Identification of a Novel Pyrrole Alkaloid from the Edible Mushroom Basidiomycetes-X (Echigoshirayukidake).

Three pyrrole alkaloid derivatives were isolated from the edible mushroom Basidiomycetes-X (Echigoshirayukidake) by water extraction followed by ethyl acetate fractionation. The chemical structures determined by MS and NMR were 4-[2-formyl-5-(hydroxymethyl)-1H-pyrrol-1-yl] butanoic acid (compound I), 4-[2-formyl-5-(hydroxymethyl)-1H-pyrrol-1-yl] butanamide (compound II), and 5-(hydroxymethyl)-1H-pyrrole-2-carboxaldehyde (compound III). Compound I was found to be the major component, followed by compound II, and compound III was the minor component. The dry powder of Basidiomycetes-X contained approximately 825 µg g-1 compound I and 484 µg g-1 compound II. Compound II was found to be a novel pyrrole aldehyde homologue not previously reported and thus is a specific component of this mushroom.

Involvement of Prokineticin 2-expressing Neutrophil Infiltration in 5-Fluorouracil-induced Aggravation of Breast Cancer Metastasis to Lung.

Adjuvant chemotherapy is used for human breast cancer patients, even after curative surgery of primary tumor, to prevent tumor recurrence primarily as a form of metastasis. However, anticancer drugs can accelerate metastasis in several mouse metastasis models. Hence, we examined the effects of postsurgical administration with 5-fluorouracil (5-FU), doxorubicin, and cyclophosphamide, on lung metastasis process, which developed after the resection of the primary tumor arising from the orthotopic injection of a mouse triple-negative breast cancer cell line, 4T1. Only 5-FU markedly increased the numbers and sizes of lung metastasis foci, with enhanced tumor cell proliferation and angiogenesis as evidenced by increases in Ki67-positive cell numbers and CD31-positive areas, respectively. 5-FU-mediated augmented lung metastasis was associated with increases in intrapulmonary neutrophil numbers and expression of neutrophilic chemokines, Cxcl1 and Cxcl2 in tumor cells, with few effects on intrapulmonary T-cell or macrophage numbers. 5-FU enhanced Cxcl1 and Cxcl2 expression in 4T1 cells in a NFκB-dependent manner. Moreover, the administration of a neutrophil-depleting antibody or a Cxcr2 antagonist, SB225002, significantly attenuated 5-FU-mediated enhanced lung metastasis with depressed neutrophil infiltration. Furthermore, infiltrating neutrophils and 4T1 cells abundantly expressed prokineticin-2 (Prok2) and its receptor, Prokr1, respectively. Finally, the administration of 5-FU after the resection of the primary tumor failed to augment lung metastasis in the mice receiving Prokr1-deleted 4T1 cells. Collectively, 5-FU can enhance lung metastasis by inducing tumor cells to produce Cxcl1 and Cxcl2, which induced the migration of neutrophils expressing Prok2 with a capacity to enhance 4T1 cell proliferation. Mol Cancer Ther; 17(7); 1515-25. ©2018 AACR.

Anti-Inflammatory Properties of Brazilian Green Propolis Encapsulated in a γ-Cyclodextrin Complex in Mice Fed a Western-Type Diet.

Ageing is often accompanied by chronic inflammation. A fat- and sugar-rich Western-type diet (WTD) may accelerate the ageing phenotype. Cell culture studies have indicated that artepillin C-containing Brazilian green propolis exhibits anti-inflammatory properties. However, little is known regarding its anti-inflammatory potential in mouse liver in vivo. In this study, female C57BL/6NRj wild-type mice were fed a WTD, a WTD supplemented with Brazilian green propolis supercritical extract (GPSE) encapsulated in γ-cyclodextrin (γCD) or a WTD plus γCD for 10 weeks. GPSE-γCD did not affect the food intake, body weight or body composition of the mice. However, mRNA levels of the tumour necrosis factor α were significantly downregulated (p < 0.05) in these mice compared to those in the WTD-fed controls. Furthermore, the gene expression levels of other pro-inflammatory markers, including serum amyloid P, were significantly (p < 0.001) decreased following GPSE-γCD treatment. GPSE-γCD significantly induced hepatic ferritin gene expression (p < 0.01), which may contribute to its anti-inflammatory properties. Conversely, GPSE-γCD did not affect the biomarkers of endogenous antioxidant defence, including catalase, glutathione peroxidase-4, paraoxonase-1, glutamate cysteine ligase and nuclear factor erythroid 2-related factor-2 (Nrf2). Overall, the present data suggest that dietary GPSE-γCD exhibits anti-inflammatory, but not antioxidant activity in mouse liver in vivo. Thus, GPSE-γCD has the potential to serve as a natural hepatoprotective bioactive compound for dietary-mediated strategies against chronic inflammation.

