The present study investigated the underlying mechanisms of Enzogenol® in reducing postprandial glucose in humans. The potential inhibitory action of Enzogenol® against digestive enzymes: α-amylase and α-glucosidase, and dipeptidyl peptidase-4 (DPP-4) enzyme was determined. Enzogenol® demonstrated the ability to inhibit all three enzymes: α-amylase enzyme activity (IC50 3.98 ± 0.11 mg/mL), α-glucosidase enzyme activity (IC50 13.02 ± 0.28 μg/mL), and DPP-4 enzyme activity (IC50 2.51 ± 0.04 mg/mL). The present findings indicate the potential for Enzogenol® to improve postprandial glycaemia by delaying carbohydrate digestion via the inhibition of digestive enzymes (α-amylase and α-glucosidase), and enhancing the incretin effect via inhibiting the dipeptidyl-peptidase-4 enzyme. The inhibitory actions of Enzogenol® on enzymes should therefore be further validated in humans for its potential use in type 2 diabetes mellitus prevention and management.The New Zealand pine bark extract (Enzogenol It is estimated that there are currently 537 million (ages 20–79 years) 10.5%) people living with diabetes worldwide, and this number is expected to increase to 783 million (12.2%) by 2045 0.5% peop.There is increasing evidence that plant-derived, antioxidant-rich extracts are able to improve glycaemia and may therefore aid in diabetes management and prevention ,8,9. In ® exhibits the ability to significantly improve postprandial glucose in healthy participants, especially those exhibiting monophasic glucose curve shapes indicative of poorer glycaemic control [® in healthy participants regardless of their glucose curve shapes. For example, in contrast to control (placebo), 50 mg of Enzogenol® significantly reduced %PG (p = 0.003) and 2hPG (p = 0.041), and at 400 mg an even higher reductions in %PG (p < 0.001) and 2hPG (p = 0.012) were observed. However, the specific glucose-lowering mechanisms of Enzogenol® have yet to be fully elucidated.We have previously demonstrated that Enzogenol control . Additio®, exert their hypoglycaemic effects in humans. Notably, inhibiting α-amylase and α-glucosidase responsible for breaking down carbohydrates may prevent or slow carbohydrate digestion during a meal [Inhibiting glucose-regulating enzymes such as α-amylase, α-glucosidase ,14,15 ang a meal ,14,15. Ig a meal ,17,18. IPinus pinaster, Pcynogenol®) and Korean pine bark (Pinus densiflora) on α-amylase and α-glucosidase enzymes, although comparative results are inconclusive due to differences in test products and methodologies [® (Pinus radiata) that is obtained from New Zealand. Similarly, no DPP-4 inhibition test to date has been conducted on Enzogenol®.Several in vitro and in vivo studies have investigated the inhibitory effects of other species of pine bark extracts, for example the French Maritime  was kindly provided by ENZO Nutraceuticals Limited. All chemicals used were of analytical grade. Ultrapure water was prepared from Millipore water purification. Stock phosphate saline buffer  was purchased from Thermo Fisher Scientific. Acarbose, α-amylase from porcine pancreas, α-glucosidase from yeast Saccharomyces cerevisiae, 3,5-dinitrosalicylic acid (DNS) reagent, p-nitro-phenyl- α-D-glucopyranoside (p-NPG), sodium carbonate solution (Na2CO3), and soluble starch (potato), were purchased from Sigma-Aldrich. The DPP-4 inhibitor screening assay kit was purchased from Abcam . The kit consisted of human recombinant DPP-4 enzyme, dipeptidyl-peptidase (DPP) substrate (H-Gly-Pro-AMC), assay buffer , and sitagliptin as a positive control inhibitor.Enzogenol® was prepared using a water-based extraction from the New Zealand Pinus radiata trees [® was solubilised in dimethyl sulfoxide (DMSO) at 20 mg/mL and diluted with pure Milli-Q water into various concentrations.Enzogenolta trees ,23. The ® was sent to Callaghan Innovation, New Zealand for analysis. Alpha-amylase, α-glucosidase and DPP-4 enzyme inhibition assays were conducted on Enzogenol® based on previous studies but with some modifications [50 values of Enzogenol® on α-amylase, α-glucosidase, and DPP-4 enzyme inhibition were also determined. The IC50 was defined as the concentration required for an inhibitor to reduce 50% of the enzyme activity. The IC50 value was obtained from a dose-dependent activity versus concentration plot, where data points of Enzogenol® in five different concentrations were fitted into a non-linear sigmoid plot, accounting for non-linear concentration dependent on enzyme-inhibitor interaction at low and high concentrations.Samples of Enzogenolications ,25. Diff® was prepared at 0.5, 1, 2.5, 5 and 10 mg/mL. Samples were incubated with α-amylase at 30 °C for 15 min before the addition of 1% starch solution. The hydrolysis of starch by α-amylase in the absence and presence of the sample was kept at 30 °C for 30 min and stopped by adding 1% DNS solution. The mixture was heated at 100 °C for 10 min and then diluted 4-fold with water before readings were measured at 540 nm. The inhibitory effect of α-amylase (%) was calculated using the following formula:Enzogenol® was prepared at 5, 10, 20, 40, and 50 μg/mL. Samples were incubated with α-glucosidase at 37 °C for 15 min. The hydrolytic kinetics by α-glucosidase in the absence and presence of the sample were started by adding p-NPG substrate at 37 °C and monitored at 405 nm for 20 min. The reaction was stopped by adding 0.5 M Na2CO3 solution and readings were measured at 405 nm. The inhibitory effect of α-glucosidase (%) was calculated using the following formula:Enzogenol® was measured at 0.25, 0.5, 1, 2.5 and 5 mg/mL. The reaction was initiated by adding DPP substrate and incubating at 37 °C for 30 min. Readings were measured at Ex 355 nm and Em 460 nm. The inhibitory effect of DPP-4 enzyme was calculated using the following formula:Enzogenoln = 3 samples of the same batch tested.The data were presented as mean ± SEM, with a minimum of ® on α-amylase, α-glucosidase and DPP-4 enzymes. The IC50 values of Enzogenol® for α-amylase, α-glucosidase and DPP-4 enzymes were 3.98 ± 0.11 mg/mL, 13.02 ± 0.28 μg/mL and 2.51 ± 0.04 mg/mL, respectively.® exhibited inhibitory effects against α-amylase, α-glucosidase, and DPP-4 enzyme. Enzogenol® had the greatest inhibition on α-glucosidase enzyme followed by DPP-4 enzyme, then α-amylase enzyme. These mechanisms may have important clinical implications for individuals with impaired glycaemic control, by preventing or slowing carbohydrate digestion [The present study demonstrated that Enzogenoligestion ,14,15 anigestion ,18.®) concluded that larger oligomers were stronger enzyme inhibitors compared to monomers [The hypoglycaemic effect of the New Zealand pine bark may be a result of its unique structural properties such as hydrogen moieties  and double bonds that determine its effective interaction with digestive enzymes, transmembrane glycoproteins such as DPP-4, apically located transporters and receptors involved in glucose metabolism during a meal . There amonomers .® exhibited considerable inhibition against both α-amylase (IC50 of 3.98 mg/mL) and α-glucosidase (IC50 of 13.02 μg/mL), with a stronger inhibition on α-glucosidase. Similarly, a Korean pine bark extract also showed a higher inhibitory potency against α-glucosidase (IC50 of 0.025 μg/mL) compared to α-amylase (IC50 of 1.69 μg/mL), although the exact phenolic composition contributing to its inhibitory action was not known [50 of 5.34 μg/mL) [Plant extracts have been shown as better inhibitors of α-glucosidase than α-amylase ,31,32. Eot known . An in v4 μg/mL) .50 values obtained [It is noteworthy that differences in test products, methods of extraction and purification, and assay conditions such as pH, reaction time and temperature, substrate concentration and source as well as enzyme concentration and source used in the different studies might have likely contributed to the different ICobtained ,33. For ®, which is a good α-glucosidase and α-amylase inhibitor, to work synergistically with acarbose to inhibit carbohydrate digestion and improve glycaemic control, whilst reducing acarbose dose to alleviate anti-diabetic drug-related adverse effects [Acarbose as an anti-diabetic drug is a strong amylase inhibitor known for causing gastrointestinal adverse effects due to the accumulation of undigested carbohydrates becoming readily available for bacterial fermentation ,34. Ther effects ,35,36.