In vitro and in vivo antiplasmodial activity and cytotoxicity of extracts of Phyllanthus niruri L. herbs traditionally used to treat malaria in Indonesia

In endemic areas where malaria is prevalent, medicinal plants are often used to treat malaria. This study was conducted to evaluate the in vitro and in vivo antiplasmodial activity and cytotoxicity of extracts of meniran (Phyllanthus niruri L.) herb traditionally used to treat malaria in Indonesia. Three extracts viz aqueous, methanolic and chloroformic extracts were obtained by maceration of the herbs. A radioactive method was used to evaluate the in vitro antiplasmodial activity of the extracts on chloroquine-resistant (FCR-3) and chloroquine-sensitive (D-10) strains of Plasmodium falciparum. In vitro antiplasmodial activity was expressed by the concentration inhibiting 50% of parasite growth (IC50). Cytotoxicity was estimated on Hela cells and the Cytotoxicity Index (CI = IC50 on HeLa cells/IC50 on FCR-3 strain) was calculated to evaluate the safety of tested extracts. A standard 4-day test on P berghei infected mice was used to evaluate the in vivo antiplasmodial activity of the extracts showing strong in vitro antiplasmodial activity, for both the methanolic and aqueous extracts. The in vivo antiplasmodial activity was expressed by the dose inhibiting 50% of parasite growth (ED50). The IC50 values obtained for these extracts against P. falciparum ranged from 2.3 to 202.4 microg/ml. The methanolic extract was the most active in vitro extract with an IC50 that ranged from 2.3 to 3.9 microg/ml and a CI that ranged from 41.3 to 57.5. This was also the most in vivo active extract with an ED50 of 9.1 mg/kg/d. Further study will be conducted to isolate and purify active compounds presented in the methanolic extract.     

The antiplasmodial and radical scavenging activities of flavonoids of Erythrina burttii

The acetone extract of the root bark of Erythrina burttii showed in vitro antiplasmodial activity against the chloroquine-sensitive (D6) and chloroquine-resistant (W2) strains of Plasmodium falciparum with IC(50) values of 0.97 ± 0.2 and 1.73 ± 0.5 μg/ml respectively. The extract also had radical scavenging activity against 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical with an EC(50) value of 12.0 μg/ml. The isoflav-3-enes burttinol-A and burttinol-C, and the 2-arylbenzofuran derivative burttinol-D were identified as the most active antiplasmodial (IC(50)<10 μM) and free radical scavenging (EC(50)ca. 10 μM) principles. The acetone extract of E. burttii at 800 mg/kg/day, in a 4-day Plasmodium berghei ANKA suppressive test, showed in vivo antimalarial activity with 52% chemosuppression. In the same in vivo test, marginal activities were also observed for the extracts of the root and stem bark of Erythrina abyssinica and the root bark of Erythrina sacleuxii.     

Fractions of an antimalarial neem-leaf extract have activities superior to chloroquine, and are gametocytocidal

The antimalarial activities of two fractions (IRDN-A and IRDN-B) of an extract from the leaves of the neem tree (Azadirachta indica) were compared with those of chloroquine, in in-vitro assays against Plasmodium falciparum. The asexual stages of a chloroquine-sensitive clone (ITG2F6) and a chloroquine-resistant isolate (W2) and the gametocytes of the NF 54 (BD-7) isolate of P. falciparum were used as the drug targets. Activity against the asexual stages was generally evaluated as the concentrations inhibiting the parasitaemias recorded in the control cultures, after an incubation of 48-72 h, by 50% (IC50) or 100% (IC100). For the ITG2F6 strain, the IC50 and IC100 (in microg/ml) were, respectively, 10(-5) and 10(-4) for IRDN-A, 10(-3) and 10(-2) for IRDN-B, and 10(-2) and 1.0 for chloroquine. The corresponding values for the W2 strain were 10(-5) and 1.0 for IRDN-A, and 10.0 and >100 for chloroquine (even at 100 microg/ml, chloroquine only inhibited the parasitaemia by 85%).Each of the two neem-leaf fractions lysed 50% and 100% of developing gametocytes, at 10(-3) and 1.0 microg/ml, respectively; and 50% and 100% of mature gametocytes at 10(-3) and 10(2) microg/ml, respectively. If they are found safe and effective in vivo, the neem-leaf fractions may form the basis of new antimalarial drugs that not only cure chloroquine-sensitive and chloroquine-resistant malaria but also markedly reduce transmission.     

