Chemical and Antiplasmodial Investigations on Eremophila-Derived Alkaloids and Semisynthetic Ether Analogues.

Microthecaline A (1), the known antiplasmodial quinoline serrulatane alkaloid from the roots of Eremophila microtheca F. Muell. ex Benth. (Scrophulariaceae), was targeted for isolation and subsequent use in the generation of a semisynthetic ether library. A large-scale extraction and isolation yielded the previously undescribed quinoline serrulatane microthecaline B (2), along with crystalline 1 that enabled the first X-ray crystallographic analysis to be undertaken on this rare alkaloid structure class. The X-ray diffraction analysis of 1 supported the absolute configuration assignment of microthecaline A, which was originally assigned by ECD data analysis. Microthecaline A (1) was converted into 10 new semisynthetic ether derivatives (3-12) using a diverse series of commercially available alkyl halides. Chemical structures of the new serrulatane alkaloid and semisynthetic ether analogues were assigned by spectroscopic and spectrometric analyses. Antiplasmodial evaluations of 1-12 showed that the semisynthetic derivative 5 elicited the most potent activity with an IC50 value of 7.2 µM against Plasmodium falciparum 3D7 (drug-sensitive) strain.

Koshidacins A and B, Antiplasmodial Cyclic Tetrapeptides from the Okinawan Fungus Pochonia boninensis FKR-0564.

Two new antiplasmodial peptides, named koshidacins A (1) and B (2), were discovered from the culture broth of the Okinawan fungus Pochonia boninensis FKR-0564. Their structures, including absolute configurations, were elucidated by a combination of spectroscopic methods and chemical derivatization. Both compounds showed moderate in vitro antiplasmodial activity against Plasmodium falciparum strains, with IC50 values ranging from 17.1 to 0.83 µM. In addition, compound 2 suppressed 41% of malaria parasites in vivo when administered intraperitoneally at a dose of 30 mg/kg/day for 4 days.

Isolation, characterisation and complement fixation activity of acidic polysaccharides from Argemone mexicana used as antimalarials in Mali.

CONTEXT: Global studies on Argemone mexicana L. (Papaveraceae) traditionally used against malaria in Mali are limited to its low-mass compounds activities, and little information on its bioactive polysaccharides is available. OBJECTIVE: This study determines the structure and the immunomodulatory activity of polysaccharides from aerial parts of A. mexicana. MATERIALS AND METHODS: Acidic polysaccharides from this plant material named HMAmA1 and HMAmA2 were isolated from water extracts. Their monosaccharide composition was determined by gas chromatography. Glycosidic linkages were determined using GC-MS. NMR was also applied. The polymers were tested for effects on the human complement system in vitro at different doses. RESULTS: The monosaccharide composition showed that the two polysaccharides contained in different amounts the following monomers: arabinose, rhamnose, galactose, and galacturonic acid. Overall structural analysis showed the presence of a low ratio of 1,2-linked rhamnose compared to 1,4-linked galacturonic acid with arabinogalactans substituted on position 4 of rhamnose. NMR data showed the presence of galacturonans alternated by rhamnogalacturonans bearing arabinose and galactose units. α-Linkages were found for l-arabinose, l-rhamnose and d-galacturonic acid, while ß-linkages were found for d-galactose. The two polysaccharides exhibited strong complement fixation activities, with HMAmA1 being the highest potent fraction. ICH50 value of HMAmA1 was 5 µg/mL, compared to the control BPII being 15.9 µg/mL. DISCUSSION AND CONCLUSIONS: Polysaccharides form A. mexicana presented a complement fixation effect. The complement system is an important part of the immune defense, and compounds acting on the cascade are of interest. Therefore, these polymers may be useful as immunodulatory agents.

Antiplasmodial diterpenoid alkaloid from Aconitum heterophyllum Wall. ex Royle: Isolation, characterization, and UHPLC-DAD based quantification.

