A comprehensive review on the effect of plant metabolites on coronaviruses: focusing on their molecular docking score and IC50 values

a Food Science Institute, Kansas State University, Manhattan, Kansas, USA b Student Research Committee, Department of Food Science and Technology, National Nutrition and Food Technology Research Institute, Faculty of Nutrition Science and Food Technology, Shahid Beheshti University of Medical Sciences, Tehran, Iran c Department of Food Science and Technology, National Nutrition and Food Technology Research Institute, Faculty of Nutrition Science and Food Technology, Shahid Beheshti University of Medical Sciences, Tehran, Iran


Introduction
Coronaviruses are considered as a family of RNA viruses that have helical nucleocapsids and extremely large genomes. Nucleotides in the coronavirus genomic RNA, determines the structural and nonstructural proteins of the virus. Some of the nonstructural proteins have pivotal role in viral RNA synthesis (replicase-transcriptase proteins), and some of them are unnecessary for virus replication but seem to have a selective benefit in vivo (niche-specific proteins). In this study we aimed to do a review on the effect of the metabolites derived from plants, on different kind of coronaviruses. Binding affinity of these compounds to a specific ligand or receptor of a target protein in virus is studied by molecular docking assay in several studies. A compound which has a lower binding energy is considered as a potential drug candidate (Chandel et al., 2020). Also IC50 value of these metabolites which is half maximal inhibitory concentration against virus is determined in different studies. Plant metabolites that are reviewed in this paper for their anti-coronavirus effects, are secondary metabolites including polyphenols (such as flavonoids, coumarins, stilbenes), alkaloids, terpenoids, organosulfur compounds saponins, saikosaponins, lectins, essential oils, nicotianamine and primary metabolites such as vitamins. 6 Polyphenols are mostly found in plant kingdom and there are many varieties of them. Flavonoids account for a large part of polyphenolic compounds. These compounds have many health benefits due to their antioxidant activity. There are also some studies on their properties, other than antioxidant activity, such as their antimicrobial, antibacterial, antifungal and antiviral activities (Tapas et al., 2008;. In addition, there are various types of flavonoids with inhibitory effect against several enzymes such as alpha-glucosidase, and alpha-amylase (Tadera et al., 2006). In this way these compounds can display their antiviral effect. Molecular docking (MolDock) simulation is used to calculate the binding of flavonoids to these enzymes.

