Antimalarial Activity of Flavonoid Compound Isolated from Leaves of Artocarpus altilis

Malaria is a potentially fatal disease transmitted through the bite of female Anopheles mosquito and affects more than 106 countries worldwide. Mortality rates of malaria have increased over the past 20 years, primarily due to resistance of P. falciparum to antimalarial drugs, especially chloroquine, artemisinin and its derivatives.1,2 Efforts are therefore needed to develop new antimalarial as that are effective, safe and have fewer side effects while also being cheap and easy to obtain, one potential source are compounds derived from plants.3 Flavonoids are secondary metabolites that are widely found in plants, and the antimalarial activity of this group has been widely reported.4


INTRODUCTION
Malaria is a potentially fatal disease transmitted through the bite of female Anopheles mosquito and affects more than 106 countries worldwide. Mortality rates of malaria have increased over the past 20 years, primarily due to resistance of P. falciparum to antimalarial drugs, especially chloroquine, artemisinin and its derivatives. 1,2 Efforts are therefore needed to develop new antimalarial as that are effective, safe and have fewer side effects while also being cheap and easy to obtain, one potential source are compounds derived from plants. 3 Flavonoids are secondary metabolites that are widely found in plants, and the antimalarial activity of this group has been widely reported. 4 Moraceae, a family of flowering plants, consists of 60 genera that include 1,400 species. The largest genus of the Moraceae family is Artocarpus which contains 50 species, 40 of which are in Indonesia. The genus Artocarpus is widely used in traditional medicines. Generally, Artocarpus species are rich in phenolic compounds. The extracts and metabolites of Artocarpus, especially leaves, barks, stems, and fruits, have been shown to be useful bioactive compounds. Several pharmacological studies on Artocarpus have proven its utilization for various diseases such as inflammation, malarial fever, diarrhea, diabetes, and tapeworm infections.
Artocarpus altilis, which in Indonesia has a local name "sukun" referred to as breadfruit, is a tropical plant. The breadfruit tree produces fruit from March to September. The synonyms of A. altilis are A. communis, and A. incises. 5,6 An ethanol extract of A.altilis leaves actively inhibited the growth of P. falciparum in vitro with IC 50 values 1.32 μg/ml, and was highly active against P. berghei in vivo with ED 50 values 0.82 mg/kg body weight. While stem bark extract from A. altilis showed a very good in vivo activity against P. berghei, and weak in vitro activity against P. falciparum. 7 The previous study showed that another species of genus Artocarpus reported that prenylflavonoid compounds isolated from A. champeden stem bark extract. A. champeden stem bark extract contains artocarpones A, artocarpone B, artoindonesianin E, heteroflavanone C, artoindonesianin R, heterophyllin, artoindonesianin A-2, cycloheterophyllin, and artonin. Heteroflavanone C had the most active inhibition against P. falciparum with IC 50 values 1 nmol/L. 8 A prenylated chalcone, isolated from A. champeden stem bark extract, namely morachalcone A, was identified as a antimalarial active marker compound. 9 Leaves and stembark extract from another species, Artocarpus heterophyllus and Artocarpus camansi, also has been reported to have good antimalarial activity against P. falciparum and P. berghei. 7,8 While A. altilis leaves extract is efficacious against malarial parasite, their mechanism of action remains to be identified. This study was conducted to search for active antimalarial compounds contained in the leaves extract of A. altilis.

MATERIALS AND METHODS
General NMR spectra were recorded on a JEOL ECS-400, using CDCl 3 as the solvent. The HPLC system also includes two LC-10AD pumps and a SCL-10A controller. An Agilent RP-18 XDB column 4.6 x 250 mm was eluted with CH 3

Plant material
The fresh leaves of Artocarpus altilis were collected from Purwodadi Botanical Garden, East Java, Indonesia in the rainy season, around September-December, and identification by a licensed botanist at Purwodadi Botanical Garden, East Java. A voucher specimen (No: B-107/IPH.06/AP.01/II/2020) of this raw material stored at the Herbarium of the Institute of Tropical Disease, Universitas Airlangga, Surabaya, Indonesia.