ETHNOPHARMACOLOGICAL RELEVANCE: Aconitum heterophyllum Wall. ex Royle is a traditionally important medicinal plant having numerous therapeutic actions as documented in Ayurveda. This plant is traditionally known for combating worm infestation, fever, respiratory tract disease, vomiting, diarrhoea, diabetes, skin disorders, anaemia, and joint disorders. Further, it has been used alone and in combination with other plants to prepare various anti-malarial formulations. However, there is no report on the assessment of its anti-plasmodial activity, and the metabolite(s) responsible for this activity. AIM OF THE STUDY: The main aim of this study was to conduct phytochemical investigation of A. heterophyllum roots for the preparation of extract, fractions, and isolation of pure molecules to identify active fractions/molecules responsible for the anti-plasmodial activity, and development of UHPLC-DAD based analytical method which can be used for the quantification of marker compounds in the extracts and fractions. MATERIALS AND METHODS: Hydroalcoholic extract (1:1 v/v) and fractions (n-hexane, chloroform, ethyl acetate, n-butanol, and water) were prepared from the dried powdered roots of A. heterophyllum. Fractions were further subjected to silica gel column chromatography to isolate pure specialized secondary metabolites from this plant. All extracts, fractions, and pure molecules were evaluated against the chloroquine resistant Pf INDO and chloroquine sensitive Pf3D7 strains in culture for calculating their IC50 values. UHPLC-DAD based analytical method was also developed for the first time for the quantification of marker compounds and quality assessment of this commercially important Himalayan medicinal plant. RESULTS: Phytochemical investigation of A. heterophyllum root led to the isolation of six specialized metabolites viz. 2-O-cinnamoyl hetisine (1), atisinium (2), 4-oxabicyclo [3.2.2] nona-1(7),5,8-triene (3), atisinium cinnamate (4), aconitic acid (5), and atisinium formate (6). Compound 1 is a new hetisine type diterpenoid alkaloid, compounds 4 and 6 are new counter ionic forms observed with atisinium ion, and compound 3 is being reported for the first time from this genus. Chloroform fraction was found to be the most active with IC50 (µg/mL) 1.01 (Pf INDO) and 1.32 (Pf3D7). The molecule 2-O-cinnamoyl hetisine (1), a new diterpenoid alkaloid isolated from chloroform fraction, showed promising antiplasmodial activities with IC50 (µM) 1.92 (Pf INDO) and 10.8 (Pf 3D7). The activity of chloroform fraction was further validated by the developed UHPLC-DAD based method as the quantity of 2-O-cinnamoyl hetisine (1) was higher in the chloroform fraction (≅200 mg/g) than in all other fractions (<7 mg/g). Atisinium (2) and 2-O-cinnamoyl hetisine (1) were found to be the main marker compounds of this plant based on quantity and antiplasmodial activity, respectively. CONCLUSION: This study provides the scientific rationale for the traditional use of this plant in treating malaria. Further, this study revealed that the anti-malarial potential of this plant might be due to the presence of diterpenoid alkaloids.

Dihydrolucilactaene, a Potent Antimalarial Compound from Fusarium sp. RK97-94.

A recently discovered secondary metabolism regulator, NPD938, was used to alter the secondary metabolite profile in Fusarium sp. RK97-94. Three lucilactaene analogues were detected via UPLC-ESI-MS analysis in NPD938-treated culture. The three metabolites were successfully purified and identified as dihydroNG391 (1), dihydrolucilactaene (2), and 13α-hydroxylucilactaene (3) via extensive spectroscopic analyses. DihydroNG391 (1) exhibited weak in vitro antimalarial activity (IC50 = 62 µM). In contrast, dihydrolucilactaene (2) and 13α-hydroxylucilactaene (3) showed very potent antimalarial activity (IC50 = 0.0015 and 0.68 µM, respectively). These findings provide insight into the structure-activity relationship of lucilactaene and its analogues as antimalarial lead compounds.

Isolation and biological activity of azocine and azocane alkaloids.

