Ginger metabolites and metabolite-inspired synthetic products modulate intracellular calcium and relax airway smooth muscle.

Asthma affects millions of people worldwide and its prevalence is increasing. It is characterized by chronic airway inflammation, airway remodeling, and pathologic bronchoconstriction, and it poses a continuous treatment challenge with very few new therapeutics available. Thus, many asthmatics turn to plant-based complementary products, including ginger, for better symptom control, indicating an unmet need for novel therapies. Previously, we demonstrated that 6-shogaol (6S), the primary bioactive component of ginger, relaxes human airway smooth muscle (hASM) likely by inhibition of phosphodiesterases (PDEs) in the ß-adrenergic (cyclic nucleotide PDEs), and muscarinic (phospholipase C, PLC) receptor pathways. However, oral 6S is extensively metabolized and it is unknown if the resulting metabolites remain bioactive. Here, we screened all the known human metabolites of 6S and several metabolite-based synthetic derivatives to better understand their mechanism of action and structure-function relationships. We demonstrate that several metabolites and metabolite-based synthetic derivatives are able to prevent Gq-coupled stimulation of intracellular calcium [Ca2+]i and inositol trisphosphate (IP3) synthesis by inhibiting PLC, similar to the parent compound 6S. We also show that these compounds prevent recontraction of ASM after ß-agonist relaxation likely by inhibiting PDEs. Furthermore, they potentiate isoproterenol-induced relaxation. Importantly, moving beyond cell-based assays, metabolites also retain the functional ability to relax Gq-coupled-contractions in upper (human) and lower (murine) airways. The current study indicates that, although oral ginger may be metabolized rapidly, it retains physiological activity through its metabolites. Moreover, we are able to use naturally occurring metabolites as inspiration to develop novel therapeutics for brochoconstrictive diseases.

Dietary Quercetin Reduces Plasma and Tissue Methylglyoxal and Advanced Glycation End Products in Healthy Mice Treated with Methylglyoxal.

BACKGROUND: Methylglyoxal (MGO), a precursor of advanced glycation end products (AGEs), has been linked to AGEs-associated diseases. OBJECTIVES: This study investigated the efficacy and mechanisms of dietary quercetin in decreasing plasma and tissue concentrations of MGO and AGEs in MGO-administered mice. METHODS: Male, 6-wk-old CD-1 mice were administered AIN-93G diet and water (Con) or 0.12% MGO in water (MGO) or MGO plus 0.2% (0.2Q) dietary quercetin for 1 wk (n = 5) (experiment 1), and water (Con), 0.12% MGO (MGO), or MGO plus 0.1% (0.1Q), 0.2% (0.2Q), or 0.4% (0.4Q) dietary quercetin for 6 wk (n = 10) (experiment 2). The plasma, kidney, and liver concentrations of MGO, quercetin, and isorhamnetin and their trapping adducts with MGO were determined by LC-MS, and AGE concentrations were measured by the fluorescent method. Furthermore, the expressions of glyoxalase I/II (GLO I/II) and aldose reductase (AR), MGO detoxification enzymes, were determined by Western blot. One-factor ANOVA and post hoc Dunnett's or Tukey's test were used to analyze the data. RESULTS: After 1 wk of treatment, the MGO concentrations in plasma (20.2%) and kidney (29.9%) in 0.2Q mice were significantly lower than those in MGO mice. After 6 wk of treatment, the concentrations of MGO in the plasma (14.7-18.6%), kidney (20-20.8%), liver (15.4-18.6%), and tissue AGEs (28-36.8%) in 0.1Q, 0.2Q, and 0.4Q mice were significantly lower than those in MGO mice. The plasma concentrations of quercetin, isorhamnetin, and their MGO adducts were dose-dependently increased after quercetin administration. In addition, after 6 wk of quercetin administration, the expressions of GLO I/II and AR in the liver and kidney were significantly upregulated to promote MGO detoxification compared with MGO-treated mice. CONCLUSIONS: Quercetin reduced plasma and tissue MGO concentrations and inhibited AGE formation by trapping MGO and regulating the MGO detoxification systems in MGO-administered healthy mice.

Avenanthramide Metabotype from Whole-Grain Oat Intake is Influenced by Faecalibacterium prausnitzii in Healthy Adults.