Method
In docking studies, the higher negative value of docking score is related to the stronger inhibitory ability of the compound. In a study by Nguyen et al., recombinant 3CLpro-a chymotrypsin-like cysteine protease which is vital to virus replication-was expressed in Pichia pastoris GS115 and was used for investigating the effect of seven flavonoids including quercetin, epigallocatechin gallate (EGCG), gallocatechin gallate (GCG), epigallocatechin (EGC), ampelopsin (AMPLS), puerarin, and daidzein on it. Among these flavonoids GCG had the strongest inhibitory effect followed by EGCG, quercetin, AMPLS, diadzein, puerin and EGC. GCG indicated several interaction with amino acid residues in the active site of 3CLpro. Also galloyl moiety at the 3-OH position of EGCG and GCG, seems to be responsible for their strong 3CLpro inhibitory effect which is not present in AMPLS and EGC. Molecular docking simulation (affinity of the compounds to bind specific receptor on enzyme) indicated that the substrate-binding pocket of 3CLpro is the site that GCG bounds. Daidzein and puerarin, showed little inhibition of 3CLpro.
The 3CLpro inhibitory effect of quercetin was 4.96-times higher than AMLSP. The difference between the structures of these polyphenols is the cause for their different inhibition activity.
EGC, by having a Hydroxyl group at 5´-position of the B ring, and lacking galloyl moiety and 4 7 and 2,3-double bounds, has the lowest inhibitory activity. IC50 of all of the mentioned compounds are shown in (Table. 1). Also inhibitory percentage for AMPLS, EGC, GGC, G, diadzein, puerarin, quercetin, and EGC was 34%, 85%, 91%, 34%, 33%, 82%, and 5.4%, at 200 µM concentration of them, respectively (Table. 2) (Nguyen et al., 2012). Hesperidin (a flavanone glycoside richly found in the citrus and citrus peel), rutin (an essential nutritional compound mostly found in tea and apples), diosmin (flavonoid mostly found in citrus fruit), apiin (flavonoid in parsely), diacetyl curcumin (synthetic derivative of curcumin), were also displayed as potential inhibitors against protease of COVID-19. According to MolDock binding score, hesperidin had the strongest affinity to block the protease of COVID-19, followed by rutin, diosmin, apiin and diacetylcurcumin (Table. 1). However, all of the mentioned compounds had acceptable binding ability which supports the prospective use of these compounds at first steps of treatment for COVID-19. It can be concluded from this study that consumption of citrus fruits, cherries and apples has the potential to increase immunity to fight against COVID-19 infections naringenin (flavanone), and daidzein (isoflavone), were examined for their inhibitory effects on 8 the SARS-CoV 3CLpro. Cell-free and cell-based assays were conducted. Only aloeemodin and hesperetin had inhibitory effect on cleavage activity of the 3CLpro in cell-free and cell-based assays which was dose-dependently. These two compounds have shown to have considerably high inhibitory effects on SARS-CoV 3CLpro with IC50 of 132 and 60 µM in cell-free assay, respectively. In cell based assay the IC50 values were 366 and 8.3 µM, respectively. Also diadzein had IC50 of 105 µM in cell-free assay but its IC50 was not significant in cell-based assay (Table. 1). Aleoemodin had inhibitory percentage of 65% at 100 µM and for hesperetin and daidzein it was around 82% and 72%, respectively (Table. 2) (Lin et al., 2005). In another study, hesperetin (which was investigated to have anti-SARS activity, was studied for its ability to inhibit Angiotensin-converting enzyme 2 (ACE2), and also its anti-2019-nCoV activity.
Results indicated that hesperetin has the ability to bind to ACE2 with the binding energy of -8.3 kcal/mol (  (IP), free theaflavin (TF1), theaflavin-3 monogallate (TF2A), theaflavin-3´ monogallate (TF2B), and theaflavin-3,3´ digallate (TF3)), were tested for their antirotaviral activity. TF1, TF2A, TF2B, and TF3 were the most active compounds having the mean effective concentration (EC50) of 0.125 mg/ml and TF2A had the least effect with an EC50 of 251.39 mg/ml against SARS-CoV (Clark et al., 1998). In a study, anthraquinone, emodin and rhein (anthraquinone compounds), chrysin (a flavonoid compound) which are produced in Rheum and Polygonum plants, and promazine which is also a phenolic compound with a similar structure to emodin, were investigated for their inhibitory effect against binding S protein (spike protein) to ACE2.
