Antiviral activity of processed Rheum palmatum L. extracts against the Influenza A H1N1 virus

1. Introduction

Influenza is a highly transmissible respiratory viral disease that causes significant morbidity and mortality worldwide (Eliopoulos et al., 2022). Influenza A virus (IAV) is the causative agent of seasonal flu epidemics and continues to be regarded as a global health concern (Xiao et al., 2022). Among its subtypes, Influenza A H1N1 (A/H1N1) has been responsible for severe pandemic infections. The 1918 “Spanish flu” resulted in an estimated 50-100 million deaths globally Liang (2023), while the 2009 “Swine flu” pandemic caused more than 284,000 deaths worldwide. Seasonal influenza continues to affect about 1 billion people annually, leading to approximately 290,000-650,000 deaths (Mostafa et al., 2023).

Vaccination remains the most effective preventive measure against influenza (Cowling et al., 2024). However, frequent viral mutations limit vaccine coverage, and immunization often fails to provide broad-spectrum protection (Li et al., 2024). Antiviral drugs, such as oseltamivir phosphate (Tamiflu) and zanamivir (Relenza), are widely used to control influenza infections (Świerczyńska et al., 2022). Despite their effectiveness, these chemical drugs may cause adverse effects and are increasingly challenged by drug resistance (Sarker et al., 2024). Moreover, recent studies have highlighted the antiviral potential of metal complexes, particularly zinc (II) complexes, which show inhibitory activity against several viruses, including influenza (Kumari et al., 2025). These limitations highlight the urgent need to explore new antiviral agents that are both safe and effective.

In this context, natural products have emerged as promising sources of bioactive compounds for influenza management. The genus Rheum has been cultivated in several countries for more than 5,000 years (Yang et al., 2024). Its edible stalks are consumed as a main component in several dishes, such as desserts, wine, and teas (Xiang et al., 2020). In traditional Chinese medicine (TCM), Rheum species have long been regarded as valuable medicinal plants owing to their diverse pharmacological properties, relatively low toxicity, and safety for human use (Mohtashami et al., 2021; Zhang et al., 2023). Among these, Rheum palmatum L. (RP) is one of the most important TCM species and has traditionally been used to treat inflammation, constipation, and digestive disorders (Yang et al., 2024). Previous studies have reported its antioxidant, anti-inflammatory, bioenergy production, and antiviral activities (Tsai et al., 2023b, 2023a). Moreover, compounds from RP have demonstrated antiviral effects against viruses such as HIV, hepatitis viruses, and SARS-CoV-2, suggesting potential antiviral activity against influenza (Chang et al., 2014).

The method of herb processing plays a crucial role in modulating the pharmacological activity of medicinal plants. In TCM, processing techniques such as steaming, stir-frying, and the use of adjuvants (e.g., vinegar, brine, wine) can modify the phytochemical composition of herbs, thereby enhancing efficacy or reducing toxicity Chen et al. (2018). Such processing strategies may broaden the therapeutic potential of crude drugs and provide a scientific rationale for evaluating their biological activities. Therefore, the present study aimed to investigate the effects of different processing methods of RP on antiviral activity against A/H1N1 in vitro and to further evaluate the possible molecular interactions of its marker compounds using in silico network pharmacology and molecular docking approaches.

2. Materials and Methods

3. Results

3.1 Antiviral activity against H1N1

Ethanol extracts of processed RP roots obtained higher antiviral activities on A/H1N1 than their water counterparts. As shown in Fig. 1, three out of eleven water extracts and eight out of eleven ethanol extracts demonstrated more than 50% viral inhibition of A/H1N1-infected cells at 1.00 mg/mL. The top five processing methods demonstrating the highest antiviral activity in decreasing order are shown as follows: oseltamivir phosphate 73.93±2.80% > RP-E10 62.27±3.05% > RP-E1 61.80±8.21% > RP-E9 57.18±7.96% > RP-E7 57.14 ±4.36% > RP-E11 57.07±5.36%. From this, ethanol extracts of raw and processing methods with vinegar (both stir-frying and soaking) offer maximum activities against H1N1.

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Fig. 1.

Antiviral effects of Rheum palmatum L. (RP) root extracts on A/H1N1-infected MDCK cells. The water (RP-W) and ethanol (RP-E) extracts of R. palmatum L. roots were prepared using the following processing methods: (1) raw extract, (2) carbonizing, (3) high-pressure steaming with water, (4) high-pressure steaming with wine, (5) soaking in wine, (6) stir-frying with wine, (7) soaking in wine with steam, (8) soaking in water with steam, (9) stir-frying with water, (10) stir-frying with vinegar, and (11) soaking in vinegar. Positive control (PC): Oseltamivir phosphate, 73.93 ± 2.80%.

