Phenotypic and genotypic characterization of Neisseria gonorrhoeae isolates from Yaoundé, Cameroon, 2019 to 2020.

This study investigated antimicrobial resistance (AMR) phenotypes and genotypes exhibited by Neisseria gonorrhoeae from Yaoundé, Cameroon. AMR to tetracycline, penicillin and ciprofloxacin was observed although none of the isolates had reduced susceptibility to azithromycin, cefixime or ceftriaxone. Whole genome sequence (WGS) data were obtained and, using a threshold of 300 or fewer locus differences in the N. gonorrhoeae core gene multilocus sequence typing (cgMLST) scheme, four distinct core genome lineages were identified. Publicly available WGS data from 1355 gonococci belonging to these four lineages were retrieved from the PubMLST database, allowing the Cameroonian isolates to be examined in the context of existing data and compared with related gonococci. Examination of AMR genotypes in this dataset found an association between the core genome and AMR with, for example, isolates belonging to the core genome group, Ng_cgc_300 : 21, possessing GyrA and ParC alleles with amino acid substitutions conferring high-level resistance to ciprofloxacin while lineages Ng_cgc_300 : 41 and Ng_cgc_300 : 243 were predicted to be susceptible to several antimicrobials. A core genome lineage, Ng_cgc_300 : 498, was observed which largely consisted of gonococci originating from Africa. Analyses from this study demonstrate the advantages of using the N. gonorrhoeae cgMLST scheme to find related gonococci to carry out genomic analyses that enhance our understanding of the population biology of this important pathogen.

In Vitro Antibacterial Potential against Multidrug-Resistant Salmonella, Cytotoxicity, and Acute Biochemical Effects in Mice of Annona muricata Leaf Extracts.

The treatment of Salmonella infections is threatened by multidrug resistance necessitating the search for alternative treatments, such as from medicinal plants. There are several reports on the antibacterial activity of Annona muricata. This study assessed the activity against multidrug-resistant (MDR) Salmonella and also the toxicity of the leaves of this plant. The hexane and methanol extracts of the leaves were screened against characterized MDR isolates by disc diffusion and microdilution methods. A cytotoxicity test was performed on monkey kidney epithelial cells; an acute toxicity test was conducted in BALB/c mice and the liver and kidney functions were assessed at the end of the test. Both extracts recorded weak activity in the disc test. Conversely, the extracts showed a wide range of activity against specific Salmonella isolates in the microdilution assay, and the lowest minimum inhibitory concentration value recorded was 0.0625 mg/mL. The hexane extract (ANOHEX) was not cytotoxic (CC50 = 57.7 µg/mL) and was also not toxic to the mice at 2000 mg/Kg bodyweight, while the methanol extract (ANOMET) was cytotoxic (CC50 = 18.44 µg/mL), and mortality was recorded at 2000 mg/Kg but not at 300 mg/Kg. There were no significant changes in biomarkers of the liver (alanine aminotransferase and aspartate aminotransferase) and kidney (creatinine and urea) functions (P > 0.05), except for ANOHEX which significantly decreased creatinine (P = 0.01), in the test mice which was not considered a toxic effect. In conclusion, this study has demonstrated high bacteriostatic activity against MDR Salmonella and a low risk of toxicity of A. muricata leaves. Hence, the leaves are a potential alternative treatment for resistant Salmonella infection. The natural products should be further investigated in vitro and in vivo.

Investigating the risk factors for seroprevalence and the correlation between CD4+ T-cell count and humoral antibody responses to Toxoplasma gondii infection amongst HIV patients in the Bamenda Health District, Cameroon.

BACKGROUND: Toxoplasmosis is caused by an obligate intracellular tissue protozoan parasite, Toxoplasma gondii that infect humans and other warm-blooded animals. Transmission to humans is by eating raw or inadequately cooked infected meat or through ingestion of oocysts that cats have passed in faeces. Studies have shown life-threatening and substantial neurologic damage in immunocompromised patients; however, 80% of humans remain asymptomatic. The aim of this study was to determine the seroprevalence of Toxoplasma gondii infection in HIV positive patients and the risk factors associated with the infection, and to investigate the correlation between CD4+ T-cell count and toxoplasma specific antibodies as possible predictors of each other amongst HIV patients in the Bamenda Health District of the North West Region of Cameroon. METHODS: A cross-sectional study was conducted, in which 325 HIV patients were recruited for administration of questionnaire, serological diagnosis of T. gondii and measurement of CD4+ T-cell count. Bivariate and multivariate logistic regression was used to identify risk factors associated with T. gondii infection while the linear regression was used to investigate the relationship between CD4+ T-cell count and antibody levels against T. gondii. RESULTS: The findings showed that, majority (45.8%) of HIV patients suffered from chronic (IgG antibody) infection, and 6.5% from acute (IgM and IgM/IgG antibody) toxoplasma infection. The overall sero-prevalence of T. gondii infection amongst HIV patients was 50.5%. On the whole, 43 men (45.7%) and 127 women (55%) presented with anti- T. gondii antibodies; however, there was no significant difference amongst males and females who were positive to T. gondii infection (p = 0.131). Marital status (p = 0.0003), contact with garden soil (p = 0.0062), and garden ownership (p = 0.009), were factors that showed significant association with T. gondii infection. There was no significant difference (p = 0.909) between the mean CD4+ T-cell count of HIV patients negative for toxoplasma infection (502.7 cells/mL), chronically infected with T. gondii (517.7 cells/mL) and acutely infected with T. gondii (513.1 cells/mL). CD4+ T-cell count was neither a predictor of IgM antibody titer (r = 0.193, p = 0.401), nor IgG antibody titer (r = 0.149, p = 0.519) amongst HIV patients acutely infected with T. gondii. CONCLUSION: The findings from this study underscore the need to implement preventive and control measures to fight against T. gondii infection amongst HIV patients in the Bamenda Health District.