There are several studies on health effects of these compounds (Huang et al., 1991;Chen et al., 2002;Kumar et al., 1998). Results of this study demonstrated that besides disrupting the viral envelope (Sydiskis et al., 1991), emodin and promazine has the potential to block the S protein of SARS-CoV and ACE2 interaction by competing the binding site of S protein with ACE2. On the other hand anthraquinone and 1,4-bis-(1-anthraquinonylamino)-anthraquinone was found to have lower effect on inhibition of the S protein and ACE2 interaction showing that the anthraquinone skeleton don't influence the S protein and ACE2 binding and the side chain has the main role in their inhibitory activity. Chrysin and rhein also had slightly inhibitory effect on mentioned binding with inhibitory percentage of less than 20% at 200 µM concentration.
Findings of this study showed that emodin and promazine have the most effect against SARS-COV S protein activity with inhibitory percentage of 50% and 55% at concentration of 200 µM, respectively (Table. 2) (Ho et al., 2006). Baicalein is mainly extracted and purified from the Chinese medicinal plant named Scutellaria baicalensis Georgi. The MolDock binding result 10 showed that baicalein have strong binding to the ACE2 enzyme, with binding energy of -8.46 kcal/mol (Table. 1), and the potential binding sites are HIS-505, ASN-149, ARG-273. Based on the anti-SARS activity of this compound and its ability to bind to ACE2, it was stated that baicalein can be a strong candidates for 2019-nCoV treatment (Chen and Du. 2020). Scutellarin is another flavone which indicated wide-ranging pharmacological effects. An in vivo study indicated that this compound is able to reduce the expression and activity of ACE in brain tissue and it was reported that it has IC50 value of 48.13 ± 4.98 μM against ACE (Wang et al., 2016;Wang and Ma, 2018). In other study by conducting a molecular docking it was found that scutellarin is able to bind to ACE2, with binding energy of -14.9 kcal/mol, and the binding sites were GLU-495, UNK-957, ARG-482 (Table. 1). It was stated that this compound can be good candidate for having anti-2019-nCoV activity (Chen and Du. 2020). As mentioned before nsP13, the SARS-CoV helicase, is also vital for viral replication and is regarded as a drug target for SARS-CoV inhibitors. In a study baicalein, scutellarin and myricetin which are natural flavonoids were studied for their inhibitory effect on nsP13. It was shown that baicalein is a strong inhibitor of the ATPase activity of nsP13 protein (with inhibition percentage of 100% at 10 μM (Table. 2)), and its pharmacological activity to inhibit ATPase activity of nsP13 is greater than that of myricetin and scutellarin. IC50 value of baicalein was shown to be 0.47 ± 0.09 μM which was lower than two other flavonoids (Table. 1) (Keum et al., 2013). In another in vitro study, the effect of EGCg which is the major active compound of tea polyphenol with different biological activities on inhibition of the bovine coronavirus (BCV) replication in Madin-Darby bovine kidney (MDBK) cells was explored. At concentrations of less than 10 μg/mL, EGCg didn't have any cytotoxicity to MDBK cells. Results of this study showed that EGCg has temperature-dependent effect on BCV and its effect at around 37°C (temperature of intestinal 11 tract) was better than the temperature of respiratory tract. These results displayed that EGCg has very good anti-BCV activity, it interacts with BCV particles and interferes with the adsorption of BCV to MDBK cells, thus EGCg can be considered as a suitable anti-BCV compound (Matsumoto et al., 2005). In another study, in which secondary metabolites of different medicinal herbs were investigated, results showed that they have inhibitory effects against novel COVID-19 protease. The, dialloyl sulfide from garlic, curcumin in turmeric spices, capsaicin in peppers, limonene and cineol in cardamom, coumarin in liquorice, verbascoside in hedge Nettle, and glucuronic acid in tragacanth were the phenolic compounds in this study that were shown to have inhibitory effect against novel COVID-19 protease. Among these compounds, curcumin had a stronger bond and high affinity with COVID-19 protease (Table. 1 (Table. 1). In this study the binding position of quercetin-3-β-galactoside for two types of SARS-CoV 3CLpro (the wild-type 3CLpro and its mutated type (3CLpro Q189A)), was also compared and the IC50 of quercetin-3-β-galactoside on SARS-CoV 3CLpro Q189A was considerably decreased to 127.89 ± 10.06 μM because of the decrease in binding affinity (Table. 1). Moreover, the inhibition percentage of this compound against SARS-CoV 3CLpro, was 41.8% at 50 μM concentraion (Table. 2). Also new derivatives of the quercetin-3-b-galactoside with some chemical variations to the binding components were devised and the results were like this: 1. Detaching the hydroxy groups of the quercetin moiety which considerably decreases the inhibitory activity 2. Addition of a large sugar on 7-hydroxy of quercetin which can be tolerated 3. Acetoxylation of sugar moiety which stops the inhibitory activity, and 4. Substitution of the galactose moiety with sugars, such as fucose which increased the inhibitory activity (2-fold), arabinose, and glucose, that had no remarkable effect on inhibitory activity (Chen et al., 2006). In other study bioflavonoids extracted from the Torreya nucifera leaves were investigated as possible anti-SARS-CoV 3CLpro. Of the isolated components, amentoflavone was documented as strong inhibitor, showing IC50 value of 8.3 μM.
