Recent developments in antimalarial drug discovery.

Although malaria remains a big burden to many countries that it threatens their socio-economic stability, particularly in the countries where malaria is endemic, there have been great efforts to eradicate this disease with both successes and failures. For example, there has been a great improvement in malaria prevention and treatment methods with a net reduction in infection and mortality rates. However, the disease remains a global threat in terms of the number of people affected because it is one of the infectious diseases that has the highest prevalence rate, especially in Africa where the deadly Plasmodium falciparum is still widely spread. Methods to fight malaria are being diversified, including the use of mosquito nets, the target candidate profiles (TCPs) and target product profiles (TPPs) of medicine for malarial venture (MMV) strategy, the search for newer and potent drugs that could reverse chloroquine resistance, and the use of adjuvants such as rosiglitazone and sevuparin. Although these adjuvants have no antiplasmodial activity, they can help to alleviate the effects which result from plasmodium invasion such as cytoadherence. The list of new antimalarial drugs under development is long, including the out of ordinary new drugs MMV048, CDRI-97/78 and INE963 from South Africa, India and Novartis, respectively.

Investigation of the Quality of the 12 Most-Used Antibiotics Available in Retail Private Pharmacies in Rwanda.

Using poor-quality antibiotics leads to increased risk of the development of microorganism-resistant strains, treatment failure, loss of confidence in health systems, and associated socio-economic impacts. The prevalence of poor-quality antibiotics has been found to be high in some of the Low and Middle-Income Countries (LMICs), but no data were available on the situation in Rwanda. This study was conducted to obtain data and inform health professionals on the quality of the 12 most-used selected antibiotics from private retail pharmacies in Rwanda. The investigation was conducted on 232 batches collected from randomly selected private retail pharmacies in all provinces of Rwanda, and concerned only with visual inspection and assay tests. Visual inspection was performed using a tool adopted by the International Pharmaceutical Federation (FIP) to identify manufacturing defects. An assay test quantified the Active Pharmaceutical Ingredient (API) in each collected batch using high-performance liquid chromatography (HPLC) coupled with an ultraviolet-visible (UV) detector, and the results were reported as the percentage content of the amount of APIs stated on the label. A total of 232 batches were analyzed, manufactured in 10 countries; the main country of manufacture was Kenya, with almost half of the batches (49.6%). The results of the visual inspection did not show the presence of counterfeit/ falsified antibiotics on the Rwandan market in this study but revealed weaknesses in labeling: more than 90% of the analyzed batches of the 12 antibiotics did not present the dosage statement on their label, and the complete list of excipients was missing in more than 20% of the analyzed batches. The assay test using HPLC confirmed the presence of APIs in 100% of the analyzed batches. However, moderate deviations from acceptable ranges of the API content defined by M. M. Nasr & C. M. Stanley in 2006 for erythromycin and the United States Pharmacopoeia 2018 for the other 11 molecules were found. The failure rate to meet the quality requirements in terms of the percentage content of active pharmaceutical ingredients declared on the labels was estimated at 8.2% in total, with 3.9% and 4.3% containing more and less than the amount of APIs stated on the labels respectively. The most-represented antibiotics on the Rwandan market were amoxicillin, co-trimoxazole and cloxacillin. No counterfeit antibiotics were found in this study. However, substandard batches with moderate deviations were found, suggesting that regular quality control of antibiotics is needed in Rwanda.

Vitamin D Levels in Mother-Baby Pairs: A Cross-Sectional Prospective Study in a Rwandan Tertiary Hospital.

