Natural Products from Medicinal Plants with Anti-Human Coronavirus Activities

molecules

Review Natural Products from Medicinal Plants with Anti-Human Coronavirus Activities

Salar Hafez Ghoran 1,* , Mohamed El-Shazly 2, Nazim Sekeroglu 3 and Anake Kijjoa 4,*

1 Department of Chemistry, Faculty of Science, Golestan University, 15759-49138 Gorgan, Iran 2 Department of Pharmaceutical Biology, Faculty of Pharmacy and Biotechnology, The German University in Cairo, 11835 Cairo, Egypt; [email protected] 3 Department of Horticulture, Faculty of Agriculture, Kilis 7 Aralik University, 79000 Kilis, Turkey; [email protected] 4 ICBAS-Instituto de Ciências Biomédicas Abel Salazar and CIIMAR, Rua de Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal * Correspondence: [email protected] (S.H.G.); [email protected] (A.K.); Tel.: +98-9144425047 (S.H.G.); +351-962712474 (A.K.)

Abstract: Since the emergence of severe acute respiratory syndrome caused by coronavirus 2 (SARS- CoV-2) first reported in Wuhan, China in December 2019, COVID-19 has spread to all the continents at an unprecedented pace. This pandemic has caused not only hundreds of thousands of mortalities but also a huge economic setback throughout the world. Therefore, the scientific communities around the world have focused on finding antiviral therapeutic agents to either fight or halt the spread of SARS-CoV-2. Since certain medicinal plants and herbal formulae have proved to be effective in treatment of similar viral infections such as those caused by SARS and Ebola, scientists have

paid more attention to natural products for effective treatment of this devastating pandemic. This review summarizes studies and ethnobotanical information on plants and their constituents used

Citation: Hafez Ghoran, S.; for treatment of infections caused by viruses related to the coronavirus family. Herein, we provide El-Shazly, M.; Sekeroglu, N.; Kijjoa, A. a critical analysis of previous reports and how to exploit published data for the discovery of novel Natural Products from Medicinal therapeutic leads to ﬁght against COVID-19. Plants with Anti-Human Coronavirus Activities. Molecules 2021, 26, 1754. Keywords: COVID-19; SARS-CoV; phytochemicals; medicinal plants; ACE2; enzyme inhibitors https://doi.org/10.3390/ molecules26061754

Academic Editor: Cédric Delattre 1. Introduction On 12 February 2020, the emergence of a severe respiratory infectious disease was Received: 23 February 2021 announced by the World Health Organization (WHO), known as Human Coronavirus Accepted: 18 March 2021 Published: 21 March 2021 Diseases 2019 (COVID-19) [1]. In the following weeks, thorough phylogenetic investigations revealed the exact identity of the virus and its similarity to the previous severe acute

Publisher’s Note: MDPI stays neutral respiratory syndrome coronavirus (SARS-CoV) with 89.1% nucleotide similarity and also with regard to jurisdictional claims in more than 95% homology with key drug targets. The new lethal and highly infectious published maps and institutional afﬁl- virus was designated as SARS-like coronavirus type 2 (SARS-CoV-2) [2,3]. To date, four iations. human coronaviruses (HCoV) have been identiﬁed viz. HCoV-OC43; β-CoV, HCoV-229E; α-CoV, HCoV-NL63, and HCoV-HKU1 that are responsible for SARS-CoV diseases [4]. The fact that SARS-CoV-2 has high phylogenic similarity to HCoV-OC43 and HCoV-229E, the main causative agents of the common cold, has guided scientists in their search for new anti-SARS-CoV-2 compounds [5]. Moreover, it was found that the angiotensin-converting Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. enzyme 2 (ACE2) in the SARS-CoV-2 host receptor is the same as that in the SARS-CoV This article is an open access article host receptor. Therefore, targeting ACE2 can be one of potentially promising approaches to distributed under the terms and hamper the current pneumonia outbreak [6–8]. In addition, previous studies highlighted conditions of the Creative Commons the importance of using drugs to inhibit enzymes involved in SARS-CoV replication such Pro Attribution (CC BY) license (https:// as SARS-CoV 3C-like protease (SARS-CoV 3CL ) and papain-like protease (SARS-CoV Pro creativecommons.org/licenses/by/ PL ). These enzyme inhibitors stop the virus from expressing key replicative enzymes 4.0/). such as RNA-dependent RNA polymerase (RdRp) and helicase (Figure1)[9–11].

Molecules 2021, 26, 1754. https://doi.org/10.3390/molecules26061754 https://www.mdpi.com/journal/molecules Molecules 2021, 26, x FOR PEER REVIEW 2 of 29 Molecules 2021, 26, 1754 2 of 28 (SARS-CoV PLPro). These enzyme inhibitors stop the virus from expressing key replicative enzymes such as RNA-dependent RNA polymerase (RdRp) and helicase (Figure 1) [9–11].

FigureFigure 1.1. GeneralGeneral overviewoverview ofof inhibition of severe acute respiratory syndrome coronavirus (SARS- CoV)CoV) replication.replication.

OnOn thethe otherother hand,hand, pathologicalpathological findingsfindings ofof COVID-19COVID-19 revealedrevealed thatthat approximatelyapproximately 20%20% ofof patientspatients are are suffering suffering from from an an acute acute respiratory respiratory distress distress syndrome syndrome (ARDS), (ARDS), which which is sparkedis sparked by by a “cytokine a “cytokine storm”, storm”, causing causing an uncontrolledan uncontrolled inflammation. inflammation. This This phenomenon phenome- isnon associated is associated with with various various inflammation-related inflammation-related cytokines, cytokines, chemokines chemokines such such asas inter-inter- leukinsleukins (IL)-1(IL)-1ββ,, IL-6,IL-6, IL-7,IL-7, IL-8,IL-8, IL-9,IL-9, IL-17,IL-17, tumortumor necrosisnecrosis factorfactor (TNF)-(TNF)-αα,, interferoninterferon (IFN)-(IFN)-γγ,, granulocyte-colonygranulocyte-colony stimulatingstimulating factorfactor (G-CSF),(G-CSF), granulocyte-macrophagegranulocyte-macrophage colony-colony- stimulatingstimulating factor (GM-CSF), (GM-CSF), and and chemokine chemokine ligands ligands 2, 3, 2, and 3, and 4 (CCL2-CCL4), 4 (CCL2-CCL4), which which have havebeen beenobserved observed in plasma in plasma obtained obtained from fromindividuals individuals suffering suffering from fromCOVID-19 COVID-19 and even and evenworse worse in dying in dyingpatients patients [12]. It [is12 worth]. It is mentioning worth mentioning that even that interferons even interferons (IFNs), including (IFNs), includingIFN-α, -β, IFN-and α-γ,-, haveβ, and been -γ, recognized have been recognizedas potential asagents potential for treatment agents for of treatmentSARS. Cinatl of SARS.et al., Cinatlin their et experiment al., in their experimentwith recombinant with recombinant interferons interferonstoward two toward clinical two strains clinical of strainsSARS-CoV-FFM-1 of SARS-CoV-FFM-1 in Vero and in VeroCaco2 and cells, Caco2 have cells, found have that found IFN- thatα significantly IFN-α significantly inhibited inhibitedthe replication the replication of SARS-CoV, of SARS-CoV, however, however, its selectivity its selectivity index (SI) index was (SI) 50–90 was 50–90times timeslower lowerthan that than of that IFN- ofβ IFN-. Despiteβ. Despite the inactivity the inactivity of IFN- ofγ IFN- in Caco2γ in Caco2 cell cultures, cell cultures, it showed it showed a better a betteractivity activity than IFN- thanα IFN- in Veroα in cell Vero cultures cell cultures [13]. [13]. TheThe increasingincreasing dailydaily infectionsinfections andand mortalitymortality ratesrates causedcaused byby COVID-19,COVID-19, combinedcombined withwith thethe lack lack of of effective effective medicines, medicines, call call for for researchers’ researchers’ urgent urgent attempts attempts to tackle to tackle this seri-this ousserious problem. problem. One ofOne the of approaches, the approaches, besides besides anti-inflammation anti-inflammation and immune-nanomedicine and immune-nano- strategiesmedicine [strategies12,14], is focusing[12,14], is on focusing medicinal on medicinal plants and plants their secondaryand their secondary metabolites metabo- since itlites is wellsince recognized it is well recognized that they have that playedthey have a crucial played role a crucial in the role discovery in the discovery of potential of anti-infectivepotential anti-infective agents. One agents. of the One most of efficient the most strategies efficient instrategies counteracting in counteracting SARS-CoV SARS- could beCoV the could use of be natural the use products of natural that products are able that to prevent are able viral to prevent adhesion. viral These adhesion. compounds These potentiallycompounds interact potentially with interact the receptor-mediated with the receptor-mediated recognition recognition and consequently and consequently block the interactionblock the interaction process between process viruses between and viruses host cells. and Anotherhost cells. alternative Another alternative could be enhancing could be theenhancing immune the system immune by takingsystem various by taking kinds various of plant kinds products of plant andproducts their relatedand their prepara- related tions like dietary supplements [15,16]. The importance of natural products to fight against preparations like dietary supplements [15,16]. The importance of natural products to fight SARS-CoV is reflected in a review article by Kumar et al. that describes synthetic and against SARS-CoV is reflected in a review article by Kumar et al. that describes synthetic naturally occurring compounds of different scaffolds that were patented from 2008–2013 and naturally occurring compounds of different scaffolds that were patented from 2008– for their anti-SARS-CoV activities with different mechanisms of action. Natural products 2013 for their anti-SARS-CoV activities with different mechanisms of action. Natural prod- patented in this period as SARS-CoV 3CLpro inhibitors include kaurane diterpenes pseurata ucts patented in this period as SARS-CoV 3CLpro inhibitors include kaurane diterpenes A(1) and liangshanin A (2), flavonols quercetagenin (3) and robinetin (4), and pentacyclic pseurata A (1) and liangshanin A (2), flavonols quercetagenin (3) and robinetin (4), and triterpenoid quinone methides celatrol (5) and tingenone (6) (Figure2)[17]. pentacyclic triterpenoid quinone methides celatrol (5) and tingenone (6) (Figure 2) [17].