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3.2 Network pharmacology

Aloe-emodin, emodin, and rhein were associated with a total of 312 proteins (as shown in Fig. 2). Of these, 145 predicted targets were related to the A/H1N1 nucleoprotein. In this constructed network analysis with Cytoscape, there are four overlapping protein-coding genes (indicated by the green triangles) that were regarded as important targets to A/H1N1 by RP compounds. This includes macrophage migration inhibitory factor (MIF), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), nuclear factor erythroid 2-related factor 2 (NFE2L2), and Toll-like receptor 4 (TLR4).

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Fig. 2.

The protein-protein interaction (PPI) network between RP and H1N1. The red edges: correlate genes between RP and H1N1; Red color: the major components of RP: Aloe-Emodin, Emodin, and Rhein; Green triangle: major target genes of RP and H1N1.

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Interactions to each gene ontology term category and KEGG pathway results are presented in Fig. 3. Table 1 shows the top 3 gene ontology terms of GO MF, BP, CC, and KEGG metabolic pathways. The A/H1N1 targets of RP compounds are involved in various biological processes (BP), such as cellular responses to chemical stimuli, protein phosphorylation, and G2/MI transition of the meiotic cell cycle. These targets are mainly distributed in the cellular compartments (CC), such as cytoplasm, plasma membrane, and receptor complexes. The enriched molecular function ontologies are implicated in protein kinase activity, adenyl nucleotide binding, and signaling receptor activity. The KEGG pathway analysis revealed that RP compounds had significant results for neuroactive ligand-receptor interaction, progesterone-mediated oocyte maturation, and the calcium signaling pathway. These pathways may most likely be responsible for the molecular mechanisms of RP in managing A/H1N1.

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Fig. 3.

The gene ontology of GO MF, GO BP, GO CC, and biological pathways of KEGG.

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Table 1. The top 3 gene ontology terms of GO MF, BP, CC, and KEGG metabolic pathways.

Source	NO.	Term name	P adj*
GO MF	1.	Protein kinase activity	2.267×10-23
	2.	Adenyl nucleotide binding	9.170×10-16
	3.	Signaling receptor activity	9.899×10-15
GO BP	1.	Response to the chemical	2.443×10-24
	2.	Protein phosphorylation	8.878×10-24
	3.	G2/MI transition of the meiotic cell cycle	2.133×10-4
GO CC	1.	Cytoplasm	3.751×10-12
	2.	Plasma membrane	6.152×10-12
	3.	Receptor complex	7.060×10-8
KEGG	1.	Neuroactive ligand-receptor interaction	3.111×10-6
	2.	Progesterone-mediated oocyte maturation	1.094×10-5
	3.	Calcium signaling pathway	1.673×10-5

* The y-axis of enrichment p-values in a negative log₁₀ scale.

Gene Ontology (GO), Molecular Function (MF), Biological Process (BP), Cellular Component (CC), Kyoto Encyclopedia of Genes and Genomes (KEGG)

3.3 Molecular docking

The binding affinity of the RP compounds towards the A/H1N1 has been represented by the LibDock score shown in Table 2. The greater values suggest stronger and more favorable interaction between the ligand and the protein (Huang et al., 2023). The 2D and 3D interactions between aloe-emodin, emodin, rhein, and oseltamivir phosphate with the A/H1N1 protein have been shown in Figs. 4-7. Based on the docking simulation results, it was found that the aloe-emodin ligand has the highest LibDock score of 96.91 and has the strongest bond to the enzyme compared to the other RP compounds (emodin and rhein). Moreover, all three compounds showed good LibDock scores (Table 2) comparable to the standard drug, oseltamivir phosphate (Table 2). Similar residue interactions were observed in oseltamivir phosphate with aloe-emodin (carbon H-bonds with GLU278) (Fig. 4) and rhein (pi-alkyl interactions with ARG225) (Fig. 6). Identical residues with Van der Waals interactions at GLU119, LEU134, SER180, ARG293, and ASN295 were observed in aloe-emodin, emodin (Figs. 4 and 5), and oseltamivir phosphate (Fig. 7). Table 3 presents the residue interactions of RP compounds with amino acid residues of A/H1N1 (4B7N).

