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Article

Screening of Medicinal Herbs Identifies Cimicifuga foetida and Its Bioactive Component Caffeic Acid as SARS-CoV-2 Entry Inhibitors

by
Ching-Hsuan Liu
1,2,†,
Yu-Ting Kuo
2,3,4,†,
Chien-Ju Lin
5,
Feng-Lin Yen
6,7,8,
Shu-Jing Wu
9 and
Liang-Tzung Lin
1,10,*
1
Department of Microbiology and Immunology, School of Medicine, College of Medicine, Taipei Medical University, Taipei 110, Taiwan
2
Department of Diagnostic Imaging, Chi Mei Medical Center, Tainan 710, Taiwan
3
Institute of Precision Medicine, College of Medicine, National Sun Yat-sen University, Kaohsiung 804, Taiwan
4
Department of Radiology, School and College of Medicine, Kaohsiung Medical University, Kaohsiung 807, Taiwan
5
School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Kaohsiung 807, Taiwan
6
Department of Fragrance and Cosmetic Science, College of Pharmacy, Kaohsiung Medical University, Kaohsiung 807, Taiwan
7
Drug Development and Value Creation Research Center, Kaohsiung Medical University, Kaohsiung 807, Taiwan
8
Department of Medical Research, Kaohsiung Medical University Hospital, Kaohsiung 807, Taiwan
9
Department of Nutritional Health, Chia-Nan University of Pharmacy and Science, Tainan 717, Taiwan
10
Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei 110, Taiwan
*
Author to whom correspondence should be addressed.
†
These authors share first authorship.
Viruses 2025, 17(8), 1086; https://doi.org/10.3390/v17081086
Submission received: 19 June 2025 / Revised: 28 July 2025 / Accepted: 1 August 2025 / Published: 5 August 2025
(This article belongs to the Section Coronaviruses)
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Abstract

The emergence of SARS-CoV-2 variants highlights the urgent need for novel therapeutic strategies, particularly entry inhibitors that could efficiently prevent viral infection. Medicinal herbs and herbal combination formulas have long been recognized for their effects in treating infectious diseases and their antiviral properties, thus providing abundant resources for the discovery of antiviral candidates. While many candidates have been suggested to have antiviral activity against SARS-CoV-2 infection, few have been validated for their mechanisms, including possible effects on viral entry. This study aimed to identify SARS-CoV-2 entry inhibitors from medicinal herbs and herbal formulas that are known for heat-clearing and detoxifying properties and/or antiviral activities. A SARS-CoV-2 pseudoparticle (SARS-CoV-2pp) system was used to assess mechanism-specific entry inhibition. Our results showed that the methanol extract of Anemarrhena asphodeloides rhizome, as well as the water extracts of Cimicifuga foetida rhizome, Xiao Chai Hu Tang (XCHT), and Sheng Ma Ge Gen Tang (SMGGT), have substantial inhibitory effects on the entry of SARS-CoV-2pps into host cells. Given the observation that Cimicifuga foetida exhibited the most potent inhibition and is a constituent of SMGGT, we further investigated the major compounds of the herb and identified caffeic acid as a bioactive component for blocking SARS-CoV-2pp entry. Entry inhibition of Cimicifuga foetida and caffeic acid was validated on both wild-type and the currently dominant JN.1 strain SARS-CoV-2pp systems. Moreover, caffeic acid was able to both inactivate the pseudoparticles and prevent their entry into pretreated host cells. The results support the traditional use of these herbal medicines and underscore their potential as valuable resources for identifying active compounds and developing therapeutic entry inhibitors for the management of COVID-19.

