Assessment of the antiprotozoal activity of Pulicaria inuloides extracts, an Algerian medicinal plant: leishmanicidal bioguided fractionation.

The lack of an effective chemotherapy for treatment of protozoan disease urges a wide investigation for active compounds, and plant-derived compounds continue to provide key leads for therapeutic agents. The current study reports the in vitro antiprotozoal evaluation of the Algerian medicinal plant Pulicaria inuloides against Leishmania amazonensis, Trypanosoma cruzi, and Acanthamoeba castellanii str. Neff. All the extracts from the aerial part showed to be present a higher leishmanicidal activity than anti-Acanthamoeba or Trypanosoma. Therefore, bioguided fractionation of the active CHCl3 extract led to the isolation and characterization of the flavonol, quercetagetin-3,5,7,3'-tetramethyl ether (1) as the main component. The structure of compound 1 was established by extensive 1D and 2D NMR spectroscopic analysis (COSY, HSQC, HMBC, and ROESY experiments), chemical transformation (derivatives 2 and 3), and comparison with data in the literature. Compound 1 and derivatives 2 and 3 were further evaluated against the promastigote and amastigote stage of L. amazonensis. Compounds 1-3 exhibited moderate leishmanicidal activity with IC50 values ranging from 0.234 to 0.484 mM and from 0.006 to 0.017 mM for the promastigote and amastigote forms, respectively, as well as low toxicity levels on macrophages (CC50 ranging from 0.365 to 0.664 mM). This study represents the first report of the antiprotozoal evaluation of Pulicaria inuloides, and the results highlight this species as a promising source of leishmanicidal agents.

Leishmanicidal activity of α-bisabolol from Tunisian chamomile essential oil.

According to the World Health Organization, leishmaniasis is considered as a major neglected tropical disease causing an enormous impact on global public health. Available treatments were complicated due to the high resistance, toxicity, and high cost. Therefore, the search for novel sources of anti-leishmania agents is an urgent need. In the present study, an in vitro evaluation of the leishmanicidal activity of the essential oil of Tunisian chamomile (Matricaria recutita L.) was carried out. Chamomile essential oil exhibits a good activity on promastigotes forms of L. amazonensis and L. infantum with a low inhibitory concentration at 50% (IC50) (10.8 ± 1.4 and 10.4 ± 0.6 µg/mL, respectively). Bio-guided fractionation was developed and led to the identification of (-)-α-bisabolol as the most active molecule with low IC50 (16.0 ± 1.2 and 9.5 ± 0.1 µg/mL for L. amazonensis and L. infantum, respectively). This isolated sesquiterpene alcohol was studied for its activity on amastigotes forms (IC50 = 5.9 ± 1.2 and 4.8 ± 1.3 µg/mL, respectively) and its cytotoxicity (selectivity indexes (SI) were 5.4 and 6.6, respectively). The obtained results showed that (-)-α-bisabolol was able to activate a programmed cell death process in the promastigote stage of the parasite. It causes phosphatidylserine externalization and membrane damage. Moreover, it decreases the mitochondrial membrane potential and total ATP levels. These results highlight the potential use of (-)-α-bisabolol against both L. amazonensis and L. infantum, and further studies should be undertaken to establish it as novel leishmanicidal therapeutic agents.

Correlation of radical-scavenging capacity and amoebicidal activity of Matricaria recutita L. (Asteraceae).

Some Acanthamoeba strains are able to cause Granulomatous Amoebic Encephalitis (GAE) and Acanthamoeba keratitis (AK) worldwide because of their pathogenicity. The treatment of Acanthamoeba infections is complicated due to the existence of a highly resistant cyst stage in their life cycle. Therefore, the elucidation of novel sources of anti-Acanthamoeba agents is an urgent need. In the present study, an evaluation of the antioxidant and anti-Acanthamoeba activity of compounds in flower extracts of Tunisian chamomile (Matricaria recutita L.) was carried out. Chamomile methanol extract was the most active showing an IC50 of 66.235 ± 0.390 µg/ml, low toxicity levels when checked in murine macrophage toxicity model and presented also antioxidant properties. Moreover, a bio-guided fractionation of this extract was developed and led to the identification of a mixture of coumarins as the most active fraction. These results suggest a novel source of anti-Acanthamoeba compounds for the development of novel therapeutic agents against Acanthamoeba infections.

Amoebicidal, antimicrobial and in vitro ROS scavenging activities of Tunisian Rubus ulmifolius Schott, methanolic extract.

