
==== Front
Heliyon
Heliyon
Heliyon
2405-8440
Elsevier

S2405-8440(24)12599-6
10.1016/j.heliyon.2024.e36568
e36568
Research Article
Amentoflavone from Selaginella tamariscina inhibits SARS-CoV-2 RNA-dependent RNA polymerase
Youn Kyoung Won a1
Lee Siyun a1
Kim Jang Hoon b
Park Yea-In a
So Jaeyeon a
Kim Chansoo a
Cho Chong Woon c
Park Junsoo junsoo@yonsei.ac.kr
a⁎
a Division of Biological Science and Technology, Yonsei University, Wonju, 26493, Republic of Korea
b Department of Herbal Crop Research, National Institute of Horticultural & Herbal Science, RDA, Eumsung, 27709, Republic of Korea
c College of Pharmacy, Chungnam National University, Daejeon, Republic of Korea
⁎ Corresponding author. Yonsei University, Division of Biological Sciences and Technology, 1 Yonseidae-gil, Wonju City, Kangwon Province, 26493, Republic of Korea. junsoo@yonsei.ac.kr
1 These authors contributed equally to this work.

20 8 2024
30 8 2024
20 8 2024
10 16 e3656814 2 2024
14 8 2024
19 8 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
The SARS-CoV-2 pandemic caused millions of deaths due to its prominent infectivity and mortality. Although the vaccines and medicines for SARS-CoV-2 are on the market, new coronavirus variants like influenza are expected to reemerge continuously. Therefore, effective and inexpensive medicines will be required to respond to SARS-CoV-2 variants. Here, we used herbal plant extracts to search for effective compounds that can interfere with SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) and found that Selaginella tamariscina extract (STE) can reduce SARS-CoV-2 RdRp activity. The HCoV-OC43 beta coronavirus model was used to examine whether STE treatment could inhibit coronavirus replication and reduce coronavirus-induced cytotoxicity. Next, we searched the active compound of STE and found that amentoflavone is the main active compound of STE. Finally, we demonstrated that amentoflavone inhibits SARS-CoV-2 RdRp and coronavirus replication. Our results collectively indicate that amentoflavone from STE is possibly beneficial in responding to coronavirus-related diseases, including SARS-CoV-2.

Highlights

• Selaginella tamariscina extract (STE) inhibits SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) activity.

• STE treatment inhibits coronavirus replication.

• Amentoflavone, a central component of STE, inhibits SARS-CoV-2 RdRp activity.

Keywords

Coronavirus
Plant
Selaginella tamariscina
Amentoflavone
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pmc1 Introduction

The latest coronavirus disease, COVID-19, has high infectivity and mortality, and its worldwide prevalence has resulted in millions of deaths. SARS-CoV-2 is an RNA virus, and the RNA virus tends to evolve into new mutant forms due to its low fidelity of RNA polymerase [1,2]. Therefore, novel forms of coronavirus will be continuously prevalent, like the influenza virus [3]. Moreover, approximately 15–30 % of common colds are associated with human coronavirus infection [4,5]. Although vaccines and medicines for SARS-CoV-2 are on the market, effective and inexpensive coronavirus therapy will be required continuously to treat coronavirus-related diseases, including SARS-CoV-2.

Viral enzymes such as polymerase and protease are typical therapeutic targets, and many viral enzyme inhibitors are developed to treat virus-related diseases [6,7]. For SARS-CoV-2 medicines, Paxlovid was developed as the inhibitor of 3CL-protease, and molnupiravir was developed as the inhibitor of coronavirus RNA-dependent RNA polymerase (RdRp) [8]. Although their efficacy and safety are approved, significant adverse effects have been reported [9]. In addition, ribavirin was developed for a wide range of RNA viruses by inducing a high mutation rate. However, recent reports showed that ribavirin was ineffective in treating coronavirus diseases.

In Asian countries, Selaginella tamariscina has been used to treat metabolic disorders, inflammatory diseases, and cancers [[10], [11], [12]]. Amentoflavone and sellaginellin are identified as bioactive compounds responsible for these activities [[13], [14], [15]]. Recent reports showed that amentoflavone inhibits SARS-CoV-2 3CL-protease, and amentoflavone also inhibits several other targets of SARS-CoV-2 [[16], [17], [18], [19]]. In addition, amentoflavone was reported to inhibit Dengue virus RNA polymerase (RdRp) [20,21]. However, the inhibitory effects of amentoflavone on SARS-CoV-2 RdRp have not been experimentally proved.

