Heme Polymerization Research Articles

Antimalarial evaluation of selected medicinal plants traditionally used for treatment of fever, by inhibition test of heme polymerization

World Health Organization (WHO) has reported 249 million cases infected by malaria worldwide and 608 thousands deaths in 2022. Investigations for new antimalarial drugs from traditional medicine have proven to be more effective and less expensive. The medicinal plants that have been used for treatment of malaria, generally known as types of fevers in traditional medicine, can be suitable candidates for evaluating antimalarial effects. The aim of the present study was to evaluate the in-vitro mechanism of antimalarial action of selected medicinal plants by inhibition test of heme detoxification (ITHD). The methanol extract and fractions were prepared through maceration of the dry powdered plants. The ITHD method was carried in 96-wells plate and the percentage of heme polymerization inhibition was determined. The cytotoxicity of the effective plants were examined on MDBK cell line by MTT assay. Bioassay guided fractionation was performed exposing to size exclusion column chromatography and liquid-liquid fractionation and was assayed by the ITHD method for the sample with the least IC50 value and lowest cytotoxic effect. The methanol fraction of Viola odorata whole plant showed the most considerable results among the tested plants with IC50 171.8 μg.mL-1 beside the lowest cytotoxic effects. This fraction by the bioassay guided fractionation led to fraction SB2 and this fraction demonstrated the most effective result with lowest IC50 = 14.8 ± 3µg.mL-1 in ITHD assay. Regarding the results of the present study and the traditional use of V. odorata for overcoming fever in Iranian traditional medicine, the final fraction of the plant could be proper candidate for further phytochemical and antimalarial studies.

Potensi Aktivitas Antimalaria Residu Hidrodestilasi Kulit Kayu Manis (Cinnamomum Burmanii) Melalui Penghambatan Polimerisasi Heme

Cinnamon (Cinnamomum burmanii) is a herbal plant from Indonesia which is widely used in cooking, cosmetics and medicine. Cinnamon bark isolate is thought to contain compounds with the structure of isoquinoline which are known to be used as antimalarials. The aim of this study was to examine the potential antimalarial activity of extracts and fractions of cinnamon bark hydrodistillation residue based on the % inhibition and IC50 value. These results will also be compared with extracts and fractions of cinnamon bark without hydrodistillation as controls. The test method is carried out in vitro by heme polymerization inhibitor. The test results showed that the n-hexane fraction, DCM fraction did not have inhibitory activity, while the ethyl acetate fraction had better potential than other extracts and fractions, where the ethyl acetate fraction after hydrodistillation (residue) had an IC50 value of 71.84 µg/mL, while the ethyl acetate fraction without hydrodistillation (control) had an IC50 value of 120.05 µg/mL. This result is better than hydroxychloroquine as positive control which has an IC50 value of 184.98 µg/mL. These results indicate that the ethyl acetate fraction from the hydrodistillation residue of cinnamon bark can be utilized and has potential as an antimalarial.

B-HEMATIN INHIBITION ACTIVITY OF THE METHANOL LEAF EXTRACT AND RESIDUAL AQUEOUS FRACTION OF FICUS ASPERIFOLIA

Chemotherapy is still regarded as the major intervention in fighting the malaria scourge despite the recent introduction of malaria vaccine. Screening of medicinal plants provides opportunities for the discovery of the potential drug molecules. Ficus asperifolia (Miq) family Moraceae has been traditionally employed in the treatment of many diseases including pain, fever and malaria. The aim of this study was to examine the anitmalarial activity of both the methanol leaf extract (MEFA) and residual aqueous fraction of Ficus asperifolia (RAF) through the determination of their heme polymerization inhibition ability. The powdered leaf was extracted using 70%

Role Transformation of HSPA8 to Heme-peroxidase After Binding Hemin to Catalyze Heme Polymerization.

