Anti-Japanese Encephalitis Virus (JEV) Activity of Triterpenes ......“tawa-tawa” Philippine Journal of Science 149 (3): 603-613, September 2020 ISSN 0031 - 7683 Date Received:

Wilanfranco C. Tayone1*, Kotaro Ishida2, Simon Goto2, Janeth C. Tayone1, Masashi Arakawa2, Eiji Morita2, and Masaru Hashimoto2

Anti-Japanese Encephalitis Virus (JEV) Activity of Triterpenes and Flavonoids from Euphorbia hirta

*Corresponding Author: [email protected]

1Institute of Agriculture and Life Sciences, Davao Oriental State College of Science and Technology, City of Mati 8200 Philippines

2Faculty of Agriculture and Life Science, Hirosaki University Hirosaki-shi, Aomori-ken 036-8560 Japan

Chemical investigation of Euphorbia hirta, a folkloric medicinal plant against dengue in the Philippines, disclosed two known flavonoids (quercetrin and myricitrin) together with seven known triterpenes (taraxerone, taraxerol, β-sitosterol, 24-hydroperoxycycloart-25-en-3β-ol, 25-hydroperoxycycloart-23-en-3β-ol, lupeol, and (23E)-cycloart-23-en-3β, 25-diol). The structures were established mainly with 1H and 13C nuclear magnetic resonance (NMR) spectroscopic analyses in comparison with the literature data. These compounds were tested against Japanese encephalitis virus (JEV). Results showed that myricitrin exhibited good inhibition on the production of the infectious viral particle at 100 µM.

Keywords: dengue, Euphorbia hirta, Japanese encephalitis virus, nuclear magnetic resonance, “tawa-tawa”

Philippine Journal of Science149 (3): 603-613, September 2020ISSN 0031 - 7683Date Received: 06 Feb 2020

INTRODUCTIONEuphorbia hirta L. is known in the Philippines as “tawa-tawa.” It is a traditional herb and abundant in open grasslands. Tawa-tawa is normally harvested by taking the whole plant at its flowering stage and the decoction is prepared by boiling it for a few minutes and then given to the patient as tea. There were several folkloric claims about the effectiveness of the said plant against dengue fever that is caused by infection of dengue virus, a member of flavivirus, but none has been substantiated yet by scientific evidence. The plant was reported by Patil’s group (2009) to be effective for the treatment of gonorrhea, dysentery, boils, pulmonary disorders, and jaundice. Other studies also claimed that it has some anti-allergic property (Singh et al. 2006), anti-tumor (Patil and Magdum 2011), antimalarial (Liu et al. 2007),

antidiabetic (Kumar et al. 2010), cytotoxic activity on HEP 2 cells (Sidambaram et al. 2011), sedative property (Lanhers et al. 1990), antifungal (Rao et al. 2010), anthelmintic (Adedapo et al. 2005), and diuretic (Johnson et al. 1999). The plant is said to be effective as an anti-inflammatory (Shih et al. 2010), antidiarrheic (Galvez et al. 1993), anti-human immunodeficiency virus (Gyuris et al. 2009), antibacterial (Ogbulie et al. 2007), anti-asthma (Ekpo and Pretorius 2007), antioxidant, and anti-proliferative (Chen and Er 2010). Moreover, it also has the potential to inhibit angiotensin-converting enzyme (Williams et al. 1997) and promote cartilage degeneration in arthritic rats (Lee et al. 2008). In the course of our previous investigations exploring biologically active metabolites from natural products, we also reported the isolation of nine metabolites from E. hirta and their potential against dengue fever (Tayone et al. 2014).

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JEV is a member of flavivirus and is one of the leading causes of severe encephalitis in Asia and the Western Pacific. Both JEV and dengue virus are transmitted by mosquitoes and share similar viral structure, genome organization, and replication strategy in the host cell. Therefore, it has been considered that anti-dengue reagent targeting basic propagation mechanism can also work as an inhibitory reagent for JEV, as well as for other members of flaviviruses. In this paper, we report the anti-JEV activities of the nine compounds isolated from the aforementioned plant and the isolation of the additional four compounds, which were not included in our earlier study (Tayone et al. 2014).

