In vitro anti-yeast activity, kinetics and mechanism of action of essential oils from two cameroonian medicinal plants.

BACKGROUND: Treatment of Candida infections have become increasingly difficult due to antifungal drug resistance, which has drawn attention toward the search for innovative and effective drugs. This study aimed to assess the activity of essential oils (EOs) from Pentadiplandra brazzeana Baillon (PB) root and Drypetes gossweileri S. Moore (DG) stem bark against Candida albicans and Candida parapsilopsis strains, and determine their antifungal mechanism when tested alone or combined. METHODS: The anticandidal activity of the EOs using the checkerboard format was assessed using the broth micro-dilution technique. The checkerboard microtiter test was performed to evaluate the interaction of the EOs. The in vitro pharmacodynamics of the EOs alone or combined, using time-kill assays, following the chequerboard technique were evaluated. The anticandidal mode of action of these EOs, combined or not, was investigated using the sorbitol protection assay, and the ergosterol binding assay. Differences (p < 0.05) between the experimental and the control groups were evaluated using one way analysis of variance (ANOVA) followed by Tukey's test for multiple comparisons. RESULTS: Essential oils (EOs) from Drypetes gossweileri (DG) stem bark showed activity with MIC value of 62.5 µg/mL against Candida albicans and Candida parapsilopsis, whereas EOs from Pentadiplandra brazzeana (PB) root exhibited MICs of 125 µg/mL and 250 µg/mL against the respective yeasts. The EOs were fungicidal with synergism on C. parapsilopsis and additivity on C. albicans, with 2 to 64-fold drop in MIC values. The MIC combination of 31.25/7.81 µg/mL and 1.95/31.25 µg/mL (DG/PB EOs) required 20 and 18 h of exposure, respectively to effectively kill 99.9% of the inoculum. This fungicidal effect was accompanied by alteration of the cell walls and membranes of yeasts. CONCLUSION: The potency of the EOs combinations indicates further directions in their investigation as potential anticandidal agents.

In vitro antiplasmodial activity-directed investigation and UPLC-MS fingerprint of promising extracts and fractions from Terminalia ivorensis A. Chev. and Terminalia brownii Fresen.

ETHNOPHARMACOLOGICAL SIGNIFICANCE: Medicinal plants from the Terminalia genus are widely used as remedies against many infectious diseases, including malaria. As such, Terminalia ivorensis A. Chev. and Terminalia brownii Fresen. are famous due to their usefulness in traditional medicines to treat malaria and yellow fever. However, further information is needed on the extent of anti-Plasmodium potency of extracts and fractions from these plants and their phytochemical profile. AIM OF THE STUDY: This study was designed to investigate the in vitro antiplasmodial activity and to determine the chemical profile of promising extracts and fractions from T. ivorensis and T. brownii stem bark. MATERIALS AND METHODS: Crude aqueous, ethanolic, methanolic, hydroethanolic and ethyl acetate extracts were prepared by maceration from the stem barks of T. brownii and T. ivorensis. They were subsequently tested against chloroquine-sensitive (Pf3D7) and multidrug-resistant (PfDd2) strains of P. falciparum using the parasite lactate dehydrogenase (PfLDH) assay. Extracts showing very good activity on both plasmodial strains were further fractionated using column chromatography guided by evidence of antiplasmodial activity. All bioactive extracts and fractions were screened for their cytotoxicity on Vero and Raw cell lines using the resazurin-based assay and on erythrocytes using the hemolysis assay. The phytochemical profiles of selected potent extracts and fractions were determined by UPLC-QTOF-MS analysis. RESULTS: Of the ten extracts obtained from both plant species, nine showed inhibitory activity against both P. falciparum strains (Pf3D7 and PfDd2), with median inhibitory concentration (IC50) values ranging from 0.13 µg/ml to 10.59 µg/ml. Interestingly, the aqueous extract of T. ivorensis (TiW) and methanolic extract of T. brownii (TbM) displayed higher antiplasmodial activities against both strains (IC50 0.13-1.43 µg/ml) and high selectivity indices (SI > 100). Their fractionation led to two fractions from T. ivorensis and two from T. brownii that showed very promising antiplasmodial activity (IC50 0.15-1.73 µg/mL) and SI greater than 100. The hemolytic assay confirmed the safety of crude extracts and fractions on erythrocytes. UPLC-MS-based phytochemical analysis of the crude aqueous extract of T. ivorensis showed the presence of ellagic acid (1) and leucodelphidin (2), while analysis of the crude methanol extract of T. brownii showed the presence of ellagic acid (1), leucodelphinidin (2), papyriogenin D (3), dihydroactinidiolide (4) and miltiodiol (5). CONCLUSIONS: The extracts and fractions from T. ivorensis and T. brownii showed very good antiplasmodial activity, thus supporting the traditional use of the two plants in the treatment of malaria. Chemical profiling of the extracts and fractions led to the identification of chemical markers and the known antimalarial compound ellagic acid. Further isolation and testing of other pure compounds from the active fractions could lead to the identification of potent antiplasmodial compounds.

