The Inhibitory Effect of Tartary Buckwheat Extracts on Adipogenesis and Inflammatory Response.

Tartary buckwheat (Fagopyrum tataricum) has been established globally as a nutritionally important food item, particularly owing to high levels of bioactive compounds such as rutin. This study investigated the effect of tartary buckwheat extracts (TBEs) on adipogenesis and inflammatory response in 3T3-L1 cells. TBEs inhibited lipid accumulation, triglyceride content, and glycerol-3-phosphate dehydrogenase (GPDH) activity during adipocyte differentiation of 3T3 L1 cells. The mRNA levels of genes involved in fatty acid synthesis, such as peroxisome proliferator-activated receptor-γ (PPAR-γ), CCAAT/enhancer binding protein-α (CEBP-α), adipocyte protein 2 (aP2), acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS), and stearoylcoenzyme A desaturase-1 (SCD-1), were suppressed by TBEs. They also reduced the mRNA levels of inflammatory mediators such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), monocyte chemoattractant protein 1 (MCP-1), and inducible nitric oxide synthase (iNOS). In addition, TBEs were decreased nitric oxide (NO) production. These results suggest that TBEs may inhibit adipogenesis and inflammatory response; therefore, they seem to be beneficial as a food ingredient to prevent obesity-associated inflammation.

Evaluation of the Biological Activity of Opuntia ficus indica as a Tissue- and Estrogen Receptor Subtype-Selective Modulator.

Phytoestrogens are selective estrogen receptor modulators (SERMs) with potential for use in hormone replacement therapy (HRT) to relieve peri/postmenopausal symptoms. This study was aimed at elucidating the molecular mechanisms underlying the SERM properties of the extract of Korean-grown Opuntia ficus-indica (KOFI). The KOFI extract induced estrogen response element (ERE)-driven transcription in breast and endometrial cancer cell lines and the expression of endogenous estrogen-responsive genes in breast cancer cells. The flavonoid content of different KOFI preparations affected ERE-luciferase activities, implying that the flavonoid composition likely mediated the estrogenic activities in cells. Oral administration of KOFI decreased the weight gain and levels of both serum glucose and triglyceride in ovariectomized (OVX) rats. Finally, KOFI had an inhibitory effect on the 17ß-estradiol-induced proliferation of the endometrial epithelium in OVX rats. Our data demonstrate that KOFI exhibited SERM activity with no uterotrophic side effects. Therefore, KOFI alone or in combination with other botanical supplements, vitamins, or minerals may be an effective and safe alternative active ingredient to HRTs, for the management of postmenopausal symptoms. Copyright © 2016 John Wiley & Sons, Ltd.

Skin Barrier-Enhancing Effects of Dermabiotics HDB with Regulation of Skin Microbiota.

In the regulation of inflammatory responses and skin homeostasis, the skin and its microbiota are closely related. Studies have reported that lactic acid bacteria extracts can improve the skin condition and microbiota. In our previous study, we developed probiotic lysates, which are efficacious in improvement of human skin cells and the skin barrier. The skin-moisturizing effect of Dermabiotics HDB (HDB) prepared with Lactiplantibacillus plantarum, and the correlation between changes in the skin microbiota and moisture contents, were evaluated and analyzed in clinical trials. The clinical parameters on the cheeks of 21 female participants were measured using biophysical tools before and after (2 weeks) using HDB or control. The skin microbes were collected and identified using 16s rRNA gene sequencing. HDB significantly improved moisture intensity, transepidermal water loss (TEWL), and hot flush level on the cheek. The beta-diversity of the skin microbiota was different from that of the control in the unweighted UniFrac principal coordinate analysis after using HDB. The genus Lawsonella demonstrated a positive correlation with TEWL and a negative correlation with the moisture contents of the keratin layer, regardless of the use of HDB and control. Conversely, after HDB use, the genus Staphylococcus was increased and associated with a lower hot flush level, while the genera of the phylum Proteobacteria tended to decrease, which is associated with an improved skin condition. Overall, HDB showed clinically proven effects, including skin moisturization with regulation of the skin microbiota.

Anti-Menopausal Effect of Heat-Killed Bifidobacterium breve HDB7040 via Estrogen Receptor (ER)-Selective Modulation in MCF-7 Cells and Ovariectomized Rats.