Thousands of known alkaloids contain a nitrogen (N) heterocycle. While five-, six- and seven-membered N-heterocycles (ie: pyrroles, imidazoles, indoles, pyridines and azepines and their saturated variants) are common, those with an eight-membered N-heterocycle are comparatively rare. This review discusses the structure and bioactivity of alkaloids that contain an azocine (or saturated azocane) ring, and the array of sources whence they originate.

Inhibition of heme detoxification pathway in malaria parasite by 3-hydroxy-11-keto-ß-boswellic acid isolated from Boswellia serrata.

Malaria eradication is still a major global health problem in developing countries, which has been of more concern ever since the malaria parasite has developed resistance against frontline antimalarial drugs. Historical evidence proves that the plants possess a major resource for the development of novel anti-malarial drugs. In the present study, the bioactivity guided fractionation of the oleogum-resin of Boswellia serrata Roxb. yielded the optimum activity in the ethyl acetate fraction with an IC50 of 22 ± 3.9 µg/mL and 26.5 ± 4.5 µg/mL against chloroquine sensitive (NF54) and resistant (K1) strains of Plasmodium falciparum respectively. Further, upon fractionation, the ethyl acetate fraction yielded four major compounds, of which 3-Hydroxy-11-keto-ß-boswellic acid (KBA) was found to be the most potent with IC50 values 4.5 ± 0.60 µg/mL and 6.25 ± 1.02 µg/mL against sensitive and resistant strains respectively. KBA was found to inhibit heme detoxification pathways, one of the most common therapeutic targets, which probably lead to an increase in reactive oxygen species (ROS) and nitric oxide (NO) detrimental to P. falciparum. Further, the induced intracellular oxidative stress affected the macromolecules in terms of DNA damage, increased lipid peroxidation, protein carbonylation as well as loss of mitochondrial membrane potential. However, it did not exhibit any cytotoxic effect in VERO cells. Under in vivo conditions, KBA exhibited a significant reduction in parasitemia, retarding the development of anaemia, resulting in an enhancement of the mean survival time in Plasmodium yoelii nigeriensis (chloroquine-resistant) infected mice. Further, KBA did not exhibit any abnormality in serum biochemistry of animals that underwent acute oral toxicity studies at 2000 mg/kg body weight.

Potent antiplasmodial alkaloids from the rhizobacterium Pantoea agglomerans as hemozoin modulators.

Global health concern regarding malaria has increased since the first report of artemisinin-resistant Plasmodium falciparum (Pf) two decades ago. The current therapies suffer various drawbacks such as low efficacy and significant side effects, alarming for an urgent need of more effective and less toxic drugs with higher patient compliance. Chemical entities with natural origins become progressively attractive as new drug leads due to their structural diversity and bio-compatibility. This study initially aimed at the targeted isolation of hydroxyquinoline derivatives following our published genomics and metabolomics study of Pantoea agglomerans (Pa). Fermentation of Pa on a pre-selected medium followed by chromatographic isolation, NMR and HRMS analyses led to the characterisation of one new hydroxyquinoline alkaloid together with another six known congeners and two known hydroxyquinolone derivatives. When screened for their antimalarial activity by high throughput screening against asexual blood-stage parasites, almost all compounds showed potent and selective sub-micromolar activities. Computational investigation was performed to identify the antiplasmodial potential targets. Ligand-based similarity search predicted the tested compounds to act as hemozoin inhibitors. Computational target identification results were further validated by competitive hemozoin inhibitory properties of hydroxyquinoline and hydroxyquinolone derivatives in vitro. The overall results suggest this natural scaffold is of potential to be developed as antimalarial drug lead.

Exploring natural microbial resources for the discovery of anti-malarial compounds.

