Antagonistic effects of indoloquinazoline alkaloids on antimycobacterial activity of evocarpine.

AIMS: The interaction of quinolone and indoloquinazoline alkaloids concerning their antimycobacterial activity was studied. METHODS AND RESULTS: The antimycobacterial and modulating activity of evodiamine (1), rutaecarpine (2) and evocarpine (3) was tested on mycobacteria including three multidrug-resistant (MDR) clinical isolates of Mycobacterium tuberculosis. Antagonistic effects were concluded from fractional inhibitory concentration (FICI) values. Interaction energies of the compounds were calculated using GLUE docking module implemented in GRID. 1 and 2 exhibited weak inhibition of rapidly growing mycobacteria, however, 1 was active against Myco. tuberculosis H37Rv (MIC = 10 mg l(-1) ) while 2 was inactive. Both 1 and 2 showed a marked antagonistic effect on the susceptibility of different mycobacterial strains to 3 giving FICI values between 5 and 9. The interaction energies between compounds 1 and 2 with compound 3 suggested the possibility of complex formation in solution. CONCLUSIONS: Indoloquinazoline alkaloids markedly reduce the antimycobacterial effect of the quinolone alkaloid evocarpine. Complex formation may play a role in the attenuation of its antimycobacterial activity. SIGNIFICANCE AND IMPACT OF THE STUDY: This study gives a striking example of antagonism between compounds present in the same plant extract which should be considered in natural product based screening projects.

Anti-Campylobacter and resistance-modifying activity of Alpinia katsumadai seed extracts.

AIMS: We tested extracts from Alpinia katsumadai seeds for anti-Campylobacter activity and investigated the roles of the CmeABC and CmeDEF efflux pumps in Campylobacter resistance to these natural phenolics. Additionally, we investigated an A. katsumadai ethanolic extract (AlpE) and other plant extracts as putative efflux pump inhibitors on Campylobacter isolates and mutants in efflux pump genes. METHODS AND RESULTS: AlpE showed antimicrobial activity against sensitive and multidrug-resistant Campylobacter isolates. CmeB inactivation resulted in the greatest reduction in resistance, while cmeF and cmeR mutations produced only moderate effects on minimal inhibitory concentrations (MICs). The chemical efflux pump inhibitors additionally reduced MICs in isolates and mutants, confirming that active efflux is an important mechanism in resistance to AlpE, with additional contributions of other efflux systems. A notable decrease in resistance to tested antimicrobials in the presence of subinhibitory concentrations of AlpE confirms its modifying activity in Campylobacter spp. CONCLUSIONS: AlpE is important anti-Campylobacter source of antimicrobial compounds with resistance-modifying activity. At least two of the efflux systems are involved in the resistance to A. katsumadai antimicrobial seed extracts. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of antimicrobial and resistance-modifying activity of AlpE from A. katsumadai seeds, demonstrating its potential in the control of Campylobacter in the food chain.

Phytochemical composition and in vitro pharmacological activity of two rose hip (Rosa canina L.) preparations.

The aim of the present study was to compare powdered rose hip with and without fruits (Rosae pseudofructus cum/sine fructibus, Rosa canina L., Rosaceae) with regard to their phytochemical profile and their in vitro anti-inflammatory and radical-scavenging properties. The two powders were subsequently extracted with solvents of increasing polarity and tested for inhibition of cyclooxygenase (COX-1, COX-2) and of 5-LOX-mediated leukotriene B(4) (LTB(4)) formation as well as for DPPH-radical-scavenging capacity. While the water and methanol extracts were inactive in the COX-1, COX-2 and LTB(4) inhibition assays, the n-hexane and the dichloromethane extracts inhibited all three enzymes. In the active extracts, the triterpenoic acids ursolic acid, oleanolic acid and betulinic acid were identified, although only in minute amounts. Furthermore, oleic, linoleic and alpha-linolenic acid were identified apart from several saturated fatty acids. Even though unsaturated fatty acids are known to be good inhibitors of COX-1, COX-2 and LT formation, no clear correlation between their concentration in the extracts and their activity was found. We suggest that other, yet unidentified, lipophilic constituents might play a more important role for the observed in vitro inhibitory activity on arachidonic acid metabolism. Some of the extracts also showed considerable DPPH radical scavenging activity, the methanolic extracts being most potent. The radical scavenging activity of the extracts correlated very well with their total phenolic content, while ascorbic acid contributes only little to the radical-scavenging activity due to its low concentration present in the extracts. In summary, extracts derived from powdered rose hip without fruits were more effective in all assays carried out compared with extracts derived from powdered rose hip with fruits.