Implementation and continued validation of the malaria Plasmodium falciparum lactate dehydrogenase-based colorimetric assay for use in antiplasmodial drug screening.

Antimalarial drug discovery has been facilitated by the development of various in vitro drug susceptibility testing methods suitable for medium-throughput or high-throughput campaigns. Among many, the Plasmodium falciparum lactate dehydrogenase (PfLDH) assay has acceptable demand on equipment, labour, technical skills and affordability and offers a good opportunity for scientists in low- and middle-income countries to participate in the global effort of discovering future antimalarial drugs. Hence, to enable our search for novel antimalarial drugs, we implemented and examined assay conditions and validated the PfLDH-based method in our laboratory using a reference set of standard antimalarial drugs with known activity against Plasmodium falciparum strains. The PfLDH assay revealed acceptable linearity profiles of R2 = 0.97 and 0.92 for Pf3D7 and PfDd2, respectively, achieved at 2% parasitaemia and 1% haematocrit. The detection and quantitation limits (DL and QL) of the PfLDH-based assay were 0.09% and 0.4% parasitemia, respectively. The assay showed an acceptable average Z-factor between 0.76 and 0.79 and was considerably robust. The average interassay reproducibility via percent coefficient of variation (%CV) was 5.47 between independent experiments. Overall, the PfLDH-based method produced a reliable and reproducible drug screening profile for in vitro assays in our setting. There were no significant interassay variability or hazards of other screening assays.

Specific sub fractions from Terminalia mantaly (H. Perrier) extracts potently inhibit Plasmodium falciparum rings, merozoite egress and invasion.

ETHNOPHARMACOLOGICAL RELEVANCE: Terminalia mantaly (H. Perrier) and Terminalia superba (Engl. & Diels) are sources of treatment for various diseases, including malaria and/or related symptoms in parts of Southwestern Cameroon. However, there is limited information on the extent of the antiplasmodial potential of their extracts. AIM OF THE STUDY: The present study was designed to investigate the antiplasmodial potential of chromatographic sub fractions (SFs) from promising fractions of Terminalia mantaly (Tm) [TmsbwChl, the chloroform fraction from water extract of Tm, IC50 (µg/mL) PfINDO: 0.56, Pf3D7: 1.12; SI > 357 (HEK/PfINDO) & 178 (HEK/Pf3D7)] and Terminalia superba (Ts) [TsrmEA, the ethyl acetate fraction from methanolic extract of Ts, IC50 (µg/mL) PfINDO: 1.82, Pf3D7: 1.65; SI > 109 (HEK/PfINDO) & 121 (HEK/Pf3D7)] obtained from previous studies. The SFs were tested against Plasmodium falciparum 3D7 (Pf3D7-chloroquine sensitive) and INDO (PfINDO-chloroquine resistant) strains in culture. Also, the phytochemical profile of potent SFs was determined and finally, the inhibition of the asexual blood stages of Plasmodium falciparum by the SFs with the highest promise was assessed. MATERIAL AND METHODS: Selected SFs were submitted to a second bio-guided fractionation using silica gel column chromatography. The partial phytochemical composition of potent antiplasmodial SFs was determined using gas chromatography coupled to mass spectrometry (GC-MS). The SYBR Green I-based fluorescence microtiter plate assay was used to monitor the growth of Plasmodium falciparum parasites in culture in the presence or absence of extracts. Microscopy and flow cytometry counting was used to assess the Plasmodium falciparum stage-specific inhibition and post-drug exposure growth suppression by highly potent extracts. RESULTS: Twenty-one of the 39 SFs afforded from TmsbwChl showed activity (IC50: 0.29-4.74 µg/mL) against both Pf3D7 and PfINDO strains. Of note, eight SFs namely, Tm25, Tm28-30, Tm34-36 and Tm38, exerted highly potent antiplasmodial activity (IC50 < 1 µg/mL) with IC50PfINDO: 0.41-0.84 µg/mL and IC50Pf3D7: 0.29-0.68 µg/mL. They also displayed very high selectivity (50 < SIPfINDO, SIPf3D7 > 344) on the two Plasmodial strains. On the other hand, 7 SFs (SFs Ts03, Ts04, Ts06, Ts09, Ts10, Ts12 and Ts13) from TsrmEA showed promising inhibitory potential against both parasite strains (IC50: 2.01-5.14 µg/mL). Sub fraction Tm36 (IC50PfINDO: 0.41 µg/mL, SIPfINDO > 243; IC50Pf3D7: 0.29 µg/mL, SIPf3D7 > 344) showed the highest promise. The GC-MS analysis of the 8 selected SFs led to the identification of 99 phytometabolites, with D-limonene (2), benzaldehyde (12), carvone (13), caryophyllene (35), hexadecanoic acid, methyl ester (74) and 9-octadecenoic acid, methyl ester (82) being the main constituents. Sub fractions Tm28, Tm29, Tm30, Tm36 and Tm38 inhibited all the three intraerythrocytic stages of P. falciparum, with strong potency against ring stage development, merozoite egress and invasion processes. CONCLUSIONS: This study has identified highly potent antiplasmodial SFs from Terminalia mantaly with significant activity on the intraerythrocytic development of Plasmodium falciparum. These SFs qualify as promising sources of novel antiplasmodial lead compounds. Further purification and characterization studies are expected to unravel molecular targets in rings and merozoites.

