Gerardiins A-L and Structurally Related Phenanthrenes from the Halophyte Plant Juncus gerardii and Their Cytotoxicity against Triple-Negative Breast Cancer Cells.

Species in the Juncaceae accumulate different types of secondary metabolites, among them phenanthrenes and 9,10-dihydrophenanthrenes in substantial amounts. These compounds have chemotaxonomic significance and also possess interesting pharmacological activities. The present study has focused on the isolation, structure determination, and pharmacological investigation of phenanthrenes from Juncus gerardii. Twenty-six compounds, including 23 phenanthrenes, have been isolated from a methanol extract of this plant. Twelve compounds, the phenanthrenes gerardiins A-L (1-12), were obtained as new natural products. Eleven phenanthrenes [effusol (13), dehydroeffusol (14), effususin A (15), compressin A, 7-hydroxy-2-methoxy-1-methyl-5-vinyl-9,10-dihydrophenanthrene, juncusol, 2-hydroxy-7-hydroxymethyl-1-methyl-5-vinyl-9,10-dihydrophenanthrene, 2,7-dihydroxy-5-formyl-1-methyl-9,10-dihydrophenanthrene, effususol A, 2,7-dihydroxy-5-hydroxymethyl-1-methyl-9,10-dihydrophenanthrene, and jinflexin C], 1-O-p-coumaroyl-3-O-feruloyl-glycerol, and the flavones apigenin and luteolin were isolated for the first time from this plant. The cytotoxicity of the 23 isolated phenanthrenes in both mouse (4T1) and human (MDA-MB-231) triple-negative breast cancer cells and in a nontumor (D3, human cerebral microvascular endothelial) cell line was tested using an MTT viability assay. The results obtained showed that the dimeric compounds gerardiins I (9), J (10), K (11), and L (12), derived biogenetically from effusol and dehydroeffusol, were cytotoxic to both tumor and nontumor cell lines, while the monomeric compounds exerted no or very low cytotoxicity. Impedance measurements were consistent with the results of the MTT assays performed.

Ecdysteroid-containing food supplements from Cyanotis arachnoidea on the European market: evidence for spinach product counterfeiting.

Phytoecdysteroids like 20-hydroxyecdysone ("ecdysterone") can exert a mild, non-hormonal anabolic/adaptogenic activity in mammals, and as such, are frequently used in food supplements. Spinach is well-known for its relatively low ecdysteroid content. Cyanotis arachnoidea, a plant native in China, is among the richest sources of phytoecdysteroids, and extracts of this plant are marketed in tons per year amounts via the internet at highly competitive prices. Here we report the investigation of a series of food supplements produced in Germany and claimed to contain spinach extracts. Twelve ecdysteroids including two new compounds were isolated and utilized as marker compounds. A comparative analysis of the products with Cyanotis and spinach extracts provides evidence that they were manufactured from Cyanotis extracts instead of spinach as stated. Based on the chromatographic fingerprints, 20-hydroxyecdysone 2- and 3-acetate are suggested as diagnostic markers for related quality control. This case appears to represent an unusual type of dietary supplement counterfeiting: undeclared extracts from alternative plants would supposedly 'guarantee' product efficacy.

Oxidized Metabolites of 20-Hydroxyecdysone and Their Activity on Skeletal Muscle Cells: Preparation of a Pair of Desmotropes with Opposite Bioactivities.

Increasing the activation of protein kinase B (Akt) has been suggested as a key signaling step in the nonhormonal anabolic activity of the phytoecdysteroid 20-hydroxyecdysone (20E) in mammals. Base-catalyzed autoxidation of this compound was shown previously to yield interesting B-ring-modified analogues. Herein is reported a thorough study on this reaction, resulting in the preparation and complete NMR spectroscopic assignments of calonysterone (5) and its previously overlooked desmotropic pair (7), along with two new sensitive metabolites of 20E. The two isomers showed considerable stability in solution. Time dependency of the reaction for yield optimization is also presented; by means of analytical HPLC, the two desmotropes can reach a maximum combined yield of >90%. The activity of these compounds on Akt phosphorylation was tested in murine skeletal muscle cells. Compounds 2 and 5 showed more potent activity than 20E in increasing Akt activation, while compound 7 exerted an opposite effect. As such, the present study provides the first direct evidence for a pair of desmotropes exerting significantly different bioactivities.

In vitro anti-diabetic activity and chemical characterization of an apolar fraction of Morus alba leaf water extract.

The tea from the white mulberry (Morus alba L.) leaf is a worldwide known traditional medicine of type II diabetes. Here, we report the investigation of the dichloromethane-soluble fraction obtained in a 0.24% m/m yield from the hot water extract of mulberry leaves. A significant, dose-dependent activity was found by means of the 24-h glucose consumption of fully differentiated adipocytes both in the absence and presence of insulin. The fraction was characterized by HPLC-DAD, GC-MS and GC-FID. The main constituent (40.3% by means of GC-FID) was isolated and identified as loliolide by EIMS, HRESIMS and NMR spectroscopy. In the active fraction benzyl alcohol, ethyl benzoate, t-cinnamic acid, p-hydroxyacetophenone, t-coniferyl alcohol and synapil alcohol were also identified by GC-MS and quantified by GC-FID (0.7, 1.3, 1.5, 2.9, 7.5 and 2.6%, respectively).