MATERIALS AND METHODS

GeneralThe structure elucidation of isolated compounds was performed with NMR spectroscopy. The 1H (500 MHz) and 13C (125 MHz) NMR spectra were recorded in CDCl3 (δH 7.24 ppm) and CD3OD (δH 3.30 ppm) on a JEOL JNM-ECA500 spectrometer. The residual CHD2OD and 13CD3OD were used as the internal standards (1H: 3.31 ppm, 13C: 49.15 ppm, respectively) as the internal standards. Splitting patterns are designated as s (singlet), d (doublet), dd (doublet of doublet), t (triplet), q (quartet), m (multiplet), and br (broad). Chemicals and solvents were purchased from Fujifilm Wako Pure Chemical Industries and Sigma-Aldrich Co. LLC. Those were used without further purification. Thin-layer chromatography (TLC) analyses were carried out using Merck silica gel TLC silica gel 60 F254 plates (No. 5715). Silica gel column chromatographies were carried out using Merck 707734.

Plant MaterialThe plant sample was collected from Mati City, Davao Oriental, Philippines (6°55′49.3″N, 126°15′12.3″E) in June 2018. The material was first identified via indigenous knowledge and comparison with images from online photos (West African plants: a photo guide; Senckenberg).

Sample Preparation, Extraction, and Isolation of Secondary MetabolitesA whole plant of E. hirta (approximately 1 kg) was collected, washed with tapped water, cut into around 30 cm long, and then air-dried. The dried plant was then soaked in 8 L of 50% EtOH/MeOH solution for 48 h and stirred frequently. A 500-mL portion of the crude extract was filtered and concentrated in vacuo until the volume became approximately 20 mL. The obtained aqueous suspension was extracted with 100 mL EtOAc and the aqueous layer was collected and concentrated in vacuo.

This extraction afforded 1.2 g. After the residue was diluted with methanol (100 mL), diatomaceous earth granule (approximately 3 g) was added. The resulting suspension was concentrated in vacuo. The residue was loaded on a Φ 1.6 x 12 cm column and attached to preparative ODS medium pressure column chromatography (Yamazen Corporation Universal™ Column, ODS-SM 50 µm 120 A, size M Φ 2.3 x 12.3 cm). The aqueous MeOH was eluted (15 mL/min) under gradient condition (10–100 % MeOH/H2O for 1 h) to afford quercetrin (1, 13.5 mg) and myricitrin (2, 14 mg). The majority of the crude extract (10 L) was filtered with cotton gauze and the filtrate was concentrated under reduced pressure until all methanol was removed. The resulting aqueous suspension was lyophilized. The obtained paste was then suspended with EtOAc and the soluble portion was concentrated to give the crude material (23.2 g). It was then suspended with H2O (1 L) and extracted with EtOAc (1 L × 3). The organic layer was concentrated to give the extract (13 g). After the residue was diluted again with EtOAc (200 mL), silica gel (20 g) was added and concentrated carefully with a rotary evaporator. The residue was placed on a silica gel column (Φ 8 x 90 cm) and then developed with EtOAc/hexane solvent system (0–100%) and gave fraction A (eluted with 100% n-hexane, 108 mg), fraction B (eluted with 30% EtOAc/hexane, 7.90 g), fraction C (eluted with 60% EtOAc /hexane, 1.60 g), fraction D (eluted with 100% EtOAc, 434 mg), and fraction E (eluted with 100% MeOH, 3.60 g). The fraction B was subjected to second silica gel column (Φ 6 x 70 cm) chromatography (0–50% EtOAc/hexane) to give fraction B-1 (363 mg), fraction B-2 (790 mg), fraction B-3 (400 mg), fraction B-4 (500 mg), fraction B-5 (4.6 g), and fraction B-6 (1.3 g). Since fraction B-2 formed solid particles, it was diluted with 10% EtOAc/hexane (150 mL). Standing the solution precipitated taraxerone (3, 56.7 mg). A portion of the fraction B-5 (2.3 g) was subjected to second silica gel column (Φ 4.5 x 70 cm) chromatography (0–30% EtOAc/hexane) to provide fractions B-5-1 (1.2 g), B-5-2 (533 mg), B-5-3 (250 mg), and B-5-4 (25 mg). Fractions B-5-1 and B-5-2 were then dissolved independently with hexane (50 mL and 30 mL, respectively) and, upon standing of the solutions, precipitated taraxerol (4, 28.7 mg) and β-sitosterol (5, 18 mg), respectively. The mother solution from fraction B-5-1 was subjected to the third silica gel column (Φ 3 x 60 cm) chromatography (0–30% EtOAc/hexane) to give 24-hydroperoxycycloart-25-en-3β-ol (6, 2.6 mg) and 25-hydroperoxycycloart-23-en-3β-ol (7, 3.0 mg) along with fractions B-5-1-1 (121 mg), B-5-1-2 (615 mg), B-5-1-5 (0.5 mg), and B-5-1-6 (43.6 mg). Further fractionation of fraction B-5-1-2 by preparative medium pressured ODS column chromatography with ODS-SM 50 µm 120 A, Φ 3.0 × 17.0 cm (Yamazen Co. Ltd.) under the same conditions mentioned above gave lupeol (8, 30 mg). The other silica gel column (Φ 3 x 60 cm)