The present study aimed to evaluate the activity of methanolic extract of Rubus ulmifolius Schott against the Acanthamoeba castellani Neff Strain as well as its antioxidant and antimicrobial effects. The tested extract has a good amoebicidal activity with low IC50 (61.785 ± 1.322 µg/ml) and also has significant activity against both Gram-positive (S. aureus, S. agalactiae) and Gram-negative bacteria (E. coli, S. typhimurium) and against C. albicans. The inhibition zones diameters (IZD) and minimal inhibitory concentration (MIC) values were in the range of 22.5-50 mm and 02.29-4.76 mg ml-1, respectively. In the other hand, the in vitro ROS scavenging activity was evaluated, the tested extract exhibited a good effect on the ·OH radical (89.99% at a concentration of 100 µg/ml) when compared to the ascorbic acid (68.81%). Moreover, the inhibition percentage of superoxide generation by R. ulmifolius extract at 100 µg/ml was greater than ascorbic acid (79.55; 64.79%, respectively). Also, the tested extract showed a high percentage of H2O2 scavenging activity (99.95% at 100 µg/ml). Our findings suggest that R. ulmifolius could be a potential source of natural antioxidant in preventing many diseases associated with oxidative stress, amoebic and bacterial infections.

Variation in Campylobacter jejuni culturability in presence of Acanthamoeba castellanii Neff.

Free-living amoebae (FLA) are protozoa that are widely distributed in the environment mainly in water and soil related habitats. These amoebae have also been reported to be associated with some bacterial pathogens for humans such as Campylobacter spp. The species C. jejuni is the causative agent of about 90% of human campylobacteriosis cases worldwide and this disease may even end up in severe autoimmune sequelae as Guillain-Barré syndrome (GBS). In this study, the interactions between the strain Acanthamoeba castellanii Neff and Campylobacter jejuni was investigated. Campylobacter jejuni was coincubated with Acanthamoeba castellanii Neff trophozoites at different temperatures, in order to evaluate the C. jejuni ability to grow in presence A. castellanii culture and Acanthamoeba Conditioned Medium (ACM). C. jejuni was coincubated with A. castellanii axenic culture at different temperatures in aerobic conditions. Our results revealed that bacteria were still cultivable (Blood Agar medium, at 37 °C, in microaerophilic atmosphere) after a 14 days C. jejuni - A. castellanii coculture, comparing with C. jejuni alone, which was only cultivable for 24 h.

In vitro interactions of Acanthamoeba castellanii Neff and Vibrio harveyi.

Free-living amoebae (FLA) are opportunistic protozoa widely distributed in the environment. They are frequently found in water and soil samples, but they have also been reported to be associated with bacterial human pathogens such as Legionella spp. Campylobacter spp or Vibrio cholerae among others. Including within Vibrio spp. V. harveyi (Johnson and Shunk, 1936) is a bioluminescent marine bacteria which has been found swimming freely in tropical marine waters, being part of the stomach and intestine microflora of marine animals, and as both a primary and opportunistic pathogen of marine animals. Our aim was to study the interactions between Vibrio harveyi and Acanthamoeba castellanii Neff. Firstly, in order to analyze changes in it cultivability, V. harveyi was coincubated with A. castellanii Neff axenic culture and with Acanthamoeba Conditioned Medium (ACM) at different temperatures in aerobic conditions. Interestingly, at 4 °C and 18-20 °C bacteria were still cultivable in marine agar, at 28 °C, in aerobic conditions, but there weren't significant differences comparing with the controls. We also noted an enhanced migration of Acanthamoeba toward V. harveyi on non-nutrient agar plates compared to controls with no bacteria.

Myrtle Berries Seeds Prevent Dyslipidemia, Inflammation, and Excessive Cardiac Reactive Oxygen Species Production in Response to High-Fat Diet-Induced Obesity.

Anthocyanins are the major polyphenols in myrtle berries seeds aqueous extract (MBSAE). This study investigates the protective potentials of MBSAE against obesity lipotoxicity and inflammation induced by a high-fat diet (HFD). It also describes the underlying mechanisms involved in its protective effects, with special attention to myocardial reactive oxygen species (ROS) production. Male Wistar rats were fed HFD for 6 weeks to induce obesity. MBSAE (100 mg/kg, b.w., p.o.) was orally administered to HFD-fed rats. Anti-obesity effects were triggered by the inhibitory action of the MBSAE against the weights of the body, its relative heart and the total abdominal fat. Treatment with MBSAE also restored the lipid profile to baseline compared with the HFD rats and lowered also the white blood cells count, including neutrophils, lymphocytes, and basophils number as well as cytokines (tumor necrosis factor-α, interleukin [IL]-6, and IL-1ß) levels in the rats serum, thus improving the tissue inflammatory status associated with obesity. Exposure of rats to HFD during 6 weeks induces a myocardial oxidative stress as assessed by deleterious effects on lipoperoxidation state, antioxidant enzyme (SOD, CAT, and GPx) activities as well as sulfhydryl groups and GSH rates. Of importance, our study shows also that HFD provokes a heart ROS (H2O2, OH•, and O2•-) overload. Of interest, all these oxidative heart disturbances were clearly ended by MBSAE treatment. Therefore, consumption of MBSAE as a natural extract may be a potential therapeutic strategy to treat obesity-associated diseases.