Identification of 3,3'-O-dimethylellagic acid and apigenin as the main antiplasmodial constituents of Endodesmia calophylloides Benth and Hymenostegia afzelii (Oliver.) Harms.

BACKGROUND: Endodesmia calophylloides and Hymenostegia afzelii belong to the Guttiferae and Caesalpiniaceae plant families with known uses in African ethno-medicine to treat malaria and several other diseases. This study aimed at identifying antiplasmodial natural products from selected crude extracts from H. afzelii and E. calophylloides and to assess their cytotoxicity. METHODS: The extracts from H. afzelii and E. calophylloides were subjected to bioassay-guided fractionation to identify antiplasmodial compounds. The hydroethanol and methanol stem bark crude extracts, fractions and isolated compounds were assessed for antiplasmodial activity against the chloroquine-sensitive 3D7 and multi-drug resistant Dd2 strains of Plasmodium falciparum using the SYBR green I fluorescence-based microdilution assay. Cytotoxicity of active extracts, fractions and compounds was determined on African green monkey normal kidney Vero and murine macrophage Raw 264.7 cell lines using the Resazurin-based viability assay. RESULTS: The hydroethanolic extract of H. afzelii stem bark (HasbHE) and the methanolic extract of E. calophylloides stem bark (EcsbM) exhibited the highest potency against both Pf3D7 (EC50 values of 3.32 ± 0.15 µg/mL and 7.40 ± 0.19 µg/mL, respectively) and PfDd2 (EC50 of 3.08 ± 0.21 µg/mL and 7.48 ± 0.07 µg/mL, respectively) strains. Both extracts showed high selectivity toward Plasmodium parasites (SI > 13). The biological activity-guided fractionation led to the identification of five compounds (Compounds 1-5) from HasbHE and one compound (Compound 6) from EcsbM. Of these, Compound 1 corresponding to apigenin (EC50 Pf3D7, of 19.01 ± 0.72 µM and EC50 PfDd2 of 16.39 ± 0.52 µM), and Compound 6 corresponding to 3,3'-O-dimethylellagic acid (EC50 Pf3D7 of 4.27 ± 0.05 µM and EC50 PfDd2 of 1.36 ± 0.47 µM) displayed the highest antiplasmodial activities. Interestingly, both compounds exhibited negligible cytotoxicity against both Vero and Raw 264.7 cell lines with selectivity indices greater than 9. CONCLUSIONS: This study led to the identification of two potent antiplasmodial natural compounds, 3,3'-O-dimethylellagic acid and apigenin that could serve as starting points for further antimalarial drug discovery.

