Aethiopinolones A-E, New Pregnenolone Type Steroids from the East African Basidiomycete Fomitiporia aethiopica.

A mycelial culture of the Kenyan basidiomycete Fomitiporia aethiopica was fermented on rice and the cultures were extracted with methanol. Subsequent HPLC profiling and preparative chromatography of its crude extract led to the isolation of five previously undescribed pregnenolone type triterpenes 1-5, for which we propose the trivial name aethiopinolones A-E. The chemical structures of the aethiopinolones were determined by extensive 1D- and 2D-NMR, and HRMS data analysis. The compounds exhibited moderate cytotoxic effects against various human cancer cell lines, but they were found devoid of significant nematicidal and antimicrobial activities.

Structure-dependent retention of steroid hormones by common laboratory materials.

The tendency of steroid molecules to adsorb to various materials, particularly plastics, has been known of for decades but has not received widespread attention in the scientific community, and a modern, systematic study is lacking. This adsorption is an important consideration for researchers working with steroid hormones as it could skew the results of various experiments. Here we show that steroids adsorb to various vessels used in experiments, including microcentrifuge tubes, glass vials, and cell culture plates, in a manner that depends on the steroid's molecular structure and on the type of vessel. The lipophilicity of steroids is a strong predictor of the degree of adsorption, with nearly 50 % of the most lipophilic steroid tested, pregnenolone, retained in a high-adsorbing microcentrifuge tube after one hour incubation of an aqueous pregnenolone solution followed by removal of the aqueous solvent. We also show the effects of other factors such as incubation time, centrifugation, and temperature on adsorption, and show that adsorption can be mostly prevented by the presence of serum proteins in steroid solutions and/or by the use of low-adsorbing tubes.

[Studies on chemical constituents of rhizomes of Ligusticum chuanxiong].

OBJECTIVE: To study chemical constitutents of the rhizomes of Ligusticum chuanxiong. METHOD: The rhizomes of L. chuanxiong were extracted with water to afford a water extract which was participated between H2O and EtOAc, n-BuOH, successively. The compounds were isolatedand purified by column chromatography from the EtOAc fraction, and identified based on spectral analyses. RESULT: Eleven compounds were isolated from the rhizomes of L. chuanxiong, and structures were characterized as (4S)-p-menth-1-ene4, 7-diol (1), aromadendrane-4alpha, 10beta-diol (2), aromadendrane-4beta, 10alpha-diol (3), aromadendrane-4beta, 10beta-diol (4), augustic acid (5), xiongterpene (6), pregnenolone (7), 3(R), 8(S), 9(Z)-falcarindiol (8), senkyunolide F (9), senkyunolide I (10), 4-hydroxyl-3-butylphthalide (11). CONCLUSION: Compounds 1-5 were obtained from the rhizomes of L. chuanxiong for the first time.

Cytotoxic constituents from Solanum lyratum.

Activity-guided fractionation of the ethanol extract of the whole plant from Solanum lyratum resulted in the isolation of a new pregnane derivative glycoside, 16-dehydropregnenolone 3-O-alpha-L-rhamnopyranosyl-(1 --> 2)-beta-D-glucopyranosid uronic acid (2), as well as other six known compounds: 16-dehydropregnenolone (1), allopregenolone (3), protocatechuic acid (4), vanillic acid (5), caffeic acid (6), and scopoletin (7). The structures of the isolated compounds were elucidated on the basis of their spectral data and chemical evidences. Compounds 1, 3, 4 were isolated for the first time from this plant. Cytotoxic activities of the isolated compounds were evaluated. Compound 1 exhibited significant cytotoxic activity against A375-S2, HeLa, SGC-7901, and Bel-7402 with IC50 values of 13.1 +/- 0.9, 21.5 +/- 1.0, 40.2 +/- 0.7, and 49.8 +/- 1.2 microg/mL, respectively.

Progesterone and its metabolites: the potent inhibitors of the transporting activity of P-glycoprotein in the adrenal gland.

P-glycoprotein (P-gp) is a transmembrane glycoprotein responsible for the multidrug resistant (MDR) phenotype in various cancer cells. It has been shown that P-gp transports various kinds of anti-cancer agents as well as hydrophobic chemicals. Although P-gp is also expressed in normal human tissues, such as liver, kidney, and adrenal gland, its function and transporting substrates in these tissues are still unknown. In previous work, we demonstrated that some compounds in human plasma modulate the transporting activity of P-gp. We also found that P-gp is expressed at a high level in the bovine adrenal gland and that this tissue contains large amount of compounds which inhibit the transporting activity of P-gp. We purified such compounds from the adrenal gland by monitoring the ability to enhance the accumulation of [3H]vincristine in MDR cells. Two major compounds were purified and identified as progesterone and pregnenolone by nuclear magnetic resonance (NMR) analysis. Progesterone was the most potent and abundant compound that inhibited the transporting activity of P-gp among the compounds extracted from bovine adrenal gland with methanol. We also found that six authentic progesterone metabolites in the 5 beta-metabolic pathway but none in the 5 alpha-metabolic pathway were able to enhance the accumulation of [3H]vincristine in MDR cells and to inhibit [3H]azidopine photolabeling of P-gp in the adrenal gland. These results indicate that some progesterone metabolites can interact with P-gp and that stereoisomerism around carbon 5 of the progesterone metabolites is important for them to be recognized by P-gp.

