Antimalarial Activity of Curcuma Caesia Against 3D7 and K1 Strains of Plasmodium Falciparum

doi:10.21203/rs.3.rs-32452/v1

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Antimalarial Activity of Curcuma Caesia Against 3D7 and K1 Strains of Plasmodium Falciparum

https://doi.org/10.21203/rs.3.rs-32452/v1

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Background: Malaria is a severe and sometimes mortal tropical disease that spreads through parasites. The purpose of the study was to evaluate in vitro and in-silicoantiplasmodial potential of Curcuma caesia extracts against Plasmodium falciparum.

Methods: Lack of a vaccine and the widespread resistance to antimalarial drugs have resulted in emphasis on novel antimalarial drugs development. Ethyl acetate and methanol extracts of Curcuma caesia were prepared and analysed for their antiplasmodial activity against Chloroquine sensitive (3D7) and resistant (K1) strains of P. falciparumusingfluorescence-based SYBR Green assay. The cytotoxicity tests were carried out using the verocell lines by MTT assay.The phosphoethanolamine methyltransferase enzyme ((PfPMT) essential for growth of Plasmodium falciparum was used as protein target for in-silicostudy.

Result: Curcuma caesia ethyl acetate extracts showedpotentantiplasmodial activitywith IC₅₀ values of 3.37 µg/ml and 1.53 µg/ml against 3D7 and K1 strain respectively.Docking results show that β-selinenol an oxygenized sesquiterpene had the free binding energy of -6.76 Kcal/mol.

Conclusion: Sesquiterpene present in the Curcuma caesia extract was responsible for antimalarial potential analyzed by molecular modeling. The present findings, however preliminary in nature. Further studies are required to proven the antimalarial efficacy C. caesia by isolating the active compounds and in vivo mechanism study that may contribute to more effective development of antimalarial drugs in the future.

Infectious Diseases

Curcuma caesia

Plasmodium falciparum

β-Selinenol

Phosphoethanolamine methyltransferase

Malaria remains an importantglobal health concern in tropical and subtropical regions and antimalarial drug resistance has emerged as alarming concern for discovery of novel antimalarial compounds. However, the increase of insecticide-resistant Anopheles mosquitos triggers not only malaria expanding to new areas but its re-emergence in places where it has historically been eradicated. In addition, chloroquine resistance currently occurs around the wholeworld. Due to increase in number of infected people in affected countries they faces extreme economic burden. An estimated 228 million cases of malaria occurred in 2018 with 405 000 deaths worldwide. About 67% children under 5 years of ages were affected with malaria. According to WHO report Plasmodium falciparum is the most rampant malaria parasite accounting for 99.7% of estimated malaria cases in 2018 [1].

Traditionally, in various cultures, medicinal plants were used to sustain their primary health care needs. The use of different extracts from medicinal plants for malaria treatment has a long and successful tradition[2]. Asan example quinine from Cinchona, and artemisinin isolated from Qinghaosu[3]. This explains why many recent studies concentrate on natural and herbal product because they can easily be purchased, can be made local and can be selected based on their pharmacological applications[4, 5].

Curcuma caesia is one of the traditional tribal medicinal plants which is used in India for treatment of fever and other ailments. It belongs to Zingiberaceae family which is commonly called as black turmeric. Plants of the family, Zingiberaceae have been documented as having antimalarial properties[6]. It is a perennial plant with bulky bluish color rhizome. The aim of this study was to analyse antimalarial activity of Curcuma caesia ethyl acetate and methanol extracts against Plasmodium falciparum along with the molecular docking study to search out the possible metabolites which interfere with plasmodium enzyme.

Plant extracts preparation and GCMS analysis

The extracts of Curcuma caesia were prepared in ethyl acetate and methanol solvent by following the method of our previous study [7].