BACKGROUND: Oat has been widely accepted as a key food for human health. It is becoming increasingly evident that individual differences in metabolism determine how different individuals benefit from diet. Both host genetics and the gut microbiota play important roles on the metabolism and function of dietary compounds. OBJECTIVES: To investigate the mechanism of individual variations in response to whole-grain (WG) oat intake. METHODS: We used the combination of in vitro incubation assays with human gut microbiota, mouse and human S9 fractions, chemical analyses, germ-free (GF) mice, 16S rRNA sequencing, gnotobiotic techniques, and a human feeding study. RESULTS: Avenanthramides (AVAs), the signature bioactive polyphenols of WG oat, were not metabolized into their dihydro forms, dihydro-AVAs (DH-AVAs), by both human and mouse S9 fractions. DH-AVAs were detected in the colon and the distal regions but not in the proximal and middle regions of the perfused mouse intestine, and were in specific pathogen-free (SPF) mice but not in GF mice. A kinetic study of humans fed oat bran showed that DH-AVAs reached their maximal concentrations at much later time points than their corresponding AVAs (10.0-15.0 hours vs. 4.0-4.5 hours, respectively). We observed interindividual variations in the metabolism of AVAs to DH-AVAs in humans. Faecalibacterium prausnitzii was identified as the individual bacterium to metabolize AVAs to DH-AVAs by 16S rRNA sequencing analysis. Moreover, as opposed to GF mice, F. prausnitzii-monocolonized mice were able to metabolize AVAs to DH-AVAs. CONCLUSIONS: These findings demonstrate that the presence of intestinal F. prausnitzii is indispensable for proper metabolism of AVAs in both humans and mice. We propose that the abundance of F. prausnitzii can be used to subcategorize individuals into AVA metabolizers and nonmetabolizers after WG oat intake. This study was registered at clinicaltrials.gov as NCT04335435.

Metabolic Interaction between Ammonia and Baicalein.

Ammonia is treated as a primary waste product of cellular metabolism in vivo and can contribute to the alteration of neurotransmission, oxidative stress, and cerebral edema and astrocyte swelling when its concentration in the brain is high. The objective of this study was to determine whether bioactive polyphenol baicalein had the capacity to trap ammonia in vitro and in vivo. Under in vitro conditions, baicalein rapidly reacted with ammonia to generate two monoaminated products and one diaminated product under different reaction times. These three major aminated products were purified from the reaction mixture, and their structures were characterized as 5-NH2-baicalein, 6-NH2-baicalein, and 5,6-di-NH2-baicalein based on the analysis of their HR-MS and 1D- and 2D-NMR data. In mice, both 5-NH2-baicalein and 6-NH2-baicalein were detected in 24 h fecal and urine samples collected from mice treated with baicalein (200 mg/kg) through oral gavage, and 6-NH2-baicalein was also detected in mouse plasma and brain samples collected at 0.5 h after baicalein treatment. Significant amounts of 6-NH2-baicalein were detected in all mouse samples including feces, urine, plasma, and brain. The levels of 6-NH2-baicalein in feces and urine were significantly higher than those of 5-NH2-baicalein. Furthermore, the average level of 6-NH2-baicalein was very close to that of baicalein (3.62 vs 3.77 ng/g) in mouse brain, suggesting it is possible that baicalein has the capacity to be absorbed rapidly into the circulation system and then cross the blood-brain barrier into the brain to detoxify ammonia in the blood and brain. In conclusion, this study confirmed that baicalein, a flavonoid with a vic-trihydroxyl structure on the A-ring, has the potential to detoxify ammonia and treat ammonia-associated chronic diseases.

Dietary Genistein Inhibits Methylglyoxal-Induced Advanced Glycation End Product Formation in Mice Fed a High-Fat Diet.

