Fisetin is a selective adenosine triphosphate-competitive inhibitor for mitogen-activated protein kinase kinase 4 to inhibit lipopolysaccharide-stimulated inflammation.

The mitogen-activated protein kinase kinase 4 (MKK4), a member of the MAP kinase kinase family, directly phosphorylates and activates the c-Jun NH2-terminal kinases (JNK), in response to proinflammatory cytokines and cellular stresses. Regulation of the MKK4 activity is considered to be a novel approach for the prevention and treatment of inflammation. The aim of this study was to identify whether fisetin, a potential anti-inflammatory compound, targets MKK4-JNK cascade to inhibit lipopolysaccharide (LPS)-stimulated inflammatory response. RAW264 macrophage pretreated with fisetin following LPS stimulation was used as a cell model to investigate the transactivation and expression of related-inflammatory genes by transient transfection assay, electrophoretic mobility shift assay (EMSA), or enzyme-linked immunosorbent assay (ELISA), and cellular signaling as well as binding of related-signal proteins by Western blot, pull-down assay and kinase assay, and molecular modeling. The transactivation and expression of cyclooxygenase-2 (COX-2) gene as well as prostaglandin E2 (PGE2) secretion induced by LPS were inhibited by fisetin in a dose-dependent manner. Signaling transduction analysis demonstrated that fisetin selectively inhibited MKK4-JNK1/2 signaling to suppress the phosphorylation of transcription factor AP-1 without affecting the NF-κB and Jak2-Stat3 signaling as well as the phosphorylation of Src, Syk, and TAK1. Furthermore, in vitro and ex vivo pull-down assay using cell lysate or purified protein demonstrated that fisetin could bind directly to MKK4. Molecular modeling using the Molecular Operating Environment™ software indicated that fisetin docked into the ATP-binding pocket of MKK4 with a binding energy of -71.75 kcal/mol and formed a 1.70 Å hydrogen bound with Asp247 residue of MKK4. The IC50 of fisetin against MKK4 was estimated as 2.899 µM in the kinase assay, and the ATP-competitive effect was confirmed by ATP titration. Taken together, our data revealed that fisetin is a potent selective ATP-competitive MKK4 inhibitor to suppress MKK4-JNK1/2-AP-1 cascade for inhibiting LPS-induced inflammation.

Comparative Analysis of Acetylated Flavonoids' Chemopreventive Effects in Different Cancer Cell Lines.

Flavonoids, a class of natural compounds with anticancer activity, exhibit varying biological activities and potencies based on their structural differences. Acylation, including acetylation of flavonoids, generally increases their structural diversity, which is closely related to the diversity of bioactivity within this group of compounds. However, it remains largely unknown how acetylation affects the bioactivity of many flavonoids. Based on our previous findings that O-acetylation enhances quercetin's bioactivity against various cancer cells, we synthesized 12 acetylated flavonoids, including seven novel compounds, to investigate their anticancer activities in the MDA-MB-231, HCT-116, and HepG2 cell lines. Our results showed that acetylation notably enhanced the cell proliferation inhibitory effect of quercetin and kaempferol across all cancer cell lines tested. Interestingly, while the 5,7,4'-O-triacetate apigenin (3Ac-A) did not show an enhanced the effect of inhibition of cell proliferation through acetylation, it exhibited significantly strong anti-migration activity in MDA-MB-231 cells. In contrast, the 7,4'-O-diacetate apigenin (2Ac-Q), which lacks acetylation at the 5-position hydroxy group, showed enhanced cell proliferation inhibitory effect but had weaker anti-migration effects compared to 3Ac-A. These results indicated that acetylated flavonoids, especially quercetin, kaempferol, and apigenin derivatives, are promising for anticancer applications, with 3Ac-A potentially having unique anti-migration pathways independent of apoptosis induction. This study highlights the potential application of flavonoids in novel chemopreventive strategies for their anti-cancer activity.

Anticancer Activity and Molecular Mechanisms of Acetylated and Methylated Quercetin in Human Breast Cancer Cells.