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Figure 2. Some natural products patented as anti-SARS-CoV agents from 2008–2013. Figure 2. Some natural products patented as anti-SARS-CoV agents from 2008–2013. The initial outbreak of COVID-19 in Wuhan quickly became a worldwide pandemic and causedThe initial hundreds outbreak of thousands of COVID-19 of lives in Wuhan and economic quickly setbacksbecame a throughout worldwide thepandemic world. Dueand tocaused the genetic hundreds similarity of thousands of SARS-CoV-2 of lives an andd economic the previously setbacks emerged throughout SARS-CoV the world. viz. CoVDue HCoVNL63,to the genetic HCoV-HKU1 similarity of and SARS-CoV-2 Middle East and respiratory the previously syndrome emerged coronavirus SARS-CoV (MERS- viz. CoV),CoV HCoVNL63, this review describes HCoV-HKU1 the exploration and Middle of some East medicinalrespiratory plants, syndrome most ofcoronavirus which are used(MERS-CoV), in traditional this medicine,review describes and their the secondary exploration metabolites of some for medicinal treatment plants, of the diseasesmost of causedwhich are by SARS-CoV.used in traditional The search medicine, engines and used their for thissecondary review metabolites are Web of Science,for treatment Scopus, of PubMed,the diseases and caused Google by scholar. SARS-CoV. The secondary The search metabolites engines coverused afor diverse this review array of are structural Web of classes.Science, Overall,Scopus, PubMed, the review and discusses Google scholar. the structures The secondary and mechanisms metabolites underlying cover a diverse their activitiesarray of structural of ca. one classes. hundred Overall, naturally the re occurringview discusses phytochemicals the structures with and anti-SARS-CoV mechanisms activitiesunderlying reported their activities to date. of ca. one hundred naturally occurring phytochemicals with anti-SARS-CoV activities reported to date. 2. Medicinal Plants with Anti-HCoV Properties 2. MedicinalThe development Plants with of newAnti-HCoV antiviral Properties drugs is an urgent issue to pursue because of life- threateningThe development viral diseases of new such antiviral as Ebola, drugs SARS, is andan urgent MERS. issue Many to plantspursue have because produced of life- phytochemicalsthreatening viral with diseases great such potential as Ebola, to combat SARS, theseand MERS. diseases. Many For plants example, have Traditional produced Chinesephytochemicals Medicine with (TCM) great specialists potential haveto comb describedat theseToona diseases. sinensis ForReom example, (also Traditional known as CedrelaChinese sinensis Medicine, Family (TCM) Meliaceae), specialists a tree have commonly described found Toona in sinensis Taiwan, Reom China, (also and known Malaysia as thatCedrela produced sinensis various, Family phenolicMeliaceae), compounds a tree commonly and sterols, found for in its Taiwan, beneficial China, effects and against Malay- HCoV-229E.sia that produced The tender various leaf phenolic extract compounds of this plant an showedd sterols,in vitrofor itsinhibitory beneficial effects effects towardagainst SARS-CoVHCoV-229E. replication The tender with leaf SIextract of 12–17 of this [18 plant]. In vitroshowedantiviral in vitro assays inhibitory of extracts effects of toward some medicinalSARS-CoV plants replication including withCimicifuga SI of 12–17 racemosa [18]. In(L.) vitro Nutt. antiviral (Ranunculaceae), assays of extractsCoptis chinensis of some Franch.medicinal (Ranunculaceae), plants including MeliaCimicifuga azedarach racemosaL. (Meliaceae),(L.) Nutt. (Ranunculaceae),Phellodendronamurense Coptis chinensisRupr. (Rutaceae),Franch. (Ranunculaceae),Sophora subprostrata Melia azedarachChun and L. (Meliaceae) T.Chen. (Fabaceae),, Phellodendron and amurensePaeonia suffruticosa Rupr. (Ru- Andrewstaceae), Sophora (Paeoniaceae) subprostrata have Chun revealed and theirT.Chen. potential (Fabaceae), as anti-SARS-CoV and Paeonia suffruticosa candidates An- for furtherdrews (Paeoniaceae) clinical studies have [19]. revealed Due to thetheir deficiency potential ofas therapeuticanti-SARS-CoV medicines, candidatesHouttuynia for fur- cordatather clinicalThunb. studies (Saururaceae) [19]. Due to was the indicated deficiency to of deal therapeutic with SARS medicines, symptoms Houttuynia because cordata of its traditionalThunb. (Saururaceae) use to relieve was lung-related indicated to abnormalities deal with SARS such symptoms as lung abscess, because mucus, of its cough, tradi- andtional pneumonia use to relieve [20]. lung-related Experimental abnormalities results showed such that as thelung aqueous abscess, extract mucus, of cough,H. cordata and + + increasedpneumonia the [20]. proliferation Experimental of CD4 resultsand showed CD8 lymphocytic that the aqueous T cells, extract whereas of H. the cordata levels in- of inflammation cytokines, IL-2 and IL-10, were effectively ameliorated. SARS-CoV 3CLPro creased the proliferation of CD4+ and CD8+ lymphocytic T cells, whereas the levels of inactivity was also inhibited by administration of the H. cordata extract [21,22]. Since out of 200 flammation cytokines, IL-2 and IL-10, were effectively ameliorated. SARS-CoV 3CLPro ac- Chinese medicinal herbs including Lycoris radiata Herb. (Amaryllidaceae), Artemisia annua tivity was also inhibited by administration of the H. cordata extract [21,22]. Since out of 200 L. (Asteraceae), Pyrrosia lingua (Thunb.) Farw. (Ploypodiaceae), and Lindera aggregata (Sims) Chinese medicinal herbs including Lycoris radiata Herb. (Amaryllidaceae), Artemisia annua Kostem (Lauraceae) have been documented for their moderate to potent antiviral activities L. (Asteraceae), Pyrrosia lingua (Thunb.) Farw. (Ploypodiaceae), and Lindera aggregata against SARS-CoV, with half maximal effective concentrations (EC ) ranging from 2.4 to (Sims) Kostem (Lauraceae) have been documented for their moderate50 to potent antiviral activities against SARS-CoV, with half maximal effective concentrations (EC50) ranging

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88.2 µg/mL, these plants have been proposed for anti-SARS-CoV remedies [23]. Another plant with various antiviral properties is Echinacea purpurea (L.) Moench (Asteraceae), whose preparation is also known as Echinaforce®, which was evaluated for its in vitro anti- SARS-CoV-2 activity [24]. The results showed that treatment with this preparation was able to irreversibly inhibit HCoV-229E activity with a half-maximal inhibitory concentration (IC50) of 3.2 µg/mL. Interestingly, this preparation is also effective against other highly pathogenic CoVs, such as those causing SARS and MERS [25]. It is important to mention also that chamomile (Anthemis hyalina DC.) flowers, black cumin (Nigella sativa L.) seeds, and Citrus sinensis L. Osbeck (Rutaceae) peels are commonly used as herbal remedies in traditional medicines in many cultures for treatment of a variety of human diseases. Ulasli et al., in their evaluation of the effects of the ethanol extracts of these three medicinal plants on the CoV replication and the expression of the transient receptor potential (TRP) genes during CoV infection, have found that treatment of HeLa-CEACAM1a (HeLa-epithelial carcinoembryonic antigen-related cell adhesion molecule 1a) cells with these plant extracts prior to infection with MHV-A59 (mouse hepatitis virus-A59) decreased the replication of the virus. Although all the extracts had an effect on IL-8 secretion, TRP gene expression and viral load after coronavirus infection, it was the flower extract of Anthemis hyalina DC. that showed the most significant difference in viral load [26]. A survey of medicinal plants on SARS and SARS-like infectious diseases revealed that Scutellaria baicalensis Georgi, Bupleurum chinense DC., and Gardenia jasminoides J. Ellis. could significantly prevent acute infection in SARS patients [27]. Following the immunotherapy approach, Sultan et al. proposed that garlic (Allium sativum L. fam.), ginger (Zingiber officinalis), Hypericum perforatum L., Camellia sinensis (L.) Kuntze, N. sativa L., and liquorice (Glycyrrhiza glabra L.) could be used for COVID-19 prevention since they are well-known immune enhancer plants [28]. Other examples of medicinal herbs that have potential for treatment or alleviate COVID-19 symptoms are presented in Table1. Molecules 2021, 26, 1754 5 of 28

Table 1. Anti-Human Coronavirus (anti-HCoV) activities of some medicinal plants.

Plant Name Family Part Used Ref. Toona sinensis Reom. Meliaceae Leaves [18] Cimicifuga racemosa (L.) Nutt. Ranunculaceae Rhizome Melia azedarach L. Meliaceae Barks Coptis chinensis Franch. Ranunculaceae Rhizomes [19] Phellodendron amurense Rupr. Rutaceae Barks Sophora subprostrata Chun & T.Chen. Fabaceae Seeds Paeonia suffruticosa Andrews Paeoniaceae Whole plant Houttuynia cordata Thunb. Saururaceae Aerial parts [20] Lycoris radiata Herb. Amaryllidaceae Stem cortex Artemisia annua L. Asteraceae Whole plant [23] Pyrrosia lingua (Thunb.) Farw. Ploypodiaceae Leaves Lindera aggregata (Sims) Kostem Lauraceae Roots Echinacea purpurea (L.) Moench Asteraceae Aerial parts [24] Anthemis hyalina DC. Asteraceae Flowers Nigella sativa L. Ranunculaceae Seeds [26] Citrus sinensis L. Osbeck Rutaceae Peels Astragalus mongholicus Bunge Fabaceae Leaves Atractylodis macrocephalae Koidz. Asteraceae Rhizome Atractylodes lancea (Thunb.) DC. Asteraceae Rhizome Glycyrrhizae uralensis Fisch. Ex DC. Fabaceae Roots Saposhnikovia divaricata (Turcz. Ex Ledeb.) Schischk. Apiaceae Flowers [29] Lonicerae japonica Thunb. Capripoliaceae Fruits Forsythia suspensa (Thunb.) Vahl Oleaceae Leaves Platycodon grandifiorus (Jacq.) A.DC. Campanulaceae Roots Agastache rugosa (Fich. & C.A.Mey.) Kuntze Lamiaceae Aerial parts Cyrtomium fortunei J. Sm. Dryopteridaceae Leaves Allium sativum L. fam. Alliaceae Bulbs Camellia sinensis (L.) Kuntze Theaceae Leaves [28] Zingiber officinalis Zingiberaceae Roots Hypericum perforatum L. Hypericaceae Aerial parts Scutellaria baicalensis Georgi. Lamiaceae Aerial parts Bupleurum chinense DC. Apiaceae Aerial parts [27] Gardenia jasminoides J. Ellis. Rubiaceae Leaves Stephania tetrandra S. Moore Menispermaceae Leaves [30] Rauwolfia spp. Apocynaceae - [31] Aesculus hippocastanum L. Sapindaceae Seeds Rheum officinale Baill. Polygonaceae Roots [32] Polygonum multiflorum Thunb. Polygonaceae Roots Scutellaria spp. Lamiaceae Aerial parts [33] Paulownia tomentosa (Thunb.) Steud. Paulowniaceae Fruits [34] Torreya nucifera L. Siebold & Zucc. Lamiaceae Leaves [35] Chamaecyparis obtusa var. formosana Cupressaceae Heartwood Juniperus formosana Hayata. Cupressaceae Heartwood [36] Cryptomeria japonica (L.f.) D.Don Cupressaceae Heartwood Rhus Chinensis Mill. Anacardiaceae Fruits [37] Laurus nobilis L. Lauraceae Berry Thuja orientalis L. Cupressaceae Fruits [38] Juniperus oxycedrus ssp. oxycedrus Cupressaceae Berry Euphorbia neriifolia L. Euphorbiaceae Leaves [39] Glycyrrhiza glabra L. Fabaceae Roots [40] Bupleurum spp. Apiaceae Aerial parts Heteromorpha spp. Apiaceae Aerial parts [41–44] Scrophularia scorodonia L. Scrophulariaceae Aerial parts The same background color refers to the same reference(s). Molecules 2021, 26, x FOR PEER REVIEW 6 of 29

The same background color refers to the same reference(s).