BACKGROUND: Methylglyoxal (MGO), an important precursor of advanced glycation end products (AGEs), circulates at high concentrations in diabetic patients' blood and plays an important role in the pathogenesis of diabetes and other chronic diseases. OBJECTIVES: The aim of this study was to determine whether dietary genistein can prevent indicators of metabolic syndrome (MetS) induced by a very-high-fat (VHF) diet or a high-fat (HF) diet plus exogenous MGO, and the accumulation of MGO and AGEs in mice. METHODS: Male, 6-wk-old C57BL/6J mice (n = 15) were fed a low-fat (LF) diet (10% fat energy) or a VHF diet (60% fat energy) alone or including 0.25% genistein (VHF-G) for 16 wk in study 1. In study 2, 75 similar mice were fed the LF diet (LF) or the HF diet alone (HF) or in combination with up to 0.2% MGO in water (HFM) and 0.067% (HFM-GL) or 0.2% (HFM-GH) dietary genistein for 18 wk. Anthropometric and metabolic data were obtained in both studies to determine the effects of MGO and genistein on variables indicative of MetS. RESULTS: Body weight gain, fat deposits, dyslipidemia, hyperglycemia, and fatty liver were ameliorated by dietary genistein in both studies. The plasma MGO concentration in VHF-G mice was 52% lower than that in VHF mice. Moreover, the AGE concentrations in plasma, liver, and kidney of VHF-G mice were 73%, 52%, and 49%, respectively, lower than in the VHF group (study 1). Similarly, the concentrations of plasma MGO and AGE in plasma, liver, and kidney of HFM-GH mice were 33.5%, 49%, 69%, and 54% lower than in HFM mice (study 2). Genistein inhibited AGE formation by trapping MGO to form adducts and upregulating the expressions of glyoxalase I and II and aldose reductase in liver and kidney to detoxify MGO in both studies. CONCLUSIONS: Our data demonstrate for the first time that genistein significantly lowers MGO and AGE concentrations in 2 mouse MetS models via multiple pathways.

One-step isolation of carnosic acid and carnosol from rosemary by centrifugal partition chromatography.

Carnosic acid and carnosol are the main bioactive components responsible for the significant antioxidant activity of Rosmarinus officinalis. Nevertheless, they are known for their instability in solutions. Separation of both compounds from crude rosemary extract was successfully achieved by one-step centrifugal partition chromatography without any degradation. A two-phase solvent system, hexane/ethyl acetate/methanol/water (3:2:3:2 v/v) was run on a preparative scale applying the elution-extrusion technique in descending mode. A 900 mg quantity of the crude extract containing 39.7% carnosic acid and 12.3% carnosol was loaded onto a 500 mL column, rotating at 1800 rpm. Carnosic acid and carnosol were obtained at purities of 96.1 ± 1% and 94.4 ± 0.9%, with recoveries of 94.3 ± 4.4% and 94.8 ± 2.3%, respectively. The compounds were identified by mass spectrometry, tandem mass spectrometry, and comparison with authentic standards.

Levels and formation of α-dicarbonyl compounds in beverages and the preventive effects of flavonoids.

ABSTRACT: Methylglyoxal (MGO) and glyoxal (GO), α-dicarbonyl compounds found in the Maillard reaction, progressively and irreversibly modify proteins. Beverages are an exogenous source of α-dicarbonyl compounds and may potentially increase MGO and GO levels in vivo. Using GC-FID method, we detected the MGO and GO contents of 86 beverages in Chinese supermarkets. The highest MGO and GO 587.5 µg/100 mL and 716.7 µg/100 mL respectively found in soyamilk and coffee. Herbal beverages, which contained bioactive components, had lower average levels of MGO (48.1 µg/100 mL) and GO (25.9 µg/100 mL). A box-and-whisker plot was used to display variation of the same group drinks, and comparing distributions between six different groups. It was further discovered that fat, protein and flavonoids, in addition to sweeteners, had notable effects on the formation of MGO and GO in soybean milk. The result of LC/MS indicated that quercetin could prevent the formation of MGO by trapping MGO to form the mono-MGO and di-MGO adducts during soybean milk manufacturing.

Trapping Methylglyoxal by Genistein and Its Metabolites in Mice.

Increasing evidence supports dicarbonyl stress such as methylglyoxal (MGO) as one of the major pathogenic links between hyperglycemia and diabetic complications. In vitro studies have shown that dietary flavonoids can inhibit the formation of advanced glycation end products (AGEs) by trapping MGO. However, whether flavonoids can trap MGO in vivo and whether biotransformation limits the trapping capacity of flavonoids remain virtually unknown. In this study, we investigated whether genistein (GEN), the major soy isoflavone, could trap MGO in mice by promoting the formation of MGO adducts of GEN and its metabolites. Two different mouse studies were conducted. In the acute study, a single dose of MGO and GEN were administered to mice via oral gavage. In the chronic study, MGO was given to mice in drinking water for 1 month and then GEN was given to mice for 4 consecutive days via oral gavage. Two mono-MGO adducts of GEN and six mono-MGO adducts of GEN phase I and microbial metabolites were identified in mouse urine samples from these studies using liquid chromatography/electrospray ionization tandem mass spectrometry. The structures of these MGO adducts were confirmed by analyzing their MS(n) (n = 1-4) spectra as well as by comparing them with the tandem mass spectra of authentic standards. All of the MGO adducts presented in their phase II conjugated forms in mouse urine samples in the acute and chronic studies. To our knowledge, this is the first in vivo evidence to demonstrate the trapping efficacy of GEN in mice and to show that the metabolites of GEN remain bioactive.

