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Background: Chalcone is a broad-spectrum natural product with anti-cancer and anti-inflammatory activities. However, low potency, low selectivity, and serious side effects limit its druggability. L-Tryptophan is an essential precursor molecule of an anti-cancer active substance. Also, the indole moiety inhibits the proliferation of tumor cells by binding to colchicine sites. A decrease in kidney cell activity caused by kidney inflammation is the primary side effect of cancer therapy. Objective: The purpose of this work was to design, synthesize, and perform bioactivity evaluation of novel chalcone derivatives possessing tryptophan moiety with dual activities of anti-cancer and partially restoring the proliferation of normal kidney cells pre-treated with cisplatin. Methods: A series of novel chalcone derivatives possessing tryptophan moiety (5a-5g, 6a-6o) were designed, synthesized, and evaluated for anti-cancer activity against four cancer cell lines (gastric (HGC-27), colon (HCT-116), prostate (PC-3), and lung (A549)), and a human normal cell line (gastric mucosal epithelial (GES-1)). The activity of restoring the proliferation of normal kidney cells pre-treated with cisplatin was evaluated by MTT assay. Cell cycle, apoptosis, and apoptosis proteins (Bax and Bcl-2) were used to evaluate the anti-cancer mechanism of the most potent compound. Moreover, a docking study was performed to explain the high anti-cancer activity of 6n. The expressions of TNF-α, IL- 6, and MCP-1 were detected by ELISA. Results: Most of the compounds exhibited high anti-cancer activity against the HGC-27 cell line and exhibited low toxicity against the normal cell line. Based on three rounds of a structure optimization, 6n was discovered as the most potent compound against HGC-27 cells with an IC50 value of 2.02 μM and an SI value of 28.47. Further studies demonstrated that 6n could induce cell cycle arrest at the G2/M phase and the apoptosis of the HGC-27 cell line by reducing the expression of Bcl-2 and improving the expression level of Bax. Molecular docking result displayed 6n bound to the colchicine site. At the same time, 6n also exhibited moderate activity of restoring the proliferation of normal kidney cells pre-treated with cisplatin by reducing the expression of inflammatory substances. Conclusion: Our findings collectively suggested that 6n should be further studied as a potential anti-cancer agent that could partially restore the proliferation of normal kidney cells pre-treated with cisplatin in gastric cancer patients by an anti-inflammatory pathway.

Objective To assess effect of licochalcone A (LicA) on amyloid-β (Aβ) peptide fragment 25–35–induced nerve injury and reveal the potential molecular mechanisms involved. Methods Viability of SH-SY5Y cells was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay after treatment with Aβ25–35 and/or LicA, following which apoptosis was detected by flow cytometry and Hoechst staining. Then, reactive oxygen species, glutathione, and superoxide dismutase were measured with flow cytometry and spectrophotometry. The ultrastructure of mitochondria was examined by transmission electron microscopy, and the biomarker proteins of autophagy, apoptosis, and phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway were measured with Western blotting. Results LicA improved cell viability and decreased lactate dehydrogenase leakage remarkably in Aβ25–35–induced injury in SH-SY5Y cells. After treatment with LicA, reactive oxygen species, glutathione, and superoxide dismutase levels in cells all were significantly decreased, which indicated that LicA has an antioxidative effect on Aβ25–35-induced oxidative injury. LicA could also significantly reduce Aβ25–35–induced autophagy in SH-SY5Y cells. In the cells injured by Aβ25–35, LicA prevented the transformation from light chain protein 3-I to light chain protein 3-II and reduced the levels of proteins GRP78, GRP94, CHOP, and Bax, but increased the levels of antiapoptotic protein and phosphorylation of PI3K, Akt, and mTOR. These effects of LicA were restored or suppressed by mTOR inhibitor rapamycin or PI3K inhibitor LY294002. Conclusions LicA protects SH-SY5Y cells against Aβ25–35–induced injury, wherein suppressed autophagy and activated PI3K/Akt/mTOR signaling pathway are involved, and mTOR-dependent autophagy at least plays some role.

Acute kidney injury (AKI) is a serious complication in critically ill patients. Accumulating evidences indicated that macrophages play an important pro-inflammatory role in AKI and isoliquiritigenin (ISL) can inhibit macrophagic inflammation, but its role in AKI and the underlying mechanism are unknown. The present study aims to investigate the renoprotective effect of ISL on AKI and the role of Formyl peptide receptors 2 (FPR2) in this process. In this study, cisplatin-induced AKI model and lipopolysaccharide-induced macrophage inflammatory model were employed to perform the in vivo and in vitro experiments. The results showed that ISL strongly relieved kidney injury and inhibited renal inflammation in vivo and suppress macrophagic inflammatory response in vitro. Importantly, it was found that FPR2 was significantly upregulated compared to the control group in AKI and LPS-induced macrophage, whereas it was strongly suppressed by ISL. Interestingly, overexpression of FPR2 with transfection of pcDNA3.1-FPR2 effectively reversed the anti-inflammatory effect of ISL in macrophage, suggesting that FPR2 may be the potential target for ISL to prevent inflammation and improve kidney injury of AKI. Take together, these findings indicated that ISL improved cisplantin-induced kidney injury by inhibiting FPR2 involved macrophagic inflammation, which may provide a potential therapeutic option for AKI.

Praziquantel is the only available drug to treat schistosomiasis, a parasitic disease that currently infects more than 240 million people globally. Due to increasing concerns about resistance and inadequate efficacy there is a need for new therapeutics. In this study, a series of 17 pyrazolines (15–31) and three pyrazoles (32–34) were synthesized and evaluated for their antiparasitic properties against ex vivo adult Schistosoma mansoni worms. Of the 20 compounds tested, six had a 50% effective concentration (EC50) below 30 μM. Our best hit, pyrazoline 22, showed promising activity against adult schistosomes, with an EC50 22 had low cytotoxicity, with selectivity index of 21.6 and 32.2 for monkey and human cell lines, respectively. All active pyrazolines demonstrated a negative effect on schistosome fecundity, with a marked reduction in the number of eggs. Structure–activity relationship analysis showed that the presence of the non-aromatic heterocycle and N-substitution are fundamental to the antischistosomal properties. Pharmacokinetics, drug-likeness and medicinal chemistry friendliness studies were performed, and predicted values demonstrated an excellent drug-likeness profile for pyrazolines as well as an adherence to major pharmaceutical companies’ filters. Collectively, this study demonstrates that pyrazoline derivatives are promising scaffolds in the discovery of novel antischistosomal agents.