Time Course Effect of R-Alpha-Lipoic Acid on Cellular Metabolomics in Cultured Hepatoma Cells.

Alpha-lipoic acid (LA) is a powerful antioxidant. LA has two enantiomers, R(+)-LA (R-LA) and S(-)-LA (S-LA). Of these, R-LA is naturally occurring and an essential cofactor in energy metabolism. R-LA treatment has been reported to affect glucose metabolism in rat hepatoma cells. This study analyzed the time course of metabolite levels in LA-treated cultured H4IIEC3 rat hepatoma cells, including a specific evaluation of the effect of R-LA and the enantioselectivity of LA. Principal component analysis showed that this experiment was well designed to observe enantioselectivity. R-LA treatment was found to inhibit the glycolysis and Thr-Gly-Ser pathways, as well as lactic acid production, leading to the inhibition of gluconeogenesis in starved H4IIEC3 cells. This study may provide mechanistic insight into how R-LA induces apoptosis in hepatoma cells.

Deficiency of the hepatokine selenoprotein P increases responsiveness to exercise in mice through upregulation of reactive oxygen species and AMP-activated protein kinase in muscle.

Exercise has numerous health-promoting effects in humans; however, individual responsiveness to exercise with regard to endurance or metabolic health differs markedly. This 'exercise resistance' is considered to be congenital, with no evident acquired causative factors. Here we show that the anti-oxidative hepatokine selenoprotein P (SeP) causes exercise resistance through its muscle receptor low-density lipoprotein receptor-related protein 1 (LRP1). SeP-deficient mice showed a 'super-endurance' phenotype after exercise training, as well as enhanced reactive oxygen species (ROS) production, AMP-activated protein kinase (AMPK) phosphorylation and peroxisome proliferative activated receptor γ coactivator (Ppargc)-1α (also known as PGC-1α; encoded by Ppargc1a) expression in skeletal muscle. Supplementation with the anti-oxidant N-acetylcysteine (NAC) reduced ROS production and the endurance capacity in SeP-deficient mice. SeP treatment impaired hydrogen-peroxide-induced adaptations through LRP1 in cultured myotubes and suppressed exercise-induced AMPK phosphorylation and Ppargc1a gene expression in mouse skeletal muscle-effects which were blunted in mice with a muscle-specific LRP1 deficiency. Furthermore, we found that increased amounts of circulating SeP predicted the ineffectiveness of training on endurance capacity in humans. Our study suggests that inhibitors of the SeP-LRP1 axis may function as exercise-enhancing drugs to treat diseases associated with a sedentary lifestyle.

Chemical Characterization, Free Radical Scavenging, and Cellular Antioxidant and Anti-Inflammatory Properties of a Stilbenoid-Rich Root Extract of Vitis vinifera.

