W2 Clone Research Articles

Antimalarial and antioxidant activities of novel artesunate-ellagic acid hybrid compound in vitro and in vivo.

Introduction: Emergence of drug resistant strains of Plasmodium species has necessitated the search for novel antimalarials with unique mechanisms of action. Synthesis of hybrid compounds has been one approach to tackling this challenge. In this study, the synthesis of artesunate-ellagic acid hybrid compound (EA31) from ellagic acid and artesunate and its evaluation for antimalarial and antioxidant activities using in vitro and in vivo models were carried out. Method: EA31 was synthesized from artesunate and ellagic acid. The activities of the hybrid compound against Plasmodium falciparum W2 and P. berghei NK65 were evaluated, and its antioxidant activities were also determined. Results: The results revealed that EA31 was more active against P. falciparum W2 (chloroquine resistant) clone and less cytotoxic to buffalo green monkey kidney cell line compared to artesunate. EA31 was also active against Plasmodium berghei NK65 in vivo. The results revealed inhibition of β-hematin formation as one of the mechanisms of action of EA31. EA31 also exhibited antioxidant activities. Conclusion: The results revealed that EA31 may exert dual action of killing malaria parasite and mopping the reactive oxygen species that mediate the secondary complications of malaria.

In Vitro and in Vivo Antimalarial Activity, Cytotoxicity and Phytochemical HRMS2 Profile of Plants from the Western Pará State, Brazilian Amazonia.

Ethnopharmacology and botanical taxonomy are valid criteria used to selecting plants for antimalarial bioprospection purposes. Based on these two criteria, ethanol extracts of 11 plants from Santarém City vicinities, Western Pará State, Brazilian Amazonia, had their in vitro antiplasmodial activity against chloroquine-resistant Plasmodium falciparum (W2 clone) assessed by the PfLDH method, whereas their cytotoxicity to HepG2-A16 cells was assessed through MTT assay. Acmella oleracea, Siparuna krukovii and Trema micrantha extracts disclosed the highest rate of parasite growth inhibition (90 %) in screening tests. In vivo antimalarial assays were conducted with these extracts against Plasmodium berghei (NK 65 strain) infected mice. Inhibition rate of parasite multiplication ranged from 41.4 % to 60.9 % at the lowest extract dose (25 mg/kg). HPLC-ESI-HRMS2 analyses allowed the putative identification of alkylamides, fatty acids, flavonoid glycosides and alkaloids in ethanol extracts deriving from these three plant species. Results pointed towards A. oleracea flowers ethanol extract as the most promising potential candidate to preclinical studies aiming the development of antimalarial phytomedicine.

Antimicrobial peptidomes of Bothrops atrox and Bothrops jararacussu snake venoms.

The worrisome emergence of pathogens resistant to conventional drugs has stimulated the search for new classes of antimicrobial and antiparasitic agents from natural sources. Antimicrobial peptides (AMPs), acting through mechanisms that do not rely on the interaction with a specific receptor, provide new possibilities for the development of drugs against resistant organisms. This study sought to purify and proteomically characterize the antimicrobial and antiparasitic peptidomes of B. atrox and B. jararacussu snake venoms against Gram-positive (Staphylococcus aureus, Methicillin-resistant Staphylococcus aureus-MRSA), Gram-negative (Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae) bacteria, and the protozoan parasites Leishmania amazonensis and Plasmodium falciparum (clone W2, resistant to chloroquine). To this end, B. atrox and B. jararacussu venom peptides were purified by combination of 3kDa cut-off Amicon®ultracentrifugal filters and reverse-phase high-performance liquid chromatography, and then identified by electrospray-ionization Ion-Trap/Time-of-Flight mass spectrometry. Fourteen distinct peptides, with masses ranging from 443.17 to 1383.73Da and primary structure between 3 and 13 amino acid residues, were sequenced. Among them, 13 contained unique sequences, including 4 novel bradykinin-potentiating-like peptides (BPPs), and a snake venom metalloproteinase tripeptide inhibitor (SVMPi). Although commonly found in Viperidae venoms, except for Bax-12, the BPPs and SVMPi here reported had not been described in B. atrox and B. jararacussu venoms. Among the novel peptides, some exhibited bactericidal activity towards P. aeruginosa and S. aureus, had low hemolytic effect, and were devoid of antiparasitic activity. The identified novel antimicrobial peptides may be relevant in the development of new drugs for the management of multidrug-resistant Gram-negative and Gram-positive bacteria.