The aim of the current study was to evaluate the protective effects and mechanisms of isoliquiritigenin (ISO) on acute renal injury. CCK-8 assays were applied to assess the effects of ISO at different doses (20, 40, and 80 μg/mL) on oxidative damage in human renal HK-2 cells incubated with high glucose. After the diabetic nephropathy (DN) rat model was established, the model animals were randomly assigned to saline-treated control, three model groups received the 10, 20 and 40 mg/kg ISO, respectively, using the healthy Sprague-Dawley (SD) rats as normal control. The blood biochemical indexes, renal functions, oxidative stress, morphological changes, fibrosis- and JAK2/STAT3-related factors in DN model rats were all assessed. The cellular viability of the renal HK-2 cells with oxidative damages were all markedly ameliorated via the incubation of ISO between 10 and 80 μg/mL compared with negative control. In addition, the significantly down-regulated ROS content and up-regulated expression levels of GSH, SOD2, and GPX1 were all observed in ISO-treated groups. Long-term administration of ISO at different doses in DN rats effectively improved general diabetic characteristics and renal morphology. Furthermore, long-term administration of ISO could ameliorate excessive oxidation stress, down-regulate the expression levels of renal fibrosis- and inflammation-related factors, as well as inhibit the JAK2/STAT3 signaling pathway. In conclusion, ISO at all three dosages could efficiently improve the renal injury induced by STZ via ameliorating renal fibrosis, oxidative stress, and inhibiting JAK2/STAT3 signaling pathways in the DN rats.

Overexpression or activation of Yes-associated protein (YAP) is common in cancer cells. Thus, targeting YAP may be a strategy for cancer therapy. Licochalcone A (LicA) is a primary active compound of licorice root and is known to have medicinal effects, such as antioxidant, antibacterial, antiviral, and anticancer effects. However, the anticancer pharmacological mechanism of LicA has not been investigated in cholangiocarcinoma. In this study, we investigated the antiproliferative effect of LicA and the underlying molecular mechanism in HCCC-9810 and RBE human cholangiocarcinoma cells. Our experiments indicated that LicA suppressed the growth of cholangiocarcinoma cells through inactivation of the Hippo pathway. Pescadillo ribosomal biogenesis factor 1 (PES1) was notably upregulated and related to carcinogenesis. We also found that LicA suppressed the expression and nuclear localization of PES1, which was associated with the inhibition of YAP expression and transcriptional activity.

Reactive oxygen/nitrogen species (ROS/RNS) are produced physiologically by several mechanisms, especially during the inflammatory response. However, their overproduction can lead to the evolution of conditions known as oxidative/nitrosative stress, resulting in the establishment of chronic inflammatory diseases. Chalcones are considered as a class of flavonoids having the molecular pattern 1,3-diaryl-2-propen-1-one. In the last few years, the antioxidant property of chalcones has been extensively studied, mainly due to their ability to inhibit the production or scavenging ROS/RNS. The antioxidant activity of chalcones, focusing on the production of ROS/RNS during the inflammatory response, is demonstrated and discussed in the present review. This literature revision was based on the modulatory effects of chalcones against different enzymes, such as superoxide dismutase (SOD), nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, catalase (CAT), myeloperoxidase (MPO) and inducible nitric oxide synthase (iNOS), and, in the scavenging of ROS/RNS. Whenever possible, the structure-activity relationship (SAR) was established. Through the analysis accomplished in this review, it can be observed that the presence of substituents, e.g. hydroxyl, methoxyl, prenyl, and halogen atoms in the chalcones scaffold, often occurs and can improve their modulatory activities, namely, in the production of ROS/RNS during the inflammatory response.

To evaluate the toxicity of three synthetic chalcones administered intraperitoneally to BALB/c mice.The median lethal dose (LD50) was estimated by Dixon's Up-and-Down method. Subchronic toxicity of chalcones was evaluated at 20 and 40 mg/kg for 21 days. Behavioral, physiological, biochemical, and histological toxic effects were evaluated.Chalcone 43 produced mucus in feces, visceral damage (liver) and alterations in organ coefficient (kidney, p = 0.037 and brain, p = 0.008) when compared to the control group. In addition, histological analysis showed that this chalcone produced edema, inflammation and necrosis in the evaluated organs, although there was no significant difference with the control. None of the biochemical parameters differed significantly between the treatment groups at 40 mg/kg dose and the control.The LD50 for all three chalcones was greater than 550 mg/kg of body weight. Chalcones 40 and 42 were found to be relatively non-toxic. Both can be considered safe for intraperitoneal application in BALB/c mice and, consequently, are potential candidates for use in the treatment of leishmaniasis.Evaluar la toxicidad de tres chalconas sintéticas administradas por vía intraperitoneal en ratones BALB/c.La dosis letal media (DL50) se estimó por el método Up-and-Down de Dixon. La toxicidad subcrónica de las chalconas se evaluó a 20 y 40 mg/kg por 21 días. Se evaluó el efecto tóxico a nivel de comportamiento, fisiológico, bioquímico e histológico.La chalcona 43 generó moco en las heces, daño visceral (hígado) y alteración en el coeficiente de órganos (riñón, p = 0,037 y cerebro, p = 0,008) en comparación con el grupo control. Además, en el análisis histológico se observó que esta chalcona produjo edema, inflamación y necrosis en los órganos evaluados, aunque no hubo diferencia significativa con el control. Todos los parámetros bioquímicos no difirieron significativamente entre los grupos de tratamiento a dosis de 40 mg/kg y el control.La DL50 para las tres chalconas fue superior a 550 mg/kg de peso corporal. Las chalconas 40 y 42 son relativamente no tóxicas. Ambas pueden considerarse seguras para la aplicación vía intraperitoneal en ratones BALB/c y, en consecuencia, son posibles candidatas para ser usadas en el tratamiento contra las leishmaniosis.