Knipholone, a selective inhibitor of leukotriene metabolism.

Inhibition of leukotriene formation is one of the approaches to the treatment of asthma and other inflammatory diseases. We have investigated knipholone, isolated from the roots of Kniphofia foliosa, Hochst (Asphodelaceae), for inhibition of leukotriene biosynthesis in an ex vivo bioassay using activated human neutrophile granulocytes. Moreover, activities on 12-lipoxygenase from human platelets and cycloxygenase (COX)-1 and -2 from sheep cotyledons and seminal vesicles, respectively, have been evaluated. Knipholone was found to be a selective inhibitor of leukotriene metabolism in a human blood assay with an IC(50) value of 4.2microM. However, at a concentration of 10microg/ml, the compound showed weak inhibition of 12(S)-HETE production in human platelets and at a concentration of 50microM it produced no inhibition of COX-1 and -2. In our attempt to explain the mechanism of inhibition, we examined the antioxidant activity of knipholone using various in vitro assay systems including free radical scavenging, non-enzymatic lipid peroxidation, and metal chelation. Knipholone was found to be a weak dose-independent free radical scavenger and lipid peroxidation inhibitor, but not a metal chelator. Therefore, the leukotriene biosynthesis inhibitory effect of knipholone was evident by its ability either to inhibit the 5-lipoxygenase activating protein (FLAP) or as a competitive (non-redox) inhibitor of the enzyme. Cytotoxicity results also provided evidence that knipholone exhibits less toxicity for a mammalian host cell.

Anti-inflammatory and anti-oxidant activity of a new class of phenyl-pyrazolone derivatives.

The anti-inflammatory activity of a new class of phenyl-pyrazolone derivatives, structurally related to phenidone, has been evaluated using the Croton oil ear test in mice as model of acute inflammation. Derivative 5h reduces the percentage of oedema similarly to indomethacin and more efficiently than phenylbutazone. The anti-inflammatory activity of these two reference drugs depends on their COX inhibition, but for the synthesized derivatives it has not been demonstrated a significant COX or LOX inhibition, as previously reported. While the anti-inflammatory activity of phenidone is correlated to its anti-oxidant properties, the redox potential of these compounds appears not decisive in the inflammatory process inhibition. In order to investigate the mechanism of action for these compounds, we quantified their anti-oxidant activity and the lipophilicity, and a relationship between the calculated logP and the percentage of oedema reduction was found. We hypothesize that the anti-inflammatory activity, recorded in vivo, could be related to lipophilic parameter of these compounds.

Anti-inflammatory activity of extracts and a saponin isolated from Melilotus elegans.

The crude methanol extract of Melilotus elegans Ser. (Fabaceae), a plant widely used in Ethiopian traditional medicine for the treatment of asthma, haemorrhoid and lacerated wounds showed a significant anti-inflammatory activity against carrageenin-induced rat paw oedema. At a dose corresponding to 333.3 mg per kg body weight of dry plant material, the methanol extract displayed a strong inhibitory effect that was comparable to the inhibitory effect of 1 mg/kg of indomethacin in the same test system. Bioassay guided fractionation of the alcoholic extract led to the isolation of an oleanene-type triterpene saponin identified as azukisaponin V (1) ((3-O-[alpha-L-rhamnopyranosyl-(1 --> 2)-beta-D-glucopyranosyl(1 --> 2)-beta-D-glucuronopyranosyl]-soyasapogenol B). The structure of the compound was identified by using MS and extensive one- and two-dimensional NMR experiments (1H, 13C, COSY, HMQC, HMBC and NOESY). One hour after injection of carrageenin, inhibition of oedema exerted by 1 was approximately ten times higher than that of indomethacin on a molar basis.

Anti-proliferative lichen compounds with inhibitory activity on 12(S)-HETE production in human platelets.

