Isolation, NMR Characterization, and Bioactivity of a Flavonoid Triglycoside from Anthyllis henoniana Stems: Antioxidant and Antiproliferative Effects on MDA-MB-231 Breast Cancer Cells.

Plant extracts are considered as a large source of active biomolecules, especially in phytosanitary and pharmacological fields. Anthyllis henoniana is a woody Saharan plant located in the big desert of North Africa. Our previous research paper proved the richness of the methanol extract obtained from the stems in flavonoids and phenolic compounds as well as its remarkable antioxidant activity. In this research, we started by investigating the phytochemical composition of the methanol extract using high performance liquid chromatography coupled with electrospray ionization mass spectrometry (LC-MS/MS). Among the 41 compounds identified, we isolated and characterized (structurally and functionally) the most abundant product, a flavonoid triglycoside (AA770) not previously described in this species. This compound, which presents no cytotoxic activity, exhibits an interesting cellular antioxidant effect by reducing reactive oxygen species (ROS) generation, and an antiproliferative action on breast cancer cells. This study provides a preliminary investigation into the pharmacological potential of the natural compound AA770, isolated and identified from Anthyllis henoniana for the first time.

Antioxidant and Antimicrobial Activities of Erodium arborescens Aerial Part Extracts and Characterization by LC-HESI-MS2 of Its Acetone Extract.

The phytochemical analysis of antioxidant and antibacterial activities of Erodium arborescens aerial part extracts constitute the focus of this research. The chemical composition of an acetone extract was investigated using LC-HESI-MS2, which revealed the presence of 70 compounds. The major identified components were tannin derivatives. Total polyphenol and total flavonoid contents were assessed in plant extracts (hexane, ethyl acetate, acetone and methanol). The results showed that the acetone extract exhibited the highest contents of polyphenols and flavonoids, 895.54 and 36.39 mg QE/g DE, respectively. Furthermore, when compared to other extracts, Erodium arborescens acetone extract was endowed with the highest antioxidant activity with 2,2-diphenyl-1-picrylhydrazyl (DPPH), ferric reducing antioxidant power (FRAP) and total antioxidant capacity (TAC) tests. In addition, the four extracts of Erodium arborescens showed variable degrees of antimicrobial activity against the tested strains, and the interesting activity was obtained with acetone and methanol extracts.

Antifungal, molluscicidal and larvicidal assessment of anemonin and Clematis flammula L. extracts against mollusc Galba truncatula, intermediate host of Fasciola hepatica in Tunisia.

OBJECTIVE: To investigate the potential of anemonin and Clematis flammula (C. flammula) extracts against infective organisms. METHODS: The molluscicidal activities of anemonin and C. flammula extracts against Galba truncatula Müll. (Lymnaeidae) and Fasciola hepatica larval stages contaminating this snail in Tunisia were assessed by testing six groups of snails in 250 mL of extracts and aqueous dechlorinated solutions with different concentrations (ranging from 2.5 to 20.0 mg/L) for 48 h. Besides, the antifungal potential of C. flammula leaves and flowers was evaluated by using the diffusion agar and broth dilution methods against four fungal strains: Aspergillus niger, Pythium catenulatum, Rhizoctonia solani and Fusarium phyllophilum. RESULTS: As a result, hexane and ethyl acetate flower extracts exhibited significant molluscicidal activities with LC50 median lethal concentrations values of 11.87 and 11.65 mg/L, respectively while LC50 value of anemonin was 9.64 mg/L after 48 h exposure. The flower extracts showed a larvicidal effect with a deterioration rate exceeding 35.39% where flower ethyl acetate residue gave a deterioration rate of cercariae close to 97%. Moreover, C. flammula extracts were not noxious to the associated fauna survival. All extracts inhibited the growth of P. catenulatum, the leaves and flowers methanolic extracts had the more important fungicide action with minimum inhibitory concentrations of 1.56 and 3.12 mg/mL together with minimum fungistatic concentrations of 3.12 and 6.25 mg/mL respectively. Only flower extracts were active against Rhizoctonia solani with minimum inhibitory concentrations varying between 0.70 and 1.56 mg/mL and 6.25 mg/mL of minimum fungistatic concentration. Phytochemical tests showed that the antifungal activity may be attributed to the presence of the flavonoids/saponins in the methanolic extracts and the molluscicide effects could be due to the richness of hexane and ethyl acetate extracts on sterols and triterpenoids. CONCLUSIONS: This study emphasizes the important molluscicidal and antiparasitic effects of flower ethyl acetate extracts and anemonin compound as well as the considerable antifungal activities of methanolic extracts. These results improve the therapeutic virtues of C. flammula aerial part extracts.