Microorganisms in nature are highly diverse biological resources, which can be explored for drug discovery. Some countries including Brazil, Columbia, Indonesia, China, and Mexico, which are blessed with geographical uniqueness with diverse climates and display remarkable megabiodiversity, potentially provide microorganismal resources for such exploitation. In this review, as an example of drug discovery campaigns against tropical parasitic diseases utilizing microorganisms from such a megabiodiversity country, we summarize our past and on-going activities toward discovery of new antimalarials. The program was held in a bilateral collaboration between multiple Indonesian and Japanese research groups. In order to develop a new platform of drug discovery utilizing Indonesian bioresources under an international collaborative scheme, we aimed at: 1) establishment of an Indonesian microbial depository, 2) development of robust enzyme-based and cell-based screening systems, and 3) technology transfer necessary for screening, purification, and identification of antimalarial compounds from microbial culture broths. We collected, characterized, and deposited Indonesian microbes. We morphologically and genetically characterized fungi and actinomycetes strains isolated from 5 different locations representing 3 Indonesian geographical areas, and validated genetic diversity of microbes. Enzyme-based screening was developed against two validated mitochondrial enzymes from Plasmodium falciparum, dihydroorotate dehydrogenase and malate:quinone oxidoreductase, while cell-based proliferation assay was developed using the erythrocytic stage parasite of 3D7 strain. More than 17 thousands microbial culture extracts were subjected to the enzyme- and cell-based screening. Representative anti-malarial compounds discovered in this campaign are discussed, including a few isolated compounds that have been identified for the first time as anti-malarial compounds. Our antimalarial discovery campaign validated the Indonesian microbial library as a powerful resource for drug discovery. We also discuss critical needs for selection criteria for hits at each stage of screening and hit deconvolution such as preliminary extraction test for the initial profiling of the active compounds and dereplication techniques to minimize repetitive discovery of known compounds.

High-throughput quantitation method for amodiaquine and desethylamodiaquine in plasma using supported liquid extraction technology.

Amodiaquine is a drug used for treatment of malaria and is often used in combination with artesunate in areas where malaria parasites are still susceptible to amodiaquine. Liquid chromatography tandem-mass spectrometry was used to quantify amodiaquine and its active metabolite, desethylamodiaquine, in plasma samples. A low sample volume of 100 µl, and high-throughput extraction technique using a supported liquid extraction (SLE+) technique on an automated liquid handler platform for faster sample processing are some of the advantages of this method. Separation of amodiaquine from desethylamodiaquine was achieved using a reversed phase Zorbax SB-CN 50 mm × 4.6 mm, I.D. 3.5 µm column with acetonitrile and 20 mM ammonium formate with 1% formic acid pH ~ 2.6 (15-85, v/v) as mobile phase. The absolute recoveries of amodiaquine and desethylamodiaquine were 66% to 76%, and their isotope label internal standard were in the range of 73% to 85%. Validation results of the developed method demonstrated intra-batch and inter-batch precisions within the acceptance criteria range of ± 15.0%. There were no matrix or carry-over effects observed. The lower limit of quantification was 1.08 ng/ml for amodiaquine and 1.41 ng/ml for desethylamodiaquine. The method showed robust and accurate performance with high sensitivity. Thus, the validated method was successfully implemented and applied in the evaluation of a clinical trial where participants received artemether-lumefantrine plus amodiaquine twice daily for three days (amodiaquine dose of 10 mg base/kg/day).

Antiplasmodial and Cytotoxic Flavonoids from Pappea capensis (Eckl. & Zeyh.) Leaves.

Ethnobotanical surveys indicate that the Masai and Kikuyu in Kenya, the Venda in South Africa, and the Gumuz people of Ethiopia use Pappea capensis for the treatment of malaria. The present study aimed to investigate the phytochemical and antiplasmodial properties of the plant leaves. The bioactive compounds were isolated using chromatographic techniques. The structures were established using NMR, HRMS, and UV spectroscopy. Antiplasmodial activity of P. capensis leaf extract and isolated compounds against chloroquine-sensitive 3D7 P. falciparum was evaluated using the parasite lactate dehydrogenase assay. Cytotoxicity against HeLa (human cervix adenocarcinoma) cells was determined using the resazurin assay. The extract inhibited the viability of Plasmodium falciparum by more than 80% at 50 µg/mL, but it was also cytotoxic against HeLa cells at the same concentration. Chromatographic purification of the extract led to the isolation of four flavonoid glycosides and epicatechin. The compounds displayed a similar activity pattern with the extract against P. falciparum and HeLa cells. The results from this study suggest that the widespread use of P. capensis in traditional medicine for the treatment of malaria might have some merits. However, more selectivity studies are needed to determine whether the leaf extract is cytotoxic against noncancerous cells.