3. Plant Secondary Metabolites with Anti-HCoV Properties For practicality, the anti-HCoV compounds are categorized according to their chemical classes while the mechanisms of actions and their docking studies are also discussed for each class of compounds when available.

3.1. Alkaloids During a screening for antiviral activities of Chinese medicinal herb extracts against SARS-CoV, using MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4- sulfophenyl)-2H-tetrazolium inner salt) assay for virus-induced cytopathic effect (CPE), Li et al. have found that the extract of Lycoris radiata Herb. showed the most potent effect with EC50 value of 2.4 μg/mL. Chemical investigation of the active fraction resulted in the isolation of the alkaloid lycorine (7) (Figure 3). Compound 7 displayed a strong inhibitory Molecules 2021, 26, 1754 effect against SARS-CoV with EC50 value of 15.7 nM and SI more than 900. It is worth6 of 28 mentioning that the authors have found that the isolated compound was more potent against SARS-CoV than the commercially available lycorine (EC50 = 48.8 nM) (Table 2) [23]. Bis-benzylisoquinoline alkaloids tetrandrine (8), fangchinoline (9), and 3.cepharanthine Plant Secondary (10) Metabolites(Figure 3), isolated with Anti-HCoV from Stephania Properties tetrandra S. Moore and other members Forof the practicality, family Menispermaceae, the anti-HCoV compounds also displayed are categorized anti-HCoV according activities. to their Since chemical HCoV- classesOC43 is while not only the most mechanisms closely related of actions to SARS-CoV and their dockingand MERS-CoV studies arebut alsoalso discussedshares several for eachfunctional class of properties compounds with when both available. of them, Kim et al. used HCoV-OC43-infected MRC-5 fi- broblasts, derived from human lung tissue, as a model to evaluate antiviral activities of 8- 3.1.10 and Alkaloids to elucidate their underlying mechanisms. They found that treatment with 8-10 Gdose-dependentlyDuring a screening increased for antiviral cell viability activities against of Chinese HCoV-OC43 medicinal infection. herb extracts Compound against 8 SARS-CoV,displayed the using most MTS potent (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4- antiviral activity with an IC50 value of 295.6 nM, whereas 9 and sulfophenyl)-2H-tetrazolium10 were less active presenting inner IC50 values salt) assay of 919.2 for virus-inducedand 729.7 nM, cytopathicrespectively. effect Therefore, (CPE), Lithe et SIs al. of have 8–10 found were that higher the extractthan 40, of 11,Lycoris and 13, radiata respectively.Herb. showed It was the also most found potent that effect anti- withHCoV-OC43 EC50 value activities of 2.4 µ ofg/mL. 8–10 Chemicalwere dose- investigation and time-dependent of the active and fraction that they resulted are most in the ef- isolationfective at of the the early alkaloid stage lycorine of the HCoV-OC43 (7) (Figure3). life Compound cycle, and 7thedisplayed timing of a treatment strong inhibitory is more effectimportant against to protect SARS-CoV against with virus-induced EC50 value ofcell 15.7 death nM than and the SI moreduration than of 900. cell exposure It is worth to mentioningthe compounds. that Compounds the authors have8–10 also found inhibited that the the isolated replication compound of HCoV-OC43 was more as potentwell as againstthe expression SARS-CoV of the than nucleocapsid the commercially (N) and available spike (S) lycorine proteins (EC (Table50 = 48.8 2) [30]. nM) (Table2)[ 23].

FigureFigure 3.3. StructuresStructures ofof lycorinelycorine ((77),), tetrandrinetetrandrine ((88),), fangchinolinefangchinoline ( 9(9),), and and cepharanthine cepharanthine ( 10(10).).

BisBy-benzylisoquinoline using a cell-based alkaloidsassay, with tetrandrine SARS-CoV (8), and fangchinoline Vero E6 cells, (9), and for cepharanthinea screening of (existing10) (Figure drugs,3), isolated natural from products,Stephania and syntheti tetrandrac S.compounds Moore and to other identify members effective of theanti-SARS family Menispermaceae,agents, Wu et al. alsohave displayed found that anti-HCoV among more activities. than Since10,000 HCoV-OC43 agents screened, is not onlyaround most 50 closely related to SARS-CoV and MERS-CoV but also shares several functional properties with both of them, Kim et al. used HCoV-OC43-infected MRC-5 ﬁbroblasts, derived from human lung tissue, as a model to evaluate antiviral activities of 8-10 and to elucidate their underlying mechanisms. They found that treatment with 8-10 Gdose-dependently increased cell viability against HCoV-OC43 infection. Compound 8 displayed the most potent antiviral activity with an IC50 value of 295.6 nM, whereas 9 and 10 were less active presenting IC50 values of 919.2 and 729.7 nM, respectively. Therefore, the SIs of 8–10 were higher than 40, 11, and 13, respectively. It was also found that anti-HCoV-OC43 activities of 8–10 were dose- and time-dependent and that they are most effective at the early stage of the HCoV-OC43 life cycle, and the timing of treatment is more important to protect against virus-induced cell death than the duration of cell exposure to the compounds. Compounds 8–10 also inhibited the replication of HCoV-OC43 as well as the expression of the nucleocapsid (N) and spike (S) proteins (Table2)[30]. By using a cell-based assay, with SARS-CoV and Vero E6 cells, for a screening of existing drugs, natural products, and synthetic compounds to identify effective anti-SARS agents, Wu et al. have found that among more than 10,000 agents screened, around 50 compounds were active at concentration of 10 µM, among which was the existing drug reserpine (11) (Figure4), an indole alkaloid produced by several members of Rauwolﬁa spp. MoleculesMolecules2021 2021, 26, 26,, 1754 x FOR PEER REVIEW 7 of7 29 of 28

compounds were active at concentration of 10 μM, among which was the existing drug reserpine (11) (Figure 4), an indole alkaloid produced by several members of Rauwolfia (Apocynaceae).spp. (Apocynaceae). Compound Compound11 displayed 11 displayed an EC an50 ECof 3.450 ofµ 3.4M, andμM, aand SI ofa SI 7.3. of Since7.3. Since11 has 11 beenhas been used used clinically, clinically, the authors the authors speculated speculated that that its related its related natural natural products products may may also also be activebe active against against SARS-CoV SARS-CoV (Table (Table2). By 2). using By us theing Internationalthe International Species Species Information Information System Sys- (ISIS)tem (ISIS) database database to search to forsear commerciallych for commercially available availabl compoundse compounds whose structures whose structures have 80% similarityhave 80% tosimilarity11, they to have 11, foundthey have that found six compounds, that six compounds, i.e., 12–17 (Figurei.e., 12–417), were(Figure related 4), were to 11related. However, to 11.12 However,–14 displayed 12–14 the displayed minimal the concentration minimal concentration of inhibition of toward inhibition SARS-CoV toward muchSARS-CoV higher much than higher reserpine than (11 reserpine), ranging (11 from), ranging 10–20 fromµM, 10–20 whereas μM, the whereas values the for values15–17 werefor 15 ca.–17 100 wereµM[ ca.31 100]. μM [31].

FigureFigure 4.4.Structures Structures ofof reserpinereserpine (11(11)) and and its its analogs analogs12 12––1717. .

3.2.3.2. AnthraquinonesAnthraquinones TargetingTargeting thethe interactioninteraction ofof SARS-CoVSARS-CoV spikespike (S)(S) proteinprotein withwith ACE2ACE2 isis ofof particular particular interestinterest inin thethe searchsearch for for anti-SARS-CoV anti-SARS-CoV agents.agents. ByBy screeningscreening ChineseChinese medicinalmedicinal herbs,herbs, supervisedsupervised by by the the Committee Committee on on Chinese Chinese Medicine Medicine and and Pharmacy Pharmacy in Taiwan, in Taiwan, Ho et Ho al. haveet al. foundhave found that three that widelythree widely used Chinese used Chinese medicinal medicinal herbs viz.herbs root viz. tubers root tubers of Rheum of Rheum officinale of- Baill.ficinale and Baill.Polygonum and Polygonum multiflorum multiflorumThunb., Thunb., vinesof vinesP. multiflorum of P. multiflorumThunb., Thunb., all belonging all belong- to theing familyto the family Polygonaceae, Polygonaceae, inhibited inhibited the interaction the interaction of S protein of S protein with ACE2with ACE2 of SARS-CoV of SARS- with IC50 values ranging from 1 to 10 µg/mL. Since emodin (18) (Figure5) is a major CoV with IC50 values ranging from 1 to 10 μg/mL. Since emodin (18) (Figure 5) is a major constituent of these herbs, this compound was evaluated for the interaction of S protein constituent of these herbs, this compound was evaluated for the interaction of S protein with ACE2. The results showed that 18 blocked the binding of S protein to ACE2, in a with ACE2. The results showed that 18 blocked the binding of S protein to ACE2, in a dose-dependent manner, with an IC50 value of 200 µM. Compound 18 also blocked the dose-dependent manner, with an IC50 value of 200 μM. Compound 18 also blocked the binding of S protein of SARS-CoV to Vero E6 cells. When compared to promazine (the binding of S protein of SARS-CoV to Vero E6 cells. When compared to promazine (the positive control), the percent inhibition of 18 was 94.12 ± 5.90% at 50 µM, whereas that of positive control), the percent inhibition of 18 was 94.12 ± 5.90% at 50 μM, whereas that of promazine was 95.6 ± 7.7% at 5 µM. Hence, the anti-SARS-CoV activity of both compounds promazine was 95.6 ± 7.7% at 5 μM. Hence, the anti-SARS-CoV activity of both com- was not due to their toxicity [32]. Compound 18 was also shown to inhibit an ion channel pounds was not due to their toxicity [32]. Compound 18 was also shown to inhibit an ion 3a protein, encoded by an open reading frame ORF-3a, in the infected cells whose activity channel 3a protein, encoded by an open reading frame ORF-3a, in the infected cells whose may influence virus release [45]. The ORF-3a is also known as “New gene” localized between “spike and envelope gene” (SNE), and has been identified in other coronaviruses including the SNE of HCoV-OC43 that shows similar ion-channel characteristics to the 3a

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protein of SARS-CoV [46]. This new observation, together with the ﬁnding that 18 may disrupt the interaction of S protein and ACE2 [32], supports the suggestion that 18 or its derivatives may become potent new therapeutic agents for treatment of SARS and other Molecules 2021, 26, x FOR PEER REVIEWcoronavirus-induced diseases. 9 of 29

FigureFigure 5.5. Structures ofof emodinemodin ((1818),), ﬂavonoids,flavonoids, andand ﬂavonoidsflavonoids glycosidesglycosides19 19––2828..

