Plants as a prospective source of natural anti-viral compounds and oral vaccines against COVID-19 coronavirus

The proposal of novel drugs and approaches for effective treatment of the novel coronavirus is a necessity after the quick outbreak of the disease. Since the commencement of the coronavirus spread, enormous efforts have been made to protect, alleviate and cure the disease, though no specific treatment has been approved. While there have been convincing results in the use of chemical drugs and interferon therapy, such therapeutic approaches have various drawbacks and lack the required performance for the treatment of the new coronavirus. Medicinal plant species can provide a solution as a source of natural antiviral compounds by the accumulation of secondary metabolites and lectins as well as acting as a platform to express the viral immunogenic proteins. This study reviews the advantages and the results of previous research for the treatment of the novel coronavirus disease and previous generations of similar coronaviruses. Several plant-derived anti coronavirus compounds have been nominated that could be targeted for further research due to the similarity of the coronavirus disease in 2003 and the current coronavirus. This review regards plant species such as Scutellaria baicalensis (Baikal skullcap), and Utrica dioica (Stinging nettle) as suitable candidates for the new coronavirus antiviral research. Furthermore, the use of plants such as Nicotiana tabacum (Tobacco) for the expression of the coronavirus viral antigens can be a target for the future vaccinal research of the new coronavirus due to the efficiency of expression and intrinsic antiviral properties.


Introduction
The Coronaviridae family is composed of enveloped single-stranded RNA viruses that were normally regarded as the origin of types of common cold, upper respiratory disorders and lower respiratory infection in the case of the elderly and patients with a weak immune system. However, in 2003, the appearance of Severe Acute Respiratory Syndrome (SARS) caused by the coronavirus agent of SARS-Cov belonging to betacoronavirus subfamily, presented an urgent necessity of research about SARS related coronavirus. Nine years later another coronavirus appeared as Middle East Respiratory Syndrom (MERS) in Saudi Arabia with 2492 total laboratory-confirmed cases and 858 fatalities [1]. At present, The current outbreak caused by a new subtype of coronavirus named SARS-Cov2 has resulted in a pandemic with thousands of deaths and infected cases. This pandemic phenomenon is maintaining a sustained progression in the world, showing an emergency international alarm for finding an effective cure.
The SARS-Cov 2, SARS Cov and MERS Cov belong to the genera of the betacoronaviruses. At the first stage of the SARS-Cov coronavirus appearance, it was attempted to cure the disease by Ribavirin which revealed positive results in curing the patients. While drugs such as Aciclovir, Levofloxacin, Vancomycin, Caspofungin, and Meropenem were not able to show a significant relief in the patients [2]. The Lopinavir/Ritonavir with Ribavirin administration to the patients at the initial stages of the disease could reduce the mortality rate [3]. However, later it was confirmed that the Ribavirin treatment was associated with considerable toxicity and this approach was not successful to show compelling results in-vitro. Additionally, it was demonstrated by a recent study that the administration of Lopinavir/Ritonavir does not have a significant effect on the cases of COVID-19 [4]. The interferon therapy, the delivery of corticosteroid and immunoglobulins are the other methods that have been applied as the common treatment of the SARS-Cov. However, none of these approaches were successful in the treatment of SARS (2003). And new targeted anti-viral drugs were necessary. Therefore, it was sought to assess the structural properties of the SARS-related betacoronaviruses. Anand et al (2003) determined a protein with three domains and 304 residues as the main protease of the SARS-Cov and following by this study various research targeted this protease for inhibition of the SARS-Cov activity [6][7][8]. Later, this protease has been named 3C Like Protease (3CL Pro ). Papain-Like protease (PL Pro ) was demonstrated as another target enzyme for the symptoms caused by SARS-Cov.
While RNA-dependent RNA polymerase has a significant role in the coding of the genomic content of coronavirus upon infection, the 3CL Pro and PL Pro are regarded as key enzymes in the processing of the nonstructural proteins of the virus (nsps) which is thought to have a main role in the establishment of the viral replication (Figure1; [9,10]. The surface of SARS-Cov is covered with the spike proteins that are able to attach to human Angiotensin-Converting Enzyme 2 receptors (ACE2). ACE2 receptors are normally expressed in the lungs, kidney, endothelium, heart, and intestine [11]. Targeting the SARS-Cov spike proteins as well as the blockage of human ACE2 proteins can be an alternative way to block the activity of the SARS-Cov.  The current methods of new coronavirus treatment   A variety of the targets considering the inhibition of the RNA transcription, RNA modification, virus   packaging enzymes, the capsid, and the surface proteins assist the virus to diffuse into the cells and can be regarded as strategies to deactivate or prohibit the propagation of RNA virus in cells and tissues [12].
The current methods of COVID-19 treatment are composed of the administration of drugs such as Remdesivir, Chloroquine, Arbidol, and Favipiravir and approaches such as interferon therapy [12,13].
Currently, studies are focusing on further investigation of the biochemical materials that could inhibit the main proteases of the virus or the compounds that could inhibit the propagation rate of the virus in the cells. The plants provide an ultimate, natural source of enzyme and viral propagation inhibitors to be implicated as a treatment method of disorders caused by SARS-Cov and SARS-Cov2.
Fortunately, there is a considerable similarity between the SARS-Cov and SARS-Cov2 virus which is more than 80% identity and 96% similarity of the genome ]15 ,14 [ . It has been confirmed that the PL Pro and 3CL Pro of the SARS-Cov and SARS-Cov2 are conserved [16]. Additionally, it is observed that there is a 76.10% identity between the mentioned viruses [17]. Based on this similarity, it is expected that the results of the studies about the SARS-Cov could be implied for the research in the SARS-Cov2 to a high degree.  Table 1 shows a selection of the approaches based on the plants, mentioning the representative agents based on our literature review that could have the value of further research for the COVID-19 disease. It is attempted to further discuss these strategies in the following sections.

