In vitro anti-Acanthamoeba activity of flavonoid glycosides isolated from Delphinium gracile, D. staphisagria, Consolida oliveriana and Aconitum napellus.

Acanthamoeba spp. are widely distributed in the environment and cause serious infections in humans. Treatment of Acanthamoeba infections is very challenging and not always effective which requires the development of more efficient drugs against Acanthamoeba spp. The purpose of the present study was to test medicinal plants that may be useful in the treatment of Acanthamoeba spp. Here we evaluated the trophozoital and cysticidal activity of 13 flavonoid glycosides isolated from Delphinium gracile, D. staphisagria, Consolida oliveriana and from Aconitum napellus subsp. Lusitanicum against the amoeba Acanthamoeba castellanii. AlamarBlue Assay Reagent® was used to determine the activity against trophozoites of A. castellanii, and cytotoxic using Vero cells. Cysticidal activity was assessed on treated cysts by light microscopy using a Neubauer chamber to quantify cysts and trophozoites. Flavonoids 1, 2, 3 and 4 showed higher trophozoital activity and selectivity indexes than the reference drug chlorhexidine digluconate. In addition, flavonoid 2 showed 100% cysticidal activity at a concentration of 50 µm, lower than those of the reference drug and flavonoid 3 (100 µm). These results suggest that flavonoids 2 and 3 might be used for the development of novel therapeutic approaches against Acanthamoeba infections after satisfactory in vivo evaluations.

Leishmanicidal activity of nine novel flavonoids from Delphinium staphisagria.

OBJECTIVES: To evaluate the in vitro leishmanicidal activity of nine flavonoid derivatives from Delphinium staphisagria against L. infantum and L. braziliensis. DESIGN AND METHODS: The in vitro activity of compounds 1-9 was assayed on extracellular promastigote and axenic amastigote forms and on intracellular amastigote forms of the parasites. Infectivity and cytotoxicity tests were carried on J774.2 macrophage cells using Glucantime as the reference drug. The mechanisms of action were analysed performing metabolite excretion and transmission electronic microscope ultrastructural alteration studies. RESULTS: Nine flavonoids showed leishmanicidal activity against promastigote as well as amastigote forms of Leishmania infantum and L. braziliensis. These compounds were nontoxic to mammalian cells and were effective at similar concentrations up to or lower than that of the reference drug (Glucantime). The results showed that 2(″)-acetylpetiolaroside (compound 8) was clearly the most active. CONCLUSION: This study has demonstrated that flavonoid derivatives are active against L. infantum and L. braziliensis.

In vitro and in vivo trypanocidal activity of flavonoids from Delphinium staphisagria against Chagas disease.

The in vitro and in vivo trypanocidal activities of nine flavonoids (1-9) isolated from the aerial parts of Delphinium staphisagria have been studied in both the acute and chronic phases of Chagas disease. The antiproliferative activity of these substances against Trypanosoma cruzi (epimastigote, amastigote, and trypomastigote forms) in some cases exhibited more potent antitrypanosomatid activity and lower toxicity than the reference drug, benznidazole. Studies in vitro using ultrastructural analysis together with metabolism-excretion studies were also performed in order to identify the possible action mechanism of the compounds tested. Alterations mainly at the level of the mitochondria may explain metabolic changes in succinate and acetate production, perhaps due to the disturbance of the enzymes involved in sugar metabolism within the mitochondrion. In vivo studies provided results consistent with those observed in vitro. No signs of toxicity were detected in mice treated with the flavonoids tested, and the parasitic charge was significantly lower than in the control assay with benznidazole. The effects of these compounds were also demonstrated with the change in the anti-T. cruzi antibody levels during the chronic stage.

Preliminary analysis of the chemical composition, antioxidant and anticholinesterase activities of Algerian propolis.

Many studies demonstrated that Algerian propolis possess a large spectrum of biological activity. However, few studies regarding its chemical composition are available on literature. We aimed in the present study to investigate the chemical composition of Algerian propolis. In addition, the antioxidants and anticholinesterase activities of propolis extracts are also reported. Chemical investigation of Algerian propolis allowed the isolation of 8 compounds. Their structures were identified on the basis of spectral data and comparison with literature. The isolated compounds are considered as markers of poplar and Citrus spp suggesting the use of both species as plant source of the tested propolis. The ethyl acetate extract of the tested propolis demonstrated the highest antioxidant activity. Among the tested extracts, only the petroleum ether and chloroform extracts exhibited an AChE inhibitory activity.