Isolation and identification of active compounds
The ethanol extract was fractionated using vacuum liquid chromatography (VLC) with a gradient of n-hexane and ethyl acetate (100%-0%). Based on thin layer chromatography (TLC) profiles, several fractions with the same profile were combined and yielded 6 fractions. Each fraction was identified with TLC methods and tested for its antimalarial activity. Fraction that contain yellow spot on its TLC profile and showed inhibition against P. falciparum, was then separated by semi-preparative High Liquid Pressure Chromatography (HPLC) methods. The semi-preparative HPLC system was run on a Shimadzu, LC-06, included two LC-10AD pumps and SCL-10A controller, with an semi preparative colomn; Agilent Eclipse XDB-C18, 5 µm (9.4 x 250 mm). Fraction were eluted in a methanol:water (8:2, v/v) mixture at flow rate of 1 mL/min, which obtained three isolates.
The active isolate profile was analyzed using TLC and HPLC. TLC was analyzed using a silica gel 60 F 254 plates, scanned by TLC Visualizer, visualized on UV 254 nm and 366 nm, and sprayed with H 2 SO 4 :water (1:9, v/v) to highlight the flavonoid compounds. The HPLC analysis system included two LC-10AD pumps and SCL-10A controller, with an analytical colomn; LiChrospher ® 100 RP-18 analytical column, 5 µm, 4.6 x 250 mm. Compounds were eluted in a methanol:water (8:2, v/v) mixture at flow rate of 0.7 mL/min. The chemical structure of each isolate was determined using a Nuclear Magnetic Resonances (JEOL ECS 400) with CDCl 3 as the solvent. The spectra were then analyzed using the MNova program. Mass spectra were identified by LC system coupled with Agilent Q-TOF/MS -MS-MS for HRESIMS analysis using methanol:water (8:2, v/v), with Agilent Zorbax Extend-C18 column, 1.8 µm, 2.1 x 50 mm In vitro cultivation of Plasmodium falciparum P. falciparum strain 3D7 was obtained from the Malaria Laboratory at The Centre of Natural Product Medicine Research and Development, Institute of Tropical Disease, Universitas Airlangga, Surabaya, Indonesia. The culture was established using the method described by Trager and Jensen with some modifications. 10 Parasite were grown in fresh human erythrocytes (human type O red blood cell) at 5% haematocrit in complete RPMI-1640 medium supplemented with 10% (v/v) human type O-positive serum, 25 mM Hepes buffer, 50 µg/mL hypoxanthine, 2 mg/mL NaHCO 3 , and 10 µg/mL gentamycin. The culture was incubated at 37°C in a modified candle jar. Parasitemia was determined by the examination of Giemsa's stained thin blood smears of infected erythrocytes. Human RBCs and serum were received from The Indonesian Red Cross, Surabaya, Indonesia.

In vitro antimalarial assay
Antimalarial assay was conducted using giemsa stained slide method. 11 Ten milligrams of tested compound and chloroquine was each diluted in 1 mL DMSO. The sample was then serially diluted in RPMI-1640 medium and prepared in dilution at a concentration of 0.01, 0.1, 1, 10, and 100 µg/mL in a 24 well plate. 500 µL parasite culture (±1% parasitemia, 5% hematocrit) was added to each well and incubated for 48 hours at 37°C. After incubation, thin blood smears were made and stained using 10% (v/v) Giemsa dye to showed infected red blood cell. The percentage of parasitemia was determined by counting infected erythrocytes per 1000 total erythrocytes under a light microscope. The percentage of growth inhibition was calculated using the following equation: Where Xe is the average parasitemia of the experimental group, and Xk is the average parasitemia of negative control.
The IC 50 values were calculated by applying four parameters logistic regression curve to the dose-response data using GraphPad Prism 7.0 software (GraphPad Co. Ltd., San Diego, CA, USA).