In vitroantimalarial activity screening

The in vitro cultures of both Chloroquine - resistant (K1) and sensitive (3D7) strains of P. falciparum were performed using RPMI-1640 medium of pH 7.2 supplemented with 25 mM HEPES, 0.5% ALBUMAX-II, 0.2% D-glucose and 0.21% sodium bicarbonate [8]. The stock (5 mg/ml) solution of test samples were prepared in DMSO and required dilutions were prepared in culture medium. The maximum concentration of extract used was 50.0 µg/ml. 1.0% parasitized cell suspension containing 0.8% parasitaemia (Asynchronous culture with more than 80% ring stages) were incubated in ⁹6 well plate with two-fold serial dilutions of test sample. The plates were kept in CO₂ incubator at 37⁰C with an atmosphere of 5% CO₂ and air mixture. After 72 hours, 100 µl of lysis buffer with 2x concentration of SYBR Green-I (Invitrogen) was added in each well and kept at 37⁰C for one hour [⁹]. The plates were examined at 485 ± 20 nm of excitation and 530 ± 20 nm of emission for relative fluorescence units (RFUs) per well, using the fluorescence plate reader (FLX800, BIOTEK). The IC₅₀ values (concentration that inhibits 50% growth of P. falciparum) were calculated by Logit regression analysis of dose-response curves. Chloroquine diphosphate (SIGMA) was used as the reference drug.

Cytotoxicity assay

The cytotoxicity of the test samples was conducted against Vero cell line (C1008, Monkey kidney fibroblast cells) using MTT assay [10]. The cells were incubated with test sample dilutions for 72 h and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) was used as reagent for detection of cytotoxicity. The highest concentration of test sample used was 200 µg/ml. MTT dye turned from yellow to purple color after cellular enzymatic reduction. CC₅₀ value (concentration that reduced the cell viability by 50%.) was calculated using dose-response curves. Podophyllotoxin (SIGMA) was used as the reference drug for cytotoxicity.

Macromolecules and ligand structure retrieval:The target protein a multifunctional enzyme phosphoethanolamine methyltransferase (PfPMT) (PDB ID: 3UJ9) was selected on the basis of the literature study. This enzyme catalyzes the methylation of phosphoethanolamine to phosphocholine which is necessary for membrane synthesis. Hence, it is vital for survival and growth of Plasmodium falciparum. As this enzyme is absent in humans, so it’s good drug target in search of new anti-malarial drug [11]. Fifty-three metabolites were obtained from a previous study[⁷] by GCMS analysis of methanol and ethyl acetate extract of Curcuma caesia were used as a ligand for docking study. Ligand structures were downloaded in SDF format from the Pubchem database and were converted into PDB format by PyMol software.

ADMET prediction

Before docking study all ligand molecules were subjected to computational ADMET analysis by using online software SwissADME[12] and ADMET-SAR [13].

Docking and visualization

AutoDock 4.2 was used to study protein and ligands interaction. AutoDock is the most widely used fully automated software for the docking study [14]. Polar hydrogen and Kollman charges were added to the protein molecule [15]. Grid box having dimensions X, Y, and Z were set to 120*120*120. Lamarck genetic algorithm was used to analyze the output for ligand conformations. The least negative ΔG specifies a strong binding and favourable conformation between ligand and protein [16]. The 3D visualization of docked structures was achieved by a graphical user interface, Discovery studio [17].

In vitro antimalarial activity screening and cytotoxicity assay

In vitro antimalarial activity of Curcuma caesiaethyl acetate and methanol extracts were analyzed for both Chloroquine-resistant (K1) and sensitive (3D7) strains of P. falciparum.IC₅₀ values of ethyl acetate extract were 3.37 µg/ml against 3D7, and 1.53 µg/ml against K1 strain.The methanol extract possesses IC₅₀ values of 8.57 µg/ml against 3D7 and 18.29 µg/ml against K1 strains (Table 1).The IC₅₀ value for promising lead compounds was considered equal to or less than 10.0 µg/ml.

Cytotoxicity was carried out againstVero cell line (C1008; Monkey kidney fibroblast cells). IC₅₀ and CC₅₀ values of both extracts against the Plasmodium falciparum sensitive and resistant strains with positive and negative control valuesalong with Selectivity index (SI) has been given in Table 1.SI was calculated by dividing the CC₅₀value with IC₅₀ and the criteria for selection is that SI value should be equal or more than 50. Higher SI value is preferable for a drug to have favourble safety and efficacy [10]. Interestingly, ethyl acetate extract of Curcuma caesia exhibited profoundin vitro antimalarial activity against 3D7 and K1 strains in comparision to methanol extract with higher SI index against K1strain.

