Four-day Suppressive Test Research Articles

In vivo antiplasmodial potential of three herbal methanolic extracts in mice infected with Plasmodium berghei NK65

ObjectiveThe present study deals with the investigation of antiplasmodial potential of leaf methanolic extract of Aegle marmelos, Aristolochia indica and Cassia auriculata against Plasmodium berghei (NK65) infected mice. MethodsThe chloroquine-sensitive parasites P. berghei (1 × 106) were inoculated into Swiss albino mice intraperitoneally. The methanol extracts of three herbal plants were orally administered in P. berghei infected mice which were further assessed using the four-day suppressive test at different doses of 150, 300 and 600 mg/kg per day. Chloroquine (CQ) was used as the standard drug with of 1.25, 2.5 and 5 mg/kg concentrations and was orally administered. ResultsThe leaves of A. marmelos, A. indica, and C. auriculata were found to suppress P. berghei parasitaemia in Swiss albino mice by (67.0 ± 4.02)%, (72.0 ± 8.44)% and (52.7 ± 2.06)% at 600 mg/kg/d with ED50 values of 284.73, 233.77 and 562.48 mg/kg, respectively. These herbal plants increased the mean survival time of infected mice and prevented body weight loss. GC-MS analysis revealed the presence of hentriacontan-16-one (C31H62O) in A. indica extract. The histopathology study showed non-toxic to kidney and liver at 600 mg/kg/body weight. ConclusionsOverall results revealed that herbal plants may be active in the development of novel and cheap antimalarial compounds.

Cysteine Protease Inhibitors from the Methanol Extract of the Root Bark of Securidaca longepedunculata with Antimalarial Potentials in Chloroquine-Resistant P. berghei Parasite

Malaria parasite resistance against Artemisinin-based Combination Therapy (ACT) in some parts of the world necessitates the search for antimalarial compounds or plant extracts with novel mode of action and active against the chloroquine-resistant strain of the parasite to serve as alternative to ACT. Cysteine protease inhibitors' fraction of the methanol extract of the root bark of Securidaca longepedunculata (CPI) was investigated for antiplasmodial activity against chloroquine-resistant P. berghei-infected mice and its inhibitory effect on papain, P. berghei cysteine proteases and heme biocystallization were also evaluated. The methanol extract of the root bark was obtained by soxhlet extraction with 1,000 mL of 70% (v/v) methanol for 48 hours and concentrated to dryness at 45∘C. CPI was obtained using PBS (pH 7) extraction followed by cold acetone precipitation. Peter's four-day suppressive and Rane's four-day curative test was employed to assess the antimalarial potentials of CPI. Data was analysed with one way ANOVA followed by Dunnett's post hoc test, differences were considered significant at p≤0.05. The suppressive effect of CPI was significant (p≤0.05) at 34 and 23 mg/kg doses. Doses of 34, 23 and 11 mg/kg produced significant (p≤0.05) dose-dependent curative effect. CPI inhibited the proteolytic activity of papain enzyme and P. berghei cysteine proteases in vitro with IC50 values of 20.1 and 5.6 μg/mL respectively. The present study showed that cysteine protease inhibitors fraction of the methanol extract of the root bark of Securidaca longepedunculata is a potential source of novel antimalarial agents that could target malaria parasite cysteine proteases.

Antimalarial Potential of the Leaf Exudate of Aloe macrocarpa Todaro and its Major Constituents against Plasmodium berghei