Role of Anti-Inflammatory, Reactive Oxygen Species Scavenging Activity and Nematicidal Properties of Myrtle Berry Seeds on Helminthiasis Treatment.

This study was undertaken to investigate the involvement of the reactive oxygen species (ROS) and proinflammatory cytokine production in the pathogenicity of gastrointestinal nematode Heligmosomoides polygyrus in mice and also the protective effect of myrtle (Myrtus communis L.) berry seeds aqueous extract (MBSAE). With this aim, Swiss Albino mice were divided into four groups of 10 animals each: control, helminthiasis, helminthiasis + MBSAE, and helminthiasis + Albendazole. The animals were infected with infective third larval stage (L3) of H. polygyrus by oral administration. Mice were treated with bidistilled water, MBSAE (100 mg/kg, b.w., p.o.) or Albendazole (100 mg/kg, b.w., p.o.). We have demonstrated that MBSAE (100 mg/kg, b.w., p.o.) significantly reduced the total mean adult worms compared with the infected and nontreated group. Moreover, hematological parameters, intestinal ROS such as hydrogen peroxide (H2O2), hydroxyl radical (OH•), and superoxide anion (O2•-), as well as serum cytokines such as interleukin (IL)-1ß and IL-6 were analyzed. We have shown that H. polygyrus infection has disrupted all these biochemical parameters and that MBSAE treatment has significantly corrected these disturbances. The effect of plant extract was comparable to the standard drug albendazole. Therefore, MBSAE appeared to be a promising agent for the control of intestinal helminthiasis.

Chamomile Methanolic Extract Mitigates Small Bowel Inflammation and ROS Overload Related to the Intestinal Nematodes Infection in Mice.

BACKGROUND: Chamomile (Matricaria recutita L.) is a plant which has been reported to be effective in treating several parasitic and digestive diseases. The present study was conducted to evaluate the anthelmintic activity of chamomile methanolic extract (CME). METHODS: In vitro, the anthelmintic activities of CME were investigated on the L3 larvae of Heligmosomoides polygyrus in comparison to albendazole. In vivo, Swiss albino mice were infected with infective third (L3) larval stage of H. polygyrus by intragastric administration. Moreover, the effect of CME and albendazole on worm eggs, adult worms, serum cytokine production, and oxidative stress was studied. RESULTS: All used doses of CME showed a potent anthelmintic activity both in vitro and in vivo and the effect being similar to treatment with albendazole. Moreover, H. polygyrus infestation was accompanied by an intestinal oxidative stress status characterized by an increased lipoperoxidation, a depletion of antioxidant enzyme activity, as well as an overload of hydrogen peroxide. We have also recorded an increase of pro-inflammatory mediator (TNF-α, IL-6, and IL-1ß) levels after treatment with CME (14 ± 0.8; 41 ± 2; 58 ± 4 pg/mg protein, respectively, with the concentration 800 mg/kg, body weight) when compared with infected control mice (20 ± 1; 59 ± 2, and 83 ± 4 pg/mg protein, respectively). However, extract treatment alleviated all the deleterious effects associated with H. polygyrus infection. CONCLUSION: These findings suggest that CME can be used in the control of gastrointestinal helminthiasis and associated oxidative stress.

Amoebicidal activity of α-bisabolol, the main sesquiterpene in chamomile (Matricaria recutita L.) essential oil against the trophozoite stage of Acanthamoeba castellani Neff.