3.3. FlavonoidsJo et al. used and a Flavonoid synthetic Glycosides peptide labelled with an Edans-Daabcyl FRET pair to search for SARS-CoVDuring the 3CL investigationpro inhibitors of the against phenolic-containing a flavonoids lib Chineserary consisting herbs used of for ten the different preven- tionscaffolds. of SARS Among during 64 its compounds, outbreaks intested China, at Lin20 μ etM, al. only examined herbacetin the anti-SARS-CoV(31), rhoifolin (32 3CL) andpro effectpectolinarin of extracts (33) of (Figure some Chinese6) were herbsfound and to exhibit herb-derived prominent phenolic inhibitory compounds activity, using with a cell-IC50 freevalues cleavage of 33.17, and 27.45 cell-based and 37.78 cleavage μM, respectively, assay. The results at a concentration showed that a ranging flavanone from hesperetin 2 to 320 (μ19M.) (FigureTo avoid5) dose-dependentlya non-specific inhibition inhibited of th theese SARS-CoVflavonoids caused 3CL Pro bycleavage aggregation activity through with ancomplexity, IC50 value the of assays 8.3 µM. in However,the presence the of capacity 0.01% Triton of 19 toX-100 inhibit were SARS-CoV-2 performed resulting replication in hasa better never inhibitory been investigated activity than (Table those2)[ with47].out On using the other Triton hand, X-100. baicalin Since SARS-CoV (20) (Figure 3CL5), apro flavonecontains glycoside one tryptophan isolated residue from a Chinese (Trp-31) medicinal at the catalytic plant Scutellaria domain, spp.a general (Lamiaceae), tryptophan- was foundbased assay to display method an antiviralwas used activityto independently against the confirm prototype the inhibitory strains (39849) activity of SARS-CoVof the three inflavonoids. foetal rhesus As kidney-4the three (fRhK-4)flavonoids cell decrease line withd anthe EC emission50 value ofintensity, 12.5 µg/mL the authors in 48 h, con- and againstcluded that10 strains there wasof SARS-CoV a complex withformation EC50 betweenvalues ranging the catalytic from domain 12.5-25 µandg/mL these in flavo- 48 h, withnoids. SI The values interaction higher thanbetween 4 to SARS-CoV 8 [48]. Both 3CL20 [pro33 ]and and the scutellarin three inhibitory (21) (Figure flavonoids5) showed were aanalyzed neuroprotective to predict effect the binding in addition affinities to a by disruption induced-fit of docking the interaction study. The of S docking protein study with ACE2of kaempferol [49]. Luteolin (34) and (22) morin and quercetin (35) (Figure (23) (Figure6) were5), also naturally performed occurring and compared flavones, were with alsoherbacetin found to(31 exhibit) to find anti-SARS-CoV structural features activity that through confer avid good interaction affinity withof 31 the. The surface results S proteinshowed of that a wild-type these compounds SARS-CoV, share and the subsequently kaempferol interfering (34) motif. the The viral docking entry study into Vero further E6 µ cellsrevealed with that EC50 thevalues phenyl of 10.6moiety and of 83.4 34 occupiesM, respectively the S1 site (Table of SARS-CoV2)[50]. through a hydrogen bondAnother with potential Glu-166, target whereas is a its viral chromen-4-one helicase. Since scaffold this enzyme locates in is essentialthe S2 site. for For viral 31, genome replication, it is currently considered as a potential target for antiviral drug devel- there are four hydrogen bonds formed within a distance of 2.33 Å in the S2 site. However, opment. Thus, in an attempt to find natural compounds with inhibitory activity against the major bonding force was due to the presence of the hydroxyl group on C-8 that binds with Glu-166 and Gln-189. These bindings were predicted to confer a good glide score (- 9.263) to 31. Due to the lack of the hydroxyl group on C-8, 34 and 35 have two hydrogen bonds less than 31. On the other hand, the binding modes of 32 and 33 are different from those of 34 and 31. Since 32 and 33 possess α-L-rhamnopyranosyl-β-D-glucopyranoside and α-L-mannopyranosyl-β-D-glucopyranoside at C-7 of the chromen-4-one scaffold, there is no hydrogen bond between the hydroxyl group on C-7 and the backbone of Ile-

Molecules 2021, 26, 1754 9 of 28

SARS-CoV, Yu et al. [51] have evaluated a series of naturally occurring compounds, including flavonoids, xanthones, terpenoids, sterols and fatty acids, against the activity of SARS helicase, nsP13, using fluorescence resonance energy transfer (FRET)-based double-strand (ds) DNA unwinding assay or a colorimetry-based ATP hydrolysis assay. Interestingly, only myricetin (24) and scutellarein (25) (Figure5) were found to inhibit the ATPase activity of nsP13 by more than 90% at a concentration of 10 µM, with IC50 values of 2.71 and 0.86 µM, respectively. The toxicity of 24 and 25 against normal breast epithelial MCF10A cells were also examined at a concentration of 2 µM and it was found that both compounds are safe at pharmacologically-effective concentrations [51]. It is well documented that naringin (26) (Figure5), a constituent of citrus fruits, exhibits a potent anti-inflammatory activity by inhibiting the expression of the pro-inflammatory cytokines such as COX-2, iNOS, IL-1β and IL-6, induced by LPS in vitro. Since 26 could also inhibit high mobility group box protein 1 (HMGB1) expression, it could restrain cytokine storm by preventing up-regulation of cytokines such as TNF-α, IL-6, IL-1β, and IL-8 [52]. Based on this observation, an attempt to identify effective antiviral and anti-inflammation compounds from Citrus flavonoids has been made to give a nutritional recommendation for the prevention and treatment of COVID-19. Some Citrus flavonoids including naringenin (27), hesperidin (28) (Figure5), neohesperidin ( 29), nobiletin (30) (Figure6), together Molecules 2021, 26, x FOR PEER REVIEW with vitamin C have found to be potential SARS-CoV-2 inhibitors 10 of whose 29 mechanisms involved ACE2 receptor inhibition and reduction of intracellular oxidative stress triggered 188. Moreover,by as inflammationthe bulky disaccharide and virus infectiongroups require [53–56]. a Inlarge addition, space27 to notaccommodate, only inhibited both HCoV- they occupy theOC43 S1 and and S2 HCoV-229E sites and the in chromen-4-one Vero E6 cells at moieties a concentration that locate of 62.5 in theµM, S2 but and also showed an S3’ sites. Therefore,inhibitory the better behavior affinity in cytopathic of 32 may effectbe due (CPE) to a concomitant at 72 h post-infection binding alongthrough with a protection S1, S2 and S3’ sitesof cells (Table from 2) SARS-CoV-2 [57]. infection at concentrations of 250 and 62.5 µM[54].

Figure 6. StructuresFigure of flavonoids 6. Structures and of flavonoid ﬂavonoids glycosides and ﬂavonoid 29–35 glycosides. 29–35.

By using molecularJo et al. docking used a syntheticstudies and peptide surface labelled plasmon with resonance an Edans-Daabcyl (SPR)/FRET- FRET pair to search pro based assays, Chenfor SARS-CoV et al. have 3CLfound thatinhibitors quercetin-3- againstβ- aD-galactoside flavonoids library(36) (Figure consisting 7) was of ten different a potent inhibitorscaffolds. of SARS-CoV Among 3CL 64pro compounds,, exhibiting testedmore than at 20 50µ%M, inhibition only herbacetin at a concen- (31), rhoifolin (32) tration of 50 μMand (IC pectolinarin50 = 42.79 μM ( 33in) a (Figure competitive6) were kinetic found mode). to exhibit Molecular prominent modeling inhibitory and activity, with Q189A mutationIC experiment50 values of revealed 33.17, 27.45 that and Gln-189 37.78 playsµM, a respectively, key role in the at abinding concentration between ranging from 2 36 and SARS-CoVto 320 3CLµMpro. In To order avoid to a elucidate non-specific structure-activity inhibition of these relationship flavonoids features, caused the by aggregation authors have synthesizedthrough complexity, eight quercetin-3- the assaysβ-D-galactoside in the presence derivatives of 0.01% (37 Triton–44) (Figure X-100 7), were performed and tested their inhibitory activities against SARS-CoV 3CLpro at a concentration of 50 μM. The results revealed that (i)-elimination of hydroxyl groups on the quercetin moiety (42– 44) significantly diminished inhibitory activity, (ii)-acetoxylation of the sugar moiety (37) decreased an inhibitory activity of the compound, and (iii)-introduction of a large sugar moiety to the hydroxyl group on C-7 of quercetin (23) did not cause any adverse effects on the ability of the inhibitor to functionally disrupt the SARS-CoV 3CLpro activity (Table 2) [58].