1. Introduction

Since its emergence in late 2019, SARS-CoV-2 has posed a significant global health threat, with over 777 million confirmed cases and over 7 million deaths reported as of December 2024 [1]. Although widespread vaccination has substantially mitigated disease severity and reduced COVID-19-related hospitalizations and mortality [2], the rapid evolution of SARS-CoV-2 has led to the emergence of multiple variants with increased transmissibility and immune evasion [3]. Studies have shown that vaccine effectiveness against infections and hospitalizations has declined over time, particularly in response to the Omicron variant [4,5]. As of June 2024, JN.1-lineage viruses have become dominant in circulation [6], and thus the U.S. FDA has recommended a monovalent JN.1-lineage-based vaccine formula for 2025–2026 [7]. As the virus continues to spread, there is an urgent need for ongoing vigilance in both preventive efforts and the development of effective therapeutic strategies to mitigate its impact.
Due to the limited availability of effective antivirals, COVID-19 management primarily focuses on symptomatic relief and supportive care. While agents like remdesivir, Paxlovid, and molnupiravir have received approval or emergency use authorization (EUA) [8], they have notable limitations: remdesivir requires intravenous administration, Paxlovid poses drug–drug interactions, and molnupiravir shows lower efficacy [9]. These challenges underscore the need for novel antiviral therapies with broad-spectrum activity, fewer drug interactions, and improved accessibility.
SARS-CoV-2 infection begins with viral attachment to host-cells’ surface receptors and co-receptors, including angiotensin-converting enzyme 2 (ACE2) [10], neuropilin-1 (NRP1) [11], CD147 [12], and HSPA5 (also known as GRP78) [13], mediated by the viral spike (S) glycoprotein. Viral entry occurs through either direct membrane fusion or endocytosis. Membrane fusion is facilitated by host serine protease TMPRSS2, which cleaves the spike protein at the S2′ site, exposing the fusion peptide and enabling viral–host membrane fusion [10,14], whereas endocytosis-mediated entry involves clathrin-dependent internalization, followed by endosomal acidification and spike cleavage by cathepsin L, leading to membrane fusion within the endosome [15,16]. Following entry, the viral genome is released into the cytoplasm and translated into polyproteins (pp1a and pp1ab), which are subsequently cleaved by viral proteases (3CLpro and PLpro) into structural and non-structural proteins [17]. Given the critical role of viral entry in SARS-CoV-2 infection, developing entry inhibitors is a promising therapeutic strategy.
Natural products and herbal medicines have received considerable attention during the COVID-19 pandemic. Numerous candidates have been proposed for their antiviral and anti-inflammatory activities, and while some have shown promising effects [18,19,20,21], not all have undergone rigorous validation. Heat-clearing and exterior-releasing medicinal herbs, such as Houttuynia Herba, Scutellaria Radix, Isatis Radix, Mori Folium, and Bupleurum Radix, are widely used in traditional Chinese medicine and Japanese Kampo medicine for the treatment of COVID-19. These medicinal herbs could therefore provide a rich resource for the identification of antiviral entry inhibitors.
This study aims to evaluate the efficacy of medicinal herbal candidates in preventing SARS-CoV-2 entry and to identify candidate compounds within herbal matrices that may serve as the basis for future development of antiviral agents. We screened three panels of herbal extracts and herbal combination formulas for their ability to inhibit SARS-CoV-2 entry using pseudotyped viral particles bearing the SARS-CoV-2 spike protein. The active compound in the most effective herbal extract, Cimicifuga foetida water extract, was also identified and validated on both WT and JN.1 pseudoparticles for its antiviral effect and potential mechanisms.

2. Materials and Methods

2.1. Cell Culture

Human hepatoma Huh-7 cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM; ThermoFisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; ThermoFisher Scientific), 1% gentamicin (ThermoFisher Scientific), and 1% amphotericin B (Sigma-Aldrich, Saint Louis, MO, USA). The 293FT cells were cultured in the same medium, supplemented with 0.5 mg/mL G418 sulfate (InvivoGen, San Diego, CA, USA) for selection. Cells were incubated at 37 °C in a 5% CO2 incubator. For all infection experiments, cells were cultured in DMEM containing 2% FBS with antibiotics as described above.

2.2. SARS-CoV-2 Pseudoparticle (SARS-CoV-2pp) Production

SARS-CoV-2 pseudoparticles (SARS-CoV-2pps) were produced as previously described [22]. The 293FT cells were seeded in 10 cm dishes and co-transfected with lentiviral vector and SARS-CoV-2 spike protein plasmids using the OMNIfect transfection reagent (Transomic Technologies, Huntsville, AL, USA). After overnight incubation, the cells were washed with Dulbecco’s phosphate-buffered saline (DPBS; Hyclone, Logan, UT, USA) to remove transfection complexes and further incubated in DMEM containing 2% FBS. The supernatants were collected after 48 and 72 h, and SARS-CoV-2pps were concentrated using PEG-8000 (Sigma-Aldrich) and resuspended in DPBS for storage. The viral stock titers were determined using the Luciferase Assay System (Promega, Madison, WI, USA), as previously described [22].

2.3. Drug Candidate Preparation

The medicinal herb candidates evaluated in this study were water, methanol, and ethanol extracts prepared from referenced medicinal plant materials [23]. The plant materials were obtained from the Hung Chuan Chinese Medicine Store (Kaohsiung City, Taiwan) and authenticated through anatomical examination and high-performance liquid chromatography (HPLC) analysis. The reference materials were deposited in the Kaohsiung Medical University Herbarium. Commercial herbal combination formulas were supplied by KO DA Pharmaceutical Co., Ltd. (Taoyuan, Taiwan), with individual components listed in their standard preparation (https://www.koda.com.tw/pro01d_e.aspx?type=02; accessed on 18 June 2025). All materials were extracted using standard protocols for hot-water extraction [24], methanol extraction [25], and ethanol extraction [26]. The water-soluble extracts were reconstituted in ddH2O, while methanol and ethanol extracts were reconstituted in dimethyl sulfoxide (DMSO; Sigma-Aldrich). Pure compounds were obtained from Sigma-Aldrich and dissolved in DMSO. For all assays, the drugs were diluted in culture media to their final working concentrations, ensuring that the DMSO content was less than 0.5%.

