However, the extracts did not strongly inhibit α-amylase. In general, the A extracts from solid-liquid extraction with 60% acetone showed stronger inhibitory potential towards a-glucosidase and GPα than other extracts whereby no correlation with TPC or TFC were observed. Additionally, DPP IV was mainly inhibited by HE extracts but the effect was not of biological relevance. Our results show that BSG is a potent source of α-glucosidase and GPα inhibitors, but further research is needed to identify these bioactive compounds within BSG extracts focusing on extracts from solid-liquid extraction with 60% acetone.With a yearly production of about 39 million tons, brewer’s spent grain (BSG) is the most abundant brewing industry byproduct. Because it is rich in fiber and protein, it is commonly used as cattle feed but could also be used within the human diet. Additionally, it contains many bioactive substances such as hydroxycinnamic acids that are known to be antioxidants and potent inhibitors of enzymes of glucose metabolism. Therefore, our study aim was to prepare different extracts—A1-A7 (solid-liquid extraction with 60% acetone); HE1-HE6  and HA1-HA3 —from various BSGs which were characterized for their total phenolic (TPC) and total flavonoid (TFC) contents, before conducting in vitro studies on their effects on the glucose metabolism enzymes α-amylase, α-glucosidase, dipeptidyl peptidase IV (DPP IV), and glycogen phosphorylase α (GPα). Depending on the extraction procedures, TPCs ranged from 20–350 µg gallic acid equivalents/mg extract and TFCs were as high as 94 µg catechin equivalents/mg extract. Strong inhibition of glucose metabolism enzymes was also observed: the IC Recently, Pratap Singh et al. tested different drying methods of BSG to find a sustainable and sensory appealing method with the aim of using BSG as protein rich snack [In recent decades, there has been growing interest in the valorization of agri-food waste and agricultural by-products as a way of achieving sustainable food production. At present, such by-products are mainly used as fuels, organic fertilizers, or animal feed. However, they could also be valuable sources of bioactive compounds. Consequently, there is ongoing research on their potential uses in the pharmaceutical industry and in functional foods . One bypch snack . Sahin efiber 30–0% w/w anch snack ,7. BSG ach snack ,9,10,11.ch snack . The antch snack ,13,14. Ich snack ,16,17, mch snack . For insch snack ,16,19, ach snack . Strong ch snack . Whole gch snack . Furtherch snack . We therch snack . The tot®, Th. Geyer GmbH & Co. KG . Methanol, acetonitrile, and acetone (HiPerSolv CHROMANORM per HPLC) were obtained from VWR . Gallic acid and glucose-6-phosphate-dehydrogenase from Saccharomyces cerevisiae  were purchased from Alfa Aesar  and formic acid from J.T. Baker . Ethyl acetate was obtained from Honeywell  and strata C18-E SPE (solid phase extraction) cartridges  were purchased from Phenomenex . 2-Chloro-4-nitrophenyl-α-D-malto-trioside (CNPG3) and Gly-Pro-7-amido-4-methylcoumarin hydrobromide (H-Gly-Pro-AMC) were obtained from Carbosynth . 4-Nitrophenyl-β-D-glucopyranoside (pNPG) was purchased from Acros Organics . Human dipeptidyl peptidase IV, native enzyme  was purchased from Active Bioscience . The drugs used as positive controls were Januvia 100 mg in which the active compound is Sitagliptin from MSD  and Glucobay®100 in which Acarbose is the active compound from Bayer Pharmaceuticals . α-Amylase from hog pancreas (EC 3.2.1.1), glycogen phosphorylase α from rabbit muscle , α-glucosidase from Saccharomyces cerevisiae (EC 3.2.1.20), and phosphoglucomutase from rabbit muscle  were purchased from Sigma-Aldrich .Chemicals were of analytical grade and obtained from Sigma-Aldrich  unless otherwise stated. Sodium dihydrogen phosphate dihydrate was purchased from Riedel de Haen . Magnesium sulfate heptahydrate, disodium hydrogen phosphate monohydrate, and potassium hydrogen phosphate were obtained from Merck . NADP disodium salt as well as glycogen from oysters and TRIS HCl were from Carl Roth . DMSO was obtained from J&K Scientific . Hydrochloric acid (HCl) and ethanol were purchased from CHEMSOLUTEThree different batches of Brewer´s spent grain (BSG) were provided by the conventional Orval brewery in Belgium  and the brewing group of the chair of bioprocess engineering at the Technische Universität Kaiserslautern . The malt used for each brewing process is specified in v/v), six HE (hydrolysis and ethyl acetate) extracts were prepared by alkaline hydrolysis followed by ethyl acetate extraction, and three HA (hydrolysis and acetone) extracts were prepared by alkaline hydrolysis followed by 60% acetone  extraction (Three different extraction processes were used including solid-liquid extraction with 60% acetone  or ultratraction . Each exv/v) at 60 °C for 30 min while stirring in a ratio of 1 g solid per 20 mL extraction solvent. The solid residues were separated from the liquid supernatants by filtration, and the residues and filtrates were processed independently thereafter.The three different BSG samples (BSG 1-3) were extracted twice with 60% acetone  and methanol was added (around 5 mL) to produce the ‘liquid´ raw extracts A1-A3. These were stored in the dark at −20 °C until purification see .g) and the supernatants were adjusted to a pH ≤ 2 with concentrated hydrochloric acid (HCl). The samples were then centrifuged again  and the supernatants were extracted three times with 200 mL of ethyl acetate, which was then removed by rotary evaporation under reduced pressure at 40 °C. The viscous residues were transferred to water (around 50 mL), methanol was added (around 5 mL) to obtain ´liquid´ raw extracts HE1-HE3, and they were stored in the dark at −20 °C before purification by SPE . Afterwards the samples were centrifuged  and one BSG sample previously defatted with isopropanol (BSG 3) were subjected to alkaline hydrolysis with 4M NaOH at a ratio of 1 g solid per 10 mL solvent over 90 min in an ultrasonic bath . The solid residues were separated from the liquid supernatants by filtration, and the filtrates and residues were processed independently thereafter.The filtrates were adjusted to a pH ≤ 2 with concentrated HCl and then filtered, after which the resulting filtrates were extracted three times with 325 mL of ethyl acetate. The extraction solvent was then removed by rotary evaporation under reduced pressure at 40 °C. The viscous residues were transferred to water (around 50 mL) and methanol was added (5 mL) to obtain the ‘liquid’ raw extracts HE4-HE6. These were stored in the dark at −20 °C until purification by SPE see .v/v) at a ratio of 1 g solid per 10 mL of extraction solvent at 60 °C  by stirring. Afterwards, the combined acetone phases were adjusted to a pH ≤ 2 with concentrated HCl, the samples were filtered, and the extraction solvent was removed by rotary evaporation under reduced pressure at 40 °C. Again, the viscous residues were transferred to water (around 50 mL) and methanol was added (around 5 mL) resulting in ´liquid´ raw extracts HA1-HA3 that were stored in the dark at −20 °C until purification by SPE . A ratio of 1 g solid per 10 mL extraction solvent was used and extraction was performed twice for 30 min each at 60°C under stirring. The samples were then filtered and the extraction solvent of the filtrates was removed by rotary evaporation under reduced pressure at 40 °C. The viscous residues were transferred to water (around 50 mL) and methanol was added (around 5 mL), giving the ‘liquid’ raw extracts A4-A7. These extracts were stored in the dark at −20 °C until purification by SPE  and two defatted BSG samples (BSG 2 + 3) were milled into a powder and then subjected to solid-liquid extraction with 60% acetone  were purified by applying a modified variant of a previously reported solid phase extraction (SPE) method  using StThe total phenolic content (TPC) of the extracts was determined by the spectrophotometric method of Folin-Ciocalteau ,29 with The total flavonoid content (TFC) of the extracts was determined by a modified aluminum chloride assay . A calib50-values  were calculated relative to the final concentrations in each well.The α-amylase inhibition assay was based on previously described spectrophotometric methods ,32. Samp50 value. Then, 20 µL of each sample, positive and negative controls, were placed in a well of a 96-well microplate and mixed with 70 µL of α-glucosidase from Saccharomyces cerevisiae (1 U/mL) dissolved in 0.1 M PBS (pH 6.8). A blank (in which the enzyme solution was replaced by PBS) was analyzed alongside each sample, allowing the samples’ intrinsic color to be accounted for in the inhibition calculation. After incubation at 25 °C for 10 min, the substrate solution, 4 mM 4-nitrophenyl-β-D-glucopyranoside (pNPG) in 0.1 M PBS at pH 6.8, was added and the samples were incubated again for 5 min at 25 °C. Finally, the absorbance was measured at λ = 405 nm using a microplate reader . Concentrations and IC50 values were calculated relative to the final concentrations in each well.The α-glucosidase inhibition assay was conducted according to Berger et al. (2020) and You et al. (2011) ,33. Samp50-values were calculated relative to the final concentrations in each well.Inhibition of DPP IV was measured using a modification of the fluorometric method reported by Connolly et al. 2014 . SamplesThe extracts’ inhibitory potential against GPα was determined by a spectrophotometric method that was adapted for use with 96-well microplates . Each saTwo buffers were used. Buffer A  was used to prepare GPα stock solutions, while Buffer B  was diluted with double distilled water and used as the assay buffer.Stock solutions of the enzymes were prepared as follows: GPα was dissolved in buffer A to a concentration of 5 U/mL. PGM and G6PDH were dissolved in double distilled water to a concentration of 500 U/mL. All three enzyme stock solutions were stored at −80 °C until use.The assay medium was prepared by mixing 5 U/mL G6PDH, 12 U/mL PGM, and 3.148 mg/mL NADP in assay buffer. The enzyme mix consisted of 93.75 mU/mL GPα and 100 mM glucose in assay buffer. Glycogen was dissolved in assay buffer at a concentration of 2 mg/mL.50 values were calculated relative to the final concentrations in each well.For the assay, 20 µL of the sample (five concentrations depending on inhibition strength), positive control, and negative control were transferred to a 96-well microplate and each mixed with 50 µL of the assay medium. Each sample was then mixed with 80 µL of enzyme mix. Each sample was analyzed alongside a blank (100 mM glucose dissolved in buffer B instead of enzyme mix) to allow the samples’ intrinsic color to be accounted for when calculating the degree of inhibition. The reaction was started by adding 50 µL glycogen (2 mg/mL) and the samples were incubated for 30 min at 37 °C. Finally, their absorbance at λ = 340 nm was measured. Concentrations and ICt test (one-sided). Differences were considered significant at the p < 0.05, p < 0.01, and p < 0.001 levels.Results are presented as means and SD of three to 83 independent experiments. Statistical analyses were performed with Origin 2019G  and Excel Office Professional Plus 2016 . Data were checked for normality (Anderson Darling test) and homogeneity of variance (Fisher test). The significance of differences from positive controls (DPP IV and GPα inhibition assays), between extraction groups (TPC assay and GPα inhibition assay), and within extraction groups  was evaluated using the one-sample The 16 different extracts  were prepared from three different BSG batches (numbered 1 to 3) from two different breweries. Two of the samples were defatted with isopropanol before extraction. The extraction processes involving SPE purification provided relatively low yields in the range of 0.8–3.5 g/100 g dried BSG see . The yiep < 0.01 and p < 0.001) more phenolics (157.5 ± 13 to 351.5 ± 20.7 µg GEq/mg extract) than those prepared by solid-liquid extraction with 60% acetone (A1-A7) (24.6 ± 3.3 to 107.2 ± 15.8 µg GEq/mg extract) and those obtained after