Background: Aloe macrocarpa Todaro is one of the Aloe species in Ethiopia where its leaf exudate is traditionally used for the treatment of various ailments, including malaria.Methods: Two pure compounds namely aloin and aloinoside were isolated from the leaf exudate. Further oxidative hydrolysis yielded another compound (aloe-emodin). Four-day suppressive test was employed to evaluate the antimalarial potential of the leaf exudates and isolated compounds as well as aloe-emodin against mice infected with Plasmodium berghei.Results: The leaf exudate of A. macrocarpa showed significant chemosuppression of P. berghei at all dose levels tested compared to the negative control group. At a dose of 400 mg/kg, the leaf exudate of A. macrocarpa, the isolated aloin as well as the semi-synthetic derivative (aloe-emodin) suppressed parasite growth by 74.3, 64.2 and 94.7%, respectively, which was statistically significant (p<0.01) compared to the negative control group. Moreover, maximal dose of aloinoside (400 mg) exhibited similar chemosuppression effect (100%) and comparable weight loss prevention with chloroquine.Conclusion: The present study indicated that aloin, aloinoside and aloe-emodin are promising antimalarial principles of A. macrocarpa, and further support the traditionally claimed use of the plant against malaria.

Antimalarial activity of selected Ethiopian medicinal plants in mice

Context: Parasites are the leading killers in subtropical areas of which malaria took the lion share from protozoan diseases. Measuring the impact of antimalarial drug resistance is difficult, and the impact may not be recognized until it is severe, especially in high transmission areas. Aims: To evaluate the in vivo antimalarial activities of hydroalcoholic extracts of the roots of Piper capense and Adhatoda schimperiana, against Plasmodium berghei in mice. Methods: Four-day suppressive and curative test animal models were used to explore the antimalarial activities of the plants. 200, 400, and 600 mg/kg of each plant extract was administered to check the activities versus vehicle administered mice. Mean survival time and level of parasitemia were the major variables employed to compare the efficacy vs. negative control. Results: In both models the 400 and 600 mg/kg doses of Adhatoda schimperiana and the 600 mg/kg dose Piper capense. showed significant parasitemia suppression and increased in mean survival time at p≤0.05. The middle dose of Piper capense had a border line inhibition where the extracts were considered active when parasitemia was reduced by ≥ 30%. Conclusions: The hydroalcoholic extracts of the roots of Adhatoda schimperiana and Piper capense possess moderate antimalarial activities, which prove its traditional claims. Thus, further studies should be done to isolate the active constituents for future use in the modern drug discovery.

Phytochemical screening and In Vivo antimalarial activities of crude extracts of Lantana trifolia root and Premna oligotricha leaves in plasmodium berghei infected mice

Resistance of malaria parasites to several antimalarial drugs mandates the need for new compounds from affordable sources. Thus the current study was conducted to justify the traditional usage of Lantana trifolia roots and Premna oligotricha leaves to combat malaria. The powdered roots of Lantana trifolia and leaves of Premna oligotricha were macerated using 70% ethanol. Secondary metabolites present in the extracts were determined using the standard chemical method. The in vivo antiplasmodial effect of the crude extracts were evaluated using the four-day suppressive test in chloroquine (CQ) sensitive strain of Plasmodium berghei infected mice. Parameters such as parasitaemia, packed cell volume, body weight change and survival time of mice were then determined as using standard tests. The ethanol extracts showed a dose-dependent chemosuppression when compared to the negative control in this study. The chemosuppressive effect produced by all doses was very significant (P <0.001). Concentrations at 350, 500 mg/kg of leaf extract and 200 mg/kg from root extract have demonstrated prevention effect on weight loss of mice (P <0.001). All the test extracts employed in this study have no significant prevention effect on PCV loss of mice at all dose levels. The present study has demonstrated the efficacy of the extracts of Premna oligotricha leaf and Lantana trifolia root traditionally used in chemotherapy of malaria infection. These observations provide the basis for the traditional use of these plants in treatments of malaria and potential for development of novel therapeutics in the conventional medicine. Key words: Antimalarial, In vivo, Lantana trifolia, parasitaemia, Plasmodium berghei, Premna oligotricha.