Hemin, a byproduct of hemoglobin degradation, inflicts oxidative insult to cells. Following its accumulation, several proteins are recruited for heme detoxification with heme oxygenase playing the key role. Chaperones play a protective role primarily by preventing protein degradation and unfolding. They also are known to have miscellaneous secondary roles during similar situations. To discover a secondary role of chaperones during heme stress we studied the role of the chaperone HSPA8 in the detoxification of hemin. In-silico studies indicated that HSPA8 has a well-defined biophoric environment to bind hemin. Through optical difference spectroscopy, we found that HSPA8 binds hemin through its N-terminal domain with a Kd value of 5.9 ± 0.04 µM and transforms into a hemoprotein. The hemoprotein was tested for exhibiting peroxidase activity using guaiacol as substrate. The complex formed reacts with H2O2 and exhibits classical peroxidase activity with an ability to oxidize aromatic and halide substrates. HSPA8 is dose-dependently catalyzing heme polymerization through its N-terminal domain. The IR results reveal that the polymer formed exhibits structural similarities to β-hematin suggesting its covalent nature. The polymerization mechanism was tested through optical spectroscopy, spin-trap, and activity inhibition experiments. The results suggest that the polymerization occurs through a peroxidase-H2O2 system involving a one-electron transfer mechanism, and the formation of free radical and radical-radical interaction. It highlights a possible role of the HSPA8-hemin complex in exhibiting cytoprotective function during pathological conditions like malaria, sickle cell disease, etc.

Evaluation of in vitro antimalarial activity of <i>Citrus aurantifolia</i> (CHRISTM.) and <i>Ziziphus mauritiana</i> (LAM) using heme polymerization inhibition assay

Citrus aurantifolia and Ziziphus mauritiana (Lam) are plants that have numerous medicinal properties and are used extensively in traditional medicine for the treatment of several diseases. Malaria parasites metabolize haemoglobin and detoxify the resulting haem by polymerizing it to form haemozoin (malaria pigment). This study aimed at evaluating the antiplasmodial activity through in vitro cell-free heme polymerization inhibition assay (HPIA) of C. aurantifolia and Z. mauritiana. The analysis was conducted using a 96-well microtiter plate reader at an absorption of 400 nm, at different concentrations (0-2 mg/ml). The IC50 was determined by non-linear regression analysis using GraphPad Prism. The result indicated that both the root extracts of C. aurantifolia and Z. mauritiana showed significant difference (p˂0.0001) with IC50 of 0.81 mg/ml and 0.77 mg/ml respectively compared to positive control. Phytochemical screening of the ethanol root extracts showed the presence of saponins, alkaloids, terpenoids, glycosides, steroids, and anthraquinone, while tannins and reducing sugars were not detected in both samples used, flavonoid was detected in C. aurantifolia. Both the extracts exhibited potential for inhibiting heme polymerization in vitro.

PROFILING OF PHYLLANTHUS AMARUS PHYTOCHEMICAL CONSTITUENTS AND EVALUATION OF ASSOCIATED ANTIMALARIAL ACTIVITY AND ANTIOXIDANT POTENTIAL IN EXPERIMENTAL MICE

The failing curative ability of antimalarials has prompted an open discussion for the use of antioxidants in combination with antimalarials for effective chemotherapy. This study profiled the phytochemical constituents of Phyllanthus amarus and evaluated their antimalarial and antioxidant activities. Phytochemical screening, antimalarial and acute oral toxicity were determined according to standard procedures. Blood antioxidant activity was assessed by measuring antioxidant enzymes and nitric oxide (NO), concentrations of malarial infected mice treated with P. amarus phytochemicals. Alkaloids, the most abundant phytochemical, demonstrated the highest malarial parasite chemosuppression and greatly improved NO concentration in infected mice. However, flavonoid extract demonstrated the highest antioxidant potential with most significant impact on catalase and malondialdehyde activity. Alkaloid may function as antimalarial agent by inhibiting haem polymerization to hemozoin as judged by its increased effect on NO concentration which bears an reverse relationship with hemozoin.