滇西地区25种药用植物抗疟活性研究

目的研究25种采自滇西地区药用植物的抗疟活性,为植物来源的新型抗疟药的研发天然产物奠定基础。方法依次用75%乙醇和水对25种药用植物进行回流提取,提取物经冷冻干燥后,采用β-羟高铁血红素形成抑制试验进行筛选,以IC50值评价其活性。结果在供试的25种药用植物中,苦参、滇白珠、马尾黄连等12种植物粗提物具有不同程度的β-羟高铁血红素形成抑制活性,其中苦参地上部分水提物和滇白珠提取物活性较好,IC50值分别为(1344.2±46.9)μg/ml和大于1 388.9μg/ml。具有抗疟活性的植物种类涉及10个科、12个属。结论苦参、滇白珠、马尾黄连等12种植物具有一定的抗疟活性,表明抗疟活性物质可能存在于多种植物中。     

Immunostimulating effect of pule (Alstonia scholaris L. R.Br., Apocynaceae) bark extracts

The immunostimulating effect of "Pule" (Alstonia scholaris (L.) R.Br., Apocynaceae) bark extracts was studied in BALB/c mouse. The extracts were administered orally, once a day for 7 consecutive days. The results showed that at the same doses (50, 100 and 200 mg/kg b.w.) the aqueous extract had higher phagocytic index (1.39-1.79) than the ethanolic extracts (0.81-0.93) in normal mice. The aqueous extract at 50 mg/kg b.w. also enhanced phagocytic activity of immunosuppressed mice significantly (P < 0.01). At 50 and 100 mg/kg b.w. the extract prevents the decrease of immune system induced by prednisone. The aqueous extract at 100 mg/kg b.w. increased lytic activity of peritoneal exudate cells against Escherichia coli significantly (P < 0.05). At the doses of 50 and 100 mg/kg b.w. the aqueous extract had no effect on primary antibody level. The aqueous extract at 50 mg/kg b.w. induced the cellular immune response while at 100 mg/kg b.w. inhibited the delayed type of hypersensitivity reaction.     

In-vivo antimalarial activity of Cassia occidentalis, Morinda morindoides and Phyllanthus niruri

The ethanolic, dichloromethane and lyophilized aqueous extracts of Cassia occidentalis root bark, Morinda morindoides leaves and whole plants of Phyllanthus niruri were evaluated for their antimalarial actvity in vivo, in 4-day, suppressive assays against Plasmodium berghei ANKA in mice. No toxic effect or mortality was observed in mice treated, orally, with any of the extracts as a single dose, of 500 mg/kg body weight, or as the same dose given twice weekly for 4 weeks (to give a total dose of 4 g/kg). No significant lesions were observed, by eye or during histopathological examinations, in the hearts, lungs, spleens, kidneys, livers, large intestines or brains of any mouse. At doses of 200 mg/kg, all the ethanolic and dichloromethane extracts produced significant chemosuppressions of parasitaemia (of > 60% for C. occidentalis root bark and Ph. niruri whole plant, and of 30% for M. morindoides leaves) when administered orally. The most active ethanolic extract, that of Ph. niruri, reduced parasitaemia by 73%. The dichloromethane extracts of M. morindoides and Ph. niruri produced similar reductions (74% and 72% chemosuppression, respectively), whereas that of C. occidentalis was slightly less active (60% chemosuppression). Each lyophilized aqueous extract was less active than the corresponding ethanolic extract.     

In vitro screening for antiplasmodial activity of isoquinoline alkaloids from Brazilian plant species

In the search for new antimalarial agents, nine Brazilian plant species were selected, from the Annonaceae (6), Menispermaceae (2) and Siparunaceae (1) families naturally occurring at the cerrado and Atlantic rainforest regions, in order to investigate their in vitro antiplasmodial activity. The ethanol and the alkaloid extracts were tested against K1, chloroquine-resistant, and Palo Alto, chloroquine-sensitive, strains of Plasmodium falciparum. The majority of the alkaloid extracts were more active than the ethanol ones, with IC(50) ranging 0.3-8.2 microg/mL. The crude Guatteria australis alkaloids were the most active against K1 with an IC(50) = 0.3 microg/mL. The most promising total alkaloid fractions for further bioguided isolation are those with the IC(50) < or = 5 microg/mL: G. australis, Cissampelos ovalifolia and Duguetia lanceolata.     