hydrolysis followed by solid-liquid extraction with 60% acetone (HA1-HA3) (60.5 ± 2.6 to 69.3 ± 6.7 8 µg GEq/mg extract). Additionally, extracts prepared from previously defatted BSG  had slightly higher TPCs than those from the corresponding non-defatted BSG samples ; this difference was even significant (p < 0.001) for the HE extracts. Extracts prepared from the same BSG batches using different extraction processes also had significantly different TPCs: A2 and A3 had significantly (p < 0.01) lower TPCs than A4 and A6. Additionally, HE extracts of the non-defatted BSG 3 sample  had significantly lower TPCs (p < 0.01 and p < 0.001) than all other HE extracts . In general, extracts of BSG 3  had lower TPCs than those prepared from other BSG batches independently of the choice of extraction process, while alkaline extracts of BSG 1 (HE1) and BSG 2  had similar TPCs. However, the TPCs of solid-liquid extracts of BSG2 (A2) were almost twice those of BSG1 (A1) leading to the assumption that, besides the extraction process, the BSG batch is also an important factor in terms of TPC.The TPC values of each extract are shown in To compare and discuss our results with literature, the obtained TPC values are related to the yields of the extracts. As our SPE method was only used as a purification step and no single substances were yet identified, no recovery rates were determined. Therefore, the relation to the yields is considered as an estimation giving adjusted TPCs ranging from 0.43 ± 0.08 to 0.86 ± 0.13 mg GEq/g BSG dw for solid-liquid extracts A1-A7, 1.7 ± 0.17 to 2.3 ± 0.11 mg GEq/g BSG for acetone extracts of hydrolysis residues (HA1-HA3), and 2.52 ± 0.21 to 3.96 ± 0.32 mg GEq/g BSG dw for extracts prepared by alkaline hydrolysis (HE1-HE6). The yield-related TPC values maintain nearly the same trend as the extract-related TPCs whereby HA extracts show slightly higher TPC values when related to the yield.p < 0.05 and p < 0.001). and as previously observed for the TPC of acetone extracts of defatted BSG, extracts of the defatted BSG batch 2 (A5) tended to have the highest TFC while those from the defatted BSG batch 3 (A7) had the lowest TFC. As was also the case for TPCs, HE extracts from the non-defatted BSG 3 (HE3 and HE5) had significantly lower TFCs (p < 0.001) than all other HE extracts .The TFC measurements showed that many extracts  had no detectable flavonoid content. The other extracts under study  showed TFCs ranging from 7.6 ± 0.7 to 93.6 ± 2.9 µg CE/mg extract . The treAs already performed for the TPC values, TFC values were calculated in relation to extracts ’yield to facilitate comparison with previously reported values. The TFCs obtained in this way ranged from 0.16 ± 0.04 to 0.27 ± 0.02 mg CE/g BSG for acetone extracts , 0.27 ± 0.02 mg CE/g BSG for acetone extracts of alkaline hydrolysis residues (HA3), and 0.22 ± 0.13 to 0.94 ± 0.03 mg CEq/g BSG for ethyl acetate extracts of alkaline hydrolysis solutions (HE1-HE5). The same trend as for TPC values was observed; HE extracts had the highest yield-related TFC whereby HA3 extract showed a slightly higher yield-related than extract-related value.50 values, i.e., the concentration at which the enzyme’s activity was reduced by 50% if the inhibition was strong enough to calculate this value. Otherwise, the extract’s inhibitory activity was considered slight to moderate.The influence of the different BSG extracts on four glucose metabolism enzymes  was investigated in vitro. The extracts’ inhibitory potential was compared to that of positive controls and expressed as IC®100) was used as positive control; its IC50 value was determined to be 35.5 ± 4.4 µg/mL. Most extracts did not detectably inhibit this enzyme even at the highest tested concentration of 1.05 mg/mL . However, extracts prepared by solid-liquid extraction with 60% acetone from BSG 1 and BSG 2  showed slight to moderate inhibition at 1.05 mg/mL, reducing the enzyme’s activity by 23.1 ± 4.2 to 49.7 ± 12.3% (data not shown); this level of inhibition is too low to permit the calculation of an IC50 value. Generally, there were some notable discrepancies between the extracts of different BSG batches as well as between the extraction processes. In particular, BSG 3 seems to contain no α-amylase inhibitors, which might be related to the malt used in the brewing process. The active compounds that are present may not be liberated by bases; they appear to be free and easily extractable with 60% acetone since all other extracts (HE and HA) did not show any inhibiting effect.The effects of the BSG extracts on α-amylase activity were investigated using a spectrophotometric in vitro assay in which the pseudo-tetra-saccharide Acarbose  as a positive control. Extracts A2-A7 and HE4-HE5 exhibited strong inhibition of the enzyme, with IC50 values ranging from 67.4 ± 8.1 µg/mL to 268.1 ± 29.4 µg/mL  showed inhibitory activity, whereas only two HE extracts  and no HA extracts  were strong inhibitors. Additionally, extracts prepared from BSG 2 by solid-liquid extraction with 60% acetone  were significantly stronger inhibitors of α-glucosidase than comparable A extracts from BSG 3  (p < 0.01 and p < 0.001). A extracts prepared from defatted material  showed stronger inhibition than those prepared from untreated (non-defatted) BSG samples ; this difference was statistically significant when comparing A6 and A7 (p < 0.001).The α-glucosidase-inhibiting potential of the 16 BSG extracts was investigated using the potent inhibitor Acarbose  strongly inhibited the enzyme, with IC50 values ranging from 290.6 ± 97.4 to 778.4 ± 95.5 µg/mL, as shown in p < 0.001) higher IC50 values than the positive control. Extracts A1 and A3-A6 (prepared by solid-liquid extraction with 60% acetone) and HA1 and HA2  exhibited slight to moderate inhibition, reducing DPP IV activity by 15.2 ± 4.1 to 49.5 ± 4.4% at a concentration of 1 mg/mL (data not shown). In general, it was mainly HE extracts which showed potent inhibition, whereby the biological relevance has to be evaluated critically due to the potent inhibition of the diabetes drug Sitagliptin.A fluorometric method using Sitagliptin (in the form of 100 mg Januvia) as a positive control was used to investigate the effects of the BSG extracts (except A2) on DPP IV. The positive control agents resulted in a very low measured IC50 = 128 ± 10 µg/mL) served as a positive control. All of the tested extracts  strongly inhibited the enzyme, with IC50 values ranging from 12.6 ± 1.1 to 261 ± 6 µg/mL (p < 0.001). Furthermore, HE extracts from alkaline hydrolysis (HE2-HE4) were significantly (p < 0.001) less inhibiting than all other tested extracts . Additionally, as also seen for the inhibition of α-glucosidase, A extracts made from defatted BSG  were significantly (p < 0.001) more potent inhibitors than the corresponding extracts prepared from non-defatted BSG , whereby the difference between the inhibition potential was even higher than as observed for α-glucosidase. In general, extracts prepared by solid-liquid extraction with 60% acetone were more potent regarding the inhibition of GPα than extracts achieved by alkaline hydrolysis (HE extracts). Especially A5 and A7 are of interest, as their effect was about ten times that of the positive control caffeine.The inhibitory effects of the BSG extracts (except A2) on GPα were investigated using a spectrophotometric in vitro assay in which the potent inhibitor caffeine (IC 6 µg/mL . ExtractHordeum vulgare), is mixed with water at temperatures of around 65 °C in the mashing process, which is followed by the lautering process in which the liquid wort is separated from the solid BSG at a higher temperature (around 75 °C). BSG thus consists mainly of the insoluble components of the barley grains, i.e., the husks, which contain most of the grains’ phenolic compounds [Sustainable and responsible food production has become increasingly important in recent years, leading to an increased emphasis on the productive use of agri-food by-products. Because BSG is rich in fiber and proteins as well as polyphenols such as hydroxycinnamic acids, BSG is both an attractive ingredient for increasing the nutritional value of food  and a poompounds ,35. Duriompounds  In generompounds ,38. SincThe extracts used in this study were produced from various BSG batches by three extraction processes including different sequences or combinations of process steps such as solid-liquid extraction with 60% acetone and alkaline hydrolysis with 4 M NaOH. All methods applied have previously been used to extract polyphenols from BSG ,39. AlkaThe TPCs of the extracts ranged from 24.6 ± 3.3 to 351.5 ± 20.7 µg GEq/mg extract and strongly depended on the extraction method. To facilitate comparison of our results with already reported values in the literature we calculated yield-related values ranging from 0.43 ± 0.08 to 0.86 ± 0.13 mg GEq/g BSG dw for solid-liquid extracts A1-A7, 1.7 ± 0.17 to 2.3 ± 0.11 mg GEq/g BSG for acetone extracts of hydrolysis residues (HA1-HA3) and 2.52 ± 0.21 to 3.96 ± 0.32 mg GEq/g BSG dw for extracts prepared by alkaline hydrolysis (HE1-HE6). The same trend as for extract-related TPC values was observed with HE extracts containing the highest number of polyphenols. This was expected since most of the polyphenols in BSG are esterified and bound to the cell wall, and can thus be released by alkaline hydrolysis or enzymatic pretreatment ,42. SimiThe trends observed in the total flavonoid contents (TFC) of the extracts were similar to those observed for the TPC. The measured TFCs were relatively low and depended on the choice of extraction process. Specifically, the TFC values ranged from not detectable  to 93.6 ± 2.9 µg CEq/mg extract Yield related values ranged from 0.16 ± 0.04 to 0.27 ± 0.02 mg CE/g BSG for acetone extracts , 0.27 ± 0.02 mg CE/g BSG for acetone extracts of alkaline hydrolysis residues (HA3), and 0.22 ± 0.13 to 0.94 ± 0.03 mg CEq/g BSG for ethyl acetate extracts of alkaline hydrolysis solutions (HE1-HE5), whereby, as already for the TPC, the same trend as for extract-related TFC was seen. In general, HE extracts (except HE5) had much higher TFCs than A and HA extracts, as was also observed for the TPC. The influence of the raw material was also similar to that in terms of TPC: defatting process  resulted in higher TFCs. In general, the TFCs were around three times lower than the corresponding TPC, which could be due to the greater specificity of the flavonoid assay. Similar trends were observed by Stefanello et al., who used a slightly different method for TFC determination and obtained TFCs of 1.24 ± 0.08 and 1.34 ± 0.03 mg quercetin equivalents (QEq)/g BSG for extracts prepared by solid-liquid extraction with acetone of defatted and non-defatted BSG, respectively, and 2.93 ± 0.22 and 4.54 ± 0.23 mg QEq/g BSG for extracts prepared by alkaline hydrolysis of defatted and non-defatted BSG samples, respectively  and TFCsv/v) extracts of barley seeds in diabetic rats, leading to a reduction in blood glucose levels after 11 days of consumption [After preparing and characterizing the extracts, their biological activity was investigated. Four enzymes catalyzing different stages involved in the glucose metabolism process were studied: the digestive enzymes α-amylase and α-glucosidase, DPP IV, which is an indirect modulator of insulin secretion, and GPα, which is involved in glycogenolysis. All four enzymes are potent targets for the treatment of type two diabetes mellitus ,51. The sumption . Howeversumption .None of the studied extracts were found to strongly inhibit α-amylase. Only some extracts prepared by solid-liquid extraction , exhibited slight to moderate inhibition of this enzyme percentage. Thus, neither alkaline hydrolysis nor solid-liquid extraction of alkaline hydrolysis residues enable the extraction of potent α-amylase inhibitors from BSG. However, there were some notable differences between the extracts of different BSG batches. Donkor et al. investigated methanolic extracts of germinated barley with a TPC of around 100 µg ferulic acid equivalents/mL and found that they induced 35% inhibition