Tayone et al.: Anti-JEV of Triterpenes and Flavonoids from E. hirta

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chromatography of fraction B-6 (0–50% EtOAc/hexane) provided (23E)-cycloart-23-en-3β, 25-diol (9, 8.2 mg). The obtained samples were characterized by the 1H and 13C NMR spectra.

Compound 1: 1H NMR (CD3OD, 500 MHz) δ: 6.27 (1H, d, J = 2.0 Hz, H-6), 6.43 (1H, d, J = 2.0 Hz, H-8), 7.42 (1H, d, J = 2.1 Hz, H-2’), 7.00 (1H, d, J = 8.3 Hz, H-5’), 7.39 (1H, dd, J = 8.3, 2.1 Hz, H-6’), 5.44 (1H, d, J = 1.5 Hz, H-1”), 4.31 (1H, dd, J = 3.3, 1.5 Hz, H-2”), 3.84 (1H, dd, J = 9.5, 3.3 Hz, H-3”), 3.38 (1H, overlap, H-4”), 3.51 (1H, dd, J = 9.6, 6.2 Hz, H-5”), 1.03 (3H, d, J = 6.2 Hz, H-6”). 13C NMR (CD3OD, 125 MHz) δ: 158.8 (C=C, C-2), 136.5 (C=C, C-3), 179.9 (C=O, C-4), 163.4 (C=C, C-5), 100.1 (C=C, C-6), 166.1 (C=C, C-7), 95.0 (C=C, C-8), 159.6 (C=C, C-9), 106.2 (C=C, C-10), 123.2 (C=C, C-1’), 117.2 (C=C, C-2’), 150.0 (C=C, C-3’), 146.7 (C=C, C-4’), 116.7 (C=C, C-5’), 123.2 (C=C, C-6’), 103.8 (O-C-O, C-1”), 72.2 (C-O, C-2”), 72.4 (C-O, C-3”), 73.5 (C-O, C-4”), 72.3 (C-O, C-5”), 17.9 (CH3, C-6”). The 1H and 13C NMR data were identical with those in the literature (Tayone et al. 2014; Ni et al. 2017; Seo et al. 2017).

Compound 1 was further reacted with 200 µL acetic anhydride (Bonner and McNamara 1968) in pyridine (500 µL) and stirred overnight at room temperature to give its expected heptaacetate derivative in 30.1% yield. This result corroborates the presence of seven hydroxy groups in 1.

Acetylation of QuercetrinA 10 mg of 1 was treated with 200 µL acetic anhydride in pyridine (500 µL) and stirred overnight at room temperature. The reaction was checked and guided by TLC analyses until the starting material has disappeared. The mixture was concentrated under reduced pressure and co-evaporated several times with toluene to remove traces of pyridine. The major product after preparative TLC gave 5 mg of the expected heptaacetate compound.

