Review Article

iMedPub Journals 2021 http://www.imedpub.com Archives of Clinical Microbiology Vol. 12 No. 2: 146

In vitro Screening of Anti-viral and Virucidal Leena Hussein Bajrai1,2*, Sherif Ali El-Kafrawy1,3, Rabie Effects against SARS-CoV-2 byHypericum Saleh Alnahas1 and Esam perforatum and Ibraheem Azhar1,3

1Special Infectious Agent Unit, King Fahd Abstract Medical Research Center, King Abdulaziz Special Infectious Agent Unit in King Fahd Medical Research Center at King University, Jeddah, Saudi Arabia Abdulaziz University, Jeddah, Saudi Arabia, has pursed the anti-viral project field 2Department of Biochemistry, King Abdulaziz to optimize the group of medicinal for human-infectious diseases. We have University, Jeddah, Saudi Arabia begun virtually in this field since COVID-19 pandemic, besides our divergence in 3Department of Medical Microbiology and the infectious agents. In this study and based on the previous review, Parasitology, King Abdulaziz University, perforatum (St. John’s Wort) and Echinacea (gaia HERBS®) were tested in vitro Jeddah, Saudi Arabia using Vero E6 cells for their anti-viral effects against the newly identified Severe Acute Respiratory Syndrome -2 (SARS-CoV-2) through its infectious cycle from 0 to 48 hours post infection. The hypericin (0.9 mg) of H. perforatum Corresponding author: and the different parts (roots, seeds, aerial) of two types of Echinacea species Leena Hussein Bajrai, Department of ( and ) were examined their efficacy in Biochemistry, King Abdulaziz University, certain concentration and under light-dependent anti-viral activities to measure Jeddah, Saudi Arabia the inhibition of the SARS-CoV-2 mRNA expression of RNA-dependent RNA polymerase (RdRP) gene and the viral load with quantitative real-time polymerase chain reaction (qRT-PCR), and to assess the neutralization of the SARS-CoV-2 spike  [email protected] receptor binding on cell culture assay. Interestingly, the mixture (H.E.) of 100 mg/ mL of H. perforatum and Echinacea was tested too on SARS-CoV-2 and showed crucial anti-viral activity competing H. perforatum then Echinacea effects as anti- viral treatment. Therefore, the results of gaia HERBS® products, H. perforatum and Citation: Bajrai LH, El-Kafrawy SA, Alnahas Echinacea species, applied in this study showed significant anti-viral and virucidal RS, Azhar EI (2021) In vitro Screening of effects in the following order of potency: H. perforatum, H.E., and Echinacea on Anti-viral and Virucidal Effects against SARS-CoV-2 infectious cycle; and will definitely require a setup of with SARS-CoV-2 by specific therapeutic protocol based on the outcome of this study. and Echinacea. Arch Clin Microbiol Vol.12 No.2:146. Keywords: SARS-CoV-2; Anti-viral assays;Hypericum perforatum; Echinacea

Received: February 15, 2020; Accepted: March 22, 2020; Published: March 28, 2020 Introduction lead ultimately to death [6]. Main protease (M pro), known as 3-chymotrypsin-like cysteine protease (3CLpro), has been (CoVs) are a large group of RNA viruses with considered as an important functional target in the viral life cycle, single-stranded RNA genomes that cause 30% of upper and and therefore as a candidate target for anti-viral against lower respiratory tract infections in humans. Four main human SARS-CoV-2 [7,8] due to its role in the release of functional coronaviruses (HCoVs) are known including severe acute polypeptides that is encoded by all HCoVs [9,10]. Updated respiratory syndrome SARS-CoV [1], HCoV-NL63 [2], HCoV-HKU1 reports about the disease management from COVID-19 have [3], and MERS-CoV in 2012 [4]. SARS-CoV-2 originated from targeted its structure, pathology, and mechanism in order to have Wuhan, Hubei Province of China and spread to the rest of the the best solution against the infection by both redesigning and world causing the global pandemic of COVID-19 with symptoms using classical anti-inflammatory agents and by targeting certain ranging from cough, fever, and dyspnea to neurological and cytokine of molecular , as per WHO recommendation [11]. vascular symptoms [5]. With advancement of the disease, For example, numbers of feasible treatment against SARS-CoV-2 intensive production of pro-inflammatory cytokines findings were proposed such as: neuraminidase inhibitors, Remdesivir, like: TNF- α, IL- 1β, IL-6, IFN- γ, CXCL10, MCP-1 resulting in Peptide (EK1), abidol, RNA synthesis inhibitors (TDF and 3TC), vasculitis, hyper-coagulability, and multi-organ damage which anti-inflammatory drugs (hormones and other molecules), and