Several lichen compounds, i.e. lobaric acid (1), a beta-orcinol depsidone from Stereocaulon alpinum L., (+)-protolichesterinic acid (2), an aliphatic alpha-methylene-gamma-lactone from Cetraria islandica Laur. (Parmeliaceae), (+)-usnic acid (3), a dibenzofuran from Cladonia arbuscula (Wallr.) Rabenh. (Cladoniaceae), parietin (4), an anthraquinone from Xanthoria elegans (Link) Th. Fr. (Calaplacaceae) and baeomycesic acid (5), a beta-orcinol depside isolated from Thamnolia vermicularis (Sw.) Schaer. var. subuliformis (Ehrh.) Schaer. were tested for inhibitory activity on platelet-type 12(S)-lipoxygenase using a cell-based in vitro system in human platelets. Lobaric acid (1) and (+)-protolichesterinic acid (2) proved to be pronounced inhibitors of platelet-type 12(S)-lipoxygenase, whereas baeomycesic acid (5) showed only weak activity (inhibitory activity at a concentration of 100 microg/ml: (1) 93.4+/-6.62%, (2) 98,5+/-1.19%, 5 14.7+/-2.76%). Usnic acid (3) and parietin (4) were not active at this concentration. 1 and 2 showed a clear dose-response relationship in the range of 3.33-100 microg/ml. According to the calculated IC50 values the highest inhibitory activity was observed for the depsidone 1 (IC50 = 28.5 microM) followed by 2 (IC50 = 77.0 microM). The activity of 1 was comparable to that of the flavone baicalein, which is known as a selective 12(S)-lipoxygenase inhibitor (IC50 = 24.6 microM).

In vitro antiprotozoal activity of extract and compounds from the stem bark of Combretum molle.

The antiprotozoal activity of the Ethiopian medicinal plant Combretum molle (R. Br. ex G. Don.) Engl & Diels (Combretaceae) was evaluated by in vitro testing against Plasmodium falciparum, Trypanosoma brucei rhodesiense, Trypanosoma cruzi and Leishmania donovani. The acetone fraction of the stem bark of this plant prepared by soxhlet extraction was inactive against the intracellular amastigotes of L. donovani and T. cruzi in murine peritoneal macrophages but showed significant activity against extracellular T. b. rhodesiense blood stream form trypomastigotes and trophozoites of P. falciparum with IC(50) values of 2.19 and 8.17 microg/mL, respectively. Phytochemical examination of the bioactive fraction resulted in the isolation of two tannins and two oleanane-type pentacyclic triterpene glycosides. One of the tannins was identified as the ellagitannin, punicalagin, whilst the structure of the other (CM-A) has not yet been fully elucidated. The saponins that were characterized as arjunglucoside (also called 4-epi-sericoside) and sericoside displayed no activity against any of the four species of protozoa tested. On the other hand, punicalagin and CM-A had IC(50) values of 1.75 and 1.50 microM, respectively, against T. b. rhodesiense and were relatively less toxic to KB cells (cytotoxic/antiprotozoal ratios of 70 and 48, respectively). The tannins also showed intermediate activity against P. falciparum, although their selectivity against these parasites was less favourable than the above. It appears that our findings are the first report of hydrolysable tannins exhibiting antitrypanosomal and antiplasmodial activities.

Investigations on antimycobacterial activity of some Ethiopian medicinal plants.

Fifteen crude extracts prepared from seven Ethiopian medicinal plants used to treat various infectious diseases were assessed for their ability to inhibit the growth of Mycobacterium tuberculosis. A preliminary screening of the crude extracts against M. tuberculosis typus humanus (ATCC 27294) was done by dilution assay using Löwenstein-Jensen medium. None of the tested extracts except the acetone fraction obtained from the stem bark of Combretum molle (R. Br. ex G. Don.) Engl & Diels (Combretaceae) showed significant inhibitory action against this strain. The acetone fraction of the stem bark of C. molle caused complete inhibition at concentrations higher than 1 mg/mL. Phytochemical analysis of the bioactive fraction led to the isolation of a major tannin and two oleanane-type pentacyclic triterpene glycosides. The tannin was identified as the ellagitannin, punicalagin, whilst the saponins were characterized as arjunglucoside (also called 4-epi-sericoside) and sericoside. All the pure compounds were further tested against the ATCC strain. Punicalagin was found to inhibit totally growth of the ATCC and also of a patient strain, which was fully sensitive to the standard antituberculosis drugs, at concentrations higher than 600 microg/mL and 1.2 mg/mL, respectively. On the other hand, the saponins failed to show any action on the ATCC strain. It appears that our findings are the first report of tannins exhibiting antimycobacterial activity.

The antioxidant activity of the essential oils of Artemisia afra, Artemisia abyssinica and Juniperus procera.