2.4. Cytotoxicity Assay

Huh-7 cells were seeded in 96-well plates (1 × 104 cells per well) and treated with various concentrations of drug candidates for 72 h at 37 °C. After treatment, cell viability was assessed using the Cell Counting Kit-8 (CCK-8; Sigma-Aldrich) according to the manufacturer’s instructions. Following the addition of the CCK-8 reagent, cells were incubated at 37 °C for 2 h, and the optical density (OD) was measured at 450 nm using a microplate reader. Cytotoxicity curves and 50% cytotoxic concentrations (CC50) for each extract were determined using non-linear regression analysis in the GraphPad Prism software (Version 9.0.2). Screening concentrations were selected based on predicted values yielding ≥90% cell viability.

2.5. Entry Inhibition Assay

Huh-7 cells were seeded in 96-well plates (1 × 104 cells per well) and inoculated with virus–drug mixtures containing SARS-CoV-2pps (MOI = 0.01) and each drug candidate at the respective screening concentration for 2 h at 37 °C. After infection, the cells were washed with DPBS and further incubated in DMEM supplemented with 2% FBS for 72 h. Cell lysates were collected, and viral infectivity was determined using the Luciferase Assay System (Promega) following the manufacturer’s protocol. Test candidates that showed statistical significance and reduced the SARS-CoV-2pp luciferase reporter signal to below 10,000 relative light units (RLU) were considered effective.

2.6. Inactivation Assay

Caffeic acid (100 μM) and SARS-CoV-2pps were mixed in an Eppendorf tube and incubated at 37 °C for 1 h. After the incubation, the virus–drug mixture was diluted 20-fold and added to Huh-7 cells seeded in 96-well plates (1 × 104 cells per well). The final MOI of SARS-CoV-2pps was 0.01. After infection for 2 h at 37 °C, the cells were washed with DPBS and further incubated in DMEM supplemented with 2% FBS for 72 h. Cell lysates were collected, and viral infectivity was determined using the Luciferase Assay System (Promega) following the manufacturer’s protocol.

2.7. Pretreatment Assay

Huh-7 cells were seeded in 96-well plates (1 × 104 cells per well) and incubated with caffeic acid (100 μM) for 24 h. After the incubation, supernatants containing the drug were removed, and the cells were infected with SARS-CoV-2pps (MOI = 0.01) for 2 h at 37 °C. After infection, the cells were washed with DPBS and further incubated in DMEM supplemented with 2% FBS for 72 h. Cell lysates were collected, and viral infectivity was determined using the Luciferase Assay System (Promega) following the manufacturer’s protocol.

2.8. Statistical Analysis

Statistical analysis was conducted using GraphPad Prism 9.0.2 (GraphPad Software, San Diego, CA, USA). Data are presented as mean ± standard deviation (SD) from three independent experiments. Statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparisons test (p < 0.05).

3. Results

3.1. Screening of Medicinal Herbal Extracts and Formulas for Viral Entry Inhibition

Alcoholic extracts and water extracts (WEs) were prepared from three panels of traditional herbal medicines. The first panel (Table 1 and Figure 1) consists of methanol extracts (MEs) and WEs from medicinal herbs that are known for their heat-clearing and detoxifying properties (Artemisia annua, Isatis indigotica Fort., Dryopteris crassirhizoma, Anemarrhena asphodeloides, Sophora tonkinensis), exterior-releasing effect (Perilla frutescens, Schizonepeta tenuifolia, Mentha canadensis, Chrysanthemum morifolium, Morus alba, Saposhnikovia divaricate, and Cimicifuga foetida), interior-warming effect (Zingiber officinale), phlegm-dispelling effect (Aster tataricus), and blood-regulating effect (Polygonum cuspidatum, Artemisia argyi) [23], which are often used for treating febrile diseases and infections. The second panel (Table 2 and Figure 2) consists of ethanol extracts (EEs) and WEs from medicinal herbs with documented antiviral activities [27]. Houttuynia cordata, Scutellaria baicalensis, Isatis indigotica Fort., and Forsythia suspensa also have heat-clearing effects [23] and have previously demonstrated in vitro antiviral activity against severe acute respiratory syndrome coronavirus (SARS-CoV) [28,29,30]. These herbs are among the components in the traditional Chinese medicine formulas used for COVID-19 treatment, such as NRICM101 [31], Shuanghuanglian [32], and Lianhuaqingwen [33]. Phyllanthus urinaria and Bupleurum kaoi, on the other hand, have demonstrated anti-hepatitis C virus (HCV) activity [34,35]. The third panel (Table 3 and Figure 3) consists of EEs and WEs from the heat-clearing herbal combination formulas Xiao Chai Hu Tang (XCHT), Huang Lian Jie Du Tang (HLJDT), Sheng Ma Ge Gen Tang (SMGGT), Long Dan Xie Gan Tang (LDXGT), and Yin Chen Hao Tang (YCHT) [23]. The cytotoxicity profiles for each extract were characterized to determine the 50% cytotoxic concentration (CC50) and the screening concentration (SC) that maintained at least 90% cell viability (Table 1, Table 2 and Table 3).
To assess the SARS-CoV-2 entry inhibitory potential of these medicinal herbs and combination formulas, a SARS-CoV-2 pseudoparticle (SARS-CoV-2pp) system that we previously established [22] was used for the inhibition assay. To validate the inhibition of viral entry, chloroquine, a known SARS-CoV-2 entry inhibitor that prevents endosomal acidification [36], was included as a positive control. Cells treated with 0.5% DMSO were used as a solvent negative control, and cells fixed with 4% paraformaldehyde (PFA) prior to SARS-CoV-2pp infection served as a non-entry negative control to account for background signal. The screening results indicated that methanol extract of Anemarrhena asphodeloides rhizome (Figure 1A), water extract of Cimicifuga foetida rhizome (Figure 1B), and water extracts of XCHT and SMGGT (Figure 3) were the most effective in blocking SARS-CoV-2pp entry, reducing the pseudoparticles’ luciferase reporter signal to below 10,000 relative light units (RLU) and similar to those of chloroquine. The percentages of inhibition were 88%, 90%, 82%, and 85%, respectively, compared to the virus-only group.