COMPARATIVE BIOLOGICAL EVALUATION OF SIX ENDOPHYTIC FUNGI ISOLATED FROM VINCA ROSEA LEAVES

In this study, a total of six endophytic fungi have been isolated from Vinca rosea (Apocynaceae) leaves growing in Egypt. The isolated fungi were identified morphologically and microscopically up to species to be; Alternaria phragmospora, Aspergillus awamori, Penicillium duclauxii, Penicillium melinii, Nigrospora sphaerica and Mucor ramosissimus. The extracts of the all identified fungi were screened biologically for antileukemic, cytotoxic, antimalarial, antileishmanial, antimicrobial, antioxidant and anti-inflammatory activities as well as for cannabinoid and opioid receptor binding affinities. Out of the six examined fungal extracts four exhibited promising antimalarial activities, four showed modeate antileukemic activities and two of them exhibited cytotoxic activities, three showed antioxidant activities, three exhibited anti-inflammatory activities, and the only one showed an antifungal activity. Alternaria phragmospora was the most active antimalarial agent inhibiting Plasmodium falciparum D6 and W2 clones with IC50 values of 1.9 and 2.1 μg/mL, respectively. Alternaria phragmospora extractwas subjected to liquid-liquid partition using 90% MeOH, hexane then BuOH and H2O. MeOH and BuOH fractions exhibited strong activities against Plasmodium falciparum D6 clone with IC50 values of 1.3 and 28 μg/mL and W2 clone with IC50 values of 1.4 and 28 μg/mL, respectively. These results may be an excellent opportunity to get a new antimalarial drug derived from endophytic fungi.

Synthesis and anti-Plasmodium falciparum evaluation of novel pyrazolopyrimidine derivatives

Nine 1-phenyl-1H-pyrazolo[3,4-d]pyrimidine derivatives with different substituents in the 4-position of the phenyl group and benzenesulfonamide moiety were synthesized and evaluated against Plasmodium falciparum. Six compounds exhibited activity in vitro against the chloroquine-resistant clone W2 with IC50 values ranging from 5.13 to 12.22 µM. The most active derivative with substituents R1 = F / R2 = CH3 exhibited an IC50 value of 5.13 µM and an IS value of 62.90, which was higher than that of the control drug sulfadoxine. For this reason, it is possible to conclude that the 1H-pyrazolo[3,4-d]pyrimidine system is promising as a prototype for further studies of antimalarial candidates.

Antimalarial and Antileishmanial Activities of Phytophenolics and Their Synthetic Analogues.

Thirty-seven phytophenolics and their synthetic analogues were evaluated for activity against two protozoal pathogens, Leishmania donovani and Plasmodium falciparum (D6 and W2 clones), respectively. 4,6-Dimethoxyaurone demonstrated the highest activity with IC50 values of 13.2 and 16.9 μm against L. donovani and P. falciparum (W2 clone), respectively, without undesired cytotoxicity against VERO cells. The moiety having two benzene rings was critical to maintain the antiprotozoal activities based on the observation that both coumaranones and chromones were inactive while other test compounds, including coumarans and aurones, remained active. There was no correlation between antiprotozoal activities and previously evaluated insect antifeedant activity against common cutworms (Spodoptera litura). Flavonoids, including aurones, pterocarpans, and lignan like dihydrobenzofurans, structural analogues of coumarans, are abundant in fruits and vegetables, so these phytophenolics may act as natural antiprotozoal agents in humans.