Molecules 2021, 26, 1754 10 of 28

resulting in a better inhibitory activity than those without using Triton X-100. Since SARS- CoV 3CLpro contains one tryptophan residue (Trp-31) at the catalytic domain, a general tryptophan-based assay method was used to independently confirm the inhibitory activity of the three flavonoids. As the three flavonoids decreased the emission intensity, the authors concluded that there was a complex formation between the catalytic domain and these flavonoids. The interaction between SARS-CoV 3CLpro and the three inhibitory flavonoids were analyzed to predict the binding affinities by induced-fit docking study. The docking study of kaempferol (34) and morin (35) (Figure6) were also performed and compared with herbacetin (31) to find structural features that confer good affinity of 31. The results showed that these compounds share the kaempferol (34) motif. The docking study further revealed that the phenyl moiety of 34 occupies the S1 site of SARS-CoV through a hydrogen bond with Glu-166, whereas its chromen-4-one scaffold locates in the S2 site. For 31, there are four hydrogen bonds formed within a distance of 2.33 Å in the S2 site. However, the major bonding force was due to the presence of the hydroxyl group on C-8 that binds with Glu-166 and Gln-189. These bindings were predicted to confer a good glide score (−9.263) to 31. Due to the lack of the hydroxyl group on C-8, 34 and 35 have two hydrogen bonds less than 31. On the other hand, the binding modes of 32 and 33 are different from those of 34 and 31. Since 32 and 33 possess α-L-rhamnopyranosyl-β-D-glucopyranoside and α-L-mannopyranosyl-β-D-glucopyranoside at C-7 of the chromen-4-one scaffold, there is no hydrogen bond between the hydroxyl group on C-7 and the backbone of Ile-188. Moreover, as the bulky disaccharide groups require a large space to accommodate, they occupy the S1 and S2 sites and the chromen-4-one moieties that locate in the S2 and S3’ sites. Therefore, the better affinity of 32 may be due to a concomitant binding through S1, S2 and S3’ sites (Table2)[57]. By using molecular docking studies and surface plasmon resonance (SPR)/FRET- based assays, Chen et al. have found that quercetin-3-β-D-galactoside (36) (Figure7) was a potent inhibitor of SARS-CoV 3CLpro, exhibiting more than 50% inhibition at a concentration of 50 µM (IC50 = 42.79 µM in a competitive kinetic mode). Molecular modeling and Q189A mutation experiment revealed that Gln-189 plays a key role in the binding between 36 and SARS-CoV 3CLpro. In order to elucidate structure-activity relationship features, the authors have synthesized eight quercetin-3-β-D-galactoside derivatives (37–44) (Figure7) , and tested their inhibitory activities against SARS-CoV 3CLpro at a concentration of 50 µM. The results revealed that (i)-elimination of hydroxyl groups on the quercetin moiety (42–44) significantly diminished inhibitory activity, (ii)-acetoxylation of the sugar moiety (37) decreased an inhibitory activity of the compound, and (iii)-introduction of a large sugar moiety to the hydroxyl group on C-7 of quercetin (23) did not cause any adverse effects on the ability of the inhibitor to functionally disrupt the SARS-CoV 3CLpro activity (Table2)[58]. Twelve geranylated flavonoids, including tomentins A–E (45–49), 30-O-methyldiplacol (50), 40-O-methyldiplacol (51), 30-O-methyldiplacone (52), 40-O-methyldiplacone (53), mimulone (54), diplacone (55) and 6-geranyl-40,5,7-trihydroxy-30,50-dimethoxyflavone (56) (Figure8) were isolated from the methanolic extract of fruits of a Chinese medicinal plant, Paulownia tomentosa (Thunb.) Steud. (Paulowniaceae). Compounds 45–56 inhibited papain-like pro protease (PL ) activity in a reversible, mixed-type mode, with IC50 values ranging from 5.0–14.4 µM (Table2)[34]. In a continuation of their work on emodin (18), Schwarz et al. have investigated whether kaempferol (34) and its glycosides can block the 3a channel protein of coronavirus by determination of their efficacy to inhibit Ba2+-sensitive current. They have found that 34, at 20 µM, was able to reduce endogenous Ba2+-sensitive current, and the degree of inhibition was independent on the voltage. In contrast to 18, which selectively inhibited the 3a-mediated current and at 20 µM already produced more than 50% block, 34 exhibited similar effects against the endogenous and the 3a-mediated components. Juglanin (57) (Figure9) seemed to be the most potent kaempferol glycoside that showed complete inhibition at 20 µM. Interestingly, the IC50 of 57 could be obtained even at a concentration Molecules 2021, 26, 1754 11 of 28

as low as 2.3 µM, indicating that 57 is about one order of magnitude more potent to block 3a-protein channel than 18. Although afzelin (58) and tiliroside (59) (Figure9) were less potent than 57, they were as effective as 18. Compound 59, at 20 µM, produced a block to 0.48 ± 0.09, whereas 57, at the same concentration, completely blocked the 3a-mediated current. Interestingly, only 10 µM of 58 (inhibition to 0.83 ± 0.01) showed a similar degree of inhibition to 20 µM of 34. Curiously, the acylated kaempferol derivatives, kaempferol-3- Molecules 2021, 26, x FOR PEER REVIEW 11 of 29 O-(2,6-di-p-coumaroyl)-glucopyranoside (60) and kaempferol-3-O-(3,4-diacetyl-2,6-di-p- coumaroyl) glucopyranoside (61) (Figure9), both containing an additional p-coumaroyl group, showed no effect on Ba2+-sensitive current at 20 µM, whereas the kaempferol triglycoside kaempferol-3-O-α-L-rhamnopyranosyl(1→2)[α-L-rhamnopyranosyl(1→6)]-β- D-glucopyranoside (62) (Figure9) exhibited about 30% inhibition at 20 µM, which is similar to the effect of 58 at 20 µM. Of note is that both compounds contain rhamnose residues. It is interesting to note also that 23, 27 and the isoﬂavone genistein (63) (Figure9), all of which are known for their antiviral potency including SARS CoV, did not exhibit any signiﬁcant Molecules 2021, 26, x FOR PEER REVIEWmodulation of the 3a-mediated current [59]. 11 of 29

Figure 7. Structures of quercetin-3-β-D-galactoside (36) and its synthetic derivatives 37–44.

Twelve geranylated flavonoids, including tomentins A–E (45-49), 3′-O-methyldiplacol (50), 4′-O-methyldiplacol (51), 3′-O-methyldiplacone (52), 4′-O-methyldiplacone (53), mimulone (54), diplacone (55) and 6-geranyl-4′,5,7-trihydroxy-3′,5′-dimethoxyflavone (56) (Figure 8) were isolated from the methanolic extract of fruits of a Chinese medicinal plant, Paulownia tomentosa (Thunb.) Steud. (Paulowniaceae) . Compounds 45-56 inhibited papain-like protease (PLpro) activity in a reversible, mixed-type mode, with IC50 Figure 7. StructuresStructures of of quercetin-3- quercetin-3-β-βD--galactosideD-galactoside (36 ()36 and) and its itssynthetic synthetic derivatives derivatives 37–4437. –44. values ranging from 5.0–14.4 μM (Table 2) [34]. Twelve geranylated flavonoids, including tomentins A–E (45-49), 3′-O-methyldiplacol (50), 4′-O-methyldiplacol (51), 3′-O-methyldiplacone (52), 4′-O-methyldiplacone (53), mimulone (54), diplacone (55) and 6-geranyl-4′,5,7-trihydroxy-3′,5′-dimethoxyflavone (56) (Figure 8) were isolated from the methanolic extract of fruits of a Chinese medicinal plant, Paulownia tomentosa (Thunb.) Steud. (Paulowniaceae). Compounds 45-56 inhibited papain-like protease (PLpro) activity in a reversible, mixed-type mode, with IC50 values ranging from 5.0–14.4 μM (Table 2) [34].

FigureFigure 8.8.Structure Structure ofof geranylatedgeranylated ﬂavonoidsflavonoids45 45––5656..

In a continuation of their work on emodin (18), Schwarz et al. have investigated whether kaempferol (34) and its glycosides can block the 3a channel protein of coronavirus by determination of their efficacy to inhibit Ba2+-sensitive current. They have found 2+ that 34, at 20 μM, was able to reduce endogenous Ba -sensitive current, and the degree of Figureinhibition 8. Structure was independent of geranylated onflavonoids the voltage. 45–56. In contrast to 18, which selectively inhibited the 3a-mediated current and at 20 μM already produced more than 50% block, 34 exhibited Insimilar a continuation effects against of their the work endogenous on emodin and (18 the), Schwarz 3a-mediated et al. components.have investigated Juglanin whether(57) (Figure kaempferol 9) seemed (34 )to and be theits glycosides most potent can kaempferol block the 3a glycoside channel proteinthat showed of corona- complete virus by determination of their efficacy to inhibit Ba2+-sensitive current. They have found that 34, at 20 μM, was able to reduce endogenous Ba2+-sensitive current, and the degree of inhibition was independent on the voltage. In contrast to 18, which selectively inhibited the 3a-mediated current and at 20 μM already produced more than 50% block, 34 exhibited similar effects against the endogenous and the 3a-mediated components. Juglanin (57) (Figure 9) seemed to be the most potent kaempferol glycoside that showed complete

Molecules 2021, 26, x FOR PEER REVIEW 12 of 29

inhibition at 20 μM. Interestingly, the IC50 of 57 could be obtained even at a concentration as low as 2.3 μM, indicating that 57 is about one order of magnitude more potent to block 3a-protein channel than 18. Although afzelin (58) and tiliroside (59) (Figure 9) were less potent than 57, they were as effective as 18. Compound 59, at 20 μM, produced a block to 0.48 ± 0.09, whereas 57, at the same concentration, completely blocked the 3a-mediated current. Interestingly, only 10 μM of 58 (inhibition to 0.83 ± 0.01) showed a similar degree of inhibition to 20 μM of 34. Curiously, the acylated kaempferol derivatives, kaempferol- 3-O-(2,6-di-p-coumaroyl)-glucopyranoside (60) and kaempferol-3-O-(3,4-diacetyl-2,6-di- p-coumaroyl) glucopyranoside (61) (Figure 9), both containing an additional p-coumaroyl group, showed no effect on Ba2+-sensitive current at 20 μM, whereas the kaempferol triglycoside kaempferol-3-O-α-L-rhamnopyranosyl(1→2)[α-L-rhamnopyranosyl(1→6)]-β-D- glucopyranoside (62) (Figure 9) exhibited about 30% inhibition at 20 μM, which is similar to the effect of 58 at 20 μM. Of note is that both compounds contain rhamnose residues. It Molecules 2021, 26, 1754is interesting to note also that 23, 27 and the isoflavone genistein (63) (Figure 9), all of 12 of 28 which are known for their antiviral potency including SARS CoV, did not exhibit any significant modulation of the 3a-mediated current [59].

