Bio-guided isolation of leishmanicidal and trypanocidal constituents from Pituranthos battandieri aerial parts.

Protozoan pathogens that cause neglected tropical diseases are a major public health concern in tropical and developing countries. In the course of our ongoing search for new lead compounds as potential antiprotozoal agents, this study aims to perform a bio-guided fractionation of Pituranthos battandieri, using an in vitro assay against Leishmania amazonensis and Trypanosoma cruzi. Two known polyacetylenes, (-)-panaxydiol (1) and (-)-falcarindiol (2) were identified from the ethanolic extract of the aerial parts of P. battandieri as the main bioactive constituents. Compounds 1 and 2 showed similar potency (IC50 values of 5.76 and 5.68 µM, respectively) against L. amazonensis to miltefosine (IC50 value of 6.48 µM), the reference drug, and low toxicity on macrophage cell lines J774. Moreover, compound 1 exhibited moderate activity (IC50 23.24 µM) against T. cruzi. In addition, three known furanocoumarins, 8-geranyloxypsoralen (3), 8-geranyloxy-5-methoxypsoralen (4), and phellopterin (5) were isolated. Their structures were elucidated by NMR and MS analysis. Compounds 1 and 2 are described for the first time in the Pituranthos genus, and this is the first report on their antiprotozoal activity. These results highlight this type of polyacetylenes as an interesting scaffold for the development of novel antiparasitic drugs.

Sesquiterpenoids and flavonoids from Inula viscosa induce programmed cell death in kinetoplastids.

Neglected tropical diseases such as leishmaniasis and American trypanosomiasis represent an increasing health problem. Current treatments are not satisfactory which remains an urgent need for novel, cheap and safe chemotherapies. In the course of our ongoing search for new potential anti-protozoal agents, this study aimed to perform a bio-guided fractionation of Inula viscosa (Asteraceae) using in vitro assays against three strains of Leishmania and Trypanosma genus. Eight known compounds were identified from the ethanolic extract of leaves, sesquiterpenoids (3 and 4) and flavonoids (5 and 6) were characterized as the main bioactive constituents. Sesquiterpene lactones 3 and 4 (IC50 values between 4.99 and 14.26 µM) showed promising antiparasitic activity against promastigotes of L. donovani, L. amazonensis and epimastigotes of T. cruzi. Their structures were successfully characterized by spectroscopic techniques including 1D and 2D NMR experiments. Furthermore, the main bioactive compounds 4, 5 and 6 displayed higher potency (IC50 values between 0.64 and 2.13 µM) against amastigotes of L. amazonensis than miltefosine (IC50 3.11 µM), and a low toxicity on macrophages cell line (SI > 45). The analysis of structure-activity relationship (SAR) of the anti-protozoal activity revealed that lactonization or oxidation enhanced the biological profile, suggesting that the hydrophobic moiety was presumably involved in the activity by increasing the affinity and/or cell membrane permeability. In order to get an insight into the mechanism of action of these compounds, programmed cell death (PCD) experiments were performed, and the obtained results suggest that the reported compounds induced PCD in the treated parasites. These results highlight that sesquiterpenoids and flavonoids from I. viscosa could constitute an interesting scaffold for the development of novel antikinetoplastid agents.

Antioxidant and Leishmanicidal Evaluation of Pulicaria Inuloides Root Extracts: A Bioguided Fractionation.

Leishmaniasis remains a major world health problem, and in particular, Algeria ranks second for the incidence of cutaneous leishmaniasis. Pulicaria inuloides is a well-known Arabian Peninsula medicinal plant. In the present study, the chloroform, ethyl acetate and n-butanol extracts from the roots of Pulicaria inuloides were analyzed for antioxidant activity and its correlation with the total phenolic and flavonoid contents. The highest antioxidant activity using a DPPH assay was showed by the ethyl acetate extract (IC50 4.08 µg/mL), which also had the highest total phenolic content (307.12 µgAGE). Furthermore, P. inuloides root extracts were evaluated against Leishmania amazonensis and Leishmania donovani. The results highlighted the chloroform extract as the most active one against both tested Leishmania strains. A bioguided fractionation of the chloroform extract led to the isolation of (8R:8S)-(75:25 er)-10-isobutyryloxy-8,9-epoxy-thymol isobutyrate as the main bioactive component, showing a potent leishmanicidal activity on L. amazonensis promatigote and amastigote stages (IC50 5.03 and 2.87 µM, respectively) and a good selectivity index on murine macrophages (CC50 19.37 µM). This study provides the first report of the antioxidant and leishmanicidal activities of P. inuloides root extracts and the results point to this species as a source of potential bioactive agents.

Evaluation of Oxasqualenoids from the Red Alga Laurencia viridis against Acanthamoeba.

Acanthamoeba genus is a widely distributed and opportunistic parasite with increasing importance worldwide as an emerging pathogen in the past decades. This protozoan has an active trophozoite stage, a cyst stage, and is dormant and very resistant. It can cause Acanthamoeba keratitis, an ocular sight-threatening disease, and granulomatous amoebic encephalitis, a chronic, very fatal brain pathology. In this study, the amoebicidal activity of sixteen Laurencia oxasqualenoid metabolites and semisynthetic derivatives were tested against Acanthamoeba castellanii Neff. The results obtained point out that iubol (3) and dehydrothyrsiferol (1) possess potent activities, with IC50 values of 5.30 and 12.83 µM, respectively. The hydroxylated congeners thyrsiferol (2) and 22-hydroxydehydrothyrsiferol (4), active in the same value range at IC50 13.97 and 17.00 µM, are not toxic against murine macrophages; thus, they are solid candidates for the development of new amoebicidal therapies.

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.

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.

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.

Anti-Acanthamoeba activity of Tunisian Thymus capitatus essential oil and organic extracts.

