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.

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.

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.

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.

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.

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.

Involvement of ERK1/2-mediated ELK1/CHOP/DR5 pathway in 6-(methylsulfinyl)hexyl isothiocyanate-induced apoptosis of colorectal cancer cells.

6-(Methylsulfinyl)hexyl isothiocyanate (6-MSITC) is a major bioactive compound in Wasabi. Although 6-MSITC is reported to have cancer chemopreventive activities in rat model, the molecular mechanism is unclear. In this study, we investigated the anticancer mechanisms using two types of human colorectal cancer cells (HCT116 p53+/+ and p53-/-). 6-MSITC caused cell cycle arrest in G2/M phase and induced apoptosis in both types of cells in the same fashion. Signaling data revealed that the activation of ERK1/2, rather than p53, is recruited for 6-MSITC-induced apoptosis. 6-MSITC stimulated ERK1/2 phosphorylation, and then activated ERK1/2 signaling including ELK1 phosphorylation, and upregulation of C/EBP homologous protein (CHOP) and death receptor 5 (DR5). The MEK1/2 inhibitor U0126 blocked all of these molecular events induced by 6-MSITC, and enhanced the cell viability in both types of cells in the same manner. These results indicated that ERK1/2-mediated ELK1/CHOP/DR5 pathway is involved in 6-MSITC-induced apoptosis in colorectal cancer cells. Abbreviations: CHOP: C/EBP homologous protein; DR5: death receptor 5; ELK1: ETS transcription factor; ERK1/2: extracellular signal-regulated kinase 1/2; JNK: Jun-N-terminal kinase; MAPK: mitogen-activated protein kinase; MEK1/2: MAP/ERK kinase 1/2; 6-MSITC: 6-(methylsulfinyl)hexyl isothiocyanate; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PARP: poly(ADP-ribose) polymerase.

DNA Microarray Profiling Highlights Nrf2-Mediated Chemoprevention Targeted by Wasabi-Derived Isothiocyanates in HepG2 Cells.

6-MSITC and 6-MTITC are sulforaphane (SFN) analogs found in Japanese Wasabi. As we reported previously, Wasabi isothiocyanates (ITCs) are activators of Nrf2-antioxidant response element pathway, and also inhibitors of pro-inflammatory cyclooxygenase-2. This study is the first to assess the global changes in transcript levels by Wasabi ITCs, comparing with SFN, in HepG2 cells. We performed comparative gene expression profiling by treating HepG2 cells with ITCs, followed by DNA microarray analyses using HG-U133 plus 2.0 oligonucleotide array. Partial array data on selected gene products were confirmed by RT-PCR and Western blotting. Ingenuity Pathway Analysis (IPA) was used to identify functional subsets of genes and biologically significant network pathways. 6-MTITC showed the highest number of differentially altered (≥2 folds) gene expression, of which 114 genes were upregulated and 75 were downregulated. IPA revealed that Nrf2-mediated pathway, together with glutamate metabolism, is the common significantly modulated pathway across treatments. Interestingly, 6-MSITC exhibited the most potent effect toward Nrf2-mediated pathway. Our data suggest that 6-MSITC could exert chemopreventive role against cancer through its underlying antioxidant activity via the activation of Nrf2-mediated subsequent induction of cytoprotective genes.

Comparison of the inhibitory effects of delphinidin and its glycosides on cell transformation.

Although anthocyanins are major forms distributed in many plant foods and promising as chemopreventive source, many molecular data are obtained from anthocyanidins, showing their low bioavailability. This study aims to clarify the inhibitory effects of delphinidin glycosides on cell transformation comparing them to those of delphinidin. Screening data revealed that delphinidin 3-sambubioside could directly bind to MAPK/ERK kinase 1. Affinity assay data confirmed that delphinidin 3-sambubioside had higher binding affinity to MAPK/ERK kinase 1 than ERK1/2 and B-Raf. Colony assay data further demonstrated that delphinidin 3-sambubioside inhibited 12-O- tetradecanoylphorbol-13-acetate-induced phosphorylation of MAPK/ERK kinase 1 and sequentially suppressed cell transformation. All of these effects caused by delphinidin 3-sambubioside were weaker than those by its aglycon, delphinidin. Our data suggested that the weaker anti- transformation activity of delphinidin glycosides compared to that of their aglycon is due to lower binding affinity to the target molecule MAPK/ERK kinase 1.