Synthesis of Calix[4]resorcinarene Derivatives as Antimalarial Agents through Heme Polymerization Inhibition Assay

Malaria is an endemic disease in tropical countries, including Indonesia, with a high annual mortality rate. Because of that, serious attention shall be given to find new antimalarial agents that are highly active for medical treatment. In this work, we designed and synthesized three calix[4]resorcinarene derivatives and evaluated them as antimalarial agents through in vitro heme polymerization inhibitory assay. The calix[4]resorcinarenes were prepared from resorcinol and corresponding aldehyde derivatives in ethanol media through a cyclo-condensation reaction. The calix[4]resorcinarene products were obtained in 31.1–85.1% yield. The synthesized compounds were subjected to structure elucidation using spectroscopy techniques. The antimalarial activity of calix[4]resorcinarene with aromatic substituent (IC50 = 0.198 mg/mL) was higher than the aliphatic ones (IC50 = 0.282–0.814 mg/mL). It was found that all calix[4]resorcinarenes in this work exhibited stronger antimalarial activity than chloroquine diphosphate as the positive control (IC50 = 1.157 mg/mL). The calix[4]resorcinarenes could interact with hydrogen bonding, thus inhibiting the heme polymerization process. These findings demonstrate that calix[4]resorcinarene derivatives are potential antimalarial agents to be developed for effective medical treatment in the near future.

Steroids from Toona sureni-derived Endophytic Fungi Stemphylium sp. MAFF 241962 and Their Heme Polymerization Inhibition Activity

Abstract Antimalarial drug resistance is a major cause of the increasing incidence of malaria worldwide, necessitating an urgent demand for the development of new antimalarial drugs. However, the availability of bioactive natural compounds derived from plants is often limited. To address this issue, continuous exploration of bioactive secondary metabolites from endophytic fungi derived from medicinal plants has been recognized as a viable alternative. Therefore, this research aimed to isolate and characterize three ergosteroids, namely isocyathisterol (1), ergosterol-5,8-peroxide (3), cerevisterol (4), and a phytosterol, β-sitosterol (2), from the rice cultures of endophytic fungus Stemphylium sp. MAFF 241962, derived from Toona sureni. Endophytic fungi species were determined using molecular analysis of the internal transcribed region (ITS) of the ribosomal DNA. After comparing the sequence data to the NCBI database using BLAST, endophytic fungi were identified as Stemphylium sp. 241962 with 100% similarity. The chemical structures were elucidated using spectroscopic methods, including 1D and 2D NMR. Antimalarial activities of compounds 1-4 were evaluated using heme polymerization inhibition activity (HPIA) method. The results showed moderate inhibition activities with IC50 values of 7.70 ± 0.11, 9.48 ± 0.09, 7.88 ± 0.10, and 8.36 ± 0.56 mg/mL, respectively, compared to positive control chloroquine diphosphate with IC50 values of 1.59 ± 0.03 mg/mL. Keywords: Steroid, Stemphylium sp., Toona sureni, Antimalarial activity, Heme polymerization inhibition

New steroid produced by Periconia pseudobyssoides K5 isolated from Toona sureni (Meliaceae) and its heme polymerization inhibition activity

A new ergostane-type steroid named (22E)-3α,6α,9α-ergosta-7,22-diene-3,6,9-triol (1), along with six known steroids 5α,8α-epidioxy-24-ethyl-cholest-6-en-3β-ol (2), ergosterol-5,8-peroxide (3), cerevisterol (4), isocyathisterol (5), 6β-hydroxystigmast-4-en-3-one (6), 6β-hydroxy-4-campesten-3-one (7), were isolated from the fermented unpolished rice media by Periconia pseudobyssoides K5 (Periconiaceae), an endophytic fungus from medicinal plant Toona sureni (Meliaceae). The fermentation takes at 28 ± 2 °C for 30 days. The structure of new steroid (1) was elucidated by extensive spectroscopic measurements (IR, HR-ESI-TOFMS, and 1D and 2D NMR) analyses. The isolated compounds (1–7) were evaluated for heme polymerization inhibition assay (HPIA). The IC50 HPIA value of 1 is 8.24 ± 0.03 mg/ml.

