The leaves of T. nucifera were collected from Jeju Island, Republic of Korea, in October 2003. A voucher specimen of the plant material was deposited in the author’s laboratory for future reference and authentication purposes (Ryu et al., 2010).

African rhesus monkey kidney (MA-104) cells were procured from the American Type Culture Collection (ATCC CRL-2373.1; Manassas, VA, United States). The MA-104 cells were cultivated in Eagle’s minimum essential medium (EMEM) accompanied by a supplementation of 5% fetal bovine serum (FBS), 100 U/mL penicillin, 100 μg/mL streptomycin, and 100 U/mL amphotericin B (Park et al., 2006). The KJ-56 (bovine rotavirus, G8P[7]) and 85A (porcine rotavirus, G5P[7]) viruses isolated from fecal samples of diarrheic Korean calves and piglets were pre-activated with 10 μg/mL trypsin (1:250; GIBCO Invitrogen Corporation, California) for 30 min at 37°C before being inoculated into confluent MA-104 cells. Subsequently, the infected cells were sustained in EMEM with 1 μg/mL trypsin.

MA-104 cells (1 × 10⁵ cells/well) were seeded into 96-well plates and incubated for 24 h. The media were removed and substituted with EMEM containing serial dilutions of the extracts (1–200 μg/mL) or compounds (1–100 μM) dissolved in DMSO (with a final concentration at 0.5% DMSO). Control groups were cultured in the presence of 0.5% DMSO. After incubation for 72 h, the media were discarded, and 5 µL of MTT (3-[4,5-dimethylthiozol-2-yl]-2,5-diphenyltetrazolium bromide; Sigma, St. Louis, MO) solution was added to each well. After 4 h at 37°C, the supernatant was removed, and 100 µL of 0.04 M HCl-isopropanol was added to each well to dissolve formazan crystals. The absorbance of each well was assessed at 540 nm by using a microplate reader. After subtracting the background absorbance at 655 nm, the 50% cytotoxic concentration (CC₅₀) of each extract was estimated using regression analysis.

The antiviral assays employed in this study have been previously described (Bae et al., 2023), and observation of these assays was carried out using the neutral red method, as described briefly. In the virucidal assay, each extract and compound were mixed with a 0.01 multiplicity of infection (MOI) of rotaviruses at various concentrations (1–100 μg/mL or 1–30 μM) and incubated at 4°C for 1 h. The mixtures were incubated in triplicate onto near-confluent MA-104 cell monolayers for 1 h, accompanied by intermittent rocking. The solution was removed and the replaced by EMEM containing 1 μg/mL trypsin. The cells were incubated for 72 h at 37°C under 5% CO₂ atmosphere until the cells in the infected, untreated control showed complete viral CPE by light microscopy. In the post-treatment assay, rotaviruses at a 0.01 MOI were inoculated onto near-confluent MA-104 cell monolayers (1 × 10⁵ cells/well) for 1 h with occasional rocking. The solution was removed and replaced by EMEM containing 1 μg/mL trypsin and various concentrations (1–100 μg/mL or 1–30 μM) of each extract or compound dissolved in DMSO (with a final concentration at 0.5% DMSO) in triplicate. Infected/untreated and uninfected/untreated cells were cultured in the presence of 0.5% DMSO. The cells were incubated for 72 h at 37°C until cells in the infected/untreated group exhibited complete viral CPE as observed under light microscopy. Neutral red solution was added to each well at 0.034% (w/v), and the plates were incubated for 2 h at 37°C in the dark. After discarding the neutral red solution, the cells were washed with PBS (pH 7.4) and followed by the addition of a destaining solution (1% glacial acetic acid, 49% H₂O, and 50% ethanol). After being incubated in darkness for 15 min at room temperature, the absorbance of each well was at 540 nm using a microplate reader. The 50% effective concentration (EC₅₀) value was calculated by regression analysis. The selectivity index (SI) was calculated using the following formula: SI = 50% cytotoxic concentration (CC₅₀)/EC₅₀.

MA-104 cells were cultured until 90% confluence, infected with rotavirus at 0.01 MOI, and cultured with 18-hydroxyferruginol (1) and 18-oxoferruginol (2). Infected/untreated cells were cultured with 0.5% DMSO. After 6 and 18 h, the culture medium was removed, and the cells were detached by scraping. Subsequently, the cell washed twice with phosphate-buffered saline (PBS) and collected by centrifugation at 500× g for 3 min. Total RNA was extracted utilizing the Qiagen RNeasy Mini Kit (QIAGEN, Hilden, Germany) following the guidelines provided by the manufacturer. Specific primers were used for the reverse transcription of viral RNA (vRNA; NSP 3 gene, 5′-ACCATCTWCACRTRACCCTCTATGAG-3’ (upstream) and 5′-GGTCAC ATAACGCCCCTATAGC -3′ (downstream). GAPDH was used as an internal control for cellular RNAs, with the primer sequences 5′-TCA​ACA​GCG​ACA​CCC​ACT​C-3′ (upstream) and 5′-CTT​CCT​CTT​GTG​CTC​TTG​CTG-3′ (downstream). cDNA was synthesized from total RNA using the cDNA Master Mix (Applied Biosystems, CA, United States). qRT-PCR was conducted using 2 μL cDNA and Power SYBR Green PCR 2 × Master Mix (Applied Biosystems, CA, United States) according to the manufacturer’s recommended thermal cycling conditions (40 cycles of 15 s at 95°C and 60 s at 60°C). qRT-PCR was conducted using the Step One Plus Real-time PCR System and the data were analyzed using StepOne software v2.1 (Applied Biosystems, Foster City, CA, United States). Relative viral RNA expression levels were normalized by GAPDH gene according to the 2^-△Ct method, and expressed as a ratio of infected controls.