Quercetin, a flavonoid polyphenol found in many plants, has garnered significant attention due to its potential cancer chemoprevention. Our previous studies have shown that acetyl modification of the hydroxyl group of quercetin altered its antitumor effects in HepG2 cells. However, the antitumor effect in other cancer cells with different gene mutants remains unknown. In this study, we investigated the antitumor effect of quercetin and its methylated derivative 3,3',4',7-O-tetramethylquercetin (4Me-Q) and acetylated derivative 3,3',4',7-O-tetraacetylquercetin (4Ac-Q) on two human breast cancer cells, MCF-7 (wt-p53, caspase-3-ve) and MDA-MB-231 (mt-p53, caspase-3+ve). The results demonstrated that 4Ac-Q exhibited significant cell proliferation inhibition and apoptosis induction in both MCF-7 and MDA-MB-231 cells. Conversely, methylation of quercetin was found to lose the activity. The human apoptosis antibody array revealed that 4Ac-Q might induce apoptosis in MCF-7 cells via a p53-dependent pathway, while in MDA-MB-231 cells, it was induced via a caspase-3-dependent pathway. Furthermore, an evaluation using a superoxide inhibitor, MnTBAP, revealed 4Ac-Q-induced apoptosis in MCF-7 cells in a superoxide-independent manner. These findings provide valuable insights into the potential of acetylated quercetin as a new approach in cancer chemoprevention and offer new avenues for health product development.

Verification of In Vitro Anticancer Activity and Bioactive Compounds in Cordyceps Militaris-Infused Sweet Potato Shochu Spirits.

Many liqueurs, including spirits infused with botanicals, are crafted not only for their taste and flavor but also for potential medicinal benefits. However, the scientific evidence supporting their medicinal effects remains limited. This study aims to verify in vitro anticancer activity and bioactive compounds in shochu spirits infused with Cordyceps militaris, a Chinese medicine. The results revealed that a bioactive fraction was eluted from the spirit extract with 40% ethanol. The infusion time impacted the inhibitory effect of the spirit extract on the proliferation of colon cancer-derived cell line HCT-116 cells, and a 21-day infusion showed the strongest inhibitory effect. Furthermore, the spirit extract was separated into four fractions, A-D, by high-performance liquid chromatography (HPLC), and Fractions B, C, and D, but not A, exerted the effects of proliferation inhibition and apoptotic induction of HCT-116 cells and HL-60 cells. Furthermore, Fractions B, C, and D were, respectively, identified as adenosine, cordycepin, and N6-(2-hydroxyethyl)-adenosine (HEA) by comprehensive chemical analyses, including proton nuclear magnetic resonance (1H-NMR), Fourier transform infrared spectroscopy (FT-IR), and electrospray ionization mass spectrometry (ESI-MS). To better understand the bioactivity mechanisms of cordycepin and HEA, the agonist and antagonist tests of the A3 adenosine receptor (A3AR) were performed. Cell viability was suppressed by cordycepin, and HEA was restored by the A3AR antagonist MR1523, suggesting that cordycepin and HEA possibly acted as agonists to activate A3ARs to inhibit cell proliferation. Molecular docking simulations revealed that both adenosine and cordycepin bound to the same pocket site of A3ARs, while HEA exhibited a different binding pattern, supporting a possible explanation for the difference in their bioactivity. Taken together, the present study demonstrated that cordycepin and HEA were major bioactive ingredients in Cordyceps militaries-infused sweet potato shochu spirits, which contributed to the in vitro anticancer activity.

Effects of Cooking Methods on Caffeoylquinic Acids and Radical Scavenging Activity of Sweet Potato.

The effects of cooking methods, including steaming, deep-frying, and baking, on the phenolic content, 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity, and isomerization of caffeoylquinic acids in sweet potato were investigated. A high correlation was observed between antioxidant capacity and total phenolic content. Deep-frying treatment resulted in higher antioxidant capacity with increasing heating time. The major phenolic components of raw sweet potat were 5-caffeoylquinic acid (CQA) and 3,5-dicaffeoylquinic acid (diCQA), which were reduced by heat treatment due to the isomerization of 5-CAQ to 3- and 4-CQA, and 3,5-diCQA to 3,4- and 4,5-diCQA. Moreover, 5-CQA was more stable than 3,5-diCQA even at 100 °C. Our results demonstrated that by controlling the cooking temperature and time, new bioactive compounds such as mono- and diCQA derivatives can be produced from sweet potato. These data indicate a potential approach for the development of new functional foods from sweet potato by controlling cooking temperature and time.