Table 2. Compounds with LibDock Scores for H1N1 (4B7N).

Compound	LibDock score
Aloe-Emodin	96.91
Emodin	96.06
Rhein	95.33
Oseltamivir phosphate*	97.09

* Control Drug

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Fig. 4.

Binding interaction of 4B7N and Aloe-Emodin (a) Ligand binding sphere and protein, (b) Two-dimensional display of the interaction.

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Fig. 5.

Binding interaction of 4B7N and Emodin (a) Ligand binding sphere and protein, (b) 2D display of the interaction.

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Fig. 6.

Binding interaction of 4B7N and Rhein (a) Ligand binding sphere and protein, (b) 2D display of the interaction.

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Fig. 7.

Binding interaction of 4B7N and Oseltamivir phosphate (a) Ligand binding sphere and protein, (b) 2D display of the interaction.

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Table 3. RP compounds interaction with H1N1 (4B7N) protein.

		Aloe-emodin				Emodin		Rhein			Oseltamivir phosphate *
Van der Waals		ARG118, GLU119, LEU134, ASP151, ARG152, ARG156, TRP179, SER180, ARG223, ARG225, SER247, GLU277, GLU278, ARG293, ASN295				GLU119, LEU134, ARG156, TRP179, SER180, GLU278, ARG293, ASN295		ARG118, GLU119, TRP179, SER180, LEU224, SER247, GLU277, TYR402			GLU119, ASP151, ARG152, SER180, LEU224, THR226, GLU228, THR243, GLU277, ARG293, ASN295, ASN344
Conventional hydrogen bond		ASP151, TRP179				ASP151, TRP179		ARG293, ARG368			ARG223, GLU278, TYR402
Carbon-hydrogen bond		ARG152, GLU278, ARG293				ARG225, SER247		-			SER247, GLU278
Pi-Alkyl	-		-	ARG223, ARG225					TRP179, ARG223, ARG225
PI-Anion	GLU228		-	ASP151, ARG152 GLU278					-
Unfavorable bump					TYR402		-			-		-
Unfavorable donor-donor					-		ARG152			-		ARG225

* Control Drug

- No Detect

4. Discussion

Plant foods with medicinal properties have gradually become relevant as appropriate alternatives for synthetic antiviral agents (Mehrbod et al., 2021). Classified as a ‘top grade’ TCM, RP has been reported to exhibit inhibitory activities against various viruses (Chang et al., 2014; Ntemafack et al., 2022). Despite these antiviral activities, the viability of RP against the influenza virus remains unclear. Thus, this study investigated the RP extracts against influenza A and evaluated the underlying mechanisms that mediate its therapeutic effects.

In this study, we examined the antiviral activities of RP prepared using various TCM processing methods. The processing methods, such as high-pressure steaming, stir-frying, and adjuvant addition, may contribute to the identity and concentration of chemical species in extracts (Tsai et al., 2023a). These processes may enhance the pharmacological activity of herbs and reduce the risks associated with crude drug toxicity (Zhang et al., 2023). Based on the results, vinegar-processed RP extracts demonstrated higher inhibition of A/H1N1-infected cells than the untreated RP extract. Chen et al. (2018) mentioned that vinegar and heat may induce chemical changes in the phytochemical metabolites of herbs, enhancing their medicinal properties. This was supported by studies of Lei et al. (2018), wherein structural changes in saikosaponins found in Bupleurum falcatum L. exhibited outstanding anti-inflammatory potentials. Wu et al. (2014) mentioned that vinegar processing of Corydalis yanhusuo root demonstrated better analgesic and cardiovascular effects correlated with higher tertiary alkaloid levels. In a recent study by Tsai et al., increased RP markers, aloe-emodin, emodin, and rhein, showed significant antioxidant and bioenergy production. This may be attributed to the effect of vinegar in enhancing the polarity of active substances, such as phenolics, flavonoids, anthraquinones, and alkaloids (Tsai et al., 2023a; Wu et al., 2014).