Oligomeric procyanidins of French maritime pine bark extract (Pycnogenol) effectively inhibit alpha-glucosidase

The standardized maritime pine bark extract (Pycnogenol) was reported to exert clinical anti-diabetic effects after peroral intake. However, an increased insulin secretion was not observed after administration of the extract to patients. Our aim was to elucidate whether the described clinical effects of Pycnogenol are related to inhibition of alpha-glucosidase. Therefore, we analyzed the inhibitory activity of Pycnogenol, green tea extract and acarbose towards alpha-glucosidase. Furthermore, we explored different fractions of Pycnogenol containing compounds of diverse molecular masses from polyphenolic monomers, dimers and higher oligomers to uncover which components exhibited the most pronounced inhibitory activity. We found that Pycnogenol exhibited the most potent inhibition (IC(50) about 5 microg/mL) on alpha-glucosidase compared to green tea extract (IC(50) about 20 microg/mL) and acarbose (IC(50) about 1mg/mL). The inhibitory action of Pycnogenol was stronger in extract fractions containing higher procyanidin oligomers. The results obtained assign a novel, local effect to oligomeric procyanidins and contribute to the explanation of glucose-lowering effects of Pycnogenol observed in clinical trials with diabetic patients.     

In vitro antimalarial activity and cytotoxicity of some selected cuban medicinal plants

Terrestrial plants have been demonstrated to be sources of antimalarial compounds. In Cuba, little is known about antimalarial potentials of plant species used as medicinals. For that reason, we evaluated the antimalarial activity of 14 plant species used in Cuba as antimalarial, antipyretic and/or antiparasitic. Hydroalcoholic extracts were prepared and tested in vitro for the antimalarial activity against Plasmodium falciparum Ghana strain and over human cell line MRC-5 to determine cytotoxicity. Parasite multiplication was determined microscopically by the direct count of Giemsa stained parasites. A colorimetric assay was used to quantify cytotoxicity. Nine extracts showed IC50 values lower than 100 μg/mL against P. falciparum, four extracts were classified as marginally active (SI < 4), one as partially active (Parthenium hysterophorus) exhibiting SI equal to 6.2 and two extracts as active (Bambusa vulgaris and Punica granatum), showing SI > 10. B. vulgaris showed the most potent and specific antiplasmodial action (IC50 = 4.7 μg/mL, SI = 28.9). Phytochemical characterization of active extracts confirmed the presence of triterpenoids in B. vulgaris and polar compounds with phenol free groups and fluorescent metabolites in both extracts as major phytocompounds, by thin layer chromatography. In conclusion, antimalarial use of B. vulgaris and P. hysterophorus was validated. B. vulgaris and P. granatum extracts were selected for follow-up because of their strong antimalarial activity.     

Nimbolide, a constituent of Azadirachta indica, inhibits Plasmodium falciparum in culture

The terpenoid lactone nimbolide, the structure of which has been unambiguously established, was found to inhibit Plasmodium falciparum in culture with a moderate potency. The EC50 against the parasite line K1 from Thailand was approximately 2.0 microM (0.95 microgram/ml). The EC50 of crude aqueous extract of Azadirachta indica var. siamensis (Sadao tree), was 115 micrograms/ml, and of crude ethanol extract was 5.0 micrograms/ml. Since nimbolide is a major constituent in these extracts, it could account substantially for their inhibitory activity. However, neither the crude extracts nor nimbolide showed any activity in vivo against Plasmodium berghei in the mouse either through ingestion (746 mg aqueous extract, 62.5 mg ethanol extract or 12.5 mg nimbolide/kg/day), or subcutaneous injection (93 mg aqueous extract, 31 mg ethanol extract or 12.5 mg nimbolide/kg/day).     

In vitro antiplasmodial activity of Central American medicinal plants

The in vitro antiplasmodial activities of 14 plant species traditionally used in Central America for the treatment of malaria or fever were evaluated. Lipophilic extracts of Piper hispidum, Siparuna andina, S. pauciflora, S. tonduziana, and Xylopia cf. frutescens, proved to be active against both a chloroquine-sensitive and a resistant strain of Plasmodium falciparum. IC50 values ranged between 3.0 microg/ml and 21.9 microg/ml; however, moderate cytotoxicity of active extracts was observed. Bioactivity-guided fractionation of Piper hispidum yielded 2',4, 6'-trihydroxy-4'-methoxydihydrochalcone (asebogenin) as an active compound.     