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Chinese [12]. Furthermore, there were extracts reported the anti-inflammatory effects [30,40-42]. several studies about the synergy between antimalarial drugs, Another study in 2009 against H5N1 HPAIV strain showed that like Chloroquine-Hydroxychloroquine, Remdesivir-Favipiravir for the extract of E. purpurea interferes with the viral entry into cells the treatment of COVID-19 [13-15]. by blocking the receptor binding activity of the virus [43]. H. perforatum (family ), or St. John’s Wort (SJW) has The application of medicinal extracts is still a controversial been very well known for a long time as an effective medicinal issue especially the plant extracts found in different manufacturing plant for a range of communicable and non-communicable methods with varying use of plants parts and species. In this diseases such as depression, bacterial and viral infections, skin study, we evaluated the efficacy of H. perforatum and Echinacea wound, and inflammation [16,17]. studies as anti-viral agents against SARS-CoV-2 in vitro. The H. perforatum demonstrate the pharmacological efficacy against microbial and Echinacea species applied in this study included aerial parts infections [16,18,19]. As an anti-viral agent, H. perforatum with high concentration of hypericin (main component 0.9 mg) activities were assessed in vitro and in vivo on infectious and three different parts of type E. purpurea (root, seed, aerial bronchitis virus (IBV), C, HIV and Coronaviruses other parts) and one part of type E. angustifolia (root) (Tables 2 and 3). than SARS-CoV-2 [20,21]. Several Studies showed its activity Naphthodianthrones against Escherichia coli, Shigella dysenteriae, Salmonella typhi, (hypericin and pseudohypericin) Bacillus cereus, and several oral bacteria [22]. Because of H. Acylphloroglucinols perforatum's metabolites from each extracted part (roots, seeds (hyperforin and adhyperforin) and aerial), they are chemically defined to naphthodianthrones Flavonol glycosides (hypericin), phloroglucinols (hyperforin), flavonoid glycosides (quercetin, quercitrin, isoquercitrin, rutin, hyperoside, (hyperoside), biflavones, and anthocyanidins (Table 1) [19]. Aerial kampferol, leteolin, myricetin) parts Therefore, the hyperforin and hypericin of H. perforatum are Biflavones shown to be effective as antibacterial compounds against various (13,118-biapigenin, 139,118-biapigenin (amentoflavone)) Gram-positive bacteria but ineffective against Gram-negative Proanthocyanidins bacteria [23-26]. Furthermore, ethyl acetate extraction section of (procycanidin B2) Hypericum showed a significant reduction on the titer of IBV [16]. Phenyl propanes (chlorogenic acid and caffeic acid) [46,47] Proprietary extract blend Amount per E Herb Table 2: Components found in Hypericum perforatum. serving Equivalency St John's Wort (Hypericum Perforatum) 500 mg Hyperocins (0.9 Echinacea angustifolia root [44] Essential oil aerial parts, St John's Wott (Hypericum mg) Polyacetylenes Perforatum) Aerial parts extract Alkamides Organic Echinacea purpurea root, 2 mL 444 mg/Ml Alkaloids Organic Echinacea angustifolia root, Caffeic acid derivatives Organic Echinacea purpurea seed, Polysaccharides and glycoproteins Organic Echinacea purpurea Aerial Echinacea purpurea root [44] Essential oil parts Alkamides Alkaloids Table 1: The products materials used from gaia herbes. Caffeic acid derivatives Another medicinal plant, which was applied for many traditional Polysaccharides and glycoproteins and common remedies like curing cold and flu symptoms and Echinacea purpurea seed [45] Essential oil boosting immune system, is Echinacea. Echinacea is known Fatty acids with nine species of several plants in the genus of Echinacea, Echinacea purpurea aerial parts Essential oil however, only three of them were used as herbal complements: [44] Alkamides E. angustifolia, E. purpurea, and E. Pallida. Echinacea contains Flavonoids and anthocyanins Caffeic acid derivatives and chemical compounds liable for medicinal properties as: Caffeic phenolic acids acid derivatives, polysaccharides, flavonoids, ketones, and Polysaccharides lipophilic alkamides (Table 1) [27-34]. According to in vitro and in vivo studies, Echinacea showed effects on the cytokine Table 3: Components found in Echinacea. production [35,36], increasing the expression of CD69 [37], an impact on natural killer cells [38], as well as reducing illness Literature Review severity, in vivo, as anti-inflammatory therapeutic agents on Analytical discussion human monocytic THP-1 cells [39]. The latter is due toits efficacy on upper respiratory tract infections especially resulted COVID-19 pandemic is causing a global challenge to the world from E. purpurea root derived polysaccharide-containing water, economic and healthcare systems. The pandemic is responsible and thereby activates phosphatidylinositol-3-kinase (PI3K) of for the death of over 1.8 million confirmed cases and 85.5 following inhibition of TLR1/2 mediated stimulation of TNF-α million infections worldwide (WHO). Although some vaccines are production [39]. Also, alkyl amides and ketones of Echinacea developed and are now released under emergency use because

