Stimulative Effects of (22E,24R)-Ergosta-7,22-diene-3.BETA.,5.ALPHA.,6.BETA.-triol from Fruiting Bodies of Tricholoma auratum, on a Mouse Osteoblastic Cell Line, MC3T3-E1.

YAOITA Y., ENDO M., TANI Y., MACHIDA K., AMEMIYA K., FURUMURA K., KIKUCHI M.

Six new sterols, 5α, 6α;8α, 9α-diepoxy-(22E, 24R)-ergost-22-ene-3β, 7α-diol (1), 5α, 6α;8α, 9α-diepoxy-(22E, 24R)-ergost-22-ene-3β, 7β-diol (2), 5α, 6α-epoxy-(22E, 24R)-ergosta-8, 22-diene-3β, 7β-diol (3), (22E, 24R)-23-methylergosta-7, 22-diene-3β, 5α, 6β-triol (4), (22E, 24R)-23-methulergosta-7, 22-diene-3β, 5α, 6β, 9α-tetrol (5) and (24S)-ergost-7-ene-3β, 5α, 6β, 9α-tetrol (6), have been isolated from seven mushrooms, Amanita pantherina, Amantia virgineoides, Lactarius piperatus, Lyophyllum shimeji, Tricholoma portentosum, Hypsizigus marmoreus and Lentinula edodes together with eighteen known ones (7-24). The structures of these new compounds were elucidated on the basis of their spectral data.

UMEHARA K., NAKAMURA M., MIYAKE T., KUROYANAGI M., UENO A.

In the previous paper, we reported the differentiation inducing activities of lignoids from Arctium Fructus (the fruits of Arctium lappa L., Compositae) against mouse myeloid leukemia cells (M1). We reinvestigated the active components of this extract and isolated three new dilignans. Furthermore, structure modifications were carried out using the most active lignan (arctigenin, 1) and its structure-activity relationship was investigated. Its aliphatic esters were more effective in inducing the differentiation of M1 cells than its aromatic esters. Especially, n-decanoate, which was the most active derivative, induced more than half of the M1 cells into phagocytic cells at a concentration of 2 microM.

KOBAYASHI M., KRISHNA M.M., ANJANEYULU V.

The lipid extract of Sinularia sp. of soft coral, collected off the coast of the Andaman and Nicobar Islands, afforded a new sterol 5, together with three known compounds 2, 3 and 4, and the aglycone (1) of 2. The structure of 5 was derived by comparison of the 1H- and 13C-NMR data with those of 2 and 4 having the same C, D- and A, B-ring substituents, respectively.

KOBAYASHI M., MRISHNA M.M., HARIBABU B., ANJANEYULU V.

Two new marine polyhydroxysterols, 23-demethylgorgost-7-ene-3β, 5α, 6β-triol (4a) and (24S)-ergostane-3β, 5α, 6β, 7β, 15β-pentol (6), were isolated from soft corals(Sinularia sp. and Lobophytum crassum, respectively) collected off the Andaman and Nicobar Islands, Indian Ocean. (24S)-Ergost-5-ene-3β, 7α-diol (1), a known synthetic compound, was isolated from Sclerophytum sp. soft coral of the same region. The structures of 4a and 6 were derived by comparison of the 1H- and 13C-NMR data with those of reference compounds having the same partial structures. The previous assignments of C-1 and C-2 of 3β, 5α, 6β-trihydroxysterol were reversed.

ISHIZUKA T., YAOITA Y., KIKUCHI M.

Four new sterols, 5α, 6α-epoxy-(22E, 24R)-ergosta-8(14), 22-diene-3β, 7β-diol (1), (22E, 24R)-ergosta-8, 22-diene-3β, 5α, 6β, 7α-tetrol (2), (22E, 24R)-ergosta-7, 9(11), 22-triene-3β, 5α, 6β-triol (3) and 3β, 5α, 6β-trihydroxy-(22E, 24R)-ergost-22-en-7-one (4), have been isolated from the fruit bodies of Grifola frondosa (FR.) S. F. GRAY (Polyporaceae)together with fourteen known ones (5-18), of which two (5 and 6) are reported for the first time from a natural source. The structures of these compounds were elucidated on the basis of spectral data.

HATA K., ISHIKAWA K., HORI K., KONISHI T.