Chalcones are an interesting class of compounds endowed with a plethora of biological activities beneficial to human health. These chemotypes have continued to attract increased research attention over the years; hence, numerous natural and synthetic chalcones have found with interesting anticancer activities through the inhibition of various molecular targets including ABCG2, BCRP, P-glycoprotein, 5α-reductase, Androgen Receptor (AR), Histone Deacetylases (HDAC), Sirtuin 1, proteasome, Vascular Endothelial Growth Factor (VEGF), Cathepsin-K, tubulin, CDC25B phosphatase, Topoisomerase, EBV, NF-κB, mTOR, BRAF, and Wnt/β-catenin. Moreover, the study of intrinsic mechanisms of action, particularly relating to specific cellular pathways and modes of engagement with molecular targets, may help medicinal chemists to develop more effective, selective, and cost-effective chalcone-based anticancer drugs. This review, therefore, sheds light on the effect of structural variations on the anticancer potency of chalcone hybrids reported in 2018-2019 alongside their mechanism of action, molecular targets, and potential impacts on effective cancer chemotherapy.

Cancer is the second death cause worldwide, with breast and colon cancer among the most prevalent types. Traditional treatment strategies have several side effects that inspire the development of novel anticancer agents derived from natural sources, like chalcone derivatives. For this investigation, twenty-three chalcones (4a-w) were synthesized and evaluated as antiproliferative agents against MCF-7 and Caco-2 cells, finding three and two compounds with similar or higher antiproliferative activity than daunorubicin, while only two chalcones showed better selectivity indexes than daunorubicin on MCF-7. From these results, we developed good-performance QSAR models (r > 0.850, q2>0.650), finding several structural features that could modify chalcone activity and selectivity. According to these models, chalcones 4w and 4t have high potency and selectivity against Caco-2 and MCF-7, respectively, which make them attractive candidates for hit-to-lead development of ROS-independent pro apoptotic agents.

Loss of neuronal tissue is a hallmark of age-related neurodegenerative diseases. Since adult neurogenesis has been confirmed in the human brain, great interest has arisen in substances stimulating the endogenous neuronal regeneration mechanism based on adult neural stem cells. Medicinal plants are a valuable source of neuroactive small molecules. In the structure-activity study presented here, the activities of prenyl- and pyranochalcones were compared to each other, using a differentiation assay based on the doublecortin promoter sequences. The latter revealed that the pyrano ring is a crucial structural element for the induction of neuronal differentiation of adult neural stem cells, while compounds with a prenyl group show significantly lower activities. Furthermore, a decrease of pro-differentiation activity was observed following structural modifications, such as substitutions on the pyrano ring and on the B-ring of the chalcone. We also initiated the elucidation of the structural characteristics of the newly discovered lead substance xanthohumol C, which correlated with the activation of the doublecortin promoter during neuronal differentiation.

Background: The inhibition of α-amylase enzyme is one of the best therapeutic approach for the management of type II diabetes mellitus. Chalcone possesses a wide range of biological activities. Objective: In the current study chalcone derivatives (1-16) were synthesized and evaluated their inhibitory potential against α-amylase enzyme. Methods: For that purpose, a library of substituted (E)-1-(naphthalene-2-yl)-3-phenylprop-2-en-1-ones was synthesized by Claisen-Schmidt condensation reaction of 2-acetonaphthanone and substituted aryl benzaldehyde in the presence of base and characterized via different spectroscopic techniques such as EI-MS, HRESI-MS, 1H-, and 13C-NMR. Results: Sixteen synthetic chalcones were evaluated for in vitro porcine pancreatic α-amylase inhibition. All the chalcones demonstrated good inhibitory activities in the range of IC50 = 1.25 ± 1.05 to 2.40 ± 0.09 μM as compared to the standard commercial drug acarbose (IC50 = 1.34 ± 0.3 μM). Conclusion: Chalcone derivatives (1-16) were synthesized, characterized, and evaluated for their α- amylase inhibition. SAR revealed that electron donating groups in the phenyl ring have more influence on enzyme inhibition. However, to insight the participation of different substituents in the chalcones on the binding interactions with the α-amylase enzyme, in silico (computer simulation) molecular modeling analyses were carried out.

Parkinson's disease is a progressive neurodegenerative disorder that affects mostly elderly people above the age of 60. Previously, we have reported that the ethoxylated chalcone derivative (E)-1-(4-ethoxyphenyl)-3-(fluorophenyl)prop-2-en-1-one (E7) showed potent, reversible, and competitive MAO-B inhibition with an IC50 value of 0.053 μm. The present study aims to investigate the anti-Parkinson activity of compound E7 in a haloperidol-induced animal model of mice. The disease was induced with haloperidol (1 mg/kg, intraperitoneal route) once daily for 21 days. E7 was given at dose levels of 10, 20, and 30 mg/kg/day for 21 days, consecutively. Behavioural tests were carried out during and at the end of the study. Biochemical analyses such as oxidative stress biomarkers and neurotransmitters were quantified on the brain homogenate at the end of the study. Behavioural results showed that there is a marked improvement in locomotor activity and motor coordination in the treatment group. Oxidative stress biomarkers such as SOD, CAT, and GSH levels were increased dose-dependently with a maximum at 30 mg/kg, whereas the dose-dependent decrease (30 mg/kg) in the MDA and nitrite levels were observed in the treatment groups. Levels of neurotransmitters, such as dopamine, serotonin, and noradrenaline, were increased in the treatment groups while dopamine and noradrenaline levels were more than in the standard treated group. MAO-B level was also decreased dose dependently in the treatment group in comparison with the control group. Based on the findings, it was concluded that the E7 compound exhibited anti-Parkinson activity which was more evident at 30 mg/kg oral dose as evaluated by the haloperidol-induced animal model of mice.

Radiation therapy is common in the current procedures of cancer treatment, but in many cases, radiation resistance of cancerous tissue limits efficacy in clinical applications. Therefore, the use of radiosensitizers has been introduced as an effective strategy to increase the efficiency of radiotherapy. Butein (2′, 3, 4, 4′-Tetrahydroxychalcone), a polyphenolic compound of flavonoids family, presents anti-cancer properties and inhibits the signaling pathways associated with radiation resistance. Therefore, we hypothesized that butein in combination with radiation may increase radiosensitivity. To evaluate the radiosensitizing effect of butein, we used MKN-45 cell line and performed several assays such as MTT, soft-agar colony formation, apoptosis, cell cycle, and comet assays. Based on obtained results, butein significantly enhanced radiosensitivity of MKN-45 cells. Butein treatment abrogated the radiation-induced G2/M cell cycle arrest, increased DNA damage, enhanced apoptosis, and reduced colony-forming ability of irradiated cells. This study on MKN-45 cells demonstrates that combination of butein with radiotherapy increases its radiosensitivity by abrogating the radiation-induced G2/M blockage, impairing DNA repair, and enhancing apoptotic and reproductive cell death. Therefore, we suggest butein as a candidate for combination with radiation therapy to decrease dose of radiation delivered to the patients and its corresponding side effects.