Compound 2: 1H NMR (CD3OD, 500 MHz) δ: 6.99 (2H, s, H-2′ and H-6 ′), 6.40 (1H, d, J = 5.0 Hz, H-8), 6.24 (1H, d, J = 5.0 Hz, H-6), 5.35 (1H, d, J = 5.0 Hz, H-1′′), 4.26 (1H, dd, J = 5.0, 2.8 Hz, H-2′′), 3.82 (1H, dd, J = 9.1, 2.8 Hz, H-3′′), 3.52 (1H, dd, J = 9.8, 6.3 Hz, H5′′), 3.34 (1H, m, H-4′′), 1.00 (3H, d, J = 5.0 Hz, H-6′′). The 1H NMR data were identical with those in the literature (Seo et al. 2017).

Compound 3: 1H NMR (CDCl3, 500 MHz) δ: 5.56 (1H, dd, J = 8.2, 3.2 Hz, H-15), 1.14 (3H, s, H-27), 1.09 (3H, s, H-23), 1.08 (3H, s, H-25), 1.07 (3H, s, H-24), 0.96 (3H, s, H-29), 0.92 (3H, s, H-28), 0.91 (3H, s, H-30), 0.83 (3H, s, H-26). The 1H NMR data were identical with those in the literature (Jamal et al. 2009).

Compound 4: 1H NMR (CDCl3, 500 MHz) δ: 5.53 (1H, dd, J = 8.2, 3.2 Hz, H-15), 3.19 (1H, m, H-3), 1.09 (3H, s, H-27), 0.98 (3H, s, H-23), 0.95 (3H, s, H-29), 0.93 (3H, s, H-24), 0.91 (3H each, s, H-26/30), 0.82 (3H, s, H-28), 0.80 (3H, s, H-25). The 1H NMR data were identical with those in the literature (Jamal et al. 2009; Feng et al. 2005).

Compound 5: 1H NMR (CDCl3, 500 MHz) δ: 5.36 (1H, d, J = 5.15 Hz, H-5), 3.53 (1H, m, H-3), 1.01 (3H, s, H-29), 0.92 (3H, d, J = 6.5 Hz, H-19), 0.85 (3H, t, J = 7.5 Hz, H-24), 0.84 (3H, d, J = 6.9 Hz, H-26), 0.81 (3H, d, J = 6.9 Hz, H-27), 0.68 (3H, s, H-28). The 1H NMR data were identical with those in the literature (Chaturvedula and Prakash 2012).

Compound 6: 1H NMR (CDCl3, 500 MHz) δ: 0.33 (1H, d, J = 4.2 Hz, H-19a), 0.55 (1H, d, J = 4.2 Hz, H-19b), 0.81 (3H, s, H-29), 0.87 (3H, d, J = 6.5 Hz, H-21), 0.89 (3H, s, H-30), 0.96 (3H, s, H-28), 0.97 (3H, s, H-18), 3.28 (1H, m, H-3), 1.73 (3H, s, H-27), 4.27 (1H, t, J = 6.5 Hz, H-24), 5.01 (1H, s, H-26a), 5.03 (1H, m, H-26b), 7.74 (1H, d, J = 5.5 Hz, -OOH); 13C NMR (CDCl3,125 MHz) δ: 32.0 (CH2, C-1), 30.4 (CH2, C-2), 78.9 (C-O, C-3), 40.5 (C, C-4), 47.1 (CH, C-5), 21.1 (CH2, C-6), 26.0 (CH2, C-7), 48.0 (CH, C-8), 20.0 (C, C-9), 26.1 (C, C-10), 26.5 (CH2, C-11), 32.9/32.87* (CH2, C-12), 45.3/45.29* (C, C-13), 48.8 (C, C-14), 35.6 (CH2, C-15), 28.1/28.08* (CH2, C-16), 52.1/52.0 (CH, C-17), 18.0 (CH3, C-18), 29.9 (CH2, C-19), 36.0/35.8* (CH, C-20), 18.2/18.16* (CH3, C-21), 27.6/27.3* (CH2, C-22), 32.0 (CH2, C-23), 90.4/90.3* (C-OOH, C-24), 143.9/143.6* (C=C, C-25), 114.2/114.7* (C=C, C-26), 16.9/17.2* (CH3, C-27), 25.4 (CH3, C-28), 14.0 (CH3, C-29), 19.3 (CH3, C-30). * signals for C-24 epimer. The 1H and 13C NMR data were identical with those in the literature (Kato et al. 1996; Ragasa and Cornelio 2013).