of porcine pancreatic α-amylase. However, their methodology differed from ours and the concentration of the extract was not reported . In anot50 values comparable to that of the positive control agent Acarbose, a well-known inhibitor of α-glucosidase and α-amylase. The other extracts  showed moderate inhibitory activity. In general, all of the studied extracts inhibited α-glucosidase in vitro to at least some degree, indicating that BSG is a potent source of inhibitors of this enzyme, whereby mainly A extracts were biological active. Such strong inhibition of α-glucosidase by BSG extracts has not previously been reported; Donkor et al. observed only slight inhibition by methanolic extracts of germinated and ungerminated barley [Inhibition of α-glucosidase was mainly observed for extracts prepared by solid-liquid extraction with 60% acetone (A2-A7), but two extracts prepared by alkaline hydrolysis (HE4-HE5) also had strong effects on α-glucosidase, with ICd barley , while Rd barley . The tot50 values were determined by those authors, the highest inhibition observed (using an extract concentration of 1.5 mg/mL) was around 40% [All of the studied extracts exhibited inhibitory activity towards DPP IV. In particular, all of the alkaline hydrolysis extracts (HE1-HE6) and most of those prepared from defatted samples (A7 and HA3) achieved strong inhibition. Other extracts  had only moderate effects. However, when evaluating the relevance of these results, it should be noted that the positive control Sitagliptin (an established anti-diabetic drug) is a 50 to 150 fold stronger DPP IV inhibitor. Most of the extracts studied here were more potent inhibitors than the BSG protein hydrolysates studied by Connolly et al.; while no ICound 40% . While tound 40% . In our All of the studied extracts were potent inhibitors of GPα with those prepared with 60% acetone generally revealing stronger effects than those prepared by alkaline hydrolysis (with the exception of HE5). All of these extracts other than HE2-HE4 inhibited the enzyme more strongly than the positive control caffeine. As already seen for α-glucosidase inhibition, extracts prepared from defatted material  were stronger inhibitors than the corresponding non-defatted extracts . GPα inhibition appeared to be uncorrelated with TFC and TPC. However, given the potent inhibitory activity of the extracts  BSG seems to be a potent source of GPα inhibitors . As the In general, HE  extracts had significantly higher TPCs and, in most cases, TFCs than A (solid- liquid extraction with acetone) and HA  extracts. Nevertheless, HE extracts were only more active than A or HA extracts towards DPP IV. Additionally, the BSG batch had no major effect on the inhibition potential. The acetone extracts were generally more potent inhibitors of α-glucosidase and GPα than the HE or HA extracts, or at least equally strong. For both enzymes, acetone extracts of defatted BSG  were stronger inhibitors than non-defatted acetone extracts . However, the differences between the three BSG batches were minor. It thus seems that the choice of extraction process affects the inhibitory activity of the extracts more than the choice of raw material.Multiple batches of BSG were extracted using three different complex extraction processes and their total phenolic as well as total flavonoid contents were determined. The extracts’ effects on the activity of four glucose metabolism enzymes were then investigated. The observed TPCs and TFCs were lower than those reported previously, although it should be noted that SPE cleanup was not applied in previously reported studies on BSG extracts and that both the malt used in the brewing process and the storage conditions of the BSG may affect the extracts’ composition. Several extracts were observed to have strong inhibitory activity, particularly towards GPα and α-glucosidase, but also towards DPP IV. However, the inhibition of DPP IV was considerably weaker than that caused by the positive control, so the biological relevance of the results for this enzyme should be evaluated critically. No appreciable inhibition of α-amylase was observed. In general, the results are not readily compared to literature data because the only relevant previous studies examined BSG protein hydrolysates or barley extracts, and the steps involved in transforming barley grains into BSG  will inevitably cause significant changes in the composition of the material .α and α-glucosidase, and that further research is warranted to identify the active compounds within these extracts. Additionally, our findings show, that solid-liquid extraction with 60% acetone resulted in more potent extracts regarding GPα and α-glucosidase inhibition than alkaline hydrolysis. Differences between the BSG batches were of minor importance; the extraction process is crucial for the biological activity determined here. Furthermore, our TPC results confirmed, that bound polyphenols account for the majority of the total phenolics in BSG. However, a high TPC and TFC was no indicator of a more pronounced inhibitory potential. HPLC-DAD and HPLC-MS/MS analysis should be performed to characterize the (phenolic) compounds in the extracts in more detail, and the activity of these compounds should be investigated in enzyme-inhibition assays.Nevertheless, our results clearly show that BSG is a source of potent inhibitors of various glucose metabolism enzymes, especially GP


Analysis of RTSE measurements track changes in the complex dielectric function spectra and structure, which indicate variations in stability influenced by the underlying material, preparation method, and perovskite composition. MAPbI3 and MA0.7FA0.3PbI3 films deposited on commercial fluorine-doped tin oxide coated glass are more stable than corresponding films deposited on soda lime glass directly. (FAPbI3)0.95(MAPbBr3)0.05 films on soda lime glass showed improved stability over the other compositions regardless of the substrate, and this is attributed to the preparation method as well as the final composition.In situ real-time spectroscopic ellipsometry (RTSE) measurements have been conducted on MAPbI ABX3 perovskites  have gained tremendous attention in photovoltaic (PV) applications due to their high-power conversion efficiency which increased from 3.8% in 2009 to 25.5% [3 perovskite is the pioneer among those used in solar cells and has been the most studied to date. Despite showing desirable optoelectronic properties, MAPbI3 degrades with exposure to humidity, oxygen, heat, and ultraviolet light [3 upon atmospheric exposure have shown phase segregation into PbI2 and MAI starting at the interfaces of the film with the ambient and substrate [3 becomes hydrated under a humid environment in the dark and forms PbI2 with humidity exposure under illumination [3, shows improved thermal stability compared to MAPbI3, however it may transform from the desired cubic, photo-conductive “black” perovskite α-phase into the “yellow” trigonal δ-phase in the presence of solvents and humidity [A cation size is critical for the formation of a cubic perovskite structure [A cation size given by Goldschmidt tolerance factor falls in the 0.8–1.0 range, which is favorable for the cubic black phase perovskite structure to form, and the corresponding material stability is improved [3 prevents ion migration in the perovskite and maintains favorable light absorption [Organic–inorganic metal halide-based to 25.5% ,2,3 receto 25.5% ,5,6. Solto 25.5% . The MAPet light ,11. Prevubstrate . It has mination . Anotherhumidity . Stabilihumidity ,15. In ptructure . When a improved ,18. Recesorption . SubstitSpectroscopic ellipsometry is a non-destructive, non-invasive measurement often used to explore the structural and optical properties of thin film materials. It measures changes in polarization state and amplitude of an incident light beam upon interaction with a sample either by reflection or transmission ,22. The 3, MA0.7FA0.3PbI3, and (FAPbI3)0.95(MAPbBr3)0.05 perovskite films deposited on soda lime glass and commercial fluorine-doped tin oxide (FTO) coated glass substrates under controlled relative humidity (RH) and temperature variations. These films are prepared using single-step spin coating and two-step solution processing [3 over time and the minimal variations for MA0.7FA0.3PbI3 and (FAPbI3)0.95(MAPbBr3)0.05 allow us to identify the factors influencing the stability of perovskites films. These factors include substrate type, mixing organic cations, incorporation of Br as a halide anion, and two-step versus single-step preparation methods. The RTSE data analysis methodology can be adopted in studying degradation of the perovskite absorber and other layers inside PV devices to assist in identifying stable materials for a large-scale industrial application.Here in situ RTSE is used to track the variation of the optical and structural properties of MAPbIocessing ,32,33. T2)-filled glove box to prevent exposure to ambient air. MAPbI3 and MA0.7FA0.3PbI3 films are prepared using single-step spin coating [2 , MAI , FAI , lead thiocyanate (Pb (SCN)2, Sigma–Aldrich, 99.5%), dimethyl sulfoxide , and N,N-dimethylformamide  are used without further purification. The perovskite precursor solutions are prepared by dissolving mixtures of MAI, FAI, PbI2, Pb (SCN)2 in DMSO/DMF (v:v = 1:9), where the perovskite concentration is 1.5 M. The perovskite precursor solution is then spin-coated onto the substrate at 500 rpm for 3 s, and then at 4000 rpm for 60 s with diethyl ether dropped on the film at 10 s of the second step. The as-prepared perovskite films are annealed on a hotplate at 65 °C for 2 min and then at 100 °C for 5 min.All perovskite films are prepared in a nitrogen 0.95(MAPbBr3)0.05, a two-step solution-processed preparation method is used as is described in [2 is dissolved in a mixed solvent of 950 μL DMF and 50 μL DMSO. 70 mg FAI, 6 mg MABr, and 7 mg MACl are then dissolved in 1 mL isopropanol (IPA). To deposit the PbI2 film, 70 μL PbI2 solution is dropped onto substrate and spin-coated at 2000 rpm for 30 s, followed by annealing at 70 °C for 2 min. Then, the IPA solution is spin-coated on the as-prepared PbI2 film at 2000 rpm for 30 s to form the perovskite phase. Next, the samples are annealed at 150 °C for 15 min at 30–40% relative humidity in ambient air.To prepare , C = sin (2ψ)cos (∆), and S = sin (2ψ)sin (∆) where the ellipsometric angles ψ and ∆ describe the relative amplitude and phase shift between the electric field components perpendicular and parallel to the plane of incidence [N, C, and S) are collected at an angle of incidence of 70° for 500 spectral points over a photon energy range from 1.25 to 6.00 eV using a single rotating compensator multichannel ellipsometer  [In situ RTSE measurements are performed to collect ellipsometric spectra in the form of ncidence . EllipsoNE, USA) . Data isNE, USA) .mod) and the experimental (exp) ellipsometric spectra is defined in terms of the unweighted mean square error (MSE) [n is the number of measured values and m is the number of fit parameters.The experimental ellipsometric spectra are analyzed using a least square regression analysis in which the quality of fit between the model (or (MSE) :(1)MSE=1ε = ε1 + iε2) spectra and structural parameters, which includes perovskite film thickness, the thickness of an interfacial layer forming between the substrate and perovskite film, and surface roughness layer thickness [ε for the perovskite film are described using a parametric optical property model, which applies physically realistic parametric dispersion relations spanning from photon energies above to below the bandgap energy [ε for these perovskites have been provided in [ε2) is described by the sum of critical-point oscillators assuming parabolic bands (CPPB) above the direct bandgap energy and an Urbach tail below the bandgap [ε2 is considered to be the direct bandgap of the perovskite film. Spectra in ε1 is calculated as the sum of a Sellmeier expression, a constant additive term to the real part of ε1 denoted by ε∞, and Kramers–Kroning integration [ε2 over this spectral