Solid-phase extraction of esculetin from the ash bark of Chinese traditional medicine by using molecularly imprinted polymers.

A molecularly imprinted polymer solid-phase extraction method is used to extract esculetin from the ash bark of Chinese traditional medicine. Ratio of ethanol and water as washing solution were investigated. Data of accumulative adsorption on molecularly imprinted polymers from the continuous loading experiment suggests that there are two different kinds of recognition sites in molecularly imprinted polymers. By selecting the washing and eluting solution a scheme was designed to separate esculetin and its analogues including esculin, coumarin, 7-methoxylcoumarin and daphnetin. Finally, by applying the revised scheme esculetin was extracted from the ash bark of Chinese traditional medicine that was purchased from two big drugstores, respectively, with both molecularly imprinted polymers and non-molecularly imprinted polymers.

Domestication Effects on Stress Induced Steroid Secretion and Adrenal Gene Expression in Chickens.

Understanding the genetic basis of phenotypic diversity is a challenge in contemporary biology. Domestication provides a model for unravelling aspects of the genetic basis of stress sensitivity. The ancestral Red Junglefowl (RJF) exhibits greater fear-related behaviour and a more pronounced HPA-axis reactivity than its domesticated counterpart, the White Leghorn (WL). By comparing hormones (plasmatic) and adrenal global gene transcription profiles between WL and RJF in response to an acute stress event, we investigated the molecular basis for the altered physiological stress responsiveness in domesticated chickens. Basal levels of pregnenolone and dehydroepiandrosterone as well as corticosterone response were lower in WL. Microarray analysis of gene expression in adrenal glands showed a significant breed effect in a large number of transcripts with over-representation of genes in the channel activity pathway. The expression of the best-known steroidogenesis genes were similar across the breeds used. Transcription levels of acute stress response genes such as StAR, CH25 and POMC were upregulated in response to acute stress. Dampened HPA reactivity in domesticated chickens was associated with changes in the expression of several genes that presents potentially minor regulatory effects rather than by means of change in expression of critical steroidogenic genes in the adrenal.

Identification of 16-dehydropregnenolone as an intermediate in 16-androstene biosynthesis in neonatal porcine testicular microsomes.

The biosynthesis of 16-androstenes has been studied in neonatal porcine testicular microsomes using 17-hydroxypregnenolone and 16-dehydropregnenolone, separately, as substrates. The metabolites formed after microsomal incubation with these substrates were purified, derivatized as O-methyloxime-trimethylsilyl ethers and analysed by capillary gas chromatography-mass spectrometry. In the incubation of 17-hydroxypregnenolone with microsomes, 16-dehydropregnenolone was identified as an intermediate in the biosynthesis of 16-androstenes. Further microsomal incubation of 16-dehydropregnenolone has established the intermediary role of this steroid in the production of 16-androstenes.

Ovarian delta-5-3-beta-hydroxysteroid dehydrogenase in aging rats.

Ovaries of rats 1, 2, 4, and 18 months old revealed significant delta-5-3-beta-hydroxysteroid dehydrogenase (3beta-OHSD) activity when dehydroepiandrosterone (DHA), pregnenolone, and 16-dehydropregnenolone served as substrates. No enzyme activity was evident in ovaries 4 and 18 months of age when the substrates 17alpha-hydroxypregnenolone, pregnenolone sulfate, and dehydroepiandrosterone sulfate were used. A marked increase in enzyme activity occurred in the granulosae of mature vesicular follicles at 18 months of age. DHA, pregnenolone, and 16-dehydropregnenolone provided similar activities. On the other hand, interstitial enzyme activity exhibited an aging decline with DHA and 16-dehydropregnenolone but increased with pregnenolone. Corpora lutea were less evident in aging ovaries but did exhibit strong 3beta-OHSD activity when DHA was used. Pregnenolone use by corpora lutea was sharply increased in aging rats but no change occurred with 16-dehydropregnenolone. Thus aging was associated with changes in 3beta-OHSD distribution and use of steroid substrate; a change in function is suggested.

Metabolism of [14C]cholesterol to C-20 isomeric [14C]pregn-5-ene-3,20-diols in the tobacco hornworm, Manduca sexta.

After injection into male and female fifth-instar larvae of Manduca sexta, [14C]cholesterol was converted to C21 steroids, [14C]pregn-5-ene-3 beta,20-diols. These metabolites were isolated from 8-day-old pupae and were identified by TLC, HPLC, and GC-MS as the C-20 isomers of pregnene-3 beta,20-diol. They also were isolated from male and female meconium fluid (of 16-day-old pupae) following injection of [14C]cholesterol into 14-day-old pupae.

