Ipecac root extracts and isolated circular peptides differentially suppress inflammatory immune response characterised by proliferation, activation and degranulation capacity of human lymphocytes in vitro.

Circular peptides are attractive lead compounds for drug development; this study investigates the immunomodulatory effects of defined root powder extracts and isolated peptides (called cyclotides) from Carapichea ipecacuanha (Brot.) L. Andersson ('ipecac'). Changes in the viability, proliferation and function of activated human primary T cells were analysed using flow cytometry-based assays. Three distinct peptide-enriched extracts of pulverised ipecac root material were prepared via C18 solid-phase extraction and analysed by reversed-phase HPLC and mass spectrometry. These extracts induced caspase 3/7 dependent apoptosis, thus leading to a suppressed proliferation of activated T cells and a reduction of the number of cells in the G2 phase. Furthermore, the stimulated T cells had a lower activation potential and a reduced degranulation capacity after treatment with ipecac extracts. Six different cyclotides were isolated from C. ipecacuanha and an T cell proliferation inhibiting effect was determined. Furthermore, the degranulation capacity of the T cells was diminished specifically by some cyclotides. In contrast to kalata B1 and its analog T20K, secretion of IL-2 and IFN- γ was not affected by any of the caripe cyclotides. The findings add to our increased understanding of the immunomodulating effects of cyclotides, and may provide a basis for the use of ipecac extracts for immunomodulation in conditions associated with an exessive immune responses.

The Genus Eriosema (Fabaceae): From the Ethnopharmacology to an Evidence-Based Phytotherapeutic Perspective?

The genus Eriosema (Fabaceae) includes approximately 150 species widely distributed across tropical and subtropical regions of the world (Africa, Neotropics, Asia and Australia). Throughout these regions, several species are used since centuries in different traditional medicinal systems, while others are used as food or food supplement. The present review attempts to critically summarize current information concerning the uses, phytochemistry and pharmacology of the Eriosema genus and to evaluate the therapeutic potential. The information published in English and French (up to September 2020) on ethnopharmacology or traditional uses, chemistry, pharmacology and toxicology of Eriosema genus was collected from electronic databases [SciFinder, PubMed, Google, Google Scholar, Scopus, Web of Science, Prelude Medicinal Plants-http://www.ethnopharmacologia.org/recherche-dans-prelude/?plant, The Plant List (http://www.theplantlist.org/), POWO (http://powo.science.kew.org/) and IUCN Red List Categories (https://www.iucnredlist.org/)], conference proceedings, books, M.Sc. and Ph.D. dissertations. The information retrieved on the ethnomedicinal indications of Eriosema genus allowed to list 25 species (∼16.6% of the genus). The majority of uses is recorded from Africa. Phytochemical analyses of 8 species led to the identification and/or isolation of 107 compounds, with flavonoids (69.2%), chromones (7.5%) and benzoic acid derivatives (3.7%) as the main chemical classes. Pharmacological investigations with crude extracts and isolated compounds showed a broad range of activities including aphrodisiac, estrogenic, anti-osteoporosis, hypolipidemic, anti-diabetic, anti-diarrheal, anti-microbial, anti-oxidant, anthelmintic, anti-cancer, and acetylcholinesterase inhibitory activities. Despite the low number of Eriosema species tested, there is convincing evidence in vitro and in vivo studies validating some traditional and ethnobotanical uses. However, the utility of several of the described uses has not yet been confirmed in pharmacological studies. Reviewed data could serve as a reference tool and preliminary information for advanced research on Eriosema species.

Regular consumption of "Nkui", a Cameroonian traditional dish, may protect against cardiovascular and bone disorders in an estrogen deficiency condition.

OBJECTIVES: There is a growing body of evidence indicating the potential of culinary herbs and spices to decrease the incidence of several chronic diseases or conditions. Because of this, the WHO recommends their regular consumption. In the Cameroonian culinary practices, "Nkui" is a famous dish made from a mixture of 10 spices. In our previous study, the ethanolic extract of this mixture exhibited promising estrogenic properties. Thus, this study aimed to evaluate its protective effects on some menopause-related cardiovascular and bone disorders. METHODS: For this purpose, a post-menopause-like model (ovariectomized rats) has been used. Animals were orally treated with the "Nkui" extract for 60 days. The investigation focused on the oxidative stress status, endothelial function (NO bioavailability), lipid profile, and bone mass, biochemical (calcium and inorganic phosphorus contents, serum alkaline phosphatase activity) and histomorphological features. RESULTS: The extract regulated lipid metabolism in a way to prevent accumulation of abdominal fat, gain in body weight and increased atherogenic indexes induced by ovariectomy. It prevented menopause-related low levels of nitric oxide and oxidative stress damage by increasing superoxide dismutase and catalase activities, while reducing glutathione and malondialdehyde levels in the heart and aorta. Moreover, it prevented ovariectomy-induced bone mass loss, bone marrow disparities and the disorganization of the trabecular network. It also increased femur calcium and inorganic phosphorus contents. CONCLUSIONS: These results suggest that a regular consumption of "Nkui" may have health benefits on cardiovascular system and osteoporosis, major health issues associated with menopause.