Compound 7: 1H NMR (CDCl3, 500 MHz) δ: 0.33 (1H, d, J = 4.2 Hz, H-19a), 0.56 (1H, d, J = 4.2 Hz, H-19b), 0.81 (3H, s, H-29), 0.87 (3H, d, J = 7.8 Hz, H-21), 0.88 (3H, s, H-30), 0.96 (3H, s, H-28), 0.97 (3H, s, H-18), 1.34 (3H, s, H-26), 1.34 (3H, s, H-27), 3.28 (1H, m, H-3), 5.52 (1H, d, J = 15.0 Hz, H-24), 5.69 (1H, ddd, J = 15.0, 8.0, 6.0 Hz, H-23), 7.27 (s, -OOH); 13C NMR (CDCl3, 125 MHz) δ: 32.0 (CH2, C-1), 30.4 (CH2, C-2), 78.9 (C-O, C-3), 40.5 (C, C-4), 47.1 (CH, C-5), 21.1 (CH2, C-6), 26.0 (CH2, C-7), 48.0 (CH, C-8), 20.0 (C, C-9), 26.1 (C, C-10), 26.4 (CH2, C-11), 32.8 (CH2, C-12), 45.3 (C, C-13), 48.8 (C, C-14), 35.6 (CH2, C-15), 28.1 (CH2, C-16), 52.1 (CH, C-17), 18.1 (CH3, C-18), 29.9 (CH2, C-19), 36.3 (CH, C-20), 18.4 (CH3, C-21), 39.4 (CH2, C-22), 130.8 (C=C, C-23), 134.4 (C=C, C-24), 82.3 (C-OOH, C-25), 24.4 (CH3, C-26), 24.3 (CH3, C-27), 25.4 (CH3, C-28), 14.0 (CH3, C-29), 19.3 (CH3, C-30). The 1H and 13C NMR data were identical with those in the literature (Kato et al. 1996).



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Compound 8: 1H NMR (CDCl3, 500 MHz) δ: 4.69 (1H, d, J = 2.4 Hz, H-29a), 4.57 (1H, dd, J = 2.4, 1.4 Hz, H-29b), 3.19 (1H, dd, J = 11.5, 4.9 Hz, H-3), 1.68 (3H, s, H-30), 1.03 (3H, s, H-26), 0.97 (3H, s, H-23), 0.94 (3H, s, H-27), 0.83 (3H, s, H-25), 0.79 (3H, s, H-28), 0.76 (3H, s, H-24). The 1H NMR data were identical with those in the literature (Chaiyadej et al. 2004).

Compound 9 : 13C NMR (CDCl3, 125 MHz) δ: 32.0 (CH2, C-1), 30.4 (CH2, C-2), 78.9 (C-O, C-3), 40.5 (C, C-4), 47.1 (CH, C-5), 21.1 (CH2, C-6), 26.0 (CH2, C-7), 48.0 (CH, C-8), 20.0 (C, C-9), 26.1 (C, C-10), 26.4 (CH2, C-11), 32.8 (CH2, C-12), 45.3 (C, C-13), 48.8 (C, C-14), 35.6 (CH2, C-15), 28.1 (CH2, C-16), 52.0 (CH, C-17), 18.1 (CH3, C-18), 29.9 (CH2, C-19), 36.4 (CH, C-20), 18.3 (CH3, C-21), 39.0 (CH2, C-22), 125.6 (C=C, C-23), 139.4 (C=C, C-24), 70.8 (C-O, C-25), 29.9 (CH3, C-26), 30.0 (CH3, C-27), 19.3 (CH3, C-28), 25.4 (CH3, C-29), 14.0 (CH3, C-30). The 13C NMR data were identical with those in the literature (Liu et al. 2010).