In this report, we searched for natural compounds that inhibit SARS-CoV-2 RdRp activity and demonstrated that Selaginella tamariscina extract (STE) inhibits SARS-CoV-2 RdRp activity. We attempted to discover the active compounds in STE and found that amentoflavone, the main active compound in STE, inhibits SARS-CoV-2 RdRp activity.

2 Materials and methods

2.1 Preparation of STE and HPLC analysis

Dried Selaginella tamariscina whole plant (4 g) was extracted with ethanol (40 mL, 3 h) two times at 40 °C with sonication equipment. Selaginella tamariscina extract (STE) was filtered and concentrated under reduced pressure to obtain 555.3 mg (yield 13.9 %).

Qualitative and quantitative analysis of extract (2.5 mg/mL) and amentoflavone (6.3–100 μg/mL) were carried out on Zarbax Eclips Plus C-18 column (Agilent, Santa Clara, CA, USA) by gradient solvent system of 0.1 % formic acid in water and 0.1 % formic acid in acetonitrile (0 min at 10 % A, 25 min at 40 % A, 50 min at 100 % A, 55 min at 100 % A, 55.1 min at 10 % A, 65 min 10 % A). The injection volume for each run was 10 μL, and the flow rate used in HPLC was 1 mL/min. Their signals were detected at 268 nm.

2.2 Coronavirus infection

For coronavirus infection, we used the HCoV-OC43 human coronavirus strain from ATCC (Rockville, MD, USA). A Human Rhabdomyosarcoma (RD) cell line (KCLB, Seoul, Korea) was used for coronavirus infection, and RD cells were cultivated in DMEM medium (Welgene, Seoul, Korea) supplemented with 10 % FBS (Thermo Fisher Scientific, Waltham, MA, USA) and 1 % antibiotic-antimycotic (Welgene). Coronavirus infection was performed at a MOI of 0.01, as described previously [22]. STE and amentoflavone were solubilized with DMSO, and an equal amount of DMSO was used as a negative control. Cells were infected with the denoted concentration of STE or amentoflavone at the same time. MTT assay was used to measure cell viability [23]. We purchased MTT from USB Corporation (Cleveland, OH, USA) and amentoflavone from Cayman (Ann Arbor, MI, USA).

2.3 RNA-dependent RNA polymerase (RdRp) assay and 3CL-protease assay

We used a SARS-CoV-2 RNA polymerase assay kit from ProFoldin (Hudson, MA, USA) to evaluate SARS-CoV-2 RdRp activity. SARS-CoV-2 3CL-protease assay was performed as previously described [24,25]. Indicated concentrations of STE or amentoflavone were added for the in vitro assay, and an equal amount of DMSO was used as a negative control.

2.4 Plaque formation assay

We used the plaque formation assay to estimate the virus titer. The virus sample was serially diluted 10-fold in MEM, added to the 12-plate wells, and cultivated for 1 h at 33 °C and 5 % CO2. The plate was overlayed with the cell culture medium containing 0.6 % agarose. After incubation, the infected cells were fixed with a 4 % paraformaldehyde solution. Plaques were visualized by staining with 0.2 % crystal violet. We determined the virus titer in the conditioned media, and the HCoV-OC43 titer was 9 × 105 PFU/mL.

2.5 Quantitative reverse transcription polymerase chain reaction (qRT-PCR)

qRT-PCR was utilized to estimate the titer of coronavirus RNA, as previously described [26]. Briefly, samples were prepared, and total RNA was purified by using Trizol method (Thermo Fisher Scientific). cDNA for qRT-PCR was prepared, and the relative amounts were evaluated utilizing the Step One Plus RT-PCR System (Thermo Fisher Scientific). The primer information of HCoV-OC43 membrane protein (M), nucleoprotein (N), and RdRp was described in the previous literature [26].

2.6 Western blotting

We used the Western blotting method to examine the expression of coronavirus protein. We collected cells and conditioned media and solubilized them in cell lysis buffer as described previously [26,27]. Anti-HCoV-OC43 antibody (Sigma-Aldrich) was utilized to detect the viral proteins in cells and conditioned media. ChemiDoc Imaging System (Bio-Rad) was used to acquire the Western blot image.