Anticancer effects of various Iranian native medicinal plants on human tumor cell lines

In this study the antineoplastic activity of methanolic extracts of six medicinal plants that are native to Iran, including Galium mite, Ferulago angulata, Stachys obtusicrena, Cirsium bracteosum and Echinophora cinerea was investigated. Different tumor cell lines were exposed to the extracts and cytotoxic analysis using MTT colorimetric assay was performed. Quantification of percentage of cells undergoing apoptotic changes by flow cytometry, and DNA fragmentation analysis on sensitive cell lines was then carried out. Results obtained indicated that almost all of the extracts more or less had the capacity to decrease the proliferation of tumor cells. Among the plants, the highest activity against K562 leukemia cell line was found for E. cinerea and C. bracteosum with IC50 less than 20 microg/ml followed by G. mite with IC50 of 39.8 microg/ml. F. angulata and E. cinerea, mostly inhibited Jurkat cells proliferation (IC50 less than 8 microg/ml). Fifty percent inhibition of Fen bladder cell carcinoma due to exposure to F. angulata and E. cinerea was found at concentrations of nearly 180 microg/ml. A549, a lung carcinoma cell, was mostly affected by S. obtusicrena (IC50 more than 200 microg/ml). In flow cytometry analysis, C. bracteosum, E. cinerea, F. angulata and S. obtusicrena extracts demonstrated no remarkable effects on the cell cycle profile of K562 and Jurkat cells. Moreover, in DNA fragmentation analysis of treated cells, no ladder formation was detected. In contrary, G. mite caused more than 40% apoptosis in the K562 and Jurkat cells. In DNA fragmentation analysis G. mite extract produced ladder formation in these cells. In conclusion, these results indicated that the extracts used in this study have anti tumor activity particularly against the leukemia cell lines and that apoptosis is the possible cause of cell death observed due to the extract of G. mite.     

Antileishmanial activity of two gamma-pyrones from Podolepsis hieracioides (Asteraceae)

Two gamma-pyrones isolated from the seeds of Podolepsis hieracioides (Asteraceae) have been tested for their in vitro leishmanicidal activity against the promastigote form of Leishmania donovani, L. major, L. infantum and L. enriettii (IC(50)=3.76-5.43 microg/ml), and against the amastigote form of L. donovani residing within RAW 264.7 macrophage-like host cells (IC(50)=8.29-8.59 microg/ml). General toxicity of the gamma-pyrones was investigated in parallel against three mammalian cell lines (RAW 264.7, SKMel, and KB; IC(50)=11.5->25.0 microg/ml). Both compounds were not active against Trypanosoma cruzi or Trypanosoma br. brucei (IC(50)>30 microg/ml).     

Larvicidal activity of latex and stem bark of Euphorbia tirucalli plant on the mosquito Culex quinquefasciatus

The methanolic, chloroform and ether extracts of Euphorbia tirucalli latex and stem bark were evaluated for larvicidal activity against laboratory-reared larvae of Culex quinquefasciatus (Diptera: Culicidae), vector of the brancroftian filariasis and worst urban nuisance mosquito. The latex extracts contain more potent larvicidal components (177.14 mg/L-326.37 mg/L) than the stem bark extracts (237.663 mg/L-513.39 mg/L). The order of toxicity (LC50) for the latex extracts was Methanol extract (177.14 mg/L) > Chloroform (200.76 mg/L) > Ether (326.37 mg/L) while the rank of order of toxicity (LC50) of stem bark extracts was Ether (237.66 mg/L) > Chloroform (343.515 mg/L) > Methanol (513.387 mg/L), Higher doses (LC90 24 h of mosquito larvae) of each extract did not cause any mortality among fishes after 24 h. The study gave a weight into the possibility of formulating suitable preparation from the latex and stem bark extracts of the plant for use in mosquito control programme.     