® has also showed inhibitory action against DPP-4 enzyme activity (IC50 of 2.51 mg/mL). Currently there is a paucity of data on similar plant extracts, making comparison difficult. The ability of Enzogenol® to inhibit DPP-4 suggests an enhanced incretin effect via a likely increase in active GLP-1 levels. This may help explain the significant improvement in postprandial glucose in healthy participants observed in our previous study [Enzogenolus study .® on enzymes involved in regulating postprandial glycaemic control. The study outcomes, in particular on DPP-4 enzymatic inhibition, add to the clinical evidence in the observed improvements in postprandial glycaemia in humans [® could therefore potentially be a useful adjunct for those at higher risk for T2D to take alone in conjunction with lifestyle advice. Equally Enzogenol® could also potentially work additively or synergistically when taken together with anti-diabetic drugs such as metformin, often prescribed as a first-line therapy for patients with prediabetes or T2D.The merits of the present study included understanding the inhibitory action of Enzogenoln humans . EnzogenNevertheless, there are limitations to the study. The present study utilised yeast α-glucosidase that is not precisely biologically similar to mammalian α-glucosidase ,37. Howe® may also be modified by several dietary factors such as the presence of macronutrients during a meal [® and its inhibitory actions on enzymes should be further validated in humans. The inhibition of Enzogenol® on α-amylase and α-glucosidase could be verified in hydrogen tests [The in vitro outcomes may also not translate into equivalent outcomes in humans, given the knowledge that plant polyphenols undergo extensive metabolism that may alter their enzymatic inhibitory effects ,42. The g a meal ,44. Theren tests  and 13C en tests ,47,48 in® inhibits α-amylase, α-glucosidase and DPP-4 enzymes. The enzymatic inhibitory effects exhibited by Enzogenol® may be one of the mechanisms leading to better glycaemic responses observed in humans. More human studies are warranted to validate the underlying hypoglycaemic mechanistic actions of Enzogenol® and its potential for use in T2D prevention and management.Enzogenol


Catha edulis Forsk) plant. Khat was found to inhibit major phase I drug metabolizing cytochrome P450 (CYP) enzyme activities in vitro and in vivo. With the upsurge of khat consumption and the potential use of cathine to combat obesity, efforts should be channelled into understanding potential cathine-drug interactions, which have been rather limited. The present study aimed to assess CYPs activity and inhibition by cathine in a high-throughput in vitro fluorescence-based enzyme assay and molecular docking analysis to identify how cathine interacts within various CYPs’ active sites. The half maximal inhibitory concentration (IC50) values of cathine determined for CYP2A6 and CYP3A4 were 80 and 90 μM, while CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP2J2 and CYP3A5 showed no significant inhibition. Furthermore, in Ki analysis, the Lineweaver-Burk plots depicted non-competitive mixed inhibition of cathine on both CYP2A6 and CYP3A4 with Ki value of 63 and 100 μM, respectively. Cathine showed negligible time-dependent inhibition on CYPs. Further, molecular docking studies showed that cathine was bound to CYP2A6 via hydrophobic, hydrogen and π-stacking interactions and formed hydrophobic and hydrogen bonds with active site residues in CYP3A4. Both molecular docking prediction and in vitro outcome are in agreement, granting more detailed insights for predicting CYPs metabolism besides the possible cathine-drug interactions. Cathine-drug interactions may occur with concomitant consumption of khat or cathine-containing products with medications metabolized by CYP2A6 and CYP3A4.Cathine is the stable form of cathinone, the major active compound found in khat ( Catha edulis Forsk plant, which is also commonly known as khat. Khat is a perennial shrub cultivated in khat-belt countries such as in Africa and the Middle East, that are ingested not to gain nutritive values but to attain psychostimulatory effects. Khat was traditionally used in social gatherings in Yemen and widely cultivated as a source of income replacing coffee cultivation despite debates that khat cultivation drains foreign investment. Khat chewing is a tradition in khat-belt countries with each khat sessions lasting 3 to 4 hours with 100-200 g of leaves chewed, which induces mild euphoria and excitation in its users. In the early 1930s, Wolfes identified cathine phenylpropanolamine) as norpseudoephedrine, which was one of the khat alkaloids that contribute to the pharmacological effects of khat leaves. It was reported that in every 100 g of fresh khat, there were 36 mg of cathinone, 120 mg of cathine and 8 mg of norephedrine present. Young stems and flowers of khat plants contain 1-phenyl-1,2-propanedione, cathinone, cathine and norephedrine. Cathinone reductase present in khat was accountable for reducing cathinone to cathine in the presence of NADPH. However, the quantity of cathine depends on the type of khat in which green khat contains a higher amount of cathine and norephedrine than red khat. Besides khat plant, cathine is also found in anorectic products. Cathinone and cathine shares closely similar chemical structures to amphetamine. Cathine was claimed to be 7-10 times less potent than cathinone but possesses a longer duration of action.Cathine (d-norpseudoephedrine (NPE)) is one of the major constituents found in the  As compared to cathinone, cathine has a slower onset of action and may not be as lipophilic to enter the central nervous system, which to some extent explains the slower metabolism of cathine. Oral administration of cathine (6.5 mg/kg) may affect adrenocortical function and was found to produce rapid synchronized cell death in human leukaemia cell lines and peripheral blood leucocytes. With its milder psychostimulatory effects, a higher dose of cathine is needed to exert its effects, and this causes severe adverse systemic effects and the inhibition of noradrenaline uptake.12 In recent years, khat use was reported in England, Wales, Rome, Amsterdam, Canada, Israel, Australia, New Zealand and the United States. Khat is chewed by individuals idling on streets in Europe accompanied sometimes by alcoholic beverages and other drugs.Over the past decades, cathine has received little experimental attention. Cathine acts as a central stimulant (indirect sympathomimetic) and an inhibitor or monoamine oxidase. Polysubstance users are also found to be using synthetic cathinone and cathine concurrently with alcohol leading to haemorrhage and intoxication. Moreover, khat use have been found to hinder antipsychotic and tuberculosis medication effects on patients who are khat users. With the widespread global use of khat together with the co-administration with clinically used drugs, the detrimental effects of khat-drug interactions are pushed towards the centre of the attention. According to the National Drug Intelligence Center, a component of the U.S. Department of Justice, the potency of harvested khat fades after 48hours as cathinone degrades into cathine (https://www.justice.gov/archive/ndic/pubs31/31482/index.htm). Thus, the effects of cathine on human drug metabolizing enzymes and the possible herb-drug interactions caused by cathine warrant further exploration.It was found that a majority of khat users used more than one other psychoactive substances in which cigarettes were mainly used to maximize the stimulation power of khat with a combination of alcohol to break the aftereffect. CYPs are predominantly expressed in the liver, besides also occurring in the small intestines, lungs, placenta and kidneys. In mammals, CYP family 1, 2, 3 and 4 are involved in detoxification, steroid and eicosanoids metabolism. In humans, CYP family 1, 2, 3 and 4 and to a lesser extent CYP family 5, 8, 19, 21 and 26 are involved in xenobiotic metabolism. The CYP enzymes are predisposed to inhibition or induction by xenobiotics including herbal