Enhanced antimalalarial activity of a prolonged release in situ gel of arteether–lumefantrine in a murine model

The World Health Organization (WHO) recommends artemisinin-based combination therapy (ACT) for treatment of falciparum malaria. Arteether (ART), an artemisinin derivative, is effective against Plasmodium falciparum, but it is available only as painful oily intramuscular (i.m.) injections. We formulated lyotropic liquid crystalline preconcentrates of ART and Lumefantrine (LUM) ACT with and without biodegradable polymer for antimalarial therapy. Following i.m. injection, both formed intact gels in situ due to rapid transition into liquid crystalline phase (LCP) which was confirmed by small angle neutron scattering (SANS), X-ray diffraction (XRD), polarization optical microscopy (POM) and rheological changes. Ex vivo release studies revealed prolong release of ART-LUM over 72 h from polymeric lyotropic liquid crystalline phases (P-LLCPr). In vitro hemolysis assay and myotoxicity studies confirmed intramuscular safety. Treatment with ART–LUM P-LLCPr conferred complete protection with no mortality at 1/40th of therapeutic dose in modified Peter’s four-day suppressive test as compared to marketed ART formulation resulted in 100% mortality within 20 days. In the clinical simulation model, P-LLCPr treatment resulted in complete cure with no recrudescence or mortality at 1/20th of therapeutic dose, while marketed formulation which resulted in 100% mortality. The high efficacy with significantly reduced dose and a single administration with single shot therapy suggest ART-LUM P-LLCPr as a promising new patient friendly alternative for antimalarial therapy.

Antimalarial Activity of Solvent and Alkaloid Fractions of &lt;i&gt;Kleinia abyssinica&lt;/i&gt;

In Ethiopia, the roots of Kleinia abyssinica (A. Rich.) A. Berger are used traditionally for the treatment of malaria. In the present study, the 80% methanol extract as well as solvent and alkaloid fractions prepared from the powdered roots of K. abyssinica were tested for their in vivo antimalarial activity using mice infected with Plasmodium berghei parasites in a four-day suppressive test. Preliminary investigation showed that the crude 80% methanol extract significantly (P < 0.05) inhibited parsitaemia of P. berghei in mice. Investigation of the activity of solvent fractions of the plant revealed that the petroleum ether and methanol fractions displayed better suppression than those of the chloroform and acetone fractions. However, the alkaloid extract obtained from the methanol fraction displayed maximum activity suppressing significantly (p < 0.05) the growth of P. berghei at dose levels of 100, 200, 400, and 800 mg/kg/day compared to the mice in the negative control group. The ED50 for the total alkaloid was found to be 60.77 mg/kg/day. In conclusion the present study confirmed the traditional uses of the plant in the treatment of malaria and that alkaloids contribute to the overall antimalarial activity. Keywords : Kleinia abyssinica , roots, four-day suppressive test, Plasmodium berghei, alkaloid fraction

THERAPEUTIC POTENTIALS OF ETHANOLIC EXTRACT OF LEAVES OF HOLARRHENA FLORIBUNDA (G. DON) DUR. AND SCHINZ (APOCYNACEAE).

Background:Holarrhena floribunda is a plant of wide usage in the Togolese folk medicine. A previous ethnobotanical survey on the latex plants of the Maritime region of the country revealed that this plant was included in several recipes curing malaria and microbial infections. Therefore, this study aimed to seek for the effectiveness of the ethanolic extract of the plant in the treatment of these diseases.Methods:The antimicrobial test was performed using the agar well-diffusion and the NCCLS broth microdilution methods, while the in vivo antimalarial activity was evaluated following the four-day suppressive test of Peters. The acute toxic effects of the extract were monitored after a single oral dose (5,000 mg/kg body weight) administration in NMRI mice.Results:The results indicated that the ethanolic extract of leaves of H. floribunda was active on Staphylococcus aureus ATCC 29213 and clinical strains of Staphylococcus aureus, Salmonella typhi and Klebsiella pneumoniae with MICs ranging from 0.62 to 1.25 mg/mL. The extract also showed significant parasitaemia suppression in a dose-dependent manner. In the acute toxicity assay, the oral administration of the extract to the mice did not affect the relative weight of vital organs, and there were no signs of toxicity or death during the study period. The LD50 of the tested extract was found to be greater than 5,000 mg/kg, indicating its safety.Conclusion:This study demonstrates the antibacterial and antimalarial activities of leaves of H. floribunda and then, supports its medicinal use in the treatment of microbial infections.

