Malignant melanoma is a highly aggressive tumor which frequently resists chemotherapy, therefore, the search for new agents for its treatment is of great importance. In this study, we purified the sesquiterpene lactones (SLs), Tomentosin and Inuviscolide from Inula viscosa (Compositae) leaves and studied their anti-cancer potency against human melanoma cell lines in order to develop new agents for melanoma treatment. SLs inhibited the proliferation of three human melanoma cell lines: SK-28, 624 mel and 1363 mel in a dose-dependent manner. We further investigated SLs mechanism of action using SK-28 as a representative cell line model. SLs caused cell-cycle arrest at G(2)/M, accompanied by the appearance of a sub-G0 fraction, indicative of apoptotic cell death. Induction of apoptosis was further confirmed by changes in membrane phospholipids, changes in mitochondrial membrane potential (DeltaPsi) and by detection of Caspase-3 activity. Rapid inhibitory phosphorylation of Cdc2 (Thr14 and Tyr15) was seen early after treatment, followed by a later decrease in the expression level of both Cyclin b1 and Cdc2. Induction of p53 and p21(waf1) proteins and phosphorylation of p53 at Ser15 were also detected early after treatment. The anti-apoptotic proteins, p65 subunit of nuclear factor kB (NF-kB), and Survivin were reduced in a dose-dependent manner. Taken together, these changes partially explain the ability of the SLs to induce G(2)/M arrest and apoptosis. Induction of apoptosis by Tomentosin and Inuviscolide in human aggressive melanoma cell lines has high pharmacological value and implies that SLs might be developed as new agents for melanoma treatment.
Inhibition of Inflammatory Cytokine Secretion by Plant-Derived Compounds Inuviscolide and Tomentosin: The Role of NFkB and STAT1. Galya Abrham, Sara Dovrat, Hanna Bessler, Shlomo Grossman, Uri Nir and Margalit Bergman.
The plant Inula viscosa has been shown to possess many important medicinal benefits, including anti-inflammatory, anti-oxidant, anti-bacterial, and anti-fungal activities, but the plant metabolites that mediate these effects and their mechanism of action are poorly understood. In a previous study, we demonstrated a reduced expression of the p65 subunit of nuclear factor kappa B (NFkB) in melanoma cells treated with the purified sesquiterpene lactone compounds, Inuviscolide (Inv) and Tomentosin (Tom), extracted from Inula viscosa leaves. In this study, we tested the in-vitro effect of these purified compounds on the secretion of pro-inflammatory cytokines from human peripheral blood mononuclear cells (PBMCs) upon stimulation with lipopolysaccharide (LPS) or phorbol myristate acetate (PMA). Their possible mechanism of action was also studied. The results showed that both agents caused decreased production of IL-2, IL-1, IFN, and slightly increased secretion of TNF, whereas secretion of IL-6 was not affected. The elevated levels of TNF did not appear to affect the viability of human PBMCs. Western blot analysis revealed a reduction in the protein level of both the transcription factor component p65/RelA of nuclear factor-kB (NFkB) and the signal transducer and activator of transcription 1 (STAT1) through proteosomal degradation. However, no change was observed in the expression level of the nuclear factor-kB component, p50 (NFkB), or the signal transducer and activator of transcription 3 (STAT3). Taken together, our results indicate the possible future use of these agents as an anti-inflammatory treatment in cases where overstimulation of cytokine secretion is the basis for the pathological symptoms.
A Mechanistic Approach to the In Vivo Anti-Inflammatory Activity of Sesquiterpenoid Compounds Isolated from Inula viscosa. Victoriano Hernández, María del Carmen Recio, Salvador Máñez, José María Prieto, Rosa María Giner, José Luis Ríos.
The present study was designed to examine the anti-inflammatory activity of the sesquiterpenoids ilicic acid and inuviscolide, isolated from Inula viscosa, on cell degranulation, leukotriene biosynthesis, neurogenic drive and glucocorticoid-like interactions. Swiss female mice were used to measure the ear oedema induced by phorbol esters or ethyl phenylpropiolate (EPP), and the paw oedema induced by phospholipase A2 (PLA2) or serotonin. Drug treatment consisted of one topically-applied dose in the ear models and a subcutaneous or intraperitoneal injection in the paw models. Quantitative analysis of leukotriene B4 (LTB4) formation was performed on rat peritoneal neutrophils by high performance liquid chromatography (HPLC). The lactone inuviscolide reduced the PLA2-induced oedema (ID50: 98 μmol/kg). The effect on serotonin-induced oedema was not changed by modifiers of the glucocorticoid response. Ilicic acid showed minor in vivo effects, but was slightly more potent than inuviscolide on the 12-O-tetradecanoylphorbol 13-acetate (TPA) acute oedema test (ID50: 0.650 μmol per ear). Inuviscolide reduced LTB4 generation in intact cells, with an IC50 value of 94 μM. On the basis of the reported results, inuviscolide is the main anti-inflammatory sesquiterpenoid from Inula viscosa, and may act by interfering with leukotriene synthesis and PLA2-induced mastocyte release of inflammatory mediators.