range. Spectra in ε for the surface roughness layer and interfacial layer are represented by Bruggeman effective medium approximations [ε = 1) and 0.5 bulk perovskite volume fractions.To fit experimental ellipsometric spectra, a parameterized optical and structural model is used  to extract the optical response of the perovskite films in terms of complex dielectric function deffeε representing the entire perovskite film, it is likely that water transport from both directions occurs simultaneously. The increase in effective thickness may be due to an increase in unit cell volume caused by the hydration of MAPbI3, which will reduce the film density [3 and MAPbI3·H2O to be 247 Å3 and 263 Å3 respectively, conversion of MAPbI3 to MAPbI3·H2O causes a volumetric lattice expansion of 6% based on the relative lattice parameters of both phases [MSE increases steadily with time using both initial static spectra in ε or allowing ε to vary for each time point. As even the model assuming changes in ε over time possesses a substantially large MSE relate to other samples 0.95(MAPbBr3)0.05 thin films deposited on soda lime glass substrates have been exposed to 26% RH at 7 °C, 26% RH at 70 °C, and 85% RH at 25 °C. ε for (FAPbI3)0.95(MAPbBr3)0.05 deposited on soda lime glass exposed to 26% RH at 7 °C. The decrease in magnitude of spectra in ε is minimal compared to both MA0.7FA0.3PbI3 and MAPbI3.To further understand the factors that influence the stability of different perovskite film compositions, 0.95(MAPbBr3)0.05 film deposited on soda lime glass substrate is exposed to the sequence of 26% humidity at 70 °C initially and then 85% RH at 25 °C later. The goal of this combined measurement is to test how this film composition resists varying exposure at both moderate humidity at high temperature and high humidity at moderate temperature after determining its stability at fixed 7 °C and 26% RH conditions. There is no change in magnitude of spectra in ε as a function of time compared to MA0.7FA0.3PbI3 and MAPbI3, and the changes are even smaller compared to those observed in this composition perovskite at a low temperature. The (FAPbI3)0.95(MAPbBr3)0.05 film at a low temperature may exhibit a small change over time as small amounts of water condense onto the film. However, even those changes are minor compared to the other perovskite compositions studied.3)0.95(MAPbBr3)0.05 film deposited on soda lime glass substrate exposed to 26% RH at 70 °C then 85% RH at 25 °C. No variation in effective thickness is observed for the film exposed to 26% RH at 70 °C but there is a minimal increase in effective thickness during 85% RH at 25 °C exposure in the reported time interval in ε obtained prior to humidity exposure is almost identical to that when spectra in ε are obtained at each time point (dynamic ε) for the time exposed to 26% RH at 70 °C. Note that the same static ε obtained prior to humidity exposure at 70 °C is used as the temperature is decreased, leading to a small increase in the MSE. This difference is expected as spectra in ε for semiconductors will vary with measurement temperature [ε measured at 70 °C provides a low MSE. This similarity in quality of fit indicates that the structural parameters of the film and the intrinsic characteristics of this perovskite reflected in ε are not affected by humidity exposure and temperature variations. Overall, temperature variations over the 7 to 70 °C ranges do not seem to have an impact on the stability of the (FAPbI3)0.95(MAPbBr3)0.05 composition perovskite films.perature ,56,57. H3)0.95(MAPbBr3)0.05 over MAPbI3 and MA0.7FA0.3PbI3 with temperature variations and humidity exposure is attributed to the incorporation of Br into the mixed MA + FA film. Previous research has shown that including a small amount of Br enhances the stability, suppresses ion migration, and reduces trap-state density [3 for solar cell absorbers, there is an improvement in open circuit voltage (VOC) from 0.87 to 1.13 V and an increase in fill factor (FF) from 0.66 to 0.74 [OC in PV devices and are more stable against degradation [3)0.95(MAPbBr3)0.05 also leads to larger grain size than in the single step approach which further enhances film stability [The improved stability for (FAPbI density . Accordi to 0.74 . Increas to 0.74 . It has  to 0.74 . Furthertability .ε above the bandgap. There is a slight decrease in bandgap when the (FAPbI3)0.95(MAPbBr3)0.05 film exposed to 26% RH at 70 °C is cooled to 85% RH at 25 °C. Previous research has shown that the bandgap decreases with decreasing temperature in these hybrid halide perovskites unlike in other semiconductors where the bandgap decreases with increasing temperature as a result of lattice dilatation [3, which exhibits substantial degradation by water molecules, retains a similar bandgap energy throughout humidity exposure although the higher energy CPs and magnitude of ε change more substantially.Bandgap energies of the studied perovskites films  are not latation ,57. Herelatation . Lack of3 degrades upon exposure to 85% RH at room temperature whereas mixed MA + FA perovskites shows improved stability when exposed to RH at various temperatures. Temperature alone in the range of 7 to 70 °C does not affect the stability of studied (FAPbI3)0.95(MAPbBr3)0.05 perovskite films. There is an influence of the substrate on the stability of these perovskites as the films deposited on FTO coated glass shows enhanced stability compared to the films deposited directly onto soda lime glass. Additionally, incorporation of bromine and the two-step preparation method are among other factors for improved stability of the mixed MA + FA cation perovskites. Among the studied perovskites, (FAPbI3)0.95(MAPbBr3)0.05 demonstrated a robust stability for the tested humidity and temperature conditions.The stability of optical and structural properties of organic–inorganic metal halides-based perovskites thin films exposed to various humidity and temperature conditions have been explored using RTSE. MAPbI


Excessive alcohol intake is associated with adverse immune response-related effects, however, acute and chronic abuse differently modulate monocyte activation. In this study, we have evaluated the phenotypic and functional changes of monocytes in acutely intoxicated healthy volunteers (HV).brightCD16-, CD14brightCD16+ and CD14dimCD16+ monocyte subsets and their TLR4 expression were analyzed by flow cytometry. Inflammasome activation via caspase-1 in CD14+ monocytes was measured upon an ex vivo in vitro LPS stimulation. Systemic IL-1β and adhesion capacity of isolated CD14+ monocytes upon LPS stimulation were evaluated.Twenty-two HV consumed individually adjusted amounts of alcoholic beverages until reaching a blood alcohol level of 1‰ after 4h (T4). Peripheral blood was withdrawn before and 2h (T2), 4h (T4), 6h (T6), 24h (T24), and 48h (T48) after starting the experiment and stained for CD14, CD16 and TLR4. CD14+ monocyte did not change following alcohol intoxication, whereas CD14brightCD16- monocyte subset significantly increased at T2 and T24, CD14brightCD16+ at T2, T4 and T6 and CD14dimCD16+ at T4 and T6. The relative fraction of TLR4 expressing CD14+ monocytes as well as the density of TLR4 surface presentation increased at T2 and decreased at T48 significantly. TLR4+CD14+ monocytes were significantly enhanced in all subsets at T2. TLR4 expression significantly decreased in CD14brightCD16- at T48, in CD14brightCD16+ at T24 and T48, increased in CD14dimCD16+ at T2. IL-1β release upon LPS stimulation decreased at T48, correlating with TLR4 receptor expression. Alcohol downregulated inflammasome activation following ex vivo in vitro stimulation with LPS between T2 and T48 vs. T0. The adhesion capacity of CD14+ monocytes decreased from T2 with significance at T4, T6 and T48. Following LPS administration, a significant reduction of adhesion was observed at T4 and T6.The percentage of CD14Alcohol intoxication immediately redistributes monocyte subsets toward the pro-inflammatory phenotype with their subsequent differentiation into the anti-inflammatory phenotype. This is paralleled by a significant functional depression, suggesting an alcohol-induced time-dependent hyporesponsiveness of monocytes to pathogenic triggers. Alcohol is one of the oldest and nowadays one of the most common addictive substances worldwide . Althougex vivo in vitro whole blood stimulation with lipopolysaccharide (LPS) increase in the first 20 minutes after drinking, suggesting an early pro-inflammatory response. Subsequent decline of monocytes, natural killer cells and interleukin (IL)-1β, IL-6 and monocyte chemoattractant protein (MCP)-1 levels in circulation and the elevation of systemic IL-10 level suggest an anti-inflammatory state in the later course 4-MyD88 and TLR-TRIF-dependent manner when stimulated with LPS in vitro (brightCD16-) are released into circulation from bone marrow and act as a precursor for intermediate monocytes (CD14brightCD16+), which in turn differentiate into non-classical monocytes (CD14dimCD16+)  . ClassicimCD16+) . Once thimCD16+) . IntermeimCD16+) , and theimCD16+) . In chroimCD16+) . Althougex vivo in vitro by suppressing the inflammasome activation.Considering that phenotypic and functional alterations of monocytes are substantially involved in the initiation and subsequent resolution of inflammation, we evaluated the phenotypic shift of monocytes following episodic excessive alcohol intake in healthy volunteers (HV). We hypothesized that acute alcohol intoxication shifts the monocytes toward the anti-inflammatory phenotype and ameliorates LPS-induced pro-inflammatory response The current study was performed pursuant to ethics committee approval (255/14) from the University Hospital of the Goethe-University Frankfurt and in accordance with the Declaration of Helsinki and following the Strengthening the Reporting of Observational studies in Epidemiology-guidelines .Twenty-two HV between 18 and 50 years of age were enrolled. Exclusion criteria included a history of chronic alcohol abuse, acute infection, pre-existing chronic inflammatory diseases, immunological disorders, human immunodeficiency virus infection, infectious hepatitis, medication and pregnancy. Renal insufficiency and changes in transaminases to identifying chronic liver diseases were excluded by blood test.Healthy volunteers received a standardized lunch (1 pizza) one hour before starting the experiment. Subsequently, HV consumed individually calculated amount of alcohol until reaching blood alcohol level of 1 ‰ after 4 hours. In detail, HV received whisky-coke cocktail  in a mixing ratio of 1:2 every 20 minutes for 4 hours. Then, the HV were monitored for further 2 hours without alcohol intake. For the individual calculation of the amount of alcohol to reach 1 ‰ after 4 hours, the modified Widmark equation including age, sex, height and weight was applied. Blood alcohol concentration (BAC) was monitored hourly until T6 and at T24 and T48. At every time point, a blood sample for the determination of the BAC was taken. Blood samples were obtained in prechilled ethylenediaminetetraacetic acid tubes  and kept on ice. Then, blood was centrifuged at 2,000 × g and at 4°C for 15 minutes and the supernatant was immediately used for the determination of BAC using the diagnostic set serum EtOH by Cobas 8000 Modular Analyzer . The participants leaved the research facility after T6 and came back at T24 and T48. During the time out of the institute, participants were allowed to eat and drink with restriction of drinking alcohol. A brief overview of the study design and sampling is shown in Peripheral blood was withdrawn in serum-gel and Li-Heparin collection tubes  before (T0) and 2 (T2), 4 (T4), 6 (T6), 24 (T24) and 48 (T48) hours after starting the alcohol consumption. Subsequently, the samples collected in serum-gel collection tubes were centrifuged directly after the collection at 2,000 × g and at 4°C for 15 minutes. Serum was stored at -80°C until further analysis. Blood in Li-Heparin collection tubes were directly processed for flow cytometry and adhesion assay.4Cl, 0.01 M