Acanthamoeba species are free-living amoebae widely distributed in the environment and which cause serious human infections. The treatment of Acanthamoeba infections is always very difficult and not constantly effective. More efficient drugs against Acanthamoeba must be developed and medicinal plants can be useful in this case. Our research focused on the examination of the anti-Acanthamoeba activity of the essential oil and the ethanolic-aqueous extract from Thymus capitatus L. The essential oil showed best activity with an IC50 of 2.73 µg/ml. The conducted Bio-guided fractionation of thyme extract result to the identification of two active compounds against the trophozoite stage of Acanthamoeba: thymol and 2,3-dihydroxy-p-cymene. The results have clearly shown that the investigated products may be successfully used against Acanthamoeba infections. These molecules that are found in plants may be an alternative for the development of new drugs.

Programmed cell death in Acanthamoeba castellanii Neff induced by several molecules present in olive leaf extracts.

Therapy against Acanthamoeba infections such as Granulomatous Amoebic Encephalitis (GAE) and Acanthamoeba Keratitis (AK), remains as an issue to be solved due to the existence of a cyst stage which is highly resistant to most chemical and physical agents. Recently, the activity of Olive Leaf Extracts (OLE) was demonstrated against Acanthamoeba species. However, the molecules involved in this activity were not identified and/or evaluated. Therefore, the aim of this study was to evaluate the activity of the main molecules which are present in OLE and secondly to study their mechanism of action in Acanthamoeba. Among the tested molecules, the observed activities ranged from an IC50 of 6.59 in the case of apigenine to an IC50 > 100 µg/ml for other molecules. After that, elucidation of the mechanism of action of these molecules was evaluated by the detection of changes in the phosphatidylserine (PS) exposure, the permeability of the plasma membrane, the mitochondrial membrane potential and the ATP levels in the treated cells. Vanillic, syringic and ursolic acids induced the higher permeabilization of the plasma membrane. Nevertheless, the mitochondrial membrane was altered by all tested molecules which were also able to decrease the ATP levels to less than 50% in IC90 treated cells after 24 h. Therefore, all the molecules tested in this study could be considered as a future therapeutic alternative against Acanthamoeba spp. Further studies are needed in order to establish the true potential of these molecules against these emerging opportunistic pathogenic protozoa.

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.

In vitro effects of triterpenic acids from olive leaf extracts on the mitochondrial membrane potential of promastigote stage of Leishmania spp.

Protozoan diseases, such as leishmaniasis, are a cause of considerable morbidity throughout the world, affecting millions every year. In this study, two triterpenic acids (maslinic and oleanolic acids) were isolated from Tunisian olive leaf extracts and their in vitro activity against the promastigotes stage of Leishmania (L.) infantum and Leishmania (L.) amazonensis was investigated. Maslinic acid showed the highest activity with an IC50 of 9.32 ± 1.654 and 12.460 ± 1.25 µg/ml against L. infantum and L. amazonensis, respectively. The mechanism of action of these drugs was investigated by detecting changes in the phosphatidylserine (PS) exposure, the plasma membrane permeability, the mitochondrial membrane potential and the ATP level production in the treated parasites. By using the fluorescent probe SYTOX® Green, both triterpenic acids showed that they produce a time-dependent plasma membrane permeabilization in the treated Leishmania species. In addition, spectrofluorimeteric data revealed the surface exposure of PS in promastigotes. Both molecules reduced the mitochondrial membrane potential and decreased the ATP levels to 15% in parasites treated with IC90 for 24h. We conclude that the triterpenic acids tested in this study, show potential as future therapeutic alternative against leishmaniasis. Further studies are needed to confirm this.

Bioassay guided isolation and identification of anti-Acanthamoeba compounds from Tunisian olive leaf extracts.

Pathogenic Acanthamoeba strains are causative agents of Granulomatous Amoebic Encephalitis (GAE) and Acanthamoeba keratitis (AK) worldwide. The existence of the cyst stage complicates Acanthamoeba therapy as it is highly resistant to antibiotics and physical agents. The aim of this study was to investigate the activity of Limouni olive leaf cultivar against the trophozoite stage of Acanthamoeba. The ethyl acetate and methanol extracts of this variety were tested against Acanthamoeba castellanii Neff. The ethyl acetate extract of olive leaf was the most active showing an IC50 of 5.11±0.71µg/ml of dry extract. Bio-guided fractionation of this extract was conducted and led to the identification of three active compounds namely oleanolic and maslinic acids and oleuropein which could be used for the development of novel therapeutic approaches against Acanthamoeba infections.

Activity of olive leaf extracts against the promastigote stage of Leishmania species and their correlation with the antioxidant activity.

Leishmaniasis is one of the neglected tropical diseases in terms of drug discovery and development. Furthermore, the chemotherapy used to treat this disease has been proved to be highly toxic and to present resistance issues. As consequent, the need for novel leishmanicidal molecules has notably increased in the recent years. In the present work an attempt was made to evaluate the antioxidant and leishmanicidal activities besides presence of compounds in leaf extracts of 5 different Tunisian olive tree varieties, used as traditional medicine in this country. The concentration of extracts needed to inhibit 50% of the parasitic growth (IC50) was estimated using different Leishmania strains. All tested extracts showed an inhibitory effect on the parasite growth with IC50 ranging from 2.130±0.023 to 71.570±4.324µg/ml, respectively for the methanolic extracts of Limouni and Zarrazi against Leishmania donovani. In fact, this activity was significantly affected by the olive cultivar and the tested Leishmania strain. Furthermore, the activities against both Leishmania tropica and major species were correlated to the total phenolic compounds. These results could suggest that olive leaf extract could carry potential new compounds for the development of novel drugs against Leishmaniasis.