Inhibition of pro-inflammatory enzymes by inuviscolide, a sesquiterpene lactone from Inula viscosa.
Máñez S, Hernández V, Giner RM, Ríos JL, Recio Mdel C.
This work concerns the pharmacological activity of inuviscolide, a sesquiterpenoid from Inula viscosa. It exerts inhibitory effects on elastase, cyclooxygenase 1 and secretory phospholipase A(2). Furthermore, it reduces the skin leukocyte infiltration in a murine model of dermatitis induced by repeated application of 12-O-tetradecanoylphorbol 13-acetate.
Antiproliferative, Antimicrobial and Apoptosis Inducing Effects of Compounds Isolated from Inula Viscosa.
The antiproliferative and antimicrobial effects of thirteen compounds isolated from Inula viscosa (L.) were tested in this study. The antiproliferative activity was tested against three cell lines using the MTT assay. The microdilution method was used to study the antimicrobial activity against two Gram positive bacteria, two Gram negative bacteria and one fungus. The apoptotic activity was determined using a TUNEL colorimetric assay. Scanning electron microscopy was used to study the morphological changes in treated cancer cells and bacteria. Antiproliferative activity was observed in four flavonoids (nepetin, 3,3′-di-O-methylquercetin, hispidulin, and 3-O-methylquercetin). 3,3′-di-O-Methylquercetin and 3-O-methylquercetin showed selective antiproliferative activity against MCF-7 cells, with IC50 values of 10.11 and 11.23 µg/mL, respectively. Both compounds exert their antiproliferative effect by inducing apoptosis as indicted by the presence of DNA fragmentation, nuclear condensation, and formation of apoptotic bodies in treated cancer cells. The antimicrobial effect of Inula viscosa were also noticed in 3,3′-di-O-methylquercetin and 3-O-methyquercetin that inhibited Bacillus cereus at MIC of 62.5 and 125 µg/mL, respectively. Salmonella typhimurium was inhibited by both compounds at MIC of 125 µg/mL. 3,3′-di-O-Methylquercetin induced damage in bacterial cell walls and cytoplasmic membranes. Methylated quercetins isolated from Inula viscosa have improved anticancer and antimicrobial properties compared with other flavonoids and are promising as potential anticancer and antimicrobial agents.
Wamidh H. Talib, Musa H. Abu Zarga and Adel M. Mahasneh.
Plants used for the treatment of diabetes in Israel.
Zohara Yaniv, Amots Dafni, Jacob Friedman, Dan Palevitch.
The genus Inula and their metabolites: from ethnopharmacological to medicinal uses. Seca AM, Grigore A, Pinto DC, Silva AM.
MATERIALS AND METHODS:
High-Level Antimicrobial Efficacy of Representative Mediterranean Natural Plant Extracts against Oral Microorganisms.
Nature is an unexplored reservoir of novel phytopharmaceuticals. Since biofilm-related oral diseases often correlate with antibiotic resistance, plant-derived antimicrobial agents could enhance existing treatment options. Therefore, the rationale of the present report was to examine the antimicrobial impact of Mediterranean natural extracts on oral microorganisms. Five different extracts from Olea europaea, mastic gum, and Inula viscosa were tested against ten bacteria and one Candida albicans strain. The extraction protocols were conducted according to established experimental procedures. Two antimicrobial assays—the minimum inhibitory concentration (MIC) assay and the minimum bactericidal concentration (MBC) assay—were applied. The screened extracts were found to be active against each of the tested microorganisms. O. europaea presented MIC and MBC ranges of 0.07–10.00 mg mL−1 and 0.60–10.00 mg mL−1, respectively. The mean MBC values for mastic gum and I. viscosa were 0.07–10.00 mg mL−1 and 0.15–10.00 mg mL−1, respectively. Extracts were less effective against C. albicans and exerted bactericidal effects at a concentration range of 0.07–5.00 mg mL−1 on strict anaerobic bacteria (Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum, and Parvimonas micra). Ethyl acetate I. viscosa extract and total mastic extract showed considerable antimicrobial activity against oral microorganisms and could therefore be considered as alternative natural anti-infectious agents.