Oat avenanthramide-C (2c) is biotransformed by mice and the human microbiota into bioactive metabolites.

BACKGROUND: Avenanthramides (AVAs), which are found exclusively in oats, may play an important role in anti-inflammation and antiatherogenesis. Although the bioavailability of AVAs has been investigated previously, little is known about their metabolism. OBJECTIVES: The aim of the present study was to investigate the metabolism of avenanthramide-C (2c), one of the major AVAs, in mice and by the human microbiota, as well as to elucidate the bioactivity of its major metabolites with the goal of finding new exposure markers to precisely reflect oat consumption. METHODS: For the mouse study, 10 CF-1 female mice were divided into control (vehicle-treated) and 2c intragastrically treated (200 mg/kg) groups (5 mice/group). Twenty-four-hour urine and fecal samples were collected with use of metabolic cages. For the batch culture incubations, 2c was cultured with fecal slurries obtained from 6 human donors. Incubated samples were collected at various time points (0, 12, 24, 48, 72, 96, and 120 h). Metabolites were identified via HPLC with electrochemical detection and LC with electrospray ionization/mass spectrometry. To investigate whether 2c metabolites retain the biological effects of 2c, we compared their effects on the growth of and induction of apoptosis in HCT-116 human colon cancer cells. RESULTS: Eight metabolites were detected from the 2c-treated mouse urine samples. They were identified as 5-hydroxyanthranilic acid (M1), dihydrocaffeic acid (M2), caffeic acid (M3), dihydroferulic acid (M4), ferulic acid (M5), dihydroavenanthramide-C (M6), dihydroavenanthramide-B (M7), and avenanthramide-B (M8) via analysis of their MS(n) (n = 1-3) spectra. We found that the reduction of 2c's C7'-C8' double bond and the cleavage of its amide bond were the major metabolic routes. In the human microbiota study, 2c was converted into M1-M3 and M6. Moreover, interindividual differences in 2c metabolism were observed among the 6 human subjects. Subjects B, C, E, and F could rapidly metabolize 2c to M6, whereas subject D metabolized little 2c, even up to 120 h. In addition, only subjects A, B, and F could cleave the amide bond of 2c or M6 to form the cleaved metabolites. Furthermore, we showed that 2c and its major metabolite M6 are bioactive compounds against human colon cancer cells. M6 was more active than 2c with the half-inhibitory concentration (IC50) of 158 µM and could induce apoptosis at 200 µM. CONCLUSION: To our knowledge, the current study demonstrates for the first time that avenanthramide-C can be extensively metabolized by mice and the human microbiota to generate bioactive metabolites.

Bioactive ginger constituents alleviate protein glycation by trapping methylglyoxal.

Considerable evidence suggests that long-term pathological diabetes is a result of the accumulation of tissue macromolecules that have been progressively modified by nonenzymatic glycation of protein. Methylglyoxal (MGO) is a highly reactive endogenous dicarbonyl metabolite derived from multiple sources such as glucose and lipids and is thought to contribute greatly to protein glycation and the formation of advanced glycation end products (AGEs). In this study, we demonstrated for the first time that both [6]-shogaol (6S) and [6]-gingerol (6G), the major active components in ginger, markedly trapped MGO in vitro and consequently formed mono-MGO adducts, 6S-MGO and 6G-MGO, which were purified from the respective chemical reaction and characterized as novel compounds by NMR experiments and LC-MS/MS approaches. We revealed that the α-carbon of the carbonyl group in the side chain of 6S or 6G is the major active site for trapping MGO. We also demonstrated that 6S and 6G could effectively inhibit the formation of MGO-induced AGEs via trapping MGO in a time-dependent manner in the human serum albumin (HSA)-MGO system. Mono-MGO adducts, 6S-MGO and 6G-MGO, were determined to be the major conjugates in 6S- and 6G-treated HSA-MGO assays, respectively, using LC-ESI-MS techniques. These findings showed the potential effects of 6S and 6G on the prevention of protein glycation, suggesting regular consumption of ginger root extract may attenuate the progression of MGO-associated diabetic complications in patients.

