Structural Assignment of the Product Ion Generated from a Natural Ciguatoxin-3C Congener, 51-Hydroxyciguatoxin-3C, and Discovery of Distinguishable Signals in Congeners Bearing the 51-Hydroxy Group.

Ciguatoxins (CTXs) stand as the primary toxins causing ciguatera fish poisoning (CFP) and are essential compounds distinguished by their characteristic polycyclic ether structure. In a previous report, we identified the structures of product ions generated via homolytic fragmentation by assuming three charge sites in the mass spectrometry (MS)/MS spectrum of ciguatoxin-3C (CTX3C) using LC-MS. This study aims to elucidate the homolytic fragmentation of a ciguatoxin-3C congener. We assigned detailed structures of the product ions in the MS/MS spectrum of a naturally occurring ciguatoxin-3C congener, 51-hydroxyciguatoxin-3C (51-hydoxyCTX3C), employing liquid chromatography/quadrupole time-of-flight mass spectrometry with an atmospheric pressure chemical ionization (APCI) source. The introduction of a hydroxy substituent on C51 induced different fragmentation pathways, including a novel cleavage mechanism of the M ring involving the elimination of 51-OH and the formation of enol ether. Consequently, new cleavage patterns generated product ions at m/z 979 (C55H79O15), 439 (C24H39O7), 149 (C10H13O), 135 (C9H11O), and 115 (C6H11O2). Additionally, characteristic product ions were observed at m/z 509 (C28H45O8), 491 (C28H43O7), 481 (C26H41O8), 463 (C26H39O7), 439 (C24H39O7), 421 (C24H37O6), 171 (C9H15O3), 153 (C9H13O2), 141 (C8H13O2), and 123 (C8H11O).

Assignment of product ions produced from ciguatoxin-3C provides a deep insight into the fragmentation mechanism of polycyclic ether compounds.

RATIONALE: Marine polycyclic ethers have drawn attention owing to their unique chemical structures and involvement in food poisoning and fish killing. To study structural diversity, we performed a structural assignment of product ions produced from a representative ladder-shaped polycyclic ether, ciguatoxin-3C, and elucidated the mechanism of generation. METHODS: The product ions used for the structural assignment were produced from a precursor ion [M + H]+ using liquid chromatography/quadrupole time-of-flight mass spectrometry, by employing an atmospheric pressure chemical ionization source. RESULTS: Three charged sites were considered at both terminals of a molecule. Typical charge-remote fragmentation was produced at the respective charge sites, yielding a hybrid spectrum. C-C bonds bordering two ethers could cleave and trigger the fission of two other bonds. Prominent ions indicating the serial loss of water molecules resulted from the simultaneous deprivation of ethereal oxygen and hydrogen atoms. The resultant double bonds formed long chains of conjugated polyenes, which stabilized charge via resonance. CONCLUSIONS: Three alternative charge sites produce a hybrid spectrum. The simultaneous fission of three bonds was explained. For the first time, intense ions due to serial dehydration were explained by the elimination of ether oxygen atoms and the subsequent conjugation of double bonds. All product ions were considered by the structural features of polycyclic ether that facilitates the formation of conjugated polyenes.

Variegatic acid from the edible mushroom Tylopilus ballouii inhibits TNF-α production and PKCß1 activity in leukemia cells.

Variegatic acid, isolated from Tylopilus ballouii dry fruiting bodies, is an inhibitor of ß-hexosaminidase release and tumor necrosis factor (TNF)-α secretion from rat basophilic leukemia (RBL-2H3) cells, with IC50 values of 10.4 µM and 16.8 µM, respectively. On the other hand, it inhibits PKCß1 activity with an IC50 value of 36.2 µM.

Peppermint (Mentha piperita L.) extract effectively inhibits cytochrome P450 3A4 (CYP3A4) mRNA induction in rifampicin-treated HepG2 cells.

Interaction between foods and drugs is an important consideration in pharmaceutical therapy. Therefore, here, we examined the suppressive effects of the extracts from seven edible herbs on the induction of CYP3A4 gene expression in rifampicin-treated HepG2 cells. We evaluated the structure and suppressive activity of the most effective active compound isolated from dried peppermint (Mentha piperita L.). The structure of the compound was identified as that of pheophorbide a based on spectroscopic data. It suppressed the induction of CYP3A4 mRNA expression by rifampicin in a dose-dependent manner. Quantitative high-performance liquid chromatography showed that 2 g of dry leaves 0.43 mg in one cup of peppermint tea. These findings demonstrate that pheophorbide a suppresses the induction of CYP3A4 mRNA expression in rifampicin-treated HepG2 cells. Pheophorbide is known to cause photosensitivity. However, the effective dose of pheophorbide a that had a suppressive effect was very low, indicating a high safety margin. Abbreviations: DAD: diode array detector; DMEM: Dulbecco's modified Eagle's medium; ELISA: enzyme-linked immunosorbent assay; HPLC: high-performance liquid chromatography; PCR: polymerase chain reaction; PXR: pregnane X receptor; CAR: constitutive androstane receptor; AHR: aryl hydrocarbon receptor; TLC: thin-layer chromatography.