Lamprini Karygianni, Manuel Cecere, Alexios Leandros Skaltsounis, Aikaterini Argyropoulou, Elmar Hellwig, Nektarios Aligiannis, Annette Wittmer and Ali Al-Ahmad.
Phytochemical investigations and antibacterial activity of selected medicinal plants from Jordan. Sawsan Abuhamdah, Rushdie Abuhamdah, Suleiman Al-Olimat and Paul Chazot.Aims: To determine the antibacterial effect of crude methanolic extracts of six selected medicinal plants grown in Jordan (Paronychia argentea Lam., Inula viscosa L., Arbutus andrachne L., Asphodelus microcarpus Salzm et Vivi, Peganum harmala L. and Aloysia citriodora Palau) against Bacillus subtilis, Staphylococcus aureus and Escherichia coli. Study Design: In vitro assessment antibacterial study. Place and Duration of Study: Department of Biopharmaceutics and Clinical Pharmacy, Faculty of Pharmacy, University of Jordan, Amman, Jordan. Between (December 2012 and January 2013). Methodology: In-vitro Laboratory experimental tests; preparation of plant extracts, phytochemical screening; susceptibility tests (zones of inhibition) and minimum inhibitory concentration (MIC) determination. Results: While the crude methanolic extract of P. argentea, A. andrachne, A. microcarpus had no antibacterial activity, crude extract of P. harmala showed good antibacterial activities against all the tested bacterial strains. MIC values for the seed and root extract of against S. aureus were 0.375 mg/ml and 1.5 mg/ml respectively while MIC values for seed and root extracts against B. subtilis were 0.375 and 6.25 mg/ml, respectively and also showed week activity against Gram negative bacteria. The crude methanolic extract of I. viscosa and A. citriodora was also active against bacterial strains S. aureus and B. subtilis and inactive against E. coli. MIC value for I. viscosa extract against S.aureus were 6.25 mg/ml and against B. subtilis 0.375 mg/ml. Meanwhile, MIC value for A. citriodora against S. aureus were 12.5 mg/ml and against B. subtilis 1.5 mg/ml. Conclusion: Results indicate the potential antibacterial activity of I. viscosa and A. citriodora towards Gram positive bacteria such as B. subtilis and S. aureus. The extracts phytochemical screening revealed the presence of terpenoids, flavonoids and phenolics. These preliminary results would be a guide in the selection of potential candidates for further pharmacological study and in search of new drug candidate for treatment of infections caused by Gram positive bacteria.
A Glycosyl Analogue of Diacylglycerol and Other Antiinflammatory Constituents from Inula viscosa. Salvador Máñez, María del Carmen Recio, Inés Gil, Cristina Gómez, Rosa-María Giner, Peter G. Waterman, and José-Luis Ríos.
Some extracts from Inula viscosa were examined for acute antiinflammatory activity in vivo. Three flavonoids: rhamnocitrin (1), 7-O-methylaromadendrin (3), and 3-O-acetylpadmatin (4); a sesquiterpene lactone, inuviscolide (2); a sesquiterpene acid, ilicic acid (5); and a digalactosyl-diacylglycerol, inugalactolipid A (6), were isolated from the CH2Cl2 extract, identified by spectroscopic methods, and characterized as the topical antiinflammatory principles of this species. All these compounds proved to be effective against 12-O-tetradecanoylphorbol-13-acetate-induced ear edema in mice, although lacking activity against arachidonic acid-induced edema. In addition, compounds 5 and, markedly, 6 showed notable effects on a multiple-dose murine chronic dermatitis model. This is the first attempt to establish a rationale concerning the documented use of the plant on various skin diseases.
Modulation of Cox-1, 5-, 12- and 15-Lox by popular herbal remedies used in southern Italy against psoriasis and other skin diseases. Ammar Bader, Francesca Martini, Guillermo R. Schinella, Jose L. Rios and Jose M. Prieto.
