The four benzo units blueshift the absorption spectrum in comparison to a solely linear nonacene, but significantly increase the stability in the solid state.In combination with bulky substituents at the core, fourfold benzannulation at the  Attachment of four benzo units electronically stabilizes a nonacene core without heteroatom substitution at/on the aromatic backbone. The four benzo units blueshift the absorption spectrum in comparison to a linear nonacene and significantly increase the stability in the solid state. Non. Other methods of stabilization allow a significant number of linearly annulated benzene rings but with starkly diminished acene character.4HepB, exhibiting hexacene‐like absorption maxima, the blueshift being a consequence of the diminished conjugation of the quadruply annulated acene core.4NonB, by employing a modification of Anthony's route.The higher acenes have been a magnificent challenge, accepted since Clar's first synthesis of pentacene.1 and an excess of p‐benzoquinone furnished 2 (Scheme 3 results in 4 (crude yield 87 %), fourfold ethynylation of which gave intermediate 5 (40 %) using a large excess of lithium acetylide (≈100 equiv). Reductive aromatization with SnCl2 furnishes tetrabenzononacene 4NonB. The concentration was adjusted to precipitate 4NonB during synthesis. 4NonB, unlike other higher acenes,Bis(bromomethyl)phenanthrene n‐hexane and MeOH on a THF solution of 4NonB under nitrogen; solvent molecules are included in the crystal packing but are heavily disordered and cannot be resolved. 4NonB crystallizes with two independent molecules per unit cell with a minor π–π interaction of two phenanthrenylenes. These molecules form 1D‐stacks and are oriented perpendicular to each other, effectively preventing dimerization but also pronounced π–π interactions in the solid state. The independent molecules’ average bond lengths of the innermost ring of the formal triphenylene units are elongated (1.44–1.47 Å) compared to that of the other aromatic C−C backbone bonds (1.38–1.46 Å).4NonB are in agreement with each other and the expected values.Single crystals were grown by subsequently layering 4NonB exhibits structured, finger‐like absorption bands with a lowest energy absorption maximum at 958 nm  due to the absence of electron‐withdrawing fluorine substituents.Non‐fluorescent m Figure , Table 14NonB is stable for more than 6 weeks under nitrogen in the solid state, although its half‐life in n‐hexane solution is only 30 min under ambient conditions (see SI) and 7 h under nitrogen atmosphere ,Similar to most higher acenes,Non, 4NonB is less stable under air and nitrogen in solution  suggests the formation of dimeric and oligomeric species, both common degradation products for higher acenes.4Non′B. The system shows NICS(1) values in accord with expectations, with the formal inner triphenylene ring being the least aromatic one—“empty” in the Clar formalism—and the other rings displaying high aromaticity. This is further illustrated in FMO calculations (see SI), in which the central triphenylene rings show small coefficients in the outer rings compared to their nonacene congeners.Figure 4NonB, in which the stabilization is due to the attachment of four benzo units at the cata‐position of the acene unit. The material displays sharp NMR resonances and a slightly blue‐shifted absorption in comparison to Anthony's nonacene Non, yet it is only one of the very few isolated and structurally characterized nonacenes. 4NonB is highly stable in the solid state. Benzo‐wings therefore should allow stabilization of other, hitherto only moderately persistent, reactive aromatics.In conclusion, we have prepared a novel, reasonably stable tetrabenzononacene, https://www.ccdc.cam.ac.uk/services/structures?id=doi:10.1002/anie.201909614 (4NonB) contains the supplementary crystallographic data for this publication. These data can be obtained free of charge from The http://www.ccdc.cam.ac.uk/. The synthetic details for precursors and remaining materials can be found in the SI.CCDC 6: (Tri‐iso‐propylsilyl)acetylene  was dissolved in n‐hexane (20 mL) and cooled to 0 °C. n‐BuLi  was added dropwise and the mixture was stirred for 1 h at room temperature. 4  was added and the suspension was stirred for 3 d at room temperature. Sat. NHCl4 solution and DCM were added, the layers were separated and the aqueous layer was extracted with DCM. The combined organic layers were dried over MgSO4, filtered and the solvent was removed under reduced pressure. The crude product was subjected to column chromatography  to yield 6 as a yellow powder . Mp: >350 °C. 1H NMR  δ=9.41 , 8.80 , 8.70 , 8.47 , 8.29 , 8.18 , 7.71 , 3.43 , 1.08 , 0.97 ppm . 13C{1H} NMR  δ=140.7, 137.7, 136.8, 135.8, 132.5, 132.3, 130.5, 130.4, 130.3, 129.5, 128.1, 127.6, 124.6, 124.6, 124.1, 123.6, 122.8, 122.0, 109.4, 108.9, 104.6, 90.3, 85.7, 69.4, 18.5, 18.5, 18.5, 11.4, 11.3 ppm. IR (neat): ν (cm−1)=2942, 2863, 1464, 1335, 1274, 1141, 1042, 888, 750, 670. HRMS : m/z calcd for C114H116F12O4Si4: [M+H]+ 1891.7988, found: 1891.7974, correct isotope distribution.Synthesis of 4NonB: 6  was dissolved in MeCN/THF  in a glove box. Anhydrous SnCl2  was added and the mixture was stirred at room temperature overnight. The resulting precipitate was filtered, washed with MeCN and dried to give almost pure 4NonB (crude yield 72 %). The material can further be purified by washing it thoroughly with DCM to yield pure 4NonB . Suitable specimen for single crystal analysis were grown by subsequently layering n‐hexane and MeOH on a THF solution under nitrogen. Mp: >350 °C. 1H NMR  δ=10.83 , 9.72 , 8.95 , 8.76 , 8.61 , 8.49 , 7.64 , 1.26 ppm . The compound was not soluble enough for 13C{1H} NMR analysis. IR (neat): ν (cm−1)=2939, 2855, 1453, 1369, 1213, 1110, 1019, 882, 753, 506. HRMS : m/z calcd for C114H105F12Si4: [M+H]+ 1823.7879, found: 1823.7891, correct isotope distribution.Synthesis of The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


Ash gourd (76.51%), brinjal (72.48%), and snake gourd (66.82%) extracts were the most effective inhibitors of angiotensin-converting enzyme (ACE), an enzyme whose excessive activities have been associated with hypertension. Brinjal also had a significantly higher renin-inhibitory activity than the other vegetable extracts. We conclude that the vegetable extracts may have the ability to reduce enzyme activities that have been associated with hyperglycemia, hyperlipidemia, and hypertension.The aim of the study was to determine the in vitro enzyme inhibition activities of aqueous polyphenolic extracts of nine popular Bangladeshi vegetables, namely ash gourd, bitter gourd, brinjal, Indian spinach, kangkong, okra, ridge gourd, snake gourd, and stem amaranth. Polyphenolic glycosides were the major compounds present in the extracts. Inhibition of α-amylase (up to 100% at 1 mg/mL) was stronger than α-glucosidase inhibition (up to 70.78% at 10 mg/mL). The Indian spinach extract was the strongest inhibitor of pancreatic lipase activity (IC Several physiological disturbances can be attributed to the effect of metabolic syndrome, which is a condition characterized by disease conditions such as obesity, dyslipidemia, hyperglycemia, and hypertension . These mSeveral epidemiological and empirical studies have reported that consumption of fruits and vegetables containing polyphenol compounds play an important role in inhibiting carbohydrate-hydrolyzing enzymes such as α-amylase and α-glucosidase . α-amylaDigestion of dietary triglycerides occurs in the small intestine by a key enzyme called pancreatic lipase (PL) to release 2-monoglyceride and fatty acids, which are then absorbed. Hence, inhibition of PL (EC 3.1.1.3) activity is regarded as a major approach for obesity prevention and treatment ,10,11. AAnother reason for the development of cardiovascular diseases is hypertension, which causes arteriosclerosis, congestive heart failure, coronary heart disease, end-stage renal diseases, myocardial infarction, and stroke . The renDifferent in vitro and in vivo studies have observed that dietary phenolic compounds have many properties beneficial for maintaining human health . In partFresh vegetables, namely ash gourd (BINA CHALKUMRA 1), bitter gourd (BARI KOROLA 1), brinjal (BARI BEGUN 8), Indian spinach (BARI PUISHAK 1), kangkong (BARI GIMAKOLMI 1), okra (BARI DHEROSH 2), ridge gourd (BARI JHINGA 1), snake gourd (BARI CHICHINGA 1), and stem amaranth (BARI DATA 1), were collected from the Bangladesh Agricultural Research Institute (BARI), Gazipur, Bangladesh. The vegetables were dried in the oven at 40 °C, ground into powders, and then stored at −20 °C. Porcine pancreatic α-amylase, porcine PL, rat intestinal acetone powder, acarbose, orlistat, 4-nitrophenyl α-D-glucopyranoside (PNP), N-(3-[2-furyl]acryloyl)-phenylalanyl glycyl glycine (FAPGG), and rabbit lung ACE were purchased from Sigma-Aldrich . A human recombinant renin inhibitor screening assay kit was purchased from Cayman Chemicals . Other analytical grade chemical reagents were obtained from Fisher Scientific Company .g, and the supernatant passed through a muslin cloth to collect filtrate #1. The residue was mixed with water (1:20) and the extraction, centrifugation, and filtration processes repeated to collect filtrate #2, which was then combined with filtrate #1. The combined filtrate was partially evaporated in a rotary evaporator  under vacuum (~24 mm Hg) at 60 °C, and the aqueous residue freeze-dried and stored at −20 °C.Water-soluble free (unbound) polyphenols were extracted using a previous method  with minm/z. The MS/MS was performed at a narrow quadruple setting (1.3 atomic mass units), using 10, 20, and 40 eV collision energy and 30–1700 m/z. The compounds were identified using MS/MS fragmentation patterns and quantified based on the MS peak area.An Agilent 1290 UHPLC system  coupled with an HSS T3 2.1 × 100 mm 1.7 µm column from Waters Corp  was used to perform UHPLC analysis of the vegetable extracts. Samples were mixed with distilled water, vortexed, and passed through a 0.2 µm filter. Then, a 5 µL portion of the filtrate was injected onto the column followed by elution at a flow rate of 0.5 mL/min using mobile phases A and B  at 40 °C. The following gradients were used: initial holding time 0.5 min, mobile phase B ramped up to 50% after 5 min, 95% after 6 min, held for 1 min, and re-equilibrated for 1.5 min. Compounds were identified using a diode array detector at a wavelength range of 230–640 nm in 2 nm increments and a frequency of 5 Hz. The mass spec was carried out in an Agilent 6550 QTOF  at 200 °C, using a drying gas pressure of 18 psi, 40 psi nebulizer and 350 °C sheath gas, a pressure of 12 psi, and 3500 V capillary with a 1000 V nozzle, and ran in positive ion electrospray at a frequency of 3Hz and acquisition from 30–1700 α-amylase inhibition assay was carried out using a previous method  with sliw/v) NaCl solution and centrifuged at 5600× g for 30 min, and the supernatant was used as the source of α-glucosidase activity. Plant extracts were dissolved  in 0.1 M sodium phosphate buffer pH 6.9 and 50 µL mixed with 50 µL of the α-glucosidase solution in a 96-well microplate followed by incubation for 10 min at 37 °C. Then, 100 µL 5 mM (PNP) also dissolved in the phosphate buffer were added to each well and the absorbance read at 405 nm in 30 s intervals for 30 min using the Synergy™ H4 microplate reader with temperature maintained at 37 °C. A blank measurement was taken without the addition of the enzyme, and its absorbance was subtracted from each well. Acarbose, an α-glucosidase inhibitor, was used as standard and assayed using the same protocol. The following equation was used to determine the α-glucosidase inhibitory activity of the samples:α-glucosidase inhibitory activity of the samples was determined according to a previously described method  with the2 and 25 µL added to the mixture containing 225 µL of a 0.5 mM 4-MUO solution and 25 µL of sample (different concentrations) to start the enzyme reaction, followed by incubation for 1 h at 37 °C. The Synergy™ H4 microplate reader was set at 400 nm and used to measure the amount of 4MU released during the reaction. Orlistat, a commonly used pharmacological agent against PL, was used as the standard.The method described by Tang et al.  with sliThe following equation was used to calculate PL inhibition:The method described by Alashi et al.  was usedThe