Antiviral Activities of Ethyl Pheophorbides a and b Isolated from Aster pseudoglehnii against Influenza Viruses

Screening of the antiviral and virucidal activities of ethanol extracts from plants endemic to the Republic of Korea revealed the inhibitory activity of a 70% ethanol extract of the whole plant of A. pseudoglehnii (APE) against influenza virus infection. Two chlorophyll derivatives, ethyl pheophorbides a and b, isolated as active components of APE, exerted virucidal effects with no evident cytotoxicity. These compounds were effective only under conditions of direct incubation with the virus, and exerted no effects on the influenza A virus (IAV) surface glycoproteins hemagglutinin (HA) and neuraminidase (NA). Interestingly, virucidal activities of ethyl pheophorbides a and b were observed against enveloped but not non-enveloped viruses, suggesting that these compounds act by affecting the integrity of the viral membrane and reducing infectivity.


Introduction
Influenza viruses belong to the Orthomyxoviridae family and are divided into A, B, and C types [1]. Among these, influenza B virus (IBV) and influenza C virus (ICV) specifically infect humans, whereas influenza A virus (IAV) is a major pathogen responsible for global pandemics owing to its zoonotic transmission. Influenza viruses display pathogenicity as well as genetic and structural differences. However, similar structures for IAV and IBV have been reported [2,3]. Their genomes contain eight negative-sense, singlestranded RNA fragments encoding more than ten proteins [4]. The eight RNA fragments are surrounded by nucleocapsid protein (NP) and form a ribonucleoprotein (RNP) complex together with RNA polymerase complex proteins (PA, PB1, PB2). RNP is surrounded by matrix protein (M1) and the nucleocapsid is encapsulated by a cell membrane-derived phospholipid bilayer. Hemagglutinin (HA) and neuraminidase (NA), spike glycoproteins that determine pathogenicity, are located on the surface of the virus membrane, while ion channels span the membrane [5,6]. NA and HA proteins are divided into subtypes according to their structural and genetic characteristics. So far, 16 subtypes for HA and 9 subtypes for NA have been classified, among which HA1, 2, and 3 and NA1 and 2 are contributory factors to epidemic and pandemic outbreaks [4,7,8]. Recent studies have revealed H1N1 and H3N2 of IAV and IBV as causative agents of seasonal flu [9]. Other important viral proteins include nonstructural protein 1 (NS1) and nonstructural protein 2 (NS2). NS1 functions to evade the immune response of cells, and NS2 is a nuclear export protein (NEP) that plays a key role in nuclear release of RNP [10].
Influenza virus infects approximately 5 million patients, resulting in 250,000-500,000 deaths each year [8]. Over the past hundred years, four pandemics have occurred, specifically, H1N1

Virucidal Activity of APE against IAV
To examine the anti-influenza virus activity of APE, viruses and cells were subjected to a time-of-drug-addition assay as shown in Figure 1A. In the first method, IAV was treated with the extract for 1 h and Madin-Darby canine kidney (MDCK) cells were infected with the mixture. At 1 h after infection, the mixture was removed (pre-treatment). In the second method, IAV was treated with the extract and MDCK cells were immediately infected with the mixture. At 1 h after infection, the mixture was removed (adsorption). In the third group, MDCK cells were infected with IAV which was removed after 1 h. Cells were subsequently treated with the extract (post-infection). At 24 h after infection, viral RNA transcript levels were evaluated via qRT-PCR. DMSO treatment was conducted as a control. Treatment with the concentrations up to 100 µg/mL of APE exhibited no significant cytotoxic activity against MDCK cells. Compared to the control, IAV replication was significantly inhibited in the pre-treatment group ( Figure 1B), confirming its virucidal activity against IAV. Virucidal activity of APE against IAV. The 70% ethanol extract of A. pseudoglehnii was used to treat IAV at a concentration of 10 µg/mL using three protocols. (A) Schematic representation of the time-of-drug-addition assay. (B) After treatments, viral RNA transcript levels were determined via qRT-PCR using primers specific for IAV M1 and GAPDH. Experiments were performed in triplicate and data expressed as GAPDH-normalized values relative to control. The viral RNA transcript level in DMSO-treated cells was set as 100%. * p < 0.05 (Student's t-test). Pre, pre-treatment; Ads, Adsorption; Post, post-treatment.

