Data on antiplasmodial and stage-specific inhibitory effects of Aromatic (Ar)-Turmerone against Plasmodium falciparum 3D7

Aromatic (ar)-turmerone is one of the aromatic constituents abundant in turmeric essential oil from Curcuma longa. Ar-turmerone exhibited anti-inflammatory properties. So far, antiplasmodial data for ar-turmerone is still not reported. The data showed the in vitro antiplasmodial effect of ar-turmerone against Plasmodium falciparum 3D7 (chloroquine-sensitive) via Plasmodium lactate dehydrogenase assay (pLDH) and cytotoxic effect against Vero mammalian kidney cells using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) colourimetric assay. Selectivity indexes of ar-turmerone were calculated based on inhibition concentration at 50% of parasite growth (IC50) from MTT and pLDH assays and the effects of ar-turmerone were compared to the antimalarial reference drug chloroquine diphosphate. The inhibitory effect of ar-turmerone at the intraerythrocytic stages of plasmodial lifecycles was evaluated via a stage-dependant susceptibility test. The antiplasmodial and cytotoxic activities of ar-turmerone revealed IC50 values of 46.8 ± 2.4 μM and 820.4 ± 1.5 μM respectively. The selectivity index of ar-turmerone was 17.5. Ar-turmerone suppressed the ring-trophozoite transition stage of the intraerythrocytic life cycle of P. falciparum 3D7.


a b s t r a c t
Aromatic (ar)-turmerone is one of the aromatic constituents abundant in turmeric essential oil from Curcuma longa . Ar-turmerone exhibited anti-inflammatory properties. So far, antiplasmodial data for ar-turmerone is still not reported. The data showed the in vitro antiplasmodial effect of ar-turmerone against Plasmodium falciparum 3D7 (chloroquine-sensitive) via Plasmodium lactate dehydrogenase assay (pLDH) and cytotoxic effect against Vero mammalian kidney cells using 3-(4, 5-dimethylthiazol-2-yl)-2, 5diphenyltetrazolium bromide (MTT) colourimetric assay. Selectivity indexes of ar-turmerone were calculated based on inhibition concentration at 50% of parasite growth (IC 50 ) from MTT and pLDH assays and the effects of ar-turmerone were compared to the antimalarial reference drug chloroquine diphosphate. The inhibitory effect of ar-turmerone at the intraerythrocytic stages of plasmodial lifecycles was evaluated via a stage-dependant susceptibility test. The antiplasmodial and cytotoxic activities of ar-turmerone revealed IC 50 values of 46.8 ± 2.4 μM and 820.4 ± 1.5 μM respectively. Value of the Data • The research data is important to understand the in vitro antimalarial and cytotoxic properties of Ar-turmerone which is useful for further animal ( in vivo ) and clinical studies. • The data obtained from this work will add to the knowledge about the medicinal value of Curcuma longa as an adjunctive therapy remedy for malaria treatment. • Parasitologists, pharmacologists, pharmacists, and nutritionists can give thought to the data obtained as it may be a new antimalarial drug template and as natural supplements for immunity against malarial infection. • The research data is useful as a reference for further drug development of experiments for malarial drug discovery involving drug pharmacology, toxicity and pharmacokinetic fields.