BACKGROUND: Exposure to the sunlight contributes largely to the production of vitamin D. However, vitamin D deficiency is a reality in tropical countries, despite enjoying enough sunlight, especially bearing women in their last trimester whose foetuses exclusively depend on their reserves. This work aimed at demonstrating the state of vitamin D in mother-baby pairs and associated factors in one of the University Hospitals in Rwanda. METHODS: This cross-sectional prospective study was performed on mother-baby pairs at Kigali University Hospital. Mother's serum 25-hydroxyvitamin D levels were considered as outcomes compared with demographic, clinical and biological markers. Correlation analysis was conducted in order to assess the association between serum 25-hydroxyvitamin D levels for the couple mothers-babies. RESULTS: Approximately 38% of women and 65% of neonates had deficiency in 25-hydroxyvitamin D (<20 ng/ml). The use of a vitamin D rich diet within 24 h recall (p < 0.01) or 1 week recall (p < 0.001) before delivery was associated with appropriate vitamin D levels in mothers. Interestingly, a strong positive correlation was found between maternal and neonatal serum 25-hydroxyvitamin D levels (r = 0.760). CONCLUSIONS: There was a high rate of vitamin D deficiency in mothers and their babies. Babies born from women with deficiency were likely to develop low levels of vitamin D. This stresses on the need to strengthen the interventions for preventing vitamin D deficiency in the couple mothers-babies such as supplement in vitamin D before and after delivery, improving the quality of meals and regular contact with sunlight.

In Vitro Antiplasmodial and Cytotoxic Activities of Compounds from the Roots of Eriosema montanum Baker f. (Fabaceae).

Malaria remains one of the leading causes of death in sub-Saharan Africa, ranked in the top three infectious diseases in the world. Plants of the Eriosema genus have been reported to be used for the treatment of this disease, but scientific evidence is still missing for some of them. In the present study, the in vitro antiplasmodial activity of the crude extract and compounds from Eriosema montanum Baker f. roots were tested against the 3D7 strain of Plasmodium falciparum and revealed using the SYBR Green, a DNA intercalating compound. The cytotoxicity effect of the compounds on a human cancer cell line (THP-1) was assessed to determine their selectivity index. It was found that the crude extract of the plant displayed a significant antiplasmodial activity with an IC50 (µg/mL) = 17.68 ± 4.030 and a cytotoxic activity with a CC50 (µg/mL) = 101.5 ± 12.6, corresponding to a selective antiplasmodial activity of 5.7. Bioactivity-guided isolation of the major compounds of the roots' crude extract afforded seven compounds, including genistein, genistin and eucomic acid. Under our experimental conditions, using Artemisinin as a positive control, eucomic acid showed the best inhibitory activity against the P. falciparum 3D7, a well-known chloroquine-sensitive strain. The present results provide a referential basis to support the traditional use of Eriosema species in the treatment of malaria.

Pentas longiflora Oliv. (Rubiaceae), a plant used in the treatment of Pityriasis Versicolor in Rwanda: Chemical composition and standardization of leaves and roots.

In Rwanda, the roots of Pentas longiflora Oliv. (Rubiaceae) have been used for a long time to treat Pityriasis versicolor. However, many people reported the use of leaves instead of roots. This research was conducted to compare the phytochemical composition and establish chromatographic methods for the standardization of roots and leaves extracts of P. longiflora. During this process, three new pentalongin glycosides (pentalonginoside A, pentalonginoside B, and pentalonginoside C) and two known glycosides of the same type (harounoside and clarinoside), as well as rutin, luteolin-7-rutinoside were isolated from methanol extract of leaves. In addition, pentalongin and psychorubrin, previously isolated from ethylacetate roots extract, were also identified in Pentas longiflora ethylacetate leaves extract. The presence of the antifungal compound pentalongin in leaves may explain the traditional use of leaves in the treatment of Pytiriasis versicolor. Furthermore, harounoside, psychorubrin, and pentalongin were selected as markers for HPLC fingerprints of MeOH extract. The accuracy and risk profile demonstrated the reliability of the validated method. In general, considerable variations of concentration in plant metabolites, including pentalongin, were observed between samples from different sites. The content in pentalongin (expressed as juglone) in collected samples ranged between 1.7 and 70.0 mg/100 g. The highest concentration (70.0 ± 17 mg/100 g) was registered in the cultivated samples from Mukoni. This important variation of pentalongin concentrations according to sampling sites, shows that in order to guarantee equivalent efficacy, finished products with P. longiflora should be standardized based on their pentalongin content.