Virucidal Activities of Ethyl Pheophorbides a and b against Influenza Virus
To determine the antiviral activities of ethyl pheophorbides a and b against IAV, viruses were treated with the compounds and experiments were performed as shown in Figure 1A. Amantadine, which inhibits the M2 ion channel and, in turn, virus uncoating, was used as a control. Similar to the results obtained with APE, IAV transcript levels were significantly reduced upon pre-treatment of the virus with both compounds, while amantadine treatment during virus adsorption to host cells induced a marked decrease in IAV replication ( Figure 3A). The IAV HA protein level was further determined with western blot analysis ( Figure 3B). Notably, expression of HA protein was significantly suppressed upon pre-treatment of viruses with ethyl pheophorbides a and b ( Figure 3B; lines 3 and 8). Amantadine treatment during adsorption also induced marked downregulation of HA protein ( Figure 3B; lines 13 and 14). Amantadine plays an important role in the fusion of viral and endosomal membranes during influenza virus entry into host cells, while ethyl pheophorbides a and b may affect virus infectivity before entry into host cells.  Figure 1A. (A) Viral RNA transcript levels were measured via qRT-PCR using primers specific for IAV M1 and GAPDH. Amantadine (Am), a M2 ion channel inhibitor (1 mM), was used as the control. Experiments were performed in triplicate and data expressed as GAPDH-normalized values relative to control. The viral RNA transcript level in DMSO-treated cells was set as 100%. * p < 0.05 (Student's t-test). (B) Equivalent amounts of whole cell extracts were subjected to western blot analysis with antibodies specific for IAV HA and tubulin. Pre, pre-treatment; Ads, Adsorption; Post, post-treatment.
To further ascertain whether ethyl pheophorbides a and b exhibit inhibitory effects against IBV, IAV and IBV were pre-treated with different concentrations of each compound for 1 h at room temperature, followed by infection of MDCK cells with the mixture containing IAV and IBV at MOI of 0.01 and 0.001, respectively. At 1 h after infection, the mixture was removed and viral titers were determined using the plaque reduction assay at 48 h after infection. Pre-treatment with ethyl pheophorbides a and b led to a significant reduction in the IAV and IBV plaque numbers ( Figure 4), with estimated 50% inhibitory concentrations (IC 50 ) of 0.086 ± 0.072 µg/mL and 0.094 ± 0.058 µg/mL for IAV ( Figure 4A), and 1.62 ± 0.86 µg/mL and 3.17 ± 0.94 µg/mL for IBV ( Figure 4B), respectively. To clarify whether the inhibitory activities of ethyl pheophorbides a and b are mediated through cytotoxicity, MDCK cells were treated with different concentrations of each compound and cell viability was determined after 48 h via the MTT assay ( Figure S7). Both ethyl pheophorbides exerted no adverse effects on cell viability up to a concentration of 10 µg/mL, indicative of virucidal activity against influenza viruses without cytotoxic effects.