Norepinephrine alkaloids as antiplasmodial agents: Synthesis of syncarpamide and insight into the structure-activity relationships of its analogues as antiplasmodial agents

Syncarpamide 1, a norepinephrine alkaloid isolated from the leaves of Zanthoxylum syncarpum (Rutaceae) exhibited promising antiplasmodial activities against Plasmodium falciparum with reported IC50 values of 2.04 μM (D6 clone), 3.06 μM (W2 clone) and observed by us 3.90 μM (3D7 clone) and 2.56 μM (K1 clone). In continuation of our work on naturally occurring antimalarial compounds, synthesis of syncarpamide 1 and its enantiomer, (R)-2 using Sharpless asymmetric dihydroxylation as a key step has been accomplished. In order to study structure-activity-relationship (SAR) in detail, a library of 55 compounds (3–57), which are analogues/homologues of syncarpamide 1 were synthesized by varying the substituents on the aromatic ring, by changing the stereocentre at the C-7 and/or by varying the acid groups in the ester and/or amide side chain based on the natural product lead molecule and further assayed in vitro against 3D7 and K1 strains of P. falciparum to evaluate their antiplasmodial activities. In order to study the effect of position of functional groups on antiplasmodial activity profile, a regioisomer (S)-58 of syncarpamide 1 was synthesized however, it turned out to be inactive against both the strains. Two compounds, (S)-41 and its enantiomer, (R)-42 having 3,4,5-trimethoxy cinnamoyl groups as side chains showed better antiplasmodial activity with IC50 values of 3.16, 2.28 μM (3D7) and 1.78, 2.07 μM (K1), respectively than the natural product, syncarpamide 1. Three compounds (S)-13, (S)-17, (S)-21 exhibited antiplasmodial activities with IC50 values of 6.39, 6.82, 6.41 μM against 3D7 strain, 4.27, 7.26, 2.71 μM against K1 strain and with CC50 values of 147.72, 153.0, >200 μM respectively. The in vitro antiplasmodial activity data of synthesized library suggests that the electron density and possibility of resonance in both the ester and amide side chains increases the antiplasmodial activity as compared to the parent natural product 1. The natural product syncarpamide 1 and four analogues/homologues out of the synthesized library of 55, (S)-41, (R)-42, (S)-55 and (S)-57 were assayed in vivo assay against chloroquine-resistant P. yoelii (N-67) strain of Plasmodium. However, none of the five molecules, 1, (S)-41, (R)-42, (S)-55 and (S)-57 exhibited any promising in vivo antimalarial activity against P. yoelii (N-67) strain. Compounds 4, 6, 7 and 11 showed high cytotoxicities with CC50 values of 5.87, 5.08, 6.44 and 14.04 μM, respectively. Compound 6 was found to be the most cytotoxic as compared to the standard drug, podophyllotoxin whereas compounds 4 and 7 showed comparable cytotoxicities to podophyllotoxin.

Synthesis, antimalarial activity in vitro, and docking studies of novel neolignan derivatives.

The absence of effective vaccines against malaria and the difficulties associated with controlling mosquito vectors have left chemotherapy as the primary control measure against malaria. However, the emergence and spread of parasite resistance to conventional antimalarial drugs result in a worrisome scenario making the search for new drugs a priority. In the present study, the activities of nine neolignan derivatives were evaluated as follows: (i) against blood forms of chloroquine-resistant Plasmodium falciparum (clone W2), using the tritiated hypoxanthine incorporation and anti-HRPII assays; (ii) for cytotoxic activity against cultured human hepatoma cells (HepG2); and (iii) for intermolecular interaction with the P. falciparum cysteine protease of falcipain-2 (F2) by molecular docking. The neolignan derivatives 9 and 10 showed activity against the blood form of the chloroquine-resistant P. falciparum clone W2 and were not cytotoxic against cultured human hepatoma cells. A molecular docking study of these two neolignans with FP2 revealed several intermolecular interactions that should guide the design of future analogs.