renin-inhibitory activity of samples was determined using a previously described method . Brieflyp < 0.05) between mean values were determined by the Duncan’s multiple range tests. The IBM SPSS statistical package  was used for all statistical analyses.A minimum of duplicate assays were used to find out the mean values and standard deviations. For statistical analysis, analysis of variance  was used, while significant differences (The major polyphenolic compounds identified in the vegetable extracts are shown in p < 0.05) better than the other extracts. The okra (40.82% ± 1.88%) and stem amaranth (36.84% ± 0.49%) showed the lowest levels of α-amylase inhibition, respectively, at 1 mg/mL.The inhibitory activity of α-amylase obtained in this study by the phenolic extracts was mostly dose-dependent, although at higher concentrations than acarbose, the standard compound . HoweverA mostly dose-dependent effect was also observed as the inhibitory activity of α-glucosidase increased with increasing concentration for almost all the samples . However50 calculation as shown in 50 values indicate stronger inhibitory activity, the results obtained show that Indian spinach (276.77 ± 4.95 µg/mL) was the most active with a significantly (p < 0.05) lower value than orlistat. Brinjal (397.22 ± 2.36 µg/mL), okra (427.94 ± 1.40 µg/mL), and kangkong (482.04 ± 0.67 µg/mL) also had strong activities, although lower than orlistat (381.16 µg/mL). It is also noticeable that the stem amaranth extract had the weakest PL inhibition (IC50 = 723.394 ± 2.36 µg/mL), just as it showed the lowest inhibitions of α-amylase and α-glucosidase inhibitory activities.PL inhibition was strong for all the vegetable extracts, which enabled ICp < 0.05) weaker inhibition than captopril, the ACE-inhibitory drug. It is also observable that ash gourd also showed a good record for inhibiting α-amylase and α-glucosidase. Stem amaranth showed the lowest activity for all the concentrations and the lowest ACE inhibition, which is consistent with the observed poor inhibitory activities against PL, amylase, and glucosidase.Different levels of ACE inhibition were obtained for the vegetable extracts but, in general, the ash gourd (76.51% ± 0.25%), brinjal (72.48% ± 0.02%), and snake gourd (66.82% ± 0.99%) were the most active . All thep > 0.05) different from the stem amaranth with no detectable renin inhibition.The highest renin inhibitory activity was exhibited by brinjal (79.64% ± 7.63%) at 0.5 mg/mL as compared to 99.11% ± 1.75% for aliskiren (drug) at 0.05 mg/mL . The snaα-amylase inhibition by bitter gourd extract [UHPLC MS/MS analysis is considered an effective analysis method because it provides high selectivity, high sensitivity, and the potentiality for robust and accurate analysis identification of compounds . As show extract . The dec extract . The hig extract . The ass extract ,32.α-glucosidase is another enzyme that makes glucose available in the body by breaking down oligo- and disaccharides, converting them into absorbable monosaccharides. Therefore, in order to reduce serum glucose levels and manage related diseases, α-glucosidase inhibition has been proposed as a suitable approach ,34. ThisLipase is secreted in the pancreas and is used as a catalyst to aid triglyceride hydrolysis in the stomach, and this hydrolysis is completed by intestinal lipase in the small intestine ,37. It hACE is responsible for the formation of angiotensin II, a powerful vasoconstrictor; excessive physiological levels of angiotensin II lead to hypertension . Therefo50 value of 59.9 µg/mL [Renin inhibition has also been shown to be an effective means of reducing blood pressure because it catalyzes the rate-determining step (conversion of angiotensinogen to angiotensin I) in the RAS pathway ,46. Howe.9 µg/mL . The higResults from this work have shown that the aqueous extracts of vegetables could inhibit enzyme activities with respect to obesity, diabetes, and hypertension. The extracts were more effective inhibitors of α-amylase than α-glucosidase, which suggests a strong potential to limit excessive glucose release during digestion because starch hydrolysis is the rate-limiting step. Ability of the extracts to also inhibit PL suggests potential use as agents to limit the release of fatty acids during intestinal digestion, which could assist in reducing hyperlipidemia. Inhibition of ACE and renin activities are indications of potential blood pressure-reducing ability, which could also make the extracts function as antihypertensive agents. Overall, Indian spinach and brinjal extracts produced the most promising inhibitory effects on the five enzymes studied in this work, whereas stem amaranth extract was the poorest. However, in vivo determination of enzyme inhibitory activities using suitable animal disease models is required to confirm potential health benefits.


The intervention included sixteen exercise sessions/four weeks with a 30-min LIE group, and eight exercise sessions/four weeks with a 1-h LIE group. Heart rate (HR) corresponding to <2 mmol∙L−1 blood lactate (La−) was controlled for LIE. Pre- and post-testing was conducted before and after 4-week LIE and tests included jogging/running speed (S), HR, and differences  in HR and S between pre- and post-testing at 1.5, 2.0, and 4.0 mmol∙L−1 La−. Only the HR at 2.0 mmol∙L−1 La− of the 30-min LIE group was decreased in the post-test compared to the pre-test (p = 0.043). The jogging/running speed of the 1-h LIE group was improved in the post-test compared to the pre-test . ∆HR at 2.0 and ∆S between the 30-min and 1-h LIE group at 1.5, 2.0, and 4.0 mmol∙L−1 La− were significantly different . Furthermore, moderate to high positive correlations between ∆HR and ∆S of all subjects at 1.5 (r = 0.77), 2.0 (r = 0.77), and 4.0 (r = 0.64) mmol∙L−1 La− were observed. The 1-h LIE group showed improved endurance not only in the low-intensity exercise domain, but also in the beginning of the moderate to high-intensity exercise domain while the 30-min LIE group was not affected by the 4-week LIE intervention. Therefore, LIE (<2.0 mmol∙L−1) for at least 1-h, twice a week, for 4 weeks is suggested to improve recovery ability in adults.Exercise is recommended to increase physical health and performance. However, it is unclear how low-intensity exercise (LIE) of different durations may affect or improve recovery ability. This study aimed to investigate how LIE-duration with the same volume affects recovery ability in adults. Twenty healthy male adults participated in this study. Participants were randomly assigned to the 30-min ( Sport2max) . Indeed, setting . −1) were developed and refined by several groups [Validated LT concepts such as anaerobic, aerobic–anaerobic threshold, maximal lactate steady state (MLSS), and the onset of blood lactate accumulation , moderate-intensity , and high-intensity exercise  [Nowadays, the blood lactate level of the LT test is used to differentiate exercise intensity areas, which include low-intensity  ,18,19,20 3; HIE) ,22,23. D 3; HIE) ,25. Ther 3; HIE) ,25. Prev 3; HIE) . −1) and LIE is commonly interpreted as recovery exercise zone [2max and 50%–60% of maximal heart rate) needs to be performed 4–6 h per week over 12 weeks in obese men, which improved fat metabolism [In comparison to HIE and MIE, fat oxidation is the predominant energy source used in LIE  impacts recovery ability in the general population because there are relatively fewer studies regarding LIE. Furthermore, previous studies have conducted different low-intensity exercises based on the percentages of estimated HRmax and VO2max. Therefore, the aim of this study was to investigate, using the LT test, how different exercise duration (30 min vs. 1 h) with the same exercise volume (4 weeks) affects recovery ability in adults.However, based on the aforementioned scientific outcomes, it is unclear as to how the duration and frequency (volume) of individualized LIE (<2 mmol·Ln = 20) participated in this study and they were recruited from regional fitness centers through official research notifications. Subjects were randomized to separate 30-min (n = 10) and 1-h LIE groups (n = 10). The anthropometric parameters of the subjects were as follows (mean ± SD): 30-min LIE group, age: 28.3 ± 2.0 years, height: 173.5 ± 4.4 cm, weight: 75.9 ± 9.9 kg, fat: 16.3 ± 3.9%, BMI: 25.1 ± 2.2 kg∙m2; 1-h LIE group, age: 28.8 ± 6.9 years, height: 173.4 ± 3.9 cm, weight: 75.4 ± 8.9 kg, fat: 17.7 ± 5.9%, BMI: 25.0 ± 2.3 kg∙m2  was controlled by HR based on the LT test at the pre-test [To achieve the same exercise volume in the different intervention groups, the four weeks consisted of sixteen exercise sessions of 30-min LIE or eight exercise sessions of 1-h LIE. Four-week exercise interventions were selected based on previous studies and in order to study the effects of maintaining the same exercise volume ,19,37,38pre-test ,37,39. Ppre-test   were estimated using a mathematical model of the interpolation that has previously been explained in detail [−1 La− between pre- and post-testing were calculated . The HR of all subjects was recorded using HR telemetry . The mean value of HR over the last 30 s of each stage was determined for statistical analysis. Capillary blood sampling for lactate analysis was taken from the earlobe (0.2 µL) immediately after each 5-min stage. All blood lactate levels were determined by an enzymatic-amperometric method  [The pre-test subjects performed an incremental exercise on a treadmill (LT test), which consisted of 5-min stages with 30 s breaks between stages. The first stage was started at 1.0 m∙shaustion ,16,37. An detail ,42,43. DGermany) ,45.t-test was used to compare the pre-test and post-test within each group. Furthermore, an independent t-test was utilized to compare the differences between the 30-min LIE and 1-h LIE groups. Box and whisker plots indicate minimum to maximum and median values. Effect sizes  were calculated for parametric tests and thresholds for small, moderate, and large effects were 0.2, 0.5, and 0.8, respectively [p < 0.05 and p < 0.01. Additionally, a Pearson’s two-tailed correlation was performed between ∆S and ∆HR at 1.5, 2.0, and 4.0 mmol∙L−1 La−. All data were analyzed using GraphPad Prism 9.0 . Parameters are presented as mean and standard deviation (SD)/standard error of the mean (S.E.M). Normal distribution was performed using the Shapiro–Wilk test. A paired ectively . Differe−1 La− of the 30-min LIE group was decreased in the post-test compared to the pre-test  (−1 La− were not affected by the 30-min LIE intervention (p > 0.05). Furthermore, the jogging/running speed at 1.5, 2.0, and 4.0 mmol∙L−1 La− showed no significant change between pre- and post-testing (p > 0.05). HR at 2.0 mmol∙LS: 0.66) . Other H−1 La− between the pre- and post-test (p > 0.05). However, the jogging/running speed of the 1-h LIE group at 1.5, 2.0, and 4.0 mmol∙L−1 La− was significantly increased post-intervention . Data are presented in There was no significant difference in HR at 1.5, 2.0, and 4.0 mmol∙L−1 La− showed no differences between short and long exercise duration (p > 0.05). However, jogging speed at 1.5 mmol∙L−1 La− of the 1-h LIE group was significantly higher compared to the 30-min LIE group   (Jogging/running speed and HR changes post-intervention at 2.0 and 4.0 mmol∙LS: 1.47) A. Delta ctively) .r = 0.77; 95% CI: 0.50–0.90; r2 = 0.59; p < 0.0001), 2.0 , and 4.0 mmol∙L−1 La−  were observed  influences the recovery ability in male adults. The major findings show that jogging/running speed at 1.5, 2.0, and 4.0 mmol∙L2.0 and ∆HR2.0 in the 30-min LIE group and between both groups, while no significant difference in jogging speed at 2 mmol∙L−1 La− was observed. HR levels of the 1-h LIE group were tended to increase with improved jogging/running speeds in the post-test, but no significant change was found. Indeed, our correlation analyses showed positive, moderate to high relationships between ∆HR and ∆S at certain lactate levels. , which resulted in increased left ventricular ejection, decreased blood pressure, and HRV interval [HR values showed only a decreased HR levels. . Increasanalyzed ,51,52. Hinterval ,52. In t−1 La− of the 30-min LIE group was not affected by the 4-week LIE intervention. In contrast, jogging/running speed at 1.5, 2.0, and 4.0 mmol∙L−1 La− of the 1-h LIE group in the post-test was significantly enhanced (see max in our study (max) for four weeks in cardiac patients could increase the running speed corresponding to 4 mmol∙L−1 La− by 5% [max; 30–60 min; eight weeks) caused a reduction in accumulated blood lactate and rate of blood lactate production during the graded exercise test by 70% and 57%, respectively [−1; zone 1) after three weeks resulted in improved time to exhaustion by 5% and increased jogging speed at 2.0 mmol∙L−1 La− by 9% [−1 La−, three days per week for 4 and 6 weeks. To improve endurance performance, the distribution of training intensity in world-class middle- and long-distance runners suggests over 87% of zone 1 (<2 mmol·L−1) per week from the general preparation phase [Jogging/running speed at 1.5, 2.0, and 4.0 mmol∙Lnced see . The recur study . Similarectively . Anothera− by 9% . Hommel a− by 9%  showed ton phase . −1 La− increased in the present study, which also indicates an improved rightward shift of the exponential lactate curve  are seen in different populations such as elite cyclists, moderately active male individuals, and individuals with metabolic syndrome [2max) [San-Millan et al.  and Yangsyndrome . Thus, te [2max) . Furthere [2max)  showed te [2max) ,25. Hence [2max) ,25. Howee [2max) ,30.Our findings indicate that 1-h LIE can improve fat oxidation and ATP re-synthesis, resulting in enhanced recovery ability and general aerobic endurance in adults. Further studies are expected to investigate how these metabolic changes are associated with 1-h LIE using other metabolic-related parameters such as respiratory exchange ratio and blood glucose.