medications that comprise mixtures of phytochemicals. Phytochemicals such as resveratrol and quercetin have been reported to significantly inhibit CYP3A4 which translates to reduced clearance and subsequent toxicity from other CYP3A4 substrates in humans.23 There are few well characterized examples including resveratrol and quercetin which were classified as clinically significant perpetrators as numerous herbal preparations interact with CYPs in vitro but showed variable in vivo interactions.Cytochrome P450 (CYP) are a crucial family of enzymes for producing cholesterol, steroids, prostacyclins and thromboxane A2 besides playing an essential role in detoxification of foreign substances and drugs.in vitro, suggesting that other active constituents in the khat plant such as merucathine, merucathinone, tannins, cathedulins and norephedrine were likely responsible for these inhibitions. Ensuing this study, it was found that KEE also inhibited other major human drug metabolizing CYP isoforms namely CYP2A6, CYP2B6, CYP2C8, CYP2C19, CYP2E1, CYP2J2 and CYP3A5 except CYP1A2 in vitro. Our ongoing studies, hence, are investigating modulatory effects of main active compounds of khat on the major drug metabolizing CYPs in vitro. The current study aimed to follow up on the in vitro inhibitory effects and mode of inhibitions of cathine, which is the second main active component present in khat plant, on several major drug metabolizing CYPs namely CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP2J2, CYP3A4 and CYP3A5. Additionally, molecular docking was performed to understand how cathine interacts with particular CYP isoforms for which its in vitro results showed significant inhibition.Investigations into the inhibitory effects of cathine on CYPs are generally lacking in the literature as most studies about khat-drug interactions have focused on cathinone, which is deemed to be the major active compound in khat. Our earlier study found that khat ethanol extract (KEE) inhibited CYP2C9, CYP2D6 and CYP3A4 significantly, but cathinone showed negligible inhibition on these CYP isoforms 1 S, 2 S)-2-amino-1-phenylpropan-1-ol.hydrochloride) was acquired from Lipomed AG . Acetonitrile was obtained from Fisher Scientific . The Vivid® CYP450 Screening kits for all CYP isoforms namely CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP2J2, CYP3A4 and CYP3A5 were obtained from Life Technologies™ . Costar Black 96-well plates and tris-base powder were obtained from Thermo Fisher Scientific  and Amresco® LLC , respectively.Cathine or d-Cathine.HCl  produced by blue (3-cyano-7-hydroxycoumarin), green (fluorescein) and cyan (7-hydroxy-4-trifluoromethylcoumarin) standards against a range of concentrations of the respective standard was plotted to derive the standard curves. The standard curve equation was used in succeeding assays to enumerate the fluorescent metabolites produced. Time curves were plotted afterwards to define the incubation time for single CYP assays namely 60 minutes for CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP2J2, CYP3A4 and 120 minutes for CYP3A5, respectively, as determined previously.® CYP450 Screening Kits for all CYP isoforms including Vivid® EOMCC CYP1A2 Blue, Vivid® CC CYP2A6 Blue, Vivid® BOMCC CYP2B6 Blue, Vivid® DBOMF CYP2C8 Green, Vivid® BOMCC CYP2C9 BLUE Vivid® EOMCC CYP2C19 Blue, Vivid® EOMCC CYP2D6 BLUE Vivid® EOMCC CYP2E1 Blue, Vivid® MOBFC CYP2J2 Cyan Vivid® BOMCC CYP3A4 BLUE and Vivid® BOMCC CYP3A5 Blue, according to manufacturer’s instructions. Using the 96-well black plates, in each well, 40 μL of respective reaction buffers  was added with 50 μL of master premix  and 0.3 μ/mL glucose-6-phosphate dehydrogenase in 100 mM potassium phosphate at pH 8.0) for incubation with shaking at room temperature for 30 minutes. 10 μL per well of a mixture of respective substrates and NADP+ were added to kick start the reaction. Total volume per well was 100 μL in the 96-well plate.The inhibitory effects of cathine on human CYP enzymes activities were determined using Vivid® BOMCC (7-benzyloxymethyloxy-3-cyanocoumarin) for CYP2B6, CYP2C9, CYP3A4 and CYP3A5; Vivid® CC (3-cyanocoumarin) for CYP2A6; Vivid® DBOMF (dibenzylmethylfluorescein) for CYP2C8; Vivid® EOMCC (ethoxymethyloxy-3-cyanocoumarin) for CYP1A2, CYP2C19, CYP2D6 and CYP2E1; Vivid® MOBFC (7-p-methoxy-benzyloxy-4-trifluorocoumarin) for CYP2J2; and 0.03 mM NADP+. The mixture was shaken at room temperature, 60 minutes for CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP2J2, CYP3A4 and 120 minutes for CYP3A5. 50 μL of 0.5 M tris-base solution was added lastly to halt the reaction. The enzyme activities were measured by using the Varioskan® Fluorescence Spectrophotometer  at excitation/emission wavelengths of 415/460 nm , 415/520 nm (cyan – CYP2J2) and 490/520 nm (green – CYP2C8).The CYP enzyme specific substrates added were as follows: Vivid50 values which were less than 100 μM, and then their Ki values were further assessed. Various concentrations of cathine  were incubated with Vivid® Fluorogenic Probe Substrates CC  for CYP2A6 and BOMCC  for CYP3A4.Cathine was dissolved in water to attain the 2.5 mM of cathine stock. The assay conditions were as described above. The assay conditions were as described above, with the exception that 40 μL of buffer was substituted using 40 μL of cathine with concentrations ranging from 0 to 1000 μM, which was obtained following two times serial dilution of the cathine stock. Cathine inhibited CYP2A6 and CYP3A4 according to their IC+ to produce NADPH during the pre-incubation. The mixture  was incubated for 30 minutes with shaking under similar range of concentrations of cathine as mentioned above for IC50 determination. Substrates were subsequently added to initiate the reaction, and fluorescence readings were taken after 60 minutes for CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP2J2, CYP3A4 and 120 minutes for CYP3A5 incubation. The IC50 shift was derived by calculating ratios of IC50 obtained from pre-incubation with and without NADPH. IC50 shift ratio >2 implies irreversible time-dependent inhibition.In mechanism-based or time-dependent inhibition, the master premix was added with NADP50 values, the remaining enzyme activity of each cathine concentration was divided by the solvent control well (without cathine but replaced by water) and multiplied the value with 100% to obtain the percent control activity (%) for each cathine concentration. The resulting percent control activity (%) were plotted against different concentrations of cathine to attain the half maximal inhibitory concentrations (IC50) curve. IC50 values of reversible and irreversible inhibition were determined by non-linear regression analysis using GraphPad Prism 9 for Windows . Data for inhibition constant (Ki) analysis and mode of inhibition were evaluated using Excel spreadsheet  and Lineweaver-Burk plots. The secondary plots of cathine concentrations against slopes of Lineweaver-Burk plots were plotted to determine the Ki values. All assays were carried out in triplicate and stated as mean ± SD.To determine IC Cathine conformations were generated using Open Babel GUI v2.3.1. The protein was prepared using AutoDock Tools 1.5.6 while AutoDock 4.2 software was used to carry out molecular docking. AutoGrid was used to create the grid box around co-crystallized ligand covering the active site of CYPs, which included the haem group. The grid size for specifying the search space was set at 60 × 60 × 60 Å (for CYP3A4) and 70 × 70 × 70 Å (for CYP2A6) with a default grid point spacing of 0.375 Å with a total of 10 docking runs. Docking simulations were performed using the Lamarckian genetic algorithm with 25 × 105 energy evaluations and 27 000 iterations per run. The best configuration and possible ligand binding was then determined based on the binding score. After docking, analysis of the ligand interactions at the binding site was performed and visualized using the PyMOL Molecular Graphics system .The crystal structures of human cytochrome P450 CYP2A6 (PDB code: 2FDV) and CYP3A4 (PDB code: 4D75) were retrieved from Protein Data Bank.R2 values are obtained: (i) blue (3-cyano-7-hydroxycoumarin) standard with Buffer I, II and III, y = 0.0083x + 0.012, R2 = 