Destabilization of FoxM1 and Inhibition of Topoisomerase I Contribute to Cytotoxicity of Prenylated Xanthones Isolated from Metaxya rostrata.

We recently isolated the prenylated xanthones 2-deprenyl-rheediaxanthone B (XB) and 2-deprenyl-7-hydroxy-rheediaxanthone B (OH-XB) from the South American tree fern Metaxya rostrata. This study explores the mechanisms underlying the FoxM1 downregulation induced by both xanthones. Analysis of cell viability and cell-death induction in SW480, HCT116, Caco-2, DLD1 and HT29 exposed to xanthones found cell-loss and activation of caspase in all cell lines except HT29 that do not have high FoxM1 protein levels. To determine the cellular mechanism of xanthone-induced FoxM1 loss, protein stability was analyzed by cycloheximide-chase experiments and showed reduction of FoxM1 stability by XB but not OH-XB. Destabilization was prevented by inhibiting proteasome activity using MG-132 and moderately by the lysosomal inhibitor bafilomycin A1 (baf A1). OH-XB had a stronger impact than XB on FoxM1 mRNA expression by qRT-PCR, and MG-132 positively affected FoxM1 protein level in OH-XB exposed cells even though no decrease in protein abundance had been induced by the xanthone. Additionally, the compound inhibited topoisomerase I causing DNA DSB and early cell cycle arrest. This may reduce FoxM1 gene expression, which may in turn compromise DNA repair and enhance xanthone-induced cell death. With regard to xanthone-induced cell death, MG-132 protected cultures from cell loss induced by both compounds, and baf A1 was active against these XB-induced effects. In summary, both destabilization of FoxM1 protein and topoisomerase I inhibition contribute to both XB and OH-XB cytotoxic activity albeit at different ratios.

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.

Prenylated xanthones from Metaxya rostrata suppress FoxM1 and induce active cell death by distinct mechanisms.

BACKGROUND: Metaxya rostrata C.Presl (Metaxyaceae) is a tree fern widespread in Central and South America and the dried rhizome is used in ethnic medicine against intestinal ulcers or tumors. An activity-guided isolation resulted in two structurally related xanthones: 2-deprenyl-rheediaxanthone B (XB) and 2-deprenyl-7-hydroxy-rheediaxanthone B (OH-XB). HYPOTHESIS/PURPOSE: This study analyzed the cytotoxic activity and underlying cellular mechanisms of OH-XB for the first time in comparison to XB. METHODS: We exposed the colorectal cancer cell line SW480 and F331 fibroblasts to XB and OH-XB and determined cell viability by neutral red uptake and nuclear morphology by staining with Hoechst dye. Cell cycle distribution and the mechanism of cell death were analyzed by FACS and western blot. Knockdown of FoxM1 expression was performed with siRNA. RESULTS: OH-XB was at least as cytotoxic as XB in the induction of cell cycle arrest and active cell death. While both compounds strongly inhibited the transcription factor FoxM1, the cellular mechanisms of growth arrest and cell death induction differed widely: OH-XB induced S-phase cell cycle arrest in contrast to a G2-M-phase arrest by XB. It caused morphological modifications typical for classical apoptosis with increased caspase 7 activity and enhanced cleavage of PARP, while XB caused caspase 2 activation and mitotic catastrophe. After knockdown of FoxM1 expression no induction of caspase activity could be observed. CONCLUSION: In summary, our data clearly showed that XB and OH-XB are promising new lead compounds for cancer therapy with distinct cellular mechanisms. Both compounds are candidates for further pre-clinical and clinical investigations.

Apigenin and Luteolin Attenuate the Breaching of MDA-MB231 Breast Cancer Spheroids Through the Lymph Endothelial Barrier in Vitro.