−1) for at least 1-h, twice a week, for 4 weeks is suggested to enhance recovery ability and endurance performance in adults. As well, LIE may be suitable not only for athletes but also pregnant women, exercise beginners, older adults and even cardiac patients, because of the very low propensity for muscle injury and cardiometabolic risk. Further studies are expected to investigate how the volume (duration and frequency) of LIE influences other specific populations such as older adults and cardiac patients.The findings of the present study indicate that the 1-h LIE group improved endurance, not only in the low-intensity exercise domain but also in the beginning of the moderate to high-intensity exercise zones . Thus, LIE (<2.0 mmol∙L


Mycobacterium avium subspecies paratuberculosis (MAP) in CD and Mycobacteria tuberculosis (MTB) continues to be under investigation. The role of toll-like-receptors (TLRs) and cytoplasmic adaptor protein (MyD88) in proinflammatory response during Mycobacterial infection has been suggested. However, the mechanism of how nicotine modulates macrophage response during infection in CD and exacerbates inflammatory response remain unclear. In this study, we elucidated the mechanistic role of nicotine in modulating MyD88-dependent/TLR pathway signaling in a macrophage system during mycobacterial infection. The data demonstrated that MAP infection in THP-1 derived macrophages was mediated through TLR2 and MyD88 leading to increase in IL-8 in expression and production. On the other hand, LPS-representing, Gram-negative bacteria mediated macrophage response through TLR4. Blocking TLR2 and TLR4 with antagonists voided the effect of MAP, and LPS, respectively in macrophages and reversed response with decrease in expression of iNOS, TNF-α and IL-8. Interestingly, nicotine in infected macrophages significantly (1) downregulated TLR2 and TLR4 expression, (2) activated MyD88, (3) increased M1/M2 ratio, and (4) increased expression and secretion of proinflammatory cytokines especially IL-8, as seen in CD smokers. We also discovered that blocking macrophages during MAP infection with MyD88 antagonist significantly decreased response which illustrates the key role for MyD88 during infection. Surprisingly, dual treatment of MAP-infected macrophages with MyD88 antagonist and nicotine absolutely impaired immune response and decreased MAP viability, which clearly validate the inflammatory role of nicotine in macrophages through TLR2/MyD88 pathway during infection. This is the first report to describe the mechanism by which nicotine modulates TLR2/MyDD88 and exacerbates inflammation in CD smokers associated with infection.Recently, we reported that cigarette smoking, and especially nicotine, increases susceptibility to mycobacterial infection and exacerbates inflammation in patients with Crohn’s disease (CD). The macrophagic response to  Mycobacterium avium subspecies paratuberculosis (MAP), adherent-invasive Escherichia coli (AIEC), and Klebsiella pneumoniae  whereas ∆∆CT = [∆CTTreated − ∆CTUntreated].RT-PCR: Gene expression analysis was performed as described earlier . BrieflyMA, USA) , and 7 µTLR4  following manufacturer’s instructions. All ELISA experiments were done in duplicates.For cell receptors, cell pellets were incubated with iced RIPA buffer  for 15 min, then centrifuged at 14,000 rpm for 20 min at 4 °C. Supernatant containing cell lysates were analyzed using ELISA kits: TLR2  and To determine if TLR2 and MyD88 have a role in MAP viability in macrophages, THP-1 macrophages were blocked by either MMG (5 µg/mL) or T6167923 (5 µg/mL) followed by MAP infection. After 24 h of infection, THP-1 macrophages were washed twice with PBS to remove extracellular bacteria, then THP-1 macrophages were collected at three time points: 0, 24, and 84 h. The cells were lysed using iced RIPA buffer , and then the samples were centrifuged at 14,000 rpm for 20 min at 4 °C. Supernatants were collected and the LIVE/DEAD™ BacLight™ Bacterial Viability Kit  was used according to the manufacturer’s protocol as described earlier [p < 0.05 and a 95% confidence interval (CI). All data collected in this study were pretested for normal distribution using the Kolmogorov–Smirnov normality test. Data is shown as (mean ± SD). p-values < 0.001 are also mentioned when achieved.Statistical analysis was performed by using GraphPad Prism 7.02 software. Significance among experiments was assessed by an unpaired two-tailed t test at TLR4 expressions on macrophages, we infected PMA-activated THP-1 with MAP for 24 h. MTB and LPS were also used as controls. Infection of macrophages with Mycobacteria increased TLR2 in gene and protein levels, respectively   A,C. A si< 0.001) B,D.TLR4 in infected macrophages, we treated infected macrophages with 4 µg/mL of pure nicotine for 24 h followed by RT-PCR and ELISA evaluation of the expression and protein levels TLR2/TLR4. As shown in p < 0.05), , or . The findings were consistent with results from protein level analysis (p < 0.05), , or . Similarly, TLR4 expression and protein levels corroborated those of TLR2 with lowest values reported after nicotine treatment of infected cells , . However, a significant elevation in MyD88 expression was observed in all mycobacterial infection without nicotine treatment compared to untreated uninfected cells, p < 0.05. Interestingly, there was no clear change in MyD88 and IL-8 expression when macrophages were treated with LPS . Pretreated macrophages with (2.5 to 10 µg/mL) gradually neutralized the effect of LPS on iNOS level and reached its optimum effect at 5 µg/mL (TNF-α and IL-8 expressions (TLR4-IN-C34 (5 µg/mL) has increased M2-macrophages expression (MMR) (TLR4-IN-C34 (5 µg/mL) + LPS: 1.10 ± 0.14 vs. LPS alone: 0.62 ± 0.06 vs. TLR4-IN-C34 (5 µg/mL) alone: 1.12 ± 0.18; p < 0.05)  with MAP infection (TLR4-IN-C34 (10 µg/mL) + MAP: 1.92 ± 0.30 vs. MAP alone: 2.5 ± 0.25; p < 0.05, for iNOS) (TLR4-IN-C34 (10 µg/mL) + MAP: 4.98 ± 0.45 vs. MAP alone: 6.50 ± 1.90; p < 0.05, for TNF-α) (TLR4-IN-C34 (10 µg/mL) + MAP: 2.67 ± 0.21 vs. MAP alone: 3.58 ± 0.51; p < 0.05, for IL-8)  B. Surprior IL-8) A,C,D.TLR4 has no role during MAP infection; thus, we examined the effect of TLR2 and its adaptor protein (MyD88) on macrophages response during MAP infection. Activated macrophages were preincubated with 0, 2.5, 5, 10 or 20 µg/mL of either MMG11 (TLR2 antagonist) or T6167923 (MyD88 inhibitor), followed by infection with MAP for 24 h. Interestingly, MMG11 at 5 µg/mL attenuated the proinflammatory status in MAP-infected macrophages and greatest decrease was observed in TNF-α, falling almost fourfold compared to MAP-infected macrophages alone (MMG11 (5 µg/mL) + MAP: 2.33 ± 0.98 vs. MAP alone: 6.50 ± 1.90; p < 0.05) (We showed in the previous experiment that  < 0.05) C.TNF-α and IL-8 to levels similar to uninfected macrophages (T6167923 (10 µg/mL) + MAP: 1.15 ± 0.35 vs. T6167923 (10 µg/mL): 1.080 ± 0.27; p < 0.05, for TNF-α) (T6167923 (10 µg/mL) + MAP: 1.10 ± 0.27 vs. T6167923 (10 µg/mL): 1.25 ± 022; p < 0.05, for IL-8)   C,D and sfor MMR) B.iNOS (4.62 ± 0.59) and IL-8 (6.40 ± 0.51) compared to all other groups, blocking MyD88 or TLR2 subverted macrophages’ response to MAP by reduction of iNOS and IL-8 expression to a level similar to uninfected macrophages regardless of nicotine treatment  or 10 µg/mL of T6167923 (MyD88 inhibitor) in the presence and absence of MAP infection and then subjected the cells to nicotine treatment. While the synergy of MAP and nicotine exhibited the highest levels of reatment A,B. The oduction C,D. It iWe previously reported that exposure of MAP-infected macrophages to nicotine maintains MAP viability and increases its burden in macrophages . Here weTLR4 have been identified to interact with pathogens through specific ligand binding [M. tuberculosis or treated with LPS. In fact, in our study we showed that nicotine seems to exacerbate inflammatory response during infection [TLR4 in macrophages during infection.MAP, a slow-growing intracellular bacterium capable of surviving within host macrophages, remains one of the most debated etiological agents of CD ,28,29,30 binding . On the  binding . We havenfection . These iIt was estimated that each tobacco cigarette contains an average of 8.4 mg of nicotine and the average nicotine concentration in a smoker’s venous blood is approximately 2 µg/mL . Of courTLR4-deficient mice were active and responded properly during MTB infection [TLR4 was reported to play a role in recognition of enteric pathogens [TLR4. We observed a significant increase in TLR2 expression and, in consequent cytokines including TNF-α and IL-8 in MAP-infected macrophages, there was no change in TLR4 expression in the same cells  and in lung epithelial cells [In our efforts to understand the effect of cigarette smoking on CD patients, we investigated possible interaction between TLR2 and nicotine in CD-like macrophages. The latter included activated macrophages infected with MAP or MTB or treated with LPS to represent Gram-negative bacteria. Initially, we did not observe any change in the expression of either TLR2 or al cells ,38, thisIL-8  and Actinobacteria (high guanine and cytosine content bacteria (Propionibacteriaceae and Bifidobacteria)) compared to healthy controls [TLR4 expression on lamina propria cells in CD patients compared to healthy controls [While MAP and LPS represent most pathogens associated with CD pathogenesis, there is no doubt that individual microbiome content plays a major factor in CD inflammatory episodes and symptoms flares. Recent studies of the microbiome in patients with CD revealed a shift in the gut microbial populations ,43. Speccontrols . One stucontrols ,43. The TLR4. MAP upregulated MyD88 expression and increased its specific correspondence of IL-8. Surprisingly, nicotine has further increased MyD88 and IL-8 expression in MAP-infected macrophages. Thus, this study illustrates the crucial role of MyD88 in upregulation of proinflammatory genes during MAP infection. Collectively, we suggested that MAP-Nicotine interaction with TLR-2 during MAP infection is MyD88-dependent. Moreover, when TLR2 or MyD88 were blocked with antagonists, there was no significant change in MAP load in macrophages even after 48 h exposure to nicotine treatment.In summary, we showed that, in MAP-infected macrophages, nicotine has significantly decreased expression of both TLR2 and Overall, the data is intriguing about the role of TLR2 and MyD88 signaling in MAP-infected macrophages in association with nicotine exposure. More studies are needed to expand on our findings. Specifically, it is important to validate our findings in primary macrophages from CD patients with and without MAP infection. Ultimately, it would be the most informative to validate our findings in mice during infection and under cigarette smoking conditions.