Menopause is induced by spontaneous ovarian failure and leads to life quality deterioration with various irritating symptoms. Hormonal treatment can alleviate these symptoms, but long-term treatment is closely associated with breast and uterine cancer, and stroke. Therefore, developing alternative therapies with novel anti-menopausal substances and improved safety is needed. In our study, heat-killed Bifidobacterium breve HDB7040 significantly promoted MCF-7 cell proliferation in a dose-dependent manner under estrogen-free conditions, similar to 17ß-estradiol. This strain also triggered ESR2 expression, but not ESR1, in MCF-7 cells. Moreover, administrating HDB7040 to ovariectomized (OVX) Sprague-Dawley (SD) female rats reduced estrogen deficiency-induced weight gain, fat mass, blood triglyceride, and total cholesterol levels. It also recovered collapsed trabecular microstructure by improving trabecular morphometric parameters (bone mineral density, bone volume per tissue volume, trabecular number, and trabecular separation) and decreasing blood alkaline phosphatase levels with no significant changes in uterine size and blood estradiol. HDB7040 also significantly regulated the expression of Tff1, Pgr, and Esr2, but not Esr1 in uteri of OVX rats. Heat-killed B. breve HDB7040 exerts an anti-menopausal effect via the specific regulation of ERß in vitro and in vivo, suggesting its potential as a novel substance for improving and treating menopausal syndrome.

(+)-Altholactone exhibits broad spectrum immune modulating activity by inhibiting the activation of pro-inflammatory cytokines in RAW 264.7 cell lines.

An evaluation of Indonesian plants to identify compounds with immune modulating activity revealed that the methanolic extract of an Alphonsea javanica Scheff specimen possessed selective anti-inflammatory activity in a nuclear factor-kappa B (NF-κB) luciferase and MTT assay using transfected macrophage immune (Raw264.7) cells. A high-throughput LC/MS-ELSD based library approach of the extract in combination with the NF-κB and MTT assays revealed the styryl lactone (+)-altholactone (2) was responsible for the activity. Compound 2, its acetylated derivate (+)-3-O-acetylaltholactone (3), and the major compound of this class, (+)-goniothalmin (1), were further evaluated to determine their anti-inflammatory potential in the NF-κB assay. Concentration-response studies of 1-3 indicated that only 2 possessed NF-κB based anti-inflammatory activity. Compound 2 reduced the LPS-induced NO production, phosphorylation of IκBα, and the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) using Western blot analysis. Further studies using qPCR indicated 2 reduced the expression of eight pro-inflammatory cytokines/enzymes (0.8-5.0µM) which included: COX-2, iNOS, IP-10, IL-1ß, MCP-1, GCS-F, IL-6 and IFN-ß. These results indicated that 2 displays broad spectrum immune modulating activity by functioning as an anti-inflammatory agent against LPS-induced NF-κB signaling. Conversely the selective cytotoxicity and in vivo anti-tumor and anti-inflammatory activity previously reported for 1 do not appear to arise from a mechanism that is linked to the NF-κB immune mediated pathway.

Effects of Saffron Extract (Affron®) with 100 mg/kg and 200 mg/kg on Hypothalamic-Pituitary-Adrenal Axis and Stress Resilience in Chronic Mild Stress-Induced Depression in Wistar Rats.

Stress-related symptoms are a global concern, impacting millions of individuals, yet effective and safe treatments remain scarce. Although multiple studies have highlighted the stress- alleviating properties of saffron extract, the underlying mechanisms remain unclear. This study employs the unpredictable chronic mild stress (CMS) animal model to investigate the impact of a standardized saffron extract, Affron® (AFN), on hypothalamic-pituitary-adrenal (HPA) axis regulation and neuroplasticity in Wistar rats following repeated oral administration. The research evaluates AFN's effects on various stress-related parameters, including hypothalamic gene expression, stress hormone levels, and the sucrose preference test. In animals subjected to continuous unpredictable CMS, repetitive administration of AFN at doses of 100 mg/kg and 200 mg/kg effectively normalized HPA axis dysregulation and enhanced neuroplasticity. Increased concentrations of AFN demonstrated greater efficacy. Following AFN oral administration, adrenocorticotropic and corticosterone hormone levels exhibited significant or nearly significant reductions in comparison to subjects exposed to stress only. These changes align with the alleviation of stress and the normalization of the HPA axis. These findings elucidate AFN's role in stress mitigation, affirm its health benefits, validate its potential as a treatment for stress-related symptoms, confirm its physiological effectiveness, and emphasize its therapeutic promise.

Myxobacteria versus sponge-derived alkaloids: the bengamide family identified as potent immune modulating agents by scrutiny of LC-MS/ELSD libraries.