Antiproliferative constituents of the roots of Conyza canadensis.

Bioassay-guided fractionation of the N-hexane and CHCl3 phases of the methanol extract of the roots of Conyza canadensis (L.) Cronquist led to the isolation of two new dihydropyranones named conyzapyranone A (1) and B (2), and the known 4 Z,8 Z-matricaria- γ-lactone (3), 4 E,8 Z-matricaria- γ-lactone (4), 9,12,13-trihydroxy-10(E)-octadecenoic acid (5), epifriedelanol (6), friedeline (7), taraxerol (8), simiarenol (9), spinasterol (10), stigmasterol, ß-sitosterol, and apigenin. The structures were determined by means of ESIMS and 1D and 2D NMR spectroscopy, including ¹H-¹H COSY, NOESY, HSQC, and HMBC experiments. The isolated compounds were evaluated for their antiproliferative activities and were demonstrated to exert considerable cell growth-inhibitory activity against human cervix adenocarcinoma (HeLa), skin carcinoma (A431), and breast adenocarcinoma (MCF-7) cells. Some of the active components, including 2, 4, and 10, proved to be substantially more potent against these cell lines than against noncancerous human foetal fibroblasts (MRC-5) and can therefore be considered selective antiproliferative natural products.

Ecdysteroids from Serratula wolffii roots.

Two new natural ecdysteroids, 20,22-didehydrotaxisterone (1) and 1-hydroxy-20,22-didehydrotaxisterone (2), were isolated from the roots of Serratula wolffii. Their structures were elucidated by 1D and 2D NMR spectroscopy and mass spectrometry. The biological activities of these compounds were determined via oral aphid (Acyrthosiphon pisum (Harris)) tests. Compound 1 was inactive and compound 2 exhibited very low toxicity in the oral aphid test. The activities of these two ecdysteroids were in agreement with those of other 22-deoxyecdysteroids.

Cloning and sequencing of cDNA encoding for the testis-specific fox (Vulpes vulpes) sperm polypeptide Vb of the cytochrome C oxidase.

Identification of fox (Vulpes vulpes) sperm antigens was carried out to assess their interest as a potential target for a contraceptive vaccine. We report here the cloning and sequencing of fSP8, a fox sperm protein of 14.7 kd. fSP8 was isoantigenic in foxes, as it was recognized by sera of both male and female foxes immunized with fox sperm proteins. No glycosylation was detected, on fSP8, as shown both by deglycosylation assay and lectin labeling. To determine the fSP8 sequence, the NH2-terminal sequence was first obtained, and a piece of cDNA was amplified from testicular RNA by Rapid Amplification of cDNA extremities polymerase chain reaction. This piece was used to screen a cDNA library from fox testis by Southern blot. A sequence of 879 base pairs was obtained, which includes a major open reading frame coding for 128 amino acids. Mass spectrometric analyses have confirmed the position of the open reading frame. Analysis of the predicted amino acids sequence revealed no apparent transmembrane regions. Comparison of the protein sequence with the Prosite database demonstrated a homology with the Zinc binding site of the subunit Vb of the cytochrome c oxidase. On the C-terminal extremity, fSP8 presents a high homology to the Vb polypeptide of the cytochrome c oxidase from bovine, mouse, and human; however the 34 amino acids on the NH2-extremity were specific to fSP8. Moreover, it was demonstrated that this sequence was testis-specific. This could contribute to the antigenicity of this protein. fSP8 is one of the first fox sperm antigens to be cloned and sequenced.

Identification of a new, testis-specific sperm antigen localized on the principal piece of the spermatozoa tail in the fox (Vulpes vulpes).

Fox (Vulpes vulpes) sperm antigens were identified to assess them as a potential target for a contraceptive vaccine. We report here the cloning and sequencing of fSP13, a fox sperm protein of 97 kDa. The fSP13 protein was both auto- and iso-antigenic in foxes; it was recognized by sera of foxes immunized with fox sperm proteins and vasectomized foxes. The NH2-terminal sequence of fSP13 was determined, and a piece of cDNA was amplified from testicular RNA by reverse transcription polymerase chain reaction. This piece was used to screen a cDNA library from fox testis by Southern blot. A sequence of 1662 base pairs was obtained, including a major open reading frame coding for 498 amino acid. Mass spectrometry analysis confirmed the position of the open reading frame and the presence of posttranscriptional modifications. Analysis of the predicted amino acid sequence revealed no apparent transmembrane regions. Comparison of the protein sequence with the Prosite database demonstrated the presence of four potential N-linked glycosylation sites. The fSP13 bears the closest amino acid similarity to two human sperm proteins: fibrousheathin 2 and testis-specific calcium binding protein 86-VII. The deduced 80 N-terminal amino acid sequence also presents similarity with the RIIalpha domain. By using a serum against fSP13, this antigen was localized on the principal piece of the fox spermatozoa. Northern blot analysis showed that fSP13 is specifically expressed in testis. The fSP13 is one of the first fox sperm antigens to be cloned and sequenced.