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.

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.

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.

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.

Structural elucidation and synthesis of vialinin C, a new inhibitor of TNF-α production.

A new inhibitor of TNF-α production (IC50=0.89 µM) named vialinin C (1) was isolated from dry fruiting bodies of an edible Chinese mushroom, Thelephora vialis. The structure of 1 was determined by high-resolution MS, NMR spectroscopic analysis, and confirmed by synthesis. Synthesis of ganbajunin B (5) obtained from the same origin was also described.

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).

Vialinin A is a ubiquitin-specific peptidase inhibitor.

Vialinin A, a small compound isolated from the Chinese mushroom Thelephora vialis, exhibits more effective anti-inflammatory activity than the widely used immunosuppressive drug tacrolimus (FK506). Here, we show that ubiquitin-specific peptidase 5/isopeptidase T (USP5/IsoT) is a target molecule of vialinin A, identified by using a beads-probe method. Vialinin A inhibited the peptidase activity of USP5/IsoT and also inhibited the enzymatic activities of USP4 among deubiquitinating enzymes tested. Although USPs are a member of thiol protease family, vialinin A exhibited no inhibitions for other thiol proteases, such as calpain and cathepsin.

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.

Design and synthesis of a vialinin A analog with a potent inhibitory activity of TNF-α production and its transformation into a couple of bioprobes.

Vialinin A (1) is an extremely potent inhibitor against tumor necrosis factor (TNF)-α production in rat basophilic leukemia (RBL-2H3) cells. This Letter describes the design and synthesis of its advanced analog, 5',6'-dimethyl-1,1':4'1″-terphenyl-2',3',4,4″-tetraol (2) with a comparable inhibitory activity (IC(50)=0.02 nM) to that of 1. The synthesis involved double Suzuki-Miyaura coupling as a key step, and required only five steps from commercially available 3,4-dimethylphenol. For identification of the target molecule, fluorescent and biotinylated derivatives of 2 were prepared through a 'click' coupling process.

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.

Effects of peanut-skin procyanidin A1 on degranulation of RBL-2H3 cells.

Peanut skin contains large amounts of polyphenols having antiallergic effects. We found that a peanut-skin extract (PSE) inhibits the degranulation induced by antigen stimulation of rat basophilic leukemia (RBL-2H3) cells. A low-molecular-weight fraction from PSE, PSEL, also had inhibitory activity against allergic degranulation. A main polyphenol in PSEL was purified by gel chromatography and fractionated by YMC-gel ODS-AQ 120S50 column. Electrospray ionization mass spectrometry (ESI-MS) analysis of the purified polyphenol gave m/z 599 [M+Na]⁺. Based on the results of ¹H-NMR, ¹³C-NMR spectra, and optical rotation analysis, the polyphenol was identified as procyanidin A1. It inhibited the degranulation caused by antigen stimulation at the IC50 of 20.3 µM. Phorbol-12-myristate-13-acetate (PMA) and 2,5,-di(tert-butyl)-1,4-hydroquinone (DTBHQ)-induced processes of degranulation were also inhibited by procyanidin A1. These results indicate that peanut-skin procyanidin A1 inhibits degranulation downstream of protein kinase C activation or Ca²âº influx from an internal store in RBL-2H3 cells.

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.

Studies on natural p-terphenyls: total syntheses of vialinin A and terrestrin B.

A powerful inhibitor of TNF-alpha production, vialinin A, was synthesized from sesamol through a series of reactions involving double Suzuki-Miyaura coupling, 2,3-dichloro-5,6-dicyano-1,4-benzoquino (DDQ) mediated de-methoxymethylation and oxidative removal of methylene acetal by lead tetraacetate. The synthetic method also made it possible to prepare a related compound, terrestrin B.

Structural revision of thelephantin G by total synthesis and the inhibitory activity against TNF-alpha production.

This paper describes the total synthesis of thelephantin G, thus revising the proposed structure 1 to 2. The key steps involved a double Suzuki-Miyaura coupling and an esterification reaction. By a similar strategy, ganbajunins D and E (3 and 4) were also prepared. Compound 2 strongly inhibited TNF (tumor necrosis factor)-alpha production in rat basophilic leukemia (RBL-2H3) cells: IC(50) = 3.5 nM, while a mixture of 1 and its regioisomer 15 showed no such activity.

In vitro inhibitory effects of pulvinic acid derivatives isolated from Chinese edible mushrooms, Boletus calopus and Suillus bovinus, on cytochrome P450 activity.

Increasing attention has been focused on food-drug interactions. We have investigated the inhibitory effect of Chinese edible mushrooms, Boletus calopus and Suillus bovinus, on cytochrome P450 (CYP) 1A2, 2C9, 2D6, and 3A4, the main drug-metabolizing enzymes. Three pulvinic acid derivatives, atromentic acid (1), variegatic acid (2), and xerocomic acid (3), isolated from Boletus calopus and Suillus bovinus, revealed nonspecific inhibitory effects on all four CYPs. Using these compounds, the maximum IC50 values obtained with CYP3A4 in vitro were atromentic acid (1), 65.1+/-3.9 microM; variegatic acid (2), 2.2+/-0.1 microM; and xerocomic acid (3), 2.4+/-0.1 microM. Variegatic acid (2) and xerocomic acid (3) were effective inhibitors, comparable to cimetidine, dicoumarol, erythromycin, safrole, and uniconazole. Variegatic acid (2) and xerocomic acid (3) efficiently reduced ferryl myoglobin in CYPs. Reduction of ferryl heme to ferric heme is likely the mechanism of the nonspecific inhibitory effects of these compounds on CYPs.