Data Description
The data on in vitro antiplasmodial and cytotoxic activities of ar-turmerone were reported in this dataset. The antiplasmodial effect of ar-turmerone against P. falciparum 3D7 (chloroquine-sensitive) using plasmodium lactate dehydrogenase (pLDH) assay showed an IC 50 of 46.8 ± 2.4 μM as demonstrated in Table 1 . The raw data source for this dataset is available through the Mendeley database link (Table S1). The data for the cytotoxic effect of arturmerone (IC 50 = 820.4 ± 1.5 μM) was also shown in Table 1 . Chloroquine diphosphate (pLDH IC 50 = 0.0 058 ±0.0 012 μM; MTT IC 50 = 1020.9 ±10.7 μM) was used as an antimalarial reference drug. The selectivity index (SI) of ar-turmerone showed a value of 17.5. This data suggested that ar-turmerone is a promising antiplasmodial compound and non-toxic. Chloroquine diphosphate exhibited high SI value (SI > 20 0 0). High SI value shown by the antimalarial drug, chloroquine diphosphate indicated that the drug is potent and non-toxic. The stage-inhibitory effect of ar-turmerone revealed 85% suppression at the ring stage, 65% suppression at the trophozoite stage, and 50% suppression at the schizont stage ( Fig. 1 ). The raw data source for this dataset is also available through the Mendeley database link (Table S2). The examination of parasite morphology changes in P. falciparum intraerythrocytic stages was presented in Fig. 2 . Positive inhibitory effects of ar-turmerone were found at the ring-trophozoite transition stage of the parasite intraerythrocytic life cycle. The rings underwent lyses and loss of cellular architectures. Morphological changes of parasites were observed after incubation with ar-turmerone or chloroquine diphosphate. Highly synchronous P. falciparum 3D7 cultures were treated with the IC 90 value of ar-turmerone (463.5 μM) or chloroquine diphosphate (0.052 μM), determined by non-linear regression analysis from pLDH assay. Chloroquine diphosphate treatment is represented as a reference drug. Arrows show the presence of parasites in the erythrocytes. The red or purple stain is referred to as erythrocytes, while blue or dark blue stain is referred to as parasites. Colour differences between the microscopic images are due to white balance settings and this does not affect the analysis of the result.
For the trophozoite-schizont transition stage, a longer transition period was observed in arturmerone treatment showing that development of the parasite intraerythrocytic morphologies from trophozoite to schizont had been slowed down due to the effect of ar-turmerone. However, some of the parasites ( > 60%) still survived indicating that the treatment of ar-turmerone during the trophozoite-schizont transition stage did not effectively eradicate the parasites compared to the ring-trophozoite transition stage. Parasites from the schizont-ring transition stage treated with ar-turmerone did not show any significant morphological changes. For an antimalarial reference drug, chloroquine diphosphate, inhibition of parasite was seen at all stages in the intraerythrocytic parasite life cycle. The data source (Table S1 and Table S2) for this data is available in Mendeley database link ( https://data.mendeley.com/datasets/wvnb772htc/draft?a= c9cba652-8b12-41dc-bcae-1bb10d27defe ).

In vitro antiplasmodial assessment
Chloroquine-sensitive strain (3D7) of P. falciparum was collected from the Malaria Research and Reference Reagent Resource Centre (MR4), Virginia, USA. The parasite was revived from cry-opreservation and cultured in growth medium at 2% haematocrit in purified O + human erythrocytes at 37 °C, 5% CO 2 . Human erythrocytes were provided by the University Health Centre. This study protocol received ethical approval from the Research Ethics Committee of Universiti Kebangsaan Malaysia (JEP-2020-334). In this study, all chemicals and reagents were purchased from Sigma-Aldrich, St. Louis, MO, USA unless stated otherwise. The growth medium contained RPMI 1640 (Gibco, Life Technologies, USA) supplemented with 0.5% Albumax I (Gibco, Life Technologies, USA), 25 mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES) (Gibco, Life Technologies, USA), 100 μM hypoxanthine, 12.5 μg/mL gentamicin and 1.77 mM sodium bicarbonate [1] . The parasitaemia level was monitored by microscopic examination using thin and thick blood smears. Susceptibility of the P. falciparum 3D7 to ar-turmerone (Cayman Chemicals, Ann Arbor, MI, USA) (CAS Number:532-65-0) was evaluated using parasite lactate dehydrogenase (pLDH) assay. PLDH assay was conducted in flat-bottomed 96-well microtiter plates [2] . Before the incubation started, the parasite cultures were synchronised to obtained > 90% rings using 5% D-sorbitol. Parasites were later plated at 2% haematocrit and 2% parasitaemia in culture media containing ar-turmerone at concentration ranged from 0.01 to 10 0 0 μM and incubated for 48 h. Chloroquine diphosphate (CAS Number: 50-63-5) was used as antimalarial reference drug at concentration ranged from 0.0 0 01 to 100 μM. Malstat reagent was prepared by mixing sodium lactate (4 g), Tris buffer (1.32 g), triton X-10 0 (40 0 μL) and 3-acetylpyridine adenine dinucleotide (APAD) (0.02 g) in 200 mL of distilled water. Malstat and nitroblue tetrazolium/phenazine ethosulfate (NBT/PES) mixture were used as substrate and colorimetric reagent in the assay system. The blood mixture was mixed with the substrate and colorimetric reagent. During the assay, changes of colour were monitored using a microplate reader at 655 nm (Fluostar Optima) after one-hour incubation. Based on the raw absorbance readings, a sigmoidal curve was plotted using GraphPad Prism 5 (GraphPad Software, Inc., San Diego, CA) to determine the 50% inhibitory concentrations (IC 50 ) of the tested compound. Experiments were performed in triplicate and repeated three times.