Cells and VirusesHEK293A and Vero cells were cultured in Dulbecco's modified Eagle medium (DMEM; Nacalai Tesque Inc., Kyoto, Japan) supplemented with 100 units/mL penicillin, 100 µg/mL streptomycin, and 10% (v/v) fetal bovine serum (FBS; Thermo Fisher Scientific Inc., MA, USA), in humidified air containing 5% CO2 at 37 °C. JEV-Chb was amplified on HEK293A cells. JEV strain AT31 (kindly provided by Prof. Konishi, Osaka University) were grown in 293A cells.

Preparation of Virus Infected Cells and Cell Culture SupernatantsCompounds 1 and 2 were dissolved and diluted in sterilized water (Nacalai) while 3, 4, 5, 6, 7, 8, and 9 were dissolved and diluted in dimethyl sulfoxide (Nacalai). HEK293A cells were seeded with a density of 1 × 104 cells/well into 96-well plates and incubated overnight. Subsequently, 1 µL of diluted compounds were added to each well. Two hours after the addition of the compounds or just solvent as a control, cells were inoculated with JEV-Chb or the mock control with a multiplicity of infection (MOI) of 0.3. Culture supernatant was taken 24, 48, or 72 h after virus inoculation (hpi). The cell lysate was prepared 72 hpi. HiBiT-mediated luciferase activity of culture supernatants and cell lysates were measured to evaluate virus proliferation.

Wildtype JEV was inoculated into sterile water (control) or 100 µM myricitrin-treated HEK293A cells with MOI of 0.3, and the cell culture supernatants were taken 48 and 72 hpi. Focus forming units of culture supernatants were determined to evaluate virus proliferation.

HiBiT Luciferase Assay Cells were lysed with lysis buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% (v/v) Triton X-100, and complete protease inhibitor cocktail (Roche Ltd., Basel, Switzerland). HiBiT-mediated luciferase activity of cell lysate or culture supernatant was measured using Nano Glo HiBiT Lytic Detection System kit (Promega) following the manufacturer’s protocol.

Cell Viability AssayHEK293A cells with a density of 1 × 104 cells/well in 96-well plates were treated with various concentrations of compounds for 72 h. Cell viability was evaluated using Cell Titer Glo 2.0 reagent (Promega) following the manufacturer’s protocol.

Virus TitrationViral titers were determined by a focus-forming assay. In brief, Vero cell monolayers were inoculated with serially diluted viruses, and cultured in DMEM supplemented with 1% methylcellulose for 36 h. The cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, and 8 mM Na2HPO4 (pH 7.4). Then, they were permeabilized and blocked with 0.1% Triton X-100 and 10% FBS in PBS. The cells were incubated with primary antibodies (rabbit anti-JEV NS3 antibody (obtained from a rabbit which is immunized with recombinant-JEV NS3 protein) in PBS for 30 min at room temperature. Then, the cells were incubated with secondary antibody (AlexaFluor 488-conjugated anti-rabbit antibodies (Jackson) in PBS for 30 min at room temperature. The infectious foci were counted using a fluorescence microscope, and the viral infectious titers were calculated as focus forming units per mL.

Statistical AnalysisValues in graphs are represented as the mean ± standard deviation. P-values were calculated by a two-tailed Student’s t-test, which was performed using Microsoft Excel (Microsoft Corp., Redmond, WA). P-values > 0.05 were considered to be significant.

RESULTS AND DISCUSSIONIn our pursuit for further investigation of E. hirta, four more compounds were isolated in addition to compounds we isolated previously (Tayone et al. 2014). The crude sample after soaking for 48 h was sequentially purified over silica gel column chromatography to afford the nine (1–9) compounds (Figure 1). Compounds 2, 6, 7, and 9 were not reported in our previous study. However, this is the first report of the isolation of 9 from E. hirta.



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Figure 1. Secondary metabolites isolated from E. hirta.

