Triterpenoidal Saponins from the Leaves of Aster koraiensis Offer Inhibitory Activities against SARS-CoV-2

Triterpenoidal saponins have been reported to be able to restrain SARS-CoV-2 infection. To isolate antiviral compounds against SARS-CoV-2 from the leaves of Aster koraiensis, we conducted multiple steps of column chromatography. We isolated six triperpenoidal saponins from A. koraiensis leaves, including three unreported saponins. Their chemical structures were determined using HR-MS and NMR data analyses. Subsequently, we tested the isolates to assess their ability to impede the entry of the SARS-CoV-2 pseudovirus (pSARS-CoV-2) into ACE2+ H1299 cells and found that five of the six isolates displayed antiviral activity with an IC50 value below 10 μM. Notably, one unreported saponin, astersaponin J (1), blocks pSARS-CoV-2 in ACE2+ and ACE2/TMPRSS2+ cells with similar IC50 values (2.92 and 2.96 μM, respectively), without any significant toxic effect. Furthermore, our cell-to-cell fusion and SARS-CoV-2 Spike-ACE2 binding assays revealed that astersaponin J inhibits membrane fusion, thereby blocking both entry pathways of SARS-CoV-2 while leaving the interaction between the SARS-CoV-2 Spike and ACE2 unaffected. Overall, this study expands the list of antiviral saponins by introducing previously undescribed triterpenoidal saponins isolated from the leaves of A. koraiensis, thereby corroborating the potency of triterpenoid saponins in impeding SARS-CoV-2 infection.


Introduction
Despite advances in vaccines and treatments, the persistent COVID-19 pandemic and the emergence of more contagious SARS-CoV-2 variants highlight the critical need for safe and effective antiviral solutions [1].To date, research has increasingly concentrated on targeting membrane fusion, a crucial phase in SARS-CoV-2 infection, as a primary strategy to combat the virus [2][3][4].SARS-CoV-2 begins its infection process by the spike glycoprotein (S protein) binding to the human angiotensin-converting enzyme 2 (ACE2) receptor, enabling entry into cells.This entry is aided either by the TMPRSS2 enzyme or through the mechanism of endocytosis [5,6].After infiltrating inside the host cell, SARS-CoV-2 utilizes the cellular machinery to replicate, translating its RNA into essential nonstructural and structural proteins for viral assembly and proliferation [2].These intricate processes, from entry to replication, are influenced by host cell proteases and the virus's capacity to modify cellular functions [7].
Consequently, there has been a significant focus on discovering phytochemicals and herbal formulations that can inhibit these proteins' activities and their interactions with cellular membranes, thereby preventing viral entry [8,9].Studies reveal that phytochemicals can effectively target and disrupt the S-protein-mediated membrane fusion.For instance, curcumin, a diarylheptanoid from Curcuma longa, was found to impede SARS-CoV-2 entry in 293T/hACE/TMPRSS2 cells by blocking pseudovirus entry [10].Similarly, emodin, an anthraquinone from Rheum officinale, significantly reduces the interaction between the S-protein and ACE2 [11] Moreover, our research, in conjunction with others, has identified a link between the chemical structure of oleanane-type saponins and their effectiveness as inhibitors of membrane fusion affected by SARS-CoV-2 [12,13].
Given the antiviral properties displayed by the A. koraiensis leaf extract, we speculated on its potential to contain a variety of saponins other than astersaponin I. Thus, this study aimed to isolate saponins previously unreported in A. koraiensis leaves and assess their antiviral potency.Repeated chromatographic processes successfully isolated three unreported triterpenoid saponins (1-3) and three known ones (4-6) from the 95% ethanol extract.Subsequently, we assessed their effect on the entry of SARS-CoV-2.Our study encompasses the saponin isolation procedure, elucidation of the structure of previously unreported compounds, and analysis of how these isolates affect SARS-CoV-2 infection.