We examined the differentiation-inducing effects of extracts of 49 wild plants, 25 types of seaweed and 26 mushrooms in Akita on the human leukemia cell line HL60 and a B16 mouse melanoma-derived sub-clone with high differentiation capability (B16 2F2). Differentiation inducers of HL60 cells such as retinoic acid, showed no effects on the differentiation of B16 2F2 cells. Furthermore, chemical compounds known to be inducers of B16 cells, did not induce differentiation of HL60 cells. Screening tests showed that the differentiation of HL60 cells was induced by extracts of 28 wild plants, 10 types of seaweed and 2 mushrooms, and melanogenesis of B16 2F2 cells was increased by extracts of 21 wild plants, 8 types of seaweed and 7 mushrooms. All of the alcoholic extracts of plants belonging to the subfamily Cichorioideae of the family Compositae caused cell differentiation of the melanoma cell line. The extracts of Chinese dandelion root, also inhibited cell growth and induced melanogenesis of B16 2F2 cells. We isolated the active compound from ethanol extracts of the crude drug. Chemical and physical data for the active compound were identical with those for lupeol, a lupane-type triterpene.

Choi E.M., Suh K.S., Kim Y.S., Choue R.W., Koo S.J.

To investigate the bioactivities of soybean, which act on bone metabolism, we studied the effect of a soybean ethanol extract on the activity of osteoblast MC3T3-E1 cells. Soy extract (0.01-0.1 g/l) dose-dependently increased survival (P

Sugimoto E., Yamaguchi M.

Daidzein is a natural isoflavone found in Leguminosae. The effect of daidzein on osteoblastic MC3T3-E1 cells was investigated. Cells were cultured in a serum-free medium for 48 hr in the presence of daidzein (10(-7)-10(-5) M). Daidzein (10(-6) and 10(-5) M) caused a significant elevation of protein content, alkaline phosphatase activity, and DNA content in cells; those increases were about 1.4-, 1.5-, and 2.0-fold, respectively. The ability of daidzein (10(-5) M) to increase protein content, alkaline phosphatase activity, and DNA content in cells was prevented completely by the presence of cycloheximide (10(-6) M), an inhibitor of protein synthesis, indicating that the isoflavone has a stimulatory effect on cellular protein synthesis. The anabolic effect of daidzein (10(-6) or 10(-5) M) in cells was not distinguishable from that of genistein (10(-6) or 10(-5) M). Meanwhile, cell protein showed an additive effect of 17beta-estradiol and daidzein, but their effects on alkaline phosphatase were not additive. Moreover, the effect of daidzein (10(-5) M) in elevating cellular protein content and alkaline phosphatase activity was inhibited completely by the presence of tamoxifen (10(-6) M), suggesting that the effect of the isoflavone is mediated partly through estrogen action. This study demonstrates that daidzein has an anabolic effect in osteoblastic MC3T3-E1 cells.

Mizutani K., Ikeda K., Kawai Y., Yamori Y.

Nutritional and pharmacological factors are needed to prevent bone loss that occurs with increasing age. The chemical compounds that act on bone metabolism as nutrients in food, however, are poorly understood. The effect of resveratrol, a natural phytoestrogen, on the proliferation and differentiation of osteoblastic MC3T3-E1 cells was studied. Resveratrol dose-dependently increased DNA synthesis (10(-9)-10(-7) M) of MC3T3-E1 cells. In addition, resveratrol increased alkaline phosphatase (ALP) activity and prolyl hydroxylase activity of MC3T3-E1 cells (10(-6)-10(-5) M). Moreover, the antiestrogen tamoxifen eliminated the stimulation of MC3T3-E1 cells on the proliferation and ALP activity by resveratrol. On the other hand, resveratrol inhibited prostaglandin E2 production in MC3T3-E1 cells (10(-8)-10(-6) M). Our present study is the first to demonstrate that resveratrol directly stimulates cell proliferation and differentiation of osteoblasts.

Nakayama Y., Takahashi K., Nqji S., Muto K., Nishijima K., Taniguchi S.

Piccialli V., Sica D.

Yoneyama T., Takahashi H., Grudniewska A., Ban S., Umeyama A., Noji M.