3.2. Dose-Dependent Antiviral Activity of Cimicifuga foetida Rhizome Water Extract

Given that the water extract of Cimicifuga foetida rhizome was the most effective in blocking SARS-CoV-2pp entry and that the herb is also an ingredient in the SMGGT formula (Puerariae Radix, Cimicifugae Rhizoma, Paeoniae Alba Radix, Glycynhizae Radix et Rhizoma, Zingiberis Rhizoma Recens) [23], we further explored the antiviral efficacy of the extract. The Cimicifuga foetida rhizome water extract showed dose-dependent inhibition of SARS-CoV-2pp entry (Figure 4A), yielding an estimated 50% effective concentration (EC50) of 151.5 μg/mL. We also examined the antiviral dose response on the currently dominant lineage JN.1 prototype and found that Cimicifuga foetida rhizome water extract also inhibited JN.1 pseudoparticle entry with an EC50 of 147.8 μg/mL (Figure 4B). These results confirm the inhibitory activity of the extract on SARS-CoV-2pp entry and highlight the extract’s potential value towards the newly emerging variants of JN.1 sublineages.

3.3. Characterization and Identification of Bioactive Antiviral Compounds

To identify the antiviral mechanism of Cimicifuga foetida rhizome water extract, we next investigated its major components, including isoferulic acid, ferulic acid, caffeic acid, and cimifugin [23,37,38]. The cytotoxicity profiles of the compounds were first determined (Table 4), and 100 μM (a non-cytotoxic concentration) was used for the entry-inhibition assay. The results indicated that, out of the four candidates, only caffeic acid blocked both WT and JN.1 SARS-CoV-2pp entry at 100 μM (Figure 5A,B) and demonstrated dose-dependent inhibition (Figure 5C). The estimated EC50’s were 58.62 μM and 88.32 μM for the WT and JN.1 strains, respectively. Additionally, caffeic acid was able to inactivate the pseudoparticles directly (Figure 5D) and also inhibit pseudoparticle entry following 24 h pretreatment of host cells (Figure 5E). These findings suggest that the compound exhibits both virucidal activity and the capacity to interact with host-cell factors involved in viral entry.