Astersaponin J Exhibits Comparable Inhibitory Activity against the Two SARS-CoV-2 Entry Pathways
We next employed SARS-CoV-2 pseudotyped viruses (pSARS-CoV-2) to assess the inhibitory activity of compounds derived from the leaves of A. koraiensis against SARS-CoV-2.This biosafety-level-2 pseudovirus incorporates the S-protein onto HIV-based lentiviral particles, providing an effective tool for evaluating viral entry and screening antiviral compounds [25].SARS-CoV-2 entry occurs through two distinct pathways: the endosomal pathway in ACE2-positive (ACE2 + ) H1299 cells and the TMPRSS2-mediated membrane fusion pathway in ACE2 and TMPRSS2 double-positive (ACE2/TMPRSS2 + ) H1299 cells.First, we investigated the effects of compounds 1-6 on pSARS-CoV-2 entry into ACE2positive cells.The pSARS-CoV-2 entry assay revealed that all the compounds except for 3 exhibited dose-dependent inhibition of pSARS-CoV-2 entry into ACE2 + H1299 with an IC 50 value of less than 10 µM (Figure 3A).Astersaponin I (4), reported as an inhibitor of infection with SARS-CoV-2 variants and syncytium formation in a previous report [13], showed the most potent inhibitory effect, with an observed IC 50 value of 1.46 µM in this study.Astersaponins J (1) and K (2) also exhibited strong antiviral effects, with IC 50 values of 2.92 and 3.16 µM.Astersaponin L (3) showed no antiviral activity until a concentration of 10 µM was reached (Figure 3A).Although less effective than astersaponin I (4), compounds

Astersaponin J Effectively Inhibits SARS-CoV-2 Entry by Blocking S-Protein-Mediated Viral Membrane Fusion
Based on our observation that astersaponin J (1) significantly inhibits both primary SARS-CoV-2 entry routes, we hypothesized that astersaponin J (1) may target the shared entry process, such as the membrane fusion between viral and host membranes.To study S-mediated membrane fusion as a representative model of viral entry, we used two stable cell lines: Spike-HEK293T cells, which overexpress S-protein with EGFP from a single bicistronic mRNA in HEK293T cells, and ACE2/TMPRSS2 + H1299 cells, which overexpress mRuby [13].Introducing Spike-HEK293 cells into a monolayer of ACE2/TMPRSS2 + H1299 cells swiftly triggers cell-to-cell fusion.The presence of S-proteins on the surface of the fused hybrid cells facilitates continuous merging with adjacent ACE2/TMPRSS2 + H1299 cells, resulting in the formation of multinucleated cells (Figure 4A).Flow cytometry analysis was conducted to assess the extent of cell-to-cell fusion after co-culturing these two cell types in a 1:10 ratio.This analysis indicated that about 8% of the total cells were double-positive for mRuby and GFP, implying that 80% of the Spike-HEK293 cells participated in the fusion process (Figure 4B).Saponins, known for their soap-like ability to form micelles, are amphiphilic, surfaceactive compounds with a wide range of pharmacological properties [26].These include hemolytic, insecticidal, anti-inflammatory, antitumor, antidiabetic, antifungal, anti-yeast, antibacterial, antiparasitic, antihyperlipidemic, and antioxidative effects [26].Particularly, triterpenoid saponins and their derivatives are noted as potential antiviral agents due to their ability to block virus-host recognition across various virus species [27].Since the antiviral effects of saikosaponins A, B 2 , C, and D have been reported as inhibitors of viral penetration into MRC-5 cells for human coronavirus (HCoV-229E), there has been significant research focusing on oleanane-type saponins and their role in blocking coronavirus membrane fusion [28].Consistent with previous findings, our study indicates that oligosaccharides with four or more sugars are likely to bind to the C28 of triterpenoidal saponins, thereby obstructing the entry of pSARS-CoV-2 into host cells [12,27,29,30].Next, we chose astersaponin J (1) to conduct a more detailed investigation, because this compound is a newly reported saponin and the most potent (except for astersaponin I, 4) in this study.The results of the pSARS-CoV-2 entry assay further demonstrated that astersaponin J (1) exhibits comparable efficiency in inhibiting pSARS-CoV-2 entry into ACE2/TMPRSS2 + H1299 and ACE2 + H1299 cells, with an IC 50 value of 2.96 µM (Figure 3B).Importantly, treatment with astersaponin J (1) up to a concentration of 10 µM did not result in cytotoxicity in H1299 cells (Figure 3C).