In vitro Antileishmanial, Antitrypanosomal, and Anti-inflammatory-like Activity of Terminalia mollis Root Bark.

This study aims at determining the in vitro antitrypanosomal, antileishmanial, antioxidant, and anti-inflammatory-like activities of Terminalia mollis root crude extracts. The antitrypanosomal and antileishmanial activities on Trypanosoma brucei brucei (strain 427) and promastigotes of Leishmania mexicana mexicana (MHOM/BZ/84/BEL46) were evaluated in vitro. The methanolic root bark extract and standards were profiled by HPLC-PDA, and the majority of compounds identified using literature data. The in vitro antioxidant and anti-inflammatory-like activities were determined by evaluating the effect of crude extracts on reactive oxygen species produced by phorbol 12-myristate 13-acetate-stimulated equine neutrophils using lucigenin-enhanced chemiluminescence and on purified equine myeloperoxidase activity measured by specific immunological extraction followed by enzymatic detection. The methanolic, aqueous crude extract, and aqueous crude extract free of tannins exhibited good growth inhibition on Trypanosoma brucei brucei (IC50 3.72, 6.05, and 4.45 µg/mL respectively) but were inactive against Leishmania mexicana mexicana (IC50 > 100 µg/mL). Suramin (IC50 0.11 µg/mL) and amphotericin (IC50 0.11 µg/mL) were used as standard respectively for the antitrypanosomal and antileishmanial activity. Very interesting antioxidant and anti-inflammatory-like activities were observed with 50% hydroethanolic, aqueous crude extracts, and aqueous crude extract free of tannins as well as with pure punicalagin, gallic, and ellagic acid (IC50 0.38 - 10.51 µg/mL for 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid), chemiluminescence, and specific immunological extraction followed by enzymatic detection assays. The study results support traditional medicinal use of the plant for the treatment of parasitical disorders and revealed for the first time the antitrypanosomal potential, anti-inflammatory-like, and antioxidant activity of Terminalia mollis root.

The Inhibition of NLRP3 Inflammasome and IL-6 Production by Hibiscus noldeae Baker f. Derived Constituents Provides a Link to Its Anti-Inflammatory Therapeutic Potentials.

The activation of NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasome and/or its components is associated with the physio-pathogenesis of many respiratory diseases including asthma, COPD (chronic obstructive pulmonary disease), SARS Cov-2 (severe acute respiratory syndrome coronavirus 2), and in several autoimmune diseases. Hibiscus noldeae Baker f. has been widely reported to be traditionally used in the treatment of different ailments, some of which are of inflammatory background such as asthma, wounds, headache, etc. However, the claims have not been supported by evidence at the molecular and functional levels. Here, we report on the bio-guided fractionation of H. noldeae and assessment of the inhibitory properties of some fractions and purified compounds on NLRP3 inflammasome and Interleukin 6 (IL-6). The activation of the NLRP3 inflammasome was determined by detecting the activity of caspase-1 and the production of Interleukin 1ß (IL-1ß) in Lipopolysaccharide (LPS) and ATP-stimulated Tamm-Horsfall Protein 1 (THP-1) macrophages, while the production of IL-6 was studied in LPS-stimulated RAW264.7 mouse macrophages. It was observed that hexane and ethyl acetate fractions of the crude extract of the aerial parts of H. noldeae, as well as caffeic acid, isoquercetin, and ER2.4 and ER2.7 fractions revealed significant inhibitory effects on Caspase-1 activities, and on IL-1ß and IL-6 production. The ER2.4 and ER2.7 fractions downregulated the production of IL-1ß and IL-6, in a similar range as the caspase-1 inhibitor AC-YVAD-CHO and the drug Dexamethasone, both used as controls, respectively. Overall, our work does provide the very first scientific based evidence for Hibiscus noldeae anti-inflammatory effects and widespread use by traditional healers in Rwanda for a variety of ailments.

Factors associated with tungiasis among primary school children: a cross-sectional study in a rural district in Rwanda.