Ergosterol is an essential component for fungi, including entomopathogenic fungi like Cordyceps. Cordyceps has been used as a traditional medicine in Japan and China and possesses various unique ergostane-type sterols, those exhibit bioactivities. In this manuscript, we reported the isolation of 2 new ergostane-type sterols, 1 and 2 along with ten sterols (3-12) from 7 strains of Cordyceps related fungal strains, Cordyceps takaomontana NBRC 101754, Metarhizium owariense NBRC 33258, Polycephalomyces formosus NBRC 109994, Cordyceps tuberculata NBRC 106948, Cordyceps tenuipes NBRC 108997, Cordyceps sp. NBRC 106954, and Tolypocladium paradoxum NBRC 106958 collected fruiting bodies of Ophiocordyceps heteropoda. In addition, the antitrypanosomal activity and antimicrobial activity of isolates were tested to find 6 showed the antitrypanosomal activity, and the minimum inhibitory concentration (MIC) value was confirmed as 1.41 µg/mL. In the antimicrobial assay, the MIC value of 8 against methicillin-resistant Staphylococcus aureus was determined to be 3.1 µg/mL.

Xu C., Li J., Su L., Tang H., Zhang W.

A new 9,11-secosteroid, (22R)-22-acetoxy-3β,6α,11-trihydroxy-9,11-seco-5α-cholest-7-en-9-one, along with twelve known analogues were isolated from the South China Sea gorgonian coral Iciligorgia sp. Their structures were determined by the spectroscopic analysis and comparison with reported data. In an in vitro test of osteoclastic differentiation, three compounds exhibited significant down-regulating effect whereas two compounds showed significant up-regulating effect at 0.5 and 1.0 μm. This is the first report of the chemical investigation of the gorgonian Iciligorgia sp. The acetoxy substitution at C-22 seems to play an important role in the regulating activity.

Grandy J.J., Onat B., Tunnicliffe V., Butterfield D.A., Pawliszyn J.

Current methods for biochemical and biogeochemical analysis of the deep-sea hydrothermal vent ecosystems rely on water sample recovery, or in situ analysis using underwater instruments with limited range of analyte detection and limited sensitivity. Even in cases where large quantities of sample are recovered, labile dissolved organic compounds may not be detected due to time delays between sampling and preservation. Here, we present a novel approach for in situ extraction of organic compounds from hydrothermal vent fluids through a unique solid phase microextraction (SPME) sampler. These samplers were deployed to sample effluent of vents on sulphide chimneys, located on Axial Seamount in the North-East Pacific, in the Urashima field on the southern Mariana back-arc, and at the Hafa Adai site in the central Mariana back-arc. Among the compounds that were extracted, a wide range of unique organic compounds, including labile dissolved organic sulfur compounds, were detected through high-resolution LC-MS/MS, among which were biomarkers of anammox bacteria, fungi, and lower animals. This report is the first to show that SPME can contribute to a broader understanding of deep sea ecology and biogeochemical cycles in hydrothermal vent ecosystems.

Osivand A., Araya H., Appiah K., Mardani H., Ishizaki T., Fujii Y.

Research Highlights: Some organisms such as plants and fungi release certain secondary metabolites, generally called allelochemicals, which can influence the organisms around them. Some of the secondary metabolites released by mushrooms may have certain effects on the growth and development of neighboring plants. Background Objectives: The purpose of the present study was to investigate the allelopathic potential of mushrooms in a forest ecosystem. To this end, 289 Japanese mushroom species were collected from the wild and tested using a modified sandwich method, which is a quick and effective bioassay technique. Materials and Methods: The collected specimens were prepared for bioassay as dried samples, and 10 mg/well (10 cm2) was added to a 6-well multidish according to the mycelia biomass, which was estimated at 700−900 kg ha−1 year−1 (7–9 mg 10 cm−2) in coniferous forests. Results: Of the screened mushroom species, 74% inhibited more than 50% of the radicle elongation in lettuce (Lactuca sativa var. Great Lakes 366) seedlings, while the average of all species was 41.1%. This result suggests that wild mushrooms have a significant regulatory effect on lettuce growth. According to our standard deviation variance analysis, 54 out of 289 species showed significant allelopathic activity. Among these species, Xeromphalina tenuipes, Cortinarius violaceus, and Clavaria miyabeana exhibited the strongest growth inhibitory activity, with radicle elongation of 5.1%, 4.3%, and 7.6% of the control, respectively. In contrast, Ischnoderma resinosum stimulated the length of radicle and hypocotyl growth by 30.6% and 42.0%, respectively. These results suggest that these species may play important roles in ecosystems. In addition, the wide range of allelopathic activities observed in mushrooms indicates that various amounts of diverse secondary metabolites from these species are involved in mushroom allelopathy. Conclusions: Our study reveals the importance of evaluating mushroom allelopathy to understand the wider ecological structures within complex ecosystems.