Tea Flavanols Block Advanced Glycation of Lens Crystallins Induced by Dehydroascorbic Acid.

Growing evidence has shown that ascorbic acid (ASA) can contribute to protein glycation and the formation of advanced glycation end products (AGEs), especially in the lens. The mechanism by which ascorbic acid can cause protein glycation probably originates from its oxidized form, dehydroascorbic acid (DASA), which is a reactive dicarbonyl species. In the present study, we demonstrated for the first time that four tea flavanols, (-)-epigallocatechin 3-O-gallate (EGCG), (-)-epigallocatechin (EGC), (-)-epicatechin 3-O-gallate (ECG), and (-)-epicatechin (EC), could significantly trap DASA and consequently form 6C- or 8C-ascorbyl conjugates. Among these four flavanols, EGCG exerted the strongest trapping efficacy by capturing approximate 80% of DASA within 60 min. We successfully purified and identified seven 6C- or 8C-ascorbyl conjugates of flavanols from the chemical reaction between tea flavanols and DASA under slightly basic conditions. Of which, five ascorbyl conjugates, EGCGDASA-2, EGCDASA-2, ECGDASA-1, ECGDASA-2 and ECDASA-1, were recognized as novel compounds. The NMR data showed that positions 6 and 8 of the ring A of flavanols were the major active sites for trapping DASA. We further demonstrated that tea flavanols could effectively inhibit the formation of DASA-induced AGEs via trapping DASA in the bovine lens crystallin-DASA assay. In this assay, 8C-ascorbyl conjugates of flavanols were detected as the major adducts using LC-MS. This study suggests that daily consumption of beverages containing tea flavanols may prevent protein glycation in the lens induced by ascorbic acid and its oxidized products.

Identification and pharmacokinetics of novel alkylresorcinol metabolites in human urine, new candidate biomarkers for whole-grain wheat and rye intake.

Biomarkers of dietary intake are prominent tools in nutritional research. The alkylresorcinol metabolites 3,5-dihydroxybenzoic acid (3,5-DHBA) and 3-(3,5-dihydroxyphenyl)propanoic acid (3,5-DHPPA) have been proposed as exposure biomarkers of whole-grain (WG) wheat and rye intake. However, the profile of alkylresorcinol metabolites is not fully understood. The aim of this study was to investigate the metabolism of alkylresorcinols in mice and in humans, while further determining urinary pharmacokinetics of the novel alkylresorcinol metabolites to explore their potential as biomarkers of WG wheat intake. Utilization of the liquid chromatography-mass spectrometry approach resulted in 10 alkylresorcinol metabolites identified in mice and in humans, including 3 phenolic acids and 7 of their phase II conjugates. Among them, 2 novel metabolites were discovered: 5-(3,5-dihydroxyphenyl)pentanoic acid (3,5-DHPPTA) and 2-(3,5-dihydroxybenzamido)acetic acid (3,5-DHBA glycine). The structures of these 2 metabolites were confirmed by comparing with authentic standards synthesized in-house. In the pharmacokinetic study, a group of 12 volunteers consumed a polyphenolic-restricted diet for 4 d before ingesting WG wheat bread containing 61 mg of alkylresorcinols. Urine samples were collected for 32 h, and alkylresorcinol metabolites were quantified with HPLC-coulometric electrode array detection. The mean urinary excretion rates and mean apparent half-life of 3,5-DHPPTA, 3,5-DHBA glycine, 3,5-DHBA, and 3,5-DHPPA at each time point were determined. Our results suggest that 3,5-DHPPTA and 3,5-DHBA glycine may be used in combination with 3,5-DHBA and 3,5-DHPPA as potential biomarkers to increase the accuracy of recording WG wheat and rye intake in epidemiologic studies. Further validation of 3,5-DHPPTA and 3,5-DHBA glycine as potential biomarkers is warranted.