Anti-inflammatory and anticancer effects of flavonol glycosides from Diplotaxis harra through GSK3ß regulation in intestinal cells.

CONTEXT AND OBJECTIVE: Diplotaxis harra (Forssk.) Boiss. (Brassicaceae) is traditionally used as an antidiabetic, anti-inflammatory or anticancer agent. In these pathologies, the glycogen synthase kinase 3 ß (GSK3ß) is overactivated and represents an interesting therapeutic target. Several flavonoids can inhibit GSK3ß and the purpose of this study was to search for the compounds in Diplotaxis harra which are able to modulate GSK3ß. MATERIALS AND METHODS: Methanol extracts from D. harra flowers were prepared and the bio-guided fractionation of their active compounds was performed using inflammatory [protease-activated receptor 2 (PAR2)-stimulated IEC6 cells] and cancer (human Caco-2 cell line) intestinal cells. 50-100 µg/mL of fractions or compounds purified by HPLC were incubated with cells whose inhibited form of GSK3ß (Pser9 GSK3ß) and survival were analyzed by Western blot at 1 h and colorimetric assay at 24 h, respectively. LC-UV-MS profiles and MS-MS spectra were used for the characterization of extracts and flavonoids-enriched fractions, and the identification of pure flavonoids was achieved by MS and NMR analysis. RESULTS: The methanol extract from D. harra flowers and its flavonoid-enriched fraction inhibit GSK3ß in PAR2-stimulated IEC6 cells. GSK3ß inhibition by the flavonoid-enriched D. harra fraction was dependent on PKC activation. The flavonoid-enriched D. harra fraction and its purified compound isorhamnetin-3,7-di-O-glucoside induced a 20% decrease of PAR2-stimulated IEC6 and Caco-2 cell survival. Importantly, normal cells (non-stimulated IEC6 cells) were spared by these treatments. CONCLUSION: This work indicates that flavonoids from D. harra display cytotoxic activity against inflammatory and cancer intestinal cells which could depend on GSK3ß inhibition.

PAR2-dependent activation of GSK3ß regulates the survival of colon stem/progenitor cells.

Protease-activated receptors PAR1 and PAR2 play an important role in the control of epithelial cell proliferation and migration. However, the survival of normal and tumor intestinal stem/progenitor cells promoted by proinflammatory mediators may be critical in oncogenesis. The glycogen synthase kinase-3ß (GSK3ß) pathway is overactivated in colon cancer cells and promotes their survival and drug resistance. We thus aimed to determine PAR1 and PAR2 effects on normal and tumor intestinal stem/progenitor cells and whether they involved GSK3ß. First, PAR1 and PAR2 were identified in colon stem/progenitor cells by immunofluorescence. In three-dimensional cultures of murine crypt units or single tumor Caco-2 cells, PAR2 activation decreased numbers and size of normal or cancerous spheroids, and PAR2-deficient spheroids showed increased proliferation, indicating that PAR2 represses proliferation. PAR2-stimulated normal cells were more resistant to stress (serum starvation or spheroid passaging), suggesting prosurvival effects of PAR2 Accordingly, active caspase-3 was strongly increased in PAR2-deficient normal spheroids. PAR2 but not PAR1 triggered GSK3ß activation through serine-9 dephosphorylation in normal and tumor cells. The PAR2-triggered GSK3ß activation implicates an arrestin/PP2A/GSK3ß complex that is dependent on the Rho kinase activity. Loss of PAR2 was associated with high levels of GSK3ß nonactive form, strengthening the role of PAR2 in GSK3ß activation. GSK3 pharmacological inhibition impaired the survival of PAR2-stimulated spheroids and serum-starved cells. Altogether our data identify PAR2/GSK3ß as a novel pathway that plays a critical role in the regulation of stem/progenitor cell survival and proliferation in normal colon crypts and colon cancer.

MOLLUSCICIDAL AND LARVICIDAL ACTIVITIES OF Atriplex inflata AERIAL PARTS AGAINST THE MOLLUSK Galba truncatula, INTERMEDIATE HOST OF Fasciola hepatica / Atividade moluscicida e larvicida das partes aéreas de Atriplex inflata contra o molusco Galba truncatula, hospedeiro intermediário da Fasciola hepatica