Effects of Ethyl Pheophorbides a and b on Major Surface Glycoproteins of Influenza Virus
We further considered the possibility that ethyl pheophorbides a and b prevent viral binding to host cells by targeting the IAV major surface glycoproteins NA and HA. To explore whether ethyl pheophorbides a and b exert inhibitory effects on NA, IAV was treated with the compounds (1 µg/mL) and the NA inhibition assay was performed ( Figure 5A). Zanamivir, a NA inhibitor was used as the control and significantly suppressed NA activity, as expected. Notably, ethyl pheophorbides a and b exerted no effects on IAV NA.
Next, to ascertain whether the two ethyl pheophorbide compounds inhibit HA, two assays using chicken red blood cells (cRBC) were employed. HA is a dimer composed of HA1 and HA2. HA1 contains a domain that binds sialic acid of cells and HA2 interacts with the cell membrane via structural rearrangement induced by low pH during fusion of the viral and cell membranes [31]. First, to establish whether the compounds target HA1, IAV was treated with ethyl pheophorbides a and b (1 µg/mL) and the hemagglutination inhibition assay was performed ( Figure 5B). In blank samples devoid of IAV, no red blood cell aggregation was observed, whereas in samples of IAV treated with DMSO, ethyl pheophorbide a or ethyl pheophorbide b, the agglutination reaction occurred ( Figure 5B). The potential inhibitory effects of ethyl pheophorbides a and b on HA2 were examined using the hemolysis inhibition assay ( Figure 5C). To this end, cRBC (2%) was mixed with IAV treated with ethyl pheophorbides a and b (1 µg/mL), followed by treatment with sodium acetate-acetic acid (0.5 M, pH 5.2) to induce hemolysis via structural changes of HA2. Compared to the blank sample without IAV, relatively high absorbance was observed in samples of IAV treated with ethyl pheophorbide a or ethyl pheophorbide b ( Figure 5C). Overall, ethyl pheophorbides a and b had no effect on the functions of HA1 and HA2, clearly suggesting inhibitory activity on infectivity of influenza viruses via a mechanism that does not involve major viral surface glycoproteins.

Effects of Ethyl Pheophorbides a and b on Enveloped Viruses
To determine whether ethyl pheophorbides a and b reduce infectivity of influenza virus via effects on the viral membrane, other enveloped viruses including hepatitis C virus (HCV), human cytomegalovirus (HCMV), Japanese encephalitis virus (JEV), and non-enveloped adenovirus were treated with the compounds. At 1 h after treatment, viral infectivity was determined using plaque assays for HCMV, JEV and adenovirus, or immunofluorescence assay for HCV. Both ethyl pheophorbide compounds significantly reduced replication of HCV, JEV and HCMV ( Figure 6A-C). The IC 50 values of ethyl pheophorbide a against HCV, HCMV and JEV were 3.04 ± 1.69 µg/mL, 0.45 ± 0.26 µg/mL, and 8.66 ± 1.44 ng/mL, respectively. The IC 50 values of ethyl pheophorbide b against HCV, HCMV and JEV were 2.19 ± 1.12 µg/mL, 0.46 ± 0.30 µg/mL, and 10.17 ± 1.79 ng/mL, respectively. Ethyl pheophorbides a and b clearly inhibited all enveloped viruses but had no effect against non-enveloped adenovirus ( Figure 6D). Based on the collective results, we propose that ethyl pheophorbides a and b exhibit broad-range virucidal activities against enveloped viruses, possibly by affecting the integrity of the viral membrane.