Ginger compound [6]-shogaol and its cysteine-conjugated metabolite (M2) activate Nrf2 in colon epithelial cells in vitro and in vivo.

In this study, we identified Nrf2 as a molecular target of [6]-shogaol (6S), a bioactive compound isolated from ginger, in colon epithelial cells in vitro and in vivo. Following 6S treatment of HCT-116 cells, the intracellular GSH/GSSG ratio was initially diminished but was then elevated above the basal level. Intracellular reactive oxygen species (ROS) correlated inversely with the GSH/GSSG ratio. Further analysis using gene microarray showed that 6S upregulated the expression of Nrf2 target genes (AKR1B10, FTL, GGTLA4, and HMOX1) in HCT-116 cells. Western blotting confirmed upregulation, phosphorylation, and nuclear translocation of Nrf2 protein followed by Keap1 decrease and upregulation of Nrf2 target genes (AKR1B10, FTL, GGTLA4, HMOX1, and MT1) and glutathione synthesis genes (GCLC and GCLM). Pretreatment of cells with a specific inhibitor of p38 (SB202190), PI3K (LY294002), or MEK1 (PD098059) attenuated these effects of 6S. Using ultra-high-performance liquid chromatography-tandem mass spectrometry, we found that 6S modified multiple cysteine residues of Keap1 protein. In vivo 6S treatment induced Nrf2 nuclear translocation and significantly upregulated the expression of MT1, HMOX1, and GCLC in the colon of wild-type mice but not Nrf2(-/-) mice. Similar to 6S, a cysteine-conjugated metabolite of 6S (M2), which was previously found to be a carrier of 6S in vitro and in vivo, also activated Nrf2. Our data demonstrated that 6S and its cysteine-conjugated metabolite M2 activate Nrf2 in colon epithelial cells in vitro and in vivo through Keap1-dependent and -independent mechanisms.

Ginger Polyphenols Reverse Molecular Signature of Amygdala Neuroimmune Signaling and Modulate Microbiome in Male Rats with Neuropathic Pain: Evidence for Microbiota-Gut-Brain Axis.

Emerging evidence shows that the gut microbiota plays an important role in neuropathic pain (NP) via the gut-brain axis. Male rats were divided into sham, spinal nerve ligation (SNL), SNL + 200 mg GEG/kg BW (GEG200), and SNL + 600 mg GEG/kg BW (GEG600) for 5 weeks. The dosages of 200 and 600 mg GEG/kg BW for rats correspond to 45 g and 135 g raw ginger for human daily consumption, respectively. Both GEG groups mitigated SNL-induced NP behavior. GEG-supplemented animals had a decreased abundance of Rikenella, Muribaculaceae, Clostridia UCG-014, Mucispirillum schaedleri, RF39, Acetatifactor, and Clostridia UCG-009, while they had an increased abundance of Flavonifactor, Hungatella, Anaerofustis stercorihominis, and Clostridium innocuum group. Relative to sham rats, Fos and Gadd45g genes were upregulated, while Igf1, Ccl2, Hadc2, Rtn4rl1, Nfkb2, Gpr84, Pik3cg, and Abcc8 genes were downregulated in SNL rats. Compared to the SNL group, the GEG200 group and GEG600 group had increases/decreases in 16 (10/6) genes and 11 (1/10) genes, respectively. GEG downregulated Fos and Gadd45g genes and upregulated Hdac2 genes in the amygdala. In summary, GEG alleviates NP by modulating the gut microbiome and reversing a molecular neuroimmune signature.

Peracetylated (-)-epigallocatechin-3-gallate (AcEGCG) potently prevents skin carcinogenesis by suppressing the PKD1-dependent signaling pathway in CD34+ skin stem cells and skin tumors.

