In vitro Anti-Leishmanial Activities of Methanol Extract of Brucea antidysenterica J.F. Mill Seeds and Its Solvent Fractions.

Introduction: Leishmaniasis is one of the neglected tropical diseases, threatening lives of about 350 million people globally. Brucea antidysenterica seeds are used for the treatment of cutaneous leishmaniasis in the traditional medicine in Ethiopia. Objective: This study aimed to evaluate Brucea antidysenterica seeds' anti-leishmanial activity in vitro. Methods: The crude (80% methanol) extract of Brucea antidysenterica seeds and its fractions were evaluated for their anti-leishmanial activities against promastigotes and intracellular amastigotes of Leishmania donovani and Leishmania aethiopica, and for their cytotoxic effects against mammalian cells. The quantitative estimations of total phenolic compounds (TPCs), flavonoids (TFCs) and alkaloids (TACs) were determined, spectrophotometrically. Median inhibitory concentration (IC50) and median cytotoxic concentration (CC50) of the extract and its solvent fractions were calculated using GraphPad Prism 9.1.0 computer software. Data was presented as mean ± standard error of the mean (SEM). Results: The crude extract and its hexane, ethyl acetate and butanol fractions showed anti-leishmanial activities, with IC50 values of 4.14-60.12 µg/mL against promastigotes, and 6.16-40.12 µg/mL against amastigotes of both Leishmania species. They showed moderate cytotoxicity against Vero cell lines and peritoneal mice macrophages, with CC50 values of 100-500 µg/mL, but >1600 µg/mL against red blood cells. Selectivity indices ranged from 7.97 to 30.97. The crude extract, and its ethyl acetate and hexane fractions possessed 54.78-127.72 mg of gallic acid equivalent TPC, 18.30-79.21 mg of quercetin equivalent TFC, and 27.62-97.22 mg of atropine equivalent TAC per gram of extracts. Conclusion: The seeds of the plant possessed anti-leishmanial activities against L. aethiopica and L. donovani that might provide a scientific justification for its use in the treatment of leishmaniasis by traditional healers. Future works are recommended to isolate, purify and identify the possible secondary metabolites attributed to the anti-leishmanial activity.

In Vitro Antileishmanial and Antischistosomal Activities of Anemonin Isolated from the Fresh Leaves of Ranunculus multifidus Forsk.

Leishmaniasis and schistosomiasis are neglected tropical diseases (NTDs) infecting the world's poorest populations. Effectiveness of the current antileishmanial and antischistosomal therapies are significantly declining, which calls for an urgent need of new effective and safe drugs. In Ethiopia fresh leaves of Ranunculus multifidus Forsk. are traditionally used for the treatment of various ailments including leishmaniasis and eradication of intestinal worms. In the current study, anemonin isolated from the fresh leaves of R. multifidus was assessed for its in vitro antileishmanial and antischistosomal activities. Anemonin was isolated from the hydro-distilled extract of the leaves of R. multifidus. Antileishmanial activity was assessed on clinical isolates of the promastigote and amastigote forms of Leishmania aethiopica and L. donovani clinical isolates. Resazurin reduction assay was used to determine antipromastigote activity, while macrophages were employed for antiamastigote and cytotoxicity assays. Antischistosomal assays were performed against adult Schistosoma mansoni and newly transformed schistosomules (NTS). Anemonin displayed significant antileishmanial activity with IC50 values of 1.33 nM and 1.58 nM against promastigotes and 1.24 nM and 1.91 nM against amastigotes of L. aethiopica and L. donovani, respectively. It also showed moderate activity against adult S. mansoni and NTS (49% activity against adult S. mansoni at 10 µM and 41% activity against NTS at 1 µM). The results obtained in this investigation indicate that anemonin has the potential to be used as a template for designing novel antileishmanial and antischistosomal pharmacophores.

Leishmania donovani Growth Inhibitors from Pathogen Box Compounds of Medicine for Malaria Venture.