Dietary stilbenoids are receiving increasing attention due to their potential health benefits. However, most studies concerning the bioactivity of stilbenoids were conducted with pure compounds, for example, resveratrol. The aim of this study was to characterize a complex root extract of Vitis vinifera in terms of its free radical scavenging and cellular antioxidant and anti-inflammatory properties. HPLC-ESI-MS/MS analyses of the root extract of Vitis vinifera identified seven stilbenoids including two monomeric (resveratrol and piceatannol), two dimeric (trans-ɛ-viniferin and ampelopsin A), one trimeric (miyabenol C), and two tetrameric (r-2-viniferin = vitisin A and r-viniferin = vitisin B) compounds which may mediate its biological activity. Electron spin resonance and spin trapping experiments indicate that the root extract scavenged 2,2-diphenyl-1-picrylhydrazyl, hydroxyl, galvinoxyl, and superoxide free radicals. On a cellular level it was observed that the root extract of Vitis vinifera protects against hydrogen peroxide-induced DNA damage and induces Nrf2 and its target genes heme oxygenase-1 and γ-glutamylcysteine synthetase. Furthermore, the root extract could induce the antiatherogenic hepatic enzyme paraoxonase 1 and downregulate proinflammatory gene expression (interleukin 1ß, inducible nitric oxide synthase) in macrophages. Collectively our data suggest that the root extract of Vitis vinifera exhibits free radical scavenging as well as cellular antioxidant and anti-inflammatory properties.

R-α lipoic acid γ-cyclodextrin complex increases energy expenditure: a 4-month feeding study in mice.

OBJECTIVE: A high-fat diet (HFD) affects energy expenditure in laboratory rodents. R-α lipoic acid cyclodextrin (RALA-CD) complex is a stable form of lipoic acid (LA) and may improve energy expenditure. The aim of this study was to determine the effect of RALA-CD on energy expenditure and underlying molecular targets in female laboratory mice. METHODS: Female C57BL/6J mice were fed a HFD containing 0.1% LA for about 16 wk. The effects on energy expenditure, gene and protein expression were assessed using indirect calorimetry, real-time reverse transcriptase polymerase chain reaction, and Western blot, respectively. RESULTS: Supplementing mice with RALA-CD resulted in a significant increase in energy expenditure. However, both RALA per se (without γ-cyclodextrin) and S-α lipoic acid cyclodextrin did not significantly alter energy expenditure. Furthermore RALA-CD changed expression of genes encoding proteins centrally involved in energy metabolism. Transcriptional key regulators sirtuin 3 and peroxisome proliferator-activated receptor-γ, coactivator 1 alpha, as well as thyroid related enzyme type 2 iodothyronine deiodinase were up-regulated in brown adipose tissue (BAT) of RALA-CD-fed mice. Importantly, mRNA and/or protein expression of downstream effectors uncoupling protein (Ucp) 1 and 3 also were elevated in BAT from RALA-CD-supplemented mice. CONCLUSION: Overall, present data suggest that RALA-CD is a regulator of energy expenditure in laboratory mice.

Photochemical stability of lipoic acid and its impact on skin ageing.

It is well known that α-lipoic acid (LA) functions as an essential co-factor of the mitochondrial multi-enzyme complex and thus plays an important role in energy metabolism. Currently, it is attracting attention as a nutritional supplement because of its unique antioxidant properties and broad spectra of cellular functions. Skin protection from photodamage and ageing is one of the functional applications of LA. Medical and cosmetic application has been widely realized in the world. However, LA has a unique structure bearing a distorted five membered 1, 2-dithiolane ring, making it quite vulnerable to UV radiation. The present article briefly reviews skin ageing from the viewpoint of oxidative stress and sun exposure and analyses the photochemical properties of LA. It also discusses the effect of LA to cellular signalling and its adequate applications to treat skin ageing caused by oxidation. Data presented in this review suggest that LA is a powerful anti-ageing agent under the appropriate usage.

Ascorbic acid partly antagonizes resveratrol mediated heme oxygenase-1 but not paraoxonase-1 induction in cultured hepatocytes - role of the redox-regulated transcription factor Nrf2.