During the process of skin tumor promotion, expression of the cutaneous cancer stem cell (CSC) marker CD34(+) is required for stem cell activation and tumor formation. A previous study has shown that activation of protein kinase D1 (PKD1) is involved in epidermal tumor promotion; however, the signals that regulate CSCs in skin carcinogenesis have not been characterized. This study was designed to investigate the chemopreventive potential of peracetylated (-)-epigallocatechin-3-gallate (AcEGCG) on 7,12-dimethylbenz[a]-anthracene (DMBA)-initiated and 12-O-tetradecanoylphorbol-13-acetate (TPA)-promoted skin tumorigenesis in ICR mice and to elucidate the possible mechanisms involved in the inhibitory action of PKD1 on CSCs. We demonstrated that topical application of AcEGCG before TPA treatment can be more effective than EGCG in reducing DMBA/TPA-induced tumor incidence and multiplicity. Notably, AcEGCG not only inhibited the expression of p53, p21, c-Myc, cyclin B, p-CDK1 and Cdc25A but also restored the activation of extracellular signal-regulated kinase 1/2 (ERK1/2), which decreased DMBA/TPA-induced increases in tumor proliferation and mitotic index. To clarify the role of PKD1 in cell proliferation and tumorigenesis, we studied the expression and activation of PKD1 in CD34(+) skin stem cells and skin tumors. We found that PKD1 was strongly expressed in CD34(+) cells and that pretreatment with AcEGCG markedly inhibited PKD1 activation and CD34(+) expression. More importantly, pretreatment with AcEGCG remarkably suppressed nuclear factor-kappaB, cyclic adenosine 3',5'-monophosphate-responsive element-binding protein (CREB) and CCAAT-enhancer-binding protein (C/EBPs) activation by inhibiting the phosphorylation of c-Jun-N-terminal kinase 1/2, p38 and phosphatidylinositol 3-kinase (PI3K)/Akt and by attenuating downstream target gene expression, including inducible nitric oxide synthase, cyclooxygenase-2, ornithine decarboxylase and vascular endothelial growth factor. Moreover, this is the first study to demonstrate that AcEGCG is a CD34(+) and PKD1 inhibitor in the multistage mouse skin carcinogenesis model. Overall, our results powerfully suggest that AcEGCG could be developed into a novel chemopreventive agent and that PKD1 may be a preventive and therapeutic target for skin cancer in clinical settings.

Cysteine-conjugated metabolite of ginger component [6]-shogaol serves as a carrier of [6]-shogaol in cancer cells and in mice.

Shogaols, a series of major constituents in dried ginger (Zingiber officinale), show high anticancer potencies. Previously, we reported that a major metabolite resulting from the mercapturic acid pathway, 5-cysteinyl-[6]-shogaol (M2), showed comparable growth inhibitory effects toward cancer cells to [6]-shogaol (6S). Here, we probe the mechanism by which M2 exerts its bioactivity. We utilized a series of chemical stability tests in conjunction with bioassays to show that thiol-conjugates display chemopreventative potency by acting as carriers of active ginger component 6S. M2 chemical degradation to 6S was observed in an environment most resembling physiological conditions, with a pH of 7.4 at 37 °C. The metabolic profiles of M2 in cancer cells HCT-116 and H-1299 resembled those of 6S, indicating that its biotransformation route was initiated by deconjugation. Further, the presence of excess glutathione significantly delayed 6S and M2 metabolism and counteracted cell death induced by 6S and M2, suggesting that increasing available free thiols exogenously both promoted the formation of 5-glutathionyl-[6]-shogaol (M13) and inhibited the production of free 6S from M2 deconjugation, resulting in delayed 6S cell entry and bioactivity. Given the chemopreventative properties of M2 and our observations in vitro, we investigated its metabolism in mice. M2 and 6S showed similar metabolic profiles in mouse urine and fecal samples. Six new thiol-conjugated metabolites (M16-M21), together with previously reported ones, were identified by LC/MS. In particular, the increase of 5-N-acetylcystenyl-[6]-shogaol (M5) and its 3'-demethylated product (M16) abundance in mouse feces after treatment with M2 indicates that in addition to acting as a carrier of 6S, M2 is also directly acetylated to M5, which is further demethylated to M16 in vivo. In conclusion, the cysteine-conjugated metabolite of [6]-shogaol M2 exerts its bioactivity by acting as a carrier of 6S in both cancer cells and in mice.

Barley Phenolamides Effectively Scavenge Harmful Methylglyoxal In Vitro and in Mice.