The binding selectivity of quercetin and its structure-related polyphenols to human serum albumin using a fluorescent dye cocktail for multiplex drug-site mapping.

Human serum albumin (HSA) is a serum protein that carries flavonoids in blood circulation. In this report, the binding selectivity and strength of interactions to HSA-binding sites (sites I or II) by flavonoids were evaluated using competition experiments and the specific fluorescent dyes, dansylamide and BD140. Most tested flavonoids bound site I preferentially, with the binding strength dependent on the mother structure in the order flavonol > flavone > flavanone > flavan 3-ols. Glycosylation or glucuronidation reduced the binding of quercetin to site I of HSA, whereas sulfation increased binding. Quercetin 7-sulfate showed the strongest binding and molecular docking simulations supported this observation. Prenylation at any position or glucuronidation and sulfation at the C-4' or C-7 position of quercetin facilitated stronger binding to site II. The binding affinity of flavonoids toward site I correlated with the partition coefficient value (logP), whereas no corresponding correlation was observed for site II.

Hydroxyl Group Acetylation of Quercetin Enhances Intracellular Absorption and Persistence to Upregulate Anticancer Activity in HepG2 Cells.

Quercetin, a flavonoid compound widely distributed in many plants, is known to have potent antitumor effects on several cancer cells. Our previous study revealed that the acetylation of quercetin enhanced its antitumor effect. However, the mechanisms remain unknown. This study aimed to elucidate the bioavailability of acylated quercetin in the HepG2 cell model based on its antitumor effect. The positions of quercetin 3,7,3',4'-OH were acetylated as 3,7,3',4'-O-tetraacetylquercetin (4Ac-Q). The inhibitory effect of 4Ac-Q on HepG2 cell proliferation was assessed by measuring cell viability. The apoptosis was characterized by apoptotic proteins and mitochondrial membrane potential shifts, as well as mitochondrial reactive oxygen species (ROS) levels. The bioavailability of 4Ac-Q was analyzed by measuring the uptake and metabolites in HepG2 cells with high performance liquid chromatography (HPLC)-photodiode array detector (PDA) and-ultraviolet/visible detector (UV/Vis). The results revealed that 4Ac-Q enhanced the inhibitory effect on HepG2 cell proliferation and induced its apoptosis significantly higher than quercetin. Protein array analysis of apoptosis-related protein indicated that 4Ac-Q increased the activation or expression of pro-apoptotic proteins, including caspase-3, -9, as well as second mitochondria-derived activator of caspases (SMAC), and suppressed the expression of apoptosis inhibiting proteins such as cellular inhibitor of apoptosis (cIAP)-1, -2, Livin, Survivin, and X-linked inhibitor of apoptosis (XIAP). Furthermore, 4Ac-Q stimulated mitochondrial cytochrome c release into the cytosol by enhancing ROS level and depolarizing the mitochondrial membrane. Finally, the analysis of uptake and metabolites of 4Ac-Q in HpG2 cells with HPLC-PDA and -UV/Vis revealed that 4Ac-Q was metabolized to quercetin and several different acetylated quercetins which caused 2.5-fold higher quercetin present in HepG2 cells than parent quercetin. These data demonstrated that acetylation of the quercetin hydroxyl group significantly increased its intracellular absorption. Taken together, our findings provide the first evidence that acetyl modification of quercetin not only substantially augments the intracellular absorption of quercetin but also bolsters its metabolic stability to elongate its intracellular persistence. Therefore, acetylation could serve as a strategic approach to enhance the ability of quercetin and analogous flavonoids to suppress cancer cell proliferation.

Apoptosis Induction in HepG2 and HCT116 Cells by a Novel Quercetin-Zinc (II) Complex: Enhanced Absorption of Quercetin and Zinc (II).