Prenylated flavonoids from the stems and roots of Tripterygium wilfordii

Phytochemical investigation on the stems and roots of Tripterygium wilfordii led to the isolation and characterization of three new prenylated flavanones, tripteryols A–C (1–3), along with (±)-5,4′-dihydroxy-2′-methoxy-6′,6″-dimethypyraro-(2″,3″:7,8)-6-methyflavanone (4), and ((2S)-5,7,4′-trihydroxy-2′-methoxy-8,5′-di(3-methyl-2-butenyl)-6-methylflavanone (5). Structures of the compounds 1–5 were elucidated using spectroscopic techniques, such as UV, IR, NMR (1D and 2D), and HRESI-MS. Tripteryols B (2) was found active in the antimicrobial assay against Cryptococcus neoformans, Pseudomonas aeruginosa, vancomycin-resistant Enterococcus faecalis (VRE) and methicillin-resistant Staphylococcus aureus (MRSA) with IC50 values in the range of 2.95–8.59μg/mL. Compounds 4 and 5 showed significant antimicrobial activities against C. neoformans, MRSA and Staphylococcus aureus with IC50 values in the range of 1.06–2.60μg/mL. Additionally, significant antimalarial activities of tripteryols A–B (1–2) against chloroquine-sensitive D6 and resistant W2 clones of Plasmodium falciparum were observed and none of the compounds 1–5 were cytotoxic to Vero cells.

Design, synthesis and anti-P. falciparum activity of pyrazolopyridine–sulfonamide derivatives

Ten 1-phenyl-1H-pyrazolo[3,4-b]pyridine derivatives connected by a linker group to benzenesulfonamide moieties with different substituents in the 4-position were synthesized and assayed against Plasmodium falciparum. These ten compounds exhibited activity in vitro against the chloroquine-resistant clone W2 with IC50 values ranging from 3.46 to 9.30μM. The most active derivatives with substituent R2=Cl or CH3 at the benzenesulfonamide moiety exhibited the lowest IC50. Compounds with an R1=CO2Et substituent at the 5-position of the 1H-pyrazolo[3,4-b]pyridine ring presented lower activity than those with a CN substituent. The 1H-pyrazolo[3,4-b]pyridine system appears to be promising for further studies as an antimalarial for overcoming the burden of resistance in P. falciparum.

In vitro and in vivo anti-malarial activity of plants from the Brazilian Amazon.

BackgroundThe anti-malarials quinine and artemisinin were isolated from traditionally used plants (Cinchona spp. and Artemisia annua, respectively). The synthetic quinoline anti-malarials (e.g. chloroquine) and semi-synthetic artemisinin derivatives (e.g. artesunate) were developed based on these natural products. Malaria is endemic to the Amazon region where Plasmodium falciparum and Plasmodium vivax drug-resistance is of concern. There is an urgent need for new anti-malarials. Traditionally used Amazonian plants may provide new treatments for drug-resistant P. vivax and P. falciparum. Herein, the in vitro and in vivo antiplasmodial activity and cytotoxicity of medicinal plant extracts were investigated.MethodsSixty-nine extracts from 11 plant species were prepared and screened for in vitro activity against P. falciparum K1 strain and for cytotoxicity against human fibroblasts and two melanoma cell lines. Median inhibitory concentrations (IC50) were established against chloroquine-resistant P. falciparum W2 clone using monoclonal anti-HRPII (histidine-rich protein II) antibodies in an enzyme-linked immunosorbent assay. Extracts were evaluated for toxicity against murine macrophages (IC50) and selectivity indices (SI) were determined. Three extracts were also evaluated orally in Plasmodium berghei-infected mice.ResultsHigh in vitro antiplasmodial activity (IC50 = 6.4–9.9 µg/mL) was observed for Andropogon leucostachyus aerial part methanol extracts, Croton cajucara red variety leaf chloroform extracts, Miconia nervosa leaf methanol extracts, and Xylopia amazonica leaf chloroform and branch ethanol extracts. Paullinia cupana branch chloroform extracts and Croton cajucara red variety leaf ethanol extracts were toxic to fibroblasts and or melanoma cells. Xylopia amazonica branch ethanol extracts and Zanthoxylum djalma-batistae branch chloroform extracts were toxic to macrophages (IC50 = 6.9 and 24.7 µg/mL, respectively). Andropogon leucostachyus extracts were the most selective (SI >28.2) and the most active in vivo (at doses of 250 mg/kg, 71 % suppression of P. berghei parasitaemia versus untreated controls).ConclusionsEthnobotanical or ethnopharmacological reports describe the anti-malarial use of these plants or the antiplasmodial activity of congeneric species. No antiplasmodial activity has been demonstrated previously for the extracts of these plants. Seven plants exhibit in vivo and or in vitro anti-malarial potential. Future work should aim to discover the anti-malarial substances present.

