTA was added to create metal complexes and reduce the amount of free metals like zinc in the extract which might also form complexes with PlTX. This might be the case as it is well known that oysters are rich in zinc content compared to other shellfish species 3+) with an isolation window of 4 Da. As collision gas nitrogen was used. The normalized collision energy (NCE) was set at 20 during fragmentation. Then after fragmentation, the ions were scanned from 187 to 1850 m/z with a resolution set at 70,000 FWHM. The automatic gain control representing the maximum capacity of the C-trap was set at a maximum of 106 ions or a maximum injection time of 200 ms for the full scan and a maximum of 2 × 105 ions or a maximum injection time of 200 ms for the MS2 scans were allowed.For method development, a Thermo Scientific UltiMate 3000 LC-system coupled to a Thermo Scientific Q Exactive focus hybrid quadrupole-orbitrap mass spectrometer  was used. In order to detect the PlTXs, electrospray ionisation in positive was used. The spray voltage in positive ionisation mode was set at 3.5 kV. The capillary temperature was set at 260 °C. For infusion experiments, PlTX was infused directly into the Mass spectrometer together with a flow of 0.1 mL min−1 was set.For measurements of shellfish extracts and the validation study, a Waters Acquity UPLC coupled to a Waters Xevo TQ-S tandem mass spectrometer  was used. The triple quad MS system was operated in positive electrospray mode and data were recorded in MRM mode using three transitions per toxin. PlTX: 898.2 > 880.2 collision energy (CE) 25 eV, 898.2 > 874.2 CE 25 eV, 897.8 > 711.9 CE 30 eV; OVTX a: 887.6 > 869.6 CE 25 eV, 887.6 > 863.6 CE 25 eV, 887.2 > 327.2 CE 30 eV; OVTX b: 902.2 > 884.2 CE 25 eV, 902.2 > 878.2 CE 25 eV, 901.9 > 371.2 CE 30 eV; OVTX c: 907.6 > 889.6 CE 25 eV, 907.6 > 883.6 CE 25 eV, 907.2 > 703.9 CE 30 eV; OVTX d and e: 892.8 > 874.8 CE 25 eV, 892.8 > 868.8 CE 25 eV, 892.5 > 327.2 CE 30 eV (specific for OVTX d) and 892.5 > 343.2 CE 30 eV (specific for OVTX e). Furthermore, a cone voltage of 30 V, a capillary voltage of 3 kV, source temperature of 150 °C, desolvation temperature of 600 °C and desolvation gas flow of 800 L hMytilus edulis), five oysters , five ensis (Ensis sp.) and five cockles (Cerastoderma edula) homogenates were spiked each with 10, 30 and 60 µg PlTX kg−1 for determination of recovery and repeatability. The same 20 blank samples were used to determine selectivity. Matrix matched standards in mussel homogenate at 0, 5, 10, 30, 60 and 100 µg PlTX kg−1 were used for quantification and linearity.Validation of the LC-MS/MS method for quantification of PlTX in shellfish was done according to the SANTE/2015/11945 guidance document . There w