Human pluripotent stem cells (hPSCs) hold great promise for applications in cell therapy and drug screening in the cardiovascular field. Bone morphogenetic protein 4 (BMP4) is key for early cardiac mesoderm induction in hPSC and subsequent cardiomyocyte derivation. Small‐molecular BMP4 mimetics may help to standardize cardiomyocyte derivation from hPSCs. Based on observations that chalcones can stimulate BMP4 signaling pathways, we hypothesized their utility in cardiac mesoderm induction. To test this, we set up a two‐tiered screening strategy, (1) for directed differentiation of hPSCs with commercially available chalcones (4’‐hydroxychalcone [4’HC] and Isoliquiritigen) and 24 newly synthesized chalcone derivatives, and (2) a functional screen to assess the propensity of the obtained cardiomyocytes to self‐organize into contractile engineered human myocardium (EHM). We identified 4’HC, 4‐fluoro‐4’‐methoxychalcone, and 4‐fluoro‐4’‐hydroxychalcone as similarly effective in cardiac mesoderm induction, but only 4’HC as an effective replacement for BMP4 in the derivation of contractile EHM‐forming cardiomyocytes., Have a little heart: A screen for mesoderm inducing chalcones in human pluripotent stem cell cultures identified 4’‐hydroxychalcone (4’HC) as an effective replacement for bone‐morphogenetic protein 4 (BMP4) in supporting the derivation of engineered heart muscle (EHM)‐formation competent cardiomyocytes.

The incidence of ulcerative colitis (UC), one of the two types of inflammatory bowel disease, is increasing in many countries. Various natural products have been demonstrated with therapeutic potentials for UC. Herein, the therapeutic effects and mechanisms of isobavachalcone (IBC), a natural chalcone, were evaluated in dextran sulfate sodium (DSS)-induced colitis mice and lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. The results demonstrated that IBC treatment significantly improved the clinical symptoms, assessed by the disease activity index (DAI) scores and the histological changes of the colon. The levels of myeloperoxidase (MPO), TNF-α, IL-6, IL-1β, and prostaglandin E2 (PGE2) in colon tissues were suppressed by IBC. The upregulation of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and NF-κB p65 in colon tissues were reversed by IBC as well. Furthermore, IBC significantly inhibited LPS-triggered secretion of TNF-α, IL-6, and nitrite, and nuclear translocation of NF-κB p65, in RAW264.7 cells. The luciferase reporter assay indicated that IBC significantly inhibited LPS-triggered transcription of toll-like receptor 4 (TLR4). Molecular docking results showed that the binding pocket of IBC was adjacent to Ser276 of p65-p50 heterodimer and IBC could form H-bond with Thr191. Collectively, these results demonstrated that IBC ameliorated colitis in mice possibly through inhibition of NF-κB p65.

Background Diabetes is one of the growing health problems worldwide, and scientists have been striving to find effective treatment methods. In this regard, chalcones have frequently been targeted by many researchers owing to their diverse biological activities. Methods Here, the Claisen–Schmidt condensation reaction was applied to synthesize five chalcone derivatives. The chalcone derivatives were evaluated for their relative antidiabetic activities in vivo using streptozotocin (STZ)-induced diabetic mice. Besides, the compounds were assessed for their reduction in postprandial hyperglycemia at 50 and 100 mg/kg dose levels against a standard drug, glibenclamide. In addition, the structure–activity relationship (SAR) was analyzed to determine the effect of structural modification in chalcones activity. Results A dose-dependent reduction in postprandial hyperglycemia was observed. The highest reduction in blood glucose level (BGL) was achieved by compound 3 at a dose of 100 mg/kg (39%). This was found to be even higher than glibenclamide (34.5%). In the STZ-induced diabetic animal model, all test compounds showed comparable efficacy with glibenclamide. The SAR analysis revealed that the incorporation of electron-donating groups at position 5 of the benzaldehyde ring and position 2 of the acetophenone ring is promising to increase the antihyperglycemic activities of chalcones. Conclusion The chalcone derivatives considered in this study could be used as potential lead compounds in the discovery of effective drugs to treat diabetes mellitus., Graphical Abstract

Background and Objectives: Despite various technological advances for the treatment of cancer, the identification of new chemical entities with potent anticancer effects remain an indispensable requirement of the time due to multi-drug resistance exhibited by previously developed anticancer drugs. Particularly, the hybrid drugs incorporating two individual bioactive pharmacophores present medicinally important structural leads, thus improving the pharmacodynamic profile of the drug molecules. The antiproliferative and pro-apoptotic activity of the carbazole-chalcone hybrids on human breast and cervical cancer cells will be examined. Materials and Methods: To overcome such complications, in the current study, we evaluated the cytotoxic effects of carbazole-chalcone hybrids on human breast adenocarcinoma (MCF-7), cervical adenocarcinoma (HeLa) cells and normal cells i.e., baby hamster kidney cells (BHK-21) using MTT (dimethyl-2-thiazolyl-2,5-diphenyl-2H-tetrazolium bromide) assay. The mechanistic studies were performed on potent compound 4g by fluorescent microscopic studies, release of lactate dehydrogenase (LDH) and mitochondrial membrane potential, activation of caspase-9 and -3 and flow cytometric analysis. Results: As revealed by MTT assay, compound 4g was identified as the most potent derivative among the tested series with IC50 values of 5.64 and 29.15μM against HeLa and MCF-7 cells, respectively. The results were compared with cisplatin. Fluorescent microscopic studies using 4′,6-diamidino-2-phenylindole (DAPI) and Propidium Iodide (PI) staining confirmed the occurrence of apoptosis in HeLa cells treated with the most active compound 4g. Moreover, compound 4g also triggered the release of Lactate Dehydrogenase (LDH) in treated HeLa and MCF-7 cells while a fluorescence assay displayed a remarkable increase in the activity of caspase-9 and -3. Moreover, flow cytometric results revealed that compound 4g caused G0/G1 arrest in the treated HeLa cells. Conclusion: Our results demonstrated that the compound 4g possesses chemotherapeutic properties against breast cancer and cervical adenocarcinoma cells, thus warranting further research to test the anticancer potential of this compound at preclinical and clinical level.