Flavonoids, present in fruits, vegetables and traditional medicinal plants, show anticancer effects in experimental systems and are reportedly non-toxic. This is a favorable property for long term strategies for the attenuation of lymph node metastasis, which may effectively improve the prognostic states in breast cancer. Hence, we studied two flavonoids, apigenin and luteolin exhibiting strong bio-activity in various test systems in cancer research and are readily available on the market. This study has further advanced the mechanistic understanding of breast cancer intravasation through the lymphatic barrier. Apigenin and luteolin were tested in a three-dimensional (3-D) assay consisting of MDA-MB231 breast cancer spheroids and immortalized lymph endothelial cell (LEC) monolayers. The 3-D model faithfully resembles the intravasation of breast cancer emboli through the lymphatic vasculature. Western blot analysis, intracellular Ca2+ determination, EROD assay and siRNA transfection revealed insights into mechanisms of intravasation as well as the anti-intravasative outcome of flavonoid action. Both flavonoids suppressed pro-intravasative trigger factors in MDA-MB231 breast cancer cells, specifically MMP1 expression and CYP1A1 activity. A pro-intravasative contribution of FAK expression in LECs was established as FAK supported the retraction of the LEC monolayer upon contact with cancer cells thereby enabling them to cross the endothelial barrier. As mechanistic basis, MMP1 caused the phosphorylation (activation) of FAK at Tyr397 in LECs. Apigenin and luteolin prevented MMP1-induced FAK activation, but not constitutive FAK phosphorylation. Luteolin, unlike apigenin, inhibited MMP1-induced Ca2+ release. Free intracellular Ca2+ is a central signal amplifier triggering LEC retraction through activation of the mobility protein MLC2, thereby enhancing intravasation. FAK activity and Ca2+ levels did not correlate. This implicates that the pro-intravasative contribution of FAK and of Ca2+ release in LECs was independent of each other and explains the better anti-intravasative effects of luteolin in vitro. In specific formulations, flavonoid concentrations causing significant anti-intravasative effects, can certainly be achieved in vivo. As the therapeutic strategy has to be based on permanent flavonoid treatment both the beneficial and adverse effects have to be investigated in future studies.

Intravasation of SW620 colon cancer cell spheroids through the blood endothelial barrier is inhibited by clinical drugs and flavonoids in vitro.

Mechanisms how colorectal cancer (CRC) cells penetrate blood micro-vessel endothelia and metastasise is poorly understood. To study blood endothelial cell (BEC) barrier breaching by CRC emboli, an in vitro assay measuring BEC-free areas underneath SW620 cell spheroids, so called "circular chemorepellent induced defects" (CCIDs, appearing in consequence of endothelial retraction), was adapted and supported by Western blotting, EIA-, EROD- and luciferase reporter assays. Inhibition of ALOX12 or NF-κB in SW620 cells or BECs, respectively, caused attenuation of CCIDs. The FDA approved drugs vinpocetine [inhibiting ALOX12-dependent 12(S)-HETE synthesis], ketotifen [inhibiting NF-κB], carbamazepine and fenofibrate [inhibiting 12(S)-HETE and NF-κB] significantly attenuated CCID formation at low µM concentrations. In the 5-FU-resistant SW620-R/BEC model guanfacine, nifedipine and proadifen inhibited CCIDs stronger than in the naïve SW620/BEC model. This indicated that in SW620-R cells formerly silent (yet unidentified) genes became expressed and targetable by these drugs in course of resistance acquisition. Fenofibrate, and the flavonoids hispidulin and apigenin, which are present in medicinal plants, spices, herbs and fruits, attenuated CCID formation in both, naïve- and resistant models. As FDA-approved drugs and food-flavonoids inhibited established and acquired intravasative pathways and attenuated BEC barrier-breaching in vitro, this warrants testing of these compounds in CRC models in vivo.

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.

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.

A critical overview on Thymus daenensis Celak.: phytochemical and pharmacological investigations.

Thymus daenensis Celak. is an herb endemic to Iran belonging to the Lamiaceae family. Growing in many parts of Iran, the plant is extensively used in folk medicine. This review was performed to compile phytochemical and pharmacological data of T. daenensis. Databases such as PubMed, Scopus, Web of Science, ScienceDirect, Scientific Information Database, Embase, IranMedex and Google Scholar were searched for the terms "Thymus daenensis" and "Avishan-e-denaii" up to 1st January 2014. Following reported ethnopharmacological uses, various T. daenensis preparations have been investigated for antimicrobial, antioxidant, insecticidal and immunomodulatory effects in recent studies. Moreover, numerous studies have been published on the composition of the herb's essential oil, focusing either on environmental parameters or preparation methods. Due to its high concentration of thymol, the plant's essential oil possesses high antimicrobial activities on human pathogenic strains. However, comprehensive studies on the toxicity and teratogenicity as well as clinical efficacy of Thymus daenensis are missing.

Preparative isolation of oleocanthal, tyrosol, and hydroxytyrosol from olive oil by HPCCC.