Table 1

*In vitro* antimalarial and cytotoxic activity of *Curcuma caesia* extracts
Test Samples	IC₅₀ (µg/ml)		CC₅₀ (µg/ml)	Selective Index (SI)
Test Samples	3D7	K1	CC₅₀ (µg/ml)	3D7	K1
C. caesia ethyl acetate extract	3.37	1.53	13.92	4.13	9.09
C. caesia methanol extract	8.57	18.29	91.20	10.64	4.98
QC-diphosphate*	0.005	0.303	175	35,000	577.5
Podophyllotoxin*	na	na	6.4	na	na
*Values are in µM

ADMET prediction

All the compounds from the GCMS study of both extracts were selected for ADMET (Absorption, Distribution, Metabolism, Excretion, and Toxicity) prediction.From the ADMET result only thirty-one compounds were selected which follows Lipinski rule with no violation and having bioavailability value of0.55 for docking study. Additional file1: Table S1 shows the bioactivity radar for rapid evaluation of drug-likeness of thirty-one metabolites.

Docking analysis of compounds

Molecular docking studies were carried out to reveal the interactions of phosphoethanolamine methyltransferase enzyme (PfPMT) of Plasmodium falciparum with thirty-one compounds identified from Curcuma caesia methanol and ethyl acetate extracts. Binding energy of all thirty-oneligands against the enzyme isdepicted in Additional file 2: Table S2. The higher negative value of binding energy corresponds to a more stable interaction of ligand to the enzyme. Among 31 ligands, β-Selinenol (-6.76 Kcal/mol), α-Eudesmol(-6.59 Kcal/mol), α –Acorenol (-5.85 Kcal/mol), Boldione(-5.55 Kcal/mol) and Xanthinin(-5.40 Kcal/mol) showed the highest binding energy (Table 2). The 2D and 3D interactions docking results were attained by introducing our result into the Discovery Studio Visualizer, enabling us to recognize important interactions between the ligands and the binding site of the receptor. Figure 1 shows the 2D interaction of all top five compounds with PfPMTwhich helpsin identifyingthe amino acids which are involved in the interaction. There are 2 amino acids involved in conventional hydrogen bond interaction between β-Selinenoland PfPMT,GLU226 and LYS 225,shown in Fig. 2(a) and with a bond length of 2.73 and 2.95. Figure 2(b) shows the surface view of the hydrogen bond.

Table 2

Binding energy of lead five ligand with PfPMT
Name	Binding energy (Kcal/mol)	No. of H-bond	Amino acid	Bond length (Ångstrom)
β-Selinenol	-6.76	2	GLU226, LYS 225	2.73 Å, 2.95 Å
α-Eudesmol	-6.59	2	ASN101,ASN74	2.55 Å, 3.23 Å
α –Acorenol	-5.85	1	GLN212	2.91 Å
Boldione	-5.55	1	LYS165	3.09 Å
Xanthinin	-5.40	3	PHE116,TRP148,GLU143	3.32 Å, 3.17 Å, 3.18 Å

Artemisinin was the backbone of antimalarial research for a decade and due to artemisinin resistance, it is necessary to identify newer antimalarial agents with higher activity and lower toxicity.Therefore, this study was conducted to explore the antimalarial activity of Curcuma caesia. Curcuma caesia belongs to the Zingiberaceae family which contains many plantswith reportedantimalarial activity like Aframomumlatifolium, Kaempferiamarginata[18],Curcuma longa, ZingiberofficinaleRoscoe, etc. Previous studies reveal that the antiplasmodial effect of ZingiberofficinaleRoscoemethanol extractagainst P. bergheiin a dose-dependent manner[19]. Duker-Eshum et al.[20] showed the significant antiplasmodial activity of the extracts ofA. latifolium and A. sceptrum. The supercritical extract of Curcuma longaalso showed the high antiplasmodialactivity [21].