In Vitro and In Vivo Anti-Salmonella Evaluation of Pectin Extracts and Hydrolysates from "Cas Mango" (Spondias dulcis).

BACKGROUND: The threat to human health posed by multidrug-resistant strains of Salmonella typhi (S. typhi) and Salmonella paratyphi (S. paratyphi) is of growing concern. Generally, there has been increasing resistance and even multidrug resistance to almost all classes of antibiotics. This has rendered treatment with antibiotics difficult and costly. The present study investigated the bioactivity of pectin and pectin hydrolysates derived from a local fruit, Spondias dulcis, against four strains of Salmonellae. METHODS: Pectin was extracted from alcohol extractives-free peel by acidic hydrolysis at a temperature of 80°C for one hour at pH 2 and 4. The pectin was precipitated with 95% alcohol at an extract to alcohol ratio of 1:10 v/v. Antimicrobial activity was determined using agar well diffusion technique. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values were determined using the broth dilution technique. An in vivo study was then carried out with the bioactive extracts against the most resistant bacteria strain, to fully establish the therapeutic effect of these extracts. Balb/C mice were used, and ciprofloxacin was the positive control antibiotic. The extracts were administered to mice at two doses, 5mg/Kg and 10mg/Kg. The efficacy of extracts in the treatment of typhoid was evaluated based on survival rate, change in body weight, and change in bacteria load. RESULTS: Only one of the extracts (crude pectin pH 2.5) was active against all the Salmonellae by well diffusion, and the growth inhibition varied from 12mm to 15mm at100 µg/ml. Three of the extracts (crude pectin pH 2.5, pH 4, 12h hydrolysate, and pH 4, 1h hydrolysate) had MIC and MBC against all four Salmonellae strains with MIC ranging from 5.68 to 44.45 µg/ml and MBC from 11.36 to 44.45 µg/mL. Three treatments, namely, the pH4-12 hr, hydrolysate at 10mg/Kg and 5mg/Kg, and the pH4-1hr, hydrolysate at 10mg/Kg, had therapeutic effects against Salmonella infection in mice. CONCLUSION: The present study highlights the potential of pectin oligosaccharides as new source of anti-Salmonella drugs. Further investigations including exploration of mechanism of action of the most active pectin extracts/hydrolysates are envisaged.

In vitro and in vivo studies on anti-malarial activity of Commiphora africana and Dichrostachys cinerea used by the Maasai in Arusha region, Tanzania.

BACKGROUND: Traditional medicinal plants are one of the potential sources of anti-malarial drugs and there is an increasing interest in the use and development of traditional herbal remedies for the treatment of malaria and other ailments. This study was carried out with the aim to investigate the phytochemical screening, cytotoxic effect and antiplasmodial activities of Dichrostachys cinerea and Commiphora africana. Both plants are used by the Maasai in Tanzania in suspected malaria and other diseases. No previous work appears to have investigated the potential anti-malarial activity of the two plants. METHODS: This study aimed to investigate the in vitro anti-malarial activity of methanol and dichloromethane extracts of the two plants against chloroquine sensitive (D6) and chloroquine resistant (Dd2) strains of Plasmodium falciparum. The anti-malarial property was assessed by the lactate dehydrogenase method (pLDH). The in vivo anti-malarial study was carried out using the Peters' 4-day suppressive test in Plasmodium berghei in Balb/c mice. Cytotoxic tests were carried out using monkey kidney epithelial cell line in [3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) assay. Qualitative phytochemical screening was carried out using standard methods of analysis. RESULTS: The phytochemical screening of plant extracts revealed the presence of alkaloids, flavonoids, tannins, steroids, triterpenoids, glycosides and saponins. However, alkaloids were absent in most plant extracts. The dichloromethane extracts of C. africana (stem bark); D. cinerea (stem bark) and methanol extracts of D. cinerea (whole stem) all showed promising in vitro anti-malarial activities. All other extracts did not show any significant anti-malarial activity. The two most promising extracts based on in vitro studies, DCM extracts of C. africana (stem bark) and D. cinerea (stems bark), equally exhibited very significant anti-malarial activities in the mouse model. They exhibited parasite suppression rates of 64.24 and 53.12%, respectively, and considerable improvement in weight and survival rate. Most plant extracts were not cytotoxic except for DCM extract of D. cinerea (whole stem) CC50 (29.44 µg/mL). CONCLUSION: The findings of this study provide scientific evidence supporting the traditional use of the plants in the treatment of malaria by the Maasai in Arusha region, Tanzania. Consequently, further work including bioassay-guided fractionation and advanced toxicity testing may yield new anti-malarial drug candidates from the two plants.