Metabolic profiling of PCM- and BCM-exposed MΦs indicated a significant increase in glycolytic metabolism, purine biosynthesis, and inositol phosphate metabolism. In addition, these metabolic patterns suggested that BCM-exposed MΦs exhibit a hyperinflammatory metabolic profile with reduced glycerol metabolism and elevated catabolism of lactate and amino acids, relative to PCM-exposed MΦs. Altogether, our study reveals novel findings concerning the metabolic modulation of human MΦs after exposure to secretory microbial products and contributes additional knowledge to the field of immunometabolism in MΦs.Macrophages (MΦs) are prevalent innate immune cells, present throughout human bodily tissues where they orchestrate innate and adaptive immune responses to maintain cellular homeostasis. MΦs have the capacity to display a wide array of functional phenotypes due to different microenvironmental cues, particularly soluble bacterial secretory products. Recent evidence has emerged demonstrating that metabolism supports MΦ function and plasticity, in addition to energy and biomolecular precursor production. In this study, 1D  The host innate immune system provides the initial line of defense against invasive microbes and infection . MacrophBiofilms are structured colonies of bacterial cells encased in a secreted extracellular polymeric matrix, which adhere to biological or abiotic surfaces ,11. BactStaphylococcus, Pseudomonas, Peptoniphilus, Enterobacter, Stenotrophomonas, Finegoldia, and Serratia spp [Pseudomonas aeruginosa, was examined in this study. P. aeruginosa is a particularly virulent pathogen that is found predominantly in venous leg ulcers [P. aeruginosa infections are correlated with increased morbidity, mortality, and worsened disease progression [While chronic wound biofilms are known to maintain a diverse flora of microbes, the most prevalent bacteria in diabetic foot, venous leg, and pressure ulcers include atia spp . In addiatia spp . This cog ulcers . Moreovegression ,23. Prevgression ,25.Notably, growing evidence has indicated that immune cell phenotypes and responses to environmental clues, including pathogen exposure, are reflected in characteristic metabolic changes and adaptations, also known as metabotypes . These rRecent studies have demonstrated that metabolites, such as itaconate and succinate, can serve as signaling molecules, in addition to being metabolic intermediates, and mediate the resultant functional phenotypes of MΦs ,35. PrioP. aeruginosa PCM and BCM exposure on primary human monocyte-derived resting (M0) MΦs, using CD14+ magnetic-activated cell sorting (MACS), in vitro MΦ differentiation and exposure schemes, 1D 1H NMR metabolomics, metabolic profiling using Chenomx Suite software, and statistical analysis. M0 MΦs were differentiated from primary human monocytes using MΦ colony-stimulating factor (M-CSF) for 9 days, prior to stimulation with PCM or BCM. Following exposure, both MΦ intra- and extracellular metabolites were extracted, 1D 1H NMR spectra were recorded, and metabolite profiling of resulting metabolite extract-derived NMR spectra was conducted. Results from this study highlight significant changes in metabolite profiles and reveal several metabolic pathways that are differentially modulated in PCM- and BCM-exposed MΦs, including glycolysis, purine biosynthesis, inositol phosphate metabolism, branched-chain amino acid catabolism, and glycerol metabolism. The functional significance of the distinct metabolic profiles found to be associated with PCM- and BCM-exposed MΦ phenotypes are also reviewed.In the present study, we aimed to investigate the metabolic impact of + monocytes were isolated from peripheral blood mononuclear cells by MACS using CD14 human microbeads , which amounted to an average purity of 97.1 ± 1.3% , and signed, informed consent from healthy, adult blood donor volunteers in Bozeman, Montana, USA. A total of 5 donors that ranged in age from 19 to 35 were included in this particular study, of whom 40% of these volunteers were female. Peripheral blood mononuclear cells were isolated by differential centrifugation in lymphocyte separation (Ficoll) media  at 800× g for 25 min at room temperature, following protocols described previously . CD14+ m1 ± 1.3%  when ana+ monocytes were seeded and cultured at a concentration of 1 × 106 cells per mL in 25 cm2 tissue culture flasks  with RPMI 1640 media containing l-glutamine , supplemented with sodium pyruvate , 1X non-essential amino acids , 10% (v/v) fetal bovine serum , and 50 ng/mL recombinant human M-CSF  for 9 days at 37 °C, 5% CO2. Media and cytokines were replenished every 3 days. Following differentiation, cell surface marker characterization of primary human monocyte-derived MΦs was performed using flow cytometry  and corresponding isotype controls. M0 MΦs were stained with anti-human antibodies directed against CD68, CD80, and CD163  and corresponding isotype controls. Cellular permeabilization was conducted on a subset of cells using Cytofix/Cytoperm  for CD68 intracellular staining. After antibody staining, cells were reconstituted in fluorescence-activated cell sorting (FACS) buffer for subsequent analysis on an LSR Fortessa flow cytometer . Analysis of FACS data was conducted using FCSalyzer software  with both monocytes and MΦs gated based upon cellular size and singlet cells. Since fluorescence levels at baseline differed between our different fluorophores, mean fluorescence intensity (MFI) values were normalized to their corresponding isotype control MFI  Brain Heart Infusion  at 37 °C and 220 rpm agitation. Following inoculation, tissue culture inserts were placed into a 6-well tissue culture plate, and 1.5 mL of 10% (v/v) BHI was added to each well. The tissue culture plate was incubated at 37 °C, and media were refreshed every 24 h to maintain biofilm viability [l-glutamine, supplemented with sodium pyruvate (1 mM), 1X NEAAs, and 10% FBS, was added to each well, and biofilms were incubated at 37 °C. BCM was harvested after 24 h of conditioning, centrifuged at 5000 rpm for 10 min at room temperature to remove cells, sterile-filtered , and stored at −20 °C. Tissue culture wells were replenished with fresh cell culture media immediately following BCM harvest. A total of five 24 h BCM collections were pooled, pH-adjusted to 7.4 using sterile, concentrated HCl or NaOH, and stored at −20 °C until further use.BCM was generated using methods similar to those described previously ,25. In biability . After 7P. aeruginosa PAO1 biofilms were determined after initial maturation (72 h) using 10% (v/v) BHI and following BCM generation using a serial dilution drop-plating method. In brief, biofilm colonies were removed from tissue culture inserts using 1X PBS, vortexed, agitated in a sonicator bath, serially diluted with PBS, plated on 100% tryptic soy agar plates, and incubated at 37 °C. Bacterial colonies were then counted, and the number of CFUs per insert was calculated.Colony-forming units (CFUs) for P. aeruginosa PAO1 was grown in 10% (v/v) BHI at 37 °C with 220 rpm agitation. This overnight culture was then centrifuged at 5000 rpm for 5 min at room temperature, and then the cells were washed using 1X PBS to remove excess media. This cell suspension was centrifuged again at 5000 rpm for 5 min at room temperature, and the cells were resuspended in MΦ culture medium, consisting of RPMI 1640 media containing l-glutamine, supplemented with sodium pyruvate (1 mM), 1X NEAAs, and 10% FBS, at a density equivalent to that of a mature PAO1 biofilm (6 × 109 CFUs/mL). This PAO1 suspension was cultured at 37 °C with 220 rpm agitation for 24 h. After the conditioning period, PCM was centrifuged at 5000 rpm for 10 min at room temperature to remove cells, sterile-filtered , pH-adjusted to 7.4 using sterile, concentrated HCl or NaOH, and stored at −20 °C until further use.PCM was generated to yield a similar proportion of CFUs per unit of fluid volume to that of BCM, as previously described ,25. In bl-glutamine, supplemented with sodium pyruvate (1 mM), 1X NEAAs, and 10% FBS, and pre-warmed to 37 °C. Control media consisting of 50% (v/v) MΦ cell culture media and 50% (v/v) 1X PBS were determined to be the most appropriate exposure media for control MΦ experiments due to its metabolic similarity to PCM and BCM compared to full strength (100% v/v) MΦ cell culture media (v/v) PCM, BCM, and control media were transferred into 25 cm2 tissue culture flasks with  and without (sham media control flasks) M0 MΦs  media A in , consistre media . Spent mMΦs B in , and theg for 1 min at room temperature to pellet non-adherent cells, decanted, and then resuspended in 0.5 mL of −20 °C 50% (v/v) aqueous methanol. An amount of 1.5 mL of −20 °C 50% (v/v) aqueous methanol was pipetted into 25 cm2 tissue culture flasks in order to synchronously quench, extract, and detach cells from the surface of the flask with the aid of a cell scraper. Cell suspensions were then transferred to clean 15 mL conical tubes, which already contained non-adherent cell suspensions, and thoroughly mixed.Following exposure, spent media  were transferred into separate, clean 2 mL lysis B matrix tubes  and lysed using 2 cycles (40 s each) on a FastPrep-24 5G homogenizer  at a speed of 6.0 m/second, with lysis tubes being stored on ice in between cycles. A 50 µL aliquot of cell lysate was stored at −80 °C for subsequent determination of protein content, and then 0.5 mL of chloroform was added to each cell lysis tube. Tubes were vortexed for 3 consecutive cycles of 10 s each, and then incubated at −20 °C for 20 min, before centrifugation at 10,000× r phases . The aquSham and spent cell culture media aliquots were filtered using 3 kDa molecular weight cutoff centrifugal filters , which had been prewashed extensively as previously described , before Sample protein content was established using a BCA (bicinchoninic acid) protein assay  and used to normalize intra- and extracellular metabolite concentrations.2PO4/Na2HPO4, 0.4 mM imidazole, 0.25 mM 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS) in 90% (v/v) H2O/10% (v/v) D2O at pH 7.0. Samples were subjected to centrifugation at 21,000 rpm for 60 s at room temperature to pellet insoluble material, and then transferred into 5 mm NMR tubes for subsequent metabolomics analysis.Intra- and extracellular metabolite extracts were reconstituted in 0.6 mL of NMR buffer, which consisted of 25 mM NaH1H Larmor frequency) AVANCE III solution NMR spectrometer, equipped with an automatic sample loading system (SampleJet), a 5 mm triple resonance , three channel inverse, liquid-helium-cooled NMR cryoprobe, and Topspin software . 