Discussion
A. pseudoglehnii is an edible plant endemic to the Republic of Korea with low reported toxicity and multiple beneficial pharmacological properties. In this study, pre-treatment of IAV with APE induced a virucidal effect and the chlorophyll derivatives ethyl pheophorbides a and b were isolated as bioactive chemical constituents.
Chlorophyll is responsible for absorbing light during photosynthesis in plants. However, excessive photosynthesis could function as a photosensitizer and cause damage to cells. Therefore, synthesis and degradation of chlorophyll is an important process in preventing cytotoxicity, and various intermediates are generated during chlorophyll degradation [32]. Ethyl pheophorbides a and b are among the intermediates that act as a photosensitizers on human cancer cells, showing anti-tumor efficacy [33]. In this study, ethyl pheophorbides a and b exhibited broad virucidal activities against enveloped viruses upon direct treatment before infection of host cells, supporting the theory that ethyl pheophorbides a and b affect the viral membrane as photosensitizers.
Chemical compounds isolated from plant extracts with virucidal activity against enveloped viruses have been previously reported. For instance, curcumin inhibits the activity of enveloped viruses by damaging the viral membrane structure, leading to increased membrane permeability. Upon insertion of curcumin into the phospholipid bilayer of the membrane, its phenol ring interacts with the hydrogen bond site of the membrane and induces structural changes [34]. Ethyl pheophorbides a and b are proposed to have a similar mechanism of action as curcumin. In addition, chlorophyll derivatives similar in structure to ethyl pheophorbides were reported to reduce the infectivity of enveloped viruses by acting as photosensitizers to mediate physicochemical changes [35]. Notably, while the membrane of influenza virus is derived from the cell membrane, the compounds were not toxic to cells and showed specific activity against enveloped viruses, which should be explored in further studies.
In this study, IC 50 values of ethyl pheophorbide a against IAV and IBV were 0.085 and 1.02 µg/mL, respectively, and antiviral activity against IBV was relatively lower. The reasons underlying the distinct virucidal activities of ethyl pheophorbides a and b against different enveloped viruses require further investigation. Interestingly, however, examination of the anti-influenza virus activity of commercially purchased pheophorbide a devoid of an ethyl group revealed similar IC 50 values against IAV and IBV (data not shown). We could not effectively compare the virucidal activities of commercially synthesized compounds with those of isolates from plant extracts. To establish whether the ethyl group serves as a critical moiety, it is necessary to synthesize these compounds to the same levels of purity and compare their virucidal effects. supplemented with 10% FBS and 1X P/S. BHK21 cells were inoculated with JEV at 37 • C for 1 h, followed by the addition of DMEM containing 2% FBS and 1X P/S. After 48 h, JEV was obtained from the supernatant. Recombinant adenovirus expressing GFP was generated by inserting the GFP gene into an adenoviral vector. The GFP gene was amplified using pEF-GFP vector (Addgene #11154) as a template. The primer sequences used were 5 -GGGGACAAGTTTGTACAAAAAAGCAGGCTTTATGGTGAGCAAGGGCGAGGAGC TGT-3 (forward) and 5 -GGGGACCACTTTGTACAAGAAAGCTGGGTTTTACTTGTACA GCTCGTCCATGCCGA-3 (reverse), and PCR was performed using 5X HOT FIREPol ® Blend Master Mix (Solis Biodyne, Tartu, Estonia). The amplified GFP gene was cloned using the Gateway system (with Gateway™ BP Clonase™ II and Gateway™ LR Clonase™ II enzyme mixtures and pAd/CMV/V5-DEST™ Gateway™ Vector for insertion of the GFP gene (Invitrogen, MA, USA)), followed by transfection into 293A cells, as described previously [36]. Upon ≥50% cell death, which was determined by observing cytopathic effects (CPE), freeze-thawing at −80 • C and 37 • C was conducted three times to obtain adenovirus expressing GFP from the supernatant. Protocols for the maintenance and propagation of human cytomegalovirus (HCMV) Towne strain, hepatitis C virus (HCV, JFH-1), human hepatocyte (Huh7.5) and primary human foreskin fibroblasts cells (HFF) have been described previously [37,38]. Aster pseudoglehnii Y. Lim, J. O. Hyun, and H. Shin (Compositae) was provided by Hantaek Botanical Garden (Kyunggi-do, Republic of Korea) in May 2020 (voucher specimen #: HTS2021-0001).

Isolation of Ethyl Pheophorbides a and b from APE
Dried whole A. pseudoglehnii (966.5 g) plants were extracted twice with 70% EtOH for seven days at room temperature. The solvent was removed in vacuo to generate a 70% EtOH extract (136.07 g), which was suspended in 1 L of water and partitioned with n-hexane and n-butanol to obtain n-hexane-(8.38 g), n-butanol-(43.28 g), and water-soluble fractions (70.35 g), respectively. The n-hexane soluble fraction displayed the most potent activity in the anti-virus assay. Consequently, this fraction (7 g) was subjected to column chromatography (CC) (4.8 × 44.5 cm) using a silica gel (70-230 mesh) column and eluted with n-hexane-EtOAc (8:2 to 0:10, v/v) into ten subfractions (HE1-HE10). Among these subfractions, HE5 and HE6 showed significant anti-influenza virus activity.

Time-of-Drug-Addition Assay
Viruses and cells were subjected to three different treatments. In the first method, IAV was treated with the extract for 1 h at room temperature and Madin-Darby canine kidney (MDCK) cells were infected with the mixture at a multiplicity of infection (MOI) of 0.01. At 1 h after infection, the mixture was removed (pre-treatment). In the second method, IAV was treated with the extract at room temperature and MDCK cells were immediately infected with the mixture at a MOI of 0.01. At 1 h after infection, the mixture was removed (adsorption). In the third group, MDCK cells were infected with IAV at a MOI of 0.01, which was removed after 1 h. Cells were subsequently treated with the extract (post-infection). At 24 h after infection, cells were harvested to extract total RNA, and viral RNA transcript levels were evaluated via qRT-PCR.