Also the three flavones (apigenin, luteolin, and quercetin) used as positive control for bioflavonoids and indicated IC50 value of 280.8, 20.2, and 23.8 μM, respectively (Table. 1). It was shown that there are interactions between the C5 hydroxyl group of amentoflavone with the nitrogen atom of the imidazole group of His163 and OH group of Leu14, with two hydrogen bonds. These groups belong to S1 site of 3CLpro. In addition, there are hydrogen bonds between the hydroxyl group in the B ring of amentoflavone and Gln189 which belongs to S2 site of 13 3CLpro. Also interactions with Val186 and Gln192 can be considered as one of the major bindings with the target site. inhibition percentage results are present in Table. 2 (Ryu et al. 2010). In a study, 720 natural compounds was investigated for their inhibitory effect against 3CLpro. Also the 3CLpro-inhibitory effect of extracts from different kinds of teas, such as black tea, oolong tea, Puer tea and green tea was investigated. Results showed that their inhibitory activities against 3CLpro of Puer and black tea extracts were greater than that of green or oolong tea extracts. Also it was indicated that (-)-epigallocatechin gallte (EGCg), catechin (C), epicatechin gallate (ECg), epicatechin (EC), theophylline (TP), epigallocatechin (EGC), and caffeine are not able to have 3CLpro inhibitory activity. Black tea polyphenols (TF1, TF2 and TF3) also displayed inhibitory activity against 3CLpro. TF3 was the most abundant polyphenol in black tea, followed by TF2A ,TF2B, and TF1. This study has discovered that TF2B, TF3 and tannic acid are effective three compounds to inhibit 3CLpro and their IC50 are less than 10 μM, but TF2A was not investigated due to its unavailability (Table. 1 (Table. 1). Moreover, the active chalcones were 2-fold more effective when tested with cell-14 based cis-cleavage assay in compare to the cell-free trans-cleavage assay. Inhibition percentage results are present in Table. 2 (Park et al., 2015). The open-reading-frame 3a of SARS coronavirus has been shown to code a protein which forms a channel with cation-selective ability that can be expressed in the infected cell. The mechanism of virus release is related to the activity of the channel. In a study the flavanols such as kaempferol, kaempferol glycosides, and acylated kaempferol glucoside derivatives were examined for their ability to block the 3a channel. Glycoside juglanin (with an arabinose residue) was found to be the most effective component to block 3a channel which had an IC50 value of 2.3 μM (Table. 1). Kaempferol derivatives which had a rhamnose residue also was likely to be quite effective in blocking 3a channel. As a conclusion, this study found out that more bioavailable compounds such as emodin and kaempferol (specifically, the glycosides of kaempferol) could be a foundation for the new antiviral drugs exploration. These compounds not only can block the 3a channel, but also they can interfere with other stages of the viral life cycle (Kaul et al., 1985) and this is an important feature to consider them as a potent antiviral agent (Schwarz et al., 2014). In another in vitro study the inhibitory activities of 64 different natural compounds were investigated against SARS helicase (nsP13). Scutellarin and myricetin showed to restrain the SARS-CoV helicase protein by influencing the ATPase activity, but they didn't have any effect on unwinding activity (inhibition of helicase activity). The IC50 values of myricetin and scutellarein were 2.71 ± 0.19 µM and 0.86 ± 0.48 µM, respectively. Moreover, the inhibitory percentage of these compounds is 100% at approximately 10 µM concentration of them (Table. 2) (Yu et al., 2012). Stilbene compounds can also be classified as polyphenols and they have a lot of biological activities.
These derivatives are assumed to be phytoalexins. In a study (E)-stilbene derivatives with hydroxyl groups were synthesized and in some of them pyridine ring was used in place of one    for these compounds and glide scores results for herbacetin, rhoifolin, and pectolinarin were -9.263, -9.565 and -8.054, respectively. Herbacetin had high binding affinity around the S1 and S2 sites and this was found to be due to the presence of additional 8-hydroxyl group. Moreover, carbohydrate groups present in rhoifolin and pectolinarin compounds were found to occupy the S1 and S2 sites and this was shown to be another cause of high affinity of these glycosylated flavonoids to SARS-CoV 3CLpro (Jo et al., 2020).