Antimalarial Activity of the Chemical Constituents of the Leaf Latex of Aloe pulcherrima Gilbert and Sebsebe.

Malaria is one of the three major global public health threats due to a wide spread resistance of the parasites to the standard antimalarial drugs. Considering this growing problem, the ethnomedicinal approach in the search for new antimalarial drugs from plant sources has proven to be more effective and inexpensive. The leaves of Aloe pulcherrima Gilbert and Sebsebe, an endemic Ethiopian plant, are locally used for the treatment of malaria and other infectious diseases. Application of the leaf latex of A. pulcherrima on preparative silica gel TLC led to the isolation of two C-glycosylated anthrones, identified as nataloin (1) and 7-hydroxyaloin (2) by spectroscopic techniques (UV, IR, 1H- and 13C-NMR, HR-ESIMS). Both the latex and isolated compounds displayed antimalarial activity in a dose-independent manner using a four-day suppressive test, with the highest percent suppression of 56.2% achieved at 200 mg/kg/day for 2. The results indicate that both the leaf latex of A. pulcherrima and its two major constituents are endowed with antiplasmodial activities, which support the traditional use of the leaves of the plant for the treatment of malaria.

Antiplasmodial, antioxidant and immunomodulatory activities of ethanol extract of Vernonia amygdalina del. Leaf in Swiss mice.

Vernonia amygdalina (V. amygdalina) leaf is locally employed in the Southern region of Nigeria in the treatment of malaria infection. This study evaluated the in vivo antiplasmodial, antioxidant and immunomodulatory effect of ethanol extract of V. amygdalina leaf. The active principles of the dried leaf were extracted with ethanol. For quality validation, chemical finger-print of the extract was performed through high performance thin layer chromatography (HPTLC). The extract was assessed for antiplasmodial activity by the standard four-day suppressive test on Plasmodium berghei (ANKA) infected male Swiss mice (six weeks old) placed into five groups of six animals each. The absorption spectra from the HPTLC revealed several peaks suggesting presence of some bioactive compounds. Results from the in vivo study showed that the ethanol extract of the plant leaf was significantly active against P. berghei in a dose-dependent manner with the minimum and maximum activity observed in the mice treated orally with 100mg/kg (% inhibition of 23.7%) and 1000 mg/kg (% inhibition of 82.3 %) of the extract, respectively, on day four of the study. There was also a dose-dependent decrease (p<0.05) in some oxidative stress indices including nitric oxide and lipid peroxidation levels in the extract treated groups as against the non-treated infected group which had high levels of these parameters. The pro-inflammatory cytokines (TNF-α and IFN-ɣ) levels were also considerably low in the extract treated groups relative to the non-treated infected group. The results suggest that ethanol extract of V. amygdalina leaf was active, with some immunomodulatory effect, against P . berghei infection.

Inhibitory effects of 19 antiprotozoal drugs and antibiotics on Babesia microti infection in BALB/c mice.

Different results have been achieved in the evaluation of antiparasitic drug activity in Mongolian jirds, hamsters, and BALB/c mice infected with Babesia microti. The aims of the present study were to find a preferable method for drug screening and to re-evaluate the activity of several drugs against B. microti. The activity of 19 drugs on B. microti-infected BALB/c mice was evaluated. The study was built on Peters' four-day suppressive test, and the pathogenicity of the blood from the treated mice was also used as indicator. The results showed that 15 of the 19 drugs had little or no in vivo effect against B. microti. The inhibitory rates of atovaquone and azithromycin were high at all doses, but the microscopy-negative blood of recovered mice was still infectious. Similar to robenidine hydrochloride at 25 and 50 mg/kg, primaquine at 100 mg/kg had a 100% inhibitory rate. Robenidine hydrochloride achieved a 100% inhibitory rate at 100 mg/kg, and the blood of recovered mice did not result in parasitemia in subpassage experiments. Parasite-negative blood from mice treated with antimalarial drugs (clinically used for babesiosis) still caused parasitemia in subpassage experiments. This suggests that these drugs cannot eradicate the parasites. Peters' four-day suppressive test and the pathogenicity of the blood from the treated mice are suitable methods for preliminary investigating possible drugs against B. microti. Considering that robenidine hydrochloride achieved the best activity against B. microti in BALB/c mice in our study, further studies are needed.