FigureFigure 9. Structure 9. Structure of flavonoids, of flavonoids, flavonoid flavonoid glycosides glycosides and andan isoflavonoid an isoflavonoid 57–6457.– 64.

pro As part of Asan investigation part of an investigation to search for to botanical search for sources botanical for SARS-CoV sources for 3CL SARS-CoVpro in- 3CL hibitors, Ryuinhibitors, et al. have Ryu found et al. that have biflavones, found that named biflavones, amentoflavone named amentoflavone(65), bilobetin (66 (65),), bilobetin ginkgetin ((6766),), and ginkgetin sciadopitysin (67), and (68 sciadopitysin) (Figure 10), isolated (68) (Figure from 10 a ),medicinal isolated plant from aTorreya medicinal plant nucifera L. SieboldTorreya and nucifera Zucc.L. (Lamiaceae), Siebold and exhibited Zucc. (Lamiaceae), an inhibitory exhibited effect against an inhibitory SARS-CoV effect against pro 3CLpro. However,SARS-CoV 65 was 3CL the most. However, potent non-co65 wasmpetitive the most anti-SARS-CoV potent non-competitive 3CLpro with anti-SARS-CoV an pro µ 65 pro IC50 value of3CL 8.3 μM.with Compound an IC50 value 65 is of 30-fold 8.3 M. more Compound potent as SARS-CoVis 30-fold more3CL potentinhibitor as SARS-CoV 3CLpro inhibitor than the parent compound apigenin (64) (Figure9) (IC = 280.8 µM), than the parent compound apigenin (64) (Figure 9) (IC50 = 280.8 μM), whereas 22 and50 23, whereas 22 and 23, whose IC values were 23.8 and 20.2 µM, respectively, were less potent whose IC50 values were 23.8 and 20.2 μM,50 respectively, were less potent than 65. Further- more, the authorsthan 65 .have Furthermore, also attempted the authors to elucidate have the also interaction attempted of to SARS-CoV elucidate the3CL interactionpro of pro pro with 65 bySARS-CoV using a three-dimensional 3CL with 65 by structure using a of three-dimensional SARS-CoV 3CLpro structure in complex of SARS-CoV with a 3CL in complex with a substrate-analogue inhibitor (coded 2z3e)24, obtained from the Protein Data Bank for modeling analysis. Computer docking analysis revealed that 65 nicely fits in the binding pocket of 3CLpro. The hydroxyl group on C-5 of 65 formed two hydrogen bonds with the nitrogen atom of the imidazole ring of His-163 (3.154 Å) and a hydroxyl group of Leu-141 (2.966 Å), both belonging to S1 site of 3CLpro, whereas the hydroxyl group in the B ring of 65 forms hydrogen bonds with Gln-189 (3.033 Å), belonging to S2 site of 3CLpro. These structure-activity relationships studies implied that interactions with Val-186 (4.228 Å) and Gln-192 (3.898 Å) are one of the key chemotypes in this inhibitor. Furthermore, the potencies of 65 and 66 correlated well with their binding energies viz 65 = 11.42 kcal/mol, and 64 = 7.79 kcal/mol. The difference in binding energy could pro explain the reason why the IC50 value of 65 against 3CL is 30-fold smaller than that of 64 (Table2)[35]. Molecules 2021, 26, x FOR PEER REVIEW 13 of 29

substrate-analogue inhibitor (coded 2z3e)24, obtained from the Protein Data Bank for modeling analysis. Computer docking analysis revealed that 65 nicely fits in the binding pro pocket of 3CLpro. The hydroxyl group on C-5 of 65 formed two hydrogen bonds with the nitrogen atom of the imidazole ring of His-163 (3.154 Å) and a hydroxyl group of Leu-141 pro (2.966 Å), both belonging to S1 site of 3CLpro, whereas the hydroxyl group in the B ring of pro 65 forms hydrogen bonds with Gln-189 (3.033 Å), belonging to S2 site of 3CLpro. These structure-activity relationships studies implied that interactions with Val-186 (4.228 Å) and Gln-192 (3.898 Å) are one of the key chemotypes in this inhibitor. Furthermore, the potencies of 65 and 66 correlated well with their binding energies viz 65 = 11.42 kcal/mol, Molecules 2021, 26, 1754 potencies of 65 and 66 correlated well with their binding energies viz 65 = 11.42 kcal/mol,13 of 28 and 64 = 7.79 kcal/mol. The difference in binding energy could explain the reason why the 50 pro IC50 value of 65 against 3CLpro is 30-fold smaller than that of 64 (Table 2) [35].

Figure 10. Structure of biflavones 65–68. FigureFigure 10.10.Structure Structure ofof biﬂavonesbiflavones65 65––6868..

3.4.3.4. LignansLignans andand NeolignansNeolignans TheThe dibenzylbutyrolactonedibenzylbutyrolactone lignans lignans hinokinin hinokinin (69 (69) )and and savinin savinin (70 (70), isolated), isolated from from the theethyl ethyl acetate acetate extract extract of the of theheartwood heartwood of Chamaecyparis of Chamaecyparis obtusa obtusa var. var.formosanaformosana, a synthetic, a syn- 0 theticphenyltetralin phenyltetralin lignan lignan 4,4ʹ-O 4,4-benzoylisolariciresinol-O-benzoylisolariciresinol (71), ( 71and), and two two naturally naturally occurring occurring bi- biphenylphenyl neolignans neolignans honokiol honokiol (72 (72) and) and magnolol magnolol (73 ()73 (Figure) (Figure 11) 11were) were assayed assayed for their for their anti- anti-SARS-CoVSARS-CoV activities activities using using a cell-based a cell-based assay assay to measure to measure a SARS-CoV-induced a SARS-CoV-induced CPE CPE on onVero Vero E6 E6cells cells [36]. [36 Compounds]. Compounds 69–7369 exhibited–73 exhibited strong strong inhibitory inhibitory activity activity in thein CPE the assays CPE µ assaysat concentrations at concentrations between between 3.3 and 20 3.3 μ andM. Interestingly, 20 M. Interestingly, 69 exhibited69 exhibited significant signiﬁcant inhibitory µ inhibitoryeffects at concentrations effects at concentrations as low as as 1 lowμM. as The 1 M.anti-SARS-CoV The anti-SARS-CoV replication replication activity, activity, meas- measuredured by an by ELISA an ELISA method, method, revealed revealed that among that among the lignans the lignans and neolignans and neolignans tested, tested, 70 ex- 70 exhibited the strongest activity, with an50 EC50 value of 1.13 µM, which is lower than hibited the strongest activity, with an EC50 value of 1.13 μM, which is lower than that of that of the reference compound valinomycin50 (EC50 = 1.63 µM), whereas 69 showed the the reference compound valinomycin (EC50 = 1.63 μM), whereas 69 showed the weakest weakest activity50 (EC50 > 10 µM). The neolignans 72 and 73 displayed signiﬁcant activity50 activity (EC50 >10 μM). The neolignans 72 and 73 displayed significant activity with EC50 withvalues EC ranging50 values from ranging 3.8 to from 7.5 3.8μM. to Moreover, 7.5 µM. Moreover, 70 also showed70 also showed inhibitory inhibitory activity activity against values ranging from 3.8pro to 7.5 μM. Moreover, 70 also showed inhibitory activity against against SARS-CoVpro 3CL with50 an IC50 value of 25 µM whereas50 the IC50 of 69 was more SARS-CoV 3CLpro with an IC50 value of 25 μM whereas the IC50 of 69 was more than 100 than 100 µM (Table2)[36]. μM (Table 2) [36].

FigureFigure 11.11.Structures Structures ofof lignanslignans69 69––7171and andneolignans neolignans72 72and and73 73. . 3.5. Gallic Acid Derivatives

By using a two-step screening method which combines a frontal affinity chromatography-

mass spectrometry (FAC/MS) and pseudotyped virus infection assay to search for drugs that can interfere with the entry of SARS-CoV into host cells, Yi et al. have found that, among small molecule libraries consisting of extracts from 121 Chinese herbs, tetra-O- galloyl-β-D-glucose (TGG, 74) (Figure 12) exhibited potent antiviral activities against the human immunodeﬁciency virus (HIV)-luc/SARS pseudotyped virus, with an EC50 value of 2.86 µM. Interestingly, 74 showed little anti-vesicular stomatitis virus (VSV) activity at the same concentration levels that can effectively inhibit the entry of HIV-luc/SARS pseudotyped virus to its host cells, indicating that 74 is highly speciﬁc against SARS-CoV. Molecules 2021, 26, x FOR PEER REVIEW 14 of 29

3.5. Gallic Acid Derivatives By using a two-step screening method which combines a frontal affinity chromatography-mass spectrometry (FAC/MS) and pseudotyped virus infection assay to search for drugs that can interfere with the entry of SARS-CoV into host cells, Yi et al. have found that, among small molecule libraries consisting of extracts from 121 Chinese herbs, tetra- O-galloyl-β-D-glucose (TGG, 74) (Figure 12) exhibited potent antiviral activities against the human immunodeficiency virus (HIV)-luc/SARS pseudotyped virus, with an EC50 Molecules 2021, 26, 1754 14 of 28 value of 2.86 μM. Interestingly, 74 showed little anti-vesicular stomatitis virus (VSV) activity at the same concentration levels that can effectively inhibit the entry of HIV- luc/SARS pseudotyped virus to its host cells, indicating that 74 is highly specific against SinceSARS-CoV.74 was Since isolated 74 was through isolated analysis through of analysis its binding of its to binding the S2 protein to the S2 of protein SARS-CoV, of SARS- this compoundCoV, this compound most likely most blocks likely the blocks entry the of HIV-luc/SARS entry of HIV-luc/SARS pseudotyped pseudotyped virus to itsvirus host to cells.its host Moreover, cells. Moreover,74 also 74 inhibited, also inhibited, in a dose-dependent in a dose-dependent manner, manner, wild-type wild-type SARS-CoV SARS- infectionCoV infection with anwith EC an50 valueEC50 value of 4.5 ofµ M4.5 (Table μM (Table2)[60 ].2) [60].

FigureFigure 12.12. StructuresStructures ofof tetra-tetra-OO-galloyl--galloyl-ββ--D-glucose-glucose (TGG)(TGG) ((74)) andand tannictannic acidacid ((7575).).

pro InIn aa screeningscreening ofof aa panelpanel ofof compoundscompounds withwith aa capacitycapacity to to inhibit inhibit SARS-3CL SARS-3CLpro activityactivity byby HPLC assay using a a protease protease inhibitor, inhibitor, NN-ethylmaleimide,-ethylmaleimide, as as positive positive control, control, Chen Chen et 0 etal. al.have have found found that that tannic tannic acid acid (75) ((Figure75) (Figure 12) and12) andtheaflavin-3 theaﬂavin-3′-gallate-gallate (TF2B, (TF2B, 76) were76) were active against 3CLpro. Fluorogenic substrate peptide assay revealed that 75, 76 and theaﬂavin-3,30-digallate (TF3, 77) (Figure 13), abundant in black tea, displayed relevant pro anti-SARS-3CL activity with IC50 values less than 10 µM (Table2)[61].

Molecules 2021, 26, x FOR PEER REVIEW 15 of 29 Molecules 2021, 26, x FOR PEER REVIEW 15 of 29 active against 3CLpro. Fluorogenic substrate peptide assay revealed that 75, 76 and theafla- active against 3CLpro. Fluorogenic substrate peptide assay revealed that 75, 76 and theafla- Molecules 2021, 26, 1754 vin-3,3′-digallate (TF3, 77) (Figure 13), abundant in black tea, displayed relevant15 anti- of 28 vin-3,3′-digallate (TF3, 77) (Figure 13), abundant in black tea, displayed relevant anti- SARS-3CLpro activity with IC50 values less than 10 μM (Table 2) [61]. SARS-3CLpro activity with IC50 values less than 10 μM (Table 2) [61].