Fasciolosis is a widespread parasitosis of farm live-stock in many developing countries. For this reason, it is necessary to search for new substances against parasitic diseases caused by flukes. Indeed, a wide variety of terrestrial plants have been subjected to chemical and pharmacological screening in order to discover their potential for human medicinal use. The molluscicidal and larvicidal activities of Atriplex inflata were tested on Galba truncatula and Fasciola hepatica larval stages infecting this snail in Tunisia. Phytochemical tests were conducted on extracts in order to establish a meaningful relationship with molluscicidal and larvicidal activities. The molluscicidal activity was evaluated by subjecting snails to sample aqueous solutions. Accordingly, hexane, ethyl acetate, methanol and methanol-water (8:2, v-v) were used as extraction solvents. As a result, hexane and ethyl acetate extracts showed potent activity, according to the World Health Organization, giving LC50 = 7.59 mg/L and 6.69 mg/L for hexane extracts of leaves and fruits, respectively. Ethyl acetate extracts gave LC50 = 5.90 mg/L and 7.32 mg/L for leaves and fruits, successively. Molluscicidal activities of powders were less potent on snails, but active according to the World Health Organization. Hexane and ethyl acetate extracts from leaves and fruits gave potent larvicidal activities with a delay rate exceeding 45.50% (45.50- 98.92%). Phytochemical tests showed that these activities may be attributed to the presence of triterpenoids and/or sterols.

Fasciolose é uma parasitose generalizada que ocorre em animais de fazendas em muitos países em desenvolvimento. Por esta razão, é necessária a busca de novas substâncias contra as doenças parasitárias causadas por vermes. De fato, uma grande variedade de plantas terrestres foi objeto de testes farmacológicos e químicos a fim de descobrir o seu potencial para utilização em terapêutica humana. As atividades moluscicida e larvicida de Atriplex inflata foram testadas contra estágios larvários de Galba truncatula e Fasciola hepatica infectando este caracol na Tunísia. Testes fitoquímicos foram realizados com extratos a fim de estabelecer uma relação significativa com as atividades moluscicida e larvicida. A atividade moluscicida foi avaliada submetendo os caracóis a soluções aquosas. Conforme o caso, hexano, acetato de etilo, metanol e metanol-água (8:2, v-v) foram utilizados como solventes de extração. Como resultado, hexano e extratos de acetato apresentaram atividades potentes de acordo com a Organização Mundial de Saúde, resultando em LC50 = 7,59 mg/L e 6,69 mg/L para extratos de hexano de folhas e frutos, respectivamente. Extratos de acetato de etilo resultaram em LC50 = 5,90 mg/L e 7,32 mg/L para as folhas e frutos sucessivamente. Atividades moluscicidas das substâncias sob a forma de pó foram menos potentes em caracóis, mas ativas de acordo com a Organização Mundial de Saúde. Hexano e extratos de acetato de folhas e frutos apresentaram atividade larvicida potente, com uma taxa de atraso superior a 45,50% (45,50-98,92%). Testes fitoquímicos mostraram que estas atividades podem ser atribuídas à presença de triterpenóides e/ou esteróis.

Protective effects of Hammada scoparia flavonoid-enriched fraction on liver injury induced by warm ischemia/reperfusion.

CONTEXT: Hepatic ischemia/reperfusion injury (IRI) is a major cause of liver damage during liver surgery and transplantation. Plants have historically been used in treating liver damage, and Hammada scoparia (Pomel) (Chenopodiaceae) has been reported to possess a broad spectrum of pharmacological and therapeutic activities. OBJECTIVE: In this study, a flavonoid-enriched fraction was used before the warm ischemia (WI) process as pharmacological preconditioning and in combination with technical postconditioning to evaluate their protective effects. MATERIALS AND METHODS: The rats were divided into five groups: a sham group; a control group (Control-IR) that was submitted to 60 min WI; a Pharmacological Preconditioning group (PreC-IR) that received flavonoid-enriched fraction (200 mg/kg body weight); a Postconditioning group (PostC) and a PreC + PostC group. RESULTS: The use of the flavonoid-enriched fraction was noted to significantly (p < 0.05) reduce liver injury, as evidenced by the decrease in liver transaminase activities (AST and ALT) and lactic dehydrogenase (LDH), alkaline phosphatase (ALP), and lipid peroxidation (TBARS), levels as well as the enhancement of antioxidant enzymes (catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx)) responses. The results also indicated that, compared with the separate application of pharmacological preconditioning and postconditioning, the combination of both treatments was more effective in reducing tissue oxidative stress levels through modulating SOD, GSH-PX, and CAT activities. Furthermore, the combined protocol further decreased the liver morphological score compared with solo treatment. DISCUSSION AND CONCLUSION: Overall, the results indicate that the H. scoparia flavonoid-enriched fraction could be a promising candidate for future application as a pharmacological preconditioning agent against hepatic IRI.