Protective activity of biflavanones from Garcinia kola against Plasmodium infection

Ethnopharmacological relevanceGarcinia kola is a medicinal plant traditionally used for malaria therapy in Central Africa. Aim of the studyTo evaluate the antimalarial potencies in vitro and in vivo of pure biflavanones from G. kola. Materials and methodsThe pure biflavanones were obtained by bioassay-guided fractionation of a 70% ethanol extract of G. kola seeds and their chemical structures were elucidated by comparison of their NMR (1H and 13C) and mass spectral data with those provided in the literature. Plasmodium falciparum (FCR-3, cycloguanil-resistant strain from Gambia) was used for in vitro assessments of antimalarial activities. Growth inhibition, intraerythrocytic development and parasite morphology were evaluated in culture by the microscopic observation of Giemsa-stained thin blood films. The cytotoxicity of the antimalarial compounds was evaluated against KB 3-1 (human epidermoid carcinoma) cells by MTT assay. In vivo antimalarial activities were determined in mice infected with Plasmodium berghei (ANKA strain) following a four-day suppressive test. ResultsThe bioassay-guided fractionation of an extract of G. kola resulted in the isolation of three biflavanones (GB-1a, GB-1, and GB-2) as its active principles. These three biflavanones displayed not only potent inhibitory activity in vitro against P. falciparum proliferation but also antimalarial potency through oral administration in mice infected with P. berghei without signs of acute toxicity. GB-1 was found to exhibit the strongest in vitro antimalarial potency on P. falciparum with an IC50 of 0.16μM, whereas it exhibited a very low in vitro cytotoxicity on KB 3-1 cells with an IC50 of greater than 150μM. During an in vivo antimalarial assay in mice infected with P. berghei, GB-1 was found to exhibit biological potency with an approximate ED50 of 100mg/kg following oral administration. GB-1 was also shown to increase the average life span of the infected mice significantly compared to that of control mice (p<0.01 Student’s t-test). ConclusionsThe antimalarial outcome of GB-1a, GB-1, and GB-2 may be related to the traditional utilization of this crude drug against malaria judging from their significant content in G. kola nuts. GB-1 showed the most potent antimalarial activity with a high selectivity index and, therefore could be exploited to identify the molecular target, which subsequently could be helpful to design novel therapeutics against malaria. GB-1 may be considered a promising antimalarial candidate for trial in vivo using higher animals infected with P. falciparum.

In vivo antiplasmodial and toxicological effect of crude ethanol extract of Echinops kebericho traditionally used in treatment of malaria in Ethiopia.

BackgroundMedicinal plants have contributed significantly to current malaria treatment. Emergence of resistance to currently available drugs has necessitated the search for new plant-based anti-malarial agents and several plant-based, pharmacologically active anti-malarial compounds have been isolated. This study was conducted to validate the traditional usage of Echinops kebericho for treating malaria in the traditional health care system of Ethiopia.MethodsThe roots of E. kebericho were collected from Masha Woreda, Sheka Zone. After collection, the plant materials were identified by a taxonomist, dried under shade and crushed to powder for extraction. The powdered roots were extracted by maceration using 70 % ethanol. Acute toxicity study of the crude extract was carried out in Swiss albino mice. The in vivo anti-malarial activity of plant extract (200, 350 and 500 mg/kg) of E. kebericho roots against a chloroquine (CQ) sensitive strain of Plasmodium berghei strain ANKA was assessed using the four-day suppressive test procedure. Parameters such as parasitaemia, packed cell volume, body weight and survival time were then determined using standard tests.ResultsOral administration of the ethanol extract showed significant (P <0.001) parasitaemia suppression at dose levels of 350 and 500 mg/kg in dose-related manner compared with the negative control. Five hundred mg/kg showed the highest (57.29 ± 1.76 %) parasitaemia suppression. The survival times of P. berghei-infected mice were also increased in a dose-dependent manner but the test material did not prevent weight loss associated with increased parasitaemia. The result also showed the plant material prevented the loss in packed cell volume associated with increased parasitaemia. Its oral LD50 was found to be greater than 5,000 mg/kg, indicating its wider safety margin in mice.ConclusionThe result revealed the ethanol extract of E. kebericho roots has anti-malarial activity against P. berghei in an animal model and lends support to the use of the plant to combat malaria in Ethiopian folk medicine. Further work is necessary to isolate, identify and characterize the active principles from the plant material.