A nuclear factor-κB (NF-κB) luciferase assay has been employed to identify the bengamides, previously known for their anti-tumor activity, as a new class of immune modulators. A unique element of this study was that the bengamide analogs were isolated from two disparate sources, Myxococcus virescens (bacterium) and Jaspis coriacea (sponge). Comparative LC-MS/ELSD and NMR analysis facilitated the isolation of M. viriscens derived samples of bengamide E (8) and two congeners, bengamide E' (13) and F' (14) each isolated as an insperable mixture of diastereomers. Additional compounds drawn from the UC, Santa Cruz repository allowed expansion of the structure activity relationship (SAR) studies. The activity patterns observed for bengamide A (6), B (7), E (8), F (9), LAF 389 (12) and 13-14 gave rise to the following observations and conclusions. Compounds 6 and 7 display potent inhibition of NF-κB (at 80 and 90 nM, respectively) without cytotoxicity to RAW264.7 macrophage immune cells. Western blot and qPCR analysis indicated that 6 and 7 reduce the phosphorylation of IκBα and the LPS-induced expression of the pro-inflammatory cytokines/chemokines TNFα, IL-6 and MCP-1 but do not effect NO production or the expression of iNOS. These results suggest that the bengamides may serve as therapeutic leads for the treatment of diseases involving inflammation, that their anti-tumor activity can in part be attributed to their ability to serve as immune modulating agents, and that their therapeutic potential against cancer merits further consideration.

Natural product libraries to accelerate the high-throughput discovery of therapeutic leads.

A high-throughput (HT) paradigm generating LC-MS-UV-ELSD-based natural product libraries to discover compounds with new bioactivities and or molecular structures is presented. To validate this methodology, an extract of the Indo-Pacific marine sponge Cacospongia mycofijiensis was evaluated using assays involving cytoskeletal profiling, tumor cell lines, and parasites. Twelve known compounds were identified including latrunculins (1-4, 10), fijianolides (5, 8, 9), mycothiazole (11), aignopsanes (6, 7), and sacrotride A (13). Compounds 1-5 and 8-11 exhibited bioactivity not previously reported against the parasite T. brucei, while 11 showed selectivity for lymphoma (U937) tumor cell lines. Four new compounds were also discovered including aignopsanoic acid B (13), apo-latrunculin T (14), 20-methoxy-fijianolide A (15), and aignopsane ketal (16). Compounds 13 and 16 represent important derivatives of the aignopsane class, 14 exhibited inhibition of T. brucei without disrupting microfilament assembly, and 15 demonstrated modest microtubule-stabilizing effects. The use of removable well plate libraries to avoid false positives from extracts enriched with only one or two major metabolites is also discussed. Overall, these results highlight the advantages of applying modern methods in natural products-based research to accelerate the HT discovery of therapeutic leads and/or new molecular structures using LC-MS-UV-ELSD-based libraries.

Gut Microbiota-Mediated Immunomodulatory Effects of Lactobacillus rhamnosus HDB1258 Cultured in the Lava Seawater in the Colitis Mouse Model.

The immunomodulatory effects of Lactobacillus rhamnosus HDB1258 were evaluated in mice with colitis induced by Klebsiella oxytoca (KO). L. rhamnosus HDB1258 was cultured in the lava seawater (LS) to improve its probiotic properties. It increased adhesive ability to mucin with mRNA expression levels of chaperone proteins (such as GroEL/ES, DnaKJ, and HtrA). In the in vivo experiments, administration of KO caused an inflammation on the colon with gut dysbiosis. LH group (oral gavage of HDB1258 1.0 × 109 colony forming units/day) showed that inflammatory biomarkers, including IL-1ß, TNF-α, IL-6, and PGE2, were significantly decreased to less than half of the KO group, and Th1 cells were decreased in the spleen, but Treg cells were not affected. In contrast, the expression levels of secretory IgA and IL-10 were significantly increased, and the composition of gut microbiota in the LH group tended to recover similar to normal mice without any effect on the α-diversity. In conclusion, L. rhamnosus HDB1258 cultured in the LS could regulate competitively pathogenic bacteria in imbalanced flora with its improved mucin adhesive ability and was an effective immunomodulatory adjuvant for treating colitis by its regulatory function on intestinal inflammation.

Click synthesis of estradiol-cyclodextrin conjugates as cell compartment selective estrogens.