Honokiol activates reactive oxygen species-mediated cytoprotective autophagy in human prostate cancer cells.

BACKGROUND: Honokiol (HNK), derived from the bark of an oriental medicinal plant (Magnolia officinalis), is a promising anticancer agent with preclinical in vitro (PC-3 and LNCaP cells) and in vivo (PC-3 xenografts) efficacy against prostate cancer. However, the mechanisms affecting anticancer response to HNK are not fully understood. METHODS: Human (androgen-independent PC-3 and androgen-responsive LNCaP) and murine (Myc-CaP) prostate cancer cells, and PC-3 tumor xenografts were used for various assays. Autophagy was assessed by transmission electron microscopy, immunofluorescence (LC3 puncta), and immunoblotting (LC3BII detection). Cell viability was determined by trypan blue assay. Apoptosis was quantitated by DNA fragmentation detection and Annexin V/propidium iodide assay. Reactive oxygen species (ROS) were detected by electron paramagnetic resonance spectrometry and flow cytometric/microscopic analysis of MitoSOX red fluorescence. RESULTS: Exposure of PC-3, LNCaP, and Myc-CaP cells to pharmacologic doses of HNK resulted in autophagy induction. The PC-3 tumor xenografts from HNK-treated mice contained higher levels of LC3BII protein compared with control tumors. Cell viability inhibition and apoptosis induction resulting from HNK exposure were significantly augmented by pharmacological inhibition of autophagy using 3-methyladenine as well as RNA interference of autophagy regulator ATG5. HNK-mediated increase in levels of LC3BII protein was partially but markedly diminished in the presence of antioxidants, including N-acetylcysteine, polyethylene glycol-conjugated (PEG)-superoxide dismutase, and PEG-catalase. On the other hand, antioxidants had no impact on HNK-induced apoptosis. CONCLUSIONS: In conclusion, the present study demonstrates, for the first time, that HNK induces ROS-mediated cytoprotective autophagy in prostate cancer cells.

Notch2 activation is protective against anticancer effects of zerumbone in human breast cancer cells.

We showed previously that zerumbone (ZER), a sesquiterpene isolated from subtropical ginger, inhibited in vitro (MCF-7 and MDA-MB-231cells) and in vivo (MDA-MB-231 cells) growth of human breast cancer cells in association with apoptosis induction. Here, we investigated the role of Notch receptors in anticancer effects of ZER (cell migration inhibition and apoptosis induction) using breast cancer cells. Western blotting was performed to determine protein expression changes. Effect of ZER on transcriptional activity of Notch was assessed by luciferase reporter assays. Transfection with small hairpin RNA or small interfering RNA was performed for knockdown of Notch2 or Presenilin-1 protein. Cell migration and apoptosis were quantitated by Boyden chamber assay and flow cytometry, respectively. Exposure of MDA-MB-231, MCF-7, and SUM159 cells to ZER resulted in increased cleavage of Notch2 in each cell line. On the other hand, levels of cleaved Notch1 and Notch4 proteins were decreased following ZER treatment. Increased cleavage of Notch2 in ZER-treated cells was accompanied by induction of Presenilin-1 protein and transcriptional activation of Notch. Inhibition of cell migration as well as apoptosis induction resulting from ZER exposure was significantly augmented by knockdown of Notch2 protein. ZER-mediated cleavage of Notch2 protein in MDA-MB-231 cells was markedly attenuated upon RNA interference of Presenilin-1. Knockdown of Presenilin-1 protein also resulted in escalation of ZER-induced apoptosis. The present study indicates that Notch2 activation by ZER inhibits its proapoptotic and anti-migratory response at least in breast cancer cells.

In vitro anticancer activity of loquat tea by inducing apoptosis in human leukemia cells.