Diketopiperazine Cyclo-(S-Pro-R-Leu) Produced by Periconia pseudobyssoides K5 Isolated from Toona sureni (Meliaceae) and its Heme Polymerization Inhibition Activity

Fungi endophytes are living microorganisms colonizing inside the internal tissue of a plant and provide ecological benefits for their host. Endophytes provide various metabolites for plant adaptation toward biotic and abiotic stresses and have tremendous pharmacological activities. Toona sureni (T. sureni) (Meliaceae) belongs to the Toona genera and is reported to have antimalarial activity. To determine the compounds produced by the endophytic fungus from this plant, we isolated a compound from Periconia pseudobyssoides (P. pseudobyssoides) K5 endophytes from the stem bark of T. sureni. Diketopiperazine cyclo-(S-Pro-R-Leu), a non-ribosomal peptide, was isolated from brown rice fermented at 28 ± 2 ℃ for 30 days. The structure was determined by spectroscopic methods including fourier-transform infrared spectroscopy, mass spectrometry, and 1D and 2D nuclear magnetic resonance techniques. This compound was evaluated for heme polymerization inhibition activity (HPIA) with an IC50 value of 9.89 ± 0.24 mmol/L compared with positive control chloroquine phosphate with an IC50 value of 3.08 ± 0.58 mmol/L. This compound has been categorized as having low activity three times lower than positive control chloroquine phosphate. This information provides new leads about the compound diketopiperazine cyclo-(S-Pro-R-Leu) produced by P. pseudobyssoides K5 endophytes having low activity in inhibiting heme polymerization. In the future, to explore the potency of this compound as antimalarial agent, the other antimalarial test such as lactate dehydrogenase assay might be useful.

THE POTENTIAL ANTIMALARIAL DRUG OF ARYL AMINO ALCOHOL DERIVATIVES FROM EUGENOL: SYNTHESIS, in-vitro AND in-silico ANALYSIS OF BIOACTIVITY

Malaria is a significant cause of death in numerous countries, and the discovery of effective drugs is crucial. In this research, a new method was employed to synthesize a derivative of aryl amino alcohol using eugenol. Initially, an epoxide compound called 2-methoxy-4-(oxiran-2-ylmethyl) phenol was synthesized from eugenol by reacting it with peroxyacetic acid in dichloromethane. Subsequently, the epoxide compound was subjected to a reaction with various amine compounds, including aniline, naphthylamine, and diphenylamine for 10 minutes under 100 W microwave conditions, resulting in the production of three derivative compounds of aryl amino alcohol. The antimalarial activity of these synthesized compounds was assessed in-vitro through an assay called heme polymerization inhibition activity (HPIA). Additionally, an in-silico analysis was conducted to evaluate the bioactivity of the three aryl amino alcohol derivatives by performing molecular docking with the Plasmepsin II enzyme. The identification of the products were determined using Fourier Transform Infrared (FTIR) spectrophotometry, proton nuclear magnetic resonance (1HNMR) spectrometry, and Electrospray ionization-Mass Spectrometry (ESI-MS). The research successfully obtained three aryl amino alcohol derivatives: 2-hydroxy-3-(phenylamino) propyl)-2-methoxyphenol, 4-(2-hydroxy-3- (naphthalene-1-ylamino) propyl)-2-methoxyphenol, and 4-(2-hydroxy-3-(diphenylamine)-2-methoxy phenol. All the synthesized compounds demonstrated lower IC50 values compared to chloroquine, indicating their potential as antimalarial drugs. Molecular docking studies revealed that these aryl amino alcohol derivatives effectively inhibited the activity of the Plasmepsin II enzyme

Evaluation of antiplasmodial activity and inhibitions of lipid peroxidation and parasite haem polymerization activity by medicinal phytochemicals of Phyllanthus amarus