Acanthus mollis (Acanthaceae), Achillea ligustica, Artemisia arborescens and Inula viscosa (Asteraceae) are used in Southern Italy against psoriasis and other skin diseases that occur with an imbalanced production of eicosanoids. We here assessed their in vitro effects upon 5-, 12-, 15-LOX and COX-1 enzymes as well as NFκB activation in intact cells as their possible therapeutic targets. All methanol crude extracts inhibited both 5-LOX and COX-1 activities under 200 µg/mL, without significant effects on the 12-LOX pathway or any relevant in vitro free radical scavenging activity. NFκB activation was prevented by all extracts but A. mollis. Interestingly, A. ligustica, A. arborescens and A. mollis increased the biosynthesis of 15(S)-HETE, an anti-inflammatory eicosanoid. A. ligustica (IC50 =49.5 µg/mL) was superior to Silybum marianum (IC50 =147.8 µg/mL), which we used as antipsoriatic herbal medicine of reference. Its n-hexane, dichloromethane and ethyl acetate fractions had also inhibitory effects on the LTB4 biosynthesis (IC50 s=9.6, 20.3 and 68 µg/mL, respectively) evidencing that the apolar extracts of A. ligustica are promising active herbal ingredients for future phytotherapeutical products targeting psoriasis.
Evaluation of the wound healing effect of some Jordanian traditional medicinal plants formulated in Pluronic F127 using mice (Mus musculus). Enam A. Khalil, Fatma U. Afifia, Maysa Al-Hussaini.The wound healing effect of the aqueous extracts of Inula viscosa, Ajuga chia, Rubia taenifolia and Parieteria diffusa, and the oil of Laurus nobilis, dispersed in water, were examined. The 10% (w/w) Pluronic F127 (PF127) was added to the applied preparations, in order to modify the aqueous extracts viscosity, and to stabilize the oil dispersion. A full thickness wound was made in the dorsal area of the mice. The wounds were treated with the different preparations with 12 h intervals for four times in two successive days. For 16 days, the wounds were visually observed, photographically documented and the wound area was measured. After day 16, the animals were sacrificed and the histology of the wound area was examined. The best wound healing activity was observed with the extract of Inula viscosa, followed by Parieteria diffusa, Laurus nobilis, Ajuga chia and the least active extract was that of Rubia taenifolia.
Antiproliferative Activity of Plant Extracts Used Against Cancer in Traditional Medicine. Talib WH, Mahasneh AM.
Forty four extracts from sixteen plants used traditionally as anticancer agents were evaluated in vitro for their antiproliferative activity against Hep-2, MCF-7, and Vero cell lines. Plants were fractionated using ethanol, methanol, chloroform, n-hexane, distilled water, and butanol. The antiproliferative activity was measured by MTT assay. TLC was used to identify active fractions. The apoptotic activity of active fractions was determined using TUNEL colorimetric assay. 20 of these extracts demonstrated significant antiproliferative activity against one or more of the cell lines. These extracts were prepared from Ononis hirta, Inula viscosa, Salvia pinardi, Verbascum sinaiticum and Ononis sicula. Methanol fractions of Ononis hirta (aerial parts) and Inula viscosa (flowers) were the most active fractions against MCF-7 cells with IC50 of 27.96 and 15.78 μg/ml respectively and they were less toxic against other cell lines. Other extracts showed lower activity against cancer cell lines. TLC analysis showed the presence of flavonoids and terpenoids in active plants while alkaloids were detected in Ononis hirta (aerial parts) extracts. Ononis hirta (aerial parts) and Inula viscosa (flowers) extracts exerted their antiproliferative activity by inducing apoptosis in cancer cell lines. Further studies are necessary for detailed chemical characterization and more extensive biological evaluation of the most active ingredients.
Antioxidant activity of anti-inflammatory plant extracts. Schinella GR, Tournier HA, Prieto JM, Mordujovich de Buschiazzo P, Rios JL.
The antioxidant properties of twenty medical herbs used in the traditional Mediterranean and Chinese medicine were studied. Extracts from Forsythia suspensa, Helichrysum italicum, Scrophularia auriculata, Inula viscosa, Coptis chinensis, Poria cocos and Scutellaria baicalensis had previously shown anti-inflammatory activity in different experimental models. Using free radical-generating systems H. italicum. I. viscosa and F. suspensa protected against enzymatic and non-enzymatic lipid peroxidation in model membranes and also showed scavenging property on the superoxide radical. All extracts were assayed at a concentration of 100 microg/ml. Most of the extracts were weak scavengers of the hydroxyl radical and C. chinensis and P. cocos exhibited the highest scavenging activity. Although S. baicalensis inhibited the lipid peroxidation in rat liver microsomes and red blood cells, the extract showed inhibitory actions on aminopyrine N-demethylase and xanthine oxidase activities as well as an pro-oxidant effect observed in the Fe3+-EDTA-H2O2 system. The results of the present work suggest that the anti-inflammatory activities of the same extracts could be explained, at least in part, by their antioxidant properties.
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