Quercetin forms complexes with various metals due to its structural attributes. It predominantly exhibits chelating activity at the 3-hydroxy/4-carbonyl group. Previously, coordination in synthetically obtained quercetin-zinc (II) complexes has been limited to this group. However, the expanded coordination observed in quercetin-iron complexes has opened avenues for diverse applications. Thus, synthesizing novel quercetin-zinc complexes with different coordination positions is a significant advance. In our study, we not only synthesized and comprehensively characterized a new quercetin-zinc (II) complex, Zn-Q, but also evaluated the structure and bioactivity of chelate complexes (Q+Zn) derived from co-treatment in cell culture mediums. The structure of the new compound Zn-Q was comprehensively characterized using 1D 1H and 2D correlation spectroscopy (COSY), nuclear magnetic resonance (NMR), Fourier-transform infrared spectroscopy (FT-IR), ultraviolet-visible spectroscopy (UV-Vis), electrospray ionization mass spectrometer (ESI-MS), and X-ray diffraction analysis (XRD) analysis. Subcellular localization and absorption of these zinc (II) complexes were determined using the ZnAF-2 DA zinc ion fluorescence probe. Throughout the experiments, both Zn-Q and Q+Zn exhibited significant antioxidant, cell growth inhibitory, and anticancer effects in HepG2 and HCT116 cells, with Zn-Q showing the highest potential for inducing apoptosis via the caspase pathway. Tracking intracellular zinc complex absorption using zinc fluorescent probes revealed zinc (II) localization around the cell nucleus. Interestingly, there was a proportional increase in intracellular quercetin absorption in conjunction with zinc (II) uptake. Our research highlights the advantages of quercetin complexation with zinc (II): enhanced anticancer efficacy compared to the parent compound and improved bioavailability of both quercetin and zinc (II). Notably, our findings, which include enhanced intracellular uptake of both quercetin and zinc (II) upon complex formation and its implications in apoptosis, contribute significantly to the understanding of metal-polyphenol complexes. Moving forward, comprehensive functional assessments and insights into its mechanism of action, supported by animal studies, are anticipated.

Ameliorative Effects of Anthocyanin Metabolites on Western Diet-Induced NAFLD by Modulating Co-Occurrence Networks of Gut Microbiome.

Anthocyanins (Acn) have been reported to have preventive effects on Western diet (WD)-induced non-alcoholic fatty liver disease (NAFLD). However, the amount of Acn that reached the bloodstream were less than 1%, suggesting that anthocyanin metabolites (Acn-M) in the gut may contribute to their in vivo effects. This study is focused on a gut microbiota investigation to elucidate the effect of two major Acn-M, protocatechuic acid (PC) and phloroglucinol carboxaldehyde (PG), on NAFLD prevention. C57BL/6N male mice were divided into five groups and fed with a normal diet (ND), WD, WD + 0.5% PC, WD + 0.5% PG and WD + a mixture of 0.25% PC + 0.25% PG (CG) for 12 weeks. The results revealed that WD-fed mice showed a significant increase in final body weight, epididymis fat weight, liver weight and fat accumulation rate, serum total cholesterol, alanine aminotransferase, monocyte chemoattractant protein 1, and 2-thiobarbituric acid reactive substances. At the same time, these indices were significantly decreased by Acn-M in the order of PG, CG > PC. In particular, PG significantly decreased serum glucose and insulin resistance. Gut microbiome analysis revealed that PG significantly increased the relative abundance of Parabacteroides, Prevotella, Prevotella/Bacteroides ratio, and upregulated glucose degradation pathway. Interestingly, the co-occurrence networks of Lachnospiraceae and Desulfovibrionaceae in the PC and PG groups were similar to the ND group and different to WD group. These data suggest that PC and PG were able to recover the gut microbiome networks and functions from dysbiosis caused by WD. Therefore, PG might act as a master metabolite for anthocyanins and prevent WD-induced NAFLD and gut dysbiosis.

Effects of Amazake Produced with Different Aspergillus on Gut Barrier and Microbiota.