Fan N.W., Chang H.S., Cheng M.J., Chan H.Y., Hsieh S.Y., Liu T.W., Chen S.W., Yuan G.F., Chen I.S.

Two new metabolites, mollisinols A and B (1 and 2), and one first isolated from nature, mollisilactone (3), along with 13 known compounds (4–16), were isolated from the EtOAc-soluble fraction of the solid-state fermentation of the endophytic fungus Mollisia sp., derived from the root bark of Ardisia cornudentata Mez (Myrsinaceae). Their structures were elucidated by spectroscopic analyses, including 1D and 2D NMR experiments, and by HR-ESI-MS mass spectrometry. Among the isolates, ergosterol peroxide (12) inhibited NO production in activated macrophages, and emodin (8) showed IL-6 inhibitory activity in LPS activated RAW 264.7 cells with IC50 values of 36.99 and 5.97 μM, respectively.

Andrade L.C., Almeida M.J., Paixão J.A., Carvalho J.F., Sá e Melo M.L.

The title compound, C(19)H(30)O(4), is an androstan-17-one derivative synthesized from the dehydro-epiandrosterone through a sequential addition of an oxidant, followed by a trans-diaxial opening of the epoxide generated, with Bi(OTf)(3) (OTf is trifluoro-methane-sulfonate). The six-membered rings have a slightly flattened chair conformation, while the five-membered ring adopts a 14-α envelope conformation. All rings are trans fused. In the crystal, the mol-ecules are connected by O-H⋯O hydrogen bonds involving the hydroxyl and carbonyl groups, forming a three-dimensional network. A quantum mechanical ab initio Roothan Hartree-Fock calculation of the free mol-ecule gives bond lengths, valency angles and ring torsion angles of the free molecule at equilibrium geometry (energy minimum) close to the experimental values.

Lee H., Hwang I., Kim J., Choi J., Jang T., Osada H., Ahn J., Na M., Lee S.

Optical rotations were measured using a JASCO DIP-1000 (Tokyo, Japan) automatic digital polarimeter. The FT-IR spectra were recorded on a JASCO FT-IR 300E spectro-photometer, and UV spectra on a JASCO V-550 spectrophoto-meter. The NMR spectra were recorded on a Bruker 250 MHz (DMX 250) spectrometer and a Varian 600 MHZ (VNS 600), respectively, using Bruker's and Varian's standard pulse pro-gram, with chemical shifts reported in ppm downfield from TMS. Column chromatography was carried out on Merck silica gel (70 - 230 mesh, Merck). TLC was performed on aluminum plates precoated with Kieselgel 60 F

Carvalho J.F., Silva M.M., Sá e Melo M.L.

A convenient, fast, and high-yielding process to synthesize 5α,6β-dihydroxysteroids directly from the correspondent Δ5-steroids is reported. The reaction protocol consists in the conjugation of a readily available and stable oxidant, magnesium bis(monoperoxyphthalate) hexahydrate, with the non-toxic bismuth(III) triflate in acetone to afford the trans-diaxial hydroxylation product in a stepwise manner and in excellent yields.

You J., Dai H., Chen Z., Liu G., He Z., Song F., Yang X., Fu H., Zhang L., Chen X.

The historical paradigm of the deep ocean as a biological 'desert' has shifted to one of a 'rainforest' owing to the isolation of many novel microbes and their associated bioactive compounds. To explore the potential of the bioactive compounds in our marine microbial natural product library, we screened it for the selective cytotoxicity of six different cancer cell lines to human normal lung fibroblast cell line HLF. The crude extract from a marine-derived fungal strain showed notable selectivity against cancer cell lines. For a bioactivity-guided fractionation and purification, a novel cyclopentenone, (-)-(4R *, 5S *)-3-ethyl-4,5-dihydroxycyclopent-2-enone (1, trichoderone), and a known compound with new activity, cholesta-7,22- diene-3 beta,5 alpha,6 beta-triol (2), were identified from a marine Trichoderma sp. that was isolated from the deep sea sediment of the South China Sea. Their structures were determined by NMR and MS data analyses. Trichoderone (1) displayed potent cytotoxicity against a panel of six cancer cell lines, whereas it did not show much cytotoxicity against normal human lung fibroblast cell line HLF even at a concentration of 7.02 mM. The selectivity index (SI) value for 1 was greater than 100. To the best of our knowledge, both compounds were isolated from marine fungi for the first time. They also exhibited bioactivities against HIV protease and Taq DNA polymerase. Optimization of the compounds would shed new light on treating cancer and infectious diseases.