0.9995, y = 0.0063x + 0.0132, R2 = 0.9983, y = 0.0093x + 0.0149, R2 = 0.998, respectively, (ii) cyan (7-hydroxy-4-trifluoromethylcoumarin) standard with Buffer II, y = 0.0003x + 0.0005, R2 = 0.9811 and (iii) green (fluorescein) standard to obtain the standard equations and R2 values, y = 0.0051x + 0.0029, R2 = 0.9978. Linear ranges of CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C19, CYP2E1 and CYP2J2 were from 0 – 60 minutes except 0 – 120 minutes for CYP3A5, and therefore, the incubation time was 1 hour for all CYP isoforms mentioned except 2 hours for CYP3A5 as determined in previous studies.Standard curves were plotted using fluorescence readings (RFU) against standard concentrations, and the following standard equations and 50 values of 80 and 90 μM as shown in non-linear graphs . A study by Bedada et al using human volunteers on one week of daily khat use (400 g) showed khat significantly inhibited CYP2D6, marginally inhibited CYP3A4, CYP2C19 and CYP1A2. An in vivo study using rats found that rats treated with both khat plus clopidogrel had significantly lower clopidogrel metabolite produced due to inhibition of CYP enzyme by khat. These studies supported that khat inhibited CYP activities but did not make any investigations on the specific compounds in khat that contributed to the CYPs inhibition. Therefore, it is plain to see that ingredients other than cathinone such as cathine and norephedrine, for instance, could be involved in the CYP inhibitory effects of khat.With regard to herb-drug interaction via CYP inhibition by khat constituents, our previous study found that khat significantly inhibited CYP2D6, CYP2C9 and CYP3A4 while cathinone showed negligible inhibitory effects.50 values  inhibition on CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP2J2, CYP3A4 and CYP3A5 . Cathine0 values  of 80 anurk plot . Non-comin vitro study, cathine, the compound which cathinone decomposes into after khat is harvested, appears to only significantly inhibit CYP2A6 and CYP3A4 out of the 11 CYP isoforms tested. CYP2A6 and CYP3A4 are abundantly expressed in the liver, with an expression rate of 3.8% and 12.0%, respectively. These CYPs played a major role in metabolizing various drugs. CYP2A6 metabolizes nicotine (the primary psychoactive component in cigarettes), cotinine, tegafur, letrozole, coumarin, methoxyflurane, efavirenz, valproic acid, pilocarpine, artemisinin, artesunate, caffeine, disulfiram, halothane, fadrozole and tyrosol,41 whereas CYP3A4 is responsible for 40% to 45% of all phase I metabolism and approximately 70% of gastrointestinal CYP activity beside being co-expressed with P-glycoprotein (PGP) in the liver and intestines.43 CYP3A4 metabolizes drugs such as paclitaxel, fentanyl, tamoxifen, tacrolimus and statins. Therefore, khat/cathine users who are on medications metabolized by CYP2A6 and CYP3A4 may experience herb-drug interactions or may fail to respond to some medications. Further human studies are warranted to investigate whether the inhibitory effects of khat/cathine on CYPs are clinically significant or otherwise.Based on our  The average content of cathine in 100 g of fresh khat leaves is 83-120 mg10. A 100-400 g of fresh khat leaves is consumed on a daily basis. Hence, assuming that khat users who took 400 g of fresh khat leaves would have consumed about 332-480 mg of cathine, therefore, an average 70 kg adult with 5 L blood would have absorbed approximately 66.4 × 10−3 to 96 ×10−3 mg/ml of cathine or in other words, about 351.7-511.5 μM. Despite the low Ki values determined in our study that ranges from 63 to 100 μM, these values may be clinically relevant since khat users commonly take huge amount of khat on a daily basis. Toennes et al reported that at a mean of 43.8 g of khat chewed, the mean ingested dose of cathine in milligrams was 32.4, while the absorbed proportion of cathine ranges from 16% to 84%. The mucosa of the oral cavity is the first absorption segment, where the major fraction of the cathine (84 ± 6%) is absorbed. Cathine could be detected in the urine up to 50-70 hours after ingestion which was about a day time more than cathinone at 22-26 hours. In addition, a study found that cathine concentration in oral fluid was higher than that of cathinone because khat users did not chew fresh leaves and cathinone easily metabolized into cathine. According to European Monitoring Centre for Drugs and Drug Addiction, hepatic first-pass metabolism of cathinone forms norephedrine and only 2% of cathinone is excreted in the urine (https://www.emcdda.europa.eu/publications/drug-profiles/khat). The elimination half-life of cathine (5.2 ± 3.4 hours) is longer than cathinone (1.5 ± 0.8 hours) (https://www.emcdda.europa.eu/publications/drug-profiles/khat). As compared to cathinone, cathine is less lipophilic and may also penetrate to the central nervous system. Nevertheless, lipophilicity of cathine should also be considered during prescribing by healthcare professionals to khat users because lipophilicity impacts cellular uptake and ADMET . High dosage accompanied by high lipophilicity compound is a detrimental combination. Although less lipophilic than cathinone, the lipophilicity of cathine may still affect metabolic activity as lipophilic compounds showed greater affinity to metabolic enzymes. Khat compounds’ lipophilicity need to be known during drug administration as they may readily cross the blood brain barrier.To our best knowledge, pharmacokinetic studies on cathine are very limited. Khat chewing releases cathine and cathinone which was subsequently absorbed by the mucous membranes of the oral cavity and followed by the stomach lining. Dried khat leaves showed an increase in cathine amount from 0.172% to 0.192% (fresh khat) to 0.184-0.198%. According to one case report, a male patient who suffered from haemorrhagic stroke with hypertension was found to have no chronic diseases history but instead daily khat chewing. The patient’s urine samples showed positive for amphetamine-like substance while liquid chromatography-tandem mass spectrometry (LC-MS/MS) detected presence of cathine and cathinone. The serum concentration of cathine was 100-fold of cathinone, suggesting that the patient’s symptoms are most likely due to cathine. The maximal plasma concentrations of cathine will be reached at 2.6 hours with a mean residence time (MRT) of 10.2 ± 2.6 hours, which was twice more than cathinone. Cathine retain in the human body for a longer period of time as compared to cathinone and could be detected in the urine samples even after approximately ≥3 days after ingestion. Since cathine is retained for a longer period of time within the human body, it may exert greater inhibitory effects on human CYPs and higher chances of causing khat-drug interactions as compared to cathinone. Nevertheless, the effects of cathine retaining within the body for a longer time have yet to be explored.However, in the early 1945-1961, there were contradicting debates by researchers that cathine has low potency and the amount of cathine in khat is insufficient to exert symptoms shown after consumption versus cathine was the only khat alkaloid responsible to give pharmacological significance.in vitro findings showed non-competitive mixed mode of inhibition, our molecular docking analysis provided a prediction of the interaction of cathine with the active site of CYP2A6 and CYP3A4 which may contribute to the mixed mode inhibition component. Intermolecular interactions namely hydrogen bonding and hydrophobic interactions were also previously established to be the chief determinants in determining the stability of energetically favoured ligands within the binding pocket. Cathine interacted with CYP2A6 via four hydrophobic interactions with Ile300, Thr305, Phe480, hydrogen bonding with Gly301 and Thr305 and single π-stacking with Phe107 and Phe118  more strongly than CYP3A4 (Ki = 100 μM). This conclusion is warranted by the binding energy value of the CYP2A6-cathine complex of −7.96 kcal mol−1 that exceeds the binding energy value of CYP3A4-cathine complex of −6.53 kcal mol−1. Non-polar or hydrophobic interactions are known to play a major role in contributing to binding forces and folding stability of the proteins or in other words the