Inflammatory bowel disease (IBD) is a chronic inflammatory disease of the gastrointestinal tract. To explore the preventive effects of dietary foods on IBD, we evaluated the effects of the traditional Japanese fermented beverage "Amazake" on gut barrier function in this study. Black koji Amazake (BA) derived from Aspergillus luchuensis MEM-C strain and yellow koji Amazake (YA) derived from Aspergillus oryzae were made in this study, and their nutrients were analyzed. Mice with mild gut barrier dysfunction induced by Western diet were administered with 10% of each Amazake for two months. Mice gut microbiota were analyzed by 16S rRNA gene sequencing. BA contained a higher amount of isomaltooligosaccharides, citric acid, and ferulic acid than YA. The animal data revealed that BA significantly induced the expressions of antioxidant factors and enzymes such as NF-E2-related factor 2 (Nfr2), heme oxygenase 1 (HO1), and superoxide dismutase-2 (SOD-2). The gut barrier protein, occludin, and fecal immunoglobulin A (IgA) were also significantly enhanced by BA. Furthermore, the levels of serum endotoxin and hepatic monocyte chemotactic protein-1 (MCP-1) were decreased in both the BA and YA groups. In gut microbiota, Lachnospiraceae was increased by BA while Akkermansia muciniphilia was increased by YA. Black koji Amazake contained a higher amount of isomaltooligosaccharides, citric acid, and ferulic acid than yellow koji Amazake and contributed to protecting gut barrier function to reduce endotoxin intrusion and inflammation.

Extraction Efficiency and Alpha-Glucosidase Inhibitory Activities of Green Tea Catechins by Different Infusion Methods.

Alpha-glucosidase is an important target for glycemic control with the aim of reducing the risk of type 2 diabetes (T2D). Green tea catechins have been reported to inhibit alpha-glucosidase activity as a potential beverage to control blood glucose levels. However, the effects of the daily infusion style of green tea on tea catechins and their activity remain unclear. In this study, the extraction efficiency of catechins was investigated for 12 green tea extracts (GTEs) infused with 70% ethanol (70% EtOH for 24 h, a favored solvent for catechin extraction), room temperature water infusion (RT H2O for 24 h, an easy way to drink tea), and hot water infusion (Hot H2O for 90 s, a standard way to drink tea). Eight catechins were quantified by HPLC, and the inhibitory effect of GTEs and their catechins on alpha-glucosidase was measured with both rat intestinal enzymes and human Caco-2 cells. The inhibitory mechanism was further analyzed in silico by docking catechins to human alpha-glucosidase using Molecular Operating Environment software. The results showed that total catechins and gallate catechins were efficiently extracted in the order of 70% EtOH, RT H2O, and Hot H2O, and the inhibitory activity against alpha-glucosidase also followed a similar order. Pearson correlation analysis indicated that the alpha-glucosidase inhibitory activity of GTEs was significantly positively correlated with the contents of total catechins, especially gallate catechins. Gallate catechins, such as EGCg and ECg, showed lower IC50 values than free catechins for the enzyme in both rats and humans. In silico simulation revealed that gallate catechins were bound to the different sites with free catechins, and the docking energy of gallate catechins was lower than that of free catechins. Taken together, our data indicated that the daily infusion style of green tea significantly impacted the extraction efficiency and alpha-glucosidase inhibitory activities of catechins, which will give us insight into the use of green tea catechins for glycemic control through efficient infusion.

Assessment of Areca Nut Bioactivities in Western Diet-Induced Mice NAFLD Model.

The areca nut is often consumed as a chewing food in the Asian region. Our previous study revealed that the areca nut is rich in polyphenols with high antioxidant activity. In this study, we further assessed the effects and molecular mechanisms of the areca nut and its major ingredients on a Western diet-induced mice dyslipidemia model. Male C57BL/6N mice were divided into five groups and fed with a normal diet (ND), Western diet (WD), WD with areca nut extracts (ANE), areca nut polyphenols (ANP), and arecoline (ARE) for 12 weeks. The results revealed that ANP significantly reduced WD-induced body weight, liver weight, epididymal fat, and liver total lipid. Serum biomarkers showed that ANP ameliorated WD-enhanced total cholesterol and non-high-density lipoprotein (non-HDL). Moreover, analysis of cellular signaling pathways revealed that sterol regulatory element-binding protein 2 (SREBP2) and enzyme 3-hydroxy-3-methylglutaryld coenzyme A reductase (HMGCR) were significantly downregulated by ANP. The results of gut microbiota analysis revealed that ANP increased the abundance of beneficial bacterium Akkermansias and decreased the abundance of the pathogenic bacterium Ruminococcus while ARE shown the opposite result to ANP. In summary, our data indicated that areca nut polyphenol ameliorated WD-induced dyslipidemia by increasing the abundance of beneficial bacteria in the gut microbiota and reducing the expressions of SREBP2 and HMGCR while areca nut ARE inhibited this improvement potential.