4. Discussion

Viral entry is the first step in viral infection; therefore, entry inhibitors represent a promising class of antivirals [39] that could not only provide prophylactic effect but also reduce secondary infections from the infected cells. In the current study, we identified several herbal candidates, including extracts of Anemarrhena asphodeloides, Cimicifuga foetida, XCHT, and SMGGT, as potential sources for the development of SARS-CoV-2-specific entry inhibitors. We have previously shown that several herbal extract candidates on our list demonstrated inhibitory effects on viral entry of other viruses. Specifically, the methanol extract of Perilla frutescens blocked viral attachment and neutralized pseudoparticles of Ebola virus (EBOV) [40]; the methanol extract of Polygonum cuspidatum was shown to inactivate dengue virus (DENV) and inhibit viral attachment and entry/fusion events [41]; the acetone extract of Phyllanthus urinaria inhibited the attachment and free viral particles of hepatitis C virus (HCV) [34]; whereas the methanol extract of Bupleurum kaoi mainly targeted the fusion step of HCV entry [35]. These extracts, however, did not inhibit SARS-CoV-2pp entry in the current study, suggesting that the reported antiviral activities are virus-specific. In addition, a mechanistic study on the formula NRICM101 suggested that Scutellaria baicalensis was able to neutralize SARS-CoV-2 in a plaque reduction neutralization test [31], yet we did not observe its effect on entry in our study. This discrepancy could potentially be explained by the different cell types (which may provide different entry routes for SARS-CoV-2) and assay conditions used. Of note, while our study employs a mechanism-specific approach to identify entry inhibitors, we cannot rule out whether the medicinal herbs and formulas could potentially harbor antiviral effects against SARS-CoV-2 through other mechanisms, including inhibitory effects on other stages of the viral replicative cycle and immunomodulation.
One of the identified candidate herbs, Anemarrhena asphodeloides, is a plant in the asparagus family. The rhizome of the herb is used in traditional Chinese medicine and contains key bioactive components, including timosaponin B-II (≥3.0%), mangiferin (≥0.7%), neomangiferin, timosaponin A-III, and isomangiferin [23,42,43,44]. Extracts from Anemarrhena asphodeloides and its major components have also been reported for their antiviral activities. Timosaponin B-II has shown anti-enterovirus 71 (EV71) effects [45]. Timosaponin A-III [46] and three additional compounds, (−)-(R)-nyasol, (−)-(R)-4′-O-methylnyasol, and broussonin A, isolated from the methanol extract of Anemarrhena asphodeloides rhizome [47] were able to inhibit respiratory syncytial virus (RSV) infection. In addition, other classes of saponins have been suggested to prevent SARS-CoV-2 entry by disrupting the viral envelope or interfering with the spike-ACE2 interaction [48]. Given the high percentage of timosaponins in Anemarrhena asphodeloides, it is plausible that these compounds may exert a similar effect on SARS-CoV-2 entry. Mangiferin, another major component of Anemarrhena asphodeloides, has demonstrated antiviral activities against herpes simplex virus type 1 (HSV-1) [49,50] and human immunodeficiency virus type 1 (HIV-1) [51]. Our finding that the methanol extract of Anemarrhena asphodeloides rhizome inhibits SARS-CoV-2pp entry warrants further investigation to identify the molecular mechanisms in targeting viral entry steps and active antiviral compounds.
The two combination candidates identified, XCHT (also known as Shosaikoto in Kampo medicine or Minor Bupleurum Combination) and SMGGT (also known as Shomakakkonto or Cimicifuga and Pueraria Combination), are classical herbal combination formulas conventionally used for treating febrile diseases, such as those from viral infections. XCHT has been documented for its immunomodulatory, anti-inflammatory, and anti-oxidant activities [52], and antiviral effects against hepatitis B virus (HBV) [53] and Coxsackie B virus type 1 (CVB1) [54]. For SMGGT, antiviral activities were reported for measles virus [55], EV71 [56], and RSV [57]. Our study is the first to demonstrate the formulas’ effects on preventing SARS-CoV-2pp entry. XCHT typically contains Bupleuri Radix, Scutellariae Radix, Ginseng Radix, Glycyrrhizae Radix et Rhizoma Praeparatum cum Melle, Pinelliae Rhizoma Praeparatum, Zingiberis Rhizoma Recens, and Jujubae Fructus; whereas SMGGT typically contains Puerariae Radix, Cimicifugae Rhizoma, Paeoniae Alba Radix, Glycynhizae Radix et Rhizoma, Zingiberis, and Rhizoma Recens [23]. Among the ingredients, the major components of Ginseng and Glycyrrhizae were reported to have direct antiviral effects on virus particles, entry, and replication of various viruses [58,59,60]. Gallic acid, methyl gallate, and pentagalloylglucose isolated from Paeoniae Alba Radix inhibit influenza A virus replication and reduce neuraminidase activity [61]. The water extract of fresh ginger (Zingiberis Rhizoma Recens), but not dried ginger, was shown to inhibit RSV entry by reducing viral attachment [62]. Interestingly, a recent study reported that the water extract of Scutellaria baicalensis inhibits SARS-CoV-2pp entry at higher concentrations (250 μg/mL) [63], but we did not observe this effect at the lower concentration used in our study (100 μg/mL). The active components responsible for SARS-CoV-2 entry inhibition and the potential of these two formulas to suppress other stages of the SARS-CoV-2 viral life cycle require further investigation.
The most potent candidate identified in our study is Cimicifuga foetida water extract. The herb and its major components have exhibited antiviral effects on various viruses. Water extracts of the herb have been shown to inhibit RSV infection and viral attachment dose-dependently [64], and cimicifugin was found to inhibit RSV attachment and internalization [65]. Another major compound, ferulic acid, possesses multiple antiviral mechanisms against both viruses and hosts [66]. Its derivatives have been shown to block replication of the coronaviruses HCoV-229E and SARS-CoV-2 [67,68]. Virtual screening studies predicted that ferulic acid could interact with SARS-CoV-2 viral proteins including spike [69] and membrane [70] proteins, which are crucial for viral attachment and virion assembly, respectively. Caffeic acid, on the other hand, demonstrated antiviral activities against influenza A virus [71], HCV [72], thrombocytopenia syndrome virus [73], and Ilhéus virus [74]. Notably, caffeic acid was shown to inhibit the replication cycle and viral attachment of HCoV-NL63 [75], a commonly used surrogate virus for SARS-CoV-2 [76].
In this study, we identified caffeic acid as a key bioactive compound in the Cimicifuga foetida rhizome extract that inhibits SARS-CoV-2pp entry. Mechanistic assays demonstrated that caffeic acid could both directly inactivate pseudoparticles and reduce viral entry when used to pretreat host cells, suggesting dual antiviral activity. These findings are supported by recent molecular docking studies, which predict that caffeic acid can bind not only to the SARS-CoV-2 spike protein [77,78], but also to host entry factors such as ACE2 and HSPA5 [78,79]. Specifically, caffeic acid binds with the spike receptor-binding domain (RBD) at residues Leu441, Tyr495, and Phe497 (binding energy = −6.43 kcal/mol, inhibition constant (Ki) = 19.36 μM); ACE2 at residues Leu73, Ala99, Leu100, Lys74, and Asn103 (binding energy = −5.31 kcal/mol, Ki = 127.93 μM); and HSPA5 substrate-binding domain β (SBDβ, which recognizes SARS-CoV-2 spike) at residues Phe451, Val453, Ile483(2), Leu480, and Ile493 (binding energy = −6.2 kcal/mol) [78,79]. Whether these are the specific target(s) of caffeic acid remains to be validated. In addition, some reports suggest that cathepsin B may be involved in the endosomal entry route of SARS-CoV-2 [80], and caffeic acid has been shown to inhibit cathepsin B activity with an IC50 of 110 ± 10 μM [81]. These studies are consistent with our experimental data, collectively indicating that caffeic acid may interfere with SARS-CoV-2 entry through multiple mechanisms, including direct viral targeting and modulation of host factors. To confirm its antiviral efficacy under physiological conditions, future studies should assess caffeic acid’s activity against infectious SARS-CoV-2 strains in vitro and in vivo. Further investigation of its pharmacokinetics, bioavailability, efficacy, and safety will also be essential for evaluating its therapeutic potential.