Despite the dedicated struggle to eliminate malaria, the current trend of the infection, particularly, in sub-Saharan Africa remains worrisome due to failing control and treatment approaches caused by a myriad of factors. Therefore, the search for new, affordable, and more potent antimalarial drugs, from plants especially, have become the frontline in recent times. This study thus, evaluates the in vitro antiplasmodial, cytotoxic and antioxidant activities of the phytochemical (alkaloids, flavonoids, tannins, saponins, cardiac glycosides and anthraquinones) extracts of Phyllanthus amarus using documented methods. Antiplasmodial and cytotoxic activities were determined by parasite growth inhibition assay and microassay technique by the lactate dehydrogenase method, respectively. Antioxidant activities were determined by measuring inhibition of both lipid peroxidation and heam polymerization by extracts. Results show that the alkaloid extract was noncytotoxic (CC = 60.26 μg/mL) and most selectable (SI = 125.5) with the highest antiplasmodial (IC = 0.48 μg/mL) and 50 50 haem polymerization inhibition (IC = 0.63 μg/mL) activities, but moderate antioxidant lipid peroxidation inhibition, (LPI; 50 IC = 500μg/mL) capacity. However, the ethanol and flavonoid phytochemical extracts displayed the highest antioxidant 50 activity in inhibition of heam polymerization (IC = 0.46 μg/mL) and lipid peroxidation (IC = 218.78 μg/mL), respectively, 50 50 with significant antiplasmodial activity (IC = 0.48 μg/mL and 1.10 μg/mL, respectively). Our results show that all 50s phytochemical extracts studied were non-cytotoxic. Although the antioxidant activities of the flavonoid and ethanol phytochemical extracts were greater in comparison to the alkaloid phytochemical, alkaloid was the most selectable active antiplasmodial phytochemical of P. amarus and one of its mechanisms of action is by inhibiting parasite haem detoxification.

Docosahexaenoic acid is potent against the growth of mature stages of Plasmodium falciparum; inhibition of hematin polymerization a possible target.

The present study investigates the potential effect of externally added unsaturated fatty acids on P. falciparum growth. Our results indicate that polyunsaturated fatty acids (PUFAs) inhibit the growth of Plasmodium in proportional to their degree of unsaturation. At higher concentration the PUFA Docosahexaenoic acid (DHA) induces pyknotic nuclei in infected erythrocytes. When Plasmodium stages were treated transiently with DHA, the ring stage culture recovered from the drug effect and parasitemia was increased post DHA removal with delayed growth of 12h, compared to untreated control. Schizont stage treated culture displayed a 36h delay in growth to infect fresh erythrocytes signifying its recovery is less than the ring stage. However the trophozoite stage failed to recover and showed a decrease in parasitemia, similar to that of continuous treated culture. PUFAs inhibited β- hematin polymerization by binding to free heme derived from hemoglobin degradation. Digestive vacuole neutral lipid bodies, which are pivotal for β- hematin polymerization, decreased and subsequently abrogated with increasing concentration of DHA in trophozoite stage treated culture. Our study concludes that DHA interacts with heme monomers and inhibits the β- hematin polymerization and growth of mature stages i.e., trophozoite and schizont stages of plasmodium.

Synthesis and Antimalarial Activity Assay of <i>C</i>- Arylcalix[4]pyrogallolarenes Using Heme Polymerization Inhibition Activity (HPIA) Method