Fresh loquat leaves have been used as folk health herb in Asian countries for long time, although the evidence supporting their functions is still minimal. This study aimed to clarify the chemopreventive effect of loquat tea extract (LTE) by investigating the inhibition on proliferation, and underlying mechanisms in human promyelocytic leukemia cells (HL-60). LTE inhibited proliferation of HL-60 in a dose-dependent manner. Molecular data showed that the isolated fraction of LTE induced apoptosis of HL-60 as characterized by DNA fragmentation; activation of caspase-3, -8, and -9; and inactivation of poly(ADP)ribose polymerase. Moreover, LTE fraction increased the ratio of pro-apoptotic Bcl-2-associated X protein (Bax)/anti-apoptotic myeloid cell leukemia 1 (Mcl-1) that caused mitochondrial membrane potential loss and cytochrome c released to cytosol. Thus, our data indicate that LTE might induce apoptosis in HL-60 cells through a mitochondrial dysfunction pathway. These findings enhance our understanding for chemopreventive function of loquat tea.

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.

In vitro and in vivo effects of phenethyl isothiocyanate treatment on vimentin protein expression in cancer cells.

We have shown previously that cancer prevention by cruciferous vegetable constituent phenethyl isothiocyanate (PEITC) in a transgenic mouse model of prostate cancer is associated with induction of E-cadherin protein expression. Because suppression of E-cadherin protein concomitant with induction of mesenchymal markers (e.g., vimentin) is a biochemical hallmark of epithelial-mesenchymal transition, a process implicated in cancer metastasis, we hypothesized that PEITC treatment was likely to suppress vimentin protein expression. Contrary to this prediction, exposure of human breast (MDA-MB-231) and prostate cancer cells (PC-3 and DU145) to PEITC resulted in a dose-dependent increase in vimentin protein level, which was observed as early as 6 h posttreatment and persisted for the duration of the experiment (24 h). RNA interference of vimentin resulted in a modest augmentation of PEITC-mediated inhibition of MDA-MB-231 and PC-3 cell migration as well as cell viability. Furthermore, the PEITC-induced apoptosis was moderately increased upon siRNA knockdown of vimentin protein in MDA-MB-231 and PC-3 cells. To our surprise, PEITC treatment caused a marked decrease in vimentin protein expression in breast and prostate carcinoma in vivo in transgenic mouse models, although the difference was statistically significant only in the breast carcinomas. The present study highlights the importance of in vivo correlative studies for validation of the in vitro mechanistic observations.

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.

D,L-sulforaphane-induced apoptosis in human breast cancer cells is regulated by the adapter protein p66Shc.

Cancer chemopreventive response to D,L-sulforaphane (SFN), a synthetic racemic analogue of broccoli constituent L-sulforaphane, is partly attributable to apoptosis induction, but the mechanism of cell death is not fully understood. The present study demonstrates a critical role for adapter protein p66(Shc) in SFN-induced apoptosis. Immortalized mouse embryonic fibroblasts (MEF) derived from p66(shc) knockout mice were significantly more resistant to SFN-induced apoptosis, collapse of mitochondrial membrane potential, and reactive oxygen species (ROS) production compared with MEF obtained from the wild-type mice. Notably, a spontaneously immortalized and non-tumorigenic human mammary epithelial cell line (MCF-10A) was resistant to SFN-induced ROS production and apoptosis. Stable overexpression of manganese superoxide dismutase in MCF-7 and MDA-MB-231 human breast cancer cells conferred near complete protection against SFN-induced apoptosis and mitochondrial membrane potential collapse. SFN treatment resulted in increased S36 phosphorylation and mitochondrial translocation of p66(shc) in MDA-MB-231 and MCF-7 cells, and SFN-induced apoptosis was significantly attenuated by RNA interference of p66(shc) in both cells. SFN-treated MDA-MB-231 and MCF-7 cells also exhibited a marked decrease in protein level of peptidyl prolyl isomerase (Pin1), which is implicated in mitochondrial translocation of p66(shc) . However, stable overexpression of Pin1 failed to alter proapoptotic response to SFN at least in MCF-7 cells. Finally, SFN-induced S36 phosphorylation of p66(Shc) was mediated by protein kinase Cß (PKCß), and pharmacological inhibition of PKCß significantly inhibited apoptotic cell death resulting from SFN exposure. In conclusion, the present study provides new insight into the mechanism of SFN-induced apoptosis involving PKCß -mediated S36 phosphorylation of p66(shc).