Background Cisplatin is an effective chemotherapy but its main side effect, acute kidney injury, limits its use. Panduratin A, a bioactive compound extracted from Boesenbergia rotunda, shows several biological activities such as anti-oxidative effects. The present study investigated the nephroprotective effect of panduratin A on cisplatin-induced renal injury. Methods We investigated the effect of panduratin A on the toxicity of cisplatin in both mice and human renal cell cultures using RPTEC/TERT1 cells. Results The results demonstrated that panduratin A ameliorates cisplatin-induced renal toxicity in both mice and RPTEC/TERT1 cells by reducing apoptosis. Mice treated with a single intraperitoneal (i.p.) injection of cisplatin (20 mg/kg body weight (BW)) exhibited renal tubule injury and impaired kidney function as shown by histological examination and increased serum creatinine. Co-administration of panduratin A (50 mg/kg BW) orally improved kidney function and ameliorated renal tubule injury of cisplatin by inhibiting activation of extracellular signal-regulated kinase (ERK)1/2 and caspase 3. In human renal proximal tubular cells, cisplatin induced cell apoptosis by activating pro-apoptotic proteins (ERK1/2 and caspase 3), and reducing the anti-apoptotic protein (Bcl-2). These effects were significantly ameliorated by co-treatment with panduratin A. Interestingly, panduratin A did not alter intracellular accumulation of cisplatin. It did not alter the anti-cancer efficacy of cisplatin in either human colon or non-small cell lung cancer cell lines. Conclusions The present study highlights panduratin A has a potential protective effect on cisplatin's nephrotoxicity.

Isoliquiritigenin (ISO) is a flavonoid extracted from the root of licorice, which serves various biological and pharmacological functions including antiinflammatory, antioxidation, liver protection, and heart protection. However, the mechanism of its action remains elusive and the direct target proteins of ISO have not been identified so far. Through cell-based screening, we identified ISO as a potent lipid-lowering compound. ISO treatment successfully ameliorated fatty acid-induced cellular lipid accumulation and improved nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) by increasing PPARα-dependent lipid oxidation and decreasing SREBPs-dependent lipid synthesis. Both these signaling required the activation of SIRT1. Knockdown of SIRT1 resulted in the reversal of ISO beneficiary effects suggesting that the lipid-lowering activity of ISO was regulated by SIRT1 expression. To identify the direct target of ISO, limited proteolysis combined with mass spectrometry (LiP-SMap) strategy was applied and IQGAP2 was identified as the direct target for ISO in regulating lipid homeostasis. In the presence of ISO, both mRNA and protein levels of SIRT1 were increased; however, this effect was abolished by blocking IQGAP2 expression using siRNA. To explore how IQGAP2 regulated the expression level of SIRT1, proteome profiler human phospho-kinase array kit was used to reveal possible phosphorylated kinases and signaling nodes that ISO affected. We found that through phosphorylation of CREB, ISO transduced signals from IQGAP2 to upregulate SIRT1 expression. Thus, we not only demonstrated the molecular basis of ISO in regulating lipid metabolism but also exhibited for the first time a novel IQGAP2-CREB-SIRT1 axis in treating NAFLD/NASH.

Background.: Inflammation is a multifactorial process reflecting the response of the organism to various stimuli and is associated with a number of disorders such as arthritis, asthma and psoriasis, which require long-lasting or repeated treatment. Objective.: The aim of this paper is to evaluate the anti-inflammatory activity of previous synthesized thiazole-based chalcone derivatives. Methods: Chalcones were synthesized via Cliazen-Schmidt condensation1-(4-methyl-2- alkylamino)thiazol-5-yl) ethanone with a corresponding aromatic aldehyde. For the evaluation of possible anti-inflammatory activity, carrageenan mouse paw edema was used. Results.: Eight out of thirteen tested chalcones showed anti-inflammatory activity in a range of 51- 55%. Prediction of toxicity revealed that these compounds are not toxic. Conclusion.: In general, it can be concluded that these compounds can be used for further modifications in order to develop more active and safe agents.

Drug resistance, including multidrug resistance resulting from different defensive mechanisms in cancer cells, is the leading cause of the failure of the cancer therapy, posing an urgent need to develop more effective anticancer agents. Chalcones, widely distributed in nature, could act on diverse enzymes and receptors in cancer cells. Accordingly, chalcone derivatives possess potent activity against various cancers, including drug-resistant, even multidrug-resistant cancer. This review outlines the recent development of chalcone derivatives with potential activity against drug-resistant cancers covering articles published between 2010 and 2020 so as to facilitate further rational design of more effective candidates.

Licochalcone A (LA), a flavonoid found in licorice, has anticancer, antioxidant, anti-inflammatory, and neuroprotective properties. Here, we explored the effect of injecting LA into the tail vein of middle-aged C57BL/6 mice on their cognitive ability as measured by the Morris water maze (MWM) test and cerebral blood flow (CBF). The related mechanisms were assessed via RNA-seq, and T (CD3e+) and B (CD45R/B220+) cells in the spleen and whole blood were quantified via flow cytometry. LA improved the cognitive ability, according to the MWM test results, and upregulated the CBF level of treated mice. The RNA-seq results indicate that LA affected the interleukin (IL)-17 signaling pathway, which is related to T- and B-cell proliferation, and the flow cytometry data suggest that LA promoted T- and B-cell proliferation in the spleen and whole blood. We also performed immune reconstruction via a tail vein injection of lymphocytes into B-NDG (NOD-PrkdcscidIl2rgtm1/Bcge) mice before treating them with LA. We tested cognitive ability by subjecting these animals to new object recognition tests and quantified the splenic and whole blood T and B cells. Cognitive ability improved after immune reconstruction and LA treatment, and LA promoted T- and B-cell proliferation in the spleen and whole blood. This study demonstrates that LA, by activating the IL-17 signaling pathway, promotes T- and B-cell proliferation in the spleen and whole blood of mice and improves cognitive ability. Thus, LA may have immune-modulating therapeutic potential for improving cognition.