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of the pandemic, the efficacy of the vaccines is still debatable especially with the emergence of new variants in the genomic structure [48-51]. Several non-specific treatment options were evaluated and entered clinical trial including the repurposing of known treatments against other diseases [52]. Therefore, improvement and investigation of an effective anti-viral therapy is for treating SARS-CoV-2 infection. In our study, the anti-SARS-CoV-2 effect of the medicinal plants H. perforatum, Echinacea and their combination was evaluated. The medicinal plants were purchased from a commercial source Figure 2: Dose-response curve analysis by Graph pad software for to ensure consistency of the composition and were tested either (H) H. perforatum, (E) Echinacea and H.E. activities. The black curve individually or combined as a single treatment. Their mode of represents inhibition of SARS-CoV-2 infection (%) and the red dots represent log concentration (µg/mL). action was evaluated using three approaches namely direct treatment of virus–infected cells, pre-treatment of cells prior viral Our results demonstrated that the H. perforatum, containing infection and virucidal approaches. The results were evaluated 0.9 mg of hypericin, can significantly reduce SARS-CoV-2 viral using qRT-PCR assay relative to the virus control (virus added to load compared to the positive control (Figures 3 and 4) through the cells without the medicinal plants) and were observed under the viral infectious cycle from 0hr to 48 hours, and clearly this the microscope for CPE effect on the cells and by colorimetric reduction in the anti-viral (treatment of virus-infected cells and staining using crystal violet (Figure 1). pre-treatment of cells prior to virus infection) and virucidal assays (Figure 5).

Figure 1: The impact of H. perforatum, Echinacea and H.E. on SARS- CoV-2 infected-Vero E6 cells by anti-viral activity essays. The cell viability and CPE through SARS-CoV-2 infectious cycle, from 24 to 48 hours post infection, were visualized by photographed images with a light microscope (magnification x100) (orange squares) and by the crystal violet assay (blue circles). The images were captured when the CPE started on the positive control at 24 hrs.

The cytotoxic effect of the tested medicinal plants was evaluated using MTT assay with results presented as percent of cytotoxicity relative to cell control (cells with no added tested medicinal plants). The IC50 of H, E and H.E. mixture was as follows 7.724, Figure 3: DThe inhibitory effect of H. perforatum, Echinacea, and not detected and 8.202; respectively (Figure 2 and Table 4). H.E. on mRNA expression levels of SARS-CoV-2 RdRP gene in Vero E6 cells was evaluated by qRT-PCR. The impact of plant materials Medicinal plant IC50 (µg/ml) Maximum non-toxic was detected by the three experimental designs: Direct Treatment concentration (µg/ml) of Virus-infected Cells. 1A: Pre-treatment of Cells Prior to Virus Infection 1B: and Virucidal. 1C: assays; treated and infected cells H 7.724 1.56 were included as negative and positive controls, respectively. So, 100 E Nd 6.25 TCID50 of the virus was added with the certain concentrations of the HE 8.202 6.25 plant materials when the cells at 80% confluence were complete. The viral inhibition percentage was calculated relatively to virus Table 4: Cellular toxicity and maximum non-toxic concentrations of H. control wells, and representatives of two independent experiments perforatum, Echinacea and H.E. mixture against SARS-CoV-2 infection. performed in triplicate are shown. Statistical analysis showed that data were significant with p<0.05 and p<0.005 (one-and two-ways ANOVA).