Structurally simple synthetic 1, 4-disubstituted piperidines with high selectivity for resistant Plasmodium falciparum.

BACKGROUND: Emergence of resistance to artemisinins and some of their combinations in chemotherapy of clinical malaria has intensified the search for novel safe efficacious antimalarial molecules. Fourteen synthetic 1, 4-disubstituted piperidines with simple molecular structures were evaluated in this study. METHODS: Antiplasmodial activity were determined against cultured chloroquine-sensitive 3D7 and resistant Dd2 strains of P. falciparum by in vitro parasite growth inhibition. A primary screen was done to identify active compounds by fluorescence microscopy followed by a secondary screen to determine IC50 and IC90 values of active compounds by the parasite lactate dehydrogenase assay. Cytotoxicity of active compounds was assessed using the MTT/formazan assay and selectivity indices (SIs) determined. Optical densities were analysed to obtain experimental results. RESULTS: The compounds produced 56 to 93% inhibition of parasite growth at 40 µg/mL. Eight compounds (2 ketone, 5 alcohol and one amine analogues) showed high activity (IC50s between 1 and 5 µg/mL). Nine compounds were highly selective for the parasite (SIs = 15 to 182). Three promising (alcohol) analogues were identified: [1-(4-fluorobenzyl) piperidin-4-yl] [4-fluorophenyl] methanol, (7), [1-(3, 4-dichlorobenzyl) piperidin-4-yl] [4- fluorophenyl] methanol (8) and [1-(4-bromobenzyl) piperidin-4-yl] [4- fluorophenyl] methanol (11) which were more active on the resistant strain (IC50 values between 1.03 to 2.52 µg/mL), than the sensitive strain (IC50 values between 2.51 to 4.43 µg/mL). CONCLUSIONS: The alcohol analogues were the most active and most selective for the parasite with three promising hit molecules identified among them, suggesting the hydroxyl group at C-7' in these alcohol analogues is contributing greatly to their antiplasmodial activity. Further exploration of the core structure using chemistry approaches and biological screening including in vivo studies in an animal model of malaria may yield important antimalarial leads.

Evaluation of in vitro culture systems for the maintenance of microfilariae and infective larvae of Loa loa.

BACKGROUND: Suitable and scalable in vitro culture conditions for parasite maintenance are needed to foster drug research for loiasis, one of the neglected tropical diseases which has attracted only limited attention over recent years, despite having important public health impacts. The present work aims to develop adequate in vitro culture systems for drug screening against both microfilariae (mf) and infective third-stage larvae (L3) of Loa loa. METHODS: In vitro culture conditions were evaluated by varying three basic culture media: Roswell Park Memorial Institute (RPMI-1640), Dulbecco's modified Eagle's medium (DMEM) and Iscove's modified Dulbecco's medium (IMDM); four sera/proteins: newborn calf serum (NCS), foetal bovine serum (FBS), bovine serum albumin (BSA) and the lipid-enriched BSA (AlbuMax® II, ALB); and co-culture with the Monkey Kidney Epithelial Cell line (LLC-MK2) as a feeder layer. The various culture systems were tested on both mf and L3, using survival (% motile), motility (T90 = mean duration (days) at which at least 90% of parasites were fully active) and moulting rates of L3 as the major criteria. The general linear model regression analysis was performed to assess the contribution of each variable on the viability of Loa loa L3 and microfilarie. All statistical tests were performed at 95% confidence interval. RESULTS: Of the three different media tested, DMEM and IMDM were the most suitable sustaining the maintenance of both L. loa L3 and mf. IMDM alone could sustain L3 for more than 5 days (T90 = 6.5 ± 1.1 day). Serum supplements and LLC-MK2 co-cultures significantly improved the survival of parasites in DMEM and IMDM. In co-cultures with LLC-MK2 cells, L. loa mf were maintained in each of the three basic media (T90 of 16.4-19.5 days) without any serum supplement. The most effective culture systems promoting significant moulting rate of L3 into L4 (at least 25%) with substantial maintenance time were: DMEM + BSA, DMEM + NCS, DMEM-AlbuMax®II, DMEM + FBS all in co-culture with LLC-MK2, and IMDM + BSA (1.5%), DMEM + FBS (10%) and DMEM + NCS (5%) without feeder cells. DMEM + 1% BSA in co-culture scored the highest moulting rate of 57 of 81 (70.37%). The factors that promoted L. loa mf viability included feeder cells (ß = 0.490), both IMDM (ß = 0.256) and DMEM (ß = 0.198) media and the protein supplements NCS (ß = 0.052) and FBS (ß = 0.022); while for L. loa L3, in addition to feeder cells (ß = 0.259) and both IMDM (ß = 0.401) and DMEM (ß = 0.385) media, the protein supplements BSA (ß = 0.029) were found important in maintaining the worm motility. CONCLUSIONS: The findings from this work display a range of culture requirements for the maintenance of Loa loa stages, which are suitable for developing an effective platform for drug screening.