INTRODUCTION: Leishmaniasis is a collective term used to describe various pathological conditions caused by an obligate intracellular protozoan of the genus Leishmania. It is one of the neglected diseases and has been given minimal attention by drug discovery and development stakeholders to narrow the safety and efficacy gaps of the drugs currently used to treat leishmaniasis. The challenge is further exacerbated by the emergence of drug resistance by the parasites. METHODS: Aiming to look for potential anti-leishmanial hits and leads, we screened Medicines for Malaria Venture (MMV) Pathogen Box compounds against clinically isolated Leishmania donovani strain. In this medium-throughput primary screening assay, the compounds were screened against promastigotes, and then against amastigote stages. RESULTS: From the total 400 compounds screened, 35 compounds showed >50% inhibitory activity on promastigotes in the initial screen (1 µM). Out of these compounds, nine showed >70% inhibition, with median inhibitory concentration (IC50) ranging from 12 to 491 nM using the anti-promastigote assay, and from 53 to 704 nM using the intracellular amastigote assay. Identified compounds demonstrated acceptable safety profiles on THP-1 cell lines and sheep red blood cells, and had appropriate physicochemical properties suitable for further drug development. Two compounds (MMV690102 and MMV688262) were identified as leads. The anti-tubercular agent MMV688262 (delamanid) showed a synergistic effect with amphotericin B, indicating the prospect of using this compound for combination therapy. CONCLUSION: The current study indicates the presence of additional hits which may hold promise as starting points for anti-leishmanial drug discovery and in-depth structure-activity relationship studies.

Antileishmanial Evaluation of the Leaf Latex of Aloe macrocarpa, Aloin A/B, and Its Semisynthetic Derivatives against Two Leishmania Species.

The currently available antileishmanial drugs are either toxic or too expensive for routine use in developing countries where the disease is most common. Local people in the Somalia region of Ethiopia use the leaves of Aloe macrocarpa Todaro for the treatment of malaria, jaundice, and skin diseases. In our ongoing search for new, efficient, and safe antileishmanial drugs, we investigated the leaf latex of Aloe macrocarpa and its acid-hydrolyzed product aloin A/B (1), as well as the semisynthesized derivatives of aloin A/B, namely, aloe-emodin (2) and rhein (3) against promastigotes and axenically cultured amastigotes of Leishmania aethiopica and L. donovani clinical isolates. Activity study was carried out based on the fluorescence characteristic of resazurin added to drug-treated cultures. Oxidative hydrolysis of aloin A/B by ferric chloride and concentrated hydrochloric acid afforded aloe-emodin (2), which was further oxidized using sodium nitrite and concentrated sulfuric acid to furnish rhein (3). Cytotoxicity study of test substances was performed against human monocytic cell line THP-1 using Alamar Blue and cell viability was measured fluorometrically. The test compounds showed lower activity (IC50 = 6.7 to 12.1 µM for promastigotes and IC50 = 3.6 to 10.2 µM for axenic amastigotes) than the reference drug amphotericin B (IC50 = 1.3 to 2.7 µM). However, amphotericin B (LC50 = 11.1 µM) was much more toxic than the test compounds (LC50 = 369.2 - 611.6 µM) towards human monocytic cell line (THP-1) despite its efficiency. As demonstrated in the current study, high selectivity indices (SIs) of the test compounds represent a remarkable advantage over the reference drug and highlight their potential use as templates for further development of safe leishmanicidal drugs.

Safety and efficacy of liposomal amphotericin B for treatment of complicated visceral leishmaniasis in patients without HIV, North-West Ethiopia.

BACKGROUND: Visceral leishmaniasis (VL) is a protozoan disease that is invariably fatal if left untreated. The disease is found in 70 countries with incidence of 0.2 - 0.4 million cases. The mainstay of treatment in resource limited countries like Ethiopia is antimonials, while use of liposomal amphotericin B is reserved for treatment of complicated VL cases. The aim of this study was to assess the safety and efficacy of liposomal amphotericin B in HIV negative VL patients diagnosed with complications. METHODS: A retrospective chart review was conducted involving records of patients admitted between January 2009 and December 2014. Baseline sociodemographic, clinical, and treatment outcome data were collected. The doses of liposomal amphotericin B and adverse events related to treatment were retrieved. Categorical and continuous variables respectively were analyzed by Chi-square and Mann-Whitney U tests. A p-value of less than 0.05 was considered statistically significant. RESULTS: A total of 147 patients with severe VL were treated with liposomal amphotericin B in total dose ranges of 20 mg/kg to 35 mg/kg. In the overall treatment outcome analysis, initial cure (30 days after start of treatment) was observed in 128 (87.1 %), treatment failures in 10 (6.8 %), interruptions in 2(1.4 %) and deaths in 7 (4.8 %) patients. Initial cure rate at high dose (24-35 mg/kg total dose) was 96.7 % (59/61) versus 80.2 % (69/86) at lower doses (<24 mg/kg); which was significantly higher (P < 0.01), OR = 4.56: 95 %, Confidence Interval (CI) = 1.17 - 20.78). Ten cases (11.8 %) of treatment failure occurred in the low dose treatment group. The most common adverse events (AEs) were hypokalemia in 39 cases (26.5 %) and infusion related reactions in 16 (10.9 %). The frequency of hypokalemia and infusion related reactions were not significantly different between the low and high dose liposomal amphotericin B. CONCLUSION: In HIV negative complicated VL patients, high dose of liposomal amphotericin B was found to have high cure rate at the end of treatment. The appropriate dose for better efficacy needs to be determined. Monitoring serum potassium level during treatment with liposomal amphotericin B should be an essential component of the clinical management of VL.