SCOPE: Methylglyoxal (MGO), a harmful reactive dicarbonyl, is involved in the pathogenesis and development of diabetes and diabetic complications. The goal of this study is to determine whether bioactive phenolamides in barley, p-coumaroylagmatine (pCAA) and feruloylagmatine (FAA), which share a similar guanidine group to diabetic drug metformin, have the capacity to detoxify MGO. METHODS AND RESULTS: In this study, the MGO-trapping abilities of these two phenolamides both in vitro and in mice are evaluated. It is found that in vitro anti-MGO capacities of pCAA and FAA are comparable to that of metformin, and both phenolamides could rapidly scavenge MGO via forming mono- and di-MGO adducts validated by in-house synthesized standards and interpretation of respective LC-MSn (n = 2-3) data. Furthermore, mono-MGO conjugates of phenolamides are detected from feces and urine of mice after oral administration of the corresponding phenolamides. CONCLUSION: These findings suggest that barley phenolamides may have the potentials to be developed as alternative therapeutics to prevent the development of MGO-associated diabetes and diabetic complications.

Fermented Oats as a Novel Functional Food.

Fermented oats are gaining popularity due to their nutritional value and the increasing consumer demand for health-conscious foods. These oats are believed to offer enhanced phytochemical and nutritional profiles compared to unfermented oats. The increased nutritional content of fermented oats is associated with various health benefits, including anti-inflammatory and antioxidant activities, which could potentially reduce the risk of chronic diseases. Further investigations are warranted to elucidate the nutritional benefits of fermented oats in human nutrition. This mini review provides a comprehensive overview of fermented oat products available on the market and the various production methods employed for fermenting oats. Furthermore, this review investigates how fermentation affects the chemical composition and biological functions of oats. Additionally, this manuscript presents some future perspectives on fermented oat products by discussing potential research directions and opportunities for further development. The findings presented in this review contribute to the expanding body of knowledge on fermented oats as a promising functional food, paving the way for future studies and applications in the field of nutrition and health.

Detoxification of the Lipid Peroxidation Aldehyde, 4-Hydroxynonenal, by Apple Phloretin In Vitro and in Mice.

4-Hydroxy-2-nonenal (4-HNE) is a secondary cytotoxic product generated from lipid peroxidation of polyunsaturated fatty acids (PUFAs). The accumulation of 4-HNE can covalently modify biomolecules, such as DNA and proteins, leading to various pathological conditions. Apple phloretin has been shown to be able to trap 4-HNE in vitro, but the trapping mechanisms of 4-HNE by phloretin are not fully understood. Moreover, whether the in vitro trapping efficacy of phloretin toward 4-HNE could be transferred into in vivo environments has never been investigated. In the present study, we observed the formation of 4-HNE conjugates of phloretin increased as phloretin decreased during the in vitro incubation. We then purified and characterized three mono-4-HNE-conjugates of phloretin using NMR and LC-MS/MS techniques. We thereafter demonstrated that apple phloretin could scavenge in vivo 4-HNE via the formation of at least three mono-4-HNE-conjugates of phloretin in a dose-dependent manner in mice after oral administration of three doses of phloretin (25, 100, and 400 mg/kg). The findings from this study pave the way to understanding how dihydrochalcones could act as effective scavengers of 4-HNE by working as sacrificial nucleophiles in vivo, thereby preventing or reducing the risk of 4-HNE-associated chronic diseases.

Absorption and Metabolism of Ginger Compounds in Broiler Chicks.

Bioavailability is critical in ensuring bioefficacy of ginger compounds, which have not been studied in chicks. In this study, day-old chicks were treated with ginger root extract at 0.0, 0.4, 0.8, 1.5, and 3.0% for 42 days. The gingerols and shogaols in chick samples were analyzed by liquid chromatography-mass spectrometry. The primary phase-I metabolic pathway for gingerols and shogaols was the reduction of ketone groups into hydroxyl groups. Shogaols were also metabolized through thiol conjugation and hydrogenation of double-bond pathways. Within the bloodstream, gingerols and their metabolites predominantly existed as glucuronidate or sulfate conjugates. However, the levels of the free form and conjugates were comparable for shogaols. In breast meat, the quantities of both the free form and conjugates for all compounds were similar. In plasma, more than 50% of absorbed 6-gingerol (6G) and 90% of absorbed 6-shogaol underwent reduction to their respective metabolites. However, in breast meat, the percentage of reduction for absorbed 6G was less than 50%, and for absorbed 6-shogaol, it was less than 60%. Ginger compounds were absorbed into chick plasma ranging from 1.4 to 8.5 µg/mL and breast meat ranging from 7.1 to 114.6 µg/100 g across the 0.4-3.0% dose range in a dose-dependent manner.