Cholesterol side-chain cleavage activity in human placenta and bovine adrenals: an one-step method for separation of pregnenolone formed in vitro.

Cholesterol side-chain cleavage (CSCC) activity towards exogenous cholesterol was quantified by an one-step reversed-phase minicolumn method for the separation of pregnenolone formed in the reaction. The assay is rapid and reproducible. The method is linear for up to 2 mg of placental mitochondrial protein and up to 1 mg of bovine adrenal mitochondrial protein in the incubata over 30 min and 5 min reaction times, respectively. Average Km and Vmax values were 14.1 microM and 3.4 pmol/min/mg for the placental preparation and 1.5 microM and 20.7 pmol/min/mg for the bovine adrenal mitochondrial preparation. In human placenta, the mitochondrial fraction contained most of the CSCC activity. Inhibition studies showed that aminoglutethimide (500 microM) inhibited both placental and bovine adrenal activities at the same level (about 80-90% inhibition) but androstenedione (500 microM), metyrapone (500 microM), benzo(a)pyrene (800 microM) and Emulgen 911 (0.05%) were more effective in human placental preparations. Neither of the activities were inhibited to any great extent by alpha-naphthoflavone (500 microM), SKF 525A (500 microM) or 7-ethoxycoumarin (1 mM).

[C21 steroidal constituents from Cynanchum hancockianum].

Two new compounds hancogenin B (V) and hancoside A (VI) and four known compounds glucogenin C (I), cynatratoside A (II), glaucogenin A (III) and anhydrohirundigenin (IV) were isolated from the roots of Cynanchum hancockianum (Maxim) Al. Iljinski. Their structures were identified on the basis of spectral evidence. The fragmentation ways of 13:14, 14:15-secopregnenes in EIMS were outlined and the antitumor activity of II and the antiendotoxic activity of VI were also preliminarily tested in vitro.

[Chemical constituents of Notopterygium incisium Ting. III: Chemical constituents isolated and identified from petroleum ether extracts of N. incisium Ting].

Six chemical compounds were isolated from the roots and rhizoma of Notopterygium incisium by column chromatography and preparative thin-layer chromatography, and identified by chemical and spectroscopic analysis as beta-sitosterol, oleic acid, linoleic acid, columbianetin, pregnenolone and ferulic acid. The presence of pregnenolone in the genus Notopterygium has not been reported.

Caught between triterpene- and steroid-metabolism: 4α-carboxylic pregnane-derivative from the marine alga-derived fungus Phaeosphaeria spartinae.

Investigation of the fungus Phaeosphaeria spartinae, an endophyte of the marine red alga Ceramium sp., led to the isolation of spartopregnenolone (1), a metabolite whose structure includes features of triterpenes and steroids, i.e. a Δ(8,9) double bond as occurring in lanosterol type triterpenoids, a carboxyl group at C-4 which is characteristic for intermediates on the way from triterpenes to steroids and an acetyl side chain as typical for pregnane type steroids. The unusual structure of compound 1 was established from extensive spectroscopic investigations and is best described as a 4α-carboxy-8,9-pregnene derivative.

Naturally occurring steroids in Xenopus oocyte during meiotic maturation. Unexpected presence and role of steroid sulfates.

In the ovary, oocytes are surrounded by follicle cells and arrested in prophase of meiosis I. Although steroidogenic activity of follicle cells is involved in oogenesis regulation, clear qualitative and quantitative data about the steroid content of follicles are missing. We measured steroid levels of Xenopus oocytes and follicles by gas chromatography-mass spectrometry. We show that dehydroepiandrosterone sulfate is the main steroid present in oocytes. Lower levels of free steroids are also detected, e.g., androgens, whereas progesterone is almost undetectable. We propose that sulfatation is a protective mechanism against local variations of active steroids that could be deleterious for follicle-enclosed oocytes. Steroid levels were measured after LH stimulation, responsible for the release by follicle cells of a steroid signal triggering oocyte meiosis resumption. Oocyte levels of androgens rise slowly during meiosis re-entry whereas progesterone increases abruptly to micromolar concentration, therefore representing the main physiological mediator of meiosis resumption in Xenopus oocyte.

Microbial transformation of 3ß-acetoxypregna-5,16-diene-20-one by Penicillium citrinum.

The biotransformation of 3ß-acetoxypregna-5,16-diene-20-one (1) by using a filamentous fungus Penicillium citrinum resulted in the production of four metabolites 2-5. The structures of these compounds were elucidated by different spectroscopic analysis (1D- and 2D-NMR) and HR-ESI-MS as 3ß,7ß-dihydroxy-pregn-5,16(17)-dien-20-one (2), 3ß-hydroxy-7α-methoxy-pregn-5,16(17)-dien-20-one (3), 3ß,7ß,11α-trihydroxy-pregn-5,16(17)-dien-20-one (4), and a known 3ß,7α-dihydroxy-pregn-5,16(17)-dien-20-one (5). The 7-O-methylation is a novel reaction in the field of microbial transformation of pregnane steroids.