Antifungal potential of extracts, fractions and compounds from Uvaria comperei (Annonaceae) and Oxyanthus unilocularis (Rubiaceae).

Phytochemical study of Uvaria comperei afforded an alkaloid, 8,9-dimethoxy-5H-phenanthridin-6-one (1), isolated and characterised (assignment of 1H and 13C NMR) for the first time from a natural source along with two flavonoids, (2S)-5-hydroxy-7,8-dimethoxyflavanone (2) and (2S)-7-hydroxy-5-methoxy-6,8-dimethylflavone (3). Clethric acid (4), oleanoic acid (5), ß-sitosterol 3-O-ß-D-glucopyranoside (9), ß-sitosterol palmitate (6) and a mixture of stigmasterol (7) and ß-sitosterol (8) were isolated from Oxyanthus unilocularis. The structures of these compounds were elucidated using modern spectroscopic techniques including1D and 2D Nuclear Magnetic Resonance (NMR) Spectroscopy (1H, 13C, 1H-1H COSY, HSQC, HMBC) and Mass Spectrometry. Some fractions and compounds from Uvaria comperei exhibited good antifungal activity against clinical isolates and standard strains of yeast species of Candida and Cryptococcus genera while extracts from Oxyanthus unilocularis displayed weak antifungal activity. The results obtained show that Uvaria comperei could be a potential source of antifungal drugs.

Spirofused tetrahydroisoquinoline-oxindole hybrids as a novel class of fast acting antimalarial agents with multiple modes of action.

Molecular hybridization of privileged scaffolds may generate novel antiplasmodial chemotypes that display superior biological activity and delay drug resistance. In the present study, we describe the in vitro activities and mode of action of 3',4'-dihydro-2'H-spiro[indoline-3,1'-isoquinolin]-2-ones, a novel class of spirofused tetrahydroisoquinoline-oxindole hybrids, as novel antimalarial agents. Whole cell phenotypic screening of these compounds identified (14b), subsequently named (±)-moxiquindole, as the most potent compound in the current series with equipotent antiplasmodial activity against both chloroquine sensitive and multidrug resistant parasite strains with good selectivity. The compound was active against all asexual stages of the parasite including inhibition of merozoite egress. Additionally, (±)-moxiquindole exhibited significant inhibitory effects on hemoglobin degradation, and disrupted vacuolar lipid dynamics. Taken together, our data confirm the antiplasmodial activity of (±)-moxiquindole, and identify 3'4'-dihydro-2'H-spiro[indoline-3,1'-isoquinolin]-2-ones as a novel class of antimalarial agents with multiple modes of action.

In Vivo Antiplasmodial Activity of Terminalia mantaly Stem Bark Aqueous Extract in Mice Infected by Plasmodium berghei.

BACKGROUND: Terminalia mantaly is used in Cameroon traditional medicine to treat malaria and related symptoms. However, its antiplasmodial efficacy is still to be established. OBJECTIVES: The present study is aimed at evaluating the in vitro and in vivo antiplasmodial activity and the oral acute toxicity of the Terminalia mantaly extracts. MATERIALS AND METHODS: Extracts were prepared from leaves and stem bark of T. mantaly, by maceration in distilled water, methanol, ethanol, dichloromethane (DCM), and hexane. All extracts were initially screened in vitro against the chloroquine-resistant strain W2 of P. falciparum to confirm its in vitro activity, and the most potent one was assessed in malaria mouse model at three concentrations (100, 200, and 400 mg/kg/bw). Biochemical, hematological, and histological parameters were also determined. RESULTS: Overall, 7 extracts showed in vitro antiplasmodial activity with IC50 ranging from 0.809 µg/mL to 5.886 µg/mL. The aqueous extract from the stem bark of T. mantaly (Tmsbw) was the most potent (IC50 = 0.809 µg/mL) and was further assessed for acute toxicity and efficacy in Plasmodium berghei-infected mice. Tmsbw was safe in mice with a median lethal dose (LD50) higher than 2000 mg/kg of body weight. It also exerted a good antimalarial efficacy in vivo with ED50 of 69.50 mg/kg and had no significant effect on biochemical, hematological, and histological parameters. CONCLUSION: The results suggest that the stem bark extract of T. mantaly possesses antimalarial activity.