Network analysis revealed that the major anthraquinones of RP (aloe-emodin, emodin, and rhein) interact with four protein-coding genes (Figs. 2 and 3 and Table 1). The macrophage migration inhibitory factor (MIF), a multipotent cytokine, has a pivotal part in cell signaling, immune responses, and inflammation (Sumaiya et al., 2022). Upon binding to its receptor, MIF activates the translocation into the nucleus of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) (Sumaiya et al., 2022). NF-κB is a transcription factor that regulates the downstream signaling of PLA2/arachidonic acid/COX-2/PGE2 and the PI3K/Akt pathways (Sumaiya et al., 2022; Vázquez et al., 2023). During influenza infection, these pathways are activated and ultimately result in heightened airway epithelial cell inflammation and enhanced virus production (Wang et al., 2024). MIF also upregulates Toll-like receptor 4 (TLR4) expression in innate and adaptive immunity (Roger et al., 2003; Sumaiya et al., 2022). TLR4 is a membrane-bound protein that recognizes components of pathogens and regulates adaptive immune responses. TLR4 activation can help resist H1N1 influenza viral infection (Roger et al., 2003). However, uncontrolled activation of TLR4 may lead to TLR4-mediated inflammatory response, which may further induce serious lung injury (Shirey et al., 2021). Studies have revealed that RP anthraquinones exhibited inhibitory activities against MIF, NF-κB, and TLR4 (Li et al., 2021; Yu et al., 2021). Thus, targeting these proteins reduces IAV-induced inflammation and lung damage both in vitro and in vivo (Liang, 2023; Yang et al., 2022).

On the other hand, nuclear factor erythroid 2-related factor 2 (NFE2L2), also known as NRF2, is involved in the KEAP1/NRF2 signaling pathway. This pathway protects the body from oxidative stress and induces antiviral and anti-inflammatory effects (Liu et al., 2023; Waqas et al., 2023). Waqas et al. (2023) discussed that the activators of NRF2 in alveolar epithelial cells decreased Influenza A viral replication. Reports have shown that anthraquinones, aloe-emodin, emodin, and rhein, improve organ fibrosis by activating the Nrf2 signaling pathway. In addition, studies showed that these metabolites promote the expression of various endogenous antioxidant and detoxifying proteins in damaged lungs in vivo (Gao et al., 2022).

The PPI network constructed agreed with the results from the gene ontology (GO) term and KEGG enrichment analyses (Figs. 2 and 3 and Table 1) (Higazy et al., 2022; Li et al., 2022; Liang, 2023). These Influenza A H1N1 targets are implicated in pathways related to inflammation, cellular survival and proliferation, immune response, and oxidative stress. These results suggest that the antiviral effect of RP may occur through inhibition of MIF, NF-κB, TLR4 activation, and stimulation of NRF2 pathways (Figs. 2 and 3 and Table 1). Further studies are necessary to deliver substantial proof of these in silico-based predictions.

Molecular docking analysis was also performed to assess the binding stability of aloe-emodin, emodin, and rhein with the A/H1N1 neuraminidase (Figs. 3-7, and Tables 2 and 3). As seen in the results, relatively strong binding interactions were observed between the major anthraquinones of RP and the protein target (Figs. 3-6, and Tables 2 and 3). This is supported by previous studies on the efficacy of aloe-emodin, emodin, and rhein to impede Influenza A infection. Aloe-emodin hindered IAV replication by amplifying galectin-3, resulting in increased antiviral genes in MDCK cells and suppressing the nuclear translocation of NF-κB (Gansukh et al., 2018). Emodin repressed IAV replication through Nrf2 activation and TLR4 attenuation (Jin et al., 2023). Rhein notably affects NF-kB and TLR4 pathways, resulting in a decline in influenza A particle production (Wang et al., 2018). With these, metabolites in RP indicate potential as complementary candidates for further investigation against A/H1N1.

5. Conclusions

In conclusion, this study demonstrated the in vitro antiviral potential of RP against the influenza A H1N1 virus. Among the different processing methods, vinegar adjuvant processing exhibited greater inhibitory activity against A/H1N1-infected MDCK cells. The underlying antiviral mechanisms of RP metabolites are likely to involve pathways linked with inflammation, cellular survival and proliferation, immune response, and oxidative stress by targeting proteins such as MIF, NF-κB, TLR4, and NFE2L2. Molecular docking analysis revealed that the compounds aloe-emodin, emodin, and rhein exhibited favorable binding to the A/H1N1 neuraminidase. Hence, the observed antiviral activity in A/H1N1-infected MDCK cells by RP extract may be attributed to the metabolite’s interaction with A/H1N1 nucleoprotein through inhibition of MIF, NF-κB, TLR4 activation, and stimulation of NRF2 pathways. Overall, the results of this study indicate that RP and its compounds possess antiviral potential against influenza A H1N1 and may warrant further investigation as candidate agents for limiting viral replication.