Plaque Reduction Assay
IAV and IBV were treated with different concentrations of ethyl pheophorbide a or b and incubated at room temperature for 1 h. MDCK cells were infected with the mixtures and the supernatant was obtained at 48 h after infection. MDCK cells were infected with serially diluted supernatant, and after 1 h, incubated with a 1:1 mixture of 2X DMEM (2X DMEM, 0.4% BSA, 25 mM HEPES, 1X P/S) and 2% agarose containing 1 µg/mL TPCK-trypsin. Adenovirus, HCMV and JEV were treated with different concentrations of ethyl pheophorbide a or b (0-10 µg/mL) and the mixtures were incubated at room temperature for 1 h. BHK21 cells were infected with serially diluted JEV for 1 h, followed by incubation in medium (DMEM, 2% FBS, 1X P/S) containing 0.5% agarose. At 48 h after incubation, cells were fixed with 10% formaldehyde at room temperature and stained with 0.3% crystal violet. 293A cells were infected with serially diluted adenovirs for 6 h, followed by incubation in medium (DMEM, 2% FBS, 1X P/S) containing 0.4% agarose. At 48 h after infection, medium containing 0.4% agarose was added. At 4 to 6 days after infection, Thiazolyl Blue Tetrazolium Bromide (MTT; Sigma-Aldrich, St. Louis, MO, USA) solution was added to the medium at a concentration of 0.5 mg/mL and plaques were observed 1 h after incubation at 37 • C. The plaque assay for HCMV has been described in a previous report [39]. The number of plaques was counted and half-maximal inhibitory concentration (IC 50 ) values were calculated using GraphPad Prism 7 (GraphPad Software, San Diego, CA, USA).

Immunofluorescence Assay
Cells were fixed with a 1:1 mixture of methanol and acetone for 10 min at −20 • C, washed with 1X PBS, and blocked with 1% BSA at room temperature for 1 h. Subsequently, cells were incubated with primary antibody overnight at 4 • C, followed by secondary antibody at room temperature for 1 h. After staining of cells with 4,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA, USA), fluorescence was examined under a Nikon TS100-F fluorescence microscope (Tokyo, Japan) equipped with a digital camera. Fluorescence images were analyzed using Nikon NIS-Elements microscope imaging software. An antibody against HCV core protein was purchased from Anogen (Mississauga, ON, Canada). Alexa 488-labeled secondary antibody was purchased from Thermo Fisher Scientific (Waltham, MA, USA).

MTT Assay
MDCK cells were treated with different concentrations of ethyl pheophorbide a or b (0-10 µg/mL) for 48 h and subsequently incubated with 0.5 mg/mL MTT solution at 37 • C. After a 4 h period, tetrazolium crystals were dissolved with DMSO and absorbance was measured at 560 nm.

Western Blot Analysis
Cells were fractionated and transferred to nitrocellulose membrane as described previously [40]. Antibodies specific for IAV H1N1 HA and tubulin were purchased from Genetex (Irvine, CA, USA) and Sigma-Aldrich, respectively. A peroxidase-labeled antimouse/rabbit immunoglobulin G antibody was purchased from Jackson ImmunoResearch Laboratories (West Grove, PA, USA).

Hemagglutination Inhibition Assay
IAV was serially diluted using 1X PBS. After incubation with chicken red blood cells (cRBCs, 0.5%) for 1 h at 4 • C, aggregation was confirmed. The lowest virus titer causing aggregation was 1 HA unit (HAU). For the experiment, 4 HAU was used. IAV was treated with DMSO, ethyl pheophorbide a (1 µg/mL), or ethyl pheophorbide b (1 µg/mL) with shaking at room temperature for 45 min. Following the addition of cRBCs (0.5%), aggregation was confirmed after 1 h at 4 • C.