Figure 13. Structures of TF2B (76) and TF3 (77). FigureFigure 13.13. Structures ofof TF2BTF2B ((7676)) andand TF3TF3 ((7777).). 3.6. Terpenoids 3.6.3.6. Terpenoids 3.6.1.3.6.1. MonoterpenoidsMonoterpenoids 3.6.1. Monoterpenoids MonoterpenesMonoterpenes areare majormajor constituentsconstituents ofof essentialessential oils.oils. LoizzoLoizzo etet al.al. havehave evaluatedevaluated Monoterpenes are major constituents of essential oils. Loizzo et al. have evaluated essentialessential oilsoils fromfrom various various members members of of the the Cupressaceae Cupressaceae family family for for their their inhibitory inhibitory activity activ- essential oils from various members of the Cupressaceae family for their inhibitory activ- againstity against SARS-CoV SARS-CoV and Herpesand Herpes simplex simplex virus typevirus 1 type (HSV-1) 1 (HSV-1) replication replicationin vitro byin visuallyvitro by ity against SARS-CoV and Herpes simplex virus type 1 (HSV-1) replication in vitro by scoringvisually of scoring the virus of induced-cytopathogenic the virus induced-cytopa effectthogenic post-infection. effect post-infection. They have found They that have the visually scoring of the virus induced-cytopathogenic effect post-infection. They have berriesfound that oil of theLaurus berries nobilis oil ofL. Laurus (Lauraceae), nobilis L. whose (Lauraceae), major constituents whose major are constituentsβ-ocimene are (78 β),- found that the berries oil of Laurus nobilis L. (Lauraceae), whose major constituents are β- 1,8-cineoleocimene (78 (79),), 1,8-cineoleα-pinene ( 80(79),), and α-pineneβ-pinene (80 (),81 and) (Figure β-pinene 14), exhibited(81) (Figure an IC14),50 exhibitedvalue of 120 an ocimene (78), 1,8-cineole (79), α-pinene (80), and β-pinene (81) (Figure 14), exhibited an µICg/mL50 value against of 120 SARS-CoV, μg/mL against with SI SARS-CoV, = 4.2, whereas withThuja SI = 4.2, orientalis whereasL. and ThujaJuniperus orientalis oxycedrus L. and IC50 value of 120 μg/mL against SARS-CoV, with SI = 4.2, whereas Thuja orientalis L. and ssp.Juniperusoxycedrus oxycedrusoils displayedssp. oxycedrus weaker oils activitiesdisplayed with weaker IC50 activitiesvalues of with 130 IC and50 values270 µg/mL of 130, Juniperus oxycedrus ssp. oxycedrus oils displayed weaker activities with IC50 values of 130 andand SI270 = μ 3.8g/mL, and and 3.7, respectively,SI = 3.8 and 3.7, when respectively, compared when with compared glycyrrhizin with (111) glycyrrhizin, as the positive (111), and 270 μg/mL, and SI = 3.8 and 3.7, respectively, when compared with glycyrrhizin (111), control.as the positive Gas chromatography/mass control. Gas chromatography/mass spectrometry (GC/MS)spectrometry analysis (GC/MS) revealed analysis that 77re- as the positive control. Gas chromatography/mass spectrometry (GC/MS) analysis re- andvealedδ-3-carene that 77 and (82) δ (Figure-3-carene 14 ()82 are) (Figure major constituents14) are major ofconstituents the T. orientalis of theoil T. whereasorientalis 80oil vealed that 77 and δ-3-carene (82) (Figure 14) are major constituents of the T. orientalis oil andwhereasβ-myrcene 80 and (β83-myrcene) are predominant (83) are predominant in the berries in oilthe of berriesJ. oxycedrus oil of ssp.J. oxycedrusoxycedrus ssp.[38 ox-]. whereas 80 and β-myrcene (83) are predominant in the berries oil of J. oxycedrus ssp. ox- Sinceycedrus many [38]. essential Since many oils essential are used inoils aromatherapy, are used in aromathera this approachpy, this could approach be promising could inbe ycedrus [38]. Since many essential oils are used in aromatherapy, this approach could be preventingpromising in SARS. preventing SARS. promising in preventing SARS.

FigureFigure 14.14. StructuresStructures ofof monoterpenesmonoterpenes78 78––8383and and sesquiterpenessesquiterpenes84 84––8686.. Figure 14. Structures of monoterpenes 78–83 and sesquiterpenes 84–86. 3.6.2. Sesquiterpenoids In the evaluation of phytochemicals for SARS-CoV activity, Wen et al. have isolated

two sesquiterpenes, cedrane-3β,12-diol (84) and α-cardinol (85) (Figure 14) from the ethyl

acetate extract of the heartwood of Juniperus formosana Hayata (Cupressaceae). Compounds

Molecules 2021, 26, x FOR PEER REVIEW 16 of 29

3.6.2. Sesquiterpenoids Molecules 2021, 26, 1754 In the evaluation of phytochemicals for SARS-CoV activity, Wen et al. have isolated16 of 28 two sesquiterpenes, cedrane-3β,12-diol (84) and α-cardinol (85) (Figure 14) from the ethyl acetate extract of the heartwood of Juniperus formosana Hayata (Cupressaceae). Com- pounds 84 and 85 exhibited inhibition of CPE of SARS-CoV on Vero E6 cells at concentra- 84 and 85 exhibited inhibition of CPE of SARS-CoV on Vero E6 cells at concentrations tions as low as 3.3 and 1 μM, respectively. Moreover, 85 inhibited SARS-CoV replication as low as 3.3 and 1 µM, respectively. Moreover, 85 inhibited SARS-CoV replication with with an EC50 of 4.44 μM (SI = 17.3) (Table 2) [36]. Interestingly, a sesquiterpene α-cedrol an EC50 of 4.44 µM (SI = 17.3) (Table2)[ 36]. Interestingly, a sesquiterpene α-cedrol (86) (86) (Figure 14), also a major component of T. orientalis L. essential oil, showed an inhibi- (Figure 14), also a major component of T. orientalis L. essential oil, showed an inhibitory tory activity against SARS-CoV with IC50 of 130 ± 0.4 μg/mL and SI = 4.2, when compared activity against SARS-CoV with IC50 of 130 ± 0.4 µg/mL and SI = 4.2, when compared to to 111 (the positive control; IC50 of 641.0 μg/mL (779 μM), SI = 1.2) [38]. 111 (the positive control; IC50 of 641.0 µg/mL (779 µM), SI = 1.2) [38].

3.6.3.3.6.3. DiterpenoidsDiterpenoids EightEight abietane abietane and and two two labdane labdane diterpenoids diterpenoids comprising comprising ferruginol ferruginol (87), dehydroabieta-(87), dehydro- 7-oneabieta-7-one (88), sugiol (88), (89 sugiol), 8β-hydroxyabieta-9(11),13-dien-12-one (89), 8β-hydroxyabieta-9(11),13-dien-12-one (91), 6,7-dehydroroyleanone (91), 6,7-dehydro- (royleanone93), pinusolidic (93), acidpinusolidic (95) (isolated acid (95 from) (isolated the ethyl from acetate the ethyl extract acetate of the extract heartwood of the heart- of C. obtusawoodvar. of C.formosana obtusa var.), cryptojaponol formosana), cryptojaponol (90) and 7β-hydroxydeoxycryptojaponol (90) and 7β-hydroxydeoxycryptojaponol (92) (isolated from(92) the(isolated heartwood from oftheCryptomeria heartwood japonica of Cryptomeria) and 3β,12-diacetoxyabieta-6,8,11,13-tetraene japonica) and 3β,12-diacetoxyabieta- (6,8,11,13-tetraene91) (isolated from ( the91) ethyl(isolated acetate from extract the ethyl of the acetate heartwood extract of ofJ. the formosana heartwoodHayata), of J. for- to- gethermosana with Hayata), foskalin together (96) (Figure with foskalin 15), were (96 evaluated) (Figure for15), their were anti-SARS-CoV evaluated for their activities anti- usingSARS-CoV a cell-based activities assay using to measurea cell-based SARS-CoV-induced assay to measure SARS-CoV-induced CPE on Vero E6 cells CPE [36 ].on Eight Vero abietaneE6 cells [36]. (87– 94Eight) and abietane two labdane (87–94 diterpenes) and two labdane (95 and 96diterpenes) showed ( strong95 and inhibitory96) showed activity strong ininhibitory the CPE assaysactivity at in concentrations the CPE assays ranging at concentrations from 3.3 to 20 rangingµM. Although from 3.3 the to SI 20 of μ theM. ﬁveAlt- abietane-typehough the SI diterpenesof the five abietane-type87, 88, 91, 92, andditerpenes94 and two87, 88 labdane-type, 91, 92, and diterpenes94 and two95 labdane-and 96 weretype 58,diterpenes 76.3, >510, 95 111,and 193,96 were >159, 58, 89.8, 76.3, none >510, of 111, the diterpenoids193, >159, 89.8, tested none inhibited of the diterpenoids SARS-CoV 3CLtestedpro inhibitedat concentrations SARS-CoV of less3CL thanpro at 100concentrationsµM. In comparison of less than with 100 the μM. positive In comparison controls, niclosamidewith the positive and valinomycin, controls, niclosamide most of the and diterpenoids valinomycin, with most potent of the activity diterpenoids against CPEwith alsopotent exhibited activity marked against inhibitoryCPE also exhibited effects on marked SARS-CoV inhibitory replication. effects The on SARS-CoV SI values for repli-87, 88cation., 91, 92 The, 94 SI– 96valuesare substantiallyfor 87, 88, 91, higher92, 94–96 than are that substantially of the positive higher control than that valinomycin of the pos- (itiveSI = 41.4control). The valinomycin authors, therefore, (SI = 41.4). proposed The auth thators, the therefore, abietane diterpenesproposed 87that–94 thepossessed abietane potentditerpenes anti-SARS-CoV 87–94 possessed activities, potent however, anti-SARS-CoV these activities activities, apparently however, did these not activities involve anap- pro inhibitionparently did of SARS-CoVnot involve 3CL an inhibition(Table2 )[of36 SARS-CoV]. 3CLpro (Table 2) [36].

FigureFigure 15.15.Structures Structures ofof abietaneabietane (87(87––94)94)and and labdane labdane ( 95(95and and96 96)) diterpenoids. diterpenoids.