Antiplasmodial and Cytotoxic Activities of Extract and Compounds from Ozoroa obovata (Oliv.) R. & A. Fern. var. obovata.

Ozoroa obovata (Oliv.) R. & A. Fern. var. obovata found in KwaZulu-Natal in South Africa was investigated for phytochemical constituents, and for antiplasmodial and cytotoxic effects. The plant leaves were collected from the University of KwaZulu-Natal (UKZN) arboretum on the Pietermaritzburg Campus, in March 2019. The inhibitory activity against 3D7 Plasmodium falciparum was determined using the parasite lactate dehydrogenase (pLDH) assay and cytotoxicity against HeLa cells was evaluated using the resazurin assay. The bioactive compounds were isolated by chromatographic purification and their structures were established with spectroscopic and spectrometric techniques. The plant leaf extract displayed significant antiplasmodial activity at 50 µg/mL and was also cytotoxic against HeLa cells. Chromatographic purification of the extract led to the isolation of two biflavonoids, four flavonoid glycosides, a steroid glycoside, and a megastigmene derivative. The compounds displayed antiplasmodial and antiproliferative activities at 50 µg/mL but the activity was substantially reduced at 10 µg/mL. The activities and compounds are being reported in O. obovata for the first time.

Developmental stages influence in vivo antimalarial activity of aerial part extracts of Schkuhria pinnata.

ETHNOPHARMACOLOGICAL RELEVANCE: Malaria remains a dire health challenge, particularly in sub-Saharan Africa. In Uganda, it is the most ordinary condition in hospital admission and outpatient care. The country's meager health services compel malaria patients to use herbal remedies such as Schkuhria pinnata (Lam.) Kuntze ex Thell (Asteraceae). Although in vivo studies tested the antimalarial activity of S. pinnata extracts, plant developmental stages and their effect at different doses remain unknown. AIM OF THE STUDY: This study aims to determine the effect of the plant developmental stage on the antimalarial activity of S. pinnata in mice and to document the acute oral toxicity profile. METHODS: Seeds of S. pinnata were grown, and aerial parts of each developmental stage were harvested. Extraction was done by maceration in 70% methanol. The antimalarial activity was evaluated using chloroquine-sensitive Plasmodium berghei on swiss albino mice, in a chemosuppressive test, at 150, 350, and 700 mg/kg, p.o. Standard drugs used were artemether-lumefantrine (0.57 + 3.43) mg/kg and chloroquine (10 mg/kg) as positive controls. Distilled water at 1 mL/100g was used as a negative control. The Lorke method was adopted to determine the acute toxicity of extracts. RESULTS: The flowering stage extract had a maximum suppression of parasitemia at 700 mg/kg (68.83 ± 4.49%). Extract at other developmental stages also significantly suppressed the parasitemia (in the ascending order) fruiting (50.71 ± 1.87%), budding (54.92 ± 7.56%), vegetative (55.39 ± 2.01%) compared to the negative control (24.7 ± 2.7%), p < 0.05. Extracts from all developmental stages increased survival time, with the flowering stage having the highest survival time at 20.33 ± 0.88 days. All extracts had an LD50 of 2157 mg/kg, implying that extracts are safe at lower doses. CONCLUSION: Together, our findings revealed that the S. pinnata extracts at the flowering stage had superior antimalarial activity compared to other plant developmental stages. Extracts from all developmental stages have demonstrated a dose-dependent suppression of malarial parasites and increased survival time with an LD50 of 2157 mg/kg. Thus, for better antimalarial activity, local communities could consider harvesting S. pinnata at the flowering stage. Further studies are needed to isolate pure compounds from S. pinnata and determine their antimalarial activity.

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.

Ethno medical knowledge and traditional use of Aristolochia bracteolata Lam. for malaria among local communities in Jubek State of South Sudan: A cross-sectional survey.