Heterogeneity in the in vitro susceptibility of Loa loa microfilariae to drugs commonly used in parasitological infections.

BACKGROUND: Co-infection with loiasis remains a potential problem in control programs targeting filarial infections. The effects of many anti-parasitic drugs often administered to Loa loa infected people are not well documented. This study compared the in vitro activity of several of these drugs on the viability of L. loa microfilariae (mf). METHODS: Human strain L. loa mf were isolated from baboon blood using iso-osmotic Percoll gradient, and cultured in RPMI 1640/10% FBS with antimalarial drugs (mefloquine, amodiaquine, artesunate, chloroquine and quinine), anthelmintics (ivermectin, praziquantel, flubendazole and its reduced and hydrolyzed metabolites), two potential trypanocidal agents (fexinidazole and Scynexis-7158) and the anticancer drug imatinib. The drug concentrations used varied between 0.156 µg/ml and 10 µg/ml. Mf motility (CR50 = 50% immotility) and a metabolic viability assay (MTT) were used to assess the effects of these drugs on the parasites. RESULTS: Mf in control cultures showed only a slight reduction in motility after 5 days of culture. Active inhibition of Loa loa motility was seen with mefloquine and amodiaquine (CR50 values of 3.87 and 4.05 µg/ml, respectively), immobilizing > 90% mf within the first 24 hours: mefloquine killed the mf after 24 hours of culture at concentrations ≥ 5 µg/ml. SCYX-7158 also induced a concentration-dependent reduction in mf motility, with > 50% reduction in mf motility seen after 5 days at 10 µg/ml. The anticancer drug imatinib reduced mf motility at 10 µg/ml from the first day of incubation to 55% by day 5, and the reduction in motility was concentration-dependent. Praziquantel and fexinidazole were inactive, and FLBZ and its metabolites, as well as ivermectin at concentrations > 5 µg/ml, had very minimal effects on mf motility over the first 4 days of culture. CONCLUSIONS: The considerable action of the anti-malarial drugs mefloquine and amodiaquine on Loa mf in vitro highlights the possibility of repurposing the existing anti-infectious agents for the development of drugs against loiasis. The heterogeneity in the activity of anti-parasitic agents on Loa loa mf supports the need for further investigation using animal models of loiasis.

Anti-plasmodial activity of Norcaesalpin D and extracts of four medicinal plants used traditionally for treatment of malaria.

BACKGROUND: Malaria is an old life-threatening parasitic disease that is still affecting many people, mainly children living in sub-Saharan Africa. Availability of effective antimalarial drugs played a significant role in the treatment and control of malaria. However, recent information on the emergence of P. falciparum parasites resistant to one of the artemisinin-based combination therapies suggests the need for discovery of new drug molecules. Therefore, this study aimed to evaluate the antiplasmodial activity of extracts, fractions and isolated compound from medicinal plants traditionally used in the treatment of malaria in Tanzania. METHODS: Dry powdered plant materials were extracted by cold macerations using different solvents. Norcaesalpin D was isolated by column chromatography from dichloromethane root extract of Caesalpinia bonducella and its structure was assigned based on the spectral data. Crude extracts, fractions and isolated compound were evaluated for antiplasmodial activity against chloroquine-sensitive P. falciparum (3D7), chloroquine-resistant P. falciparum (Dd2, K1) and artemisinin-resistant P. falciparum (IPC 5202 Battambang, IPC 4912 Mondolkiri) strains using the parasite lactate dehydrogenase assay. RESULTS: The results indicated that extracts of Erythrina schliebenii, Holarrhena pubescens, Dissotis melleri and C. bonducella exhibited antiplasmodial activity against Dd2 parasites. Ethanolic root extract of E. schliebenii had an IC50 of 1.87 µg/mL while methanolic and ethanolic root extracts of H. pubescens exhibited an IC50 = 2.05 µg/mL and IC50 = 2.43 µg/mL, respectively. Fractions from H. pubescens and C. bonducella roots were found to be highly active against K1, Dd2 and artemisinin-resistant parasites. Norcaesalpin D from C. bonducella root extract was active with IC50 of 0.98, 1.85 and 2.13 µg/mL against 3D7, Dd2 and IPC 4912-Mondolkiri parasites, respectively. CONCLUSIONS: Antiplasmodial activity of norcaesalpin D and extracts of E. schliebenii, H. pubescens, D. melleri and C. bonducella reported in this study requires further attention for the discovery of antimalarial lead compounds for future drug development.