The essential oils of Artemisia afra Jacq., Artemisia abyssinica Schultz-Bip. and Juniperus procera Hoechst ex Endl. were examined for their potential radical scavenging activities. First a rapid evaluation of antioxidants was made using a TLC screening method. The abilities of the volatile oils to act as nonspecific donors for hydrogen atoms or electrons were checked in the diphenylpicrylhydrazyl assay. Oils from all three species showed positive results and were examined further. The oils of A. afra and J. procera were also effective hydroxyl radical scavenging agents when assessed in the deoxyribose degradation assay, whilst oils from A. abyssinica exhibited a paradoxical effect. In the in vitro assay for non-enzymatic lipid peroxidation in liposomes, the oils of A. afra and J. procera also displayed antioxidant potential. It was not possible to measure the effect of A. abyssinica oil in this system because certain components, e.g. alk-2-enals, interfered with the assay. The compounds that contribute to the radical scavenging activities of A. afra and J. procera were identified and then assessed for their effects in the various test systems. Finally, the qualitative and quantitative compositions of the essential oils were studied by GC-MS.

Antiviral activity against human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) of ethnobotanically selected Ethiopian medicinal plants.

Ethiopian medicinal plants used for the treatment of a variety of ailments including infectious diseases were screened for activity against human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2). Seventy-one polar and nonpolar extracts derived from 21 plants belonging to 14 families were tested for inhibition of viral replication using HIV-1 (III(B)) and HIV-2 (ROD) strains. Selective inhibition of viral growth was assessed by the simultaneous determination of the in vitro cytotoxicity of each of the extracts against MT-4 cells. Six extracts made from the root bark of Bersama abyssinica Fresen, the leaves of Combretum paniculatum Vent., and Dodonaea angustifolia L.f., and the stem bark of Ximenia americana L. displayed antiviral activity at concentrations that were nontoxic to MT-4 cells. The highest selective inhibition of HIV-1 replication was observed with the acetone fraction of C. paniculatum and the methanol fraction of D. angustifolia which showed selectivity indices (ratio of 50% cytotoxic concentration to 50% effective antiviral concentration) of 6.4 and 4.9, and afforded cell protection of viral induced cytopathic effect of 100% and 99%, respectively, when compared with control samples. The greatest degree of antiviral activity against HIV-2 was achieved with the acetone extract of C. paniculatum (EC(50): 3 microg/mL), which also showed the highest selectivity index (32). The 50% cytotoxic concentration ranged from 0.5 microg/mL for the hexane extract of D. angustifolia L.f., the most cytotoxic of the extracts tested, to >250 microg/mL for some extracts such as the methanol fraction of Alcea rosea L., the least toxic tested. Only the polar extracts that were obtained by extraction with hydroalcohol, methanol or acetone exhibited inhibition of viral growth at subtoxic concentrations. The results obtained in this study enable the selection of extracts which show some specificity of action and support the further investigation of these extracts for their potential as new lead antiretroviral compounds.

Antioxidant activity of Nigella sativa essential oil.

The essential oil of black cumin seeds, Nigella sativa L., was tested for a possible antioxidant activity. A rapid evaluation for antioxidants, using two TLC screening methods, showed that thymoquinone and the components carvacrol, t-anethole and 4-terpineol demonstrated respectable radical scavenging property. These four constituents and the essential oil possessed variable antioxidant activity when tested in the diphenylpicrylhydracyl assay for non-specific hydrogen atom or electron donating activity. They were also effective.OH radical scavenging agents in the assay for non-enzymatic lipid peroxidation in liposomes and the deoxyribose degradation assay. GC-MS analysis of the essential oil obtained from six different samples of Nigella sativa seeds and from a commercial fixed oil showed that the qualitative composition of the volatile compounds was almost identical. Differences were mainly restricted to the quantitative composition.

Triterpenoid saponins and sapogenin lactones from Albizia gummifera.

The structures of two new monodesmosidic and bisdesmosidic triterpenoid saponins (1 and 2) and the known compound delta 5-stigmasterol-3-O-beta-D-glucopyranoside (3) as well as two new oleanane type triterpene lactone glycosides 4, 5 and a new sapogenin lactone 6 isolated from the stem bark of Albizia gummifera C.A. Smith (Mimosaceae) have been elucidated as 3-O-¿beta-D-glucopyranosyl(1-->2)-[alpha-L-arabinopyranosyl(1-->6) ]-beta-D- glucopyranosyl¿-oleanolic acid (1), beta-D-glucopyranosyl(1-->2)-beta-D-glucopyranosyl 3-O-¿beta-D-glucopyra-nosyl(1-->2)-[alpha-L-arabinopyrano syl(1-->6)]-beta-D- glucopyranosyl¿-oleanolate (2), 3 beta-¿O-D-glucopyranosyl-(1-->2)-[O-alpha-L-arabinopyranosyl(1-->6 )] beta-D-glucopyranosyloxy¿-machaerinic acid gamma-lactone (4), 3 beta-O-beta-D-glucopyranosiduronic acid (1-->2)-beta-D-glucopyranosyloxy]-machaerinic acid gamma-lactone (5), and A-homo-3a-oxa-5 beta-olean-12-en-3-one-28-oic acid (6), respectively. The complete assignment of the 1H and 13C resonances of 1, 2, 4 and 6 and of the peracetate of 5 were achieved by means of 2D-NMR studies.