KHCO3, 0.1 mM ethylenediaminetetraacetic acid (EDTA)) were added and incubated in the dark at 4°C. Washing step was repeated. Monocyte population was defined by gating the corresponding forward and side scatter scan. From each sample a minimum of 3.0 x 104 cells were measured, which were subsequently analyzed. The percentage of CD14brightCD16- , CD14brightCD16+ (intermediate subset) and CD14dimCD16+  as well the percentage and mean fluorescent units of TLR4 out of CD14+ viable cells were assessed by flow cytometric analyses using a BD FACS Canto 2™ and FACS DIVA™ software (BD Bioscience). Unspecific binding of the antibodies was excluded by using isotype controls.100 µl heparinized blood was transferred into polystyrene FACS tubes (BD Pharmingen™). The samples were incubated with 5 µl CD14 , 5 µl CD16  and 5 µl TLR4  in the dark at 4°C for 10 minutes. Subsequently, the samples were washed with 2 ml FACS buffer  in phosphate-buffered saline (PBS)) and centrifuged at 350 × g at 4°C for 5 minutes. For red blood cells lysis, 3 ml lysis buffer -1-piperazineethanesulfonic acid, 10% heat-inactivated fetal bovine serum (FBS)] and LPS at final concentration of 10 µg/ml (For the detection of IL-1β levels in directly collected serum (see Blood Sampling) and the supernatants after whole blood stimulation with LPS (see Whole Blood Stimulation With LPS), ELISA DuoSet kit  was used according to the provider's instructions.E. coli O127:B8 strain; Sigma Aldrich, Germany), 100 µM BzATP   were added and incubated in the dark at 4°C for 10 minutes. Cells were washed with 2 ml FACS buffer and centrifuged at 200 × g at 4°C for 5 minutes. Following pellet resuspension in 500 µl FACS buffer, caspase-1 expression was assessed by flow cytometric analyses using a BD FACS Canto 2™ and FACS DIVA™ software (BD).Active caspase-1 was quantified by using a FAM-FLICA Caspase-1 (YVAD) Assay Kit  . 50 µl bGermany)  and 10 µ+ monocyte we described already elsewhere (2+ and Ca2+), cells were resuspended in 500 µl RPMI 1640 media with supplements. Mononuclear cells were stimulated with 1 µg/ml LPS at 37°C and 5% CO2 for 90 minutes and subsequently washed with MACS buffer.The isolation of CD14lsewhere . Periphe+ labeled monocytes were magnetically isolated by using LS columns  according to the manufacturer’s protocol. Following a further washing step with 10 ml FACS buffer, cell pellet was resuspended in 500 µl of RPMI 1640 media with supplements. Only cell cultures with a purity and viability of ≥95% were used for further experiments. Cell population purity was evaluated by flow cytometry according its size and CD14 expression. Cell viability was proved by Türk’s solution exclusion assay .For CD14 labeling, cell pellet was resuspended in 75 µl MACS buffer and the cell suspension was incubated with 25 µl magnetic CD14 microbeads  at room temperature for 15 minutes. Following a washing step with 3 ml MACS buffer, CD14+ monocytes were seeded in 6-well flat-bottom plate , where adherent A549 pulmonary epithelial cells to the density of 80% were seeded a day before, and, incubated at 37°C and 5% CO2 for 35 minutes. Cells were washed twice with RPMI 1640 media with supplements and following fixation by 1 ml 1% glutaraldehyde for 3 minutes, monocytes were washed once more with PBS. Adherent monocytes on plates were stored in 2 ml PBS in the dark and at 4°C until evaluation. The adherent monocytes were then counted in 5 different fields of a defined size (5×0.25 mm2) using a phase contrast microscope (×20 objective). The mean cellular adhesion rate was calculated.The adhesion assay was performed as described before . 200,000GraphPad Prism 6.0 software  was used to perform the statistical analysis. Data are given as mean ± standard error of the mean (SEM). The data distribution was tested by the D’Agostino-Pearson test, and the data is not normally distributed. Since we have 6 groups with matched or repeated measures, thus paired data, the non-parametric Friedman’s test was applied. Dunn-Bonferroni post-hoc test for multiple comparisons was used. T0 is the base condition to which the others were compared. The Wilcoxon matched-pairs signed rank test as a nonparametric statistical tool comparing two paired groups in From 22 healthy volunteers, 12 female and ten male with a mean age of 25 (± 4) years were enrolled. All included individuals exerted normal ranges of transaminases excluding chronic e.g. liver diseases, since the rages at T0 were: 15-20 U/l GGT, 22 U/l GPT and 23 U/l GOT. Thus, no individuals with chronic liver diseases or malfunction were included. Furthermore, the BAC increased to 0.46±0.02‰ at T2, with further at T4 (1.11±0.05‰) reaching the target BAC of 1‰. At T6 BAC decreased to 0.83±0.06‰ and was not further detectable at T24 and T48.ex vivo in vitro labeled circulating monocytes were gated as it is shown in the + monocytes did not change during the whole observational period of 48 hours compared to the counts before the experiment start (T0)  with subsequent decrease to the baseline at T24 and T48 . Following LPS stimulation, we have shown that the acute ethanol intoxication did not have an impact on IL-1β concentration at T2, T4 and T6 compared to IL-1β concentration before starting binge drinking (T0)   Synthesis of inactive pro-IL-1β, 2) enzymatic cleavage of pro-IL-1β into active IL-1β by caspase-1 with subsequent secretion of IL-1β into extracellular space . Since LEx vivo in vitro stimulation of monocytes with LPS and ATP significantly increases their caspase-1 level. During and following the excessive alcohol intake, caspase-1 expression is upregulated but significantly less induced compared to non-intoxicated HV. This is in line with an in vitro study, that we have published recently showing that ethanol pre-treatment (170 mM) of human HepG2 liver cells downregulated caspase-1 expression following LPS and ATP administration  on monocytes and the most in vitro studies focus on the long-term impact of glucose on monocyte functionality (diabetes models). Afshar et al. have shown in line with the data presented in this study that acute alcohol intoxication by ethanol mixed with chilled sugar-free flavored seltzer water led an early pro-inflammatory immune response 20 minutes after reaching blood alcohol concentration of 1‰  would significantly improve these findings. This was not possible for technical and time reasons. Second, including a placebo group would immensely enhance the validity of the results. Until now, there are no short-term on of 1‰ . Two andon of 1‰ . Furthercapacity , 47. Altcapacity , 49, we capacity –52.Ex vivo in vitro LPS stimulation of monocytes shows a downregulation of NLRP3 expression in samples obtained from severely injured patients compared to healthy subjects  from the University Hospital of the Goethe-University Frankfurt. The patients/participants provided their written informed consent to participate in this study.BR designed the study, obtained the ethical approval, performed statistical analyses, and revised the manuscript. RS, FH, BX, and AJ collected the samples and performed the experiments. AJ and RS performed the statistical analyses, and AJ wrote the original draft. AG, ID, CN, AN, and PC contributed to analyses and revised the manuscript. ID, CN, and IM contributed intellectually to the completion of the study. All authors contributed to the article and approved the submitted version.The study was supported by grants from the Deutsche Forschungsgemeinschaft .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Epilobium parviflorum, Cardiospermum halicacabum, and Melilotus officinalis, their antioxidant, antiinflammatory effects, and their mechanism of action. Methods: plant samples were macerated in 40% ethanol or hot/ cold glycerate and assessed for polyphenols content. The antioxidant activity was investigated by DPPH radical scavenging assay and H2DCFDA test in LPS-stimulated RAW264.7 macrophages and N9 microglial cells. MTS experiments and antiinflammatory properties verified cellular toxicity through NO assay. Interaction with A2A adenosine receptors was evaluated through binding assays using [3H]ZM241385 radioligand. Results: Polyphenols were present in 40% ethanol plant extract, which at 0.1–10 µg/µL achieved good antioxidant effects, with a DPPH radical scavenging rate of about 90%. In LPS-stimulated cells, these plant extracts, at 1μg/μL, did not affect cell vitality, displayed significant inhibition of H2DCFDA and NO production, and inhibited ZM 241385 binding in CHO cells transfected with A2A receptors. RAW 264.7 and N9 cells presented a density of them quantified in 60 ± 9 and 45 ± 5 fmol/mg of protein, respectively. Conclusion: Epilobium parviflorum, Cardiospermum halicacabum, and Melilotus officinalis extracts may be considered a source of agents for treating disorders related to oxidative stress and inflammation.Background: We investigated the phenolic content characterizing different plant extracts from  All other reagents were from Sigma Aldrich .Whatman GF/B glass fiber filters were from PerkinElmer . ZM 241385 (specific activity 20 Ci/mmol), a potent and selective A2A receptor ligand, was used in both saturation and competition binding experiments ZM 241385. To determine non-specific binding, 1 µM of ZM 241385 was added. For saturation experiments increasing concentrations of [3H]ZM 241385 (0.1–10 nM) were incubated with membranes from RAW 264.7 and N9 cells. After 1 h at 4 °C, bound and free radioactivity were separated through a filtration method with Brandel Whatman using GF/B glass fiber filters . The radioactivity was quantified by a Tri-Carb Packard 2500 TR scintillation counter .ZM 241385 binding to A2A adenosine receptors of 40 ± 5% and 19 ± 3%, respectively. The 10 µg/µL Melilotus officinalis displaced 86 ± 9% of the [3H]ZM 241385 binding, suggesting a very high affinity of this compound for A2A adenosine receptors. The effect was still significant with the 1 µg/µL concentration (49 ± 6%). Finally, the binding inhibition of Cardiospermum halicacabum was higher when diluted 1 µg/µL (47 ± 5%) in comparison to when diluted 10 µg/µL concentration (20 ± 4%)  .2A adenosine receptors in RAW 264.7 and N9 cells, binding experiments with [3H]ZM 241385 were carried out. In particular, cellular membranes obtained from RAW 264.7 and N9 cells were incubated with increasing concentrations of the selective and high-affinity radioligand antagonist in the range 0.1–10 nM for 1 h. Following the filtration procedure to separate bound and free radioactivity, the results revealed that these cells presented A2A adenosine receptors with a density of 60 ± 9, 45 ± 5 fmol/mg of proteins and [3H]ZM 241385 had an affinity value of 0.9 ± 0.2 and 1.2 ± 0.2 nM to them, respectively.To evaluate the expression of AEpilobium parviflorum, Melilotus officinalis, and Cardiospermum halicacabum are used as herbal medicines in several countries for their antioxidant and antiinflammatory effects. Still, their mechanism of action is not fully understood. For the present research, specific parts of them were selected. In particular, the dried aerial part of Epilobium parviflorum, aerial flower part of Melilotus officinalis, and flowering tops of Cardiospermum halicacabum were chosen based on the richest parts containing bioactive substances [Epilobium parviflorum, Melilotus officinalis, and Cardiospermum halicacabum plants through the DPPH assay. The solvent significantly affects the phytochemical pattern of a given herbal extract. Because the chemistry modulates the biological activity, different solvent systems were used, including hot and cold glycerate and 40% ethanol, to prepare plant extracts. By comparing the different types of extraction, all the plant extracts demonstrated a good radical scavenging activity. Still, the most significant effect was observed with 40% ethanol plant extracts, and the percentage of inhibition correlated with extracts concentration. It is well known that ethanol is a polar solvent able to extract a significant amount of polyphenolic compounds like the flavonoids and tannins responsible for the observed antiradical activity