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reducing the viral load, up to 48 hours, compared to Echinacea and H.E. mixture that had 3.30 and 31.36%, up to 36 hours, respectively. The anti-viral effect of the H. perforatum (with concentration of 1.56 g/mL) was higher than Echinacea and H.E. mixture (with concentration of 6.25 µg/mL for both), and the effect was noted at lower IC50 and a higher reduction in viral load of SARS CoV-2. H. perforatum showed the highest inhibitory effect of in all three anti-viral assays (Figure 6), while Echinacea showed the lowest inhibitory effect. The H.E. mixture showed lower inhibition than H. perforatum alone that would be expected as the active compounds were diluted by the addition of Echinacea.

Figure 4: The effect of H. perforatum, Echinacea, and H.E. on viral load of SARS-CoV-2 infection in Vero E6 cells was evaluated by qRT-PCR. The impact of plant materials was detected by the three experimental designs: Direct Treatment of Virus-infected Cells 1A: Pre-treatment of Cells Prior to Virus Infection 1B: and Virucidal 1C: Assays; treated and infected cells were included as negative and positive controls (PC), respectively. So, 100 TCID50 of the virus was added with the certain concentrations of the plant materials when the cells at 80% confluence were complete. The effect of the plant materials was observed clearly with reduction in viral load at 1.56, 6.25, and 6.25 μg/mL, respectively. The viral load was calculated relatively to virus control wells, and representatives of two independent experiments performed in triplicate are shown. Statistical analysis showed that data were significant with p< 0.005 (one-and two-ways ANOVA). Figure 6: The effect of H. perforatum, Echinacea, and H.E. on viral load of SARS-CoV-2 infection in Vero E6 cells was evaluated by qRT-PCR. The impact of plant materials was detected by the three experimental designs: Direct Treatment of Virus-infected Cells 1A: Pre-treatment of Cells Prior to Virus Infection ; 1B: Virucidal; 1C: assays, and Positive Control (PC). The effect of the plant materials was observed clearly with reduction in viral load at 1.56 μg/mL for H. perforatum, then for H.E. at 6.25 μg/mL, and finally for Echinacea at 6.25 μg/mL. Hypericins were reported to be the active anti-viral compounds in H. perforatum extract against IBV in vitro and in vivo [14]. The H. perforatum ethylacetate (HPE) extract includes: hyperoside, quercitrin, quercetin, pseudohypericin, and hypericin. The anti- viral effect of HPE was reported previously against bovine virus (BVDV) in vitro [52] and hepatitis C virus (HCV) in vivo [53] with hypericin of H. perforatum extract defined as the active Figure 5: Inhibitory effects of H. perforatum, Echinacea and H.E. on SARS-CoV-2 mRNA expression level of RdRP gene through infection ingredient. Other studies in 2001, 2009, and 2012 showed that in Vero E6 cells. H. perforatum extract had an anti-viral effect against A virus and HIV [54-56], suggesting that HPE could be developed Noticeably the results showed that the highest anti-viral effect and used as an anti-viral drugs. In addition, the anti-viral effect of was through the virucidal mechanism (Figures 4 and 6) compared H. perforatum extract and HPE had a remarkable decrease in the to the other two mechanisms with the highest inhibition concentration of IL-6 and TNF-α through the NF-κB in lung tissue observed at 36 hours after infection as shown in (Figure 3). While, of mice infected with influenza a virus [56] and in the trachea H. perforatum has the strongest inhibitory effect (35.77%) and 4 This article is available in: https://www.acmicrob.com/ 2021 Archives of Clinical Microbiology Vol. 12 No. 2: 146

and kidney for chickens infected with IBV, mainly from hypericin and Echinacea for patients in different-time administration. content [14]. Alternatively, H. perforatum is preferable single used as anti- viral treatment for the mild to severe case-patients because Echinacea, as indicated by the supplier insert is composed of two it demonstrated a high efficacy against SARS-CoV-2 infection. types (E. purpurea (root, seed, aerial parts) and E. angustifolia On the other hand, either Echinacea or H.E. is suggested to be (root)). The beneficial effects of Echinacea were reported admitted as prophylactic treatment with considering the dose, previously in both in vitro and in vivo studies as preventive side effects, drug interactions, and toxicity. and treatment for respiratory tract viruses due to its active components such as: polysaccharides, glycoproteins, caffeic acids, and alkamides [57-60]. 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