Alkaloids
Plants Emotin is one of the major alkaloids that is found in ipecacuanha or ipecac root. In other study In a study it was indicated that tryptanthrin (a plant alkaloid) which is among the major active compounds in S. cusia leaf is able to inhibit HCoV-NL63 replication independently. Findings showed that tryptanthrin has a greater antiviral activity against HCoV-NL63 in compare to indigodole B which has an extra ethyl moiety at C5a in the place of double bond in tryptanthrin.
In addition, in particular, tryptanthrin changes the antigenic configuration of viral S proteins and in this way it inhibits the PLP2's cleavage activity (with IC50 of less than 0.1 μM). Therefore, tryptanthrin can be used as one of the successful compounds against human coronaviruses.

Saponins and saikosaponins
Glycyrrhizin is another phytochemical belong to saponins, which is derived from Chinese Medicine herb licorice root (Glycyrrhiza radix). Glycyrrhizin was shown to be a good treatment for SARS by influencing the viral adsorption and penetration before (Cinatl et al., 2003). In other study docking results demonstrated that glycyrrhizin has the potential to bind to ACE2 with binding energy of -9 kcal/mol, and the binding sites were ARG-559, GLN-388, ARG-393, ASP-

Organosulfur Compounds
Garlic essential oil is very good source of organosulfur compounds which are known for their therapeutic properties. Also these compounds are expected to interact with ACE2 protein's amino acids. In a study, researchers investigated organosulfur compounds' effect against SARS-CoV-2. Results of MolDock study showed that allyl disulfide and allyl trisulfide which are the most abundant compounds in garlic essential oil, have the best binding affinity to the 6LU7 protein of SARS-Cov-2, followed by diallyl tetrasulfide. Trisulfide, 2-propenyl propyl and diallyl tetrasulfide was found to have the best interaction with ACE2 receptor.
Moreover, organosulfur compounds in garlic showed stronger interaction with 6LU7.
Generally, Results exhibited that the essential oil extracted from garlic is a precious natural antivirus source (Thuy et al., 2020).

Lectin
Lectins are proteins which can bind to carbohydrates to form glycoproteins. They can agglutinate cells and in this way lead to precipitation of glycoconjugates (Vasconcelos and Oliveira, 2004).
In addition to this, they can make binds with carbohydrates, reversibly. Lectin proteins have been showed to make hydrophobic and hydrogen bonding and van der Waals interactions (Singh, et al., 1999

Herb extracts and essential Oils
Essential oils (EOs) have widely used for thrapeutic purposes due to their antiviral, antimicrobial  nM (Li et al., 2005). However, it has been verified that vitamin A has immune supporting roles such as cytokine expression, antibody production, cell killing like macrophages or monocytes ( Specifically, vitamin B2 (riboflavin) is essential for energy metabolism of cells (Powers, 2003).

Micronutrients and phytonutrients found in fruits and vegetables mainly vitamins (vitamin
It had been reported that vitamin B2 may decrease the titer of MERS-CoV within human plasma (Keil et al., 2016). Moreover, vitamin B3 (nicotinamide) might be used as a treatment for ventilator induced lung injuries by inhibiting the neutrophil infiltration of the lungs, but conversely it can cause hypoxemia (Jones et al., 2015). Generally, it has been approved that vitamin B groups can be selected as a supporting treatment for COVID-19 and deficiencies in this vitamin can cause weakness in human's immune system (Zhang and Liu, 2020).
Moreover, it has been found that vitamin C may also be used as treatment for respiratory diseases (Hemilä, 2017). Ascorbic acid (water soluble vitamin C) plays a vital role in collagen synthesis and it is accepted as natural antioxidant. It is also known as protector against respiratory infections such as coronavirus (Hemilä, 2003). It has been reported that vitamin C had the effect to enhance resistance of chick embryo tracheal organ cultures against coronavirus infection (Atherton et al., 1978). Furthermore, trials on humans have been revealed that vitamin C usage has declined respiratory tract infections. Therefore, it can be deduced that vitamin C may decrease respiratory tract symptoms in recent COVID-19 infection (Zhang and Liu, 2020).
Fat soluble vitamin E containing tocopherols and tocotrienols also has significant effect to decrease oxidative stress by binding to free radicals due to its antioxidant property (Galmés et al., 2018). It has been reported that vitamin E deficiency might lead to myocardial injuries

Nicotianamine
Nicotianamine (a Fe chelator in plants) which is rich in soybean was investigated for its inhibitory effect on ACE2 and was shown to inhibit the ACE2 activity, with IC50 value of 84 nM (Takahashi et al., 2015). Molecular docking of nicotianamine was conducted to determine its affinity to ACE2 enzyme, it was shown that binding energy of nicotianamine to ACE2 is -5.1 kcal/mol (Chen and Du. 2020).