Antiplasmodial and anti-inflammatory effects of an antimalarial remedy from the Wayana Amerindians, French Guiana: Takamalaimë (Psidium acutangulum Mart. ex DC., Myrtaceae)

Ethnopharmacological relevanceField investigations highlighted the use of Psidium acutangulum Mart. ex DC (syn. P. persoonii McVaugh), a small tree used by the Wayana Amerindians in Twenke–Taluhwen and Antecume–Pata, French Guiana, for the treatment of malaria, and administered either orally in the form of a decoction or applied externally over the whole body. This use appears limited to the Wayana cultural group in French Guiana and has never been reported anywhere else. Our goal was to evaluate the antimalarial and anti-inflammatory activities of a P. acutangulum decoction to explain the good reputation of this remedy. Materials and methodsInterviews with the Wayana inhabitants of Twenke–Taluhwen and Antecume–Pata were conducted within the TRAMAZ project according to the TRAMIL methodology, which is based on a quantitative and qualitative analysis of medicinal plant uses. A decoction of dried aerial parts of P. acutangulum was prepared in consistency with the Wayana recipe. In vitro antiplasmodial assays were performed on chloroquine-resistant FcB1 ([3H]-hypoxanthine bioassay) and 7G8 (pLDH bioassay) P. falciparum strains and on chloroquine sensitive NF54 ([3H]-hypoxanthine bioassay) P. falciparum strain. In vitro anti-inflammatory activity (IL-1β, IL-6, IL-8, TNFα) was evaluated on LPS-stimulated human PBMC and NO secretion inhibition was measured on LPS stimulated RAW murine macrophages. Cytotoxicity of the decoction was measured on L6 mammalian cells, PBMCs, and RAW cells. A preliminary evaluation of the in vivo antimalarial activity of the decoction, administered orally twice daily, was assessed by the classical four-day suppressive test against P. berghei NK65 in mice. ResultsThe decoction displayed a good antiplasmodial activity in vitro against the three tested strains, regardless to the bioassay used, with IC50 values of 3.3µg/mL and 10.3µg/mL against P. falciparum FcB1 and NF54, respectively and 19.0µg/mL against P. falciparum 7G8. It also exhibited significant anti-inflammatory activity in vitro in a dose dependent manner. At a concentration of 50µg/mL, the decoction inhibited the secretion of the following pro-inflammatory cytokines: TNFα (−18%), IL-1β (−58%), IL-6 (−32%), IL-8 (−21%). It also exhibited a mild NO secretion inhibition (−13%) at the same concentration. The decoction was non-cytotoxic against L6 cells (IC50>100µg/mL), RAW cells and PBMC. In vivo, 150µL of the decoction given orally twice a day (equivalent to 350mg/kg/day of dried extract) inhibited 39.7% average parasite growth, with more than 50% of inhibition in three mice over five. The absence of response for the two remaining mice, however, induced a strong standard deviation. ConclusionsThis study highlighted the in vitro antiplasmodial activity of the decoction of P. acutangulum aerial parts, used by Wayana Amerindians from the Upper-Maroni in French Guiana in case of malaria. Its antioxidant and anti-inflammatory potential, which may help to explain its use against this disease, was demonstrated using models of artificially stimulated cells.