For the provision of oleocanthal (OLC), a phenolic compound with very promising pharmacological properties, isolation from olive oil is a very important option. Due to the compound's sensitivity to decomposition upon exposure to oxygen and light, a very gentle isolation method has been developed under use of high performance countercurrent chromatography (HPCCC). By partition of olive oil between hexane and methanol, an extract enriched in phenolics was prepared and subjected to a two-step HPCCC separation under use of heptane-EtOAc-MeOH-H2O mixtures in normal-phase and reverse phase mode, respectively. With this method, the isolation of tyrosol, hydroxytyrosol, and the mixture of (3S,4E)- and (3S,4Z)-OLC was achieved in approx. 70 min for each step. By one- and two-dimensional NMR-experiments and LC-MS, the equilibrium of (3S,4E)- and (3S,4Z)-OLC in such olive oil extracts has unambiguously been proven for the first time.

Phytochemical and pharmacological aspects of Salvia mirzayanii Rech. f. & Esfand.

Salvia mirzayanii Rech. f. & Esfand is an endemic herbaceous plant belonging to the Lamiaceae family. The plant grows in the center and south of Iran and is broadly used in folk medicine. This review focuses on phytochemical and pharmacological data of S mirzayanii. Databases such as PubMed, Scopus, Web of Science, Scientific Information Database (Iranian database involving English and Persian articles), and Google Scholar were searched for terms "Salvia mirzayanii," "Iranian sage," "Marv-e-Talkh," and "Moor-e-Talkh" in both Persian and English up to December 10, 2013. In line with the ethnopharmacological uses, antioxidant, immunomodulatory, anti-inflammatory, antimicrobial, and insecticidal activities of different preparations of S mirzayanii have been shown in recent studies. The antimicrobial activity against human pathogenic strains has mainly been attributed to the essential oil of S mirzayanii, which in numerous analyses has been investigated either in plants of different origin or prepared by differing extraction methods. Despite the studies on antioxidant or antimicrobial activities, profound research on the toxicity or clinical properties of the herb is missing.

Lupinalbin A as the most potent estrogen receptor α- and aryl hydrocarbon receptor agonist in Eriosema laurentii de Wild. (Leguminosae).

BACKGROUND: Eriosema laurentii De Wild. (Leguminosae) is a plant used in Cameroon against infertility and gynecological or menopausal complaints. In our previous report, a methanol extract of its aerial parts was shown to exhibit estrogenic and aryl hydrocarbon receptor agonistic activities in vitro and to prevent menopausal symptoms in ovariectomized Wistar rats. METHODS: In order to determine the major estrogen receptor α (ERα) agonists in the extract, an activity-guided fractionation was performed using the ERα yeast screen. To check whether the ERα active fractions/compounds also accounted for the aryl hydrocarbon receptor (AhR) agonistic activity of the crude methanol extract, they were further tested on the AhR yeast screen. RESULTS: This study led to the identification of 2'-hydroxygenistein, lupinalbin A and genistein as major estrogenic principles of the extract. 2'-hydroxygenistein and lupinalbin A were, for the first time, also shown to possess an AhR agonistic activity, whereas genistein was not active in this assay. In addition, it was possible to deduce structure-activity relationships. CONCLUSIONS: These results suggest that the identified compounds are the major active principles responsible for the estrogenic and AhR agonistic activities of the crude methanol extract of the aerial parts of Eriosema laurentii.

Safety profile and gender specific differences of a methanol extract of Eriosema laurentii (Leguminosae) in acute and subchronic (28 days) oral toxicity studies in Wistar rats.

Despite widespread use of Eriosema laurentii De Wild (Leguminosae) in West and Central Africa as herbal medicine and food additive the toxicity of this plant is unknown. Therefore, we performed the safety evaluation of a methanol extract (AEL). In acute toxicity, single oral administration of 2000mg/kg AEL caused neither toxicological symptoms nor mortality and the LD50 was estimated >5000mg/kg. In the subchronic oral toxicity, AEL induced no phenotypical signs of toxicity during and after treatment. Only a delayed decrease of relative spleen weight in males at the highest dose of 400mg/kg occurred. High density lipoprotein (HDL) increased significantly in females at 200 and 400mg/kg. Non-persistent increases in alanine aminotransferase activity within normal ranges were noted at 200mg/kg in males and at all doses in females. In males, AEL induced a decrease of white blood cell count at 400mg/kg, whereas lymphocytes increased at 200 and 400mg/kg and granulocytes at 400mg/kg. In females, no differences in haematological parameters occurred. Neither differences in bilirubin, creatinine and total protein levels were observed nor histological alterations in organs. The results indicate a broad safety margin for AEL.