of plant extracts used in this study [Cardiospermum halicacabum, the 40% ethanol extracts contained higher amounts of both polyphenols and flavonoids in comparison to hot and cold glycerate fractions. As for Epilobium parviflorum and Melilotus officinalis, these chemical classes of compounds were not significantly more concentrated in 40% ethanol extracts, suggesting that other non-flavonoid components may be responsible for the higher antioxidant activity [Epilobium parviflorum and Cardiospermum halicacabum 40% ethanol plant extracts showed toxic effects in RAW 264.7 and N9 cell lines, respectively, when tested at 2.5 µg/µL, but were safe at 1 µg/µL and 0.1 µg/µL concentrations. On this basis, 1 µg/µL and 0.1 µg/µL concentrations were chosen for the next experiments to evaluate their ability to reduce radical oxygen and NO production in in vitro cellular models. The activation of macrophages and microglia by the bacterial surface molecule LPS leads to the production of free oxygen and NO radicals, which exert important roles in inflammation, affecting many age-related diseases such as Alzheimer’s pathology [Epilobium parviflorum and Cardiospermum halicacabum, at 0.1 µg/µL concentration, reduced free radical and NO production only in RAW 264.7 cells, confirming their ability to cope up with oxidative stress, according to literature data [2A adenosine receptor subtype, having a crucial role in reducing inflammation [2A receptor-deficient mice presented increased inflammation and tissue damage in models of acute liver injury, endotoxin-associated sepsis, and infected wound model, suggesting a non-redundant role in the attenuation of inflammation and tissue damage in vivo [2A adenosine receptors to produce beneficial effects, caffeine and resveratrol have been described [2A antagonist radioligand ZM 241385, in CHO cells transfected with A2A adenosine receptors, being Melilotus officinalis the most potent extract, suggesting their interaction with this membrane receptor subtype. Therefore, radioligand binding experiments demonstrated the expression of A2A adenosine receptors in both RAW 264.7 and N9 cells, with a density of 60 ± 9 and 45 ± 5 fmol/ mg of protein, as potential targets of Epilobium parviflorum, Melilotus officinalis, and Cardiospermum halicacabum to counteract inflammation. In conclusion, the results of this study show that the ethanolic extracts from the dried aerial part of Epilobium parviflorum, aerial flower part of Melilotus officinalis, and flowering tops of Cardiospermum halicacabum are characterized by the presence of several polyphenols, in particular flavonoids and condensed tannins, and may be considered as a potential source of agents for the treatment of disorders related to oxidative stress and inflammation.Natural products exert a relevant role in human health as well as in drug discovery . Extractbstances ,28,29. Fis study ,35. Indeactivity . Therefoathology . As the athology ,38. Therure data ,39,40. Iammation ,42. Inde in vivo ,44. Targ in vivo . As an eescribed ,47. Inte


To evaluate the image quality of dual energy CT (DECT) of the shoulder after arthrography and of virtual non-contrast (VNC) 3D reformats of the glenoid and to compare glenoid measurements on VNC 3D reformats and on 2D CTs.DECT arthrography (80 kV/140 kV) was performed in 42 shoulders of 41 patients with instability using diluted iodinated contrast media (80 mg/ml). VNC images and VNC 3D reformats of the glenoid were calculated using image postprocessing. Dose parameters, CT values of intraarticular iodine and muscle, image contrast (iodine/muscle), and image quality  were evaluated. Two independent readers assessed glenoid morphology and performed glenoid measurements on 2D and 3D images.P > 0.99) with no difference for measurement of the glenoid diameter with mean 28.3 mm (± 2.8 mm) vs. 28.4 mm (± 2.9 mm) (P = 0.5), width of the glenoid defect with 3.2 mm (± 2.1 mm) vs. 3.1 mm (± 2.3 mm) (P = 0.84), surface area with 638.5 mm2 (± 127 mm2) vs. 640.8 mm2 (± 129.5 mm2) (P = 0.47), and surface area of the defect with 46.6 mm2 (± 44.3 mm2) vs. 47.2 mm2 (± 48.0 mm2) (P = 0.73), respectively.Calculation of VNC images and VNC 3D reformats was successful in 42/42 shoulders (100%). The effective dose was mean 1.95 mSv (± 0.9 mSv). CT values of iodine and muscle were mean 1014.6 HU (± 235.8 HU) and 64.5 HU(± 8.6 HU), respectively, and image contrast was mean 950.2 HU (± 235.5 HU). Quality of cross-sectional images, VNC images, and VNC 3D reformats was rated good (median 4 (4–5), 4 (3–4), 4 (3–5), respectively). Detection of an osseous defect was equal on 2D and 3D images (13/42, DECT shoulder arthrography is feasible and allows successful iodine removal with generation of VNC images and accurate VNC 3D reformats of the glenoid for assessment of bone loss.The online version contains supplementary material available at 10.1007/s00256-021-03916-3. The 3D reformats of shoulder CT examinations generated with the volume rendering technique (VRT) are routinely used for assessment of the glenoid morphology and for quantitative measurements and are highly accurate. They are often used during preoperative planning in shoulder instability procedures, especially as the 3D visualization is preferred by the surgeons over the 2D CT reformats –7. The 3However, for SECT scans after arthrography, calculation of 3D VRT reformats is practically useless, because intraarticular iodinated contrast material overlays bone and cannot be successfully separated. In contrast to SECT, dual energy CT (DECT) allows for characterization of tissues  according to different attenuation values at different energy levels , 9. DECTWe set out to use dual energy CT after arthrography of the shoulder for clinical routine examinations. Our hypothesis was that DECT arthrography provides good image quality and allows for successful calculation of virtual non-contrast images and of accurate VNC 3D VRT reformats of the glenoid for assessment of bone loss.Therefore, the purpose of this study was to evaluate the image quality of DECT scans of the shoulder after arthrography and of VNC 3D VRT reformats of the glenoid. Furthermore, to compare glenoid measurements on VNC 3D VRT reformats and on 2D CTs.The cantonal ethics committee approved this single center retrospective study which was conducted according to the Declaration of Helsinki.A search of the picture archiving and communication system (PACS) of Balgrist University Hospital was performed to find patients who received a clinical dual energy CT scan of the shoulder after arthrography. Inclusion criteria were males and females aged 18 years or older with shoulder instability. Patients with an incorrect dosage of intraarticular contrast material at arthrography or with metallic screws in the glenoid after surgery were excluded.z-axis. Both CT scanners operated with automated tube current modulation , a collimation width of 0.6 mm, a rotation time of 0.5 s, and a pitch of 0.8. The dose settings of the DECT scan were adjusted to the parameters of a single energy scan of the shoulder at 120 kV (reference 150 mAs). The applied total dose was split automatically between the 80 kV and the 140 kV scan by the CT machine.All patients received a dual energy CT scan of the shoulder at Balgrist University Hospital either on a 128-slice CT scanner  or on a 64-slice CT scanner  within 15 min after arthrography. The scan protocol was adapted from the protocol for the liver VNC application since no shoulder-specific manufacturer settings were available: All scans were performed in sequential technique with a 80 kV scan followed by a second scan with 140 kV of the same coverage in Injection of diluted iodinated contrast material into the glenohumeral joint at Balgrist University Hospital is routinely performed under conventional fluoroscopy using an anterior approach through the rotator cuff interval . As therBoth the 80 kV and 140 kV scans were reconstructed in the axial image plane (0.75 mm) using a bone (Br 51) and a soft tissue kernel (Qr 40). Furthermore, a blended axial CT arthrogram (CT-A) dataset (0.75 mm) was calculated from the 80 kV and 140 kV scans in bone kernel (Br 51) using a mixing ratio of 0.3:0.7, from which axial (2 mm), sagittal oblique (2 mm), sagittal en face (1 mm), and coronal oblique (2 mm) CT arthrogram images were reconstructed.For the calculation of 3D VRT reformats, both the axial 80 kV and 140 kV dataset with a 0.75-mm section thickness in soft tissue kernel (Qr 40) were loaded into the dual energy viewer of syngo.via . First, virtual non-contrast images with a 0.75-mm section thickness were calculated with the shoulder VNC application which was adapted from the liver VNC application to display higher Hounsfield units. Second, 3D osseous reformats of the glenoid were generated from the VNC images using the volume rendering tool in syngo.via  with 7 and M.M. (reader 2) with 6 years of experience) on a PACS workstation. Both readers were blinded to each other and were blinded to clinical information and imaging results.k for the adult chest of 0.014 mSv/mGy*cm to estimate the effective dose [Scan length and CT dose parameters were extracted from the dose report: tube current–time product (mAs), volume CT dose index (CTDIvol), and dose length product (DLP). The DLP was multiplied with a standard conversion factor ive dose .2). The copy and paste function of the PACS was used, allowing to position the ROIs at the identical location in both datasets. Furthermore, CT values (HU) of the deltoid muscle were also measured on the blended axial CT-A images using ROIs of the same size. Image contrast of blended CT-A images was calculated as the difference of CT values of intraarticular iodinated contrast material and deltoid muscle.CT values (HU) of the intraarticular iodinated contrast material were measured by reader 1 on reconstructed axial 0.75-mm images of the 80 kV scan in soft tissue kernel and on blended axial CT arthrogram images (0.75 mm) using regions of interest (ROI) of equal size , image quality of virtual non-contrast images , and image quality of virtual non-contrast 3D VRT reformats of the glenoid . Ratings are defined in Supplementary Table In a sub-analysis, the image quality was evaluated separately for the different CT systems.Furthermore, CT arthrogram images of patients with surgical correlation were assessed for rotator cuff and labrum tears and for cartilage defects.2) for both 2D and 3D images [Glenoid measurements were performed on sagittal en face 2D arthrogram images (1 mm) and on en face VNC 3D VRT reformats of the glenoid by both readers. The best fit-circle method was applied for all measurements, which assumes that the inferior shape of the glenoid fits a perfect circle , 20. TheWe used general descriptive statistics and reported ordinal data as median with 25th percentile (Q1) and 75th percentile (Q3) and continuous data as mean with standard deviation (SD). To test for normal distribution, the Shapiro–Wilk test was used.Prevalence of an osseous defect was evaluated on reconstructed 2D CT-A images and on VNC 3D VRT reformats, and the McNemar test was used for comparison. We used Bland–Altman plots  and the P.SPSS  was used for statistical analysis with a level of significance of < 0.05 for any value of The PACS query revealed 52 patients with shoulder instability who received a clinical dual energy CT of the shoulder after arthrography. Six patients declined informed consent and were therefore excluded. Four patients were excluded because of metal implants and one patient because of incorrect dosage of intraarticular contrast material at arthrography. The resulting study group included 41 patients  and 42 shoulders .CT parameters of the DECT of the shoulder after arthrography are listed in Table Calculation of the blended CT arthrogram images, of virtual non-contrast images and of VNC 3D VRT reformats of the glenoid, was successful for all shoulders .CT values of the intraarticular iodinated contrast material were mean 1542.4 HU (± 369 HU) for the 80 kV scan in soft tissue kernel and 1014.6 HU (± 235.8 HU) for the blended CT