Antileishmaniasis activity of flavonoids from Consolida oliveriana.

A set of flavonoids from Consolida oliveriana, kaempferol (1), quercetin (2), trifolin (3), and acetyl hyperoside (5) and their O-acetyl derivatives (1a, 2a, 3a), and octa-O-acetylhyperoside (4) showed leishmanicidal activity against promastigote as well as amastigote forms of Leishmania spp. The cellular proliferation, metabolic, and ultrastructural studies showed that the acetylated compounds 2a, 3a, and 4 were highly active against Leishmania (V.) peruviana, while 2a as well as 4 were effective against Leishmania (V.) braziliensis. These compounds were not cytotoxic and are effective at similar concentrations up to or lower than the reference drugs (pentostam and glucantim).

Trifolin acetate-induced cell death in human leukemia cells is dependent on caspase-6 and activates the MAPK pathway.

In the present study we demonstrated that the flavonoid derivative trifolin acetate (TA), obtained by acetylation of naturally occurring trifolin, induces apoptosis. Associated downstream signaling events were also investigated. TA-induced cell death was prevented by the non-specific caspase inhibitor z-VAD-fmk and reduced by the presence of the selective caspase inhibitors z-LEHD-fmk (caspase-9), z-DEVD-fmk (caspase-3) and z-VEID-fmk (caspase-6). The apoptotic effect of TA was associated with (i) the release of cytochrome c from mitochondria which was not accompanied by dissipation of the mitochondrial membrane potential (DeltaPsi(m)), (ii) the activation of the mitogen-activated protein kinases (MAPKs) pathway and (iii) abrogated by the over-expression of Bcl-2 or Bcl-x(L). TA-induced cell death was attenuated by inhibition of extracellular signal-regulated kinases (ERK) 1/2 with U0126 and inhibition of p38(MAPK) with SB203580. In contrast, inhibition of c-Jun NH(2)-terminal kinase (JNK) by SP600125 significantly enhanced apoptosis. Although reactive oxygen species (ROS) increased in response to TA, this did not seem to play a pivotal role in the apoptotic process since different anti-oxidants were unable to provide cell protection. The present study demonstrates that TA-induced cell death is mediated by an intrinsic-dependent apoptotic event involving mitochondria and MAPK, and through a mechanism independent of ROS generation.

DPPH radical scavenging activity of two flavonol glycosides from Aconitum napellus sp. lusitanicum.

The DPPH radical scavenging activity of two flavonol glycosides obtained from ethanolic extracts of Aconitum napellus sp. lusitanicum was studied. The results showed a high DPPH antiradical activity of compound 1 (quercetin 3-O-(6-trans-caffeoyl)-beta-glucopyranosyl-(1-->2)-beta-glucopyranosyl-7-O-alpha-rhamnopyranoside) when compared with compound 2 (quercetin-3-sophoroside-7-rhamnopyranoside), rutin and ascorbic acid. The relationship between the caffeoyl and rhamnopyranoside groups in the flavonol glycosides structures and the DPPH antiradical activity was also discussed.

In vitro activity of C20-diterpenoid alkaloid derivatives in promastigotes and intracellular amastigotes of Leishmania infantum.

The in vitro anti-proliferative effects are described of several atisine-type diterpenoid alkaloids against the protozoan parasite Leishmania infantum, which causes human visceral leishmaniasis and canine leishmaniasis in the Mediterranean basin, as well as human cutaneous leishmaniasis throughout the Mediterranean region. From a total of 43 compounds tested, including C19- and C20-diterpene alkaloids from several chemical classes, only 15,22-O-diacetyl-19-oxo-dihydroatisine, azitine and isoazitine were highly active against cultures of the parasite (promastigote form) with IC50 values within the range of the reference drug pentamidine-isothionate (7.39-12.80 mg/L for the test compounds, 11.32 mg/L for the positive control). These compounds were not toxic to the host cell. When treated with a dosage of 5 microg/mL of the active compounds (half of their IC50), the promastigote forms lost 80% of their infection capacity and the multiplication of extracellular forms of L. infantum was severely affected. The study showed that atisine-type C20-diterpenoid alkaloids exhibited promising anti-leishmanial properties with strong molecular selectivity. These might have implications for other intracellular pathogens- or phylogenetically related parasites, such as Trypanosoma spp.