2.7 Statistical analysis

To judge the statistical significance, we used a two-tailed Student's t-test utilizing the Microsoft Excel program (Redmond, WA, USA). For the computation of the half maximal inhibitory concentration (IC50), we used the AAT Bioquest program (Web address, https://www.aatbio.com/tools/ic50-calculator).

3 Results

3.1 Selaginella tamariscina extract inhibits SARS-CoV-2 RNA-dependent RNA polymerase activity

RNA-dependent RNA polymerase (RdRp) is a typical target of antivirals. We aimed to find the potential chemicals to inhibit SARS-CoV-2 RdRp and used the herbal plant extracts to examine the inhibitory activity against SARS-CoV-2 RdRp. We used the SARS-CoV-2 RdRp assay kit and various herbal plant extracts and found that Selaginella tamariscina extract (STE) potentially inhibits the SARS-CoV-2 RdRp (data not shown). To confirm the screening result, we repeated the RdRp assay and found that STE treatment inhibits SARS-CoV-2 RdRp in a dose-dependent manner (Fig. 1A). The IC50 of STE for SARS-CoV-2 RdRp was 98.04 μg/mL (Fig. 1B). These results indicate that STE contains a potential chemical that inhibits the SARS-CoV-2 RdRp activity.Fig. 1 STE inhibits SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) assay. (A) STE inhibits SARS-CoV-2 RdRp activity in a dose-dependent manner. To examine the inhibitory effect, SARS-CoV-2 RdRp assay kit was used, and the RdRp assay was performed in triplicate. The graph shows the mean and standard errors. Control vs. STE treatment, **: p < 0.005, ***: p < 0.001. (B) IC50 of STE was computed and shown in the graph. (C) STE treatment decreases coronavirus-induced cytotoxicity. RD cells were infected with either mock or coronavirus, and the infected cells were treated with the denoted concentration of STE. Seventy-two hours after infection, cell viability was examined with MTT assay. The MTT assay was carried out in triplicate. Means and standard errors are shown in the graph. Control vs. STE treatment, **: p < 0.005, ***: p < 0.001. (D) Coronavirus-infected cells with STE treatment were examined by a light microscope. RD cells were infected with either mock or coronavirus and cultivated with the denoted concentration of STE. Seventy-two hours after infection, the images were obtained by the inverted microscope, scale bar, 10 μm.

Fig. 1

Coronavirus infection results in cytopathic effects by inducing cell death [26]. Because STE treatment interferes with coronavirus replication, we examined whether STE treatment ameliorates the coronavirus-induced cytopathic effects. We utilized the human coronavirus OC43 strain, which belongs to the human beta coronavirus family. While coronavirus infection dramatically decreases cell viability, STE treatment decreases the coronavirus-induced cytotoxicity in a dose-dependent manner (Fig. 1C and D). These experimental results indicate that STE treatment inhibits coronavirus-induced cytopathic effect.

3.2 STE treatment inhibits coronavirus replication

Because STE treatment inhibits SARS-CoV-2 RdRp activity, we hypothesized that STE interferes with coronavirus replication. We evaluate the inhibitory effect of coronavirus (HCoV-OC43) replication by STE treatment. Human RD cells were used for coronavirus infection, and the level of coronavirus replication was evaluated by Western blot using an anti-HCoV-OC43 antibody. While STE treatment did not significantly reduce the expression level of coronavirus protein in the cell lysate (data not shown), STE treatment decreased the viral protein in the conditioned media significantly (Fig. 2A and B). The Western blot results suggest that STE treatment inhibits coronavirus production and release in the infected cells.Fig. 2 STE treatment interferes with coronavirus replication. (A) STE treatment lowers the expression of coronavirus proteins. RD cells were infected with coronavirus (10−3 dilution of conditioned media) and cultivated with the denoted concentration of STE. Three days after the coronavirus infection, the conditioned media were collected and examined using a Western blot assay. (B) The level of coronavirus proteins was quantified and shown in a graph. Control vs. STE treatment, *: p < 0.05 (C) RD cells were infected with coronavirus (10−3 dilution of conditioned media), and we evaluated the levels of M, N, and RdRp gene in the conditioned media by qRT-PCR. (D) Experimental design of plaque formation assay. (E) The STE treatment decreased the number of plaques in the medium.