Cyclodextrin (CD) is a well known drug carrier and excipient for enhancing aqueous solubility. CDs themselves are anticipated to have low membrane permeability because of relatively high hydrophilicity and molecular weight. CD derivatization with 17-beta estradiol (E(2)) was explored extensively using a number of different click chemistries and the cell membrane permeability of synthetic CD-E(2) conjugate was explored by cell reporter assays and confocal fluorescence microscopy. In simile with reported dendrimer-E(2) conjugates, CD-E(2) was found to be a stable, extranuclear receptor selective estrogen that penetrated into the cytoplasm.

A Mixture Containing Fermented Achyranthes japonica Nakai Ameliorates Osteoarthritis in Knee Joints of Monoiodoacetate-Injected Rats.

We demonstrated the effect of a mixture containing fermented Achyranthes japonica Nakai (FS) in the context of a monosodium iodoacetate (MIA)-induced osteoarthritis animal model. The mineralization, anabolic and catabolic factors, and the amount of cytokines within the articular cartilage of rats were measured after administration of MIA. We found that dietary supplementation with methylsulfonylmethane (positive control) and FS (FS 100 mg/kg body weight [b.w.] and FS 300 mg/kg b.w.) effectively suppressed pathological changes in the knee joint and inhibited changes in the architectural and mineralization parameters. In addition, prostaglandin E2 (PGE2) and proinflammatory cytokines in the serum and catabolic factors, including matrix metalloproteinase (MMP)-3 and MMP-7 in articular cartilage, were decreased by dietary supplementation with FS in MIA-induced osteoarthritis. Based on these findings, we suggest that FS can be used for the development of potential therapies for osteoarthritis.

Development of a liquid chromatography electrospray ionization tandem mass spectrometry method for analysis of stable 4-hydroxyequilenin-DNA adducts in human breast cancer cells.

Estrogen-DNA adducts are potential biomarkers for assessing cancer risk and progression in estrogen-dependent cancer. 4-Hydroxyequilenin (4-OHEN), the major catechol metabolite of equine estrogens present in hormone replacement therapy formulations, autoxidizes to a reactive o-quinone that subsequently causes DNA damage. The formation of stable stereoisomeric cyclic 4-OHEN-DNA adducts has been reported in vitro and in vivo, but their removal by DNA repair processes in cells has not been determined. Such studies have been hampered by low yields of cyclic adducts and poor reproducibility when treating cells in culture with 4-OHEN. These problems are attributed in part to the instability of 4-OHEN in aerobic, aqueous media. We show herein that low yields and reproducibility can be overcome by 4-OHEN diacetate as a novel, cell-permeable 4-OHEN precursor, in combination with a sensitive LC-MS/MS method developed for detecting adducts in human breast cancer cells. This method involves isolation of cellular DNA, DNA digestion to deoxynucleosides, followed by the addition of an isotope-labeled internal standard (4-OHEN-(15)N(5)-dG adduct) prior to analysis by LC-MS/MS. A concentration-dependent increase in adduct levels was observed in MCF-7 cells after exposure to 4-OHEN diacetate. The chemical stabilities of the adducts were also investigated to confirm that adducts were stable under assay conditions. In conclusion, this newly developed LC-MS/MS method allows detection and relative quantification of 4-OHEN-DNA adducts in human breast cancer cells, which could be adapted for adduct detection in human samples.

Fermented Castanea crenata Inner Shell Extract Increases Fat Metabolism and Decreases Obesity in High-Fat Diet-Induced Obese Mice.

The anti-obesity effects of fermented Castanea crenata inner shell extract (FCCE) were investigated using high-fat diet (HFD)-induced obese mice. In the FCCE intake groups, body weight gain and adipocyte area were significantly reduced, especially body weight gain in the 250 mg/kg FCCE group (G4) decreased by 37%, respectively, compared with negative control group (G2, HFD group). After oral administration of the FCCE, the increase of serum low-density lipoprotein (LDL)-cholesterol induced by HFD was suppressed significantly, as well as the level of aspartate aminotransferase, and alanine aminotransferase, which are markers of hepatitis induced by obesity. Serum leptin in G4 group was significantly decreased to less than that of G2 group. Also, in G4 and 500 mg/kg FCCE group (G5), enzymes-related lipogenesis, citrate synthase, and ATP citrate lyase were decreased, whereas the level of enoyl-CoA hydratase used for ß-oxidation was significantly increased in comparison with normal diet group. Furthermore, the FCCE stimulated the expression of lipolytic regulators, especially AMP-activated protein kinase. In conclusion, we suggest that the FCCE may ameliorate in diet-induced obesity by regulating lipid metabolism.

