Biosynthetic Potential of the Endophytic Fungus Helotiales sp. BL73 Revealed via Compound Identification and Genome Mining.

Endophytic fungi have been recognized as prolific producers of chemically diverse secondary metabolites. In this work, we describe a new representative of the order Helotiales isolated from the medicinal plant Bergenia pacumbis. Several bioactive secondary metabolites were produced by this Helotiales sp. BL 73 isolate grown on rice medium, including cochlioquinones and isofusidienols. Sequencing and analysis of the approximately 59-Mb genome revealed at least 77 secondary metabolite biosynthesis gene clusters, of which several could be associated with detected compounds or linked to previously reported molecules. Four terpene synthase genes identified in the BL73 genome were codon optimized and expressed, together with farnesyl-, geranyl-, and geranylgeranyl-pyrophosphate synthases, in Streptomyces spp. An analysis of recombinant strains revealed the production of linalool and its oxidized form, terpenoids typically associated with plants, as well as a yet unidentified terpenoid. This study demonstrates the importance of a complex approach to the investigation of the biosynthetic potential of endophytic fungi using both conventional methods and genome mining. IMPORTANCE Endophytic fungi represent an as yet underexplored source of secondary metabolites, of which some may have industrial and medical applications. We isolated a slow-growing fungus belonging to the order Helotiales from the traditional medicinal plant Bergenia pacumbis and characterized its potential to biosynthesize secondary metabolites. We used cultivation of the isolate with a subsequent analysis of compounds produced, bioinformatics-based mining of the genome, and heterologous expression of several terpene synthase genes. Our study revealed that this Helotiales isolate has enormous potential to produce structurally diverse natural products, including polyketides, nonribosomally synthesized peptides, terpenoids, and ribosomally synthesized and posttranslationally modified peptides (RiPPs). Identification of meroterpenoids and xanthones, along with establishing a link between these molecules and their putative biosynthetic genes, sets the stage for investigation of the respective biosynthetic pathways. The heterologous production of terpenoids suggests that this approach can be used for the discovery of new compounds belonging to this chemical class using Streptomyces bacteria as hosts.

Endophytic Akanthomyces sp. LN303 from Edelweiss Produces Emestrin and Two New 2-Hydroxy-4 Pyridone Alkaloids.

In the search for new antibiotics, several fungal endophytes were isolated from the medicinal plant Leontopodium nivale subsp. alpinum (Edelweiss). The extract from one of these fungi classified as Akanthomyces sp. displayed broad-spectrum antibiotic activity against gram-negative bacteria and fungi. Further investigation into the composition of this extract using bioactivity-guided fractionation, HRMS, and nuclear magnetic resonance revealed two new 4-hydroxy-2-pyridone alkaloids (1, 2) and emestrin (3), an epidithiodioxopiperazine not previously known to be produced by a member of Cordycipitaceae. Further testing of purified compounds 1 and 2 proved that they are devoid of antibiotic activity, and all the activities observed in the crude extract could be assigned to emestrin (3), whose configuration was confirmed by crystallographic data. This study demonstrates, for the first time, that endophytic fungi from Edelweiss can produce new compounds, prompting further investigation into them for drug discovery.

Viennamycins: Lipopeptides Produced by a Streptomyces sp.

Extracts from Streptomyces sp. S4.7 isolated from the rhizosphere of edelweiss, an alpine medicinal plant, exhibited activity against Gram-positive bacteria. LC-HRMS analyses of the extracts resulted in the detection of two unknown, structurally related lipopeptides that were assumed to be responsible for the antibiotic activity. LC-MS guided isolation and structure elucidation of viennamycins A and B (1 and 2) by HR-MS/MS, 1D and 2D NMR, and Marfey's analyses revealed them to be novel compounds, with viennamycin A containing cysteic acid, a unique feature for lipopeptides. Tests for antibacterial, antifungal, and cytotoxic activities of purified viennamycins, both with and without divalent cations, did not reveal any bioactivity, suggesting that their biological function, which could not be determined in the tests used, is atypical for lipopeptides. The genome of Streptomyces sp. S4.7 was sequenced and analyzed, revealing the viennamycin biosynthetic gene cluster. Detailed bioinformatics-based analysis of the viennamycin gene cluster allowed elucidation of the biosynthetic pathway for these lipopeptides.