MA-104 cells were cultured on 8-well chamber slides (LAB-TEK, NUNC, United States), and the cells were infected with rotavirus at 0.01 MOI for 1 h. The supernatant was removed and replaced with EMEM containing 1 μg/mL trypsin and 25 μM of the compounds. The cells were incubated for 24 h at 37°C in a 5% CO₂ atmosphere, washed three times with PBS (pH 7.4), and fixed in 4% paraformaldehyde solution for 15 min at room temperature. After three washes with PBS (pH 7.4), the cells were incubated at 37°C for 1 h with a monoclonal antibody against rotavirus VP6 protein (Santa Cruz, California) diluted 1:50 in PBS (pH 7.4). After washing with PBS (pH 7.4), the cells were incubated at 37°C for 1 h with a FITC-conjugated goat anti-mouse IgG antibody (Santa Cruz) diluted 1:100 in PBS (pH 7.4). Cells were washed with PBS (pH 7.4), stained with 500 nM 4′,6-diamidino-2-phenylindole (DAPI) solution for 10 min at room temperature, and washed thrice with PBS (pH 8.0). Slides were mounted using the SlowFade Gold antifade reagent (Invitrogen, CA, United States). Confocal fluorescence imaging was performed using the Carl Zeiss LSM 510 META con-focal microscope (Carl Zeiss Inc., Jena, Germany). Confocal fluorescence imaging was quantified using ImageJ software 1.8.0.

All experiments were performed in triplicate. Data are expressed as mean ± SE. Statistical analysis was performed using Sigma Plot Statistical Analysis software. Differences between group mean values were determined using a one-way analysis of variance, followed by a Tukey’s test, assuming equal variances.

The structures of 18-hydroxyferruginol (1) and 18-oxoferruginol (2) are presented in Figure 1. Diterpenoid compounds were isolated from the ethanol extracts. The cytotoxicity of T. nucifera extracts and compounds was evaluated using the MTT assay at 50% cell cytotoxicity (CC₅₀). Confluent MA-104 cells were incubated with EMEM in the absence or presence of a two-fold diluted EtOH extract (1–200 μg/mL) or compounds (1–100 μM) for 72 h, and MTT reagents were added to the cells. The cytotoxicity of the extracts and compounds differed at different concentrations. The EtOH extract of T. nucifera showed a high CC₅₀ at 133.8 μg/mL (Table 1). The cytotoxicities of compounds 1 and 2 were determined as CC₅₀ values of 62.7 and 40.7 μM (Table 1). Hence, at concentrations of compounds 1 and 2 < 25 μM, no toxic effects were observed on MA-104 cells. Subsequent experiments designed to assess the antiviral efficacy of the compounds were conducted at minimally toxic concentrations, resulting in >95% cell viability.

Time-of-addition experiments were performed to determine the viral replication stage at which the compounds exerted their maximum inhibitory effects. Our findings showed that 18-hydroxyferruginol (1) demonstrated the most antiviral activity against KJ-56 (G8P[7]) (EC₅₀ = 24.7 μM, SI = 2.52) and 85A (G5P[7]) (EC₅₀ = 21.1 μM, SI = 2.95) in the post-treatment assay. Moreover, 18-oxoferruginol (2) exerted antiviral activity against all strains, with KJ-56 (G8P[7]) (EC₅₀ = 23.2 μM, SI = 1.75) and 85A (G5P[7]) (EC₅₀ = 22.6 μM, SI = 1.80) (Table 1). These results indicate that both compounds 1 and 2 can effectively inhibit rotavirus during the post-treatment stage.

As rotavirus viral RNA is synthesized in the early (6 h) and late (18 h) stages, we tested the effects of these compounds on both synthesis stages in infected cells using qRT-PCR. As shown in Figure 2, viral RNA levels (NSP3 gene) were decreased by 76.6% (KJ-56) and 63.15% (85A) by compound 1 and by 13.25% (KJ-56) and 15.91% (85A) by compound 2 at 6 h. We showed that rotavirus viral RNA levels were markedly decreased by 81.5%–86.3% by compound 1% and 77.2%–95.0% by compound 2 (25 μM) in all virus strains at 18 h compared with infected/untreated cells (0.5% DMSO) (Figure 2). These results indicate that compounds 1 and 2 exerted a stronger inhibitory effect against KJ-56 (Bovine, G8P[7]) and 85A (Porcine, G5P[7]) on the late stage of viral replication than on the early stage. Notably, we examined the intracellular trafficking of the rotavirus VP6 protein in virus-infected cells using immunofluorescence. The VP6 protein was found mainly to show green fluorescence in the cytoplasm of rotavirus-infected cells (Figures 3B, F) but not in mock-infected MA-104 cells (Figures 3A, E). However, cell treatment with 25 μM of compounds 1 (Figures 3C, G) or 2 (Figures 3D, H) after infection reduced the number of fluorescence-positive cells in the cytoplasm, indicating that these compounds disturbed viral protein production (Figure 3I).