1D 1H-NMR spectral acquisitions were performed using the Bruker-supplied ‘zgesgp’ pulse sequence [1H spectral window. Free induction decays were recorded with 32,000 data points and a dwell time interval of 52 microseconds, equating to a total data acquisition time of 1.7 s. Recovery delay times between data acquisitions were fixed at 1 s, amounting to an overall 2.7 s relaxation recovery delay in between scans [1H-1H total correlation spectroscopy (TOCSY) NMR as previously described [NMR spectra were collected at 25 °C (298 K) on a Bruker 600 MHz  NMR spectrometers; manual adjustments were performed to attain optimal spectral pattern fits based upon metabolite intensity and peak cluster location [Additional processing of baseline ,47. All location . In addit-test with Welch’s correction.Metabolite concentrations were normalized to protein content and NMR buffer volume before multivariate statistical analysis using MetaboAnalyst 4.0 ,50. AfteP. aeruginosa PCM and BCM exposure on primary human MΦs, it was imperative to first generate P. aeruginosa PCM and BCM. For this purpose, P. aeruginosa biofilms were grown on 6-well tissue culture inserts and used to produce BCM. The initial cell density of each insert immediately following inoculation was 6.50 ± 1.22 × 107 CFUs/insert, and after 72 h of growth in 10% (v/v) BHI the biofilms had reached a cell density of 2.10 ± 0.51 × 109 CFUs/insert (9 CFUs/mL (To assess the metabolic impact of s/insert . These m CFUs/mL A. The pHP. aeruginosa was grown in 10% (v/v) BHI for 16 h at 37 °C with 220 rpm agitation, resulting in a cell density of 2.94 ± 0.89 × 109 CFUs/mL. These planktonic cells were then centrifuged, washed, resuspended in cell culture media, and incubated for 24 h at 37 °C with 220 rpm agitation, resulting in a final cell density of 1.04 ± 0.49 × 1010 CFUs/mL  NMR spectrometer, and corresponding 1D 1H NMR spectra were processed and profiled using Topspin and Chenomx NMR Suite software, respectively. Representative 1D 1H NMR spectra acquired from these metabolite extracts revealed clear signal intensity differences and distinct metabolic profiles between PCM-exposed, BCM-exposed, and control primary human MΦs  and inositol 1,3,4,5-tetrakisphosphate P4). While the latter product, InsP4, suppresses the function of pleckstrin homology (PH) domain-containing protein activity, such as Akt serine/threonine kinase 1, the former product, PtdInsP3, binds to PH domain-containing proteins (3, which rapidly accumulates at the site of phagocytosis and ensures membrane delivery to distending pseudopods [Phox, an essential subunit of reduced nicotinamide adenine dinucleotide phosphate oxidase, has been shown to be recruited to type I PI3K signals [Our data also indicate that PCM- and BCM-exposed MΦs consumed a significantly greater amount of myo-inositol from the cell culture media relative to control MΦs ; howeverproteins  and as aeudopods . Moreove signals . We therP. aeruginosa PCM and BCM in vitro have demonstrated that BCM-exposed MΦs possess unique metabolic characteristics when compared to PCM-exposed MΦs, including suppressed glycerol metabolism, increased lactate catabolism, and elevated amino acid metabolism, which may be indicative of a hyperinflammatory metabolic state. Furthermore, we have observed several immunometabolic response trends shared by PCM- and BCM-exposed MΦs compared to control MΦs, including elevated consumption of glycolytic substrates, increased purine biosynthesis, and enhanced inositol phosphate metabolism. These findings emphasize the importance of elucidating the immunometabolic modulatory differences between host cell interactions with planktonic versus biofilm bacteria. Further characterization of these types of interactions may aid in the development of novel therapeutics to rescue dysfunctional hyperinflammatory MΦ behaviors and resolve chronic inflammation in wounds that are often colonized by P. aeruginosa bacterial biofilms, including venous leg ulcers.In conclusion, our NMR-based metabolic data obtained on primary human monocyte-derived MΦs exposed to


Bufadienolide compounds occur in many plants and animal species and have strong cardiac and anti-inflammatory properties. The compounds have been recently investigated for cytotoxic and antitumor activity.– a bufadienolide steroid occuring in plants from Kalanchoe genus (Crassulaceae), was evaluated with cervical cancer HeLa cells in vitro.The cytotoxic effect of bersaldegenin-1,3,5-orthoacetate The cytotoxic activity of the compound (at 0.1–20.0 μg/mL) on the cells was determined by Real-Time Cell Analysis (RTCA) system for 24 h. The estimation of cell cycle arrest, reactive oxygen species (ROS) production, reduction of mitochondrial membrane potential (MMP), and caspases-3/7/9 activity in the HeLa cells treated with the compound was done by flow cytometry and luminometric technique. DNA damage in the cells was estimated by immunofluorescence staining and the comet assay with etoposide as a positive control.50 = 0.55 μg/mL) by the suppression of HeLa cells proliferation in G2/M phase of cell cycle and induction of cell death through double-stranded DNA damage and reactive oxygen species overproduction. Furthermore, we did not observe an increase in the activity of caspase-3/7/9 in the treated cells as well as a decrease in cellular mitochondrial membrane potential. Gene expression analysis revealed the overexpression of NF-Kappa-B inhibitors genes (>2-fold higher than control) in the treated cells.The compound had strong effect on the cells (ICBersaldegenin-1,3,5-orthoacetate induces cell cycle arrest and caspase-independent cell death through double-stranded DNA damage. These results are an important step in further studies on cell death signalling pathways induced by bufadienolides. Bufo (the toad) which contains bufadienolides   Wollenw. (Nectriaceae), F. sporotrichioides Sherb. , and Rhabdophis (Colubridae) , Tylecodon (Crassulaceae), Helleborus (Ranunculaceae), Scilla (Hyacinthaceae), Cotyledon (Crassulaceae), Urginea (Hyacinthaceae), Mimosa (Fabaceae), Millettia (Fabaceae), and Drimia genus (Hyacinthaceae)  and lung cancer (A549) cell lines  Pers.) has strong cytotoxic activity on the KB cell line  revealed toxic activity on lung (A549), prostate (PC-3), epidermoid (A431), and ovarian (1A9) cancer cell lines  Raym.-Hamet & H. Perrier showed antitumor-promoting activity on Burkitt’s lymphoma Raji cells , lung adenocarcinoma (A549), promyelocytic leukaemia (HL-60), and melanoma (A2058) cell lines  with 80% (v/v) aqueous methanol and the bufadienolide-rich fraction was obtained. This fraction was then partitioned between water and butanol to afford n-BuOH extract, which was applied on a Sephadex LH-20 column , eluted with MeOH, and afforded subfractions, which were separated on a Lichroprep RP-18 column , eluted with a linear gradient of 15%–40% (v/v) aqueous MeOH in a 0.1% (v/v) HCOOH. Individual bufadienolides were purified isocratically by a semi-preparative HPLC on a C18 column , using different concentrations of aqueous methanol solutions with 0.1% (v/v) HCOOH. The one from the isolated compounds was bersaldegenin-1,3,5-orthoacetate. The yield of this compound was 0.32 mg (0.032%) in 1 g of dry mass of the root. Its structure elucidation was performed using ESI-MS and NMR spectral data analyses, optical rotation, circular dichroism and IR spectroscopy. The compound was dissolved in sterile DMSO at a concentration 20 mg/mL.The method of isolation and structure elucidation of bersaldegenin-1,3,5-orthoacetate was described by Moniuszko-Szajwaj et al. . Briefly2 incubator (37 °C) in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% (v/v) foetal bovine serum (FBS), 100 units/mL of penicillin, and 100 µg/mL of streptomycin .The human cervical adenocarcinoma cell line (HeLa S3) was obtained from the American Type Culture Collection . The cell line was cultured in a humidified 5% CO4 cells/well. After 24 h, bersaldegenin-1,3,5-orthoacetate was added to the cells at a concentration range of 0.1–20.0 µg/mL for 24 h. Additionally, DMSO – as a solvent of the compound, was added to HeLa cells (a control sample) at a concentration of 0.25% (v/v). Vinblastine sulphate was used as a positive control. The RTCA software v. 1.2.1. calculated IC50 values. All the experiments were performed in duplicate, in three independent repeats (n = 6).To monitor HeLa cell viability and proliferation we used the xCELLigence Real-Time Cell Analyzer Dual Plate  as described in our previous study  in a control sample was 0.25% (v/v). Then, the cells were stained with the Hoechst dye (1.0 µg/mL) and observed under a fluorescent microscope .To show the effect of bersaldegenin-1,3,5-orthoacetate in HeLa cell nuclei, we stained the cells with the blue fluorescent Hoechst 33342 dye . HeLa cells were seeded in 12-well plates at a density of 1 × 105 cells/well and treated with bersaldegenin-1,3,5-orthoacetate at concentrations of 0.1, 0.5, 1.0, 2.0, and 5.0 µg/mL. The concentration of DMSO in a control sample was 0.25% (v/v). After 3 h, 24 h, and 48 h of incubation, the cells were harvested and prepared according with Muse MitoPotential Assay Kit  protocol. The amount of depolarized/live/dead cells was determined by Muse Cell Analyzer (Merck Millipore). All the experiments were independently repeated three times.HeLa cells were seeded in 12-well plates at a density of 1 × 105 cells/well) with the compound at concentrations of 0.1, 0.5, 1.0, 2.0, and 5.0 µg/mL for 24 h. The DMSO concentration in a control sample was 0.25% (v/v). After 24 h of incubation, the cells were stained with Muse Oxidative Stress Kit (Merck Millipore), according with the manufacturer’s protocol. All samples were analysed by Muse Cell Analyzer. The experiments were repeated three times, independently.To determine the effect of bersaldegenin-1,3,5-orthoacetate on oxidative stress production in HeLa cells, we treated the cells (1 × 105 cells/well) and treated with bersaldegenin-1,3,5-orthoacetate at concentrations of 0.1, 0.5, 1.0, 2.0, and 5.0 µg/mL. The concentration of DMSO added to a control sample did not exceed 0.25% (v/v). After 24 h, the cells were harvested and prepared according with the protocol of Muse Caspase-3/7 Assay Kit (Merck Millipore). Then, the cells were analysed