Isolation and Characterization of Acetylcholinesterase Inhibitors from Piper longum and Binding Mode Predictions.

Restoration of cholinergic function is considered a rational approach to enhance cognitive performance. Acetylcholinesterase inhibitors are still the best therapeutic option for Alzheimer's disease. The fruits of Piper longum have been used in traditional medicines for the treatment of memory loss. It was demonstrated that the dichloromethane extract of these fruits is able to inhibit acetylcholinesterase. Thus, the aim of this study was to identify the contained acetylcholinesterase inhibitors. The active zones were presented via TLC-bioautography, and five compounds were isolated in the process of a bioassay-guided phytochemical investigation. Their structures were characterized as piperine, methyl piperate, guineenisine, pipercide, and pellitorine using spectroscopy and spectrometry methods (UV, IR, MS, 1H-, and 13C-NMR). In vitro acetylcholinesterase inhibitory activities of the isolates and their IC50 values were determined via a colorimetric assay. Three of them exhibited enzyme inhibitory activities, with piperine being the most potent compound (IC50 of 0.3 mM). In order to investigate the binding mode of the tested compounds, docking studies were performed using the X-ray crystal structure of acetylcholinesterase from Tetronarce californica with the Protein Data Bank code 1EVE. The content of the active compounds in the extract was determined by a developed HPLC method. Piperine was present in the maximum quantity in the fruits (0.57%), whereas methyl piperate contained the minimum content (0.10%).

In Vitro Digestion of Grape Seed Oil Inhibits Phospholipid-Regulating Effects of Oxidized Lipids.

The intake of dietary lipids is known to affect the composition of phospholipids in gastrointestinal cells, thereby influencing passive lipid absorption. However, dietary lipids rich in polyunsaturated fatty acids, such as vegetable oils, are prone to oxidation. Studies investigating the phospholipid-regulating effect of oxidized lipids are lacking. We aimed at identifying the effects of oxidized lipids from moderately (18.8 ± 0.39 meq O2/kg oil) and highly (28.2 ± 0.39 meq O2/kg oil) oxidized and in vitro digested cold-pressed grape seed oils on phospholipids in human gastric tumor cells (HGT-1). The oils were analyzed for their antioxidant constituents as well as their oxidized triacylglycerol profile by LC-MS/MS before and after a simulated digestion. The HGT-1 cells were treated with polar oil fractions containing epoxidized and hydroperoxidized triacylglycerols for up to six hours. Oxidized triacylglycerols from grape seed oil were shown to decrease during the in vitro digestion up to 40% in moderately and highly oxidized oil. The incubation of HGT-1 cells with oxidized lipids from non-digested oils induced the formation of cellular phospholipids consisting of unsaturated fatty acids, such as phosphocholines PC (18:1/22:6), PC (18:2/0:0), phosphoserine PS (42:8) and phosphoinositol PI (20:4/0:0), by about 40%-60%, whereas the incubation with the in vitro digested oils did not affect the phospholipid metabolism. Hence, the gastric conditions inhibited the phospholipid-regulating effect of oxidized triacylglycerols (oxTAGs), with potential implications in lipid absorption.

Antiplasmodial activity of triterpenes isolated from the methanolic leaf extract of Combretum racemosum P. Beauv.