In Vitro Antioxidant Activity of Areca Nut Polyphenol Extracts on RAW264.7 Cells.

Chewing areca nuts is a popular hobby in the Asian region, and areca nuts are rich in polyphenols, although some alkaloids are included. In this study, we explored the antioxidant activity of areca nut polyphenols (ANP) in lipopolysaccharides (LPS)-stimulated RAW264.7 cells. The results revealed that ANP reduced the level of reactive oxygen species (ROS) in LPS-stimulated RAW264.7 cells and enhanced the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase 1 (HO-1). RNA-seq analysis showed that ANP down-regulated the transcription of genes related to the cancer pathway at 160 µg/mL, and the inflammatory pathway as well as viral infection pathway at 320 µg/mL. The cellular signaling analysis further revealed that the expressions of these genes were regulated by the mitogen-activated protein kinase (MAPK) pathway, and ANP downregulated the activation of the MAPK signaling pathway stimulated by LPS. Collectively, our findings showed that ANP inhibited the MAPK pathway and activated the Nrf2/HO-1 antioxidant pathways to reduce ROS generation induced by LPS.

Natural Garlic Organosulfur Compounds Prevent Metabolic Disorder of Lipid and Glucose by Increasing Gut Commensal Bacteroides acidifaciens.

A number of reports of the effects of garlic on gut microbiota revealed that the active garlic organosulfur compounds (OSCs) are destabilized by the action of alliinase during garlic preparation. In this study, garlic alliinase was deactivated to obtain stable garlic OSCs. Experiments with C57BL/6J mice fed with lipid and glucose metabolic disorder-inducing Western diet (WD) revealed that stable garlic OSCs prevented the disorder by increasing the relative abundance of gut Bacteroides acidifaciens. Molecular analysis indicated that garlic OSCs inhibited dyslipidemia and fatty liver by increasing taurine and subsequently promoting hepatic fatty acid ß-oxidation. In parallel, garlic OSCs could meliorate glucose homeostasis by inhibiting dipeptidyl peptidase-4 (DPP-4) and hepatic gluconeogenesis. In vitro bacterial culture experiments revealed that garlic OSCs directly increased the growth of gut Bacteroides acidifaciens. The results of this study demonstrate that the molecular mechanism of the preventive effect of garlic OSCs on the WD-induced metabolic disorder is attributed to the enhanced growth of Bacteroides acidifaciens and the consequent increase in taurine.

Highly efficient protein expression of Plasmodium vivax surface antigen, Pvs25, by silkworm and its biochemical analysis.

Plasmodium vivax ookinete surface protein, Pvs25, is a candidate for a transmission-blocking vaccine (TBV) for malaria. Pvs25 has four EGF-like domains containing 22 cysteine residues forming 11 intramolecular disulfide bonds, a structural feature that makes its recombinant protein expression difficult. In this study, we report the high expression of recombinant Pvs25 as a soluble form in silkworm, Bombyx mori. The Pvs25 protein was purified from hemolymphs of larvae and pupae by affinity chromatography. In the Pvs25 expressed by silkworm, no isoforms with inappropriate disulfide bonds were found, requiring no further purification step, which is necessary in the case of Pichia pastoris-based expression systems. The Pvs25 from silkworm was confirmed to be molecularly uniform by sodium dodecyl sulfate gel electrophoresis and size-exclusion chromatography. To examine the immunogenicity, the Pvs25 from B. mori was administered to BALB/c mice subcutaneously with oil adjuvant. The Pvs25 produced by silkworm induced potent and robust immune responses, and the induced antisera correctly recognized P. vivax ookinetes in vitro, demonstrating the potency of Pvs25 from silkworm as a candidate for a malaria TBV. To the best of our knowledge, this is the first study to construct a system for mass-producing malaria TBV antigens using silkworm.