Evaluation of subchronic toxicity of dietary administered Cry1Ab protein from Bacillus thuringiensis var. Kurustaki HD-1 in F344 male rats with chemically induced gastrointestinal impairment.

Bacillus thuringiensis (Bt) proteins are developed for genetically modified crops and the Bt proteins demonstrate no evidence of toxicity by the oral route in traditional animal models. However, the possible toxicity of Bt proteins under conditions of reduced gastric acid secretion and/or small intestinal damage has not been investigated. In the present study, we therefore evaluated following four F344 rat groups with a purified Bt protein Cry1Ab from B. thuringiensis var. Kurustaki HD-1. Gastrointestinal impairment (GI) alone and GI+Bt protein fed (GI+Bt) groups were given i.p. injections of famotidine to reduce gastric acid secretion twice a day at 30mg/kg body weight in weeks 2 and 4. GI and GI+Bt groups were additionally fed diets containing 80ppm indomethacin for induction of intestinal damage during weeks 1 and 3. Bt alone and GI+Bt groups were also fed diet containing Bt protein Cry1Ab at a concentration of 10ppm in weeks 2 and 4. A no treatment control group was also included. At the end of week 4, all animals were euthanized under ether anesthesia, blood samples were collected for hematology and serum biochemistry and a complete necropsy was performed. No significant changes indicative of toxicity of the Bt protein Cry1Ab used here were noted with any of the parameters investigated. In conclusion, no significant toxicological effects were detected in this subchronic gastrointestinal impairment rat model.

First total synthesis of vialinin A, a novel and extremely potent inhibitor of TNF-alpha production.

Vialinin A, a powerful inhibitor (IC50 90 pM) of TNF-alpha production, was synthesized from sesamol in 11 steps with 28% overall yield. The key reactions include a double Suzuki coupling of electron-rich aryl triflate with phenylboronic acid and an oxidative deprotection of bis-MOM ether. In addition, the related synthetic studies also suggest the necessity for structural revision of ganbajunin C, a positional isomer of vialinin A.

A new medium-term rat colon bioassay applying neoplastic lesions as endpoints for detection of carcinogenesis modifiers-validation with known modifiers.

We have established a medium-term colorectal carcinogenesis rat model initiated with 1,2-dimethylhydrazine (DMH) followed by dextran sodium sulfate (DSS) treatment, featuring induction of neoplastic lesions within 10 weeks. In the present study, we examined its ability to detect modification of colon lesion development with 10- or 20-week experimental periods. F344 male rats were given three subcutaneous injections of DMH (40 mg/kg b.w.) in a week followed by free access to drinking water containing 1% DSS for a week. One week after this regimen, basal diet alone, basal diet containing 0.04% nimesulide or 2% lactoferrin as known inhibitors, 0.3% deoxycholic acid (DCA) as a promoter or 1.5% 1-hydroxyanthraquinone (1-HA) as a carcinogen were supplied. At week 10, the incidence and multiplicity of combined adenomas and adenocarcinomas were significantly (P < 0.05 or 0.01) decreased by nimesulide and lactoferrin, and values for adenomas were significantly (P < 0.01) increased in the 1-HA group. There was no clear change in the DCA group. At week 20, multiplicity and volume of the tumors were significantly (P < 0.01 or 0.05) decreased by nimesulide, but no effect was now evident with lactoferrin. Multiplicity and volume of tumors were significantly (P < 0.01) increased in 1-HA group and a similar tendency was apparent (P = 0.08) with DCA. It is concluded that this system offers a useful tool for detection of colorectal carcinogenesis modifiers within 10-20 weeks, pending further studies for verification employing other model chemicals.

Indomethacin induces small intestinal damage and inhibits amitrole-associated thyroid carcinogenesis in rats initiated with N-bis(2-hydroxypropyl)nitrosamine.

Effects of intestinal damage on thyroid carcinogenesis due to amitrole (AT) were examined in F344 male rats initiated with N-bis(2-hydroxypropyl)nitrosamine (DHPN). In experiment 1, rats were provided with diet containing 0.03% AT for 20 weeks after a single subcutaneous injection of DHPN (2800 mg/kg body weight), and concomitantly received 0.01% indomethacin (IM) in the diet to cause small intestinal damage or 1% dextran sodium sulfate (DSS) in the drinking water for induction of colitis following a schedule of intermittent 1-week administration and 1-week withdrawal for a total of 10 times. Groups without AT- and/or IM or DSS treatment were also included. Histopathological examination revealed significant reduction in the incidence and multiplicity of follicular cell adenomas and adenocarcinomas in the group concomitantly treated with IM, but no change in the DSS group, as compared with the AT alone group. In experiment 2, rats were similarly fed diet containing AT for 3 weeks with concomitant IM or DSS treatment after a DHPN initiation, and serum thyroid stimulating hormone levels were found to be significantly elevated only in the IM case. The increase in thyroid follicular cell proliferation due to AT was also clearly suppressed in the group concomitantly treated with IM. From these findings, IM-induced intestinal damage may inhibit thyroid carcinogeneisis in rats, although contributions of other factors, such as a direct inhibitory effect of IM to thyroid follicular cell proliferation cannot be ruled out.