BACKGROUND: Both resveratrol and vitamin C (ascorbic acid) are frequently used in complementary and alternative medicine. However, little is known about the underlying mechanisms for potential health benefits of resveratrol and its interactions with ascorbic acid. METHODS: The antioxidant enzymes heme oxygenase-1 and paraoxonase-1 were analysed for their mRNA and protein levels in HUH7 liver cells treated with 10 and 25 µmol/l resveratrol in the absence and presence of 100 and 1000 µmol/l ascorbic acid. Additionally the transactivation of the transcription factor Nrf2 and paraoxonase-1 were determined by reporter gene assays. RESULTS: Here, we demonstrate that resveratrol induces the antioxidant enzymes heme oxygenase-1 and paraoxonase-1 in cultured hepatocytes. Heme oxygenase-1 induction by resveratrol was accompanied by an increase in Nrf2 transactivation. Resveratrol mediated Nrf2 transactivation as well as heme oxygenase-1 induction were partly antagonized by 1000 µmol/l ascorbic acid. CONCLUSIONS: Unlike heme oxygenase-1 (which is highly regulated by Nrf2) paraoxonase-1 (which exhibits fewer ARE/Nrf2 binding sites in its promoter) induction by resveratrol was not counteracted by ascorbic acid. Addition of resveratrol to the cell culture medium produced relatively low levels of hydrogen peroxide which may be a positive hormetic redox-signal for Nrf2 dependent gene expression thereby driving heme oxygenase-1 induction. However, high concentrations of ascorbic acid manifold increased hydrogen peroxide production in the cell culture medium which may be a stress signal thereby disrupting the Nrf2 signalling pathway.

Simple HPLC evaluation of lipoamidase activity in tissue using a newly synthesized fluorescent substrate, dansyl-α-lipoyllysine.

α-Lipoic acid (LA) is a naturally occurring disulfide-containing compound used as an antioxidant supplement which also has been used as a medicine for diabetic neuropathy in Europe. Physiologically LA acts as a coenzyme of mitochondrial multienzyme complex in its protein bound form but it is not yet clear how the externally administrated LA is incorporated into other proteins in the same protein-bound form or why the bound form is active as an antioxidant. The binding and cleavage of LA to or from the protein is mediated by lipoamidase and thus determines LA distribution in tissues. We have developed a simple sensitive assay for lipoamidase using a fluorescent substrate, dansyl-α-lipoyllysine (DLL). Lipoamidase in tissues cleaves the amide bond between LA and the ε-amino-lysine moiety to release dansylated lysine (DL). A HPLC comparison of the fluorescence intensity between DLL and DL was used to quantify the enzyme activity. The hydrolytic reaction did not occur when the tissue was heat-treated before incubation with DLL and was inhibited by free LA, especially by the R-enantiomer of LA (physiologically active form). N(ε)-Acetyl-L-lysine did not compete with DLL in the cleavage reaction. The method was applied for the determination of lipoamidase activity levels in various rat tissues. It was revealed the spleen had the highest activity followed by the kidney, heart, lung and liver. The activity in the brain was below the detection limit of the assay.

Cancer cell cytotoxicity of extracts and small phenolic compounds from Chaga [Inonotus obliquus (persoon) Pilat].

Previously, we studied the antioxidant potential of Chaga mushroom [Inonotus obliquus (persoon) Pilat] extracts and isolated several small (poly)phenolic compounds as the major antioxidant components in the 80% methanol (MeOH) extract. In the present study, these isolated phenolic ingredients together with several other types of Chaga extracts were examined for cytotoxic effects against normal (IMR90) and cancer (A549, PA-1, U937, and HL-60) cell lines. Results revealed decoctions from both the fruiting body (FB) and sclerotium (ST) parts of Chaga, especially the ST part, showed considerable cytotoxicity toward tumor cells, but the cytotoxicity appeared to be stronger against normal cells than cancer cells. The 80% MeOH ST extract also showed the same trend. On the other hand, the 80% MeOH extract of FB showed significant cytotoxicity towards tumor cell lines without affecting normal cells, for example, the 50% lethal dose was 49.4 +/- 2.9 microg/mL for PA-1 cells versus 123.6 +/- 13.8 microg/mL for normal cells. The phenolic components isolated from the 80% MeOH extracts had markedly greater cancer cell toxicity than the extracts themselves. In particular, two out of seven compounds showed strong cytotoxicity towards several tumor cell lines without giving rise to significant cell toxicity toward normal cells. For example, the 50% lethal dose for 3,4-dihydroxybenzalacetone was 12.2 micromol/L in PA-1 cells but was 272.8 micromol/L in IMR90 cells. Fluorescence-activated cell sorting analysis further revealed these phenolic ingredients have high potentiality for apoptosis induction in PA-1 cells.