Astersaponin J Effectively Inhibits SARS-CoV-2 Entry by Blocking S-Protein-Mediated Viral Membrane Fusion
Based on our observation that astersaponin J (1) significantly inhibits both primary SARS-CoV-2 entry routes, we hypothesized that astersaponin J (1) may target the shared entry process, such as the membrane fusion between viral and host membranes.To study S-mediated membrane fusion as a representative model of viral entry, we used two stable cell lines: Spike-HEK293T cells, which overexpress S-protein with EGFP from a single bicistronic mRNA in HEK293T cells, and ACE2/TMPRSS2 + H1299 cells, which overexpress mRuby [13].Introducing Spike-HEK293 cells into a monolayer of ACE2/TMPRSS2 + H1299 cells swiftly triggers cell-to-cell fusion.The presence of S-proteins on the surface of the fused hybrid cells facilitates continuous merging with adjacent ACE2/TMPRSS2 + H1299 cells, resulting in the formation of multinucleated cells (Figure 4A).Flow cytometry analysis was conducted to assess the extent of cell-to-cell fusion after co-culturing these two cell types in a 1:10 ratio.This analysis indicated that about 8% of the total cells were doublepositive for mRuby and GFP, implying that 80% of the Spike-HEK293 cells participated in the fusion process (Figure 4B).
In contrast, when control GFP-HEK293 cells (lacking S-protein) were co-cultured with ACE2/TMPRSS2 + cells, double-positive cells were barely detected, highlighting the requirement of S-protein for cell-to-cell fusion (Figure 4B).To assess the potential antifusion activity of astersaponin J (1), we pre-treated the cells with varying concentrations of astersaponin J (1) for 1 h before the addition of Spike-HEK293 cells.Remarkably, at a concentration of 20 µM, astersaponin J (1) demonstrated a robust blockade of the fusion event.Only 24% of the cells participated in the generation of cell fusion hybrids, whereas 76% of the cells did not engage in fusion (Figure 4C).Due to the significant size difference between HEK293T cells and viral particles, the fusion efficiency between HEK293T cells and H1299 cells was much higher than between viral particles and H1299 cells.As a result, a higher concentration of astersaponin J (1) was needed to block this fusion process compared to that required to inhibit viral fusion (Figures 3A and 4C).Next, we evaluated the effect of astersaponin J (1) on the interaction between the SARS-CoV-2 S protein and ACE2.To assess this, we used a recombinant protein comprising the receptor-binding domain (RBD) of the S-protein fused with GFP (S-RBD-GFP) and introduced it into ACE2 + H1299 cells (Figure 4D).Flow cytometry analysis revealed that more than 97% of the cells exhibited binding of S-RBD-GFP (Figure 4E), indicating the attachment of the SARS-CoV-2 S protein to the cellular receptor required for viral entry.Notably, when ACE2 + H1299 cells were pre-treated with astersaponin J (1), there was no notable impact on the binding of S-RBD-GFP to ACE2 within the applied concentration range, although a minor peak shift was observed at 20 µM in the binding profile.These findings indicate that astersaponin J (1) specifically disrupts the S-protein-mediated membrane fusion process without altering the protein-protein interaction between the S-protein and ACE2.This distinct mechanism of action contributes to effectively inhibiting both major entry pathways of SARS-CoV-2.In this process, a cell suspension of Spike+ HEK293T cells, which stably express GFP, is added to a monolayer of ACE2/TMPRSS2 + H1299 cells, which exhibit mRuby fluorescence.This interaction between the two cell types results in cell-to-cell fusion, generating multinuclear cells.(B) The number of cells that were double-positive for GFP and mRuby, indicating cell-to-cell fusion, was quantified using flow cytometry.A control experiment was conducted using HEK293T cells expressing only GFP (without the spike protein).(C) ACE2/TMPRSS2 + H1299 cells were pre-treated with the indicated concentrations of 1 for 1 h prior to adding Spike+ HEK293T cells, followed by flow cytometry.The result are presented as representative data from triplicates.(D) A diagram depicting the interaction between the SARS-CoV-2 spike receptor binding domain (RBD) fused to the GFP (S-RBD-GFP) and ACE2 protein, which is overexpressed in H1299 cells.(E) The effect of 1 on the interaction between S-RBD-GFP and ACE2 on the surface of H1299 cells, observed by flow cytometry after treatment with the predetermined concentrations of 1 for 1 h.The grey peaks in the flow cytometry graphs represent control experiments conducted without the addition of RBD-GFP.