BACKGROUND: Tungiasis is a relatively frequent ectoparasitosis in low-income settings, yet its morbidity and social impact are still not well understood due to the scarcity of information. In Rwanda, data on the magnitude and conditions leading to the tungiasis is rare. This study sought to determine the prevalence and factors associated with tungiasis among primary school children in Rwandan setting. METHOD: A descriptive cross-sectional study utilising systematic random sampling method was adopted to select 384 children from three primary schools. From July to October 2018, data were collected on socio-demographic characteristics of children, parents, and households. Logistic regression was applied to analyse socio-demographic factors associated with tungiasis with a level of significance set at P-value< 0.05. RESULTS: Prevalence of tungiasis among three primary schools was 23%. Factors associated with tungiasis included walking barefoot (AOR: 78.41; 95% CI: 17.91-343.10), irregular wearing of shoes (AOR: 24.73; 95% CI: 6.27-97.41), having dirty feet (AOR: 12.69; 95% CI: 4.93-32.64), wearing dirty clothes (AOR: 12.69; 95% CI: 4.18-38.50), and living in a house with earthen plastered floor (AOR: 28.79; 95% CI: 7.11-116.57). Children infected with tungiasis attended class less frequently (AOR: 19.16, 95%CI: 7.20-50.97) and scored lower (AOR: 110.85, 95%CI: 43.08-285.20) than those non-infected. The low school attendance and poor performance could be partly explained by difficulty of walking, lack of concentration during school activities, and isolation or discrimination from classmates. CONCLUSION: Tungiasis was a public health challenge among school going children in a rural Rwandan setting. This study revealed that children affected with tungiasis had poor hygiene, inadequate housing environments and consequently poor school attendance and performance. Improving socio-economic conditions of households with special emphasis on hygiene of family members and housing conditions, would contribute to preventing tungiasis.

An ethnobotanical survey and inhibitory effects on NLRP3 inflammasomes/Caspase-1 of herbal recipes' extracts traditionally used in Rwanda for asthma treatment.

ETHNOPHARMACOLOGICAL RELEVANCE: Respiratory diseases and asthma, in particular, are nowadays a global health problem. In Rwanda, some traditional healers claim to treat asthma with plant-based recipes, though there is no scientific proof so far. AIM OF THE STUDY: Our study aimed at evaluating the toxicity and the anti-inflammatory effect of plant recipes used in Rwanda against asthma in order to select potential candidates for further characterization of the active compounds. MATERIALS AND METHODS: Water (aqueous) and methanol-dichloromethane (organic) extracts from selected folkloric recipes were submitted for toxicity test on THP-1 derived macrophages using CellTiter-Glo Luminescent Cell Viability Assay. The evaluation of the anti-inflammatory effect of the plant extracts was carried out using the Caspase-Glo 1 Inflammasome assay on THP-1 -derived macrophages. RESULTS: Most of both organic and aqueous extract showed more than 95% of cell viability up to 200 µg/ml, except for R03Cn organic extract that inhibited 25% of the cell viability. Plant extracts inhibited caspase-1 activation in THP-1 derived macrophages in a dose-dependent manner. Four extracts (R03Cn and R07Kn aqueous extracts, R10MK and R19Sz organic extracts) strongly downregulated the activation of caspase-1 (more than 70% at 50 µg/ml). In general, organic extracts exhibited better caspase-1 inhibitory effects than their aqueous counterparts. CONCLUSIONS: The inhibition of inflammasome/caspase-1 is one of key mechanisms of action in asthma. Some traditional recipes are active on this mechanism and are thus strong candidates for the treatment of asthma and other inflammasome-mediated diseases. Further investigations are needed to characterize active molecules.

In vitro and in vivo antiplasmodial activity of three Rwandan medicinal plants and identification of their active compounds.