5. Conclusions

This study highlights the potential of the extracts from Anemarrhena asphodeloides, Cimicifuga foetida, XCHT, and SMGGT as effective inhibitors of SARS-CoV-2 entry. Furthermore, caffeic acid was identified as a bioactive compound from Cimicifuga foetida rhizome. Our findings support further investigation and development of these medicinal herbs, herbal formulas, and compound as candidates for COVID-19 prevention and promising sources for developing novel entry inhibitors against SARS-CoV-2.

Author Contributions

Conceptualization, C.-H.L., Y.-T.K. and L.-T.L.; methodology, C.-H.L., C.-J.L., F.-L.Y., S.-J.W. and L.-T.L.; formal analysis, C.-H.L. and L.-T.L.; investigation, C.-H.L., C.-J.L., F.-L.Y. and S.-J.W.; resources, C.-J.L., F.-L.Y., S.-J.W. and L.-T.L.; writing—original draft preparation, C.-H.L. and L.-T.L.; writing—review and editing, C.-H.L., Y.-T.K. and L.-T.L.; supervision, L.-T.L.; funding acquisition, Y.-T.K. and L.-T.L. All authors have read and agreed to the published version of the manuscript.

Funding

L.-T.L. and Y.-T.K. were supported by grants from Chi Mei Medical Center and Taipei Medical University (111CM-TMU-03) and from the National Science and Technology Council (NSTC) of Taiwan (NSTC 113-2314-B-384-003). L.-T.L. additionally received support from the NSTC of Taiwan (NSTC 114-2923-B-038-001-MY3, NSTC 114-2320-B-038-044).

Data Availability Statement

Data is contained within the article.