General Experimental Procedures
The general experimental procedures are provided in Supplementary Data.

Plant Material
The A. koraiensis leaves were collected in Pyeongchang, Republic of Korea, in 2017.The plant's origin was verified by Prof. Dae Sik Jang (D.S.J.), and a reference sample (ASKO1-2017) has been stored at the College of Pharmacy, Kyung Hee University, Republic of Korea.

Absolute Configurations of Sugars
To perform the absolute structural analysis of sugars, acid hydrolysis was conducted.Each sample (1-3; 3.0 mg) was diluted in 2 M D 2 SO 4 and heated at 60 • C for 3 h.Subsequently, the reaction crude was concentrated and partitioned with H 2 O and BuOH.The water-soluble fraction was dissolved in a solution containing 10 mg/mL L-cysteine methyl ester and pyridine, and the reaction was conducted at 60 • C in a water bath for 1 h.Then, 20.0 µL of o-tolyl isothiocyanate was added for the consequent reaction at 60 • C for 1 h.The identification of sugars was performed using LC-ESI-MS.
A Thermo Hypersil GOLD C18 column (Thermo Fisher Scientific, Waltham, MA, USA) was employed to separate the derivatized mixture at 35 • C for 20 min under isocratic conditions with a mobile phase consisting of 0.5% formic acid (FA) in H 2 O and 0.5% FA in acetonitril in a ratio of 80:20.The determination of L-arabinose (t R 9.0 min), D-glucose (t R 11.0 min), D-xylose (t R 12.2 min), L-rhamnose (t R 18.3 min), and D-apiose (t R 16.8 min) was achieved by comparing their retention times (t R ) and full MS spectrum with authentic standards (Figures S20-S23).

Cell Culture
H1299 cells (KCLB, Seoul, Republic of Korea, 25803) were acquired from the Korean Cell Line Bank and maintained in RPMI 1640 medium (Gibco, Grand Island, NY, USA).HEK293T cells (CRL-3216) were purchased from the American Type Culture Collection and cultured in Dulbecco's Modified Eagle's Medium (DMEM; Corning, Glendale, AZ, USA).Both cell lines were maintained with 10% fetal bovine serum (FBS) (Gibco, Grand Island, NY, USA) and 1× penicillin-streptomycin solution (HyClone, Logan, UT, USA).The cells were cultured at 37 • C in a humidified incubator with 5% CO 2 .

Cell Viability Assay
H1299 cells were plated in a 96-well plate at a density of 5 × 10 3 cells per well.The following day, the cells were treated with various concentrations of astersaponin J as indicated.After 24 h of incubation, WST-8 solution (Biomax, Guri-si, Gyeonggi-do, Republic of Korea) was added to each well and incubated for 2 h at 37 • C in a CO 2 incubator.The absorbance of each well was measured at 450 nm using a SpectraMax iD5 Multi-Mode Microplate Reader (Molecular Devices, San Jose, CA, USA).To determine the 50% cytotoxic concentration (CC 50 ), the mean absorbance values of the drug-treated samples were compared to those of cells treated with DMSO, and the ratio between the two was calculated.