ETHNOPHARMACOLOGICAL RELEVANCE: Combretum racemosum showed activity in previous ethnopharmacological investigations of some Combretum species used in malaria treatment in parts of West Africa. AIM OF THE STUDY: This study aimed at confirming the antimalarial potential of this plant by an activity-guided isolation of its active principles. MATERIALS AND METHODS: A crude methanolic leaf extract of Combretum racemosum and fractions thereof obtained by partition with chloroform and n-butanol were investigated for antiplasmodial activity against chloroquine-sensitive (D10) and chloroquine-resistant (W2) strains of Plasmodium falciparum. Repeated chromatographic separations were conducted on the chloroform fraction to isolate bioactive compounds for further tests on antiplasmodial activity. The characterization of the isolated substances was performed by applying NMR- and MS-techniques (ESI-MS, HR-ESIMS, 1D and 2D NMR). RESULTS: The chloroform fraction (D10: IC50 = 33.8 ±â€¯1.5 µg/mL and W2: IC50 = 27.8 ±â€¯2.9 µg/mL) exhibited better antiplasmodial activity than the n-butanol fraction (D10: IC50 = 78.1 ±â€¯7.3 µg/mL and W2: IC50 = 78 ±â€¯15 µg/mL) as well as the methanolic raw extract (D10: IC50 = 64.2 ±â€¯2.7 µg/mL and W2: IC50 = 65.8 ±â€¯14.9 µg/mL). Thus, the focus of the phytochemical investigation was laid on the chloroform fraction, which led to the identification of four ursane-type (19α-hydroxyasiatic acid (1), 6ß,23-dihydroxytormentic acid (4), madecassic acid (8), nigaichigoside F1 (10)) and four oleanane-type (arjungenin (2), combregenin (5), terminolic acid (7), arjunglucoside I (11)) triterpenes, as well as abscisic acid (9). Compounds 1 and 2, 4 and 5, 7 and 8 as well as 10 and 11 were isolated as isomeric mixtures in fractions CR-A, CR-C, CR-E and CR-H, respectively. All isolated compounds and mixtures exhibited moderate to low activity, with madecassic acid being most active (D10: IC50 = 28 ±â€¯12 µg/mL and W2: IC50 = 17.2 ±â€¯4.3 µg/mL). CONCLUSION: This paper reports for the first time antiplasmodial principles from C. racemosum and thereby gives reason to the traditional use of the plant.

Simultaneous Analysis of Epoxidized and Hydroperoxidized Triacylglycerols in Canola Oil and Margarine by LC-MS.

The progress of lipid oxidation in foods is evaluated by measuring the peroxides and their scission products. However, hydrogen abstraction-independent pathways are not considered by commonly applied methods despite the known reactivity of epoxides toward biomolecules. Herein, a novel liquid chromatography tandem-mass spectrometry method was developed to detect hydroperoxidized and epoxidized triacylglycerols (TAGs) without derivatization or hydrolyzation of food samples. Epoxidized TAGs could be detected in refined canola oil at concentrations of 96.8 ± 2.08 µM, while only 5.77 ± 0.04 µM hydroperoxidized TAGs could be determined. In contrast to canola oil, margarine was more resistant to lipid oxidation since generation of epoxidized TAGs could only be marginally enhanced from 21.7 ± 0.48 to 28.8 ± 0.64 µM in margarine after treatment at 180 °C for 60 min, as also reflected by a peroxide value of 0.80 ± 0.00 mequiv O2/kg, which remained unchanged. The new method allows the assessment of food safety by the simultaneous measurement of hydroperoxidized and epoxidized TAGs without hydrolysis and laborious sample preparation.

Lanostane Triterpenes from Gloeophyllum odoratum and Their Anti-Influenza Effects.

In an in vitro screening for anti-influenza agents from European polypores, the fruit body extract of Gloeophyllum odoratum dose-dependently inhibited the cytopathic effect of the H3N2 influenza virus A/Hong Kong/68 (HK/68) in Madin Darby canine kidney cells with a 50% inhibitory concentration (IC50) of 15 µg/mL, a noncytotoxic concentration. After a chromatographic work-up, eight lanostane triterpenes (1: -8: ) were isolated and their structures were elucidated based on high-resolution electrospray ionization mass spectrometry analyses, and one- and two-dimensional nuclear magnetic resonance experiments. Constituents 1: (gloeophyllin K) and 2: (gloeophyllin L) are reported here for the first time, and compounds 5: , 7: , and 8: have not been described for the investigated fungal material so far. The highest activity was determined for trametenolic acid B (3: ) against HK/68 and the 2009 pandemic H1N1 strain A/Jena/8178/09 with IC50 values of 14 and 11 µM, respectively. In a plaque reduction assay, this compound was able to bind to cell-free viruses and to neutralize their infectivity.