In vitro antitrypanosomal activity,antioxidant property and phytochemical constituents of aqueous extracts of nine Nigerian medicinal plants

ObjectiveTo study the in vitro antitrypanosomal activity, antioxidant property and phytochemical constituents of aqueous extracts of nine Nigerian medicinal plants.MethodsIn vitro antitrypanosomal activity test was carried out on aqueous extracts of dried leaves of Acacia albida (A. albida), Artemisia absinthium, Bryophyllum pinnatum, Gongronema latifolium, Holarrhena floribunda, Leptadenia hastata, Pericopsis laxiflora (P. laxiflora) and dried stem barks of A. albida and P. laxiflora. The phytochemical constituents and composition of the extracts and the in vitro antioxidant activity of the extracts were subsequently measured using the α,α-diphenyl-β-picryl-hydrazyl (DPPH) radical scavenging assay, Ferric reducing antioxidant power (FRAP) assay, thiobarbituric acid (TBA) activity assay and H₂O₂ radical scavenging activity assay.ResultsFrom the study, it was discovered that the stem bark extracts of A. albida and P. laxiflora were most active against both Trypanosoma evansi and Trypanosoma congolense. There was complete cessation of motility in both trypanosomes within 5 min at 40 mg/mL of the stem bark extract of A. albida and complete cessation of motility within 25 min and 40 min at 40 mg/mL with P. laxiflora stem bark extract for Trypanosoma congolense and Trypanosoma evansi, respectively. Quantitative analysis of the phytochemical constituents of the aqueous extracts of the plant parts such as alkaloids, saponins, flavonoids and phenols revealed that the stem barks of A. albida, P. laxiflora and leaves of Leptadenia hastata contained relatively high amount of all the phytochemicals quantified. The stem bark extracts of A. albida, P. laxiflora and leaves of Gongronema latifolium possess more scavenging capacity when compared to other extracts in relation to vitamin C, the reference antioxidant.ConclusionsThis study provides scientific evidence for the use of A. albida, and P. laxiflora for the treatment of trypanosomosis and diseases associated with oxidative stress.     

Lipopolysaccharide binds to and activates A(1) adenosine receptors on human pulmonary artery endothelial cells

Previously, it was reported that A(1) adenosine receptor antagonists prevent endotoxin-induced acute lung injury and pulmonary arterial endothelial cell damage. In competition radioligand binding experiments in membranes prepared from human pulmonary artery endothelial cells (PAECs), lipopolysaccharides (LPSs) of Escherichia coli, Salmonella typhimurium, Klebsiella pneumoniae, and Pseudomonas aeruginosa displaced the binding of a selective A(1) adenosine receptor antagonist [(125)I]-BWA844U (IC(50) values: 195 ng/ml, 290 ng/ml, 602 ng/ml, and 693 ng/ml, respectively) in a dose-dependent, competitive manner. There was no displacement of this radioligand by enterotoxin (< or = 10 microg/ml), diphosphoryl lipid A (< or = 10 microg/ml), and glycolipids, monosialoganglioside (< or = 1 microg/ml), lactocerebroside (< or = 100 microg/ml), or NBD galactocerebroside (< or = 100 microg/ml). Based on calculated IC(50) values, LPS (E. coli, IC(50) 111 ng/ml) displaced the selective A(1) adenosine receptor agonist, [(3)H]-2-chloro, N(6)-cyclopentyladenosine (CCPA) in human PAECs with a potency profile, CCPA > LPS > 2-phenylaminoadenosine (CV 1808), a selective A(2) adenosine receptor agonist. The potency profile for displacement of the selective A(2a) adenosine receptor agonist [(3)H]-CGS 21680 was CV 1808 > CCPA. LPS (E. coli 0.1 pg/ml-10 microg/ml) did not displace [(3)H]-CGS 21680 binding. In human PAECs, IL-6 and TXA(2) release induced by LPS (0-1 microg/ml) or CCPA (0-1 microM) at high doses was significantly reduced by the selective A(1) adenosine receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 1 microM). These data suggest that LPS binds to and activates A(1) adenosine receptors on human PAECs to induce the release of IL-6 and TXA(2). Activation of A(1) adenosine receptors on human PAECs by LPS, may contribute to the pathophysiology of acute lung injury associated with Gram-negative septicemia and endotoxemia.     