CYP enzyme while charged residues interacting with polar groups in the form of hydrogen bonds reinforce specificity. Both cathine-CYP complexes showed similar hydrogen bonds on one end of the cathine molecule . In CYP2A6, cathine overlaps very well with aldrithiol while ketoconazole is much bigger in size and has more interactions as compared to cathine in CYP3A4. In the active site of CYP2A6, aldrithiol formed hydrogen bonds with Asn297, π-stacking with Phe107, Phe111, and Phe118 and hydrophobic bonds with Phe209, Ile300, Gly301, Thr305 and Phe408. Ketoconazole formed π-stacking with Phe304, and strong ionic interaction with Fe2+ of the haem group of CYP3A4 and hydrophobic bonds with Phe57, Arg105, Ser119, Leu210, Phe241, Ile301, Ala305, Ala370, Arg372, Glu374, Gly481 and Leu482. Some similar interactions seen from our docking outcome such as (1) in CYP2A6, cathine and aldrithiol  both have a series of π-stacking interactions with Phe107, Phe118 and Phe480 to stabilize them, hydrophobic interactions with Thr305 and (2) in CYP3A4, cathine and ketoconazole both have π-stacking with Phe304 formed.We have performed docking using known inhibitors of CYP2A6 (PDB 2FDY) – aldrithiol and CYP3A4 (PDB 2V0M) – ketoconazole from Protein Data Bank to compare with cathine’s interactions. Cathine overlaps well with experimentally determined binding modes of aldrithiol and ketoconazole  of the substrate (e.g. cathine). Further studies should employ docking combined with molecular dynamics simulations to investigate effects of SOM flexibility of CYPs. The in vitro mixed mode inhibition results have raised another limitation of this study especially when mixed mode of inhibition suggests multiple possible binding modes but could not be predicted from our docking study. What is more, the allosteric sites of CYP enzymes are an area of active research, although locations among helices C, E and H have recently been proposed.The current study utilizes high-throughput fluorescence-based assays for detection of enzyme-drug interactions.in vitro and docking predictions are lacking in biotransformation capabilities, and the lack of in vivo biological mechanisms has probably stronger impact on the accuracy of toxicity evaluations as cathine inhibitory effects on CYPs were performed in vitro. This study answers some previous questions regarding the inhibitory effects of cathine on major human drug metabolizing CYP enzymes but also raises new questions to explore. As mentioned earlier that cathine is retained in the human body for a longer period of time as compared to cathinone, therefore future studies should consider effects of cathine on CYP1A1 that are present in large amount in gut cells, shedding light to enteric drug interaction potential. Research has also shown that (1R)(2S)-norephedrine was not the final product produced from the phenypropylamino alkaloids pathway in khat plant as claimed in earlier studies, but instead the metabolic pathway continues downstream of cathine to form oxazolidine derivates. Therefore, future studies are recommended to look into the inhibitory effects in vitro and in silico of other khat constituents including norephedrine and oxazolidine derivatives. This area of research remains widely unexplored, giving room for further research on the unique phytochemistry of khat to better understand the khat-drug interactions besides detecting possible similar phytoconstituents that are also present in other herbal products, thus indirectly helping in understanding therapeutic potential of other herbs as well. Future in vivo studies using human subjects and animal models are also warranted to look into the effect of specific khat contents, for instance, cathinone, cathine, norephedrine, merucathine, merucathinone, cathedulins and tannins.It is worth mentioning that in vitro inhibition determined as cathine inhibited CYP2A6 with Ki value of 63 μM which was stronger than CYP3A4 with Ki value of 100 μM. This deduction is justified by the binding energy value of the CYP2A6-cathine complex of −7.96 kcal mol−1 that exceeds the binding energy value of CYP3A4-cathine complex of −6.53 kcal mol−1. Additional π-stacking with nearby phenylalanine residues in CYP2A6 may explain why cathine showed more stable and stronger affinity on CYP2A6 than CYP3A4. In silico, in vitro and in vivo research on cathine are scarcely explored, and this study offers useful insights into the molecular interaction of cathine with CYPs besides directing future studies in the khat-drug interaction area. This study is a preliminary screening that must be validated by means of various approaches. Nevertheless, in view of the budding khat and cathinone derivatives’ use, knowledge on khat-drug, cathinone-drug or cathine-drug interactions are urgently needed. The current in vitro preliminary findings of this study require more diverse models  before a comprehensive exploration in clinical trials. The current study showed synergistic results from both in vitro and in silico docking evaluation of cathine as the inhibitor of CYPs enzyme activities. Our study proved that cathine exhibited negligible in vitro inhibitory effects on CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP2J2 and CYP3A5, and significant inhibition on CYP2A6 and CYP3A4. Despite this preliminary in vitro screening outcome, it would be best to recommend healthcare experts and patients to not co-administer khat or cathine-containing products with clinical drugs to minimize the possible cathine-drug interactions, especially those metabolized by CYP2A6 and CYP3A4.Cathine reversibly inhibited CYP2A6 and CYP3A4 activities. Docking results of CYP2A6 and CYP3A4 is aligned to the Click here for additional data file.In Vitro Inhibitory Effects of Cathine on 11 Major Human Drug Metabolising Cytochrome P450s by Sharoen Y. M. Lim, Jason Siau Ee Loo, Mustafa Alshagga, Mohammed A. Alshawsh, Chin E. Ong, and Yan Pan in International Journal of ToxicologySupplemental Material for Protein-Ligand Identification and


According to the ROC curves, IL-1beta may be considered an effective predictor of IgE-mediated FA in AD children . In the delayed-type FA group, the concentration of most cytokines had slightly decreased compared to the control group. The obtained results suggest that FA influences the Th17-related cytokine profile in the serum of AD children. More advanced studies are needed to confirm the involvement of Th17 cytokines in the allergic inflammatory response and to prove their usefulness in clinical practice.The main risk factor for the development of food allergies (FAs) in children is atopic dermatitis (AD). AD is usually recognized as the Th1/Th2 paradigm of allergic disease. Recently, the Th1/Th2 paradigm in allergy and autoimmunity has been revised, including the role of the Th17 cell population and related cytokines. However, there are only a few studies that have found Th17 cytokine involvement in the allergic inflammatory response, especially with food allergens. This research aimed to analyze the serum profile of cytokines involved in the T-helper cell type 17 immune response pathway in young, atopic children with an IgE-mediated and delayed-type FA. The study involved 76 children (0–5 years old) with chronic AD. We used the Bio-Plex system to simultaneously determine the concentrations of 15 different cytokines in one experiment. In accordance with complete dermatological and allergological examination, including OFC testing and ALEX2 assays, participants were divided into 3 groups: IgE-mediated FA, delayed-type FA, and the control group. Data were analyzed using univariate statistical tests. In the IgE-mediated FA group, the circulating levels of tested cytokines had increased compared with those of other patients; however, a statistically significant difference was only obtained for IL-1beta ( Allergic diseases constitute a huge challenge for contemporary medicine . The serThe main risk factor for the development of FA is atopic dermatitis (AD) ,21. AccoTherefore, this research aimed to characterize the serum profile of the cytokines involved in the T-helper cell type 17 immune response pathway in young, atopic children with IgE-mediated and delayed-type FA. Cytokines are important mediators of immunity, and