In vitro synergistic effect of fluoroquinolone analogues in combination with artemisinin against Plasmodium falciparum; their antiplasmodial action in rodent malaria model.

BackgroundEmergence of drug-resistant parasite strains has surfaced as a major obstacle in attempts to ameliorate malaria. Current treatment regimen of malaria relies on the concept of artemisinin-based combination therapy (ACT).MethodsFluoroquinolone analogues, compounds 10, 12 and 18 were investigated for their anti-malarial interaction in combination with artemisinin in vitro, against Plasmodium falciparum 3D7 strain, employing fixed-ratio combination isobologram method. In addition, the efficacy of these compounds was evaluated intraperitoneally in BALB/c mice infected with chloroquine-resistant Plasmodium berghei ANKA strain in the Peters’ four-day suppressive test.ResultsPromising results were obtained in the form of synergistic or additive interactions. Compounds 10 and 12 were found to have highly synergistic interactions with artemisinin. Antiplasmodial effect was further verified by the convincing ED50 values of these compounds, which ranged between 2.31 and 3.09 (mg/kg BW).ConclusionsIn vivo studies substantiated the potential of the fluoroquinolone derivatives to be developed as synergistic partners for anti-malarial drug combinations.

Investigating the In-vivo Antiplasmodial Properties of Aqueous Extract of Moringa oleifera Lam (Moringaceae) Leaves

Aim: The study investigated Moringa oleifera Lam (Moringaceae), for its in-vivo antiplasmodial properties, using a murine model involving Plasmodium berghei. Study Design: Experimental. Places of Study and Duration: Department of Pharmaceutical Chemistry, KNUST, Department of Immunology, NMIMR and Department of Animal Experimentation, NMIMR, May – November, 2013. Methodology: Phytochemical investigation was conducted on powdered plant material and aqueous leaf extract (ML), to determine the presence of secondary metabolites using standard methods. Using 4-day suppressive and 7-day curative tests respectively, ML at 250-1000 mg/kg, was evaluated for in-vivo antiplasmodial activity in P. berghei infected male ICR mice (25-30 g), using Artemether-Lumefantrine (A/L) at 4 mg/kg as reference drug. The effect of ML on the body weights and the survival of the mice, as well as the effect on improvement in clinical signs was also monitored. Results: ML extract was found to contain alkaloids, saponins, flavonoids and tannins. The extracts (250-1000 mg/kg) produced significant reduction in parasitemia in the four-day suppressive test (F6,49 = 4.309; p =.0014). However, 250 mg/kg (69.31%; p .05). In the curative test, similar results were obtained with significant parasitemia reduction for 250 mg/kg (AUC = 52.52±6.732; p< .01) and 500 mg/kg (AUC = 49.62 ± 3.804; p< .01) compared to the positive control group (AUC = 101.3±14.32). Piloerection, lethargy and decreased locomotion were observed to be progressive in all infected experimental groups except A/L. Survival data showed that, although 750 mg/kg and 1000 mg/kg groups recorded relatively higher mortalities, statistical analysis didn’t indicate significant difference. Conclusion: ML extract demonstrated in-vivo antiplasmodial activity; probably due to the alkaloids, saponins, flavonoids and tannins in its composition.

English

Pyronaridine is a partner drug in, Pyramax&reg;, a combination of artesunate (ASN)-pyronaridine (PRD) which was recently prequalified by WHO drug as a potential alternative for treatment of malaria in African setting. Pyronaridine is a mannich base, with a long half-life, thus predisposed to resistance. In this study, we selected pyronaridine resistance by submitting Plasmodium berghei ANKA line in vivo to increasing pyronaridine concentration for 20 successive passages over a period of six months. The effective doses that reduce parasitaemia by 50% (ED50) and 90% (ED90) determined in the standard four-day suppressive test for the parent line were 1.83 and 4.79 mgkg-1, respectively. After 20 drug pressure passages, the ED50 and ED90 increased by 66 and 40 fold, respectively. After dilution cloning, the parasites were grown in the absence of drug for five passages and cryo-preserving them at -80&deg;C for at least one month, the resistance phenotypes remained stable. Thus, the resistant phenotype line could be used to explore genetic determinants associated with pyronaridine resistance; therefore, this strain represents a vital tool to study the mechanisms of resistance. Key words: Malaria, pyronaridine, Pyramax&reg;, resistance, Plasmodium berghei ANKA.