by Muse Cell Analyzer. The experiments were performed at least in three independent repeats.The activity of caspase-3/7 was measured according with method described previously . The activity of caspase-9 was measured in the cells after 1, 2, 3, 4, 14, and 24 h of incubation the cells with the compound. We used Caspase-Glo 9 Assay Kit  and Glomax Multi + Detection System (Promega), according to the manufacturer’s instruction. The experiments were repeated three times, independently.5 cells/well) and incubated with the compound at concentrations of 0.1, 0.5, 1.0, 2.0, and 5.0 µg/mL for 48 h. DMSO concentration in a control sample was 0.25% (v/v). Next, the cells were harvested and stained with Muse Cell Cycle Assay Kit (Merck Millipore), following the manufacturer’s protocol. The amount of HeLa cells in G0/G1, S, and G2/M phases of cell cycle was determined by Muse Cell Analyzer. The experiments were independently repeated three times.To determine the HeLa cell cycle arrest after treatment with bersaldegenin-1,3,5-orthoacetate, we seeded the cells . Slides were immersed in the lysis solution for 1 h at 4 °C, after which they were placed in the alkaline unwinding solution for 1 h at 4 °C. Slides were next placed in the electrophoresis slide tray and immersed in the alkaline electrophoresis solution in the CometAssay ES unit (Trevigen). Slides were submitted to electrophoresis at 1 V/cm for 30 min. Following electrophoresis, slides were washed and then stained with Sybr Gold  for 30 min at RT. Slides were analysed under a fluorescence microscope (Nikon PCM-2000) and images of at least 50 comets per slide were analysed with the TriTek CometScore 2.0 software. Two regions of the comet were selected, the whole cellular DNA and the region of DNA within the comet head. The densities of the regions were defined and the results are presented as tail moment expressed as the percentage of DNA in the tail multiplied by the tail length. Each treatment was normalized to that of the control.The evaluation of DNA damage was analysed with the alkaline comet assay using the Reagent Kit for Single Cell Gel Electrophoresis Assay  according to the manufacturer’s instructions. HeLa cells were seeded at a density of 105 cells/well) and treated with bersaldegenin-1,3,5-orthoacetate at concentrations of 1 and 5 µg/mL. DMSO was added to the cells (control) at a concentration of 0.25% (v/v). A positive control was etoposide added to the cells at concentration of 10 μM. After 6 h and 24 h, the cells on the coverslips were fixed with 4% formaldehyde (v/v) at room temperature for 10 min and then, permeabilized with 0.1% Triton-X100 (Sigma-Aldrich) for 15 min. Blocking step was performed with 5% bovine serum albumin  for 1 h. Subsequently, the cells were incubated with the anti-phospho-H2A.X (Ser139), clone JBW30, Alexa Fluor 555 Conjugate antibody  in 1% BSA (w/v) for 1 h. After three steps of washes, the cells on the coverslips were incubated with Hoechst 33342 dye (2.0 µg/mL) at room temperature for 20 min and observed under a fluorescence microscope. Images of at least 100 nuclei per slide were analysed.HeLa cells were seeded in a 6-well plate with coverslips (5 × 106 cells/well) in 6-well plates and treated with bersaldegenin-1,3,5-orthoacetate at a concentration of 1.0 μg/mL for 24 h. The control sample was treated with DMSO at a concentration 0.05% (v/v). Next, the cells were harvested and the isolation of total RNA was performed with RNeasy Mini Kit  according with the manufacturer’s instruction. The quality and concentration of isolated RNA was measured by Agilent Technologies 4200 TapeStation , according with the manufacturer’s protocol. Next, we performed synthesis of cDNA using Maxima First Strand cDNA Synthesis Kit , following with the manufacturer’s protocol. Obtained cDNA was applied on the Applied Biosystems TaqMan Array Human Apoptosis 96-well FAST Plates  that contain 92 assays for cell death associated genes and 4 assays for endogenous control genes  (Table S1). The PCR reactions were performed on StepOnePlus Real-Time PCR System (Life Technologies). All the obtained data were analysed by StepOne software v.2.3. The experiments were performed three times, independently.We seeded HeLa cells . All data are expressed as mean values ± standard deviation (SD). For comparison studies, Student’s 50 value of bersaldegenin-1,3,5-orthoacetate (0.55 µg/mL), calculated in our previous study  generation in HeLa cells, we treated the cells with different concentrations of the compound. After 24 h, we analysed the cells by flow cytometry and observed significant increase in amount of ROS positive cells above the compound concentration of 1.0 µg/mL. The percentage of ROS (+) cells were 4.52 ± 0.5, 7.39 ± 0.59, 8.84 ± 0.22, 15.18 ± 0.55, 23.21 ± 1.68% for 0.1, 0.5, 1.0, 2.0, and 5.0 µg/mL, respectively .To study the effect of the compound on the mitochondrial membrane potential (MMP) in HeLa cells, we treated the cells with different concentrations of the compound (0.1–5.0 µg/mL). The obtained results indicate that bersaldegenin did not significantly affect MMP in living cells. We observed depolarization only in the dead cells after 24 h of incubation. The percentage of depolarized/dead cells was 0.67 ± 0.16, 2.7 ± 0.22, 5.02 ± 0.43, 9.89 ± 0.71, 12.41 ± 0.69% at the compound concentrations of 0.1, 0.5, 1.0, 2.0, and 5.0 µg/mL, respectively. After 48 h, the percentage of depolarized/dead cells was comparable to the control. Then, in the amount of depolarized/live cells we observed slight increase . After 3 h, we did not observe any changes in decrease in mitochondrial potential in the living and dead cells after treatment with the compound in comparison to the control sample .The activity of caspase-9 was measured in HeLa cells after treatment with bersaldegenin-1,3,5-orthoacetate for 1, 2, 3, 4, 14 and 24 h. The caspase-9 activity estimated by luminometric method revealed that the tested compound did not increase the activity level of this caspase in the cells in all the measurement points in comparison to the control. Furthermore, we observed significant decrease in the activity of caspase-9 after 24 h of treatment the cells with the compound .Furthermore, the activity of caspase-3/7 is indicated by an increase in population of early apoptotic cells. In the treated cells with the compound after 24 h and 48 h, the activity of caspase-3/7 was slightly higher than in the untreated cells. Also, we observed a significant increase in the population of late apoptotic and dead cells what indicated that the main pathway of the HeLa cell death is caspase-3/7 independent .The effects of bersaldegenin-1,3,5-orthoacetate on the induction of DNA damage in HeLa cells were evaluated with the alkaline comet assay. The effects of the compound at the concentrations of 1.0 and 5.0 µg/mL were evaluated after 6 h and 24 h of treatment. The degree of DNA damage was determined by defining the extent of DNA migration in the comet and results are presented as tail moment, which is the product of the percentage of total DNA in the tail and tail length. The results of the comet assay revealed increase in DNA damage as determined by the increase in tail moment. After 6 h of treatment, 5-fold and 18-fold increases in DNA damage were observed, as compared to control samples, at the concentrations of bersaldegenin-1,3,5-orthoacetate 1.0 and 5.0 µg/mL, respectively. At longer time treatment, a further increase in DNA damage was observed, and the tail moment increased 20-fold and 42-fold at the concentrations of 1.0 and 5.0 µg/mL, respectively .Additionally, to confirm induction of DNA damage by bersaldegenin orthoacetate in HeLa cells we performed immunofluorescence staining. The cells were treated with the compound at concentrations of 1.0 and 5.0 μg/mL for 6 h and 24 h. The obtained results showed that the amount of the cells with phospho histone H2A.X (γH2AX) increased after treatment with the compound and γH2AX signal was higher in these nuclei than in DMSO control . Most ofAfter 48 h of treatment the cells with the compound, we estimated cell cycle distribution by flow cytometry. The obtained results indicate that bersaldegenin significantly arrested cell cycle in G2/M phase and slightly in S phase . The higThe analysis of genes expression in HeLa cells treated with the compound (1.0 μg/mL) showed that the higher expression was observed for BCL2A1 (Bcl-2-Related Protein A1), BIK (Bcl-2-Interacting Killer), CASP3 , MCL1 (Myeloid Cell Leukaemia Sequence 1), NFKBIA (NF-Kappa-B Inhibitor Alpha), NFKBIB (NF-Kappa-B Inhibitor Beta), NFKBIZ (NF-Kappa-B Inhibitor Zeta), PA15, PMAIP1 (Phorbol-12-Myristate-13-Acetate-Induced Protein 1), RELB , TNFRSF10 (Tumor Necrosis Factor Receptor Superfamily Member 10), and TNFRSF10B  genes. The expression of these genes was at least two times higher in comparison to the control sample (0.05% DMSO). The genes – BCL2A1, BIK, and NFKBIZ were the most up-regulated. We also observed down-regulation for 24 genes which expression was at least two times lower than in the control sample . The genIn this study, we estimated the cytotoxic activity of bersaldegenin-1,3,5-orthoacetate on HeLa cells with determination of selected factors that play a role in the cell death pathway. Our results indicate that the compound is strong cytotoxic agent and its effect is time- and dose-dependent.The antiproliferative and cytotoxic effect of bufadienolides is known, however available data concerning these activities are limited. Han et al. tested a few bufadienolides  on human liver cancer cells (HepG2). The results indicated that these compounds have significant antiproliferative effects in a concentration range below 150 ng/mL  family factors which regulate physiological and pathological processes. These proteins are located in the cytoplasm and are associated with a family of inhibitors of NF-кB proteins (IкBs). After stimulation, IкB undergoes degradation in a proteasome, and the free NF-кB translocates to the nucleus and regulates genes transcription  U266 cells and caused pro-apoptotic effects through ROS-mediated activation of ERK, JNK, and p38 MAPK pathways and the activation of caspase-3 in the cells (Baek et al. In conclusion, bersaldegenin-1,3,5-orthoacetate strongly induces caspase-independent cell death in cancer HeLa cells which is associated with cell cycle arrest in G2/Mphase, ROS overproduction, and double-stranded DNA damage.Click here for additional data file.