Involvement of AMP-activated Protein Kinase α/Nuclear Factor (Erythroid-derived 2) Like 2-iniatived Signaling Pathway in Cytoprotective Effects of Wasabi 6-(Methylsulfinyl) Hexyl Isothiocyanate.

6-(Methylsulfinyl) hexyl isothiocyanate (6-MSITC) is an active ingredient present in Wasabi, which is a popular pungent spice used in Japanese cuisine. Our previous studies suggested that the primary antioxidant activity of 6-MSITC may link to other biological activity. This study aimed to clarify how the antioxidant activity of 6-MSITC contributes to preventing overloaded lipid stress in hepatic cell model. HepG2 cells were treated with 6-MSITC at defined concentrations and times in normal medium or in combined fatty acids (CFA) medium, and the targeted proteins were detected by Western blotting. The kinetic data revealed that 6-MSITC activated AMP-activated protein kinase α (AMPKα) and nuclear factor (erythroid-derived 2) like 2 (Nrf2), and then enhanced the protein expression of Forkhead box protein O1 (FOXO1) and Sirtuin1 as well as that of the Nrf2 target proteins, NAD(P)H:quinone oxidoreductase 1 (NQO1) and heme oxygenase (HO-1). Furthermore, lipid metabolic stress was mimicked in HepG2 cells by overloading CFA. 6-MSITC significantly alleviated CFA-induced formation of thiobarbituric acid reactive substances and fat accumulation. Signaling analysis data revealed that 6-MSITC enhanced phosphorylation of AMPKα, upregulated the expression of Nrf2, NQO1, heme oxygenase 1, FOXO1, and Siruin1, and downregulated the expression of PPARα. Taken together, our results suggested that the AMPKα/Nrf2-mediated signaling pathways might be involved in the cytoprotective effects of Wasabi 6-MSITC against metabolic lipid stress.

Bilberry Anthocyanins Ameliorate NAFLD by Improving Dyslipidemia and Gut Microbiome Dysbiosis.

Non-alcoholic fatty liver disease (NAFLD) is a manifestation of metabolic syndrome closely linked to dyslipidemia and gut microbiome dysbiosis. Bilberry anthocyanins (BA) have been reported to have preventive effects against metabolic syndrome. This study aimed to investigate the protective effects and mechanisms of BA in a Western diet (WD)-induced mouse model. The results revealed that supplementation with BA attenuated the serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), low-density lipoprotein cholesterol (LDL-c), fat content in liver, 2-thiobarbituric acid reactive substances (TBARS) and α-smooth muscle actin (α-SMA) caused by WD. Furthermore, gut microbiota characterized by 16S rRNA sequencing revealed that BA reduced remarkably the ratio of Firmicutes/Bacteroidetes (F/B) and modified gut microbiome. In particular, BA increased the relative abundance of g_Akkermansia and g_Parabacteroides. Taken together, our data demonstrated that BA might ameliorate WD-induced NAFLD by attenuating dyslipidemia and gut microbiome dysbiosis.

Ameliorative effects and molecular mechanisms of vine tea on western diet-induced NAFLD.