Characterization of Soybean Germinated Embryo Extract as an Estrogen Receptor Subtype-Selective and Tissue-Specific Modulator.

Phytoestrogens possess beneficial effects in the management of menopausal symptoms with few side effects. Soybeans are major natural sources of isoflavones, with high estrogen receptor (ER)-ß selectivity. The objective of this study therefore was to develop a solvent-mediated extraction method for soybean germinated embryos (SGEs) and to investigate the biological activities of the extract. Ethanolic extraction yielded the SGE extract (SGEE), which had a unique composition of biologically active aglycones and soyasaponins. SGEE showed a proliferative effect in MCF7 cells and ERß-selective transcriptional activities in human embryonic kidney cells. In addition, oral administration of SGEE to ovariectomized rats resulted in the induction of ERß and estrogen-responsive genes in the uterus and a decrease in tail skin temperature and uterus weight. Our data suggest that germination and ethanolic extraction are effective measures for producing isoflavone-rich food supplements, which may be useful as alternative menopausal hormone therapy.

Anti-inflammatory, antiproliferative, and cytoprotective activity of NO chimera nitrates of use in cancer chemoprevention.

Nonsteroidal anti-inflammatory drugs (NSAIDs) have shown promise in colorectal cancer (CRC), but they are compromised by gastrotoxicity. NO-NSAIDs are hybrid nitrates conjugated to an NSAID designed to exploit the gastroprotective properties of NO bioactivity. The NO chimera ethyl 2-((2,3-bis(nitrooxy)propyl)disulfanyl)benzoate (GT-094), a novel nitrate containing an NSAID and disulfide pharmacophores, is effective in vivo in rat models of CRC and is a lead compound for design of agents of use in CRC. Preferred chemopreventive agents possess 1) antiproliferative and 2) anti-inflammatory actions and 3) the ability to induce cytoprotective phase 2 enzymes. To determine the contribution of each pharmacophore to the biological activity of GT-094, these three biological activities were studied in vitro in compounds that deconstructed the structural elements of the lead GT-094. The anti-inflammatory and antiproliferative actions of GT-094 in vivo were recapitulated in vitro, and GT-094 was seen to induce phase 2 enzymes via the antioxidant responsive element. In the variety of colon, macrophage-like, and liver cell lines studied, the evidence from structure-activity relationships was that the disulfide structural element of GT-094 is the dominant contributor in vitro to the anti-inflammatory activity, antiproliferation, and enzyme induction. The results provide a direction for lead compound refinement. The evidence for a contribution from the NO mimetic activity of nitrates in vitro was equivocal, and combinations of nitrates with acetylsalicylic acid were inactive.

Protein Expression Level of Skin Wrinkle-Related Factors in Hairless Mice Fed Hyaluronic Acid.

The aim of this study was to evaluate the wrinkle improving effect of hyaluronic acid intakes. Wrinkles were induced by exposing the skin of hairless mice to ultraviolet B (UVB) irradiation for 14 weeks. Hyaluronic acid was administered to the mice for 14 weeks including 4 weeks before experiments. Skin tissue was assayed by enzyme-linked immunosorbent assay to determine protein expression of wrinkle-related markers. The group supplemented with high concentrations of hyaluronic acid appeared significantly better than control group for collagen, matrix metalloproteinase 1, interleukin (IL)-1ß, and IL-6 assay. Transforming growth factor-ß1 (TGF-ß1) and hyaluronic acid synthase 2 (HAS-2) were not shown to be significantly different. In conclusion, hyaluronic acid administration regulated expression levels of proteins associated with skin integrity, and improved the wrinkle level in skin subjected to UVB irradiation.

Anti-inflammatory effects of chicanine on murine macrophage by down-regulating LPS-induced inflammatory cytokines in IκBα/MAPK/ERK signaling pathways.

Schisandra chinensis Baill is a Chinese traditional medicine with multiple pharmacological activities. In this study, chicanine, one of the major lignan compounds of S. chinesis, was investigated for suppressive effects on lipopolysaccharide (LPS)-induced inflammatory responses in murine macrophages (RAW 264.7 cells). Chicanine was found to have anti-inflammatory properties with the inhibition of nitric oxide (NO) and Prostaglandin E (2) (PGE2) production and nuclear factor-κB (NF-κB) signaling in LPS-stimulated RAW 264.7 cells with no cytotoxic effects. Treatment of RAW 264.7 cells with chicanine down-regulated LPS-induced expression of pro-inflammatory cytokines including TNFα, IL-1ß, MCP-1, G-CSF, cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS). These inhibitory effects were found with the blockage of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinases 1 and 2 (ERK 1/2), and also IκB-α phosphorylation. These results indicated that anti-inflammatory actions of chicanine in macrophages involved inhibition of LPS-induced TLR4-IκBα/MAPK/ERK signaling pathways.