Malaria is one of the most devastating and widespread tropical parasitic diseases in developing countries with high prevalence. Furthermore, antimalarial drug resistance results in a global resurgence of malaria. Thus there is an urgent need to find new and active antimalarial agents. In this work, we reported the synthesis of C-arylcalix[4]pyrogallolarenes and their in vitro activity assay as new antimalarial agent candidates. The C-arylcalix[4]pyrogallolarenes were prepared in high yields through a condensation reaction between pyrogallol and aromatic aldehydes (i.e., benzaldehyde, 4-hydroxy-3-methoxybenzaldehyde, and 2-chlorobenzaldehyde) under acidic conditions. The antimalarial activity of C-arylcalix[4]pyrogallolarenes was tested using the Heme Polymerization Inhibition Activity (HPIA) method with chloroquine diphosphate as the positive control. The heme polymerization inhibitory activity was reflected from the IC50 values in which the IC50 values were obtained from probit analysis using IBM SPSS statistics 25 software. The result showed that the IC50 values of C-arycalix[4]pyrogallolarene derivatives were in a range of 0.238–1.268 mg/mL, which were lower than chloroquine diphosphate (IC50 = 2.788 mg/mL). This finding reveals that the C-arylcalix[4]pyrogallolarenes are potential antimalarial agents to be developed in the future.

Synthesis of <i>N</i>-Phenylpyrazoline Derivative from 4-Chlorobenzaldehyde and 4-Chloroacetophenone and its Activity as an Antimalarial Agent

This study was aimed to synthesize N-phenylpyrazoline derivative and evaluate its antimalarial activity. The pyrazoline was synthesized from chalcone derivative through a cyclo- condensation reaction. Moreover, chalcone derivative was prepared from 4-chlorobenzaldehyde and 4-chloroacetophenone through Claisen-Schmidt condensation. The structure elucidation of the products was performed by FTIR, GC-MS, 1H- and 13C-NMR spectrometers. The antimalarial assay was conducted by inhibition of heme polymerization. The chalcone was obtained as a white solid in 83% yield, while N-phenylpyrazoline derivative was obtained as brown solid in 70% yield. The IC50 values of chalcone and N-phenylpyrazoline derivatives as well as a positive control (chloroquine diphosphate) were 98.66, 20.83, and 3.54 mg/mL, respectively. The results showed that both chalcone and N-phenylpyrazoline derivatives had lower antimalarial activity than the positive control. Molecular docking studies were also performed to explain the mode of action of chalcone and N-phenyl pyrazoline as antimalarial agent.

Design and synthesis of harmiquins, harmine and chloroquine hybrids as potent antiplasmodial agents

Malaria remains one of the major health problems worldwide. The lack of an effective vaccine and the increasing resistance of Plasmodium to the approved antimalarial drugs demands the development of novel antiplasmodial agents that can effectively prevent and/or treat this disease.Harmiquins represent hybrids that combine two moieties with different mechanisms of antiplasmodial activity in one molecule, i.e., a chloroquine (CQ) scaffold, known to inhibit heme polymerization and a β-carboline ring capable of binding to P. falciparum heat shock protein 90 (PfHsp90). Here we present their synthesis, evaluation of biological activity and potential mechanism of action. The synthesized hybrids differed in the type of linker employed (triazole ring or amide bond) and in the position of the substitution on the β-carboline core of harmine. The antiplasmodial activity of harmiquins was evaluated against the erythrocytic stage of the Plasmodium life cycle, and their cytotoxic effect was tested on HepG2 cells. The results showed that harmiquins exerted remarkable activity against both CQ-sensitive (Pf3D7) and CQ-resistant (PfDd2, PfK1, and Pf7G8). P. falciparum strains. The most active compound, harmiquine 32, displayed single-digit nanomolar IC50 value against Pf3D7 (IC50 = 2.0 ± 0.3 nM). Importantly, it also showed significantly higher activity than CQ against the resistant Plasmodium strains and had a very high selectivity index (4450). Harmiquins may act through the inhibition of heme polymerization and binding to the ATP binding site of the PfHsp90, which would explain their increased activity against the CQ-resistant Plasmodium strains. These results establish harmiquins as valuable antiplasmodial hits for future optimization.

Synthesis, characterization, molecular docking, antimalarial, and antiproliferative activities of benzyloxy-4-oxopyridin benzoate derivatives.

