Porcine epidemic diarrhea (PED) is an acute intestinal illness with high contagiousness caused by the porcine epidemic diarrhea virus (PEDV) [1], which is accompanied by clinical signs such as severe diarrhea, vomiting, and dehydration. PEDV was first found in Britain in 1971. It belongs to the alphacoronavirus genus in the coronaviridae family. It can infect pigs of all ages, but suckling piglets could die after being infected [2,3]. The PEDV genome is comprised of 5’UTR - replicase polyprotein 1a/b (ORF1a/b) - spike (S) - ORF3 - envelope (E) - membrane (M) - nucleocapsid (N) - 3’UTR [4]. The immune pressure from the vaccination causes mutations in the S gene to maintain the ability to evade the immune system [5]. Recent research has shown that the commercial vaccinations cannot provide adequate protection against epidemic strains due to genetic mutations [4]. These issues emphasize the necessity for alternative methods of disease control.

S protein is in control of attachment and fusion of the virus and host membrane, which allows the virus to enter host cells because it is the main protein on the viral surface, and it is an important target for antiviral drug discovery [6]. The S1 and S2 regions of S protein are the primary targets of neutralizing antibodies, and certain epitopes have been verified to be neutralizing [7,8]. At present, neutralization epitopes identified in S protein include COE (aa 499–638), SS2 (aa 748–755), SS6 (aa 764–771), and 2c10 (aa 1368–1374). Moreover, many epitopes have been identified, such as S1° (aa 1–219), E10E-1-10 (aa 435–485), S1B (aa 510–640), S1D (aa 636–789) and P4B-1 (aa 575–639) [4]. On one hand, immune pressure from vaccinations consistently alters the S protein to help it to evade the immune system [5]; on the other hand, vaccinations cannot offer sufficient protection against strains that have the frequent mutations in the neutralizing epitopes of epidemic strains. However, mutation of S protein could lead to antigenic drift and shift. Therefore, it is crucial to identify effective drugs to control PEDV infection [9].

Researchers around the world are increasingly interested in natural antiviral drugs derived from traditional Chinese medicine. Many natural compounds have been found to have antiviral properties to date [[10], [11], [12], [13], [14], [15], [16], [17], [18]]. Ginsenoside Rg1 derived from ginseng and notoginseng inhibits porcine circovirus type 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV) infection via NF-κB signaling pathway [11]. Apigenin inhibits protein synthesis and replication of African swine fever virus (ASFV) in a dose-dependent manner [15,16]. It has also been discovered that single components of traditional Chinese medicine have anti-PEDV activities. Monolaurin has anti-inflammatory and anti-PEDV activities by preventing virus replication and decreasing the level of expression of IL-6 and IL-8. Monolaurin can relieve diarrhea and enhance immune function by promoting the healing of intestinal villi [10]. Triacetyl Resveratrol efficiently inhibits PEDV replication by triggering the mitochondria-related caspase cascade and promoting apoptosis [12]. Puerarin prevents PEDV infection by modulating the interferon and NF-κB signaling pathway [17]. Wogonin has clinical therapeutic effects on animals and demonstrates inhibitory effects against PEDV variant strains by targeting the viral Mpro protein [18]. Baicalein and baicalin target 3C-like protease to suppress PEDV replication in Vero and LLC-PK1 cells, as indicated by reductions in viral RNA, protein, and titer [19]. In vitro assays, hypericum japonicum extract could directly inactivate PEDV strains; moreover, it inhibited the proliferation of PEDV strains in Vero cells at its non-cytotoxic concentrations [20].

Matrine (MT) is a naturally occurring alkaloid and belongs to important bioactive compounds derived from Chinese herbs such as sophorae flavescentis radix (SFR) and sophora flavescens [21]. MT has the advantages of known, distinct chemical structure [22]. In terms of quality control, single component provide several advantages over injections of traditional Chinese medicine [23]. According to pharmacological studies, sophorae flavescentis extracts (SFE) and constituent compounds are mostly alkaloids of quinolizidine type, including oxymatrine (OMT) and MT [24]. In previous studies, OMT was converted to MT in the intestines after oral administration [25]. MT has a variety of medicinal benefits, including sedative, anti-inflammatory, antibacterial, antiparasitic, antiviral, and anti-tumor characteristics [26]. Further research has revealed that MT dramatically reduces the expression of N protein in Marc-145 cells and prevents PRRSV-induced apoptosis by inhibiting activation of Caspase-3 [27]. MT exhibits antiviral activities by preventing viral replication and controlling immunological responses in mice after concurrent PRRSV and PCV2 infections [28].

The primary organizational system in medicine nowadays is organ-by-organ [29]. The symptom-centric nature of modern medicine and the concept of the one disease, one target, one drug severely hampers drug innovation [30]. Therefore, we need help understanding the molecular mechanisms of disease, which leads to treating the symptoms without ever achieving long-term healing of the disease [31]. Redefining diseases and shifting the focus from organ and symptom to mechanism and causation are fundamental and intellectual breakthroughs [30]. Network pharmacology focuses on treating the root causes of diseases rather than just the symptoms [[29], [30], [31]]. It will change the method by which diseases are identified, diagnosed, treated, and cured. Molecular docking is a computer technique based on structural design, which study the conformation and orientation (collectively together as the “pose”) of drug-protein into the binding domain of macromolecular target [[4], [5], [6], [7], [8], [9],32,33]. Network pharmacology and molecular docking are widely used in component-based Chinese medicine to predict bioactive components and clarify mechanisms of action [32].

In this work, antiviral effects and mechanism of action of MT were studied using Vero cells and IPEC-J2 cells by network pharmacology and experimental validation. We used computer-aided drug design (CADD) to analyze the pharmacophore with the help of the MT and PEDV protein binding sites discovered using molecular docking. The docking region “GLY434” of MT and S protein is conservative through sequence comparison. All of these findings motivate exploring the precise molecular processes based on the underlying anti-PEDV activity of MT.