Bisorbicillinol inhibits Lyn tyrosine kinase for allergic response on RBL-2H3 cells.

Bisorbicillinol, which is isolated from Trichoderma sp. USF2690, is an inhibitor of ß-hexosaminidase release and tumor necrosis factor (TNF)-α, and Interleukin (IL)-4 secretion from rat basophilic leukemia (RBL-2H3) cells, with IC50 values of 2.8 µM, 2.9 µM and 2.8 µM respectively. We showed that the inhibitory mechanism of ß-hexosaminidase release and TNF-α secretion involved inhibition of Lyn, a tyrosine kinase. The inhibitory activities of bisorbicillinol indicate that this compound is a new candidate anti-allergic agent.

Vialinin A and thelephantin G, potent inhibitors of tumor necrosis factor-α production, inhibit sentrin/SUMO-specific protease 1 enzymatic activity.

Several p-terphenyl compounds have been isolated from the edible Chinese mushroom Thelephora vialis. Vialinin A, a p-terphenyl compound, strongly inhibits tumor necrosis factor-α production and release. Vialinin A inhibits the enzymatic activity of ubiquitin-specific peptidase 5, one of the target molecules in RBL-2H3 cells. Here we examined the inhibitory effect of p-terphenyl compounds, including vialinin A, against sentrin/SUMO-specific protease 1 (SENP1) enzymatic activity. The half maximal inhibitory concentration values of vialinin A and thelephantin G against full-length SENP1 were 1.64±0.23µM and 2.48±0.02µM, respectively. These findings suggest that p-terphenyl compounds are potent SENP1 inhibitors.

Ubiquitin-specific peptidase 5, a target molecule of vialinin A, is a key molecule of TNF-α production in RBL-2H3 cells.

Tumor necrosis factor alpha (TNF-α), a central mediator of the inflammatory response, is released from basophilic cells and other cells in response to a variety of proinflammatory stimuli. Vialinin A is a potent inhibitor of TNF-α production and is released from RBL-2H3 cells. Ubiquitin-specific peptidase 5 (USP5), a deubiquitinating enzyme, was identified as a target molecule of vialinin A and its enzymatic activity was inhibited by vialinin A. Here we report production of TNF-α is decreased in USP5 siRNA-knockdown RBL-2H3 cells, compared with control cells. The finding of the present study strongly suggests that USP5 is one of the essential molecules for the production of TNF-α in RBL-2H3.

Inhibitory effects of vialinin A and its analog on tumor necrosis factor-α release and production from RBL-2H3 cells.

Vialinin A is an extremely potent inhibitor of tumor necrosis factor (TNF)-α release from RBL-2H3 cells. The present study investigated in detail the inhibitory effects of vialinin A and its analog, 5',6'-dimethyl-1,1':4',1″-terphenyl-2',3',4,4″-tetraol (DMT), on TNF-α. Vialinin A and DMT inhibited the release of TNF-α from RBL-2H3 cells in a dose-dependent manner, but had no effect on ß-hexosaminidase activity. Also, vialinins had little effect on TNF-α mRNA levels. Intriguingly, vialinins inhibited TNF-α production at low concentrations, but not shown a dose-dependency. The potent inhibitory activities of vialinins against TNF-α production and release suggest promising new candidate pathways for anti-inflammatory agents.

Effective cytochrome P450 (CYP) inhibitors isolated from tarragon (Artemisia dracunculus).

Two effective cytochrome P450 (CYP) inhibitors were isolated from tarragon, Artemisia dracunculus. Their structures were spectroscopically identified as 2E,4E-undeca-2,4-diene-8,10-diynoic acid isobutylamide (1) and 2E,4E-undeca-2,4-diene-8,10-diynoic acid piperidide (2). Both compounds had dose-dependent inhibitory effects on CYP3A4 activity with IC50 values of 10.0 ± 1.3 µM for compound 1 and 3.3 ± 0.2 µM for compound 2, and exhibited mechanism-based inhibition. This is the first reported isolation of effective CYP inhibitors from tarragon (Artemisia dracunculus) purchased from a Japanese market.

Effective cytochrome P450 (CYP) inhibitor isolated from thyme (Thymus saturoides) purchased from a Japanese market.

A highly polymethylated flavone that effectively inhibited cytochrome P450s (CYPs) 1A2 and 3A4 (IC(50) = 2.41 and 1.71 µM) in vitro was isolated from thyme leaves (Thymus saturoides) purchased from a Japanese market. Its structure was spectroscopically identified as 4',5-dihydroxy-3',6,7,8-tetramethoxy flavone (8-methoxycirsilineol, 1). This is the first report describing a strong inhibitor of CYP1A2 and 3A4 isolated from Thymus saturoides.

Evaluation of the effect of flavangenol on serum lipid peroxide levels and development of atherosclerosis in spontaneously hyperlipidemic B6.KOR-Apoeshl mice.