Utrica dioica agglutinin lectin
Stinging nettle (Utrica dioica) has been used in various countries as traditional medicine for years. This plant has been reported to have therapeutic effects on cardiovascular, immunity, neuronal and digestive systems [36]. The agglutinin lectin is extracted from the rhizomes of the common stinging nettle.
Previously it was indicated that from the lectins that have been extracted from the selective plant species, the Allium porrum Agluttinin (APA) of Leek, Utrica dioica Agglutinin (UDA) of stinging nettle and NICTABA lectin of Tobacco (Nicotiana tabacum) was able to show the highest inhibition tendency to prohibit the proliferation rate of the virus to 50% (EC50) at less than 1.3 µg/ml pure extract with low toxicity in-vitro [19]. Similarly, further studies confirmed that the UDA has an impact on the inhibition of the SARS-Cov virus in-vitro [20]. Experiments have proposed that the lectins will interrupt the viral attachment and the effect of lectins has the highest performance when they are delivered at the early stages of the infection cycle [19]. Later the antiviral effect of the UDA has been assessed in-vivo in Bulb C mouse models and it was found that the UDA significantly decreased the mortality caused by SARS and reduced the weight loss [21]. This study also proposed that the UDA prevents the virus attachment by inhibition of the SARS-CoV spike (S) glycoprotein [21]. The evidence from the previous research shows that UDA can be a suitable compound for further research in the COVID-19 arena.
The secondary metabolites of the plants

Glycyrrhizin
Glycyrrhizin is the major component in the licorice (Glycyrrhiza glabra) root. This compound has been used traditionally for the treatment of gastritis, bronchitis, and jaundice and is reported to have antioxidant and anti-inflammatory activity that can stimulate the formation of interferons in the body [37]. It has been proposed that Glycyrrhizin can decrease the attachment of the SARS-Cov agents to the cells especially during the initial phase of the viral infection cycle [39]. Glycyrrhizin is composed of Flavonoids, Glycyrrhetinic acid, β-sitosterol and hydroxyl coumarins [37] and has been observed to have a significant anti-SARS-Cov activity by Cinatl et al (2003). Later Pilcher (2003)  Interferon-beta 1a and considerably low toxicity concentration for the cell lines in-vitro. The anti-viral activity of this compound was found so important that later a grant for the patent of anti-SARS drug production with this biochemical compound was approved [26]. The in-vitro assessment of the extracted flavonoids from the Scutellaria baicalensis also showed significant anti-viral activity on the Lipopolysaccharide activated cells while the oral administration of the compound significantly increased the survival rate of influenza A virus-infected mice [25].  [28,29]. It was also noticed that quercetin 3-β-D-glucoside has the potential to inhibit the 3CL Pro of the MERS-Cov [47]. This compound is found to have low toxicity to the cells in-vitro, and it is one of the major natural components that is targeted for the treatment of the COVID-19 disease.