ETHNOPHARMACOLOGICAL RELEVANCE: The recognized challenges to access conventional antimalarial medicines could have contributed to the extensive use of Aristolochia bracteolata Lam. (Aristolochiaceae)to manage malaria in South Sudan traditionally. However, the use and acquired experiences are notwell documented. AIM OF THE STUDY: This study aimed to document the traditional use of A. bracteolata for malaria treatment and experiences among the local communities in Jubek State of South Sudan. METHODS: We performed a cross-sectional study in four counties in Jubek State and interviewed 396 community members, including traditional healers, using a semi-structured questionnaire. Four focused group discussions (FGDs) were also conducted using the interviewer guide. The inclusion criteria were; adults 18 years and older, men andwomen participants, at least one year residing in the study area before the study, and those with a history of medicinal plant use. Data were summarized and presented as proportions. Qualitative data were analyzed using a thematic content approach. The major themes that emerged were discussed. We applied the Pearson Chi-Square test at α = 0.05 to test the study's significant differences in responses. The statistical package for social sciences version 21 software was used for data analysis. RESULTS: Women accounted for 208 (52.5%) of participants, with the majority 321 (81.1%) were between 18 and 45 years. Interestingly, most 312 (78.8%) had formal education. Moreover, about 208 (52.5%) participants collect the plant in their vicinity, where leaves were the most commonly used part 277 (46.4%), followed by the roots, seeds, and stems at 245(41.0%), 71 (11.9%), and 4 (0.7%), respectively. Furthermore, about 63 (15.9%) of the participants experienced side effects, including early abortions, heartburns, sweating, and stomach discomforts. Conversely, a total of 387 (96.0%) reported getting cured of malaria. Generally, the quantity of medicine taken per day differs concerning parts of the plant, with leaves ranging from 1 to 10 pieces, roots at 0.4-1 g, and seeds at 0.1-0.5 g. The locals used these plant parts to prepare infusion and decoction traditional dosage forms for oral use. CONCLUSION: The documented medicinal plant's therapeutic uses provided critical information on the traditional use of A. bracteolata by the community in Jubek state of South Sudan to treat malaria. Although most users reported getting cured of malaria, a notable proportion of them experienced side effects, including early-stage abortion and stomach discomforts. Thus, the use of A. bracteolata preparations, particularly in pregnant women, should be avoided. Finally, further studies are needed to devise a strategy to neutralize the toxic compounds and create community awareness on best practices to minimize side effects.

Plants used traditionally as antimalarials in Latin America: Mining the tree of life for potential new medicines.

ETHNOPHARMACOLOGICAL RELEVANCE: Malaria remains a serious and challenging disease. Traditional antimalarial medicines are largely based on plants, and ethnopharmacological research has inspired the development of antimalarial pharmaceuticals such as artemisinin. Antimalarial drug resistance is an increasing problem in Plasmodium species, and new therapeutic strategies to combat malaria are needed. Although the number of malaria cases has been decreasing in Latin America, malaria remains a significant threat in many regions. Local people in Latin America have been using numerous plant species to treat malaria, some of which have been scientifically studied, but many others have not. AIM OF THE STUDY: Our principal objective is to harness ethnobotanical data on species used traditionally to treat malaria, combined with phylogenetic approaches, to understand how ethnobotany could help identify plant genera as potential sources of new medicines. MATERIALS AND METHODS: Plants used to treat malaria in Latin America were compiled from published and grey literature, unpublished data, and herbarium specimens. Initial assessment of potentially important species/genera/families included compiling the number of species used within the genus, the number of use reports per genus and species, and the geographic distribution of their use. The analysis of taxonomic distribution of species reported as antimalarial in Latin America (excluding the Southern Cone) was conducted, to determine which genera and families with reputed antimalarial properties are over-represented, and phylogenetic analyses were performed to identify if there was evidence for antimalarial species being dispersed/clustered throughout the tree or at its tips. This approach enabled 'hot-nodes' in certain families to be identified, to predict new genera with potential antimalarial properties. RESULTS: Over 1000 plant species have been used to treat malaria in Latin America, of which over 600 species were cited only once. The genera with the highest number of antimalarial species were Aspidosperma, Solanum, Piper, Croton and Aristolochia. In terms of geographic distribution, the most widely used genera were Aspidosperma, Momordica, Cinchona, Senna and Stachytarpheta. Significant phylogenetic signal was detected in the distribution of native species used for malaria, analysed in a genus-level phylogenetic framework. The eudicot and magnoliidae lineages were over-represented, while monocots were not. CONCLUSION: Analysis of ethnobotanical use reports in a phylogenetic framework reveals the existence of hot nodes for malaria across the Latin American flora. We demonstrate how species and genera currently lacking such reports could be pinpointed as of potential interest based on their evolutionary history. Extending this approach to other regions of the world and other diseases could accelerate the discovery of novel medicines and enhance healthcare in areas where new therapeutic strategies are needed.