Fig. 2

Next, we evaluated the expression of coronavirus RNA by qRT-PCR. We infected coronavirus into RD cells and examined whether STE treatment affects coronavirus RNA expression. We evaluated the RNA level of membrane protein (M), nucleoprotein (N), and RdRp genes in a conditioned media. qRT-PCR results showed that STE treatment decreases the level of coronavirus RNA in a dose-dependent manner (Fig. 2C). Because STE treatment reduces the viral protein and RNA in the conditioned media, we examined whether STE treatment results in decreased coronavirus in the conditioned media. The conditioned media were added to the cell, and we evaluated the infectious virus by plaque formation (Fig. 2D and E). STE-treated conditioned media results in decreased plaque formation, indicating that the conditioned media contains less infectious coronavirus. (Fig. 2E). These experiment results indicate that STE inhibits coronavirus replication.

3.3 Amentoflavone, a central component of STE, inhibits SARS-CoV-2 RdRp

According to the results above, STE showed an inhibitory effect on SARS-CoV2 RdRp and also inhibited the proliferation of HCoV-OC43 in cells. Since these efficacies of STE are related to its main component, we attempted to find the active compound in STE, and it was reported in the literature that amentoflavone is the main component in STE [13]. Qualitative analysis was carried out to determine the content of amentoflavone in STE. As shown in Fig. 3A, the primary signal of STE was 28.9 min and had the same retention time as amentoflavone. This compound was found to be present at 33.21 ± 0.02 mg in 1 g of STE (Fig. 3A and B). Next, we examined whether amentoflavone inhibits SARS-CoV-2 RdRp activity. Amentoflavone treatment decreases SARS-CoV-2 RdRp activity significantly (Fig. 3C and D). Previous in silico studies suggested that amentoflavone is a prospective inhibitor of the coronavirus 3CL-protease enzyme [19]. Thus, we examined whether amentoflavone treatment decreases SARS-CoV-2 3CL protease activity, and our results showed that amentoflavone decreased SARS-CoV-2 3CL protease activity (Fig. 3E and F). Amentoflavone IC50 for RdRp assay was much lower than IC50 for 3CL-protease (Fig. 3D and F). These results support that amentoflavone is more effective in inhibiting coronavirus RdRp activity.Fig. 3 Amentoflavone treatment inhibits SARS-CoV-2 RdRp activity. (A) The signals are amentoflavone (a) and extract (b) of STE, respectively. (B) The structure of amentoflavone. (C) Amentoflavone inhibits SARS-CoV-2 RdRp activity. (D) The IC50 of amentoflavone was computed and shown in the graph. (E) Amentoflavone inhibits the activity of SARS-CoV-2 3CL-protease. (F) The IC50 of amentoflavone was computed and shown in the graph.

Fig. 3

3.4 Amentoflavone treatment decreases the level of coronavirus protein

Because amentoflavone is a central component of STE, we expected that amentoflavone treatment would decrease virus protein expression in the infected cell. We examined whether amentoflavone treatment decreases the level of coronavirus proteins in the coronavirus-infected cells (Fig. 4A and B). Interestingly, STE treatment did not decrease the coronavirus expression in the infected cells. However, amentoflavone treatment reduced the coronavirus protein expression in the infected cells and conditioned media (Fig. 4A and B). These experimental results suggest that amentoflavone interferes with the coronavirus replication. We also examined the cytotoxicity of amentoflavone and found that a higher concentration of amentoflavone treatment showed a slight decrease in RD cell viability (Fig. 4C).Fig. 4 Amentoflavone treatment decreases coronavirus replication. (A, B) Amentoflavone decreases the expression of coronavirus proteins. RD cells were infected with either mock or coronavirus and treated with the denoted concentration of amentoflavone. Cell lysates and conditioned media were harvested and subjected to Western blot. (C) Cytotoxicity of amentoflavone was evaluated using an MTT assay. Control vs. amentoflavone: ∗p < 0.05.

Fig. 4

4 Discussion

In this report, we demonstrated that amentoflavone from Selaginella tamariscina can interfere with coronavirus RNA-dependent RNA polymerase (RdRp) activity and coronavirus replication. Initially, we used various herbal extracts to examine the inhibitory activity of RdRp and found that Selaginella tamariscina extract (STE) can inhibit RdRp activity. We also showed that STE can inhibit the replication of human coronavirus. Previous studies showed that STE contains amentoflavone as an active compound. We also showed that STE contains up to 3.3 % of amentoflavone in this study. Although STE's IC50 for RdRp activity was up to 100 μg/mL, the IC50 of amentoflavone is far lower than STE's IC50 (7.1 μg/mL (equal to 13.17 μM)) (Fig. 3). These experimental results indicate that amentoflavone is the active compound of STE.