Althaea officinalis L. , is used for the treatment of cough since centuries. Application of medicinal extracts of marshmallow roots shows immediate effects like a protective film on the inflamed mucosa. Because the soothing layer reduce irritation of the mucous system, a faster regeneration is supported by defense mechanisms required to protect the respiratory tract from environmental injury. Macrophages (MΦ), which belong to a group of multipurpose defensive cells, provide the first line of defense against mucosal invasive pathogens. The present study was performed to investigate, whether the herbal medicinal product has anti-inflammatory or anti-oxidative effects on pro-inflammatorily activated MΦ or after oxidative stress induction. Special attention should be payed to elucidate the effects of A. officinalis on the mechanism of intracellular defense as well as on migratory capacity of the MΦ.The medicinal plant marshmallow ® increased their viability without affecting the cell number. Phytohustil® or root extracts of A. officinalis (REAo) – an active component of Phytohustil® – were able to protect human MΦ against H2O2-induced cytotoxicity and H2O2-induced ROS production. Phytohustil®, REAo or diclofenac used as anti-inflammatory reference substance, inhibited the LPS-induced release of tumor necrosis factor-alpha (TNF-α) as well as of IL6 in MΦ. Treatment with Phytohustil®, its excipients or REAo did not impair the mitochondrial membrane potential (MMP). Finally, Phytohustil® and REAo activated the migratory capacity of MΦ.Treatment of PMA-differentiated human THP-1 MΦ with Phytohustilin vitro investigations indicate protective, i.e., anti-oxidative and anti-inflammatory effects of REAo and Phytohustil®, additionally improving the migratory capacity of MΦ. These antiinflammatory effects were similar or even better than diclofenac. Thus, our data support and may explain the positive effect of Phytohustil® observed in patients during the therapy of inflamed buccal mucosal membranes or treatment of cough.The present  Althaea officinalis L. , also called marshmallow, is known as a medicinal plant from ancient time for the treatment of the irritation of laryngopharyngeal mucosa and hence associated dry cough. Many compounds have been extracted from A. officinalis, including starch (25–35%), pectins (11%), saccharose (10%), mucilage (5%), flavonoids, caffeic acid, p-coumaric acid, isoquercitrin, coumarins, phytosterols, tannins, etc., as well as many amino acids  contains water-miscible polysaccharides (acidic polysaccharides), mostly galacturorhamnans, arabinans, glucans, and arabinogalactans  cell line , cultured in RPMI complete medium .The A. officinalis in purified water  with a drug-extract-ratio (DER) of 3-9:1 after drying. 100 g Phytohustil® cough syrup  contains 35.6 g liquid REAo as active ingredient at a DER 19.5-23.5:1 according to DAC (German Drug Codex) 1999. Steigerwald Arzneimittelwerk GmbH also provided excipients, ethanol, propyl-4-hydroxybenzoate EP, methyl-4- hydroxybenzoate EP, sucrose. The concentration of Phytohustil® was calculated in μg/ml dry extract.Steigerwald Arzneimittelwerk GmbH  provided STW42-H marshmallow (REAo) as active constituent. REAo was prepared by maceration of the roots of The REAo was previously characterized and published . A repre4) seeded in 100 μl medium/well in 96-well plates  were incubated overnight in RPMI complete medium, and differentiated into MΦ by incubation with of 0.1 μg/ml phorbol-12-myristate-13-acetate  for 3–5 days; afterward the medium was changed and different concentrations of the REAo, Phytohustil® or its excipients were added. After 48 h treatment viability and cell number were measured as described below.THP-1 cells . PrestoBlue® was directly added to the cells (into the culture medium) at a final concentration of 10% and measured according to the manufacturer’s specifications. Results were expressed in % of viability (OD570nm/600nm of samples × 100/OD570nm/600nm of untreated control). After the PrestoBlue® reaction, the cells were fixed with 4% PFA/PBS and stained with crystal violet (Merck/Sigma-Aldrich Chemie GmbH) solution  and washed; afterward the cells were lysed in a 1% sodium dodecyl sulfate  solution. The crystal violet absorbance was measured at 595 nm (reference 655 nm) to spectrophotometrically determine the total cell number.As a control (=100% viability), we used cells cultured with medium alone (∼ untreated control). Cell viability was assessed using PrestoBlue® or its excipients against cytotoxicity induced by H2O2 treatment was determined using the PrestoBlue® viability assay (Fisher Scientific GmbH) and crystal violet cell quantification assay as described above. In detail, 3 × 104 THP-1 cells were seeded in 100 μl medium/well using 96-well plates  and differentiated with PMA in MΦ, the medium was changed and the MΦ were pre-treated for 48 h with non-cytotoxic concentrations of the REAo, Phytohustil® or its excipients. Afterward, the MΦ were treated with or without 5 mM H2O2 (3 h) and quantification of viability and cell number was performed.The protective effect of the REAo, Phytohustil4 PMA-differentiated MΦ as described above by using 10 μM of the fluorescent mitochondrial dye JC-10  in black Lumox multi well plates . After treatment with the REAo, Phytohustil® or its excipients, the cells were incubated (5–15 min) in serum-free medium containing JC-10 dye loading solution (according to the manufacturer’s protocol). JC-10 accumulates in mitochondria, selectively generating an orange J-aggregate emission profile (590 nm) in healthy cells. However, upon cell injury, as membrane potential decreases, JC-10 monomers are generated, resulting in a shift to green emission (525 nm) to detect subtle changes in MMP. The MMP was measured, considering the fluorescence intensity ratio or relative fluorescence units (RFU), of changes measured at 490 nm excitation/525 nm emission  divided by 540 nm excitation/590 nm emission , using the CytationTM 3 Cell Imaging Multi-Mode Reader (BioTek Instruments). The MMP RFU490/525/RFU540/590 was normalized against the Hoechst 33342 RFU (RFU of cell nuclei) quantified at 350 nm excitation/461 nm emission. Treatment with 12.5 mM H2O2 was used as control of mitochondrial membrane depolarization.Mitochondrial membrane potential was measured in 3 × 104 PMA-differentiated MΦ were seeded in 100 μl medium/well using 96-well plates ; thereafter, the medium was changed and the MΦ were pre-treated for 48 h with different concentrations of REAo, Phytohustil® or its excipients contained in the corresponding concentrations of Phytohustil®. Afterward, the cells were activated for 3 h with 0.01 μg/ml (TNF-α experiments) or 1.0 μg/ml (IL6 experiments) ultrapure lipopolysaccharide (LPS-EB) from E. coli O111:B4  – that is only recognized by toll-like receptor 4 (TLR4). Human TNF-α or IL6 were determined in the culture medium using the assay Duo Set ELISA Development kit  following the manufacturer’s instructions. Afterward the cells were fixed with 4% PFA/PBS and stained with crystal violet and the absorbance was measured as described above. The TNF-α or IL6 OPD absorbance (490 nm and 655 nm reference) was normalized to the crystal violet absorbance. The results were expressed as % of the released TNF-α or IL6 after stimulation with LPS which was considered as 100% release. Additionally, diclofenac sodium salt was used as anti-inflammatory reference substance in our experimental setting (The release of TNF-α or IL6 was determined using enzyme-linked immunosorbent assay (ELISA). In detail, 3 × 10 setting .4 PMA-differentiated MΦ after pre-treatment with REAo, Phytohustil® or its excipients, and afterward with 12.5 mM H2O2 during 3 h. The production of ROS was detected by using 10 μM of the fluorescent DCFDA incubated during 30 min. Total ROS were quantified, considering the fluorescence intensity RFU, measured at 495 nm excitation/529 nm emission and normalized against the Hoechst 33342 RFU (RFU of cell nuclei) measured at 350 nm excitation/461 nm emission using the CytationTM 3 Cell Imaging Multi-Mode Reader (BioTek Instruments).The production of ROS was measured by detecting the fluorescent intensity of the oxidant-sensitive probe 2′-7′-dichlorofluorescin diacetate . ROS were measured in 3 × 10® or its excipients (24 h). The scratch was photographed at time 0 and after 24 h, using an inverted microscope Axiovert 135, equipped with motorized stage and a digital AxioCam MRc camera . Effects on MΦ migration were plotted as a percentage of closure of the scratch (% of the scratch closure = [(Δt) × 100%]/At 0 h; where “At 0 h” is the area of the scratch measured immediately after scratching, “At 24 h” indicates the area of the scratch measured 24 h after scratching and Δt = At 0 h–At 24 h  of REAo, Phytohustil–At 24 h .t test or Mann–Whitney U-test. Data was shown as mean + SEM. p < 0.05 was considered as statistically significant.The SigmaPlot 12 software  was used to carry out statistical analyses by the unpaired Student’s ® cell viability reagent. PrestoBlue® reactivity is based on resazurin, which functions as a cell viability indicator based on mitochondrial enzyme activity  increased the viability by +7.0 to +11.0% in comparison with the negative untreated control  (® and afterward treatment with 0.01 μg/ml LPS significantly (p < 0.05) increased the viability by 10.7% or 13.0%  . Pre-treor 13.0% . The preiability  and the 2O2 and found that 5 mM significantly (p < 0.001) inhibited the viability by 10.4% in comparison with the untreated control (® led to a significant concentration-dependent inhibition of H2O2-induced cytotoxicity by 6.7% (p < 0.01), 9.5% (p < 0.001), and 11.4% (p < 0.001) in comparison to 3 h treatment with 5 mM H2O2 (p < 0.05) 6.3% inhibition of H2O2-induced cytotoxicity (p < 0.001) attenuated the 5 mM H2O2-mediated cytotoxicity by 10.9% (2O2 we found a significantly (p < 0.01) decreased cell number by 16.4% when compared to the untreated control, considered as 100% of CV absorbance (as 100% cell quantity) (® significantly inhibited the H2O2-mediated decreasing cell number by 19.5% (p < 0.01), 19.7% (p < 0.01), and 12.4% (p < 0.05) (2O2 induced-cytotoxicity (2O2-treated cells (We investigated the cytotoxic effects of H control . Pre-tre mM H2O2 . Pre-tretoxicity . Pre-incby 10.9% . After tuantity) . Pre-tre < 0.05) . Incubattoxicity . Pre-treed cells .®, its excipients or REAo on the MMP. We utilized the JC-10 dye, which is useful for determining the MMP by fluorescence microscopy. H2O2 (12.5 mM) is used as a control, which significantly (p < 0.001) increased the MMP depolarization by 76.7% compared to untreated control (∼ 100% MMP integrity)  . Treatme control .® (100 μg/ml), its excipients or REAo (100 μg/ml) did not affect the ROS level compared to untreated control (2O2 (12.5 mM) significantly (p < 0.01) increased the ROS production by 96.9% compared to untreated control  inhibited the ROS production by 52.4% or by 58.7%. compared to MΦ stimulated with 12.5 mM H2O2 alone  the TNF-α release compared to untreated control (p < 0.001) increased TNF-α release of 87.8% compared to untreated control (® (50–500 μg/ml), its excipients or REAo and additionally activated with LPS  to measure TNF-α release. Pre-treatment with Phytohustil® significantly inhibited LPS-induced TNF-α release by 21.6% , 34.8% , 47.3% , 45.2% , and 52.4%  in comparison with LPS-treated MΦ (p < 0.05), 21.5% , and 23.3%  in comparison with LPS-treated MΦ , 28.9% , 23.7% , and 29.1%  (p < 0.001) higher than the untreated control (Treatment (48 h) of MΦ with different concentrations  of Phytohustil control . Incubat control , 7A. Fureated MΦ . Pre-treeated MΦ . In gene0 μg/ml) . Incubat control .® and REAo we determined the inhibition of the IL6 release of LPS-activated MΦ. Treatment of MΦ with REAo or Phytohustil®  alone did not affect the IL6 release compared to untreated control (®’s excipients significantly (p < 0.001) induced the release of IL6 compared to untreated control (p < 0.001) increased IL6 release of 86.3% compared to untreated control , 69.0% , 78.5% , 63.3% , 67.7%  in comparison to LPS-treated MΦ (p < 0.05), 34.7% , 49.3% , and 56.46%  in comparison with LPS treated MΦ  and 44.5%  and 43.7%  but not by 400 μg/ml or 500 μg/ml , 45.0% (p < 0.001), 34.2% (p < 0.05), or 33.1% (p < 0.05) in comparison with LPS treated MΦ  increase of the TNF-α release compared to the untreated control (p < 0.01) compared to LPS. Pre-treatment with 200 μg/ml Phytohustil® as well as REAo showed an inhibition of 31.1% (p < 0.01) and 17.8% (p < 0.01) compared to LPS. Whereas Phytohustil® excipients did not inhibit the LPS-induced TNF-α release when compared to LPS (® was significantly (p < 0.05) 6.7% more effective than 200 μg/ml diclofenac, whereas TNF-α release of REAo was similar to diclofenac  increase of the IL6 release compared to the untreated control (p < 0.001) inhibited the LPS-induced IL6 release by 83.6%. Pre-treatment with 200 μg/ml Phytohustil® revealed similar anti-inflammatory effects like 200 μg/ml diclofenac  induced an IL6 release (The treatment with 0.1 μg/ml LPS (3 h) induced a significant 85.0%  using the scratch assay. The results indicate that the treatment with Phytohustil® significantly stimulated the migratory capacity and scratch closure after 24 h treatment in a concentration-dependent manner by 2.0-fold , 2.8-fold , 3.0-fold  compared to untreated control (p < 0.01), 2.9-fold (p < 0.001) or 3.2-fold compared to untreated control  in vitro.Monocyte migration plays an important role in physiological and pathological processes that include wound healing, repairing mucosal damage and resolution of inflammation . During  healing . These r healing . Pectic  healing , a similin vitro investigations show a significant anti-oxidant and anti-inflammatory activity of Phytohustil® or REAo- an active component of Phytohustil® – in MΦ, with additional effects on cellular integrity and migratory capacity. The anti-inflammatory effects of Phytohustil® or REAo were similar or even better than effects of diclofenac. These findings may support the therapeutical effects of Phytohustil® observed in patients during the treatment of irritated mucosal membranes and appropriate for symptomatic treatment of dry cough.The present All datasets generated for this study are included in the article/GB and RK were responsible for the conception and design of the study. KB, PH, and HS for the data collection, analysis, and image processing. E-UH, performed the HPLC. GB drafted the manuscript and RK revised it critically for important intellectual content. JM, CF, and HA-K discussed the study concept and were responsible for the final approval of the version to be submitted. All authors read and approved the final manuscript.CF, HA-K, and JM are employed by Steigerwald Arzneimittelwerk GmbH. We confirm that we have the correct permissions from the copyright holder to publish a research article about this product. The remaining 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.