their response due to an imbalance or deficiency in the cytokine network may largely determine immune disease susceptibility and severity . ResearcThe study was approved by the Bioethical Commission of Poznan University of Medical Sciences. All participants received an explanation of the main objectives and possible benefits of the study. Informed consent was obtained from all volunteers and, in the case of children, from their parents.The scope of the research covered the diagnosis of major FAs in children aged 0 to 5 years. The study involved 76 volunteers, in whom there was a likely association between chronic symptoms of AD (L20) and exacerbations with food exposure. In addition, some patients developed acute symptoms following food exposure in the form of urticaria (L50), angioedema (T78.3), and/or anaphylaxis (T78.0). The complete characteristics of the demographic profile of patients are presented in All participants underwent a detailed medical examination, and their parents were obligated to carefully fill in a questionnaire. The information contained in the questionnaire allowed for the inclusion/exclusion of patients from the study and helped to assign them to the appropriate group. The criteria for including participants in the study and the rationale for selecting the group of children aged 0 to 5 are presented in After the patients’ parents or legal guardians were acquainted with the nature of the research and consented to participate in the study, venous blood was collected from all children. The serum samples for testing were collected at the Individual Specialist Medical Practice of MD Joanna Matysiak in Kalisz, and then stored at −80 °C until analysis.A complete dermatological and allergological examination of the participants, including the examination of the sensitization to molecular components and extracted “whole” allergens, was performed using ALEX2 Allergy Explorer, Polycheck Allergy Diagnostics Multiparameter Technology (Pediatric 30-I) and singleplex ImmunoCAP testing. Based on the results of allergological in vitro tests, medical history, and oral food challenge tests (OFC), the participants were divided into target groups. The study group contained 36 young children suffering from AD and diagnosed with IgE-mediated FA , while the control group consisted of 40 atopic patients without IgE-mediated FA . In the next stage of the experiment, based on clinical symptoms and the OFC test, from the group of patients with negative sIgE results, we distinguished 15 children with delayed-type FA  and 25 participants without FA . The sera of all patients were subjected to Bio-Plex examination to characterize the pathophysiological conditions associated with IgE-mediated FA and delayed-type food hypersensitivity.We examined IgE-mediated FA in the Polish population of young children with chronic symptoms of AD, determining the concentration of IgE antibodies specific to 330 allergens, concerning both the extracted “whole” allergens and the molecular components. We used the Allergy Explorer ALEX 2 test : a macroarray containing 295 allergens, the Polycheck Pediatric 30-I test : a screening panel of the 30 most common pediatric allergens, and singleplex ImmunoCAP assays . In this experiment, the ImmunoCAP system was used to determine the level of IgE antibodies that were specific to 5 allergens: cow’s milk extract and its components, i.e., alpha-lactalbumin, beta-lactoglobulin, casein, and Bovine serum albumin.The positive results of the allergen-specific IgE assays were confirmed by the OFC test. Based on physical examination and clinical history, the patients were subjected to the OFC test selectively. Children with a history of anaphylaxis and detectable levels of IgE specific to a suspected food were excluded from being tested with the oral food challenge. The conducted analyzes allowed for the division of participants into appropriate groups .The principle of all in vitro analyzes was based on the ELISA method. The tested allergens were immobilized in a solid phase made of a 3D cellulose polymer. Cellulose-immobilized antigens reacted with specific antibodies present in the serum of patients. Unbound, free antibodies remained flushed away, thus preventing non-specific binding. Antibodies labeled with enzymes directed against the specific IgE were added sequentially. After the incubation step, the unbound p-sIgE antibodies were washed away. Then, an incubation step with the appropriate substrate was performed. The antibody-conjugated enzyme  was reacted with the colorless substrate to form a colored product. Finally, the degree of fluorescence of the enzymatic reaction product was measured. The color intensity/optical density was measured at 450 nm. The intensity of the color indicated the amount of the allergen-specific antibodies present .Diagnosis of delayed-type FA was based on observations of allergic symptoms after the intake of the suspected food. The most reliable clinical procedure for diagnosing IgE-mediated FA and non-IgE mediated FA is the oral food challenge test. The OFC comprises an oral administration of the suspected allergen in a controlled and standardized setting ,54.According to procedure, the suspected food allergen, e.g., cow’s milk protein, was completely excluded from the patient’s diet for a period of 4–6 weeks. Then, the parents of the patients were instructed in detail on how to reintroduce the previously excluded allergen into the patient’s diet. The diet extension continued until the allergy symptoms recurred or until the suspected food allergen was completely introduced, which confirmed or ruled out delayed-type FA. During the next follow-up visit, a detailed clinical interview was conducted, and a physical examination of the patient was performed. During the OFC, the suspected food allergen was administered daily, in gradually increasing doses.Children undergoing the OFC had no history of anaphylaxis, no detectable high levels of IgE specific to suspected foods, no conditions that might affect their safety, and were not taking drugs, which might interfere with the assessment or affect safety.Using the Bio-Plex Pro Human Th17 Cytokine Assay , one experiment evaluated a comprehensive mix of fifteen pro-inflammatory factors that are involved in the T-helper cell type 17 immune response pathway  according to the manufacturer’s instruction. The xMAP technology used in the study is based on three basic elements: fluorescently dyed microspheres/magnetic separation, a dedicated flow cytometer, and a high-speed digital signal processor. The kit consists of ready-to-use reagents, a 96-well plate, standards, and quality controls recommended for the assay. The Bio-Plex assay is essentially an immunoassay formatted on magnetic beads. The assay principle is similar to that of a sandwich ELISA. In simple terms, 50 µL of serum, standards, and quality control samples were added to consecutive wells containing antibody-conjugated magnetic beads, and then the mixture was incubated for one hour at room temperature. After the incubation and washing steps were completed, the biotinylated detection antibody was added to the magnetic beads to form a sandwich complex. The final reaction mixture was obtained by adding the fluorescent streptavidin-phycoerythrin conjugate. Cytokine concentrations were determined by flow cytometry using a Bio-Plex Matrix Reader , which is equipped with two LEDs; the first emits red light with a wavelength of 635 nm, while the light emitted by the second diode is green with a wavelength of 532 nm. Data acquisition was performed using Bio Plex Manager 6.0 software . Before the final analysis, the software was verified and fully calibrated. The standard curve was designed based on the standards provided by the manufacturer. The concentrations of the tested factors were presented in picograms per milliliter (pg/mL) based on the standard curves. Two of the ninety-six wells were filled with Bio-Rad diluents and considered as blank for the experiment. In order to verify the correctness of the analysis, quality controls (low and high level) were used.p < 0.05 was considered statistically significant. Data were analyzed using univariate statistical tests. Depending on the type of data distribution