Antioxidative flavonoids are used to reduce the risk of cardiovascular diseases in humans. However, the precise mechanism for the anti-atherosclerotic actions of flavonoids remains to be elucidated. In the present study, to assess the mechanism for the action of antioxidative flavonoids on atherosclerosis, we investigated the effect of flavangenol, one of the most potent antioxidants currently known, on spontaneously hyperlipidemic B6.KOR-Apoeshl mice. Flavangenol was orally administered to B6.KOR-Apoeshl mice ad libitum (6 mg flavangenol/mouse/day). After 6 months, serum levels of lipids (total cholesterol, triglyceride, HDL-cholesterol and LDL-cholesterol) and lipid peroxide were measured, and histopathological changes (lipid accumulation and inflammatory cell infiltration) in the aortic root were evaluated. Serum levels of total cholesterol and LDL-cholesterol were markedly increased, and HDL-cholesterol levels were decreased in B6.KOR-Apoeshl mice compared to C57BL/6 mice used as a control (p<0.001). Among these serum lipids, only HDL-cholesterol levels were significantly increased by flavangenol administration (p<0.05). Moreover, Oil Red O staining (lipid accumulation) was significantly increased in B6.KOR-Apoeshl mice compared to C57BL/6 mice (p<0.001). Notably, flavangenol administration significantly suppressed the increase in Oil Red O staining (p<0.01). Similarly, inflammatory cell infiltration into the intima was significantly increased in B6.KOR-Apoeshl mice compared to C57BL/6 mice (p<0.01), and flavangenol administration significantly suppressed the inflammatory cell infiltration (p<0.01). Importantly, flavangenol administration significantly reduced the increase of serum lipid peroxide levels in B6.KOR-Apoeshl mice (p<0.05). Together, these observations indicate that flavangenol, one of the most potent antioxidants, exerts its anti-atherosclerotic action on spontaneously hyperlipidemic and atherosclerotic B6.KOR-Apoeshl mice, possibly by increasing HDL-cholesterol levels and reducing lipid peroxide levels, thereby suppressing the lipid accumulation (formation of atherosclerotic lesions) and inflammatory cell infiltration (chronic inflammation) in the intima of the aortic root.

Enhancement of lung carcinogenesis initiated with 4-(N-hydroxymethylnitrosamino)-1-(3-pyridyl)-1-butanone by Ogg1 gene deficiency in female, but not male, mice.

The present study was conducted to assess involvement of oxidative stress in lung adeno-carcinogenesis, using mice deficient in the 8-hydroxyguanine DNA glycosylase 1 (Ogg1) gene encoding an enzyme that repairs an oxidative DNA injury 8-oxoguanine (8-oxoG). Furthermore, for comparison with the human case, mutations of mouse epidermal growth factor receptor (Egfr) and K-ras genes were examined. The homo- and heterozygously Ogg1 gene-deficient and wild-type mice (C57BL6/J origin), 6 weeks old, were administered 4-(N-hydroxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) by continuous subcutaneous infusion using an osmotic pump at a total dose of 6 mg/mouse for 1 week, then treated with one of 4 antioxidants (phenyl N-tert-butyl nitrone 0.13% in drinking water, resveratrol 20 ppm in diet, lactoferrin 2% in diet and bilberry powder 2% in diet) or no supplement for 33 weeks. Development of lung adenomas and preneoplastic atypical hypreplasias was significantly enhanced by the homo- and heterozygous Ogg1 gene deficiency only in female mice with intralesion accumulation of 8-oxoG. All antioxidants tended to inhibit enhanced adeno-carcinogenesis. The Egfr and K-ras gene mutations were detected at sites also found in human lung cancers with low incidences, while the Egfr gene mutation was detected for the first time in chemical lung carcinogenesis of animals. It is indicated that the Ogg1 gene deficiency enhances lung adeno-carcinogenesis in mice by virtue of accelerated oxidative stress. The presently utilized Ogg1 gene-deficient mice model may be useful to draw mechanism-based strategies to control human lung adenocarcinomas, especially in women.

Vialinin A, a novel potent inhibitor of TNF-alpha production from RBL-2H3 cells.

Thelephora vialis is a mushroom that grows in symbiosis with pine trees in Yunnan, China, a place known to have some of the richest and most diverse bioresources in the world. This is one of the most favored edible mushrooms, due to its flavor. Our screening for bioactive compounds from these mushrooms isolated a novel potent antioxidant, vialinin A, together with known compounds, from the dry fruiting bodies of T. vialis. Vialinin A is a terphenyl derivative and was elucidated by spectroscopic and chemical methods. Vialinin A showed anti-allergic activities, inhibition of beta-hexosaminidase, tumor necrosis factor (TNF)-alpha, interleukin 4 and monocyte chemotactic protein 1 release from RBL-2H3 cells, whereas atromentin and an inseparable mixture of ganbajunins D and E showed no such effects. Vialinin A displayed potent inhibition of TNF-alpha production from RBL-2H3 cells (IC50, 0.09+/-0.01 nM), indicating stronger inhibition than tacrolimus for organ transplantation (IC50, 0.25+/-0.03 nM). The potent inhibitory activities of these compounds against TNF-alpha production indicate promising new candidates for anti-allergic agents.