Antiplasmodial activity of iron(II) and ruthenium(II) organometallic complexes against Plasmodium falciparum blood parasites.

This work reports the in vitro activity against Plasmodium falciparumblood forms (W2 clone, chloroquine-resistant) of tamoxifen-based compounds and their ferrocenyl (ferrocifens) and ruthenocenyl (ruthenocifens) derivatives, as well as their cytotoxicity against HepG2 human hepatoma cells. Surprisingly with these series, results indicate that the biological activity of ruthenocifens is better than that of ferrocifens and other tamoxifen-like compounds. The synthesis of a new metal-based compound is also described. It was shown, for the first time, that ruthenocifens are good antiplasmodial prototypes. Further studies will be conducted aiming at a better understanding of their mechanism of action and at obtaining new compounds with better therapeutic profile.

Ethnopharmacological evaluation of medicinal plants used against malaria by quilombola communities from Oriximiná, Brazil

Ethnopharmacological relevanceMalaria is the most important parasitic disease in the world, including in the Amazon region, due to its high incidence. In addition, malaria is difficult to control because of the geographical characteristics of the endemic Amazon region. The quilombola communities of Oriximina, located in remote rainforest areas, have extensive experience with medicinal plants due to their close contact with and dependence on local biodiversity as a therapeutic resource. To search for active bioproducts against malaria, based on in vitro tests using blood culture-derived parasites and plants selected by an ethno-directed approach in traditional quilombola communities of Oriximiná, in the Amazon region of Brazil. Materials and methodsEthnobotanical data were collected from 35 informants in the quilombola communities of Oriximiná, Brazil, by a free-listing method for the survey of species locally indicated to be effective against malaria and related symptoms. Data were analyzed by salience index (S) and major use agreement. The activity of extracts from 11 plants, selected based on their Salience values (four plants with S>1; seven plants with S<0.1), was measured in vitro in cultures of W2 clone Plasmodium falciparum parasites resistant to chloroquine. ResultsThirty-five ethnospecies comprising 40 different plants belonging to 23 botanical families and 37 genera were listed as antimalarials by the ethno-directed approach. Among these, 11 species selected based on their S values were assayed against P. falciparum. The most active plant extracts, with an IC50 as low as 1.6μg/mL, were obtained from Aspidosperma rigidum (Apocynaceae), Bertholletia excelsa (Lecythidaceae) and Simaba cedron (Simaroubaceae), all of which displayed an S value>1. ConclusionA strong correlation between the consensus of the informants from quilombola communities living in a malaria endemic area and the salience index indicating antiplasmodial activity was observed, where the ethnospecies mostly cited to be effective against malaria produced the most active plant extracts in vitro. It was also evident from the data that these groups approached the treatment of malaria with an holistic view, making use of purgative, depurative, emetic and adaptogen plants.

Aryltetralols from Holostylis reniformis and syntheses of lignan analogous

Two new lignans, an aryltetralol and its methyl ether analogous, were isolated from Holostylis reniformis (Aristolochiaceae) together with futokadsurin C and (−)-8′-epi-aristoligone. The latter was also obtained as an enantiomeric mixture by synthesis and was transformed into aryltetralols and aryltetralenes that were subjected to chiral-HPLC separations. The compound structures were determined by spectroscopic methods. Several of these lignans had their antiplasmodial activity (against Plasmodium falciparum, W2 clone, anti-HRPII) and toxicity to mammalian kidney cells (MDL50) evaluated. (−)-Cyclogalgravin and (−)-aristoligol exhibited activity (IC50∼10.8 and 8.4μM, respectively), the latter exhibited lower toxicity.

Minor oxygenated cannabinoids from high potency Cannabis sativa L.

Nine oxygenated cannabinoids were isolated from a high potency Cannabis sativa L. variety. Structure elucidation was achieved using spectroscopic techniques, including 1D and 2D NMR, HRMS and GC–MS. These minor compounds include four hexahydrocannabinols, four tetrahydrocannabinols, and one hydroxylated cannabinol, namely 9α-hydroxyhexahydrocannabinol, 7-oxo-9α-hydroxyhexa-hydrocannabinol, 10α-hydroxyhexahydrocannabinol, 10aR-hydroxyhexahydrocannabinol, Δ9-THC aldehyde A, 8-oxo-Δ9-THC, 10aα-hydroxy-10-oxo-Δ8-THC, 9α-hydroxy-10-oxo-Δ6a,10a-THC, and 1′S-hydroxycannabinol, respectively. The latter compound showed moderate anti-MRSa (IC50 10.0μg/mL), moderate antileishmanial (IC50 14.0μg/mL) and mild antimalarial activity against Plasmodium falciparum (D6 clone) and P. falciparum (W2 clone) with IC50 values of 3.4 and 2.3μg/mL, respectively.