Successful Treatment of Human Visceral Leishmaniasis Restores Antigen-Specific IFN-γ, but not IL-10 Production.

One of the key immunological characteristics of active visceral leishmaniasis (VL) is a profound immunosuppression and impaired production of Interferon-γ (IFN-γ). However, recent studies from Bihar in India showed using a whole blood assay, that whole blood cells have maintained the capacity to produce IFN-γ. Here we tested the hypothesis that a population of low-density granulocytes (LDG) might contribute to T cell responses hyporesponsiveness via the release of arginase. Our results show that this population is affected by the anticoagulant used to collect blood: the frequency of LDGs is significantly lower when the blood is collected with heparin as compared to EDTA; however, the anticoagulant does not impact on the levels of arginase released. Next, we assessed the capacity of whole blood cells from patients with active VL to produce IFN-γ and IL-10 in response to antigen-specific and polyclonal activation. Our results show that whole blood cells produce low or levels below detection limit of IFN-γ and IL-10, however, after successful treatment of VL patients, these cells gradually regain their capacity to produce IFN-γ, but not IL-10, in response to activation. These results suggest that in contrast to VL patients from Bihar, India, whole blood cells from VL patients from Gondar, Ethiopia, have lost their ability to produce IFN-γ during active VL and that active disease is not associated with sustained levels of IL-10 production following stimulation.

Phytochemistry and antileishmanial activity of the leaf latex of Aloe calidophila Reynolds.

Leishmaniasis is a major protozoal disease threatening the lives of 350 million people throughout the world. However, the therapeutic options for the disease are limited. In the present study, the antiprotozoal activity of the latex obtained from the Ethiopian plant Aloe calidophila Reynolds was evaluated by in vitro testing against Leishmania aethiopica and Leishmania major. It was found that the latex possesses moderate activity against both parasites with IC50 values of 64.05 and 82.29 µg/mL, respectively. Phytochemical investigation resulted in the isolation of three anthrones identified as aloinoside, aloin, and microdontin on the basis of IR, MS, (1) H NMR, and (13) C NMR spectral data. The isolated compounds showed strong antileishmanial activity with IC50 values ranging from 1.76 to 6.32 µg/mL against L. aethiopica and from 2.09 to 8.85 µg/mL against L. major. Although these values were higher than those of amphotericin B (IC50 = 0.109 and 0.067 µg/mL), the selectivity indices (813.35 and 694.90, respectively, against L. aethiopica and L. major) of aloinoside were much better than those of the standard drug (423.49 and 688.96). The results indicate that the isolated compounds have the potential to be used as a scaffold for the development of safe and cost-effective antileishmanial agents.

Constituents, antileishmanial activity and toxicity profile of volatile oil from berries of Croton macrostachyus.

The chemical composition of the volatile oil from berries of Croton macrostachyus Hochst. ex Del. was determined by GC and GC/MS. The oil was tested for its in vitro antileishmanial activity on two Leishmania strains, and its toxicity on the human monocytic leukemia (THP-1) cell line and erythrocytes from sheep blood. The main constituents of the oil were benzyl benzoate (51.8%), linalool (10.1%), gamma-muurolene (9.3%), (E,E)-alpha-farnesene (3.2%), delta-cadinene (2.8%) and alpha-curcumene (2.7%). The oil was effective against L. donovani and L. aethiopica promastigotes (MIC = 0.08 microL/mL and 0.16 microL/mL, respectively) and axenic amastigote stages (EC50 = 20.00 nL/mL and 6.66 nL/mL, respectively). The CC50 value for the oil was 10.00 nL/mL on THP-1 cells with selectivity index values of 0.5 for L. donovani and 1.5 for L. aethiopica. The median lethal concentration (LC50) of the oil was 2.45 microL/mL. Thus the observed high efficacy and moderate toxicity of the volatile oil from C. macrostachyus, makes the plant a promising source of new lead compounds in the search for safe and effective antileishmanial drugs.