between the study group and the control group, the obtained values were compared using the Mann-Whitney test, the Student’s t-test or the Welch test. The Shapiro-Wilk test was used to check the normality of the distribution of the results. Variables with a normal distribution had their equality of variance tested with Levene’s test, while variables without a normal distribution were compared using the Mann-Whitney U test. If Levene’s test results were not statistically significant (p > 0.05), a Student’s t-test was conducted. The Welch test was applied if Levene’s test results were statistically significant (p < 0.05). Additionally, the Kruskal-Wallis one-way ANOVA test was used to compare the data between the three groups. A univariate receiver operating characteristic (ROC) curve was calculated using the MedCalc software . For each tested analyte, a standard univariate ROC curve was calculated to graphically present the correlation between sensitivity and specificity.Statistica 13.0  and MedCalc  were used to conduct statistical analyzes. A numerical value of The adoption of a novel methodology based on the Luminex/xMAP technology allowed for the simultaneous determination of several markers of the inflammatory process in the serum during one experiment. We successfully measured the serum concentrations of nine out of the fifteen determined cytokines  from seventy-five out of the seventy-six patients. The values for the six analyzed proteins were below the detection level for all analyzed samples or were only detected in parts of the samples. These cytokines were excluded from the data analysis. Finally, the serum concentrations of the nine cytokines from seventy-five patients were statistically analyzed.The concentrations of inflammatory factors in the serum of 35 patients with IgE-mediated hypersensitivity ) and 40 patients without IgE-mediated FA (control group (0)) were measured. The determined concentration values are presented in p < 0.05) compared to that in the control group. Similar differences were observed in the levels of other inflammatory factors between the study groups, although these differences were not statistically significant.The inflammation profile of the AD patients diagnosed with IgE-mediated hypersensitivity ) was compared to those without IgE-mediated FA (control group (0)) using a U Mann-Whitney test. The results of the statistical analysis are presented in In the next stage of the experiment, based on the results of allergological in vitro examination, medical history, clinical symptoms and OFC testing, all participants were divided into three groups: the IgE-mediated FA (35 patients); the delayed-type FA (15 patients); the control group (25 participants). The measured cytokine concentrations for each group are shown in p < 0.05) higher in the IgE-mediated allergy group when compared to the other groups. In turn, among patients with delayed-type hypersensitivity, the level of interleukin 1 beta was significantly  higher when compared to the control group and significantly (p < 0.05) lower in comparison with IgE-mediated allergy patients. More detailed results are presented in To compare the results between the three groups, a Kruskal-Wallis one-way ANOVA was performed. Statistically significant differences occurred in the concentration of one inflammatory marker. The concentration of IL-1beta was significantly   .p < 0.05). Among patients with delayed-type hypersensitivity, the concentrations of most cytokines were slightly lower, while the level of interleukin 1 beta was significantly (p < 0.05) higher compared to the control group and significantly (p < 0.05) lower in comparison with IgE-mediated allergy patients. Based on the univariate ROC curve, which graphically shows the sensitivity and specificity of the analyzed marker, interleukin 1 beta can be considered a prognostic factor for IgE-mediated FA.In this study, we focused on the analysis of the serum profile of inflammatory factors in young children with food allergy (FA) and chronic symptoms of atopic dermatitis (AD). We successfully measured the concentration of nine cytokines involved in the T-helper cell type 17 immune response pathway: IL-1 beta, IL-4, IL-17A, IL-22, IL-23, IL-25, IL-31, sCD40L, and TNF-alpha. In one experiment, we examined several serum inflammatory markers using the Bio-Plex method (Bio-Rad). The Bio-Plex system utilizes xMAP technology to enable the multiplexing of up to 100 different analytes . Multiplp < 0.05) elevated levels of IL-1 beta in AD patients diagnosed with IgE-mediated FA compared to AD patients with delayed FA and AD participants without FA. We also observed a slightly increased concentration of this interleukin in the group of atopic patients with non-IgE-mediated allergy in comparison with the atopic children without allergies. According to our data, elevated levels of IL-1 beta in atopic children diagnosed with IgE-mediated FA suggest its involvement in the development of an allergic inflammatory response to food allergens, which may lead to an exacerbation of AD. There is a hypothesis that states that exposure to the allergen through the skin causes the release of IL-1 beta from damaged tissues, contributing to the production of Th2 cells [Interleukin-1 beta (IL-1 beta) is a highly pro-inflammatory cytokine expressed in antigen-presenting cells (APC) and was discovered and first described as a lymphocyte activating factor ,60 or leh2 cells . Assessmh2 cells . At the h2 cells . Cheungih2 cells . These fh2 cells . Eosinoph2 cells ,82,83,84h2 cells . In patih2 cells . On the h2 cells ,42,46. Th2 cells . The indh2 cells . On the h2 cells . Based oIn the development of allergic disorders, a special role is ascribed to immunological processes. In the future, the inhibition of the activity of pro-inflammatory cytokines may be helpful in treating children with chronic AD and IgE-mediated FAs. Nowadays, some inflammatory diseases are treated by blocking IL-1 beta . IL-1 beIn this study, we observed elevated levels of the remaining eight cytokines  in the serum of patients with IgE-mediated hypersensitivity in comparison to other participants; however, the obtained differences were not statistically significant. According to data from the literature, most of these inflammatory factors are involved in the course of allergic conditions . IL-4 anThis is the first study to use Bio-Plex to determine the serum concentration of multiple inflammatory cytokines involved in the T-helper cell type 17 immune response pathway in atopic children with food hypersensitivity, making it impossible to accurately compare the results with other researchers. We assume that the identification of changes in serum concentration  constitutes the basis for further clarification and differentiation of the molecular mechanisms of FAs. Currently, there is no specific biological indicator for IgE-mediated and delayed-type FA in patients with chronic symptoms of AD. According to the literature, total serum IgE level is not a specific marker of IgE-mediated allergies . A breakIn the future, identification of the presence of both Th17 cells and Th17-related cytokines in the course of FA and AD may reveal a common effector pathway for these diseases. AD appears to precede and trigger the development of FA in young children . NeverthCytokines are critical in allergic intercellular communication networks and contribute to disease pathology by recruiting and activating pro-inflammatory leukocytes. According to our results, cytokines involved in the T-helper cell type 17 immune response pathway can contribute to the pathogenesis of classically recognized Th2-mediated FA. Increased serum concentrations of IL-1 beta may suggest the development of IgE-mediated FA in young children suffering from chronic AD without additional comorbidities. In the future, there is a great need to investigate the involvement of both Th17 cells and Th17-related cytokines in the allergic inflammatory response and demonstrate their usefulness in clinical practice. Our results are the basis for further, more advanced analysis of allergy mechanisms and the search for new therapeutic targets and markers, differentiating the types of food hypersensitivity at the proteomic level.