RdRp is a typical antiviral target of RNA viruses, and several RdRp inhibitors for SARS-CoV-2 have already been developed and are on the market. However, RNA viruses produce resistant variants quickly due to their high mutation rate. Therefore, various RdRp inhibitors should be developed to protect against viruses with resistant variants. Amentoflavone can be one of the candidate antivirals for coronavirus, and amentoflavone can be tested to examine its inhibitory activity against other RNA viruses.

Previous reports showed that amentoflavone can inhibit the 3CL-protease of coronavirus [17,19]. 3CL-protease belongs to the typical antiviral targets of coronavirus, and several coronavirus inhibitors have been developed using 3CL-protease. We also examined whether amentoflavone can inhibit coronavirus 3CL-protease and found that amentoflavone can inhibit coronavirus 3CL-protease. However, the IC50 for 3CL-protease is more than 100 μM, whereas the IC50 for RdRp is 13.17 μM (Fig. 3). These results suggest that amentoflavone is more potent in inhibiting coronavirus RdRp than 3CL-protease. However, amentoflavone can inhibit RdRp and 3CL-protease simultaneously in the coronavirus-infected cells. Previous in silico studies predicted that amentoflavone inhibits viral enzymes by molecular docking [20,28]. Therefore, the molecular docking of amentoflavone with RdRp is one of the possible mechanisms to inhibit the coronavirus RdRp activity.

When we treated STE in the coronavirus-infected cells, we did not observe a decrease in coronavirus proteins in the infected cells (data not shown). However, we found that STE can inhibit the virus in the conditioned medium (infected cell medium) (Fig. 2A). The produced coronavirus will be released from the cells, and these proteins will be detected in the conditioned media. Therefore, these results suggest that STE treatment interferes with the release of coronavirus from the infected cells. Similarly, when we treated amentoflavone to the cells, the expression of coronavirus proteins in the conditioned medium was not detected upon 10 μM of amentoflavone treatment. However, the coronavirus protein expression in the cell lysates was detected upon 10 μM of amentoflavone treatment. These results suggest that amentoflavone treatment interferes with the release of coronavirus from the infected cells, similar to STE treatment.

Recent studies showed that SARS-CoV-2 replication mainly occurs in the respiratory organs and intestine [29,30]. Previous coronavirus studies with natural compounds like EGCG showed that high concentrations of EGCG, like micromolar concentration, are present in the intestines [31]. Although amentoflavone can be effective in micromolar concentration, amentoflavone can inhibit coronavirus replication in the intestines and ameliorate coronavirus-induced diarrhea or other intestinal symptoms. Moreover, we identified a single active compound, and more effective derivatives can be developed to inhibit coronavirus RdRp by modifying the structure of amentoflavone. Therefore, further research will be required to examine the efficacy of amentoflavone in vivo and to develop more active derivatives.

5 Conclusions

This study found that Selaginella tamariscina extract (STE) can inhibit SARS-CoV-2 RdRp activity. We also demonstrated that STE treatment inhibits coronavirus replication and decreases coronavirus-induced cytotoxicity. Amentoflavone is the main active compound of STE, and we demonstrated that amentoflavone inhibits SARS-CoV-2 RdRp activity and coronavirus replication. Amentoflavone can inhibit coronavirus replication at a much lower concentration than STE. Our results indicate that amentoflavone from Selaginella tamariscina is potentially helpful in treating SARS-CoV-2 and coronavirus-related diseases.

Funding

This study was supported by the Cooperative Research Program (RS-2022-RD010239 ) of the 10.13039/501100003627 Rural Development Administration , Republic of Korea.

Data availability statement

Data included in article/supp. material/referenced in article.

CRediT authorship contribution statement

Kyoung Won Youn: Writing – original draft, Investigation. Siyun Lee: Investigation. Jang Hoon Kim: Writing – original draft, Investigation. Yea-In Park: Investigation. Jaeyeon So: Investigation. Chansoo Kim: Investigation. Chong Woon Cho: Methodology. Junsoo Park: Writing – original draft, Funding acquisition.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A Supplementary data

The following is the supplementary data to this article:Multimedia component 1

Multimedia component 1

Acknowledgements

Professor Seo Young Yang at Kyungpook University kindly provided STE.

Appendix A Supplementary data to this article can be found online at https://doi.org/10.1016/j.heliyon.2024.e36568.
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