Antimalarial activity of 4-amidinoquinoline and 10-amidinobenzonaphthyridine derivatives.

Chloroquine (CQ) has been used as first line malaria therapeutic drug for decades. Emergence of CQ drug-resistant Plasmodium falciparum malaria throughout endemic areas of the world has limited its clinical value. Mefloquine (MQ) has been used as an effective malaria prophylactic drug due to its being long-acting and having a high potency against blood stage P. falciparum (Pf). However, serious CNS toxicity of MQ has compromised its clinical value as a prophylaxis drug. Therefore, new and inexpensive antimalarial drugs with no cross-resistance to CQ or CNS toxicity are urgently needed to combat this deadly human disease. In this study, a series of new 4-amidinoquinoline (4-AMQ) and 10-amidinobenzonaphthyridine (10-AMB) derivatives were designed, prepared, and assessed to search for new therapeutic agents to replace CQ and MQ. The new derivatives displayed high activity in vitro and in vivo, with no cross-resistance to CQ, and none were toxic in mice up to 160 mpk × 3. The best compound shows IC50 < 1 ng/mL against D6, W2 and C235 Pf clones, low inhibitory activity in hERG K(+) channel blockage testing, negativity in the Ames test, and 5/5 cure @ <15 mpk × 3 in mice infected with Plasmodium berghei. In addition to these desirable pharmacological profiles, compound 13b, one of the most active compounds, is metabolically stable in both human and mouse liver microsomal preparations and has a plasma t(1/2) of 50 h in mice, which made it a good MQ replacement candidate.

Anti-malarial activity and toxicity assessment of Himatanthus articulatus, a plant used to treat malaria in the Brazilian Amazon.

BackgroundPlasmodium falciparum has become resistant to some of the available drugs. Several plant species are used for the treatment of malaria, such as Himatanthus articulatus in parts of Brazil. The present paper reports the phyto-chemistry, the anti-plasmodial and anti-malarial activity, as well as the toxicity of H. articulatus.MethodsEthanol and dichloromethane extracts were obtained from the powder of stem barks of H. articulatus and later fractionated and analysed. The anti-plasmodial activity was assessed against a chloroquine resistant strain P. falciparum (W2) in vitro, whilst in vivo anti-malarial activity against Plasmodium berghei (ANKA strain) was tested in mice, evaluating the role of oxidative stress (total antioxidant capacity - TEAC; lipid peroxidation – TBARS, and nitrites and nitrates - NN). In addition, cytotoxicity was evaluated using the HepG2 A16 cell-line. The acute oral and sub-chronic toxicity of the ethanol extract were evaluated in both male and female mice.ResultsPlumieride was isolated from the ethyl acetate fraction of ethanol extract, Only the dichloromethane extract was active against clone W2. Nevertheless, both extracts reduced parasitaemia in P. berghei-infected mice. Besides, a significant reduction in pulmonary and cerebral levels of NN (nitrites and nitrates) was found, as well as in pulmonary TBARS, indicating a reduced oxidative damage to these organs. The ethanol extract showed low cytotoxicity to HepG2 A16 cells in the concentrations used. No significant changes were observed in the in vivo toxicity studies.ConclusionsThe ethanol extract of H. articulatus proved to be promising as anti-malarial medicine and showed low toxicity.

Drying anti-malarial drugs in vitro tests to outsource SYBR green assays.