The 1H and 13C NMR data are summarized below. The spectral comparison of our data with those in literature had established their structures.

Anti-JEV ActivityTo screen antiviral compounds isolated from E. hirta, we used JEV-Chb (Goto et al., unpublished data), a recombinant JEV strain carrying a sequence encoding a reporter peptide HiBiT fused to the capsid structural protein (Schwinn et al. 2018). The effects of the nine isolated compounds on the growth of JEV-Chb were

examined. We used N2,N4-dibenzylquinazoline-2,4-diamine, a known inhibitor of JEV propagation (Tamura et al. 2018) as a positive control. JEV-Chb was inoculated onto cell culture supplemented with each compound and incubated for 24, 48, or 72 h. The amount of extracellular viral particles and intracellular pro-viral particles were estimated by measuring the HiBiT luciferase activities from culture supernatant and cell lysate, respectively. After the initial screening, we found that compounds 2, 6, and 8 inhibited the accumulation of JEV-Chb in culture supernatant in a dose-dependent manner (Figure 2). Compounds 6 and 8 also inhibited the accumulation of JEV-



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Figure 2. Dose-dependent effect of the isolated compounds on the accumulation of capsid in JEV-Chb-infected culture supernatant. HEK293T cells treated with compounds isolated from E. hirta were infected with JEV-Chb (MOI = 0.3). At 24, 48, or 72 hpi, HiBiT activity in culture supernatant were measured. Error bar represents the standard deviation (n = 3).

Chb in cells while 2 did not (Figure 3), suggesting that 2 affects viral assembly but not the protein production. These two compounds also showed significant cytotoxic effects (Figure 4), suggesting that the apparent inhibition of JEV-Chb propagation was a result of cell death. In conclusion, out of the nine compounds we tested, only 2 was able to successfully inhibit the propagation of JEV-Chb (Figure 3) without detectable cytotoxicity (Figure 4). The recombinant JEV-Chb strain was initially used in the anti-viral study for faster and easier detection in real-time fluorescent microscopy. After 2 showed activity in the preliminary screening against JEV-Chb, its anti-viral activity was then verified with the nonrecombinant or wildtype strain. Results

further confirmed a similar inhibitory effect of 2 on the propagation of a wildtype JEV strain at 100 µM (Figure 5). However, 2 is weaker compare to baicalein, a flavonoid with potent in vitro anti-JEV effects at all different stages of JEV infection (Johari et al. 2012).

In our experiments, 2 reduced the level of capsid in the culture supernatant (Figure 2) but not in the cells (Figure 3), suggesting that 2 affects assembly or secretion of the viral particle rather than viral protein production. Compound 2, being the most polar of the nine isolated metabolites, was the most active. Minor structural differences of 1 and 2 lead to significant differences in the ability to inhibit the production of the viral particle. Independent studies



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Figure 3. Dose-dependent effect of the isolated compounds on the accumulation of capsid in JEV-Chb-infected cells. HEK293T cells treated with compounds isolated from E. hirta were infected with JEV-Chb (MOI = 0.3). At 72 hpi, HiBiT activity in the cells were measured. Error bar represents the standard deviation (n = 3).



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Figure 4. Dose-dependent effect of the isolated compounds on cell viability. HEK293T cells were treated with compounds isolated from E. hirta for 72 h. Cell viability was evaluated using Cell Titer Glo 2.0 reagent (Promega). Error bar represents the standard deviation (n = 3).



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Figure 5. Effect of 2 on wildtype-JEV propagation and infected cell viability. HEK293T cells treated with 100 µM myricitrin were infected with WT-JEV (MOI = 0.3). At 48 hpi, JEV infectious titer in the culture supernatants was measured by performing focus forming assay (left panel), and cell viability was evaluated using Cell Titer Glo 2.0 reagent (right panel). Error bar represents the standard deviation (n = 3).

have demonstrated that 2 has an antioxidative activity (Lei 2017; Domitrović et al. 2015). Other studies also showed that flavivirus infection induces oxidative stress. This oxidative stress resulted in the intracellular accumulation of reactive oxygen species (ROS), and a decrease in ROS levels through chemical or genetic inhibition weakened the innate immune responses to flavivirus infection and facilitated flavivirus replication. On the other hand, cells infected with flavivirus induce oxidative-stress responses to control the antiviral, inflammatory, and anti-apoptotic properties by inducing antioxidant gene expression (Valadão et al. 2016; Zhang et al. 2014).