An evolutionary approach to identify potentially protective B cell epitopes involved in naturally acquired immunity to malaria and the role of EBA-175 in protection amongst denizens of Bolifamba, Cameroon.

BACKGROUND: The search for a vaccine against malaria caused by Plasmodium falciparum has lasted for more than 100 years, with considerable progress in the identification of a number of vaccine candidates. The post-genomic era offers new opportunities for an expedited search using rational vaccine design and prioritization of key B-cell epitopes involved in natural acquired immunity. METHODS: Malaria vaccine candidate genes that have reached clinical trial were searched on an evolutionary relationship tree, to determine their level of lineage-specificity. Ten other genes with similar protein features and level of lineage specificity to the vaccine candidates were randomly selected, and computationally evaluated for the presence of B-cell epitopes. The protein fragment with maximum probability of putative epitopes were synthesized and used in an ELISA experiment to determine the presence of antibodies to these peptides, in the serum of malaria patients and healthy malaria uninfected inhabitants from a malaria endemic region (Bolifamba), alongside with a vaccine candidate EBA-175. RESULTS: Two peptide fragments of 25 and 30 amino acid length from PF3D7_1233400 and PF3D7_1437500 respectively, coded as PF4-123 and PF4-143 were shown to contain B-cell epitope(s). Total IgG antibodies to these peptides were not significantly different between sick and healthy participants, but cytophilic antibodies to these peptides were significantly higher in healthy participants (p < 0.03). Total IgG to the vaccine candidate EBA-175 was significantly higher in sick participants than in healthy participants, likewise cytophilic antibodies (p < 0.04). Antibodies to the peptides PF4-123 and PF4-143 correlated negatively (p = 0.025 and 0.008 and r = -0.291 and -0.345, respectively) to parasite load. Total IgG antibodies to EBA-175 showed a negative correlation to parasite load (r = -0.144), which was not significant (p = 0.276). Duration of stay in Bolifamba also negatively correlated with parasite load (p = 0.026, r = -0.419) and total IgG to PF4-143 was significantly associated with prolonged duration of stay in the locality of Bolifamba, Cameroon (p = 0.006, r = 0.361). CONCLUSIONS: The present study has identified two genes PF3D7_1233400 and PF3D7_1437500 containing peptide fragment (PF4-123 and PF4-143) with B-cell epitopes that are correlated with naturally acquired immunity to malaria. A pipeline has been developed for rapid identification of other B-cell epitopes involved in naturally acquired immunity.

The current status of malaria epidemiology in Bolifamba, atypical Cameroonian rainforest zone: an assessment of intervention strategies and seasonal variations.

BACKGROUND: Global malaria has been on the decline over the past decade due to expansion of interventions. The present study aimed at determining the current status of malaria epidemiology in the context of sustained interventions and seasonal variations in Bolifamba, which represents a typical semi-urban malaria endemic community in the Cameroonian rainforest. METHODS: A monthly cross-sectional survey was carried out in Bolifamba, a multi ethnic semi-urban locality on the eastern flanks of Mt Cameroon, for a year during which blood samples were collected from participants and examined for malaria parasites by microscopy. Correlation analysis of seasonal/monthly malaria prevalence was done with weather data from Ekona, a nearby village with a meteorological station. Intervention strategy such as use of Insecticide Treated Bed Net (ITBN) and risk factors such as duration of stay in the locality, age and housing type were also investigated. RESULTS: The results revealed a malaria prevalence of 38.3 % in the rainy season, which was significantly higher than 24.4 % observed in the dry season (P < 0.0001). A high prevalence of asymptomatic malaria which was more than double the prevalence of symptomatic malaria on a monthly basis was observed, 30.7 % vs 17.8 % in the rainy and dry season respectively (p < 0.0001) and asymptomatic malaria was significantly associated with anemia (p < 0.005). April was the peak month of malaria prevalence and coincided with peak periods of both asymptomatic and symptomatic malaria. The Plasmodium falciparum parasite rates in the 2- up to 10-years age group (PfPR(2-10)) was 40.8 %. The regular use of ITBN was significantly associated with low prevalence of 31.7 % as opposed to irregular or non-usage of ITBN 38.2 % (p < 0.05). Log of parasite load was found to initially increase to 2.49 with less than 5 years of stay, and decreased gradually with increasing duration of stay in the locality (p = 0.046). Climatic factors were significantly and positively associated with monthly malaria prevalence and the strongest predictors of malaria prevalence were rainfall and minimum temperature with r values of 0.563 and 0.6 respectively. CONCLUSIONS: The study highlights the role of seasonal change in modifying malaria prevalence during the year and the beneficial effect of ITBN. It also underscores a sublime problem of asymptomatic malaria associated with anemia, and indicates that partial immunity is acquired with prolonged stay in Bolifamba. This preliminary result is the basis of ongoing work to identify the antigens involved in acquired immunity.

Control of malaria and other vector-borne protozoan diseases in the tropics: enduring challenges despite considerable progress and achievements.

Vector-borne protozoan diseases represent a serious public health challenge, especially in the tropics where poverty together with vector-favorable climates are the aggravating factors. Each of the various strategies currently employed to face these scourges is seriously inadequate. Despite enormous efforts, vaccines-which represent the ideal weapon against these parasitic diseases-are yet to be sufficiently developed and implemented. Chemotherapy and vector control are therefore the sole effective attempts to minimize the disease burden. Nowadays, both strategies are also highly challenged by the phenomenon of drug and insecticide resistance, which affects virtually all interventions currently used. The recently growing support from international organizations and governments of some endemic countries is warmly welcome, and should be optimally exploited in the various approaches to drug and insecticide research and development to overcome the burden of these prevalent diseases, especially malaria, leishmaniasis, Human African Trypanosomiasis (HAT), and Chagas disease.

AfroDb: a select highly potent and diverse natural product library from African medicinal plants.

Computer-aided drug design (CADD) often involves virtual screening (VS) of large compound datasets and the availability of such is vital for drug discovery protocols. We assess the bioactivity and "drug-likeness" of a relatively small but structurally diverse dataset (containing >1,000 compounds) from African medicinal plants, which have been tested and proven a wide range of biological activities. The geographical regions of collection of the medicinal plants cover the entire continent of Africa, based on data from literature sources and information from traditional healers. For each isolated compound, the three dimensional (3D) structure has been used to calculate physico-chemical properties used in the prediction of oral bioavailability on the basis of Lipinski's "Rule of Five". A comparative analysis has been carried out with the "drug-like", "lead-like", and "fragment-like" subsets, as well as with the Dictionary of Natural Products. A diversity analysis has been carried out in comparison with the ChemBridge diverse database. Furthermore, descriptors related to absorption, distribution, metabolism, excretion and toxicity (ADMET) have been used to predict the pharmacokinetic profile of the compounds within the dataset. Our results prove that drug discovery, beginning with natural products from the African flora, could be highly promising. The 3D structures are available and could be useful for virtual screening and natural product lead generation programs.

New antimalarial hits from Dacryodes edulis (Burseraceae)--part I: isolation, in vitro activity, in silico "drug-likeness" and pharmacokinetic profiles.

The aims of the present study were to identify the compounds responsible for the anti-malarial activity of Dacryoedes edulis (Burseraceae) and to investigate their suitability as leads for the treatment of drug resistant malaria. Five compounds were isolated from ethyl acetate and hexane extracts of D. edulis stem bark and tested against 3D7 (chloroquine-susceptible) and Dd2 (multidrug-resistant) strains of Plasmodium falciparum, using the parasite lactate dehydrogenase method. Cytotoxicity studies were carried out on LLC-MK2 monkey kidney epithelial cell-line. In silico analysis was conducted by calculating molecular descriptors using the MOE software running on a Linux workstation. The "drug-likeness" of the isolated compounds was assessed using Lipinski criteria, from computed molecular properties of the geometry optimized structures. Computed descriptors often used to predict absorption, distribution, metabolism, elimination and toxicity (ADMET) were used to assess the pharmacokinetic profiles of the isolated compounds. Antiplasmodial activity was demonstrated for the first time in five major natural products previously identified in D. edulis, but not tested against malaria parasites. The most active compound identified was termed DES4. It had IC50 values of 0.37 and 0.55 µg/mL, against 3D7 and Dd2 respectively. In addition, this compound was shown to act in synergy with quinine, satisfied all criteria of "Drug-likeness" and showed considerable probability of providing an antimalarial lead. The remaining four compounds also showed antiplasmodial activity, but were less effective than DES4. None of the tested compounds was cytotoxicity against LLC-MK2 cells, suggesting their selective activities on malaria parasites. Based on the high in vitro activity, low toxicity and predicted "Drug-likeness" DES4 merits further investigation as a possible drug lead for the treatment of malaria.

Bioactive natural products derived from the Central African flora against neglected tropical diseases and HIV.

This review discusses the medicinal potential of bioactive metabolites isolated from medicinal plants in Central Africa for the treatment of neglected tropical diseases and HIV. A correlation is established between the biological activities of the isolated compounds and the uses of the plants in traditional medicine. Insight is provided on how secondary metabolites from medicinal plants in Central Africa could be exploited for drug discovery.