arthrogram images. The mean CT value of the deltoid muscle measured on CT-A images was 64.5 HU (± 8.6 HU). The image contrast of CT-A images was mean 950.2 HU (± 235.5 HU) between intraarticular iodinated contrast material and deltoid muscle.CT arthrogram images, VNC images, and VNC 3D VRT reformats were of good quality: The overall image quality of the CT-A images was median 4 (4–5), the image quality of virtual non-contrast images was median 4 (3–4), and the image quality of virtual non-contrast 3D VRT reformats of the glenoid was median 4 (3–5) Figs.  and 3.FiFor CT 1 and CT 2, the image quality of CT arthrogram images, VNC images, and VNC 3D VRT reformats were median 4 (4–5) and 5 (4–5), 4 (3–4) and 4.5 (4–5), and 4 (3–4.25) and 5 (3.25–5), respectively. However, the number of patients scanned on CT 1 was more than 4 times higher than on CT 2 (34 vs. 8 patients).n = 8) revealed labrum tears in 5 patients, rotator cuff tears in 1 patient, and cartilage defects in 3 patients, all of which were confirmed surgically.Sub-analysis of CT arthrogram images of patients with shoulder surgery (P = 0.5). The glenoid surface area was also not different with mean 638.5 mm2 (± 127 mm2) on 2D images vs. mean 640.8 mm2 (± 129.5 mm2) on VNC 3D images (P = 0.47).Glenoid measurements were successful on all sagittal en face 2D CT arthrogram images  and on all en face VNC 3D VRT reformats of the glenoid . No difference was observed for the glenoid diameter with mean 28.3 mm (± 2.8 mm) measured on 2D images compared to mean 28.4 mm (± 2.9 mm) measured on VNC 3D VRT reformats (P > 0.99) with no discrepant cases. There was no difference for the width of the osseous glenoid defect with mean 3.2 mm (± 2.1 mm) measured on 2D images compared to 3.1 mm (± 2.3 mm) measured on VNC 3D images (P = 0.84), nor was the surface area of the osseous glenoid defect measured different with mean 46.6 mm2 (± 44.3 mm2) and 47.2 mm2 (± 48.0 mm2) (P = 0.73), respectively  or on CT 2 .Detection of an osseous defect was equal on sagittal en face 2D CT-A images  and on en face VNC 3D VRT reformats   and excellent on VNC 3D VRT reformats (ICC 0.98).We showed that DECT shoulder arthrography is feasible for clinical routine examinations and allows successful iodine removal using material decomposition with generation of virtual non-contrast (VNC) images and accurate 3D VRT reformats of the glenoid for assessment of bone loss.With a concentration of 80 mg iodine per ml contrast solution injected into the joint we were able to acquire DECT arthrogram images in diagnostic quality and successfully calculate both cross-sectional VNC images as well as VNC 3D VRT reformats for all clinical examinations. Subhas et al. showed diagnostic image quality for CT scans of human knee cadavers performed in single energy technique with 80 kV after arthrography using 60 mg iodine per ml solution. Furthermore, they demonstrated that the saturation point of the CT detector was reached at 93 mg iodine per ml solution for both the 80 kV SECT and the 80 kV scan of the 80 kV/140 kV DECT scan in a phantom .An et al. determined in a 2014 phantom study the optimal contrast dose at 60 mg iodine/ml and the energy level at 72 keV for DECT arthrography of the shoulder using virtual monochromatic spectral imaging. The optimized protocol was then applied to 23 patients, which resulted in 36% reduction of image noise and in 45% reduction of beam-hardening artifacts compared to the standard protocol . The autn = 8), the labrum, rotator cuff, and cartilage lesions identified on CT-A images were surgically confirmed. In their study with 47 shoulder patients, Foti et al. demonstrated that DECT arthrography is equal to magnetic resonance arthrography in detecting labral tears [In our study, evaluating 42 shoulders in 41 patients, we observed excellent image contrast with a mean difference of 950 HU between the intraarticular iodinated contrast material and the soft tissues on CT-A images, which allows to confidently assess the labrum, the rotator cuff, and the articular cartilage for pathology. In all of our patients with subsequent shoulder surgery  at 75 mg iodine/ml. In their study, subtraction and image calculation failed for all higher contrast agent dosages, because the demonstrable upper HU limit of the CT detector was reached and consequently the subtraction algorithm of the VNC application was unable to recognize the material as iodine , 13. Ourr = 0.875) and on 2D CT (r = 0.831), respectively, with comparable prediction errors for 3D CT and 2D CT. They recommend to use 3D CT as the most accurate, reliable, and reproducible imaging modality to estimate anteroinferior glenoid bone loss [Our results showed that detection of an osseous defect of the glenoid was accurate on VNC 3D VRT reformats, with no discrepant cases compared to 2D CT. In a study with 7 shoulder cadavers, Rerko et al. also demonstrated a high correlation between true anteroinferior glenoid bone loss vs. predicted bone loss on 3D CT , the glenoid surface area (P = 0.47), the width of the glenoid defect (P = 0.84), or the surface area of the glenoid defect (P = 0.73). Therefore, we conclude that the subtraction algorithm of the VNC application works properly and 3D VRT reformats are accurate.Margarelli et al. discovered 97% agreement between 2D CT and 3D VRT reformats in detecting an osseous defect of the glenoid in 100 patients with unilateral anterior glenohumeral instability. Furthermore, they showed a mean difference of 0.62% between the 2 methods for measurement of the percentage of glenoid bone loss using the Pico method [With a CTDIvol of mean 11.7 mGy and an effective dose of mean 1.95 mSv, the dual energy CT scans of our study were below the radiation dose reported in the literature. An et al. reported a CTDIvol of 17.8 mGy for DECT arthrography of the shoulder in 23 patients using virtual monochromatic spectral imaging . Biswas A limitation of our study was that patients were scanned on 2 different CT scanners. However, acquisition parameters were adjusted to equal dose, and the same dual energy protocols were used, which resulted in comparable image quality and no difference in glenoid measurements according to a sub-analysis for examinations performed on CT 1 and 2. Furthermore, the dual energy CT protocol incorporated a 70-s delay between the 80 kV and the 140 kV scan since it was derived from the liver VNC protocol. In order for the VNC application to work, deactivation of the delay was not possible. However, we did not experience significant patient motion during the two scans which could possibly negatively affect image quality.In summary, dual energy CT arthrography of the shoulder for clinical routine examinations is feasible and showed good image quality at a dose comparable to single energy shoulder CT. With image postprocessing, diagnostic virtual non-contrast 3D VRT reformats of the glenoid were successfully calculated, which allow for 3D assessment of glenoid morphology and bone loss and accurate glenoid measurements.Supplementary file1 (DOCX 85 KB)Below is the link to the electronic supplementary material.


Trichoderma longibraciatum was isolated from an industrial soil sample in Dammam, Saudi Arabia using the enrichment method of mineral salt's medium (MSM) amended with 2,6-DCP. Morphological and molecular identification  of the 2,6-DCP tolerating fungal isolate were charactraized. The fungal isolate has demonstrated a tolerance to 2,6-DCP up to 300 mg/L. Mycelial growth and fungal sporulation were reduced with increasing 2,6-DCP concentrations up to 96 h incubation period. However, after 168 h incubation period, the fungal isolate recorded maximum growth at all the tested 2,6-DCP concentrations up to 150 mg/L. Carboxy methyl cellulase production by tested fungus was decreased by increasing 2,6-DCP concentration up to 75 mg/L. The biodegradation pattern of 2,6-DCP in GM liquid medium using GC–mass analysis as well as the degradation pathway was presented. This study provides a promising fungal isolate that could be used in the bioremediation process for chlorinated phenols in soil.2,6-Dichlorophenol  is an aromatic compound with industrial importance in making insecticides, herbicides, and other organic compounds. However, it poses serious health and ecological problems. Microbial degradation of 2,6-DCP has been widely applied due to its effectiveness and eco-friendly characteristics. In this study,  These compounds are used as mothproofing agents, miticides, germicides, algicides, fungicides and wood preservatives, as well as in the synthesis of dyes and drugs3. They are also discharged into the environment through industrial, urban and agricultural wastewaters. These halogenated organic products are source of pollution for soil and ground water with regard to their high toxicity, strong odor emission and persistence4. In addition, Cps are characterized by their carcinogenic, mutagenic, and cytotoxic effect on human as showed by the World Health Organization5.Chlorophenols (CPs) are aromatic compounds with at least one chlorine atom and one hydroxyl group6. But considering the hazardous byproducts, the high cost and the limited concentration range of these processes, attention has been focused on the environmentally safe, cost effective and efficient bioremediation methods7. To illustrate, bacterial species including genera of Pseudomonas, Ralstonia, Rhodococcus, Alcaligenes and Herbaspirillum are capable of utilizing CPs as sole carbon and energy sources8. Furthermore, white rot fungi are promising organisms because of their ability to completely degrade aromatic xenobiotics9. This ability is associated with the production of extracellular oxidoreductases  that can remove chlorophenols from wastewater through an oxidative coupling reaction. For instance, Phanerochaete chrysosporium has been shown to degrade chlorinated aromatic compounds such us 2–4 dichlorophenol, pentachlorophenol and 2,4,5-trichlorophenoxiacetic acid using a degradation system consisting of peroxidases, laccases, cellobiose dehydrogenase and transmembrane methyl transferase10. Members of the genus Trametes are also excellent candidates for these activities with confirmed bioremediation capacity13. Fungi from other classes showed also various extent of chlorophenols degradation15. Although fungi of the genus Trichoderma have been studied mainly as biological control agent against plant diseases, species of Trichoderma has been shown to metabolize CPs. For instance, Cser-jesi and Johnson,16 reported the degradation of pentachlorophenol by T. irgatum. Furthermore, T. harzianum isolated from water samples of Lake Bonney, South of Australia mineralized a minor percentage (2–3%) of spiked radiolabeled pentachlorophenol and partially dehalogenated (46%) spiked tetrachloroguaiacol in mineral salts medium17. Additionally, PCP was completely degraded co-metabolically by T. longibrachiatom DSM 1651718. Chakroun et al.19, demonstrated the transformation of 2,4-dichlorophenoxyacetic acid and 4-chlorophenol in the presence of 2,2′-azinobis-(3-ethylbenzthiazoline-6- sulphonate) as mediator by the laccase purified from T. atroviride. The production of laccases by Trichoderma species is very interesting because it is known to produce a variety of cellulolytic enzymes including carboxymethyl cellulase (CMCase)21. Therefore, the cooperation between lignocellulolytic enzymes could enlarge the potential applications of such strain in various industries and in the treatment of agricultural and industrial wastes.The removal of chlorinated phenols from contaminated sites involves several chemical and physical methods including oxidation by chemical, photochemical or photocatalytic processes and adsorption respectivelyThe present study aimed to (i) isolate fungi from an industrial soil sample in Dammam, Saudi Arabia to screen these fungal strains for 2,6-DCP degradation, (ii) identify the isolated 2,6-DCP tolerant fungus molecularly, (iii) investigate the transformation pattern and pathway of 2,6-DCP and (iv) study the effect of 2,6-DCP on its growth and carboxy methyl cellulase activity .17 