Immune response plays a vital role in the pathogenesis of neuropathic pain. Immune response-targeted therapy becomes an effective strategy for treating neuropathic pain. Licochalcone A (Lic-A) possesses anti-inflammatory and neuroprotective effects. However, the potential of Lic-A to attenuate neuropathic pain has not been well explored. To investigate the protective effect and evaluate the underlying mechanism of Lic-A against neuropathic pain in a rat model. Chronic constriction injury (CCI) surgery was employed in rats to establish neuropathic pain model. Rats were intraperitoneally administrated with Lic-A (1.25, 2.50 and 5.00 mg/kg) twice daily. Mechanical withdrawal threshold and thermal withdrawal latency were used to evaluate neuropathic pain. After administration, the lumbar spinal cord enlargement of rats was collected for ELISA, Western blot and immunofluorescence analysis. Mechanical withdrawal threshold and thermal withdrawal latency results showed that Lic-A significantly attenuated CCI-evoked neuropathic pain in dose-dependent manner. Lic-A administration also effectively blocked microglia activation. Moreover, Lic-A suppressed p38 phosphorylation and the release of inflammatory factors such as tumor necrosis factor-α, interleukin-1 and interleukin-6. Our findings provide evidence that Lic-A may have the potential to attenuate CCI-evoked neuropathic pain in rats by inhibiting microglia activation and inflammatory response.

Food additives are widely used in our daily life, and the side-effects caused by them have gained extensive attention around the world. Notably, constituent-oriented metabolites, in some sense, always contribute to pharmacological changes, inducing toxicity, therapeutic effects, etc. Characterization of the metabolites and their potential functions is of great importance to the practical applications. In this work, an integrated strategy by combining metabolite profiling and network pharmacology was applied to characterize the metabolic features and reveal pharmacological changes of neohesperidin dihydrochalcone (NHDC) in vivo to demonstrate its pharmacological mechanism and potential functions. As a result, a total of 19 metabolites (3 in plasma, 19 in urine, 8 in feces, 3 in heart, 5 in liver, 0 in spleen, 1 in lung, 2 in kidneys and 2 in brain) were screened and 18 of them were characterized for the first time. Phase I metabolic reactions of hydrolysis and phase II reactions of glucuronidation, sulfation, glutamylation, N-butyryl glycylation and lactylation were the main metabolic reactions of NHDC in vivo. Moreover, the results analyzed by network pharmacology revealed that, in addition to common pathways (steroid hormone biosynthesis) of NHDC, metabolites' targets were involved in pathways in cancer, ovarian steroidogenesis, proteoglycans in cancer, PI3K-Akt signaling pathway and progesterone-mediated oocyte maturation, indicating that these functional changes might result in potential novel functions or other side-effects, such as a disorder of steroid hormones. Our work provided the metabolic features and functional modifications of NHDC in vivo for the first time, and meaningful information for further pharmacological validations or potential functions is supplied.

Background: Liver fibrosis leads to loss of liver function. Xiaoyaosan decoction (XYS) as a classical Traditional Chinese Medicine (TCM) formula is used to treatment liver fibrosis in clinical and alleviated CCl4-induced liver fibrosis in our previous study.Xiaoyaosan decoction was composed of many ingredients, and the active ingredients is not clear. The Aim of this study is to explore the clarified compounds or compound combinations to treat liver fibrosis.Methods: Network pharmacology combined with transcriptomics analysis were used to analyze the Xiaoyaosan decoction (XYS) and liver depression and spleen deficiency syndrome liver fibrosis. This consisted constructed XYS-Syndrome-liver fibrosis network, the predict formula named LLAAF was develop from the network by topological analysis according to network stability. The anti-fibrosis effect was evaluated by in vitro and in vivo study.Results: According to the network XYS-Syndrome-liver fibrosis network, luteolin, licochalcone A, aloe-emodin, and acacetin formula (LLAAF) was predicted from 8 key compounds and 255 combinations in XYS, and LLAAF had a synergistic effect on the proliferation inhibition of hepatic stellate cells (HSCs) compared to each individual compound alone. The treatment of XYS and LLAAF showed a similar anti-liver fibrotic effect that reduced histopathological changes of liver fibrosis, Hyp content and levels of α-SMA and collagen I in CCl4-induced liver fibrosis in rat. Transcriptomics analysis revealed LLAAF regulated PI3K-Akt, AMPK, FoxO, Jak-STAT3, P53, cell cycle, focal adhesion, and PPAR signaling. Furthermore, LLAAF was confirmed to regulate Jak-STAT and PI3K-Akt-FoxO signaling in vitro and in vivo.Conclusions: This study developed a novel anti-liver formula, LLAAF from XYS demonstrated the anti-liver fibrotic activity may be involved in the regulation of Jak-STAT and PI3K-Akt-FoxO signaling.

Background Recent years, survival rates of human with high-risk acute myeloid leukaemia (AML) have not raised substantially. This research aimed to investigate the role of 4′-O-Methylbroussochalcone B, for the treatment of human AML. Methods Firstly, we evaluated the effects of six chalcones on AML cells activity by MTT assay. Immunofluorescence staining, tubulin polymerization assay and N,N′-ethylenebis (iodoacetamide) (EBI) competition assay were performed on ML-2 cells. Transwell and apoptosis assay were also utilized in ML-2 cells and OCI-AML5 cells. The expressions of migration-related proteins, apoptosis-related proteins and Wnt/β-catenin pathway were detected by Western Blot. Results The results found six chalcones exhibited the anti-proliferative activity against different AML cell lines. Based on the results of immunofluorescence staining, tubulin polymerization assay and EBI competition assay, 4′-O-Methylbroussochalcone B was discovered to be a novel colchicine site tubulin polymerization inhibitor. 4′-O-Methylbroussochalcone B could induce apoptosis, inhibit proliferation and migration of ML-2 cells and OCI-AML5 cells. The cells were arrested in the G2-M phase by the treatment of 4′-O-Methylbroussochalcone B. In addition, 4′-O-Methylbroussochalcone B regulated MAPK and Wnt/β-catenin pathways in AML cells. Conclusion 4′-O-Methylbroussochalcone B might inhibit proliferation and migration of the AML cells by MAPK and Wnt/β-catenin pathways as a tubulin polymerization inhibitor. It is promising for 4′-O-Methylbroussochalcone B to become a new drug to treat AML.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that affects 50 million people worldwide. The current medicines have modest benefits in preventing or curing AD. Thus, it is urgent to discover drugs with the potential to change the progression of the disease. The primary clinical symptoms are memory loss and anxiety, while the critical pathological characteristics are Aβ plaques and hyperphosphorylated tau tangles. In this study, isobavachalcone (ISO), isolated from Psoralea corylifolia, was administered to 3×Tg-AD mice. It has been shown that this compound could significantly improve anxiety, memory and recognition deficits in the AD mice, attenuate the accumulation of Aβ oligomers, reduce the hyperphosphorylation of tau, and prevent the production of tau filaments. The metabolomic analysis implicates that the most probable pathways affected by ISO were bile secretion, tyrosine metabolism, and purine metabolism. In summary, ISO possesses the potential for further development as a drug candidate for AD.