Secondary metabolites from lichen as potent inhibitors of advanced glycation end products and vasodilative agents.

Secondary metabolites from lichens are known for exhibiting various biological effects such as anti-inflammatory, antioxidant and antibacterial activities. Despite this wide range of reported biological effects, their impact on the formation of advanced glycation end products (AGEs) remains vastly unexplored. The latter are known contributors to lifestyle and age-related diseases such as Alzheimer and Parkinson. Moreover, the development of atherosclerosis and arterial stiffness is causally linked to the formation of AGEs. With this in mind, the present work evaluated the inhibitory effects of secondary lichen metabolites on the formation of pentosidine-like AGEs' by using an in vitro, Maillard reaction based, fluorescence assay. Overall, thirty-seven natural and five synthetically modified compounds were tested, eighteen of which exhibiting IC50 values in the range of 0.05 to 0.70 mM. This corresponds to 2 to 32 fold of the inhibitory activity of aminoguanidine. Targeting one major inhibiting mechanism of AGEs formation, all compounds were additionally evaluated on their radical scavenging capacities in an DPPH assay. Furthermore, as both AGEs' formation and hypertension are major risk factors for atherosclerosis, compounds that were available in sufficient amounts were also tested for their vasodilative effects. Overall, and though some of the active compounds were previously reported cytotoxic, present results highlight the interesting potential of secondary lichen metabolites as anti-AGEs and vasodilative agents.

Resveratrol Inhibits Key Steps of Steroid Metabolism in a Human Estrogen-Receptor Positive Breast Cancer Model: Impact on Cellular Proliferation.

The role of resveratrol (RES) in preventing breast cancer is controversial, as low concentrations may stimulate the proliferation of estrogen-receptor alpha positive (ERα+) breast cancer cells. As metabolism is the key factor in altering cellular estrogens, thereby influencing breast tumor growth, we investigated the effects of RES on the formation of estrogen metabolites, namely 4-androstene-3,17-dione (AD), dehydroepiandrosterone (DHEA), dehydroepiandrosterone-3-O-sulfate (DHEA-S), estrone (E1), estrone-3-sulfate (E1-S), 17ß-estradiol (E2), 17ß-estradiol-3-O-(ß-D-glucuronide) (E2-G), 17ß-estradiol-3-O-sulfate (E2-S), 16α-hydroxy-17ß-estradiol (estriol, E3), and testosterone (T) in ERα- MDA-MB-231 and ERα+ MCF-7 cells. Incubation of both of the cell lines with the hormone precursors DHEA and E1 revealed that sulfation and glucuronidation were preferred metabolic pathways for DHEA, E1 and E2 in MCF-7 cells, compared with in MDA-MB-231 cells, as the Vmax values were significantly higher (DHEA-S: 2873.0 ± 327.4 fmol/106 cells/h, E1-S: 30.4 ± 2.5 fmol/106 cells/h, E2-S: 24.7 ± 4.9 fmol/106 cells/h, E2-G: 7.29 ± 1.36 fmol/106 cells/h). RES therefore significantly inhibited DHEA-S, E1-S, E2-S and E2-G formation in MCF-7, but not in MDA-MB-231 cells (Kis: E2-S, 0.73 ± 0.07 µM < E1-S, 0.94 ± 0.03 µM < E2-G, 7.92 ± 0.24 µM < DHEA-S, 13.2 ± 0.2 µM). Suppression of these metabolites subsequently revealed twofold higher levels of active E2, concomitant with an almost twofold increase in MCF-7 cell proliferation, which was the most pronounced upon the addition of 5 µM RES. As the content of RES in food is relatively low, an increased risk of breast cancer progression in women is likely to only be observed following the continuous consumption of high-dose RES supplements. Further long-term human studies simultaneously monitoring free estrogens and their conjugates are therefore highly warranted to evaluate the efficacy and safety of RES supplementation, particularly in patients diagnosed with ERα+ breast cancer.