Docking analysis
The molecular structure of the falcipain-2 enzyme was downloaded from the RCSB protein data bank website (http://www.rcsb.org/pdb/ home/home.do). In this study, the falcipain-2 was chosen with the code PDB: 3BPF. This receptor was chosen because showed strongest interaction with compound-1. The ligand (was prepared by making 2D, and 3D structures of compound-1 using the ChemBioOffice program Ultra 11.0 and its energy was minimized using MMFF94. The ligand with the minimum energy that has been measured is then saved in the form of anmol2 {SYBYL2 (*.Mol2)}. The doctoring process of the enzyme falcipain-2 pdb.3BPF uses a Molegro Virtual Docker Version 5.5 program. The results obtained in the form of the Rerank Score (RS) value, which is the energy needed in the ligand-receptor interaction process, and from these values antimalarial activity can be predicted. 13

Isolation and identification of active compound
An ethanol extract of A. altilis was fractionated by the VLC method using the gradient solvent of n-hexane and ethyl acetate (100%-0%) yielded six fractions (fraction 1-6). Identification with TLC method showed that fraction-3 gave the yellow spot that indicated a flavonoid compound. Fraction-3 was further separated by semi-preparative HPLC using acetonitrile:water (7:3 v/v) and yielded five isolates (isolate [1][2][3][4][5]. carbon quartener at δ C 78.8 (C-3") indicates a 2.2-dimethylchromene ring which is substituted in ring B. A chelated hydroxyl group at at δH 12.74 (1H, s, 2'-OH), and two hydroxyl signal showed at aromatic ring at δH 7.25 (4'-OH) and δH 5.58 (4-OH). The methylene group signal at δ H 3.10 (2H, q, H-α) and δ H 2.97 (2H, m, H-β) substituted to C7 shows the correlation between ring A and ring B. The characteristics of chalcone pyrano-O-prenylated are shown by the methylene group signal at δH 3.1 (2H, q, H-alfa) and δH 2.97 (2H, m, H-beta) which have a correlation with carbonyl at δC 203.8. The prenyl group signal at δ C 5.08 (1H, t, H-6"), δ C 1.66 (3H, s, H-9") and δ C 1.56 (3H, s, H-8") substituted with C6". The 13 C NMR spectrum of isolate-2 shows 25 wellseparated signals for 25 carbon atoms, consisting of one conjugated carbonyl carbon at δ C 203.8 (C=O), four oxyaryl carbon bond with two aromatic ring at δ C 139.6 (C-3), δ C 143.2 (C-4), δ C 165.3 (C-2'), δ C 162.3 (C-4'), one 2.2 dimethylcromen ring at δ C 78.7 (C-3"), and one isoprenyl at δ C 132.1 (C-7"). By conducting a references study on compounds that have been isolated from A. altilis, and comparing the NMR spectrum with the references, it is concluded that isolate-2 (Compound-1) is a flavonoid compound, class of dihydrochalcone, similar to [1-(2,4- 16 The molecular structur of Compound-1 has been confirmed it mass spectra which were identified by Agilent  In vitro antimalarial assay showed that isolate-2 inhibited P. falciparum growth with IC 50 of 0.48 μg/ml. Isolate-2 was analysed by TLC method using silica gel 60 RP-18 F254, mobile phase acetonitrile:water (7:3 v/v)= 7:3 v/v, scanned on UV 254 and 366 nm, and sprayed with a specific reagent for flavonoid compound. The results showed that isolate-2 had the yellow spot, typical of a flavonoid group, with Rf 0.4. Analysis of isolate-2 with HPLC method (Figure 1), at concentration 0.5 mg/ml, using isocratic solvent acetonitrile: water (7:3 v/v) showed that isolate-2 had a major peak, with retention time at 15.74 minutes. The UV spectrum of the peak showed maximum absorption with λ max 248, 275, 300, and 315 nm (Figure 2). The UV spectra of most flavonoids consists of two major absorption, for band II in the range of 240-285 nm and band I in the range of 300-400 nm. In general terms the band II absorption may be considered as having originated from the A-ring benzoyl system and band I from the B-ring cinnamoyl system. 14 The UV spectrum of major peak showed a characteristic absorption peak of the flavonoid compound, class of dihydrochalcone. 15 Isolate-2 was then identified based on proton and carbon signals in the   Compound-1 (Figure 3). 16 Yellow gum, m/z 409.2100, the 1H-NMR spectral data were identical to those in reference : 1