BackgroundMeasurement of anti-malarial drug efficacy and resistance relies mainly on in vivo clinical trials, in vitro/ex vivo assays and molecular markers detection. The existing in vitro/ex vivo assays, in particular those that are using non-radioactive devices, need to be standardized and adapted to field conditions. SYBR Green assay offers a rapid and cheap alternative to other in vitro assays, but it requires tools not commonly available in field laboratories. Here is described a modified SYBR green I protocol to perform the parasite growth test with blood samples in endemic areas, followed later by the SYBR green fluorescence assay performed at a specialized laboratory level.MethodsIn vitro susceptibility of Plasmodium falciparum clones HB3, 3D7, W2 and 7G8 to chloroquine (CQ), dihydroartemisinin (DHA), pyronaridine (PYD) and piperaquine (PPQ) was tested. Fresh isolates of P. falciparum from imported malaria cases were collected for ex vivo assays. The parasite suspension was added in 96-well plates predosed with anti-malarial drugs and incubated for 72 hours at 37°C, 5% CO2. SYBR green I protocol was modified to dry the plates after freeze-thawed process to mimic storage and shipping conditions. The plates were rehydrated with 200 μl of complete RPMI medium for fluorescence assay.ResultsThere were no significant differences in IC50 values of CQ, DHA, PYD and PPQ, determined by the modified protocol, compared to standard protocol. Longer storage did not affect the IC50 values.ConclusionThe SYBR green I modified protocol produced reliable results and could be a suitable method for in vitro/ex vivo assays in field.

Investigation of antimalarial activity, cytotoxicity and action mechanism of piperazine derivatives of betulinic acid.

To semisynthesise piperazine derivatives of betulinic acid to evaluate antimalarial activity, cytotoxicity and action mechanism. The new derivatives were evaluated against the CQ-sensitive Plasmodium falciparum 3D7 strain by flow cytometry (FC) using YOYO-1 as stain. Cytotoxicity of 4a and 4b was performed with HEK293T cells for 24 and 48 h by MTT assay. The capability of compound 4a to modulate Ca(2+) in the trophozoite stage was investigated. The trophozoites were stained with Fluo4-AM and analysed by spectrofluorimetry. Effect on mitochondrial membrane potential (ΔΨm) was tested for 4a by FC with DiOC6 (3) as stain. For β-haematin assay, 4a was incubated for 24 h with reagents such as haemin, and the fluorescence was measured by FlexStation at an absorbance of 405 nm. Antimalarial activity of 4a and 4b was IC50 = 1 and 4 μm, respectively. Compound 4a displayed cytotoxicity with IC50 = 69 and 29 μm for 24 and 48 h, respectively, and 4b was not cytotoxic at the tested concentrations. Addition of 4a leads to an increase in cytosolic Ca(2+) . We have measured ΔΨm after treating parasites with the compound. Data on Figure 4a show that mitochondria were not affected. The action mechanism for 4a, inhibition of β-haematin formation (17%), was lower than CQ treatment (83%; IC50 = 3 mm). Compound 4a showed excellent antimalarial activity, and its action mechanism is involved in Ca(2+) pathway(s).

Blood shizonticidal activities of phenazines and naphthoquinoidal compounds against Plasmodium falciparum in vitro and in mice malaria studies.

Due to the recent advances of atovaquone, a naphthoquinone, through clinical trials as treatment for malarial infection, 19 quinone derivatives with previously reported structures were also evaluated for blood schizonticide activity against the malaria parasite Plasmodium falciparum. These compounds include 2-hydroxy-3-methylamino naphthoquinones (2-9), lapachol (10), nor-lapachol (11), iso-lapachol (12), phthiocol (13) and phenazines (12-20). Their cytotoxicities were also evaluated against human hepatoma and normal monkey kidney cell lines. Compounds 2 and 5 showed the highest activity against P. falciparum chloroquine-resistant blood-stage parasites (clone W2), indicated by their low inhibitory concentration for 50% (IC50) of parasite growth. The therapeutic potential of the active compounds was evaluated according to the selectivity index, which is a ratio of the cytotoxicity minimum lethal dose which eliminates 50% of cells and the in vitro IC50. Naphthoquinones 2 and 5, with activities similar to the reference antimalarial chloroquine, were also active against malaria in mice and suppressed parasitaemia by more than 60% in contrast to compound 11 which was inactive. Based on their in vitro and in vivo activities, compounds 2 and 5 are considered promising molecules for antimalarial treatment and warrant further study.