Potent antiplasmodial extracts and fractions from Terminalia mantaly and Terminalia superba.

BACKGROUND: The emergence and spread of malaria parasites resistant to artemisinin-based combination therapy stresses the need for novel drugs against malaria. Investigating plants used in traditional medicine to treat malaria remains a credible option for new anti-malarial drug development. This study was aimed at investigating the antiplasmodial activity and selectivity of extracts and fractions from Terminalia mantaly and Terminalia superba (Combretaceae) that are used in Cameroon to treat malaria. METHODS: Twelve methanolic (m) and water (w) extracts obtained by maceration of powdered dried leaves (l), stem bark (sb) and root (r) of Terminalia mantaly (Tm) and Terminalia superba (Ts) and 12 derived fractions of hexane, chloroform, ethyl acetate and 4 final residues of selected extracts were assessed for antiplasmodial potential in vitro against the chloroquine-resistant PfINDO and the chloroquine-sensitive Pf3D7 strains of Plasmodium falciparum using the SYBR green I-based fluorescence assay. The cytotoxicity of potent extracts and fractions was evaluated in vitro using the MTT assay on HEK239T cell line. RESULTS: The antiplasmodial IC50 of extracts from both plants ranged from 0.26 to > 25 µg/mL. Apart from the extracts Tmrm and Tsrw that exerted moderate antiplasmodial activities (IC50: 5-20 µg/mL) and Tmrw that was found to be non-active at the tested concentrations (IC50 > 25 µg/mL), all other tested crude extracts exhibited potent activities with IC50 < 5 µg/mL. The aqueous extracts from the stem bark of Terminalia mantaly (Tmsbw) and the leaf of Terminalia superba (Tslw) displayed the highest antiplasmodial activities (IC50: 0.26-1.26 µg/mL) and selectivity (SI > 158) on both resistant PfINDO and sensitive Pf3D7 strains. Four fractions upon further extraction with chloroform and ethyl acetate (TmlwChl, TmsbwChl, TmsbwEA, TsrmEA) afforded from three selected crude extracts (Tmlw, Tmsbw, Tsrm) exhibited highly potent activities against both P. falciparum strains (IC50 < 2 µg/mL) and high selectivity (SI > 109). CONCLUSIONS: The results achieved in this work validate the reported traditional use of Terminalia mantaly and Terminalia superba to treat malaria. Moreover, the highly potent and selective fractions warrant further investigation to characterize the active antiplasmodial principles and progress them to rodent malaria models studies if activity and selectivity are evidenced.

Ethnobotanical survey of medicinal plants used as insects repellents in six malaria endemic localities of Cameroon.

BACKGROUND: The combined efforts to combat outdoor/indoor transmission of malaria parasites are hampered by the emerging vector resistance in a wide variety of malaria-endemic settings of Africa and the rest of the world, stressing the need for alternative control measures. This study aimed at documenting insect's repellent plant species used by indigenous populations of 6 localities of East, South, West and Centre regions of Cameroon. METHODS: Information was gathered through face-to-face interviews guided by a semi-structured questionnaire on the knowledge of medicinal plants with insect repellent properties. RESULTS: A total of 182 informants aged from 25 to 75 years were recruited by convenience from May to June 2015. The informants had general knowledge about insects' repellent plants (78.6%). A total of 16 plant species were recorded as insects' repellents with 50% being trees. The most cited plants were Canarium schweinfurthii (Burseraceae) (in four localities, 58/182), Elaeis guineensis (Arecaceae) (in three localities, 38/182), Chromolaena odorata (Compositae) (16/182) and Citrus limon (Rutaceae) (11/182) in two localities each. Among the repellent plant species recorded, 50% were reported to be burnt to produce in-house smokes, 31.2% were mashed and applied on the body, and 18.8% were hung in the houses. The leaf was the most commonly used plant part (52.9%), followed by the bark (17.6%). CONCLUSIONS: This study has shown that rural populations of the 6 targeted localities possess indigenous knowledge on repellent plants that are otherwise cost-effective and better choice for repelling insects including malaria-transmitting mosquitoes. Meanwhile, such practices should be validated experimentally and promoted as sustainable malaria transmission control tools in the remotely located communities.