For mephedrone and its metabolites, calibration curves were constructed in a calibration range from 0.2 to 5 mmol·L−1; limits of detection, limits of quantification, precision, and repeatability were calculated, and according to Mandel’s fitting test, the linear calibration ranges were determined.Mephedrone, a psychoactive compound derived from cathinone, is widely used as a designer drug. The determination of mephedrone and its metabolites is important for understanding its possible use in medicine. In this work, a method of capillary electrophoresis for the chiral separation of mephedrone and its metabolites was developed. Carboxymethylated β-cyclodextrin was selected as the most effective chiral selector from seven tested cyclodextrin derivates. Based on the simplex method, the optimal composition of the background electrolyte was determined: at pH 2.75 and 7.5 mmol·L Mephedrone, a psychostimulant drug first synthesized in the 1920s, is classified as a synthetic cathinone , for exaThe most commonly used techniques for isolating new psychoactive substances from biological samples are liquid-to-liquid extraction (LLE) and solid-phase extraction (SPE) . LLE is Simple colorimetric tests are used for rapid orientation detection of drugs. However, the identification is very indicative, detecting only the presence of certain structural motifs . HoweverOf the advanced chromatographic techniques, gas and liquid chromatography with a mass spectrometer (GC-MS and LC-MS) are the most suitable for the analysis of synthetic cathinones. In general, the GC-MS technique is used more for drug analysis, mainly due to shorter elution times ,17,18,19The first enantioseparation of cathinone derivatives by capillary electrophoresis (CE) was performed by Mohr et al. in 2012 using sulfated β-cyclodextrin as a chiral selector . As withMephedrone is referred to as a catecholamine reuptake inhibitor ,36,37, bS)-mephedrone is about 50 times more potent as a serotonergic mediator. Nevertheless, the (R)-enantiomer is primarily responsible for the euphoric effects ..N-demeth1; 4-methyl methcathinone) are 4-methylcathinone  and 4-hydroxymethyl methcathinone . These, together with 4-methylephedrine , 4-carboxymethylcathinone , 4-methylnorephedrine , and normephedrone-ω-carboxylic acid (3′-OOH-4-MC), were identified in the urine of users by ultra-high-performance liquid chromatography‒mass spectrometry (UHPLC-MS) [5; 4-CE), 4-MNE (7), 4-hydroxymethylcathinone  [9; 4-CNE), 4-carboxycathinone (4-CC), and 4-hydroxymethylnorephedrine (4-OH-MNE) [The major metabolites of mephedrone (HPLC-MS) . Other m4-OH-MC) , 4-carbo-OH-MNE)  were fouThe aim of this work was to find the optimal conditions for the separation of mephedrone and its metabolites by capillary electrophoresis , using −1, either in a 50 mmol·L−1 phosphate buffer at pH 2.5 (−1 acetate buffer at pH 5 (O-methyl)-β-cyclodextrin (Me-β-CD), 2-hydroxypropylated β-cyclodextrin (HP-β-CD), sulfated β-cyclodextrin (S-β-CD), γ-CD, and carboxymethylated γ-cyclodextrin (CM-γ-CD), CM-β-CD, which is often used for the separation of basic nitrogenous substances [−1 compared to 19 mg·mL−1). Thus, in the search for optimal separation conditions, it is possible to achieve higher concentrations if necessary.The first optimization step was to select a suitable CD. Based on our previous experience with CDs , analyset pH 2.5  or in a  at pH 5 . From th at pH 5 , i.e., βbstances ,53, seemKa CM-β-CD is 3.0–3.5 [Ka of mephedrone is 8.77 [−1.The enantioselective separation of ionic analytes with ionic CDs is controlled by at least two mechanisms. The first is the interaction of the hydrophobic cavity of the CD derivative with the analyte; the second is the electrostatic interaction between the ionic groups of the CD derivative and the analyte . The app 3.0–3.5 . Cathino is 8.77 , for met is 8.77 , for phe is 8.77 , and for is 8.77 . In addi is 8.77 . The sep is 8.77 , we have−1 CM-β-CD; (2) pH 3.5 and 5 mmol·L−1 CM-β-CD , which served as a compared response for the simplex procedure. Simplex followed the rules of the equilateral simplex procedure. After calculating the coordinates of point (8), the simplex looped. Thus, the chiral separation proceeded best under the initial conditions chosen. The simplex procedure is summarized in A mixture of analytes 1–9  with a c−1 CM-β-CD. The other two points correspond to: (2) pH2 = 2.5 − 0.5·0.5 = 2.25 and c2 = 5 + √3⁄2·2.887 = 7.5; (3) pH3 = 2.5 + 0.5·0.5 = 2.75 and c3 = c2, where the values 0.5 and 2.887 correspond to half the sizes of the edges of the first simplex.A half-simplex was designed to more accurately determine the optimal chiral separation conditions B. The fiRkr = 12.5, it is possible in a situation where the simplex forms points (3), (4), and (5), to proceed in two directions, namely to point (6) and point (6′), which gives a small response. Points (3) and (4) also provide the maximum value of the criterion Rkr. Point (3) was chosen as the point meeting the optimal conditions, as in the second case the analysis was extended to more than 100 min. The simplex procedure is summarized in −1 CM-β-CD  and (4) provide the same response,  CM-β-CD .Ka is 4.21, it can be assumed that at pH 2.75 the carboxyl group is 4% deprotonated. Thus, it is clear that pH plays an important role in the separation. A lower or higher pH than 2.75 affects the degree of deprotonation of the used CM-β-CD, and thus the separation efficiency.At pH 2.75, approximately 65–85% of CM-β-CD is in a protonated form. At a low pH, most of the silanol groups on the capillary wall are protonated, leading to low electroosmotic flow, and CM-β-CD carries an approximately one-fold negative charge. At pH 2.75, the amine groups of all analytes are practically 100% protonated. Metabolites 5, 6, and 9 carry a carboxyl group on the aromatic nucleus in the para position. Upon approximation with benzoic acid, whose pThe concentration of CM-β-CD is also an important factor as it affects how much analyte will be complexed with CD. For a given racemic mixture, there is an optimal concentration at which the individual enantiomers will be separated as best as possible ,61. This−1 CM-β-CD and pH 2.75. Reduced peak areas (area/migration time) were subjected to the Dean–Dixon test to exclude outliers, which is adapted to small datasets with unknown distributions.Calibration solutions were measured under the optimized BGE composition, namely at 7.5 mmol·La in the regression equation y = bx + a. In the case of analytes 1, 2, 5, and 7, the size of the intercept is insignificant according to the test. Subsequently, the confidence intervals for the intercept a and the slope b of all calibration equations were calculated. If the significance test of the parameter did not allow for ignoring the intercept, the number of degrees of freedom for calculating tcrit was equal to n − 2, and in the case of a zero intercept n − 1. Ignoring the intercept improved the values of the coefficient of determination R2 and thus allowed us to reduce the confidence intervals , hydrochloric acid (30%), γ-cyclodextrin , carboxymethylated γ-cyclodextrin sodium salt , and heptakis-β-cyclodextrin . Psychoactive amines 1–9 (The following substances were used: ortho-phosphoric acid (50%), β-cyclodextrin , carboxymethylated β-cyclodextrin sodium salt , 2-hydroxypropylated β-cyclodextrin , sulfated β-cyclodextrin , acetic acid (99%), thiourea (99%) , ultrapure water , 1 mol·Lines 1–9  in the fCE separations were performed with an Agilent CE instrument 7100  equipped with a UV-Vis diode-array detector. A bare fused-silica capillary of 375/75 μm od/id and 58.5/50 cm total/effective length was obtained from Polymicro Technologies  was used.−1 sodium phosphate buffer, pH 2.5 (50 mmol·L−1 orthophosphoric acid adjusted to appropriate pH with 1 mol·L−1 NaOH) or acetate buffer, pH 5.0 (50 mmol·L−1 acetic acid adjusted to appropriate pH with 1 mol·L−1 NaOH), and 10 mmol·L−1 of the studied CDs . The background electrolyte used for the optimizing of the chiral selector concentration and pH consisted of 46.6 mmol·L−1 hydrochloric acid (pH 1.5) or 50 mmol·L−1 phosphate buffer  and 0–10 mmol·L−1 of CM-β-CD.The BGE used for the optimizing of the chiral selector consisted of 50 mmol·L−1 NaOH for 30 min, then with H2O for 30 min. Between the runs, the capillary was rinsed at 99.4 kPa first with 0.1 mol·L−1 NaOH for 2 min, then with H2O also for 2 min, and finally with the running buffer again for 2 min . The analytes were injected hydrodynamically at a pressure of 1.5 kPa for 5 s. Separations were performed at 10 kV, 20 kV (anode at the injection capillary end), or −20 kV with a voltage ramp time of 12 s. Detection was carried out at 207 nm during the optimization step and 258 nm for analytes 1, 2, 3, and 6; 236 nm for analyte 5; and 214 nm for analytes 4 and 7  during the calibration step. The capillary was thermostated at 25 °C during the analyses.A new fused-silica capillary was first rinsed with 1 mol·L−1 concentration. For optimizing the chiral selector, the analyte solutions were further mixed and diluted with water to a final concentration of 1 mmol·L−1. For calibration, seven different mixtures of analytes 1–7  were prepared and each mixture was analyzed five times. Individual analytes were dissolved in water at a 20 mmol·Lytes 1–7  at conceRcr was introduced and calculated as follows. For resolution between two neighborhood peaks i and j, if  < 0.5 then Rcr,ij = 0, if 0.5 < Rs,ij < 1.5 then Rcr,ij = 0.5, and if Rs,ij > 1.5 then Rcr,ij = 1. The sum of Rcr,ij = Rcr.For optimizing the BGE composition, the two-dimensional simplex method was used . The opt−1. A total of nine analytes were present in the mixture, and 18 distinguishable peaks were found. Thus, all analytes were separated.In this work, the composition of the basic electrolyte for the chiral separation of mephedrone and its selected metabolites was optimized. A total of seven cyclodextrin derivatives were selected as potential chiral selectors. From the seven tested cyclodextrins, carboxymethylated β-cyclodextrin was selected as the most suitable. Cyclodextrin concentration and pH were determined based on the simplex method for two parameters. The optimized pH was found to be 2.75 and the concentration of carboxymethylated β-cyclodextrin was 7.5 mmol·Ly-axis was neglected in some cases. Finally, linear concentration ranges according to Mandel’s test and detection limits, limits of determination, precision, and repeatability were determined.Furthermore, the data for the construction of calibration dependences were measured. According to the Dean‒Dixon test, outliers were excluded, and regression parameters of calibration lines were calculated. Based on the test of significance of the parameter, the intercept on the