Norditerpene and diterpene alkaloids from Aconitum variegatum.

Aerial parts of Aconitum variegatum L. from the Pyrenees furnished four norditerpene alkaloids, 16 beta-hydroxycardiopetaline, 8-ethoxysachaconitine, 14-acetylgenicunine B, N-deethyl-N-19-didehydrosachaconitine, five diterpene alkaloids 15-veratroyldictizine, 15-veratroyl-17-acetyldictizine, 15-veratroyl-17-acetyl-19-oxodictizine, N-ethyl-1 alpha-hydroxy-17-veratroyldictizine, variegatine and the known alkaloids sachaconitine, 14-O-acetylsachaconitine, karakoline, talatizamine, 10-hydroxytalatizamine, 14-acetyltalatizamine, 14-acetyl-10-hydroxytalatizamine, N-methylarmepavine, pengshenin B, delsoline, dihydrodelsoline, delcosine and genicunin B. Structures of the alkaloids were established by MS, 1D- and 2D-NMR techniques.

Dianthramide glucosides from tissue cell cultures of Delphinium staphisagria L.

Tissue cell cultures of Delphinium staphisagria L. produced three dianthramide glucosides N-(2'-beta-glucopyranosylsalicyl)-5-hydroxyanthranilic acid methyl ester, N-(2'-beta-glucopyranosyl-5'-methoxysalicyl)-5-hydroxyanthranilic acid methyl ester and N-(2'-beta-glucopyranosyl-5'-hydroxysalicyl)-5-hydroxy-6-methoxyanthranilic acid methyl ester, together with known methyl esters of N-salicylanthranilic acid and N-(2'-beta-glucopyranosyl-5'-hydroxysalicyl)-5-hydroxyanthranilic acid. Structures of the glucosides were established by MS, 1-D and 2-D NMR techniques.

Alkaloids from Delphinium pentagynum.

Aerial parts of a collection of Delphinium pentagynum Lam. from Niebla, Southern Spain, furnished one diterpene alkaloid, 2-dehydrodeacetylheterophylloidine, two norditerpene alkaloids, 14-demethyl-14-isobutyrylanhweidelphinine and 14-demethyl-14-acetylanhweidelphinine, the known alkaloids 14-deacetylnudicauline, methyllycaconitine, 14-deacetyl-14-isobutyrylnudicauline, 14-acetylbrowniine, browniine, delcosine, lycoctonine, 18-methoxygadesine, neoline, karakoline and the aporphine alkaloid magnoflorine. Structures of the alkaloids were established by MS, 1D and 2-D NMR techniques.

Antifeedant C20 diterpene alkaloids.

We have tested the insect antifeedant and toxic activity of 21 C20 diterpenoid alkaloids on Spodoptera littoralis and Leptinotarsa decemlineata. The antifeedant effects of the test compounds were structure- and species-dependent. The most active antifeedants to L. decemlineata and S. littoralis were the rearranged form of hetisine (20; EC50 = 1.7 microg/cm2) and 19-oxodihydroatisine (9; EC50 = 0.1 microg/cm2), resp. Glandulosine (8) moderately affected orally injected S. littoralis larvae. A few compounds (13-oxocardiopetamine (4), 9, and atisinium chloride (13)) had cytotoxic effects to insect-derived Sf9 cells with varying degrees of selectivity with respect to mammalian CHO cells. Compounds 4 and 15,22-O-diacetyl-19-oxodihydroatisine (10) increased Trypanosoma cruzi mortality. Our results support the plant protective role of C20 diterpenoid alkaloids and open a new field for parasite control strategies.

Astragalin heptaacetate-induced cell death in human leukemia cells is dependent on caspases and activates the MAPK pathway.

Flavonoids are naturally occurring polyphenolic compounds and are among the most promising anticancer agents. Here we demonstrate that the flavonoid derivative astragalin heptaacetate (AHA) induces cell death. This was prevented by the non-specific caspase inhibitors z-VAD-fmk and Q-VD-OPH, and reduced by the selective caspase-4 inhibitor z-LEVD-fmk. AHA-induced cell death was found to be: (i) associated with the release of cytochrome c, (ii) suppressed by the overexpression of Bcl-x(L), (iii) amplified by inhibition of extracellular signal-regulated kinases (ERKs) 1/2 and c-jun NH(2)-terminal kinases/stress activated protein kinases (JNK/SAPK) signaling, and (iv) completely abrogated by the free-radical scavenger N-acetyl-l-cysteine.