Acknowledgments

The authors would like to acknowledge Ming-Hong Yen (Kaohsiung Medical University, Kaohsiung, Taiwan) for technical support.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Inhibition of SARS-CoV-2pp entry by the extracts of heat-clearing and detoxifying medicinal herbs. (A) Methanol extracts (MEs) and (B) water extracts (WEs) of each herb were examined. SARS-CoV-2pp entry was quantified by luciferase reporter activity, expressed as relative light units (RLU). Extracts that reduced the luciferase signal to below 10,000 RLU were considered effective (red bar). Chloroquine (CQ) was used as a positive control (dark grey bar). Cells treated with 0.5% DMSO served as a solvent negative control (white bar), while cells fixed with paraformaldehyde (PFA) before infection served as a non-entry negative control. Data shown are mean ± SD from three independent experiments.
Figure 1. Inhibition of SARS-CoV-2pp entry by the extracts of heat-clearing and detoxifying medicinal herbs. (A) Methanol extracts (MEs) and (B) water extracts (WEs) of each herb were examined. SARS-CoV-2pp entry was quantified by luciferase reporter activity, expressed as relative light units (RLU). Extracts that reduced the luciferase signal to below 10,000 RLU were considered effective (red bar). Chloroquine (CQ) was used as a positive control (dark grey bar). Cells treated with 0.5% DMSO served as a solvent negative control (white bar), while cells fixed with paraformaldehyde (PFA) before infection served as a non-entry negative control. Data shown are mean ± SD from three independent experiments.
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Figure 2. Inhibition of SARS-CoV-2pp entry by the ethanol extracts (EEs) and water extracts (WEs) of antiviral medicinal herbs. SARS-CoV-2pp entry was quantified by luciferase reporter activity, expressed as relative light units (RLU). Extracts that reduced the luciferase signal to below 10,000 RLU were considered effective. Chloroquine (CQ) was used as a positive control (dark grey bar). Cells treated with 0.5% DMSO served as a solvent negative control (white bar), while cells fixed with paraformaldehyde (PFA) before infection served as a non-entry negative control. Data shown are mean ± SD from three independent experiments.
Figure 2. Inhibition of SARS-CoV-2pp entry by the ethanol extracts (EEs) and water extracts (WEs) of antiviral medicinal herbs. SARS-CoV-2pp entry was quantified by luciferase reporter activity, expressed as relative light units (RLU). Extracts that reduced the luciferase signal to below 10,000 RLU were considered effective. Chloroquine (CQ) was used as a positive control (dark grey bar). Cells treated with 0.5% DMSO served as a solvent negative control (white bar), while cells fixed with paraformaldehyde (PFA) before infection served as a non-entry negative control. Data shown are mean ± SD from three independent experiments.
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Figure 3. Inhibition of SARS-CoV-2pp entry by the ethanol extracts (EEs) and water extracts (WEs) of herbal combination formulas. SARS-CoV-2pp entry was quantified by luciferase reporter activity, expressed as relative light units (RLU). Extracts that reduced the luciferase signal to below 10,000 RLU were considered effective (red bars). Chloroquine (CQ) was used as a positive control (dark grey bar). Cells treated with 0.5% DMSO served as a solvent negative control (white bar), while cells fixed with paraformaldehyde (PFA) before infection served as a non-entry negative control. Data shown are mean ± SD from three independent experiments.
Figure 3. Inhibition of SARS-CoV-2pp entry by the ethanol extracts (EEs) and water extracts (WEs) of herbal combination formulas. SARS-CoV-2pp entry was quantified by luciferase reporter activity, expressed as relative light units (RLU). Extracts that reduced the luciferase signal to below 10,000 RLU were considered effective (red bars). Chloroquine (CQ) was used as a positive control (dark grey bar). Cells treated with 0.5% DMSO served as a solvent negative control (white bar), while cells fixed with paraformaldehyde (PFA) before infection served as a non-entry negative control. Data shown are mean ± SD from three independent experiments.
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Figure 4. Dose-dependent inhibition of SARS-CoV-2pp entry by Cimicifuga foetida rhizome water extract. Entry of SARS-CoV-2pps bearing the (A) wild-type (WT) or (B) JN.1 spike was quantified by luciferase reporter activity and normalized to the drug = 0 μg/mL group. Data shown are mean ± SD from three independent experiments. A least-squares-fit non-linear regression model was used to predict the 50% effective concentration (EC50).
Figure 4. Dose-dependent inhibition of SARS-CoV-2pp entry by Cimicifuga foetida rhizome water extract. Entry of SARS-CoV-2pps bearing the (A) wild-type (WT) or (B) JN.1 spike was quantified by luciferase reporter activity and normalized to the drug = 0 μg/mL group. Data shown are mean ± SD from three independent experiments. A least-squares-fit non-linear regression model was used to predict the 50% effective concentration (EC50).
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Figure 5. Mechanistic evaluation of the antiviral activity of Cimicifuga foetida rhizome water extract. (A,B) Inhibitory effects of the major compounds (100 μM) on entry of (A) WT and (B) JN.1 variant SARS-CoV-2pp entry. (C) Dose–response curve of caffeic acid against SARS-CoV-2pp entry. (D) Virucidal activity of caffeic acid (100 μM) assessed by inactivation assay. (E) Inhibition of SARS-CoV-2pp entry following pretreatment of host cells with caffeic acid (100 μM). Statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparisons test (* p < 0.05; ** p < 0.01; **** p < 0.0001).
Figure 5. Mechanistic evaluation of the antiviral activity of Cimicifuga foetida rhizome water extract. (A,B) Inhibitory effects of the major compounds (100 μM) on entry of (A) WT and (B) JN.1 variant SARS-CoV-2pp entry. (C) Dose–response curve of caffeic acid against SARS-CoV-2pp entry. (D) Virucidal activity of caffeic acid (100 μM) assessed by inactivation assay. (E) Inhibition of SARS-CoV-2pp entry following pretreatment of host cells with caffeic acid (100 μM). Statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparisons test (* p < 0.05; ** p < 0.01; **** p < 0.0001).
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Table 1. List of heat-clearing and detoxifying medicinal herb candidates.
Table 1. List of heat-clearing and detoxifying medicinal herb candidates.
SpeciesPart (s)CC50 (μg/mL)SC (μg/mL)
MEWEMEWE
Artemisia annuaHerba284.2>8001005
Perilla frutescensFolium167.9>800125200
Zingiber officinaleRhizoma (dried)51.75>8004820
Schizonepeta tenuifoliaHerba727.6>80020050
Mentha canadensisHerba330>800160200
Chrysanthemum morifoliumFlos1082>800200100
Morus albaFolium505>800200100
Saposhnikovia divaricateRadix972>800200200
Cimicifuga foetidaRhizoma46.17>80020200
Isatis indigotica Fort.Folium624.3>800200200
Polygonum cuspidatumRadix133.8617.940200
Dryopteris crassirhizomaRhizoma1723>800200200
Anemarrhena asphodeloidesRhizoma766.6>800200200
Sophora tonkinensisRadix402.5>8005030
Aster tataricusRadix et rhizoma882.2>800200200
Artemisia argyiFolium208.2595.650250
CC50, 50% cytotoxic concentration; SC, screening concentration; ME, methanol extract; WE, water extract.
Table 2. List of antiviral medicinal herb candidates.
Table 2. List of antiviral medicinal herb candidates.
SpeciesPart (s)CC50 (μg/mL)SC (μg/mL)
EEWEEEWE
Houttuynia cordataHerba>20>10020100
Scutellaria baicalensisRadix>7.8>1001.5100
Isatis indigotica Fort.Radix>125>125105
Forsythia suspensaFructus109.7>1251050
Bupleurum kaoiRadix80.92>8408
Phyllanthus urinariaHerba>7.8>125540
CC50, 50% cytotoxic concentration; SC, screening concentration; EE, ethanol extract; WE, water extract.
Table 3. List of herbal combination formula candidates.
Table 3. List of herbal combination formula candidates.
FormulaCC50 (μg/mL)SC (μg/mL)
EEWEEEWE
Xiao Chai Hu Tang (XCHT; Minor Bupleurum Combination)233>12510080
Huang Lian Jie Du Tang (HLJDT; Coptis & Scute Combination)185>1255050
Sheng Ma Ge Gen Tang (SMGGT; Cimicifuga & Pueraria Combination)154.9>1258045
Long Dan Xie Gan Tang (LDXGT; Gentiana Combination)>250>12520040
Yin Chen Hao Tang (YCHT; Capillaris Combination)104.3>1252030
CC50, 50% cytotoxic concentration; SC, screening concentration; EE, ethanol extract; WE, water extract.
Table 4. Cytotoxicity profile of major compounds from Cimicifuga foetida rhizome water extract.
Table 4. Cytotoxicity profile of major compounds from Cimicifuga foetida rhizome water extract.
CompoundCC50 (μM)
Cimifugin4206
Caffeic acid882.8
Ferulic acid4843
Iosferulic acid6710
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MDPI and ACS Style