Antiplasmodial activities of indole alkaloids from Tabernaemontana penduliflora K. Schum (Apocynaceae).

Five undescribed carboxy-indole alkaloids with corynanthe skeleton, penduflorines A-E (1-3) as well as a voacangine-N-oxide alkaloid, tabernaemontine (4), were isolated along with eight other known compounds (5-12) from the trunk bark of Tabernaemontana penduliflora K. Schum (Apocynaceae). Their structures were determined by means of spectroscopic and spectrometric methods such as UV, IR, NMR and HR-ESI-MS. Antiplasmodial activities of new isolates were evaluated against two strains of Plasmodium falciparum 3D7 and Dd2 by the Sybr green I-based fluorescence assay setup. Those compounds showed good in vitro activities. Among them, penduflorines A and B (1a and 1b) as well as tabernaemontine (4) showed significant inhibitory activities against the two strains with IC50 values ranged between 1.85 and 7.88 µg/mL. This is the first report of quaternary-N-indole alkaloids (1a, 1b, 2, 3a, 3b and 4) occurring in the form of zwitterion from Tabernaemontana genus.

In-vivo anti-plasmodial activity of phosphate buffer extract of Calotropis procera latex in mice infected with Plasmodium berghei.

ETHNOPHARMACOLOGICAL RELEVANCE: Malaria is a global health problem with the greatest burden in sub-Saharan Africa (sSA). The resistance to available antimalarial agents necessitate for the development of new and safe drugs for which medicinal plants provides credible alternative sources for discovering new and cheap therapeutic agents. Calotropis procera is used in several folk or traditional medicines for the treatment of various diseases across different regions of the world. In Nigeria traditional medicine, C. procera latex is used either alone or in combination with other herbs to cure common diseases including malaria. In Malaka district (Indonesia), Calotropis gigantea (a member of Apocyanceae), is one of the most used herbs to treat malaria patient via the massage method. AIM OF THE STUDY: This study aimed to evaluate the anti-plasmodial activity of phosphate buffer extract of Calotropis procera latex in mice infected with Plasmodium berghei. MATERIALS AND METHODS: The plant's anti-plasmodial agent was extracted using 0.2 M-phosphate buffer (pH 7.0), followed by precipitation using acetone. 90 (ninety) mice were divided into three main groups of 30 (thirty) mice each, used for the curative, suppressive and prophylactic tests, respectively. The 30 (thirty) mice in each of the main groups were sub-divided into five groups of 6 (six) mice. The mice in the group 1, 2 and 3 (test groups) were made to receive graded doses of 25 mg/kg, 50 mg/kg and 75 mg/kg of the extract of C. procera latex intraperitoneally; group 4 (negative control group) received 0.2 ml of normal saline; while group 5 (positive control group) were administered with 5 mg/kg chloroquine. The phytochemical constituents of the plant and its intraperitoneal median lethal dose (LD50) were also undertaken. RESULTS: The freeze-dried acetone extract exhibited acute toxicity with median lethal dose (LD50) of 745 mg/kg body weight in mice. The highest percentage parasite suppression (61.85%), percentage parasite cure (50.26%), and percentage parasite prophylaxis (65.47%), were obtained for the groups treated with 75 mg/kg bodyweight/day of the extract. The least percentage parasite suppression (44.74%), percentage parasite cure (35.21%), and percentage parasite prophylaxis (45.21%), were obtained for the groups treated with 25 mg/kg body weight of the extract. Also, a dose-dependent percentage parasite suppression (53.03%), percentage parasite cure (39.70%), and percentage parasite prophylaxis (49.82%) were obtained for the groups treated with 50 mg/kg body weight. This is comparable to the groups treated with standard chloroquine. The extract also produced a significant elevation in body weight of the animals for suppressive and curative tests. However, there were observable significant decreases in body weight of the animals in the case of prophylactic test. CONCLUSION: This study showed that the phosphate buffer extract of C. procera latex possess anti-plasmodial activity. The results of this study can be used as a basis for further phytochemical investigations in the search for new and locally affordable antimalarial agents.