Other plant metabolites and products
Scutellarein is another ample natural flavone in the Scutellaria genus. This compound is observed to have the anti-viral, anti-inflammatory and antioxidant ability. As this metabolite is also discovered in This study proposes that the antiviral activity of the extracts is due to the presence of Lycorine in the L.radiata plant [49]. Hesperetin is another flavonoid that is observed to have an inhibitory effect on the SARS-Cov 3CL Pro [50]. Hesperetin is normally found in the species of citrus (Lemons) genus in-vitro.    [72], studies have mainly focused on S and N proteins. The genome of S1 or N protein can be mounted to a vector and be delivered to the host organism for transcription, expression, and accumulation of the viral antigen [74]. The introduction of the new piece of codon to the plant will finally produce a transgenic plant that is able to express and accumulate the viral N or S proteins in the inner segments and organelles of the plant cells of plant tissue. The general overview of the required considerations and challenges for the production of plant vaccines (mainly in seed oral vaccines) has been discussed previously by our group [74]. There are mainly two types of SARS-Cov virus viral antigen proteins that have been aimed to be expressed in the recombinant plant platform, S1 and N proteins.
The expression of N protein N protein is the nucleocapsid protein of the SARS-Cov ( Figure 2). It is demonstrated that the antibodies against the N and S protein were clearly detected in the patients of the early stages of SARS-Cov disease [34]. It is also discovered that the SARS-Cov N protein was able to induce both temporal and longterm memory T-cell response [33]. For this reason, the N protein is regarded as one of the effective inductors of the humoral and cellular responses in the SARS-Cov contamination process [33]. Studies The expression of S1 protein The expression of the spike glycoprotein of the SARS-Cov is followed by the creation of a large polypeptide that is finally cleaved by proteases that are encoded by the host or virus to S1 and S2 subunits. S1 is the peripheral part of the virus that is believed to contain the main receptor-binding domain (RBD) on the N-terminal part. It is noticed that the antibodies against S1 protein are strongly efficient in the RBD interaction. Therefore, it is aimed to target the S1 genes to the part of the cells and organelles within the plants such as tobacco, tomato and lattice platforms.
The previous research for expression of the S1 protein in the plants proved compelling evidence of the plant's potential to be used as a bioreactor for expression of SARS-Cov viral antigen proteins. The S1 proteins were expressed in the cytosol [30] or chloroplast [31] with an accumulation rate of up to a maximum of 10.2% of Total Soluble Protein (TSP). The oral delivery of the S1 proteins in the platform of tomato resulted in a significant IgA increase while the injection of the S1 protein from the dried root of tobacco resulted in a significant detection of the IgG in western blot and ELISA results [32].
On the whole, the transgenic plants have this potential to provide an approach for the production of plant vaccines against COVID-19 disease. However, the usage of plant cells as a bioreactor is subjective to several ethical and environmental considerations. As this process will result in the generation of a new transgenic plant, it is probable that the propagation of the plant species as the result of the uncontrolled breeding might affect the species within the ecosystems, create an imbalanced niche or even cause poisoning to the species that might consume the plant randomly. Consequently, the transgenic plant culture should be done under restricted control [74].

Controlling the plants' anti-viral compounds expression via environmental conditions
Based on the fact that the expression of the lectins and secondary metabolites of the plants are highly dependent on the environmental conditions. Alternatively, it is possible to increase the expression of an anti-viral compound within the plant by changing the environmental circumstances to induce a more stressful condition for the plant. As mentioned earlier in the article some plant species such as tobacco can express anti-SARS lectins that tend to inhibit viral propagation [19]. It is also found that the expression of the lectins in the species such as tobacco can be increased by the presence of pathogens, pest insects and other biochemical materials that could induce the immunogenic response in the plant species. In this case, it is possible to increase the effective lectin content for a higher yield in case of the attitude for non-oral delivery or to increase the effective dose in case of oral delivery [74]. For this reason, the type of pest or invader species [75], the variations in the stress environment [76] and the biochemical (such as Jasmonates) compounds [77] can alter the expression of the lectins in the plants especially in the expression of Nictaba of tobacco. It is worth mentioning that the application of the plant biotechnology technics can also provide the ability of the overexpression of the lectin genes such as UDA and Nictaba agglutinin to provide a higher yield and dosage of the anti-viral compound in the plants.
The effectivity of the anti-viral plants' oral delivery In this review, we have recommended various plants that can have the anti-SARS Cov2 potential.
However, the oral delivery of such compounds might be further advantageous as oral delivery is facile and non-invasive. In the case of the usage of plants as the vaccines for expression of the anti-SARS Cov agents, it was observed that the oral delivery of vaccines to animal model was able to induce the mucosal immune responses [32,33]. Meanwhile, the oral delivery of the plant baicalin was shown to successfully reduce the mortality in the influenza-infected mice [25]. However, the question is whether the direct oral delivery of the compounds will be also effective in terms of having antiviral activities. It was confirmed by several studies that most of the plant anti-SARS agents can inhibit the connection of the viral agents to the cells [39]. Therefore it can be postulated that most plant anti-viral agents might apply a protective behavior against the entrance and propagation of the viral agents in the host body. In this case, adding the edible plant species that produce anti-SARS-Cov compounds such as leek, onion, garlic Furthermore, tobacco contains NICTABA lectin which is a strong antiviral agent. Overal, more studies are needed in order to further assess the anti-viral ability of plant species.