Background and purpose:Malaria and cancer are two major health issues affecting millions of lives annually. Maltol complexes and derivatives have been extensively investigated as chemotherapeutic and antimalarial activities. In this study, the design, synthesis, biological activities, and docking study of a novel series of pyridinones derivatives were reported.Experimental approach:The chemical structures of synthesized compounds were approved by FTIR, 1HNMR, 13CNMR, and mass spectroscopies. The antimalarial activity was evaluated through β-hematin inhibition assay and the cytotoxicity activities were evaluated against PC12 and fibroblast cell lines via MTT and cell uptake assays. To theoretically investigate the ability of compounds to inhibit hemozoin formation, the synthesized compounds were docked in a heme sheet to explore their binding mode and possible interactions.Findings/Results:β-Hematin inhibition assay showed acceptable activity for 7f, 7c, and 7d compounds and the molecular docking study showed 7h and 7f had effective interactions with the heme sheet. The cytotoxic study revealed compound 4b (IC50 = 18 μM) was significantly more active against PC12 cells than docetaxel (IC50 = 280 μM). The observations of cell uptake images were also shown both cell penetration and monitoring potential of synthesized compounds.Conclusion and implications:The compounds showed a moderate ability to inhibition of heme polymerization and also good interaction with heme through molecular docking was observed. Additionally, some of them have a good cytotoxic effect on the study2 cell line. So further study on these compounds can lead to compounds that can be considered as anti-malarial and/or anticancer agents.

Phytochemical Screening and Haem Polymerization Inhibitory Activity of Root Extract and Fractions from Strychnos lucida R. Br.

Phytochemical Screening and Haem Polymerization Inhibitory Activity of Root Extract and Fractions from Strychnos lucida R. Br.

The Antagonizing Role of Heme in the Antimalarial Function of Artemisinin: Elevating Intracellular Free Heme Negatively Impacts Artemisinin Activity in Plasmodium falciparum.

The rich source of heme within malarial parasites has been considered to underly the action specificity of artemisinin. We reasoned that increasing intraparasitic free heme levels might further sensitize the parasites to artemisinin. Various means, such as modulating heme synthesis, degradation, polymerization, or hemoglobin digestion, were tried to boost intracellular heme levels, and under several scenarios, free heme levels were significantly augmented. Interestingly, all results arrived at the same conclusion, i.e., elevating heme acted in a strongly negative way, impacting the antimalarial action of artemisinin, but exerted no effect on several other antimalarial drugs. Suppression of the elevated free heme level by introducing heme oxygenase expression effectively restored artemisinin potency. Consistently, zinc protoporphyrin IX/zinc mesoporphyrin, as analogues of heme, drastically increased free heme levels and, concomitantly, the EC50 values of artemisinin. We were unable to effectively mitigate free heme levels, possibly due to an unknown compensating heme uptake pathway, as evidenced by our observation of efficient uptake of a fluorescent heme homologue by the parasite. Our results thus indicate the existence of an effective and mutually compensating heme homeostasis network in the parasites, including an uncharacterized heme uptake pathway, to maintain a certain level of free heme and that augmentation of the free heme level negatively impacts the antimalarial action of artemisinin. Importance: It is commonly believed that heme is critical in activating the antimalarial action of artemisinins. In this work, we show that elevating free heme levels in the malarial parasites surprisingly negatively impacts the action of artemisinin. We tried to boost free heme levels with various means, such as by modulating heme synthesis, heme polymerization, hemoglobin degradation and using heme analogues. Whenever we saw elevation of free heme levels, reduction in artemisinin potency was also observed. The homeostasis of heme appears to be complex, as there exists an unidentified heme uptake pathway in the parasites, nullifying our attempts to effectively reduce intraparasitic free heme levels. Our results thus indicate that too much heme is not good for the antimalarial action of artemisinins. This research can help us better understand the biological properties of this mysterious drug.

Haem polymerization inhibitory activity and cytotoxicity of six medicinal plants used in cameroon for the management of malaria

Haem polymerization inhibitory activity and cytotoxicity of six medicinal plants used in cameroon for the management of malaria