Acanthamoeba genus includes opportunistic pathogens which are distributed worldwide and are causative agents of a fatal encephalitis and severe keratitis in humans and other animals. Until present there are not fully effective therapeutic agents against this pathogen and thus the need to search for novel anti-amoebic compounds is urgent. Recently, essential oils of aromatic and medicinal plants have shown activity against Acanthamoeba strains. Therefore, this study was aimed to evaluate the activity of main component of chamomile essential oil (a sesquiterpene) namely α-bisabolol against the Acanthamoeba castellani Neff strain. After evaluation of the activity and toxicity of this molecule, IC50 values of 20.839 ± 2.015 for treated amoebae as well as low citotoxicty levels in a murine macrophage cell line was observed. Moreover, in order to elucidate mechanism of action of this molecule, changes in chromatin condensation levels, permeability of the plasmatic membrane, the mitochondrial membrane potential and the ATP levels in the treated amoebic strains were checked. The obtained results revealed that α-bisabolol was able to induce apoptosis, increase the permeability of the plasmatic membrane and decrease both mitochondrial and ATP levels in the treated amoebae. Therefore, and given the obtained results, α-bisabolol could be used a future therapeutic agent against Acanthamoeba infections.

First report of the in vitro nematicidal effects of camel milk.

Antipathogenic properties of camel milk have been investigated to substitute for drugs hence overcome drug resistance. The main objective of this present study was to investigate the anthelmintic activity of camel milk in relationship to its chemical composition. In vitro anthelmintic effects of camel milk against Haemonchus contortus from sheep were ascertained by egg hatching and worm motility inhibitions in comparison to milks from cow, ewe and goat as well as a reference drug albendazole. Chemical composition revealed that camel milk has higher contents of protective protein (lactoferrin) and vitamin C than other species' milk. It showed ovicidal activity at all tested concentrations and completely inhibited egg hatching at a concentration close to 100mg/mL (inhibitory concentration (IC50)=42.39mg/mL). Camel milk revealed in vitro activity against adult parasites in terms of the paralysis and/or death of the worms at different hours post treatment. After 8h of exposure, it induced 100% mortality at the highest tested concentration. There was 82.3% immobility of worms in albendazole 8h post-exposition. No such effects were seen with the other species' milks. Bioactive compounds such as lactoferrin and vitamin C may be involved in such an effect. To our knowledge, these results depict for the first time that camel milk possesses in vitro anthelmintic properties and further in vitro and in vivo trials against different parasite species and stages are required to make use of this milk for the control of gastrointestinal nematode parasites.

Correlation of polyphenolic content with radical-scavenging capacity and anthelmintic effects of Rubus ulmifolius (Rosaceae) against Haemonchus contortus.

Phenolic content, antioxidant and anthelmintic activities of herbal extracts are of particular interest to drug industry; plant extracts with significant anthelmintic activity have the potential to be used as alternatives to conventional chemical drugs. In the present study, Rubus ulmifolius fruit extracts obtained using solvents of increasing polarity (water, methanol, chloroform and hexane) were examined for their antioxidant and anthelmintic activities in correlation with their polyphenolic content. In vitro antioxidant activity of all extracts was carried out using free radical-scavenging activity by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azinobis-(3-ethilenebenzotiazolin)-6-sulfonic acid (ABTS) radical cation. In vitro anthelmintic activities were investigated on the egg and adult worms of Haemonchus contortus from sheep in comparison to albendazole. Total polyphenol content of R. ulmifolius was higher in more polar extract, ranging from 64.5 in aqueous extract to 1.57 mg gallic acid equivalents per gram of dry weight (GAE/g DW) in hexanic extract. Likewise, highest amounts of flavonoids and condensed tannins were found in aqueous extract (28.06 mg QE/g and 7.42 mg CE/g DW, respectively) compared to hexanic extract (0.71 mg QE/g and 0.29 mg CE/g DW, respectively) (p<0.05). Both DPPH and ABTS antioxidant assays showed that all tested extracts possess free radical scavenging activity, while the inhibitory concentration 50% (IC50) range values were similar for both assays (2.13-45.54 µg/mL and 1.2-43.82 µg/mL, respectively). All plant extracts showed ovicidal activity at all tested concentrations. Fruit methanolic (IC50=2.76mg/mL) and aqueous (IC50=2.08 mg/mL) extracts showed higher inhibitory effects than chloroformic (IC50=7.62 mg/mL) and hexanic (IC50=12.93 mg/mL) extracts on egg hatching (p<0.05). There was a significant correlation of total polyphenol, flavonoids and tannins content with scavenging of either DPPH (r=0.722, 0.764 and 0.752, p<0.01, respectively) or ABTS radicals (r=0.893, 0.765 and 0.722, p<0.01, respectively) and with inhibition of egg hatching (r=0.874, 0.883 and 0.862, p<0.01, respectively). Highest inhibition of motility (100%) of worms was observed 8h post-exposure in aqueous and methanolic extract at 8 mg/mL. To our knowledge, these results depict for the first time that R. ulmifolius possesses in vitro anthelmintic properties.