Antiinflammatory activity of extracts of Biophytum sensitivum in carrageenin-induced rat paw oedema.

The antiinflammatory activity of aqueous and methanol extracts of aerial parts, an aqueous extract of roots as well as ultrafiltration fractions of a methanol extract of roots of Biophytum sensitivum were evaluated in the carrageenin induced rat paw oedema model. All the extracts except the methanol extract of aerial parts exhibited antiinflammatory activity, but inhibition of oedema was found to be maximum with aqueous extracts.

5-Methylflavasperone and rhamnetin from Guiera senegalensis and their antioxidative and 5-lipoxygenase inhibitory activity.

From the methylene chloride extract of the leaves of Guiera senegalensis a novel naphthopyran, 5-methylflavasperone (5,8,10-trimethoxy-2-methyl-4 H-naphtho[1,2-b]pyran-4-one), as well as rhamnetin were isolated. Rhamnetin showed a strong inhibitory activity on 5-lipoxygenase from porcine leucocytes with an IC50 value of 0.7 microM whereas 5-methylflavasperone revealed only a weak inhibitory activity (IC50 > 200 microM). In the deoxyribose assay both compounds exerted similar antiradicalar effects (IC50 15.8 and 16.7 microM, respectively). In another assay only rhamnetin inhibited peroxidation of phospholipid liposomes (IC50 = 2.4 microM). For 5-methylflavasperone no dose dependent inhibition could be observed.

Amentoflavone from Biophytum sensitivum and its effect on COX-1/COX-2 catalysed prostaglandin biosynthesis.

Amentoflavone (I3', II8-biapigenin) was isolated from the roots of Biophytum sensitivum DC. (Oxalidaceae) and proved to be a selective inhibitor of cyclooxygenase (COX)-1 catalysed prostaglandin biosynthesis when tested in vitro with an IC (50) value of 12.4 microM (standard: indomethacin, IC (50) = 1.1 microM). Doses of up to 37 microM showed only a slight inhibition in the corresponding COX-2 assay. Quantification of amentoflavone was carried out by reversed phase HPLC in methanolic and aqueous extracts of the roots, stems and leaves. Highest amounts of amentoflavone were detected in methanolic extracts of roots and stems (0.26-0.35%), while considerably lower amounts were detected in the corresponding water extracts.

Studies of Epilobium angustifolium extracts on growth of accessory sexual organs in rats.

Hexane and water extracts, as well as an ultrafiltration fraction (MW < 1000 Da) obtained from the water extract from Epilobium angustifolium L. (Onagraceae) were administered perorally (p.o.) on intact male rats as well as on testosterone stimulated castrated rats and changes in growth of prostate, musculus laevator ani and seminal vesicles were determined. In intact rats the water extract decreased the weight of seminal vesicles whereas in castrated rats an increased weight of all accessory sexual organs was observed. In intact rats the ultrafiltration fraction (MW < 1000 Da) exhibited the same but smaller effect than the water extract, in testosterone stimulated castrated rats it showed no effect in comparison with the control group. The hexane extract exhibited no significant effects, both in intact and testosterone stimulated castrated rats. For characterization the hexane extract was analyzed for the first time for its fatty acid composition by GC-MS.

Flavone glucuronides of Lycopus virginicus.

From the ethanolic extract of the aerial parts of Lycopus virginicus (L.) Michx. (Lamiaceae) the 7- O-beta- D-glucuronides of apigenin, acacetin, and luteolin as well as the methyl ester of apigenin 7- O-beta- D-glucuron-ide could be isolated. Quantification of luteolin 7- O-beta- D-glucuronide by HPLC in crude methanolic extracts from L. virginicus, L. europaeus, and L. exaltatus showed contents of 0.017-0.22%.