Non-alcoholic fatty liver disease (NAFLD) is a disease that is prevalent worldwide, and its prevention by dietary administration has recently been considered as an important strategy. In this study, we administered mice with vine tea polyphenol (VTP) extracted from Ampelopsis grossedentata, a Chinese herb, to investigate the preventive effect on western diet (WD)-induced NAFLD. Male C57BL/6N mice were fed either a normal diet (ND) or WD with or without VTP for 12 weeks. The results revealed that VTP supplementation decreased the serum levels of cholesterol and triglycerides, and reduced the accumulation of hepatic lipid droplets caused by WD. Molecular data revealed that VTP enhanced fatty acid oxidation by reactivating the WD-suppressed phosphorylation of AMP-activated protein kinaseα (AMPKα) and the expressions of peroxisome proliferator-activated receptor alpha (PPARα), carnitine palmitoyl transferase IA (CPT1A) and cytochrome P450, family 4, subfamily a1 (CYP4A1). VTP inhibited hepatic lipogenesis by reducing the WD-enhanced level of mature sterol regulatory element-binding protein 1 (SREBP1) and fatty acid synthase (FAS). Moreover, VTP activated nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-mediated expressions of hemeoxygenase-1 (HO-1) and quinone oxidoreductase (NQO1), and reduced hepatic TBARS levels to prevent hepatic oxidative stress. On the other hand, VTP also increased intestinal zonula occludens-1 (ZO-1) expression and the relative abundance of gut Akkermansia, and reduced the ratio of Firmicutes/Bacteroidetes. Thus, VTP might prevent WD-induced NAFLD by balancing fatty acid oxidation and lipogenesis, hepatic oxidative stress, and gut microbiome, at least. These results suggest that vine tea, containing a high content of the bioactive compound dihydromyricetin, is a potential food resource for preventing NAFLD.

Modulation of Allicin-Free Garlic on Gut Microbiome.

The allicin diallyldisulfid-S-oxide, a major garlic organosulfur compound (OSC) in crushed garlic (Allium sativum L.), possesses antibacterial effects, and influences gut bacteria. In this study, we made allicin-free garlic (AFG) extract and investigated its effects on gut microbiome. C57BL/6N male mice were randomly divided into 6 groups and fed normal diet (ND) and high-fat diet (HFD) supplemented with or without AFG in concentrations of 1% and 5% for 11 weeks. The genomic DNAs of feces were used to identify the gut microbiome by sequencing 16S rRNA genes. The results revealed that the ratio of p-Firmicutes to p-Bacteroidetes increased by aging and HFD was reduced by AFG. In particular, the f-Lachnospiraceae, g-Akkermansia, and g-Lactobacillus decreased by aging and HFD was enhanced by AFG. The g-Dorea increased by aging and HFD decreased by AFG. In addition, the ratio of glutamic-pyruvic transaminase to glutamic-oxaloacetic transaminase (GPT/GOT) in serum was significantly increased in the HFD group and decreased by AFG. In summary, our data demonstrated that dietary intervention with AFG is a potential way to balance the gut microbiome disturbed by a high-fat diet.

Antioxidant Properties of a Traditional Vine Tea, Ampelopsis grossedentata.

Ampelopsis grossedentata, also called vine tea, has been used as a traditional beverage in China for centuries. Vine tea contains rich polyphenols and shows benefit to human health, but the chemical and antioxidant properties of vine tea polyphenols from different locations remain unclear. This study aims to investigate the chemical and antioxidant properties of vine tea from three major production areas in China including Guizhou, Hunan, and Guangxi Provinces. The highest amount of polyphenol from vine tea was extracted by 70% ethanol at 70 °C for 40 min with ultrasonic treatment. The major compound in vine tea polyphenols (VTP) was determined as dihydromyricetin (DMY) by high-performance liquid chromatography (HPLC) and the content was estimated as 21.42%, 20.17%, and 16.47% of dry weight basis from Hunan, Guizhou, and Guangxi products, respectively. The antioxidant activities were investigated in vitro and in culture hepatic cells. VTP and DMY showed strong 1,1-Diphenyl-2-picrylhydrazyl free radical (DPPH) scavenging ability and high oxygen radical absorption capacity (ORAC) value in vitro. VTP and DMY also increased the level of nicotinamide adenine dinucleotide phosphate (NADPH):quinone oxidoreductase (NQO1) in HepG2 cells. Moreover, VTP and DMY enhanced the level of nuclear factor erythroid 2-related factor 2 (Nrf2) and reduced the level of Kelch-like ECH-associated protein 1 (Keap1). Taken together, our data demonstrated that the extraction of vine tea by 70% ethanol with ultrasonic treatment is a novel method to efficiently obtain components possessing stronger antioxidant activity. Furthermore, the results from the culture cells suggest that the bioactive component of vine tea might exert the antioxidant activity by activating the cellular Nrf2/Keap1 pathway.