CONCLUSIONTaken all together into account, myricitrin showed inhibition of JEV at 100 µM. A more thorough study is needed on its cellular antiviral stress response mechanisms and in vivo investigation to demonstrate myricitrin’s potential. Moreover, further investigations are crucial in understanding and establishing at what stage (early or late) of the cycle does 2 inhibits JEV replication.

ACKNOWLEDGMENTSThis study was funded by the Japan Society for the Promotion of Science (JSPS). W.C. Tayone is a JSPS International Research Fellow (L18541).

STATEMENT ON CONFLICT OF INTEREST The authors declare no conflicts of interest.

NOTES ON APPENDICES The 1H, 13C, and other 2D NMR data of the isolated compounds are available as supporting information and accessible at http://philjournsci.dost.gov.ph.

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APPENDIX

Table I. NMR data of the compounds.

Compound 1H 13C COSY HSQC HMBC

quercetrin (1) 25 26

acetylated quercetrin 27 28 29 30 31

myricitrin (2) 32

taraxerone (3) 33

taraxerol (4) 34

β-sitosterol (5) 35

24-hydroperoxycycloart-25-en-3β-ol (6) 36 37 38

25-hydroperoxycycloart-23-en-3β-ol (7) 39 40 41

lupeol (8) 42

(23E)-cycloart-23-en-3β, 25-diol (9) 43 44 45

Figure I. 1H NMR of quercetrin (1) in CD3OD, 500 MHz.

Figure II. 13C NMR of quercetrin (1) in CD3OD, 125 MHz.

Figure III. 1H NMR of acetylated quercetrin in CDCl3, 500 MHz.

Figure IV. 13C NMR of acetylated quercetrin in CDCl3, 125 MHz.



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Figure V. COSY of acetylated quercetrin in CDCl3.

Figure VI. HSQC of acetylated quercetrin in CDCl3.

Figure VII. HMBC of acetylated quercetrin in CDCl3.

Figure VIII. 1H NMR of myricitrin (2) in CD3OD, 500 MHz.

Figure IX. 1H NMR of taraxerone (3) in CDCl3, 500 MHz.

Figure X. 1H NMR of taraxerol (4) in CDCl3, 500 MHz.

Figure XI. 1H NMR of β-sitosterol (5) in CDCl3, 500 MHz.

Figure XII. 1H NMR of 24-hydroperoxycycloart-25-en-3β-ol (6) in CDCl3, 500 MHz.



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Figure XVI. 13C NMR of 25-hydroperoxycycloart-23-en-3β-ol (7) in CDCl3, 125 MHz.

Figure XIII. 13C NMR of 24-hydroperoxycycloart-25-en-3β-ol (6) in CDCl3, 125 MHz.

Figure XIV. COSY of 24-hydroperoxycycloart-25-en-3β-ol (6) in CDCl3.

Figure XV. 1H NMR of 25-hydroperoxycycloart-23-en-3β-ol (7) in CDCl3, 500 MHz.

Figure XVII. COSY of 25-hydroperoxycycloart-23-en-3β-ol (7) in CDCl3.

Figure XVIII. 1H NMR of lupeol (8) in CDCl3, 500 MHz.

Figure XIX. 1H NMR of (23E)-cycloart-23-en-3β,25-diol (9) in CDCl3, 500 MHz.

Figure XX. 13C NMR of (23E)-cycloart-23-en-3β,25-diol (9) in CDCl3, 125 MHz.



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Figure XXI. COSY of (23E)-cycloart-23-en-3β,25-diol (9) in CDCl3.



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Anti-Japanese Encephalitis Virus (JEV) Activity of Triterpenes ......“tawa-tawa” Philippine Journal of Science 149 (3): 603-613, September 2020 ISSN 0031 - 7683 Date Received: