Ergostane-Type Sterols from King Trumpet Mushroom (Pleurotus eryngii) and Their Inhibitory Effects on Aromatase

Two new ergostane-type sterols; (22E)-5α,6α-epoxyergosta-8,14,22-triene-3β,7β-diol (1) and 5α,6α-epoxyergost-8(14)-ene-3β,7α-diol (2) were isolated from the fruiting bodies of king trumpet mushroom (Pleurotus eryngii), along with eight known compounds (3–10). All isolated compounds were evaluated for their inhibitory effects on aromatase. Among them, 4 and 6 exhibited comparable aromatase inhibitory activities to aminoglutethimide.


Introduction
Estrogen is responsible for breast cancer growth. The target genes of an estrogen receptor are in control of cancer cell development in estrogen-dependent breast tumors. Binding of estrogen receptor to estrogen triggers transcription of its target genes [1]. Aromatase is the rate-limiting enzyme in estrogen biosynthesis [1]. This enzyme converts androgens (testosterone and androtestosterone) into estrogens (estradiol and estrone, respectively) [2]. Aromatase inhibitors (AIs) are adjuvant in hormone treatments commonly prescribed for breast cancers that are hormone receptor-positive in the early stage [3]. However, the currently used AIs have several side effects of menopausal symptoms such as hot flashes, vaginal dryness, sexual dysfunction, musculoskeletal symptoms, osteoporosis, bone fracture, fatigue, mood disturbance, nausea, and vomiting [3]. Therefore, natural compounds obtained from safe food resources might be useful in the search for promoter-specific AIs with few side effects [4].

General Methods
Dibenzylfluorescein (DBF) and Human CYP19 + P450 Reductase SUPERSOMES (human recombinant aromatase) were obtained from BD Biosciences (Heidelberg, Germany). The physical data were obtained by the following instruments: a Yanagimoto micro-melting point apparatus for melting points (uncorrected); a JASCO DIP-1000 digital polarimeter for Optical rotations; a Perkin-Elmer 1720X FTIR spectrophotometer for IR spectra; an Agilent-NMR-vnmrs600 for the 1

Materials
The fruiting bodies of P. eryngii were purchased from HOKUTO Corp. They were cultivated in Kagawa, Japan (Sample 1 in 2011, and Sample 2 in 2014). A voucher material has been deposited in the Herbarium of the Laboratory of Medicinal Chemistry, Osaka University of Pharmaceutical Sciences.

Inhibitory Effects against Human Recombinant Aromatase
Inhibitory assay against human recombinant aromatase was performed as described previously [28,29].

Statistics
Values are described as the mean ± standard error of the mean (S.E.M.). Statistical analysis was performed by one-way analysis of variance, followed by Dunnett's test. Probability (p) values less than 0.05 were regarded as significant.