The Degradation and Regeneration of alpha-Lipoic Acid under the Irradiation of UV Light in the Existence of Homocysteine.

alpha-Lipoic acid (LA) is the one of the strongest antioxidants to be utilized in supplement, skin ointment and so on. The distorted five membered dithiolane ring of LA, which is necessary structure to work as a cofactor of enzyme, is considerably vulnerable to UV irradiation. LA is easily decomposed by photoirradiation resulting in the loss of its characteristic absorption band at 333 nm. The photodegradation of LA means loss of its physiological activity, so that protection of LA from UV light is eagerly desired. Thiol compounds can be regarded as a potential candidate. In order to pursue the possibility of the thiol compounds in prevention of LA degradation, we examined the photoirradiation of LA in the presence and absence of homocysteine.

Simultaneous comparison of relative reactivities of twelve major anthocyanins in bilberry towards reactive nitrogen species.

The reactivities of twelve major anthocyanins identified in bilberry extracts towards nitric oxide (NO.) and peroxynitrite (ONOO-) were studied in vitro using capillary zone electrophoresis (CZE). The reactivities of the anthocyanins towards NO. were slightly weak compared with that of (+)-catechin as a reference antioxidant under anaerobic conditions except delphinidin glycosides (Dp3glys). The reactivities of other anthocyanins were not significantly affected by either the aglycon structure or the type of sugar moiety. Under aerobic conditions, all anthocyanins and catechin showed significant enhancement of the reactivity, indicating that they reacted with other reactive species secondarily generated from NO. . Dp3glys showed rather extraordinally high reactivity towards ONOO- compared to other anthocyanins which showed approximately two times low reactivity than catechin when compared with IC50. Structural divergence in the reactivity was also small for all these anthocyanins.

Structural dependence of HPLC separation pattern of anthocyanins from Bilberry (Vaccinium myrtillus L.).

An HPLC method using isocratic elution was established for the analysis of fifteen anthocyanins contained in bilberry (Vaccinium myrtillus L.). Separation was attained by using an aqueous solution of 20% methanol containing 0.5% TFA as the mobile phase with a flow rate of 2 ml/min. The detection limit was 0.3 pmol for delphinidin 3-O-beta-D-glucopyranoside, which is a major anthocyanin in bilberry extract. The reproducibility was 0.19-3.85% (S.E.M) for peak area and 0.64-0.77% (S.E.M) for relative mobility normalized by the elution position of the solvent peak. When the relative elution volumes of each anthocyanins were correlated to their corresponding anthocyanin structures, a characteristic pattern was observed. From this pattern, the structures of unknown anthocyanins could be predicted from their elution times. Therefore, the present method is useful for the study of anthocyanins from various biological sources.

Complete assignment of bilberry (Vaccinium myrtillus L.) anthocyanins separated by capillary zone electrophoresis.

Capillary zone electrophoresis (CZE) mobilities of fifteen anthocyanins in bilberry extract were completely characterized. Four minor anthocyanins in bilberry extract (malvidin 3-O-alpha-L-arabinopyranoside (Mv 3-ara), peonidin 3-O-beta-D-galactopyranoside (Pn 3-gal), peonidin 3-O-alpha-L-arabinopyranoside (Pn 3-ara), and petunidin 3-O-alpha-L-arabinopyranoside (Pt 3-ara)) that remained unidentified in our previous CZE study were isolated from the bilberry extract, and the chemical structures were assigned by NMR and MS. Their CZE mobilities were then precisely examined together with those of other major anthocyanins in the extract. When the CZE mobilities of the fifteen anthocyanins assigned here were plotted against their molecular weight/numbers of free phenolic group, it was found that separation of anthocyanins by CZE is primarily determined by the type of conjugated sugar present, and secondly by the aglycon structure.