Antioxidant and antidiabetic profiles of two African medicinal plants: Picralima nitida (Apocynaceae) and Sonchus oleraceus (Asteraceae).

BACKGROUND: Diabetes mellitus (DM) is a metabolic disease characterized by chronic hyperglycaemia generally associated with oxidative stress. The present study aims at evaluating the antioxidant and antidiabetic potential of methanol and hydroethanol extracts of the stem bark and leaves of Pricralima nitida and the Sonchus oleraceus whole plant respectively. METHODS: The in vitro antioxidant activity was assessed using 1,1-Diphenyl-2-picrilhydrazyl (DPPH) for free radical-scavenging properties of the extracts, and the Folin-Ciocalteu method in determining their phenol contents. The antidiabetic activity was tested in mice following streptozotocin diabetes induction, and selected oxidative stress markers (Malondialdehyde, Hydrogen peroxides and Catalase) were measured in order to evaluate the level of oxidative stress in treated animals. RESULTS: The in vitro antioxidant activity using DPPH showed IC50 ranging from 0.19 ± 0.08 to 1.00 ± 0.06 mg/mL. The highest activity was obtained with the hydroethanol extracts of S. oleraceus (0.19 mg/mL and P. nitida (0.24 mg/mL). Polyphenol contents ranged from 182.25 ± 16.76 to 684.62 ± 46.66 µg Eq Cat/g. The methanol extract of P. nitida showed the highest activity, followed by the hydroethanol extract of S. oleraceus (616.89 ± 19.20 µEq Cat/g). The hydroethanol extract of whole plants (150 mg/Kg) and methanol leave extract of P. nitida (300 mg/Kg) exhibited significant antidiabetic activities with 39.40% and 38.48% glycaemia reduction, respectively. The measurement of stress markers in plasma, liver and kidney after administration of both extracts showed significant reduction in MDA and hydrogen peroxide levels, coupled with a substantial increase in catalase activity. CONCLUSIONS: These findings suggest that S. oleraceus whole plant and P. nitida leaves possess both antidiabetic and antioxidant properties, and therefore could be used as starting point for the development of herbal medicines and/or source of new drug molecules against diabetes.

Anti-hyperglycaemic globulins from selected Cucurbitaceae seeds used as antidiabetic medicinal plants in Africa.

BACKGROUND: The recent epidemic of diabetes mellitus (DM) in Africa, coupled with rampant poverty, is an indication of the urgent need to develop new efficacious, cheaper and more available drugs to face this growing public health challenge. A number of plants products among which the protein-rich Cucurbitaceae seeds are commonly used in traditional medicine with increasing acclaimed efficacy against DM. The aim of this study was to analyse and evaluate the hypoglycaemic activity of storage proteins of five species of Cucurbitaceae, which include Telfairia occidentalis, Citrullus lanatus, Lagenaria siceraria, Cucumeropsis mannii and Cucurbita moschata. METHODS: The different families of storage proteins were extracted following differential solubility, and their contents were estimated using the Bradford method. The analysis of these proteins was done by electrophoresis in non-denaturing and denaturing conditions. The evaluation of hypoglycaemic properties of various globulins extracted was performed on male Wistar rats by the oral glucose tolerance test. RESULTS: The results showed that among the proteins extracted, globulins constitute the most abundant class of storage proteins in all five species selected. Citrullus lanatus and Cucurbita moschata presented the highest levels of globulin (275.34 and 295.11 mg/g dry matter, respectively). The results of electrophoresis showed that all species possess acidic and neutrals albumins and globulins, with molecular weight of protein subunits ranging from 6.36-44.11 kDa for albumins, 6.5-173.86 kDa for globulins and 6.5-49.66 kDa for glutelins. The 6.36 kDa of albumin subunit protein and the 6.5 kDa of globulin subunit protein were present in all the species. The oral glucose tolerance test showed that the globulins of the seeds of all species except Cucumeropsis mannii caused significant drop in blood sugar (88 - 137.80%, compared to the controls, p<0.05). CONCLUSIONS: These findings showed that the selected Cucurbitaceae seeds contained globulins with significant anti-hyperglycaemic activity. It is therefore highly encouraged to pursue investigations towards development of peptide-drugs and/or phytomedicines from these bioactive proteins which could be used as affordable alternative therapy against DM.

Antimalarial drug interactions of compounds isolated from Kigelia africana (Bignoniaceae) and their synergism with artemether, against the multidrug-resistant W2mef Plasmodium falciparum strain.