Allspice and Clove As Source of Triterpene Acids Activating the G Protein-Coupled Bile Acid Receptor TGR5.

Worldwide, metabolic diseases such as obesity and type 2 diabetes have reached epidemic proportions. A major regulator of metabolic processes that gained interest in recent years is the bile acid receptor TGR5 (Takeda G protein-coupled receptor 5). This G protein-coupled membrane receptor can be found predominantly in the intestine, where it is mainly responsible for the secretion of the incretins glucagon-like peptide 1 (GLP-1) and peptide YY (PYY). The aim of this study was (i) to identify plant extracts with TGR5-activating potential, (ii) to narrow down their activity to the responsible constituents, and (iii) to assess whether the intestinal microbiota produces transformed metabolites with a different activity profile. Chenodeoxycholic acid (CDCA) served as positive control for both, the applied cell-based luciferase reporter gene assay for TGR5 activity and the biotransformation assay using mouse fecal slurry. The suitability of the workflow was demonstrated by the biotransformation of CDCA to lithocholic acid resulting in a distinct increase in TGR5 activity. Based on a traditional Tibetan formula, 19 plant extracts were selected and investigated for TGR5 activation. Extracts from the commonly used spices Syzygium aromaticum (SaroE, clove), Pimenta dioica (PdioE, allspice), and Kaempferia galanga (KgalE, aromatic ginger) significantly increased TGR5 activity. After biotransformation, only KgalE showed significant differences in its metabolite profile, which, however, did not alter its TGR5 activity compared to non-transformed KgalE. UHPLC-HRMS (high-resolution mass spectrometry) analysis revealed triterpene acids (TTAs) as the main constituents of the extracts SaroE and PdioE. Identification and quantification of TTAs in these two extracts as well as comparison of their TGR5 activity with reconstituted TTA mixtures allowed the attribution of the TGR5 activity to TTAs. EC50s were determined for the main TTAs, i.e., oleanolic acid (2.2 ± 1.6 µM), ursolic acid (1.1 ± 0.2 µM), as well as for the hitherto unknown TGR5 activators corosolic acid (0.5 ± 1.0 µM) and maslinic acid (3.7 ± 0.7 µM). In conclusion, extracts of clove, allspice, and aromatic ginger activate TGR5, which might play a pivotal role in their therapeutic use for the treatment of metabolic diseases. Moreover, the TGR5 activation of SaroE and PdioE could be pinpointed solely to TTAs.

Acetylated Furostene Glycosides from Solanum gilo Fruits.

In continuation of our work on a traditional mixture of spices called "Nkui", used in Cameroon for its influence on women's reproductive health, we investigated the chemical composition of Solanum gilo, one component of "Nkui". A methanolic extract was studied in detail. After dereplication of several known compounds, two furo-5-stene-derived saponin glycosides with acetylated sugar moieties were isolated. By extensive 1- and 2D NMR experiments and HR-MS and GC-MS methods, the structures were elucidated as 26-[(3‴,4‴,6‴-tri-O-acetyl)-ß-D-glucopyranosyloxy]-22-hydroxyfurost-5-en-3ß-yl-O-α-L-rhamnopyranosyl-(1″→2')-ß-D-glucopyranoside (A) and 26-[(3‴,4‴,6‴-tri-O-acetyl)-ß-D-glucopyranosyloxy]-22-hydroxyfurost-5-en-3ß-yl-[O-α-L-rhamnopyranosyl-(1''''→4')-O-α-L-rhamnopyranosyl-(1″→2')]-ß-D-glucopyranoside (B), both new natural compounds.

Isolation of eudesmanes from Pluchea odorata and evaluation of their effects on cancer cell growth and tumor invasiveness in vitro.