Evaluation of Anti-Plasmodial Effect of Azadrachta Indica in Plasmodium Berghi-Infected Mice

The emergence and spread of strains of P.falciparium to most of all available antimalarial drugs constitute major problem to control of malaria. Thus there is need to search for novel, cheap, effective and safe antimalarial drugs. This study was designed to evaluate the antimalarial activities of both the leaf and the bark of Azadrachta indica. A hundred apparentelly healthy albino mice (27-30g comprising of both test and control groups) were infected intraperitoneally with Plasmodium berghei. Acute and sub-acute toxicity studies of the extracts were carried out by giving up to 800mg/kg to apparentelly healthy non-infected mice. Weight loss, change in general behaviour and mortality were used as indicators of toxicity. Doses of 200, 600 and 800mg/kg/day of each extract of the leaf, the bark and their combination were given orally to infected mice after three hours.Following the four-day suppressive test procedure, none of the extracts caused symptoms of toxicity at the given doses within 24hours. Each extract showed variable level of parasitaemia suppression depending on the dose. The crude aqeous extract of A.indica leaf exerted 70% suppression at the dose of 800mg/kg. The crude aqeous extract of the bark and the leaf of A.indica swed the highest (79.68%) suppression of parasitaemia at the dose of 800mg/kg. Furthermore, the crude aqeous extract of the bark of A.indica induced 66.32% suppression, whereas 25mg/kg of chloroquine exerted 94.65% suppression of parasitaemia. The study showed that crude aqeous extracts of Azadrachta indicacaused strong clearance against P.berghei indicating that they contain some chemical constituents that possibly lead to antimalarial drug development.

The in vivo antimalarial activity of methylene blue combined with pyrimethamine, chloroquine and quinine

The effectiveness of methylene blue (MB) combined with pyrimethamine (PYR), chloroquine (CQ) or quinine (Q) was examined in a classical four-day suppressive test against a causative agent of rodent malaria, Plasmodium berghei. A marked potentiation was observed when MB was administered at a non-curative dose of 15 mg/kg/day in combination with PYR (0.19 mg/kg/day) or Q (25 mg/kg/day). No synergy was found between MB (15 mg/Kg) and CQ (0.75 mg/Kg). Our results suggest that the combination of MB with PYR or Q may improve the efficacy of these currently used antimalarial drugs.

Clotrimazole nanoemulsion for malaria chemotherapy. Part II: Stability assessment, in vivo pharmacodynamic evaluations and toxicological studies

The aim of present investigation was to evaluate the potential of clotrimazole as antimalarial drug. Due to poor aqueous solubility and high lipophilicity, it was previously formulated in a nanoemulsion based system. The intrinsic effects of nanoemulsion on improvement of antimalarial activity of clotrimazole were assessed in mice infected with Plasmodium berghei and compared to its suspension formulation. In four-day suppressive test, mice treated with 10mg/kg clotrimazole nanoemulsion showed the highest suppression of parasitemia and; parasitemia was significantly lower than that of 10mg/kg clotrimazole suspension. In onset of activity and recrudescence test, percent reduction of parasitemia was significantly higher in 10 and 15mg/kg clotrimazole nanoemulsion groups compared to 15mg/kg suspension group. In both murine models, survival of mice treated with nanoemulsion was significantly prolonged compared to suspension at equivalent doses. The inhibition of parasite growth by clotrimazole in the nanoemulsion was dose dependent as determined by test for linear trend. In repeated dose oral toxicity, levels of serum liver enzymes and biomarkers of hepatotoxicity did not vary significantly from control. Six-month stability testing of the clotrimazole nanoemulsion exhibited no changes in various physiochemical attributes of drug product compared to initial analysis.