In vitro cytotoxic test and selectivity indexes calculation
Vero (ATCC: CCL-81), normal epithelial monkey kidney cells were obtained from the American Type Culture Collection (ATCC), USA. Cytotoxicity of ar-turmerone was evaluated using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay in Vero cells seeded at 2 × 10 4 cells/mL in DMEM complete medium (Gibco, Life Technologies, USA) [3] . The concentration of ar-turmerone and chloroquine diphosphate ranged from 0.1 to 10 0 0 0 μM was used in the assay system. Untreated cell cultures were used as a control. The cell culture was incubated in the presence of test compounds for 48 h (37 °C, 5% CO 2 ). MTT reagent was used as a colorimetric reagent in this assay and then the reagent was added to each well before 3 h incubation. The medium in the mixture was then removed and MTT formazan product was then dissolved in dimethyl sulphoxide (DMSO). Absorbance was measured at 540 nm (Fluorostar OP-TIMA). The concentration of test compounds at 50% growth inhibition (IC 50 ) was determined from a sigmoidal curve generated from a statistical analysis software, GraphPad Prism 5. Selectivity indexes were measured as the ratio of cytotoxic over antiplasmodial activity, each one expressed with IC 50 (ratio IC 50 MTT/IC 50 pLDH assay) [4] . When a compound exhibits an SI value more than 10 (SI > 10), the compound is generally considered as a promising antimalarial agent, non-toxic and safe which indicating that the pharmacological effect shown is not due to the cytotoxic effect [4] . Lower SI value suggests that the compound has low antiplasmodial activity and high toxicity.

Stage-specific susceptibility test
The stage-specific developmental inhibition of ar-turmerone was studied according to an established protocol [5] . Briefly, P. falciparum 3D7 was exposed to ar-turmerone at the concentra-tion of IC 90 (463.5 μM) for 5 h at each stage; ring stage (at 0 to 5 h), trophozoite stage (at 28 to 33 h), and schizont stage (at 42 to 47 h). Chloroquine diphosphate was used as a reference drug at the IC 90 value (0.052 μM). The IC 90 value used in the study was to evaluate the maximum parasite inhibitory effect of ar-turmerone and to compare the compound potency with the reference antimalarial drug, chloroquine diphosphate. After incubation, parasites were washed with incomplete medium (medium without Albumax I) to remove the test compound and were then further cultured using normal culture conditions (growth medium supplemented with Albumax I). Untreated synchronous cultures served as a control and were processed in the same way as the treated cultures. After the next ring cycle, blood smears were stained with Field A (methylene blue) and B (eosin) staining. Parasitaemia level in treated cultures was measured by counting the number of new rings formed in a total of 2 0 0 0 cells during the second intraerythrocytic cycle. The parasitaemia counting was carried out to observe the effect of the treatments given at the different stages (ring, trophozoite and schizont). Chemo-suppression percentage was calculated from the parasitaemia level of treated cultures as compared to a control. The stage counting was done blinded to treatment. Morphological changes were observed at each stage by microscopic examination of thin and thick blood smear of the treated and untreated cultures at 10 0 0 × magnification using immersion oil.

Declaration of Competing Interest
The authors declare that they have no known competing for financial interests or personal relationships exist.