Rivera A., Benavides O.L., Rios-Motta J.

From the ethanolic extract of Lentinus edodes (Shiitake) eight known sterols were isolated and identified: ergosterol, ergosterol peroxide, (22E)-ergosta-5,7,9(11), 22-tetraen-3β-ol, (22E)-ergosta-7,9(11),22-trien-3β-ol, (22E)-ergosta-6,8,22-trien-3β-ol, (22E)-norergosta-5,7,9,22-tetraen-3β-ol, 3β,5α-dihydroxy-(22E)-ergosta-7,22-dien-6-one, (22E)-ergosta-4,6,8(14),22-tetraen-3-one and, for the first time in mushrooms, (22E)-ergosta-6,22-diene-3β,5α,8α-triol.

Nieminen P., Kärjä V., Mustonen A.-.

The yellow tricholoma (Tricholoma flavovirens or Tricholoma equestre) is a wild mushroom species that was previously considered edible and tasty. Recently, it caused several cases of delayed rhabdomyolysis in humans and elevated serum creatine kinase (CK) activities in laboratory mice (Mus musculus) in a dose-response study. The present study continued to examine the effects of prolonged T. flavovirens consumption at 12 g freshly frozen mushroom kg(-1)d(-1) on the plasma clinical chemistry and organ histology of mice. The plasma CK and CK-MB activities and the plasma bilirubin concentrations were higher in the exposed mice than in the controls. In addition, pericardial lymphocyte infiltrates were present in the mice that had consumed the mushroom. The results indicate myo-, cardio- and hepatotoxic effects of T. flavovirens.

Papoutsi Z., Kassi E., Mitakou S., Aligiannis N., Tsiapara A., Chrousos G.P., Moutsatsou P.

Acteoside and martynoside are plant phenylpropanoid glycosides exhibiting anticancer, cytotoxic and antimetastatic activities. We investigated their potential to activate estrogen receptor isoforms ERalpha and ERbeta in HeLa cells transfected with an estrogen response element (ERE)-driven luciferase (Luc) reporter gene and an ERalpha or ERbeta expression vector. Their estrogenic/antiestrogenic effects were also assessed in breast cancer cells (MCF7), endometrial cancer cells (Ishikawa) and osteoblasts (KS483), by measuring IGFBP3 levels, cell viability and number of mineralized nodules, respectively, seeking for a natural selective estrogen receptor modulator (SERM). Acteoside and martynoside antagonized both ERalpha and ERbeta (p

Kassi E., Papoutsi Z., Fokialakis N., Messari I., Mitakou S., Moutsatsou P.

To prevent bone loss that occurs with increasing age, nutritional and pharmacological factors are needed. Traditional therapeutic agents (selective estrogen receptor modulators or SERMs, biphosphonates, calcitonin) may have serious side effects or contraindications. In an attempt to find food components potentially acting as SERMs, we submitted four plant aqueous extracts derived from Greek flora (Sideritis euboea, Sideritis clandestina, Marticaria chamomilla, and Pimpinella anisum) in a series of in vitro biological assays reflective of SERM profile. We examined their ability (a) to stimulate the differentiation and mineralization of osteoblastic cell culture by histochemical staining for alkaline phosphatase and Alizarin Red-S staining, (b) to induce, like antiestrogens, the insulin growth factor binding protein 3 (IGFBP3) in MCF-7 breast cancer cells, and (c) to proliferate cervical adenocarcinoma (HeLa) cells by use of MTT assay. Our data reveal that all the plant extracts studied at a concentration range 10-100 microg/mL stimulate osteoblastic cell differentiation and exhibit antiestrogenic effect on breast cancer cells without proliferative effects on cervical adenocarcinoma cells. The presence of estradiol inhibited the antiestrogenic effect induced by the extracts on MCF-7 cells, suggesting an estrogen receptor-related mechanism. In conclusion, the aqueous extracts derived from Sideritis euboea, Sideritis clandestina, Marticaria chamomilla, and Pimpinella anisum may form the basis to design "functional foods" for the prevention of osteoporosis.