The traditionally used Central American medicinal plant Pluchea odorata, known as an anti-inflammatory and cancer cell growth-inhibiting remedy, was subjected to bioassay-guided isolation. Structure elucidation by 1D- and 2D-NMR and MS techniques supported by ECD and UV spectroscopic data revealed seven structurally previously undescribed and eight known eudesmane-type sesquiterpenes. Furthermore, one previously undescribed and one known phytol-like alcohol were identified. All compounds were tested for their cytotoxicity in cancer cells and for their anti-invasive effects. Among the eudesmanes, 3α-(2',3'-epoxy-2'-methylbutyryloxy)-4α-hydroxy-11-hydroperoxy-eudesm-6-en-8-one exhibited the most potent cytotoxic activity with an IC50 value of 8.8 µM (after 48 h). Also in an in vitro model measuring the tumor-triggered breaching of the adjacent lymph endothelial cell barrier (3S*,4R*,5S*,10S*,2'R*,3'R*)-3-(2',3'-epoxy-2'-methylbutyryloxy)-4,7-dihydroxy-eudesm-11-en-8-one (IC75 = 47 µM) and (3S*,4R*,5R*,10S*,2'R*,3'R*)-3-(2',3'-epoxy-2'-methylbutyryloxy)-4-acetyloxy-6-methoxy-11-hydroxy-eudesm-6-en-8-one (IC75 = 73 µM) showed inhibitory activities. Furthermore, preliminary structure-activity relationships (SARs) of the eudesmanes were developed.

Metabolism of Curcumin in Human Breast Cancer Cells: Impact of Sulfation on Cytotoxicity.

Curcumin is a natural polyphenol with promising anticancer properties that undergoes pronounced metabolism in humans. In order to determine whether metabolism of curcumin also occurs in tumor cells and whether biotransformation has any impact on cytotoxicity, metabolism experiments were conducted with hormone-dependent ZR-75-1 and hormone-independent MDA-MB-231 human breast cancer cells. By using HPLC-ESI-Qq-TOF-MS, it was possible to identify one main metabolite, namely curcumin sulfate, in both cell lines. Its concentration in the cytoplasm and culture medium was 1.6- to 1.7-fold higher in ZR-75-1 cells than in MDA-MB-231 cells, concomitant with a 2-fold higher IC50 value in the ZR-75-1 cell line (14 µM compared to 7.3 µM). The net result of sulfation seems to lower the intracellular concentration of curcumin, thereby decreasing its growth inhibitory activity. Interestingly, for the first time, we also found the formation of a curcumin dimer in the cytoplasm but not in the cellular medium of both cell lines. Compared to curcumin sulfate, however, its maximal intracellular concentrations were up to 4-fold lower, indicating only a minor contribution to the overall curcumin clearance. In conclusion, our data elucidated the metabolism of curcumin in breast cancer cells, which must be considered in humans following oral uptake of dietary curcumin as a chemopreventive agent.

HPTLC Bioautography Guided Isolation of α-Glucosidase Inhibiting Compounds from Justicia secunda Vahl (Acanthaceae).

INTRODUCTION: α-Glucosidase inhibitors form an essential basis for the development of novel drugs in diabetes type 2 treatment. Searching for α-glucosidase inhibitors in plants, TLC bioautographic assays have been established and improved within the last years. In traditional medicine, extracts from the leaves of Justicia secunda Vahl are used to treat diabetes mellitus symptoms. OBJECTIVE: To screen for α-glucosidase inhibitors in J. secunda via HPTLC bioautography. Methodology - Extracts from the leaves of J. secunda and fractions thereof were evaluated in terms of their α-glucosidase inhibiting potential by subjecting them to HPTLC bioautography. The aqueous (AQ) fraction deriving from the methanol extract was further fractionated via column chromatography on polystyrene Diaion® HP-20. Two AQ subfractions revealed active compounds, which were isolated via preparative HPTLC and semipreparative HPLC. Their identification and structure elucidation was achieved employing HPLC-ESI-MSn , HRESI-MS, and NMR analyses. RESULTS: α-Glucosidase inhibitors were visualised as white zones on violet background on the TLC plate. The crude water extract, the methanol extract, and the methanol extract derived AQ fraction showed α-glucosidase inhibiting effects. In the latter, two diastereomeric mixtures responsible for the α-glucosidase inhibition were enriched. They were identified as the novel 2-caffeoyloxy-4-hydroxy-glutaric acid and the diastereomers secundarellone B and C. CONCLUSION: The current study presents the α-glucosidase inhibiting potential of J. secunda supporting its traditional medicinal use in diabetes mellitus treatment. HPTLC bioautography screening for α-glucosidase inhibitors provides a simple and effective method for the investigation of complex samples, such as plant extracts. Copyright © 2016 John Wiley & Sons, Ltd.