Structure determination of monohydrated trifolin (kaempferol 3-O-ß-D-galactopyranoside) from laboratory powder diffraction data.

The crystal structure of monohydrated trifolin (kaempferol 3-O-ß-D-galactopyranoside) (an important biologically active compound, which was isolated from the aerial part of Consolida oliveriana) has been determined from conventional laboratory X-ray powder diffraction data. Variable counting time technique was used during measurement and crystal structure was solved by means of Monte Carlo algorithm. The final structure was achieved by Rietveld refinement using both constraints and restraints on interatomic bond lengths and angles.

Acylated flavonol tetraglycosides from Delphinium gracile.

An ethanol extract of the aerial parts of Delphinium gracile DC. yielded five flavonol glycosides quercetin-3-O-{[beta-d-xylopyranosyl (1-->3)-4-O-(E-p-caffeoyl)-alpha-l-rhamnopyranosyl (1-->6)][beta-d-glucopyranosyl (1-->2)]}-beta-d-glucopyranoside (1), quercetin-3-O-{[beta-d-xylopyranosyl (1-->3)-4-O-(E-p-coumaroyl)-alpha-l-rhamnopyranosyl (1-->6)][beta-d-glucopyranosyl (1-->2)]}-beta-d-glucopyranoside (2), quercetin-3-O-{[beta-d-xylopyranosyl (1-->3)-4-O-(Z-p-coumaroyl)-alpha-l-rhamnopyranosyl (1-->6)][beta-d-glucopyranosyl (1-->2)]}-beta-d-glucopyranoside (3), kaempferol-3-O-{[beta-d-glucopyranosyl (1-->3)-4-O-(E-p-coumaroyl)-alpha-l-rhamnopyranosyl (1-->6)][beta-d-glucopyranoside-7-O-(4-O-acetyl)-alpha-l-rhamnopyranoside (4) kaempferol-3-O-{[beta-d-glucopyranosyl (1-->3)-4-O-(E-p-coumaroyl)-alpha-l-rhamnopyranosyl (1-->6)][beta-d-glucopyranoside-7-O-(4-O-acetyl)-alpha-l-rhamnopyranoside (5) in addition to 4-(beta-d-glucopyranosyloxy)-6-methyl-2H-pyran-2-one (6) and rutin. Structures were elucidated by spectroscopic methods.

Cytotoxic activities of flavonoid glycoside acetates from Consolida oliveriana.

The flavonoids kaempferol, quercetin, trifolin, hyperoside 2''- and 6''- acetates, 7-glucotrifolin, biorobin and robinin were isolated from the aerial parts of Consolida oliveriana. Their derivatives kaempferol tetraacetate, quercetin pentaacetate, trifolin heptaacetate and hyperoside octaacetate exhibited significant cytotoxicity IN VITRO against three human cell lines HL-60, U937 and SK-MEL-1 while hyperoside 2''-acetate, hyperoside-6''-acetate, glucotrifolin decaacetate and heptamethyltrifolin were inactive.

Sesquiterpene lactones from Anthemis plutonia.

Aerial parts of Anthemis plutonia furnished three guaianolides, one of them new.

Five new alkaloids from the leaves of Remijia peruviana.

Three new indolylquinuclidine-type alkaloids, remijinine (1), epiremijinine (2), and 5-acetyl-apocinchonamine (3), and two new cinchonine-derived alkaloids, N-acetyl-deoxycinchonicinol (4) and N-acetyl-cinchonicinol (5), as well as the known alkaloids quinamine, conquinamine, cinchonine, and quinidine were isolated from the leaves of Remijia peruviana. The structures of the new alkaloids were elucidated on the basis of spectroscopic analysis, including homonuclear and heteronuclear correlation NMR experiments (COSY, ROESY, HMQC, and HMBC). The relative configuration at C-7 for remijinine (1) and, in consequence, for epiremijinine (2) was established by X-ray crystal structure analysis of the former.