Essential-oil composition, antileishmanial, and toxicity study of Artemisia abyssinica and Satureja punctata ssp. punctata from Ethiopia.

Essential oils of Artemisia abyssinica and Satureja punctata ssp. punctata from Ethiopia were analyzed by GC and GC/MS, and screened for leishmanicidal activity against promastigote and axenic amastigotes of Leishmania donovani and L. aethiopica, including toxicity studies on human monocytic leukemia cells (THP-1) and erythrocytes in vitro. GC/MS of A. abyssinica oil revealed 67 compounds (99.94%) with the major constituents yomogi alcohol (38.47%), artemisyl acetate (24.88%), and artemisia alcohol (6.70%), and oxygenated monoterpenes (84.00%) as the dominant group. The oil of S. punctata contained 67 compounds (99.49%) with the main constituents geranial (27.62%), neral (21.72%), alpha-bisabolol (13.62%), and (E)-nerolidol (4.82%), of which oxygenated mono- and sesquiterpenes (58.39 and 26.91%, resp.) showed highest abundance. Both oils showed effect on promastigotes (MIC 76.5 to 312.5 nl/ml) and amastigotes (EC(50) 4.06 to 131.00 nl/ml) of L. donovani and L. aethiopica, and varying toxicities on THP-1 cells (CC(50) 0.013 to 350 nl/ml with selectivity index between 0.001 and 28) and erythrocytes (with LC(50) 0.35 to 1.52 microl/ml). S. punctata oil exerted highest activity against both Leishmania sp. and toxicity. The revealed antileishmanial activities support further isolation and investigation of oil constituents for in vitro/in vivo evaluation.

Local suppression of T cell responses by arginase-induced L-arginine depletion in nonhealing leishmaniasis.

The balance between T helper (Th) 1 and Th2 cell responses is a major determinant of the outcome of experimental leishmaniasis, but polarized Th1 or Th2 responses are not sufficient to account for healing or nonhealing. Here we show that high arginase activity, a hallmark of nonhealing disease, is primarily expressed locally at the site of pathology. The high arginase activity causes local depletion of L-arginine, which impairs the capacity of T cells in the lesion to proliferate and to produce interferon-gamma, while T cells in the local draining lymph nodes respond normally. Healing, induced by chemotherapy, resulted in control of arginase activity and reversal of local immunosuppression. Moreover, competitive inhibition of arginase as well as supplementation with L-arginine restored T cell effector functions and reduced pathology and parasite growth at the site of lesions. These results demonstrate that in nonhealing leishmaniasis, arginase-induced L-arginine depletion results in impaired T cell responses. Our results identify a novel mechanism in leishmaniasis that contributes to the failure to heal persistent lesions and suggest new approaches to therapy.

Prevalence of onchocercal skin disease and infection among workers of coffee plantation farms in Teppi, southwestern Ethiopia.

In June 1996, a study on the economic impacts of onchocercal skin disease was initiated in southwestern Ethiopia. We made parasitological and clinicoepidemiological investigations among 1619 workers of a coffee plantation firm in Teppi, south-western Ethiopia. Sixty percent of the workers were included in the study. The prevalence of onchocercal skin disease (OSD) was 85.3%. Severe OSD (SOSD) was found in 17.3% of the study subjects. This was 1/5 of all OSD cases. The overall nodule carrier rate was 44.2%, which differed significantly by age classes from a rate of 12.3% to 73.0%. This rate varied by sex, 51.7% in males and 22.6% in females. Microfilarial carrier rate (MFCR) was 77.6%. This rate did not vary neither with severity of disease nor with presence or absence of pruritus or onchodermatitis. Mean microfilarial count was determined to be 38.1 per mg of skin snip or 44.4 per mg of infected skin snips. The geometric mean of microfilarial load per infected skin was 23.8. The community microfilarial load (CMFL) was estimated to be 14.0 per mg skin snip. The study showed that SOSD is prevalent in Teppi and affects a substantial number of the working population. An intervention program is called for.