Defined differently from apoptosis, necrosis, and autophagy, ferroptosis has been implicated in acute kidney injury (AKI) such as ischemia-reperfusion injury induced AKI, folic acid caused AKI and cisplatin induced AKI. However, whether ferroptosis is involved in LPS induced AKI could be remaining unclear and there is still a lack of therapies associated with ferroptosis in LPS induced AKI without side effects. This study aimed to elucidate the role of isoliquiritigenin (ISL) in ferroptosis of LPS-induced AKI. We used LPS to induce renal tubular injury, followed by treatment with ISL both in vitro and in vivo. Human renal tubular HK2 cells were pretreated with 50 μM or 100 μM ISL for 5 h before stimulation with 2 μg/mL LPS. Mice were administered a single dose of either 50 mg/kg ISL orally or 5 mg/kg ferroptosis inhibitor ferrostatin-1 intraperitoneally before 10 mg/kg LPS injection. We found that LPS could induce mitochondria injury of renal tubular presented as the shape of mitochondria appeared smaller than normal with increased membrane density and are faction or destruction of mitochondrial crista through scanning electron microscope. Ferrostatin-1 significantly protected mice against renal dysfunction and renal tubular damage in LPS-induced AKI. ISL inhibited Fe2+ and lipid peroxidation accumulation in LPS-stimulated HK2 cells. It also increased the expression of GPX4 and xCT, reduced the expression of HMGB1 and NCOA4 then attenuated mitochondria injury in renal tubular following LPS stimulation. These results indicated the potential role of ISL against ferritinophagy-mediated ferroptosis in renal tubular following LPS stimulation.

The objectives of this study were to investigate the effects of marein, a major bioactive compound in functional food Coreopsis tinctoria, in hypertrophic H9c2 cells. Treating angiotensin II/hypoxia-stimulated H9c2 cells with marein led to decreasing cell surface area, intracellular total protein, atrial natriuretic peptide, and free fatty acids levels, but increasing glucose level. Marein treatment decreased hypoxia inducible factor-1α (HIF-1α), peroxisome proliferator activated receptor γ (PPARγ), medium chain acyl-coenzyme A dehydrogenase, glucose transporter-4, and glycerol-3-phosphate acyltransferase protein expressions, and increased PPARα, fatty acid transport protein-1, carnitine palmitoyltransferase-1, and pyruvate dehydrogenase kinase-4 protein expressions. Similar results were observed in HIF-1α-overexpressing H9c2 cells, whereas these effects were abolished in siRNA-HIF-1α-transfected H9c2 cells. It was concluded that marein could ameliorate abnormal glucolipid metabolism in hypertrophic H9c2 cells, and the effects could be attributable to reduction of HIF-1α expression and subsequent regulation PPARα/γ-mediated lipogenic gene expressions.

The in vitro antimicrobial properties of some chalcones (1a–1c ) and chalcone tethred 1,4-disubstituted 1,2,3-triazoles (2a–2u) towards different microbial strains viz. Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Aspergillus niger and Candida albicans are reported. Compounds 2g and 2u exhibited better potency than the standard Fluconazole with MIC values of 0.0063 µmol/mL and 0.0068 µmol/mL, respectively. Furthermore, molecular docking was performed to investigate the binding modes of two potent compounds 2q and 2g with E. coli topoisomerase II DNA gyrase B and C. albicans lanosterol 14α-demethylase, respectively. Based on these results, a statistically significant quantitative structure activity relationship (QSAR) model was successfully summarized for antibacterial activity against B. subtilis.

Hydroxylated chalcones are phytochemicals which are biosynthetic precursors of flavonoids and their 1,3-diaryl-prop-2-en-1-one structure is used as a scaffold for drug development. In this study, the structure-dependent activation of aryl hydrocarbon receptor (AhR)-responsive CYP1A1, CYP1B1, and UGT1A1 genes was investigated in Caco2 colon cancer cells and in non-transformed young adult mouse colonocytes (YAMC) cells. The effects of a series of di- and trihydroxychalcones as AhR agonists was structure dependent with maximal induction of CYP1A1, CYP1B1, and UGT1A1 in Caco2 cells observed for compounds containing 2,2′-dihydroxy substituents and this included 2,2′-dihydroxy-, 2,2′,4′-trihydroxy-, and 2,2′,5′-trihydroxychalcones. In contrast, 2′,4,5′-, 2′3′,4′-, 2′,4,4′-trihydroxy, and 2′,3-, 2′,4-, 2′,4′-, and 2′,5-dihydroxychalcones exhibited low to non-detectable AhR activity in Caco2 cells. In addition, all of the hydroxychalcones exhibited minimal to non-detectable activity in YAMC cells, whereas 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induced CYP1A1, CYP1B1, and UGT1A1 in Caco2 and YAMC cells. The activity of AhR-active chalcones was confirmed by determining their effects in AhR-deficient Caco2 cells. In addition, 2,2′-dihydroxychalcone induced CYP1A1 protein and formation of an AhR-DNA complex in an in vitro assay. Simulation and modeling studies of hydroxylated chalcones confirmed their interactions with the AhR ligand-binding domain and were consistent with their structure-dependent activity as AhR ligands. Thus, this study identifies hydroxylated chalcones as AhR agonists with potential for these phytochemicals to impact AhR-mediated colonic pathways.

Diabetic nephropathy (DN) as a global health concern is closely related to inflammation and oxidation. Isoliquiritigenin (ISL), a natural flavonoid compound, has been demonstrated to inhibit inflammation in macrophages. Herein, we investigated the effect of ISL in protecting against the injury in STZ-induced type 1 DN and in high glucose-induced NRK-52E cells. In this study, it was revealed that the administration of ISL not only ameliorated renal fibrosis and apoptosis, but also induced the deterioration of renal function in diabetic mice. Mediated by MAPKs and Nrf-2 signaling pathways, respectively, upstream inflammatory response and oxidative stress were neutralized by ISL in vitro and in vivo. Moreover, as further revealed by the results of molecular docking, sirtuin 1 (SIRT1) binds to ISL directly, and the involvement of SIRT1 in ISL-mediated renoprotective effects was confirmed by studies using in vitro models of SIRT1 overexpression and knockdown. In summary, by reducing inflammation and oxidative stress, ISL has a significant pharmacological effect on the deterioration of DN. The benefits of ISL are associated with the direct binding to SIRT1, the inhibition of MAPK activation, and the induction of Nrf-2 signaling, suggesting the potential of ISL for DN treatment.