containing (in grams of ingredient per liter): K2HPO4, 0.75; KH2PO4, 0.2; MgSO4, 0.1and FeSO4.7H2O, 0.06. The final pH was adjusted to 7.022. The tested pollutant 2,6-Dichlorophenol was added at the concentration 100 mg/L as sole carbon and energy source. The cultures were placed on a shaker (120 rpm) for a week at 30 ºC. Five ml of cultures fluid was subculture into fresh sterile MSM containing the tested 2,6-DCP. The pollutant 2,6-DCP concentrations were increased gradually: 0, 25, 50, 75, 100 and 150 mg/L. Fungal Colonies becoming visible after 3–4 days of incubation at 30 °C were picked up and sub-cultured to ensure purity. Pure cultures were grown in MSM containing 100 mg/L 2,6-DCP slants and stored at 4 °C for further investigations.Hydrocarbons polluted soil samples were collected from an automobile oil change station in industrial zones at Dammam city, Saudi Arabia, on 16 March 2019. One gram of polluted soil was inoculated into 100 ml of a Mineral Salt's Medium (MSM)−1 of 2, 6-DCP were characterized morphologically and molecularly. The morphological appearances of the selected fungal isolates grown on Potato Dextrose Agar (PDA) plates were characterized by visual observation and micro-morphological techniques23.Fungal isolates that tolerated up 100 mg L24 and ITS4: (5′-TCC TCC GCT TAT TGA TAT GC-3′)25 to amplify the internal transcribed spacer (ITS) rRNA genes. PCR reaction was run under the following thermocycler parameters: an initial denaturation at 95 °C for 10 min, followed by forty-five cycles of: denaturation at 95 °C for 30 s, 55 °C annealing for 30 s, and 72 °C extension for 1 min, with a final extinction step at 72 °C for 10 min.For molecular identification, genomic DNA was extracted from pure colonies of grown fungal mycelia using the column-based method of QlAamp DNA Mini Kit  following manufacturer’s instructions with additional steps: . The DNA quality and quantity were determined spectrophotometrically using NanoDrop, 2000c spectrophotometer .PCR reactions of 50 µl were performed including the following:  and 2 µM of the previously described primers; ITS1: (5′-CTT GGT CAT TTA GAG GAA GTA A-3′)Amplicons obtained from previous PCR step were purified using ExoSAP-IT  following manufacturer’s recommendations. Using 10 µM of either forward (ITS1) or reverse (ITS4) primer used in the initial amplification step, samples were amplified using BigDye Terminator v3.1 Cycle Sequencing Kit . Following the amplification,. Purification was conducted using Cycle sequencing purification- BigDye X-Terminator purification kit  to prepare the samples for sequencing, which were carried out using the series Genetic Analyzer 3500 .23.The rate of fungal growth on PDA medium supplemented with various concentrations of 2,6-DCP  was tested. Agar plates were inoculated with 5 mm plug of fungal mycelium and incubated at 28 °C. Four replicates were prepared for each concentration. Radial mycelial growth was measured after 1, 2, 3, 4 and 7 days of incubation26. The GM medium used contained (g/l): 1.0 K2HPO4, 0.01 ZnSO4.7H2O, 0.005 CuSO4.5H2O, 0.5 MgSO4.7H2O, 0.01 FeSO4.7H2O, 0.5 KCl, 10 glucose and 3.0 NaNO3 as an energy source of nitrogen. The pH of the liquid medium was adjusted to 5.5 before autoclaving at 121 °C for 15 min. Two pieces of agar plugs (5 mm in diameter) of a 4-day old fungal isolate grown on PDA at 28 °C were used as inoculums in each of the 20 mL liquid medium in 100 mL flasks. The cultures were grown under static condition for 12 day at 28 °C. A flask containing 2,6-DCP without fungal inoculum was prepared as control. All the experiments were done in triplicate. Fungal growth was estimated after filtration and overnight drying in an over at 70 °C. The experiments of biodegradation products were carried out with the culture filtrate of 2,6-DCP (100 mg/L) treatment after 12 days incubation period.Biodegradation of 2,6-DCP  was determined in Glucose Minimal (GM) liquid medium described by–1. The injector temperature and the ion-source temperature were set to 250 °C. The oven temperature was set to range from 50 to 300 °C, with a hold time of 3 min and a total run duration of 29 min. ACQ Mode Scan range: 35 m/z to 500 m/z, scan speed: 1666.A Gas Chromatography-Mass Spectrometry (GC–MS) Model QP2010 SE (Shimadzu-Japan) was used to quantify the biodegradation of 2,6-DCP and its breakdown products. The GC–MS was outfitted with a 5 Sil MS capillary column with a length of 30 m, an internal diameter of 0.25 mm, and a film thickness of 0.25 m. Helium was used as the carrier gas at a flow rate of 2.5 ml min27. Conidia of Trichoderma were produced by transferring agar discs (7 mm in diameter) from PDA culture to flasks each containing 20 ml of minimal synthetic medium (MSM) with the following composition (g/l): MgSO4.7H2O 0.2; K2HPO4 0.9; KCl 0.2; NH4NO3 1.0; FeSO4.7H2O 0.002; MnSO4 0.002 and ZnSO4 0.002. supplemented with 1% CMCase and incubated at 30 °C for 7 days. Enzyme activity was estimated by measuring the accumulation of soluble sugars using the phenol sulphuric acid as following: 0.5 ml of the diluted glucose standard was added to each tube along with 0.5 ml at 5% phenol solution. Likewise, 0.5 ml of the sample's supernatant was added to each respective tube with 0.5 ml of 5% phenol solution. All tubes were vortexed to thoroughly mix the contain 2.5 ml of Conc. H2SO4 was added to each tube. The tubes were sealed and were incubated for 30 min at room temperature before reading the absorbance at 485 nm. After 7-day incubation period, the fungal mycelia were filtrated dried at 80 °C for constant weight. The fungal growth was reported as mg/20 ml.Carboxymethyl Cellulase (CMCase) production by fungal isolate and the effect of 2,6-DCP at different concentrations was assayed according to Dubois et al.www.ncbi.nlm.nih.gov/genbank/). The determined nucleotide sequences of the isolated fungi in this study and other fungi with 2,6-DCP degredation caractaristics were subjected to multiple nucleotide sequence alignment using Cluster Omega tool . From the multiple alignment data, a phylogenetic tree was constructed using Interactive Tree Of Life (iTOL) tool (Letunic and Bork28 Nucleic Acids Res https://doi.org/10.1093/nar/gkab301) to analyze the relationship between the fungi species. Tree construction was performed using the neighbor joining method and distance divergence (%) was determined using the Molecular Evolutionary Genetics Analysis 7 (mega7) software package29.Nucleotide sequences of the internal transcribed spacer (ITS) rRNA gene region of the fungi known for their 2,6-DCP degredation were retrieved from the DNA database of GenBank  (https://www.rstudio.com/) was used to develop analytics and visualizations graphics.Data presented in this study were means of three replications, One-way ANOVA test was used in Statistical Package for Software Science (SPSS) statistic program at This article does not contain any studies with human participants or animals performed by any of the authors.Trichoderma (Ascomycetes). Fast growing colony that fills up 9 cm Petri dish in 4–5 days. Cottony mycelial mat formed were white at first and becoming dark grey with conidiospores development  followed by Cladosporium sp. (62% of sequence identities) and highly diverse from Trichosporon sp. (36% of sequence identities)  at retention time 14.4 min, and its fragments at m/z 162 [M-1]+ (2), 128 [M-Cl]+ (3), and 92 [M-2Cl]+(4) at retention time 12.01 min. Other fragments were found at m/z 145, 117, 100, and 65 with their relative intensities as [CP-Cl + OH] (5), [HOOCCH2CH2COOH] (6) [CP-CO]+(7), and [CP-CO-Cl]+(8), respectively (Figs. The release of one chloride ions per mol of 2,6-DCP during the biodegradation was indicated in Fig. Data in Table 31. The disadvantages of these methods, such as the potential of forming hazardous byproducts, have been extensively demonstrated33. On the other hand, biological degradation methods are considered an attractive alternative phenol elimination approach. Bioremediation and phytoremediation exploiting microbes are innovative technologies that can alleviate a variety of environmental pollution problems. Fungi have been found to be effective in the degradation of chlorophenol found in polluted sites. In particular, Trichoderma sp. is one of the most effective groups of fungi employed in bioremediation36.The removal of dichlorophenol compounds, which are toxic, hardly degraded, and tend to accumulate in soil and water, has been primerily achieved using physical and chemical methodsTtrichoderma longibraciatum.In light of the importance of exploring biological treatment alternatives agents, the current study aimed to investigate the ability of the isolated fungi in degrading 2,6-DCP and study the transformation and pathway patterns of 2,6-DCP. Moreover, the effect of 2,6-DCP on the fungal growth and carboxy methyl cellulase activity was investigated. Using morphological and molecular identifications, we determined that the isolated tolerant fungus strain reported in our investigation belonged to T. longibraciatum ability to grow and tolerate 2,6-DCP at various concentrations ranging from 25 to 150 mg/L was also examined. Fungal growth was determined by the radial growth and the dry weight of T. longibraciatum to predict the impact of 2,6-DCP concentrations. Findings demonstrated that the isolated fungi exhibited different degrees of sensitivity towards 2,6-DCP during the first 4 days incubation period, with a reduction in the growth associated with elevated 2,6-DCP concentrations compared with control. The inhibition of the growth may be attributed to the reduction of the ergosterol, a derivative of cholesterol essential for the regulation of fungal membrane fluidity and integrity targeted by fungicide37. Although the growth was reduced, the T. longibraciatum exhibited controlled growth after 7 days, even with the highest tested concentrations. Our results are supported by previous studies, which have demonstrated the ability of microorganisms to utilize the degraded chlorophenols as their sole carbon and energy source39.39. Despite the fact that chlorophenol compounds are poorly biodegradable and persistent in the environment, various studies have shown that aerobic breakdown of chlorophenol congeners is possible38. Oxidase must be engaged in the aerobic breakdown of chlorophenols for bacteria to integrate ambient oxygen into their substrates43. T. longibraciatum longibraciatum was incubated to 2,6-DCP for 12 days in order to confirm the degradation intermediates and hypothesize the degradation routes. The mass spectrum obtained for the control sample of 2,6-DCP in the positive-ion mode is illustrated in Fig. − ion (Fig. Chlorophenols compounds are degraded and transformed in both abiotic and biotic environmentsion Fig. D. It demTrichoderma longibraciatum which reflects the majority of the compounds observed by GC–MS. The initial pathway for 2,6-DCP degradation, OH° radicals attack the aromatic ring and simultaneously release chlorine ions into solution and [M-Cl]+, this compound was clearly observed in Fig. +, and [CP-Cl + OH] products. In the second and third routes, 2,6-DCP is continually attacked by hydroxyl radicals, resulting in the formation of certain intermediates such as [CP-CO]+, [CP], and [CP-CO–Cl]+, the latest was observed in Fig. 46.Based on the intermediates identified in GC–MS, the underlying degradation pathways were listed in Fig. T. longibraciatum has the ability to tolerate high concentrations of 2,6-DCP; while also using it as an energy source. In addition, a potential pathway of 2,6-DCP degradation was proposed. The resistance of T. longibraciatum to high concentrations of 2,6-DCP, and its ability to degrade this hazardous and toxic compound makes it an essential fungal genus to be explored and employed in bioremediation of pesticide-contaminated sites. It is critical to investigate the production of oxygenases by strains known for their ability to tolerate evaluated levels of 2,6-DCP.Here we investigated the use of tolerant fungus strain isolated from industrial soil sample in degrading 2,6-DCP. Findings revealed that