Lipophilic stinging nettle extracts possess potent anti-inflammatory activity, are not cytotoxic and may be superior to traditional tinctures for treating inflammatory disorders.

Extracts of four plant portions (roots, stems, leaves and flowers) of Urtica dioica (the stinging nettle) were prepared using accelerated solvent extraction (ASE) involving water, hexanes, methanol and dichloromethane. The extracts were evaluated for their anti-inflammatory and cytotoxic activities in an NF-κB luciferase and MTT assay using macrophage immune (RAW264.7) cells. A standardized commercial ethanol extract of nettle leaves was also evaluated. The methanolic extract of the flowering portions displayed significant anti-inflammatory activity on par with a standard compound celastrol (1) but were moderately cytotoxic. Alternatively, the polar extracts (water, methanol, ethanol) of the roots, stems and leaves displayed moderate to weak anti-inflammatory activity, while the methanol and especially the water soluble extracts exhibited noticeable cytotoxicity. In contrast, the lipophilic dichloromethane extracts of the roots, stems and leaves exhibited potent anti-inflammatory effects greater than or equal to 1 with minimal cytotoxicity to RAW264.7 cells. Collectively these results suggest that using lipophilic extracts of stinging nettle may be more effective than traditional tinctures (water, methanol, ethanol) in clinical evaluations for the treatment of inflammatory disorders especially arthritis. A chemical investigation into the lipophilic extracts of stinging nettle to identify the bioactive compound(s) responsible for their observed anti-inflammatory activity is further warranted.

The naphthol selective estrogen receptor modulator (SERM), LY2066948, is oxidized to an o-quinone analogous to the naphthol equine estrogen, equilenin.

o-Quinone forming estrogens and selective estrogen receptor modulators (SERMs) have been associated with carcinogenesis. LY2066948, a novel SERM in development by Eli Lilly for the treatment of uterine fibroids and myomas, has structural similarity to the equine estrogen equilenin present in hormone replacement formulations; both contain a naphthol group susceptible to oxidative metabolism to o-quinones. LY2066948 was synthesized and assayed for antiestrogenic activity, and in cell culture was confirmed to be a more potent antiestrogen than the prototypical SERM, 4-hydroxytamoxifen. Oxidation of LY2066948 with 2-iodoxybenzoic acid gave an o-quinone (t(1/2)=3.9 ± 0.1h) which like 4-hydroxyequilenin-o-quinone (t(1/2)=2.5 ± 0.2 h) was observed to be exceptionally long-lived with the potential to cause cytotoxicity and/or genotoxicity. In model reactions with tyrosinase, the catechol metabolites of LY2066948 and equilenin were products; interestingly, in the presence of ascorbate to inhibit autoxidation, these catechols were formed quantitatively. Tyrosinase incubations in the presence of GSH gave the expected GSH conjugates resulting from trapping of the o-quinones, which were characterized by LC-MS/MS. Incubations of LY2066948 or equilenin with rat liver microsomes also gave detectable o-quinone trapped GSH conjugates; however, as observed with other SERMs, oxidative metabolism of LY2066948 mainly occurred on the amino side chain to yield the N-dealkylated metabolite. CYP1B1 is believed to be responsible for extra-hepatic generation of genotoxic estrogen quinones and o-quinone GSH conjugates were detected in equilenin incubations. However, in corresponding incubations with CYP1B1 supersomes, no o-quinone GSH conjugates of LY2066948 were detected. These studies suggest that although the naphthol group is susceptible to oxidative metabolism to long-lived o-quinones, the formation of these quinones by cytochrome P450 can be attenuated by the chemistry of the remainder of the molecule as in the case of LY2066948.

Azonazine, a novel dipeptide from a Hawaiian marine sediment-derived fungus, Aspergillus insulicola.

Azonazine, a unique hexacyclic dipeptide, was isolated from a Hawaiian marine sediment-derived fungus eventually identified as Aspergillus insulicola. Its absolute configuration, 2R,10R,11S,19R, was established using NMR, HRESIMS, and CD data plus insights derived from molecular models. A possible route for its biogenesis is proposed, and biological properties were explored against cancer cell lines and in an NFκB inhibition assay.