Liu, C.-H.; Kuo, Y.-T.; Lin, C.-J.; Yen, F.-L.; Wu, S.-J.; Lin, L.-T. Screening of Medicinal Herbs Identifies Cimicifuga foetida and Its Bioactive Component Caffeic Acid as SARS-CoV-2 Entry Inhibitors. Viruses 2025, 17, 1086. https://doi.org/10.3390/v17081086

AMA Style

Liu C-H, Kuo Y-T, Lin C-J, Yen F-L, Wu S-J, Lin L-T. Screening of Medicinal Herbs Identifies Cimicifuga foetida and Its Bioactive Component Caffeic Acid as SARS-CoV-2 Entry Inhibitors. Viruses. 2025; 17(8):1086. https://doi.org/10.3390/v17081086

Chicago/Turabian Style

Liu, Ching-Hsuan, Yu-Ting Kuo, Chien-Ju Lin, Feng-Lin Yen, Shu-Jing Wu, and Liang-Tzung Lin. 2025. "Screening of Medicinal Herbs Identifies Cimicifuga foetida and Its Bioactive Component Caffeic Acid as SARS-CoV-2 Entry Inhibitors" Viruses 17, no. 8: 1086. https://doi.org/10.3390/v17081086

APA Style

Liu, C.-H., Kuo, Y.-T., Lin, C.-J., Yen, F.-L., Wu, S.-J., & Lin, L.-T. (2025). Screening of Medicinal Herbs Identifies Cimicifuga foetida and Its Bioactive Component Caffeic Acid as SARS-CoV-2 Entry Inhibitors. Viruses, 17(8), 1086. https://doi.org/10.3390/v17081086

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