Characterization of the molluscicidal activity of Bauhinia variegata and Mimusops elengi plant extracts against the fasciola vector Lymnaea acuminata

The molluscicidal activity of Bauhinia variegata leaf and Mimusops elengi bark was studied against vector snail Lymnaea acuminata. The toxicity of both plants was time and concentration-dependent. Among organic extracts, ethanol extracts of both plants were more toxic. Toxicity of B. variegata leaf ethanolic extract (96h LC50- 14.4 mg/L) was more pronounced than M. elengi bark ethanolic extract (96h LC50-15.0 mg/L). The 24h LC50 of column purified fraction of B. variegata and M. elengi bark were 20.3 mg/L and 18.3 mg/L, respectively. Saponin and quercetin were characterized and identified as active molluscicidal component. Co-migration of saponin (Rf 0.48) and quercetin (Rf 0.52) with column purified bark of M. elengi and leaf of B. variegata on thin layer chromatography demonstrate same Rf value i.e. 0.48 and 0.52, respectively. The present study clearly indicates the possibility of using M. elengi and/or B. variegata as potent molluscicide.     

The in-vivo antimalarial activities of Uvaria chamae and Hippocratea africana

The antimalarial activities of ethanolic root extracts of two plants used traditionally as malarial remedies in southern Nigeria, Uvaria chamae (Annonaceae) and Hippocratea africana (Hippocrateaceae), were studied in vivo, in mice infected with Plasmodium berghei berghei. The extract of U. chamae, when given orally at 300-900 mg/kg.day, exhibited significant antimalarial activity against both early and established infections. When established infections were treated, the mean survival time of the mice observed with this extract at 900 mg/kg.day was similar to that seen with the positive control: chloroquine at 5 mg/kg.day. The extract of H. africana, tested at oral doses of 200-600 mg/kg.day, also demonstrated promising blood schizontocidal activity, both in early and established infections. Although the question of their toxicities has still to be fully addressed, it is clear that both U. chamae and H. africana possess considerable antimalarial activity and they, or drugs based on their antimalarial constituents, may prove useful in the treatment of human malaria.     

Antimalarial activity of Ageratum conyzoides in combination with chloroquine and artesunate

ObjectiveTo determine the suppressive and curative activity of aqueous leaf extract of Ageratum conyzoides (A. conyzoides) in combination with chloroquine and artesunate, respectively against Plasmodium berghei infection in mice.MethodsUsing malaria (Plasmodium berghei) infected albino mice of both sexes, aqueous extracts of A. conyzoides in combination with chloroquine and artesunate were tested for antimalarial activity, respectively. Four-day suppressive test and Rane's curative test were carried out.ResultsSuppressive tests showed significant dose dependent reduction in parasitemia level produced by the extract-chloroquine and extract-artesunate combinations. Suppressive activities of both extract-drug combinations were greater than the individual drugs alone. Extract-chloroquine (100:5) produced the highest suppressive effect (98% suppression). Curative tests showed absolute survival in two extract-drug combinations. Two extract-drug combinations produced higher curative effects than the individual drugs alone. The highest dose combinations of extract-chloroquine (100:5) and extract-artesunate (100:5) produced absolute parasitemia clearance (cure) in the infected mice.ConclusionsThe study indicated that aqueous extract of A. conyzoides had the ability to potentiate the antimalarial activity of chloroquine and artesunate against induced plasmodiasis in mice. It contributes a lot in the malaria endemic and poverty stricken tropics.     

Synergism of benflumetol and artemether in Plasmodium falciparum.

Using a continuous in vitro culture system, the sensitivity of Plasmodium falciparum to artemether and a new antimalarial drug, benflumetol (lumefantrine), alone and in combination, was investigated with a multiresistant strain (T-996) from Thailand and a chloroquine-resistant strain (LS-21) from India. Both strains showed similar 50% inhibitory concentration (IC50) levels with artemether alone or benflumetol alone, but the IC90 was higher in strain T-996 compared with strain LS-21: for artemether, 34.45 and 7.11 nmol/L (10.28 and 2.12 ng/ml of erythrocyte-medium mixture [EMM]), and for benflumetol, 293.03 and 95.61 nmol/L (154.69 and 50.47 ng/ml of EMM). When tested in association at artemether:benflumetol (mol: mol) ratios between 100:1 and 1:100, substantial synergism was seen in both strains, especially at the IC90 and IC99 levels. This phenomenon resembles the synergistic interaction of artemisinin derivatives and mefloquine, first observed in laboratory models and later confirmed in clinical experience.