Rare phenolic structures found in the aerial parts of Eriosema laurentii De Wild.

Recent pharmacological and toxicological investigations of Eriosema laurentii (Leguminosae) have underlined the plausibility of the use of this plant in traditional African medicine. A very complex pattern of phenolic compounds was detected in the tested extracts. Based on these preceding results a detailed phytochemical study was performed and resulted in the isolation and identification of eleven compounds. All are reported in this plant for the first time and four of those are previously undescribed secondary metabolites: 3,4',6,8-tetrahydroxyflavanone-7-C-glucoside and 3,4',6,8-tetrahydroxyflavone-7-C-glucoside with an extremely rare substitution pattern as well as 1-[2,4-dihydroxy-3-(2-hydroxy-3-methylbut-3-en-1-yl)phenyl]-3-phenylpropan-1-one and 1-[2,4-dihydroxy-3-(2-hydroxy-3-methylbut-3-en-1-yl)phenyl]-3-(4-methoxyphenyl)-propan-1-one. Their structures were elucidated unambiguously by extensive MS- and NMR-experiments.

Estrogenic properties of spices of the traditional Cameroonian dish "Nkui" in ovariectomized Wistar rats.

BACKGROUND: Besides the basic role to flavor and color foods, several health benefits have been attributed to spices. The traditional Cameroonian food "Nkui" is prepared using several spices (Afrostyrax lepidophyllus Mildbr., Capsicum frutescens Linn., Fagara leprieurii Guill. et Perr., Fagara tessmannii Engl., Mondia whitei Hook. F. Skell., Pentadiplandra brazzeana Baill., Solanum gilo Raddi., Tetrapleura tetraptera Taub. and Xylopia parviflora A. Rich. Benthane) that are believed to have a positive impact on the female reproductive physiology. Aiming to determine the potential effect of this food on the female reproductive tract, we evaluated the estrogenic properties of aqueous and ethanol extracts of Nkui using a 3-day uterotrophic assay in ovariectomized (OVX) rats. METHODS: OVX female Wistar rats were randomly separated in several groups of five animals each and submitted to a 3-day uterotrophic assay (per os). At the end of treatment, animals were sacrificed and uterus, vagina and mammary gland collected and fixed in 10 % formalin for histological analysis. RESULTS: These extracts increased the uterine wet weight, the uterine and vaginal epithelial heights, and the lumen and diameter of alveoli in the mammary glands. They also altered the estradiol-induced increase of uterine wet weight. The dichloromethane and methanol fractions of the ethanol extract exhibited estrogenic properties as well by increasing uterine and vaginal endpoints. CONCLUSIONS: These results suggest that the spices of "Nkui" contain estrogenic phytoconstituents and this traditional food may be considered as functional.

Quantitation of phenylpropanoids and iridoids in insulin-sensitising extracts of Leonurus sibiricus L. (Lamiaceae).