Antimalarial activity
In vitro antimalarial test was conducted using a synchronized P. falciparum strain 3D7, which is sensitive to chloroquine. This antimalarial activity test was conducted to determine the potential inhibition of P. falciparum growth in general, meaning that no inhibition was observed in certain phases. Chloroquine was use as a positive control.
Antimalarial assay of the Compound-1 (Table 1) revealed that this compound inhibited the growth of P. falciparum with the IC 50 value of 0.48 μg/ml (calculated 1.05 μM). The IC 50 value indicated that this compound possesses a potent antimalarial activity. This evidence suggests for further biochemical assay of the compound to determine the mechanism of action. The fact that the compound was originated from the leave extract also indicate its high availability and sustainability as we do not need to cut the tree source.

Docking analysis
To predict the mechanism of action, an in silico study was performed using the Molegro Virtual Docking (5.5) program to determine the possible interaction of compounds with the protein target. We evaluated several proteins from Protein Data Base, which have been reported to have an interaction with falcipain (www.rcsb.org). As a result, the isolated compound was predicted to have strong interaction with receptor 3BPF, falcipain-2, a protein that can be found in the food vacuole of P. falciparum involved in hemoglobin hydrolysis. The in silico study was performed by compari ng Compound-1 with drug references, chloroquine. The re-rank score of Compound-1 was -71.2844 kcal/mol, while the rerank score of choloroquine was -66.3610 kcal/mol. A lower value of the re-rank score indicates a stronger interaction to a receptor 13 . Hydrogen bond interaction was predicted between falcipain 2 and Compound-1 group with Gln 36* (2 bonds), Asn 173*, and Gly 83. In addition, van der Walls interactions were predicted between falcipain-2 and Compound-1 group with Gln 36, cys 39, Ser 41, Gly 40, Cys 42, Leu 172, Asn 173, Trp 206, His 174, and Val 152. It seems to contribute to the binding interaction of 3BPF and Compound-1 ( Figure 4). While, chloroquine revealed hydrogen binding to Gly 83, Gln 36, Cys 42, and His 174, and steric van der Walls to Cys 42, Ser 41, and Gly 82 ( Table 2). The interaction is described in the 3D profile ( Figure 5).
The study showed that compound-1 have a strong interaction with 3.BPF protein, a facipain-2 receptor. It has been reported that Falcipain-2 is a cysteine protease that can be found in food vacuole of plasmodium. 22 This enzyme is a key member of the hemoglobin degradation pathway, a process that is required at erythrocytic stages of P. falciparum to obtain amino acids. 23,24 Falcipain-2 is an attractive target for drug discovery. 25,20 Nowadays, the search for potent falcipain-2 inhibitors is a hot topic in the search for potential malaria drugs. 26 Other studies have also shown that trophozoites falcipain-2 knock out cause a decrease in cysteine protease activity, accumulation of hemoglobin that cannot be degraded in food vacuoles, and increase the sensitivity of cysteine and aspartic protease inhibitors. 27 In silico study predicted that the target site of Compound-1 is in parasitic food vacuoles as a cysteine protease inhibitor. Based on our study, we concluded that prenylated dihydrochalcones acted by inhibiting the activity of Plasmepsin II enzyme, resulting in the inability of hemoglobins to be degraded into large fragment. Meanwhile, compound-1 acted by inhibiting the activity of falcipain-2 enzyme, hence disabling the degradation of large fragments into amino acids. Din Syafruddin was trained in medicine and doctoral degree in the parasite cell biology, he is currently a senior research fellow at the Malaria and Vector Resistance Laboratory at the Eijkman Institute for Molecular Biology, and Professor and chairman of the Department of Parasitology, Faculty of Medicine, Hasanuddin University. His research was to elucidate the role of the extrachromosomal DNAs in the assembly of the mitochondrial energy transducing membrane of the malarial parasite. His current research include molecular mechanisms underlying Plasmodium resistance to antimalarial drug, antimalarial drug discovery, molecular taxonomy of the mosquito vector and studies on the vector resistance to insecticides.