Suppressive, curative, and prophylactic potentials of an antimalarial polyherbal mixture and its individual components in Plasmodium berghei-Infected mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Malaria remains one of the most prevalent infectious diseases in tropical regions of the world, particularly in sub-Saharan Africa, where it remains epidemiologically holoendemic. The absence of effective vaccines and Plasmodium resistance to antimalarial drugs have been the major challenges to malaria control measures. An alternative strategy could be the application of validated and standardized herbal formulations. AIM OF THE STUDY: To evaluate the antimalarial activity of a polyherbal mixture (APM) and compare it to those of its individual constituent plants. METHODS: APM consisted of stem barks of Mangifera indica (MI), Azadirachta indica (AI), Nauclea latifolia (and roots, NL) and roots of Morinda lucida (ML). Dihydroartemisinin-piperaquine (DHP) and pyronaridine-artesunate (PA) served as positive controls. Antimalarial activity was evaluated using suppressive, curative and prophylactic assays in mice infected with Plasmodium berghei. RESULTS: All the herbal mixtures, individually and in combination, showed significant (p < 0.05) antiplasmodial activities in the various assays. They produced considerable parasite suppression (>50%), substantial clearance (>70%), and notable prophylaxis (>60%, except for NL: 35%). APM (95.4-98.7%) and AI (92%), respectively, elicited greater and comparable suppression relative to DHP (88%) and PA (87.3%). However, all the herbal decoctions, individually (72-93.6%) and in combination (82.5-91%), showed lower parasite clearance than DHP (100%) and PA (99.5%). Meanwhile, APM showed relatively greater suppression and prophylaxis than its constituent plants, suggesting that the combination produced synergistic or additive effects. CONCLUSION: These findings could substantiate the use of these plants, singly or in combination, as traditional remedies for malaria. Further studies are recommended to evaluate their clinical usefulness.

Antiplasmodial Compounds from Deep-Water Marine Invertebrates.

Novel drug leads for malaria therapy are urgently needed because of the widespread emergence of resistance to all available drugs. Screening of the Harbor Branch enriched fraction library against the Plasmodium falciparum chloroquine-resistant strain (Dd2) followed by bioassay-guided fractionation led to the identification of two potent antiplasmodials; a novel diterpene designated as bebrycin A (1) and the known C21 degraded terpene nitenin (2). A SYBR Green I assay was used to establish a Dd2 EC50 of 1.08 ± 0.21 and 0.29 ± 0.02 µM for bebrycin A and nitenin, respectively. Further analysis was then performed to assess the stage specificity of the inhibitors antiplasmodial effects on the Dd2 intraerythrocytic life cycle. Exposure to bebrycin A was found to block parasite maturation at the schizont stage if added any time prior to late schizogony at 42 hours post invasion, (HPI). In contrast, early life cycle exposure to nitenin (prior to 18 HPI) was identified as crucial to parasite inhibition, suggesting nitenin may target the maturation of the parasite during the transition from ring to early trophozoite (6-18 HPI), a novel property among known antimalarials.