INTRODUCTION: Leonurus sibiricus L. is regularly used in traditional Mongolian medicine including for the treatment of symptoms of diabetes mellitus. OBJECTIVES: To provide a validated quantitation method for the quality control of Leonurus sibiricus and to prove in vitro insulin-sensitisation, thereby supporting the traditional use of Leonurus sibiricus. METHODOLOGY: Pulverised Leonurus sibiricus material was either extracted with methanol or methanol:water (25:75, v/v). HPLC-CAD (charged aerosol detector) separations were performed on a Luna Phenyl-Hexyl column with water and acetonitrile (both modified with 0.1% formic acid) as mobile phase. Gradient elution was employed using theophylline as internal standard. Tentative peak identification was facilitated by HPLC-MS. Validation was carried out according to ICH (International Conference on Harmonisation) guidelines. Potential insulin-sensitisation of accordant extracts was assessed in glucose uptake experiments in C2C12 myocytes and protein tyrosine phosphatase 1B (PTP1B) enzyme assays. RESULTS: Thirty-six compounds were tentatively identified based on their retention times, UV spectra, MS fragments and data from literature. They comprise phenolcarboxylic acids, flavonoids, iridoid glycosides, and phenylpropanoids, among which acetylharpagide, ajugoside, lavandulifolioside, and verbascoside were selected for quantitation. The methanol extract contained 0.42% combined iridoids, and 1.58% combined phenylpropanoids. Validation showed good accuracy, intermediate precision and robustness. The methanol extract of Leonurus sibiricus led to a 1.5 fold increase in insulin-stimulated cellular glucose uptake and inhibition of PTP1B by 40% at a concentration of 10 µg/mL. CONCLUSION: HPLC-CAD analysis allowed sensitive quantitation of the selected marker compounds in Leonurus sibiricus, thereby providing a reliable tool for its quality control.

Triterpenoic Acids from Apple Pomace Enhance the Activity of the Endothelial Nitric Oxide Synthase (eNOS).

Pomace is an easy-accessible raw material for the isolation of fruit-derived compounds. Fruit consumption is associated with health-promoting effects, such as the prevention of cardiovascular disease. Increased vascular nitric oxide (NO) bioavailability, for example, due to an enhanced endothelial nitric oxide synthase (eNOS) activity, could be one molecular mechanism mediating this effect. To identify compounds from apple (Malus domestica Borkh.) pomace that have the potential to amplify NO bioavailability via eNOS activation, a bioassay-guided fractionation of the methanol/water (70:30) extract has been performed using the (14)C-L-arginine to (14)C-L-citrulline conversion assay (ACCA) in the human endothelium-derived cell line EA.hy926. Phytochemical characterization of the active fractions was performed using the spectrophotometric assessment of the total phenolic content, as well as TLC, HPLC-DAD-ELSD, and HPLC-MS analyses. Eleven triterpenoic acids, of which one is a newly discovered compound, were identified as the main constituents in the most active fraction, accompanied by only minor contents of phenolic compounds. When tested individually, none of the tested compounds exhibited significant eNOS activation. Nevertheless, cell stimulation with the reconstituted compound mixture restored eNOS activation, validating the potential of apple pomace as a source of bioactive components.

A eudesmane-type sesquiterpene isolated from Pluchea odorata (L.) Cass. combats three hallmarks of cancer cells: Unrestricted proliferation, escape from apoptosis and early metastatic outgrowth in vitro.

Pluchea odorata is ethno pharmaceutically used to treat inflammation-associated disorders. The dichloromethane extract (DME) was tested in the carrageenan-induced rat paw oedema assay investigating its effect on inflammation that was inhibited by 37%. Also an in vitro anti-neoplastic potential was reported. However, rather limited information about the bio-activity of purified compounds and their cellular mechanisms are available. Therefore, two of the most abundant eudesmanes in P. odorata were isolated and their anti-neoplastic and anti-intravasative activities were studied. HL-60 cells were treated with P. odorata compounds and metabolic activity, cell number reduction, cell cycle progression and apoptosis induction were correlated with relevant protein expression. Tumour cell intravasation through lymph endothelial monolayers was measured and potential causal mechanisms were analyzed by Western blotting. Compound PO-1 decreased the metabolic activity of HL-60 cells (IC50 = 8.9 µM after 72 h) and 10 µM PO-1 induced apoptosis, while PO-2 showed just weak anti-neoplastic activities at concentrations beyond 100 µM. PO-1 arrested the cell cycle in G1 and this correlated with induction of JunB expression. Independent of this mechanism 25 µM PO-1 decreased MCF-7 spheroid intravasation through the lymph endothelial barrier. Hence, PO-1 inhibits an early step of metastasis, impairs unrestricted proliferation and induces apoptosis at low micromolar concentrations. These results warrant further testing in vivo to challenge the potential of PO-1 as novel lead compound.