For decades, drug resistance has been the major obstacle in the fight against malaria, and the search for new drugs together with the combination therapy constitutes the major approach in responding to this situation. The present study aims at assessing the in vitro antimalarial activity of four compounds isolated from Kigelia africana stem bark (atranorin - KAE1, specicoside - KAE7, 2ß,3ß,19α-trihydroxy-urs-12-20-en-28-oic acid - KAE3, and p-hydroxy-cinnamic acid - KAE10) and their drug interactions among themselves and their combination effects with quinine and artemether. The antiplasmodial activity and drug interactions were evaluated against the multidrug-resistant W2mef strain of Plasmodium falciparum using the parasite lactate dehydrogenase assay. Three of the four compounds tested were significantly active against W2mef: specicoside (IC(50) = 1.02 ± 0.17 µM), 2ß,3ß,19α-trihydroxy-urs-12-en-28-oic acid (IC(50) = 1.86 ± 0.15 µM) and atranorin (IC(50) = 1.78 ± 0.18 µM), whereas p-hydroxy-cinnamic acid showed a weak activity (IC(50) = 12.89 ± 0.87 µM). A slight synergistic effect was observed between atranorin and 2ß,3ß,19α-trihydroxy-urs-12-en-28-oic acid (Combination index, CI = 0.82) whereas the interaction between specicoside and p-hydroxy-cinnamic acid were instead antagonistic (CI = 2.67). All the three compounds showed synergistic effects with artemether, unlike the slight antagonistic interactions of atranorin and 2ß,3ß,19α-trihydroxy-urs-12-en-28-oic acid in combination with quinine. K. africana compounds are therefore likely to serve as leads in the development of new partner drugs in artemether-based combination therapy.

Hypericum lanceolatum (Hypericaceae) as a potential source of new anti-malarial agents: a bioassay-guided fractionation of the stem bark.

BACKGROUND: Malaria is a major public health threat in Africa, and traditional medicine continues to play a key role in its control especially in rural areas. A bioassay-guided fractionation was carried out in order to evaluate the anti-malarial potential and the safety of the methanol extract of the Hypericum lanceolatum stem bark. METHODS: The anti-plasmodial activity was assayed by the lactate dehydrogenase method (pLDH) against the multidrug-resistant W2mef laboratory strain, and a field isolate (SHF4) of Plasmodium falciparum. Cytotoxicity tests were carried out using the LLC-MK2 monkey kidney epithelial cells. RESULTS: Five compounds were isolated from the most active and least cytotoxic ethylacetate sub-extract: betulinic acid (HLT1), 2,2',5,6'-tetrahydroxybenzophenone (HLT2), 5-hydroxy-3-methoxyxanthone (HLT3), 3-hydroxy-5-methoxyxanthone (HLT4) and HLT0 (yet to be identified). Three of the tested compounds presented significant anti-plasmodial activities (with 50% inhibitory concentration, IC50 < 5 µM), with 5-hydroxy-3-methoxyxanthone exerting the highest activity, followed by HLT0 and betulinic acid. All the compounds with significant anti-plasmodial activity were non-cytotoxic, except betulinic acid which showed a 50% cytotoxic concentration, CC50 of 25 µg/mL. CONCLUSIONS: These findings justify the use of H. lanceolatum stem bark as anti-malarial by traditional healers of Western Cameroon, and could constitute a good basis for further studies towards development of new drug candidates or phytomedicines for malaria.

In vitro antiplasmodial activity and cytotoxicity of crude extracts and compounds from the stem bark of Kigelia africana (Lam.) Benth (Bignoniaceae).

In order to assess the potential of the stem bark of Kigelia africana (Lam.) Benth as source of new anti-malarial leads, n-hexane and ethyl acetate (EtOAc) extracts and four compounds isolated from the stem bark were screened in vitro against the chloroquine-resistant W-2 and two field isolates of Plasmodium falciparum using lactate dehydrogenase assay. The products were also tested for their cytotoxicity on LLC/MK2 monkey kidney cells. The EtOAc extract exhibited a significant antiplasmodial activity (IC(50) = 11.15 µg/mL on W-2; 3.91 and 4.74 µg/mL on field CAM10 and SHF4 isolates, respectively), whereas the n-hexane fraction showed a weak activity (IC(50) = 73.78 µg/mL on W-2 and 21.85 µg/mL on SHF4). Three out of the four compounds showed good activity against all the three different parasite strains (IC(50) <5 µM). Specicoside exhibited the highest activity on W-2 (IC(50) = 1.54 µM) followed by 2ß, 3ß, 19α-trihydroxy-urs-12-en-28-oic acid (IC(50) = 1.60 µM) and atranorin (IC(50) = 4.41 µM), while p-hydroxycinnamic acid was the least active (IC(50) =53.84 µM). The EtOAc extract and its isolated compounds (specicoside and p-hydroxycinnamic acid) were non-cytotoxic (CC(50) > 30 µg/mL), whereas the n-hexane extract and two of its products, atranorin and 2ß, 3ß, 19α-trihydroxy-urs-12-en-28-oic acid showed cytotoxicity at high concentrations, with the last one being the most toxic (CC(50) = 9.37 µg/mL). These findings justify the use of K. africana stem bark as antimalaria by traditional healers of Western Cameroon, and could constitute a good basis for further studies towards development of new leads or natural drugs for malaria.