SARS-CoV-2 S-Pseudotyped Lentivirus Production and pSARS-CoV-2 Entry Assay
To generate SARS-CoV-2 S-pseudotyped lentiviruses (pSARS-CoV-2), we employed a second-generation lentiviral packing system following a previously described method [13].Briefly, HEK293T cells were transfected with a lentiviral plasmid containing the fly luciferase gene, psPAX2 packing plasmid, and SARS-CoV-2 S plasmid using Lipofectamine 3000 transfection reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions.Culture supernatants were collected at 24 h and 48 h post-transfection, centrifuged to remove cellular debris, and stored at 4 • C until further use.For the pSARS-CoV-2 entry assay, supernatants containing pSARS-CoV-2 virus particles were added to ACE2 + and ACE2/TMPRSS2 + H1299 cells, along with each compound.Following 24 h of incubation, the efficiency of viral entry was measured by quantifying the firefly luciferase activity in the cell lysates using a luciferase assay system (Promega, Madison, WI, USA) and SpectraMax iD5 Multi-Mode Microplate Reader (Molecular Devices, San Jose, CA, USA).The dose-response curves were created utilizing Prism v.9.0.0 software (GraphPad, CA, USA).The IC 50 and CC 50 values were determined through nonlinear regression analysis, employing the log of inhibitor concentration versus response using a variable slope equation: Y = bottom + (top − bottom)/(1 + 10[(logIC50 − X) HillSlope ]).All IC 50 and CC 50 values were obtained in triplicate measurements.
3.9.Cell-to-Cell Fusion Assay ACE2/TMPRSS2 + H1299 cells co-expressing mRuby2 were seeded in a 12-well plate at a density of 2 × 10 5 cells per well and allowed to adhere overnight.The following day, the cell cultures were treated with either DMSO or the indicated concentration of astersaponin J for 1 h.Subsequently, Spike + HEK293T cells co-expressing GFP were added to the wells at a density of 2 × 10 4 cells.After incubation for 1 h, the co-cultures were harvested using trypsin-EDTA (Gibco, USA).Flow cytometry analysis was performed using an LSRFortessa™ flow cytometer (BD Biosciences, San Jose, CA, USA) to determine the percentage of cells that were double-positive for GFP and mRuby, indicating cell-to-cell fusion.A total of 1 × 10 4 cells were analyzed, and the data were analyzed using FlowJo (v9) software (Tree Star Inc, Ashland, OR, USA).

Figure 4 .
Figure 4. Astersaponin J (1) hinders SARS-CoV-2 envelope fusion with the host cell membrane without affecting the S-ACE2 interaction: (A) Schematic illustration depicting the process of cell fusion mediated by the SARS-CoV-2 S protein.In this process, a cell suspension of Spike+ HEK293T cells, which stably express GFP, is added to a monolayer of ACE2/TMPRSS2 + H1299 cells, which exhibit mRuby fluorescence.This interaction between the two cell types results in cell-to-cell fusion, generating multinuclear cells.(B) The number of cells that were double-positive for GFP and mRuby, indicating cell-to-cell fusion, was quantified using flow cytometry.A control experiment was conducted using HEK293T cells expressing only GFP (without the spike protein).(C) ACE2/TMPRSS2 + H1299 cells were pre-treated with the indicated concentrations of 1 for 1 h prior to adding Spike+ HEK293T cells, followed by flow cytometry.The result are presented as representative data from triplicates.(D) A diagram depicting the interaction between the SARS-CoV-2 spike receptor binding domain (RBD) fused to the GFP (S-RBD-GFP) and ACE2 protein, which is overexpressed in H1299 cells.(E) The effect of 1 on the interaction between S-RBD-GFP and ACE2 on the surface of H1299 cells, observed by flow cytometry after treatment with the predetermined concentrations of 1 for 1 h.The grey peaks in the flow cytometry graphs represent control experiments conducted without the addition of RBD-GFP.

Aglycon Position Sugar δ H Multi (J in Hz) δ C δ H Multi (J in Hz) δ C
a Overlapped signals.
a Overlapped signals.
a Overlapped signals.