Idiopathic generalized epilepsy (IGE) was characterized by 3–6 Hz generalized spike-wave discharges (GSWDs), and extensive altered interactions in subcortical-cortical circuit. However, the dynamics and the causal relationship among these interactions were less studied. Using resting-state functional magnetic resonance imaging (fMRI) data, the abnormal connections in the subcortical-cortical pathway in IGE were examined. Then, we proposed a novel method of granger causal analysis based on the dynamic functional connectivity, and the predictive effects among these abnormal connections were calculated. The results showed that the thalamus, and precuneus were key regions representing abnormal functional network connectivity (FNC) in the subcortical-cortical circuit. Moreover, the connectivity between precuneus and adjacent regions had a causal effect on the widespread dysfunction of the thalamocortical circuit. In addition, the connection between the striatum and thalamus indicated the modulation role on the cortical connection in epilepsy. These results described the causality of the widespread abnormality of the subcortical-cortical circuit in IGE in terms of the dynamics of functional connections, which provided additional evidence for understanding the potential modulation pattern of the abnormal epileptic pathway. Idiopathic generalized epilepsy (IGE) was typically characterized by 3–6 Hz generalized spike-wave discharges (GSWDs). The mainstream concept believed that the burst of GSWD was related to the inter-regional interactions in the subcortical-cortical circuit , 2005, iRegarding the origin of generalized epileptic discharge, previous studies have proposed some different discharge model hypotheses, with two representative concepts of the cortical focus theory and the thalamic pacemaker theory. The hypothesis of cortical focus theory assumed that epileptic discharges originated in a certain local area of the cortex and then spread through the interaction of subcortical structures with the cortex, thus recruiting specific neuronal networks into typical oscillatory behavior . Under tOther structures under the cortex, such as the striatum and cerebellum, also played an important role in regulating epilepsy. Some animal studies have shown that the striatum, whose fibers connected to the substantia nigra reticulum, was involved in the regulation of epileptic discharges . PreviouIn this study, we investigated the altered subcortical-cortical pathway of IGE, and the causality relationship among these abnormal connections was examined using the dynamic functional of the resting-state network. The dynamic interactions within the abnormal epileptic networks may provide evidence for the origin and modulation of epileptic activity.In total, seventy-eight patients with IGE  including 32 patients with juvenile myoclonic epilepsy (JME) and 46 patients with generalized tonic–clonic seizures (GTCSs) were recruited in this study. Diagnosis and classification was made by neurologists in accordance with the International League Against Epilepsy (ILAE) guidelines . RoutineIn this study, the MRI data of all the participants were recorded from the 3T MRI scanner  with an eight channel-phased array head coil in UESTC. Resting-state fMRI data were acquired using a gradient-echo echo planar imaging sequences . A total of 255 volumes were collected in the resting-state scan for each participant. During fMRI scanning, all the participants were instructed to keep still and close their eyes without sleeping. In addition, high-resolution T1-weighted images were also acquired using a 3D fast spoiled gradient echo (T1-3D FSPGR) sequence .1 and NIT toolboxes2 , and also cerebellum network, each of network consisting of a few components, that is the ROIs. The MNI coordinates for the selected ROIs were shown in Spatial ICA was performed to the group fMRI, and the functional networks identified by the ICs were obtained. Here, the number of ICs was 100 and determined according to the previous studies. In ICASSO,min = 0.01 Hz). Then, the variation of the weights for the dynamic FNC in the time course was calculated, representing the dynamics of the connections in the subcortical–cortical circuit.The static FNC and dynamic FNC was constructed among ROIs. Static FNC was computed by the Pearson’s correlation of the time courses between any two selected components. Based on the static FNC, we calculated the degree of FNC nodes by summarizing the weights of the connections of each node. Dynamic FNC was also computed using correlations between windowed time-courses of different ROIs. Here, the length of the slide window was 50TR, with the sliding step width 1TR. The selection of the window length was constrained by the minimum frequency of the BOLD signal .Finally, According to the previous studies, we selected the components in DMN, SMN, BGN, SN, and cerebellum from 100 ICs obtained in ICA analysis. Thus, each network was constituted of a few components, i.e., the ROIs. Here, DMN included the frontal_sup_medial area, the angular, cingulum_post (PCC), and two precuneus components. SMN included bilateral post-central areas, pre-central, paracentral_lobule, and supp_motor_area (SMA). BGN was comprised of the thalamus, putamen, and caudate. SN involved the left and right frontal_mid area, the cingulum_ant (ACC), cingulum_mid (MCC), and insula. The cerebellum network included the cerebellum posterior lobe and the declive. The maps of these five networks were illustrated in t-test was conducted to the static FNC, and the difference of FNC between IGE and HC was shown in P < 0.001). In between-group comparisons, age and gender were regressed out as the covariates. The results showed that thalamus, precuneus were the key nodes representing significant alteration in the subcortical–cortical circuit in epilepsy. Increased connectivity between thalamus and striatum, as well as the insula, and increased connectivity between precuneus and motor area was found in IGE comparing to HC. Decreased connectivity between the thalamus and frontal area, between precuneus and the frontal area, and also the thalamus/striatum was found in IGE. In addition, striatum showed increased connectivity with insula and cerebellum in epilepsy brain. Moreover, we compared the degree of FNC nodes between IGE and HC, and the thalamus, precuneus, ACC and post-central areas had decreased static FNC in IGE (P < 0.05)  .Besides, comparison of static FNC was also performed between two epilepsy groups, i.e., JME and GTCS. Comparing to GTCS, JME showed decreased connectivity in thalamus-precuneus, and thalamus-frontal connections. The results may be related to the longer epilepsy duration of these patients with JME in this study.P < 0.05) (P < 0.01) were shown in After the abnormal connections of static FNC in the epilepsy group were labeled, the dynamic changes of these connections were extracted according to the weights of the dynamic FNC matrices. The variation of the dynamic FNC showed that the connections of thalamus-frontal_mid, thalamus-precuneus, precuneus-frontal_mid, and frontal_mid-post_central had increased dynamics in IGE ( < 0.05) . Then, tTo sum up, patients with epilepsy had increased cerebellum-striatal-thalamic connections and widespread decrease in thalamocortical circuits. The causal relationship between these abnormal connections was summarized in This study investigated the subcortical–cortical pathway in IGE, and showed that thalamus, precuneus were key nodes representing abnormal functional connectivity in subcortical–cortical circuit. Furthermore, the causality relationship among these abnormal connections in IGE was examined. It was shown that the increased connections between precuneus and the adjacent regions had the causal effect on the widespread decreased connections in thalamocortical circuit. In addition, the two-way causal effect of the connection between the thalamus and the striatum on the cortical connection indicated the modulation role of subcortical circuit in epilepsy.Numerous studies have shown that there were abnormalities in a wide range of functional connections in IGE . AlteredAccording to the hypothesis of generalized epileptic discharges , the epiIn one of our previous study, we detected the synchronous network during the stage of epileptic discharges, and found the connectivity within frontal areas and between frontal–parietal areas was the main network pattern after the discharge onset . Also, eTo re-test the stability of the causal analysis, we also conducted the multivariate GCA to describe the directed causal effect from this seed connection to the other connections, as well as the reverse impact intensities from other connections to this seed connection. The results showed similar directed patterns with the bivariate GCA, including the predictive effect from precuneus-related connections to the thalamocortical connections, and then to the cortical–cortical connections. Moreover, much extensive effects were found from the striatum-thalamus to both the thalamocortical and the cortical–cortical connections. The results were shown in To sum up the results, we concluded the causal relationship among the abnormal functional connectivity in epileptic brain, which described the trigger role of precuneus with its extensive hyperconnectivity, and the widely distributed dysconnectivity of thalamocortical circuit, and also the following alteration of frontal and motor-related connectivity. This causality in epileptic circuit provided additional evidence for the theory of epileptic origin and propagation on the level of macro functional connectivity. However, the main limitation of this study is that the causal relationship of the abnormal connectivity was based on the resting-state data rather than the discharging state. Therefore, the result provided the potential modulation pattern in terms of the epileptic activity, and the inference of the epileptic propagation circuit in this study was urgently needed to test by the physical stimulation and clinical evaluation.This study investigated the subcortical–cortical pathway in IGE, and showed that thalamus, precuneus were key regions of the abnormal FNC in epileptic brain. Furthermore, the causality analysis among these abnormal connections demonstrated the predictive effect of precuneus on the widely distributed thalamocortical dysconnectivity. In addition, the increased connectivity between thalamus and the striatum indicated the modulation role of subcortical circuit on the cortical connection in epilepsy. These results provided additional evidence for the widespread abnormality in epilepsy brain based on the dynamics of functional connections, and the predictive relationship in the subcortical–cortical circuit enlightened the theory of epileptic origin and propagation.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Ethics Committee of the University of Electronic Science and Technology of China. The patients/participants provided their written informed consent to participate in this study.YQ, DY, and CL conceived and designed the work. YQ and SL acquired the data. YQ analyzed the data. YQ and CL wrote the manuscript. All authors revised the work for important intellectual content and read, and approved the manuscript.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.