Several terpenes were identified as having antimalarial activity from different plant sources. For example, Artemisinin (sesquiterpene lactone) andpaclitaxelare used clinically for malarial treatment. Goulart et al.[22] demonstrated that farnesol and linalool both are terpenesthat can inhibit the biosynthesis of many intermediates and end products of the isoprenoids pathway of Plasmodium falciparum.Limonene hinders the protein isoprenylation in the intraerythrocytic stages of P. falciparum[23]. Kenmogne et al. [24] isolated Zambisiacolactone B labdane, aditerpenoids from A. zambesiacumwhich was the most active against chloroquine-resistant strainP. falciparum with an IC₅₀ of 4.97 µM. Curcumin from Curcuma longa has antimalarial potential.GCMS analysis of C. caesia extracts revealed the presence of many sesquiterpene.

Modern studies have opened the way to identifying new drug target in search of effective and novel antimalarial drug with less time consumption. The phosphoethanolamine methyltransferase, PfPMT, of the human malaria parasite Plasmodium falciparum, a member of a novely identified family of phosphoethanolamine methyltransferases (PMT) found exclusively in nematodes, protozoa and plants. It is involved in the synthesis of the major membrane phospholipid, phosphatidylcholine. But PMT from these organisms use multiple methyltransferase domains for the S-adenosylmethionine (AdoMet) reactions which is a three-stepS-adenosylmethionine-dependent methylation of the nitrogen atom of phosphoethanolamine to phosphocholine. In P. falciparum, this enzymatic reaction is a limiting step in the pathway of synthesis of phosphatidylcholine from serine, hence it is potent new drug target for antimalarial drug discovery.and plays an important role in the development, replication and survival of the parasite within human red blood cells [11^,25].

To explore the metabolite involved in the antimalarial activity of Curcuma caesia in silico study against phosphoethanolamine methyltransferase enzyme (PfPMT) of Plasmodium falciparum was done. In molecular docking the least binding energy revealed the stronger docking berween the ligand and parasitic target. The docking results showed that β-Selinenol and α-Eudesmolpresent in C. caesia havingleast binding energy. Both are the isomeric form. β-Selinenol displays considerable in vitro antiplatelet activity, antiangiogenic activity, anti-hepatotoxic effects and antitumoral effects [26].Earlier reports suggest many sesquiterpene as a antiparasitic effect like Artemisinin, Lactucin, Lactucopicrinetc[27]. Antimalarial activity of C. caesia may be due the presence of sesquiterpenes.

The development and identification of new malaria molecules is a growing need for multi-drug resistant malaria. Current study is designed for search of the new and effective antimalarial drug by in vitro antimalarial activity of Curcuma caesia extracts against Chloroquine sensitive (3D7) and resistant (K1) strains of P. falciparum strains. Although Curcuma caesia has been used in traditional medicine systems for fever and various infections, the antimalarial activity has been established for the first time. Further studies are required to proven the antimalarial efficacy C. caesia by isolating the active compounds and evaluate its potential as antimalarial drug.

Acknowledgments

Authors would like to thank ICAR-Indian Institute of Spices Research, Kozhikode, Kerala for providing the sample. Authors would also like to thank the Council of Scientific & Industrial Research (CSIR) - Central Drug Research Institute, Lucknow 226031, Uttar Pradesh, India for conducting in vitro antimalarial and cytotoxic study..

Authors’ contributions

Monika Chaturvedi has prformed experimentation work, data collection, and drafted the manuscript. Reena Rani has made significant involvement in the interpretation of data and revising the manuscript. Dushyant Sharma participated in the design of the study and performed the statistical analysis. Jaya Parkash Yadav helped in designed the study and manuscript.All authors read and approved the final manuscript.

Funding

Author would like to thank University Grant Commission (UGC), New Delhi for financial support by UGC SAP [F. 20/2012(SAP II)] project fund.

Availability of data and materials

The data supporting the conclusions of this article are provided within the article and its additional files. The original datasets analysed in this current study are available from the corresponding author upon request.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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Antimalarial Activity of Curcuma Caesia Against 3D7 and K1 Strains of Plasmodium Falciparum

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Abstract

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Background

Methods

Results

ADMET prediction

Docking analysis of compounds

Discussion

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References

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