In our previous study, we reported the interesting in vitro antiplasmodial activity of some Rwandan plant extracts. This gave rise to the need for these extracts to also be evaluated in vivo and to identify the compounds responsible for their antiplasmodial activity. The aim of our study was, on the one hand, to evaluate the antiplasmodial activity in vivo and the safety of the selected Rwandan medicinal plants used in the treatment of malaria, with the objective of promoting the development of improved traditional medicines and, on the other hand, to identify the active ingredients in the plants. Plant extracts were selected according to their selectivity index. The in vivo antiplasmodial activity of aqueous, methanolic, and dichloromethane extracts was then evaluated using the classical 4-day suppressive test on Plasmodium berghei infected mice. The activity of the plant extracts was estimated by measuring the percentage of parasitemia reduction, and the survival of the experimental animals was recorded. A bioguided fractionation was performed for the most promising plants, in terms of antiplasmodial activity, in order to isolate active compounds identified by means of spectroscopic and spectrometric methods. The highest level of antiplasmodial activity was observed with the methanolic extract of Fuerstia africana (> 70 %) on days 4 and 7 post-treatment after intraperitoneal injection and on day 7 using oral administration. After oral administration, the level of parasitemia reduction observed on day 4 post-infection was 44 % and 37 % with the aqueous extract of Terminalia mollis and Zanthoxylum chalybeum, respectively. However, the Z. chalybeum extract presented a high level of toxicity after intraperitoneal injection, with no animals surviving on day 1 post-treatment. F. africana, on the other hand, was safer with 40 % mouse survival on day 20 post-treatment. Ferruginol is already known as the active ingredient in F. Africana, and ellagic acid (IC50 = 175 ng/mL) and nitidine (IC50 = 77.5 ng/mL) were identified as the main active constituents of T. mollis and Z. chalybeum, respectively. F. africana presented very promising antiplasmodial activity in vivo. Although most of the plants tested showed some level of antiplasmodial activity, some of these plants may be toxic. This study revealed for the first time the role of ellagic acid and nitidine as the main antimalarial compounds in T. mollis and Z. chalybeum, respectively.

17-O-acetyl,10-hydroxycorynantheol, a selective antiplasmodial alkaloid isolated from Strychnos usambarensis leaves.

In the course of our investigations on Strychnos usambarensis leaves in order to isolate isostrychnopentamine, the main alkaloid responsible for the antiplasmodial activity of the plant, a new tertiary indolic alkaloid has been isolated: 17-O-acetyl,10-hydroxycorynantheol 1. Its structure was determined by means of spectroscopic and spectrometric methods such as UV, IR, CD, NMR, and ESI-MS. 17-O-acetyl,10-hydroxycorynantheol 1 is one of the most active monomeric indole alkaloids known to date showing an in vitro activity against Plasmodium falciparum close to 5 µM and a high selectivity.

Antiplasmodial and cytotoxic activities of Rwandan medicinal plants used in the treatment of malaria.

AIM OF THE STUDY: In our study, methanol, dichloromethane and aqueous extracts of 13 Rwandan medicinal plants used in the treatment of malaria were tested for in vitro antiplasmodial activity. MATERIALS AND METHODS: The growth inhibition of chloroquine-sensitive Plasmodium falciparum strain (3D7) was evaluated using the measurement of lactate dehydrogenase activity. The active extracts were also tested against the chloroquine-resistant Plasmodium falciparum strain (W2) and for cytotoxicity assay using human normal foetal lung fibroblasts (WI-38). RESULTS: The majority of the plants tested showed an antiplasmodial activity and the best results were observed with dichloromethane leaf and flower extracts of Tithonia diversifolia, leaf extract of Microglossa pyrifolia and root extract of Rumex abyssinicus, methanol leaf extract of Fuerstia africana, root bark extracts of Zanthoxylum chalybeum and methanol bark extract of Terminalia mollis. Those extracts were active (IC(50)<15mug/ml) on both chloroquine-sensitive and resistant strains of Plasmodium falciparum. Zanthoxylum chalybeum, Solanecio mannii and Terminalia mollis presented the best selectivity index. CONCLUSIONS: The traditional use of most of the plant evaluated was confirmed by the antiplasmodial test. This study revealed for the first time the antiplasmodial activity of two plants: Terminalia mollis and Rumex abyssinicus.