We developed a NASH‐on‐a‐chip where human primary hepatocytes (HCs), Kupffer cells (KCs), and hepatic stellate cells (HSCs) were cultured in the 3D hydrogel scaffold, while a blood vessel mimetic endothelialized inlet channel was engineered using human liver sinusoidal endothelial cells (LSECs), via which the system was exposed to metabolic and inflammatory stressors. Exposing liver chips to a lipotoxic environment led to the gradual development of NASH phenotypic characteristics, including intracellular lipids accumulation, hepatocellular ballooning, HSC activation, and elevation of inflammatory and profibrotic molecular markers. We demonstrate that the NASH‐on‐a‐chip can be used to test the efficacy of pharmacological agents to inhibit the development of NASH., Nonalcoholic steatohepatitis (NASH), an advanced stage of nonalcoholic fatty liver disease (NAFLD), is a rapidly growing and global health problem compounded by the current absence of specific treatments. A major limiting factor in the development of new NASH therapies is the absence of models that capture the unique cellular structure of the liver microenvironment and recapitulate the complexities of NAFLD progression to NASH. Organ‐on‐a‐chip platforms have emerged as a powerful approach to dynamically model diseases and test drugs. Herein, we describe a NASH‐on‐a‐chip platform. Four main types of human primary liver cells (hepatocytes [HCs], Kupffer cells, liver sinusoidal endothelial cells, and hepatic stellate cells [HSCs]) were cocultured under microfluidic dynamics. Our chip‐based model successfully recapitulated a functional liver cellular microenvironment with stable albumin and urea secretion for at least 2 weeks. Exposing liver chips to a lipotoxic environment led to gradual development of NASH phenotypic characteristics, including intracellular lipid accumulation, hepatocellular ballooning, HSC activation, and elevation of inflammatory and profibrotic markers. Further, exposure of the chip to elafibranor, a drug under study for the therapy of NASH, inhibited the development of NASH‐specific hallmarks, causing an ~8‐fold decrease in intracellular lipids, a 3‐fold reduction in number of ballooned HCs, a significant reduction in HSC activation, and a significant decrease in the levels of inflammatory and profibrotic markers compared with controls. Conclusion: We have successfully developed a microfluidic NASH‐on‐a‐chip platform that recapitulates the main NASH histologic endpoints in a single chip and that can emerge as a powerful noninvasive, human‐relevant, in vitro platform to study disease pathogenesis and develop novel anti‐NASH drugs.

Cardamonin, a natural chalcone, is reported to induce apoptosis and inhibit cancer cell growth. However, the mechanisms underlying the therapeutic effects of cardamonin remain to be established. Here, we have focused on cardamonin-induced apoptosis in ovarian cancer cells, both in vitro and in vivo. The effects of cardamonin on cell cycle patterns and apoptotic responses of cells were assessed in this study. Western blot was employed to determine the effects of cardamonin on expression of cell cycle- and apoptosis-related proteins. Our results indicate that cardamonin suppresses cancer cell growth by inducing G2/M phase arrest and apoptosis through targeted inhibition of NF-κB and mTOR pathways. The collective findings provide novel insights into the pathways responsible for the anticancer effects of cardamonin and support its potential utility as a clinical therapeutic agent for ovarian cancer.

Mouse somatic cells can be chemically reprogrammed into pluripotent stem cells (CiPSCs) through an intermediate extraembryonic endoderm (XEN)-like state. However, it is elusive how the chemicals orchestrate the cell fate alteration. In this study, we analyze molecular dynamics in chemical reprogramming from fibroblasts to a XEN-like state. We find that Sox17 is initially activated by the chemical cocktails, and XEN cell fate specialization is subsequently mediated by Sox17 activated expression of other XEN master genes, such as Sall4 and Gata4. Furthermore, this stepwise process is differentially regulated. The core reprogramming chemicals CHIR99021, 616452 and Forskolin are all necessary for Sox17 activation, while differently required for Gata4 and Sall4 expression. The addition of chemical boosters in different phases further improves the generation efficiency of XEN-like cells. Taken together, our work demonstrates that chemical reprogramming is regulated in 3 distinct “prime–specify–transit” phases initiated with endogenous Sox17 activation, providing a new framework to understand cell fate determination., Yang, Xu, Gu et al. demonstrate that activation of endogenous Sox17 pushes fibroblasts to an extraembryonic endoderm-like state in chemically induced reprogramming of somatic cells into stem cells. This study provides insights into how chemicals prime the transition of somatic cells into stem cells.

Background Alcohol (EtOH) is reported to adversely affect one of the most crucial roles of the blood-brain barrier (BBB), the regulation of its permeability, thereby compromising the stability of the homeostatic environment of the brain. The central component of the BBB, endothelial cells (ECs), regulates BBB transcellular transport, while their paracellular pathways are made virtually impermeable by molecular structures called tight junctions (TJs). These TJs are composed of proteins, such as claudin-5, a protein involved in the regulation of paracellular permeability and of key interest in this study. Methods and results The working hypothesis of this study postulated that the high levels of antioxidants (AOs) in the fermented Aspalathus linearis (Rooibos; Rf) tincture may protect the ECs of the BBB against oxidative stress induced by EtOH exposure. Cells were exposed for 24 hours to selected concentrations of EtOH (25 and 100 mM), Rf (containing an antioxidant equivalence of 1.9 nM Aspalathin), and cotreatments of EtOH and Rf. Cell viability, live cell number, and toxicity were analyzed using the trypan blue exclusion assay. RT-qPCR was implemented to quantify claudin-5 transcription. In addition, permeability (Transepithelial Electrical Resistance) of bEnd5 monolayers was measured. The experimental timeline for the above-mentioned parameters was 24 and 48 hours. Conclusions Our study showed that simultaneous exposure of Rf and EtOH was able to negate the effects of EtOH on cell viability and cell proliferation, but was not able to reverse or reduce the effects of EtOH on claudin-5 transcription and paracellular permeability. Furthermore, a novel finding in this study suggests that very low concentrations of AOs in tinctures such as Rooibos tea could profoundly alter the redox status of brain ECs.