SixSix abietaneabietane diterpenes,diterpenes, 18-hydroxyferruginol18-hydroxyferruginol ((9797),), hinokiolhinokiol ((9898),), ferruginolferruginol ((8787),), 18-18- oxoferruginoloxoferruginol ( 99(99),),O -acetyl-18-hydroxyferruginolO-acetyl-18-hydroxyferruginol (100 (100), methyl), methyl dehydroabietate dehydroabietate (101 ),(101 one), primarane diterpene, isopimaric acid (102), and one labdane diterpene, kayadiol (103) (Figure 16), isolated from the n-hexane fraction of leaves of T. nucifera L. Siebold and Zucc. (Lamiaceae), were investigated for their inhibitory activities against SARS-CoV 3CLpro by using a FRET assay. Compounds 97–103 displayed inhibition against SARS-CoV 3CLpro at Molecules 2021, 26, x FOR PEER REVIEW 17 of 29

Molecules 2021, 26, 1754 one primarane diterpene, isopimaric acid (102), and one labdane diterpene, kayadiol17 ( of103 28) (Figure 16), isolated from the n-hexane fraction of leaves of T. nucifera L. Siebold and Zucc. (Lamiaceae), were investigated for their inhibitory activities against SARS-CoV 3CLpro by using a FRET assay. Compounds 97–103 displayed inhibition against SARS-CoV 3CLpro at concentrationsconcentrations upup to to 100 100µ μM,M, whereas whereas89 89exhibited exhibited signiﬁcantly significantly higher higher inhibitory inhibitory effects effects on pro 3CLon 3CLwithpro with an an IC 50IC50= = 49.6 49.6µ μM.M. Moreover, Moreover,89 89was was nearly nearly 4-fold 4-fold more more potent potent than thana a parentparent abietaneabietane diterpene,diterpene, abieticabietic acidacid ((104104)) (IC(IC5050 = 189.1 μµM) (Table 2 2))[ [35].35].

FigureFigure 16.16. StructuresStructures ofof abietaneabietane ((9797––101101and and104 104),), primarane primarane ( 102(102),), and and labdane labdane ( 103(103)) diterpenoids. diterpenoids. 3.6.4. Triterpenoids 3.6.4.Betulinic Triterpenoids acid (105 ) and betulonic acid (106) (Figure 17), isolated from the ethyl acetate extractBetulinic of the heartwood acid (105) of andJ. formosana betulonicHayata, acid (106 also) (Figure showed 17), strong isolated inhibitory from activitythe ethyl in ace- the CPEtate extract assays of at concentrationsthe heartwood betweenof J. formosana 3.3 and Hayata, 20 µM. also Compound showed strong105 exhibited inhibitory inhibitory activity pro effectsin the onCPE SARS-CoV assays at 3CL concentrations(IC50 = 10 betweenµM) 10-fold 3.3 strongerand 20 μ thanM. Compound106 (IC50 > 10105µ M)exhibited. Com- pound 105 showed a competitive inhibition mode of action with Ki value of 8.2 ± 0.7 µM. inhibitory effects on SARS-CoV 3CLpro (IC50 = 10 μM) 10-fold stronger than 106 (IC50 > 10 Docking study revealed that 105 ﬁtted nicely into the substrate-binding pocket of SARS- μM). Compoundpro 105 showed a competitive inhibition mode of action with Ki value of 8.2 CoV± 0.7 3CLμM. Docking. Moreover, study therevealed binding that was 105 strengthened fitted nicely into by a the hydrogen substrate-binding bond formed pocket by the hydroxyl group on C-3 of 105 with the oxygen atom of the carbonyl group of Thr-24, of SARS-CoV 3CLpro. Moreover, the binding was strengthened by a hydrogen bond located at the N-terminus of domain I (residues 8–101) of the 3CLpro. On the contrary, 106 formed by the hydroxyl group on C-3 of 105 with the oxygen atom of the carbonyl group showed only hydrophobic interaction but did not form any intermolecular bonds with the of Thr-24, located at the N-terminus of domain I (residues 8–101) of the 3CLpro. On the enzyme (Table2)[36]. Molecules 2021, 26, x FOR PEER REVIEWcontrary, 106 showed only hydrophobic interaction but did not form any intermolecular 18 of 29 bonds with the enzyme (Table 2) [36]. Chang et al. have evaluated the anti-HCoV-229E activity of 22 triterpenoids including four friedelanes, three glutinanes, one lupane, three taraxeranes, two oleananes, one dammarane, one ocotillone, two simiarenanes, three cycloartanes, and one cycloeucalane, isolated from leaves of Euphorbia neriifolia L. (Euphorbiaceae). However, only 3β- friedelanol (107), 3β-acetoxyfriedelane (108), friedelin (109) and epitaraxerol (110) (Figure 17) exhibited anti- HCoV-229E activity. Compounds 107-109 exhibited more potent activity than the positive control, actinomycin D. The percent of inhibition were in the following descending order: 107 (132.4%) > 110 (111.0%) > 109 (109.0%) > 108 (80.9%) > (actinomycin D 69.5%). Most significantly, exposure to these compounds led to an improvement in cell viability [39].

FigureFigure 17.17. StructuresStructures ofof triterpenoidstriterpenoids 105105––110110..

3.6.5. Saponins Glycyrrhizin (111) (Figure 18), a triterpenoid saponin mainly isolated from roots of Glycyrrhiza glabra L. (Fabaceae), is also known to exhibit a myriad of biological activities including antiviral effects against HSV-1, varicella-zoster virus (VZV), hepatitis A (HAV) and B virus (HBV), HIV, Epstein-Barr virus (EBV), human cytomegalovirus and influenza virus, and has been considered as a potential treatment for patients with chronic hepatitis C [62]. Cinatl et al. [62], in their evaluation of the antiviral potential of 111, ribavirin, 6- azauridine, pyrazofurin and mycophenolic acid against two clinical isolates of coronavirus (FFM-1 and FFM-2) from patients with SARS, have found that 111 was the most potent inhibitor of SARS-CoV replication in Vero cells, with SI = 67, whereas ribavirin and mycophenolic acid did not affect SARS-CoV replication. On the other hand, 6-azauridine and pyrazofurin, inhibitors of orotidine monophosphate decarboxylase, inhibited replication of SARS-CoV at non-toxic doses with SI = 5 and 12, respectively. Compound 111 was also found to inhibit adsorption and penetration of the virus, which are the early steps of the replicative cycle. Moreover, 111 was less effective when added during the adsorption period than when added after virus adsorption (EC50 600 mg/L vs 2400 mg/L, respectively) and was most effective when given during and after the adsorption period (EC50 300 mg/L) [40]. Due to the efficacy of 111, Hoever et al. have obtained 15 semisynthetic analogs of glycyrrhizin (112–126) (Figure 18) and tested them to find more potent compounds against SARS-CoV. They have found that the introduction of N-acetylglucosamine into the glycoside chain of glycyrrhizin (112) increased the anti-SARS-CoV activity about 9 times when compared to 111. Compound 112 inhibited SARS-CoV replication at an EC50 of 40 μM with SI > 75. Compound 112 showed no CPE at 500 μM. The authors suggested that the introduction of N-acetylglucosamine moiety into the carbohydrate portion of 111 increases its hydrophilic property, which might be important for the interaction of glycyrrhizin with viral proteins. Moreover, the authors have speculated that the viral entry is inhibited by binding of N-acetylglycosamine to the carbohydrates of the viral S protein. Although the analogs 120–123 (Figure 18) were active against SARS-CoV, with EC50 values ranging from 5 to 50 μM, they also exhibited high cytotoxicity when compared to 111 and the glycopeptide derivatives 112 and 113, resulting in low SI values (2 to 5). Compounds 113 and 114, containing L-Cys(SBn) and Gly-Leu, respectively, were active against SARS-CoV. Com- pound 113 was 10-fold more active against SARS-CoV than 111, with an EC50 = 35 μM (SI = 41), whereas 114 exhibited an EC50 = 139 μM (SI = 2). On the contrary, the glycopeptide analogs 115–119 did not exhibit any activity against SARS-CoV in concentrations up to 1 mM. Therefore, it was concluded that a free carboxylic acid group at C-30 of the triterpene

Molecules 2021, 26, 1754 18 of 28

Chang et al. have evaluated the anti-HCoV-229E activity of 22 triterpenoids including four friedelanes, three glutinanes, one lupane, three taraxeranes, two oleananes, one dammarane, one ocotillone, two simiarenanes, three cycloartanes, and one cycloeucalane, isolated from leaves of Euphorbia neriifolia L. (Euphorbiaceae). However, only 3β-friedelanol (107), 3β-acetoxyfriedelane (108), friedelin (109) and epitaraxerol (110) (Figure 17) exhibited anti- HCoV-229E activity. Compounds 107–109 exhibited more potent activity than the positive control, actinomycin D. The percent of inhibition were in the following descending order: 107 (132.4%) > 110 (111.0%) > 109 (109.0%) > 108 (80.9%) > (actinomycin D 69.5%). Most signiﬁcantly, exposure to these compounds led to an improvement in cell viability [39].

3.6.5. Saponins Glycyrrhizin (111) (Figure 18), a triterpenoid saponin mainly isolated from roots of Glycyrrhiza glabra L. (Fabaceae), is also known to exhibit a myriad of biological activities including antiviral effects against HSV-1, varicella-zoster virus (VZV), hepatitis A (HAV) and B virus (HBV), HIV, Epstein-Barr virus (EBV), human cytomegalovirus and influenza virus, and has been considered as a potential treatment for patients with chronic hepatitis C [62]. Cinatl et al. [62], in their evaluation of the antiviral potential of 111, ribavirin, 6-azauridine, pyrazofurin and mycophenolic acid against two clinical isolates of coronavirus (FFM-1 and FFM-2) from patients with SARS, have found that 111 was the most potent inhibitor of SARS-CoV replication in Vero cells, with SI = 67, whereas ribavirin and mycophenolic acid did not affect SARS-CoV replication. On the other hand, 6-azauridine and pyrazofurin, inhibitors of orotidine monophosphate decarboxylase, inhibited replication of SARS-CoV at non-toxic doses with SI = 5 and 12, respectively. Compound 111 was also found to inhibit adsorption and penetration of the virus, which are the early steps of the replicative cycle. Moreover, 111 was less effective when added during the adsorption period than when added after virus adsorption (EC50 600 mg/L vs. 2400 mg/L, respectively) and was most effective when given during and after the adsorption period (EC50 300 mg/L) [40]. Due to the efficacy of 111, Hoever et al. have obtained 15 semisynthetic analogs of glycyrrhizin (112–126) (Figure 18) and tested them to find more potent compounds against SARS-CoV. They have found that the introduction of N-acetylglucosamine into the glycoside chain of glycyrrhizin (112) increased the anti-SARS-CoV activity about 9 times when compared to 111. Compound 112 inhibited SARS-CoV replication at an EC50 of 40 µM with SI > 75. Compound 112 showed no CPE at 500 µM. The authors suggested that the introduction of N-acetylglucosamine moiety into the carbohydrate portion of 111 increases its hydrophilic property, which might be important for the interaction of glycyrrhizin with viral proteins. Moreover, the authors have speculated that the viral entry is inhibited by binding of N-acetylglycosamine to the carbohydrates of the viral S protein. Although the analogs 120– 123 (Figure 18) were active against SARS-CoV, with EC50 values ranging from 5 to 50 µM, they also exhibited high cytotoxicity when compared to 111 and the glycopeptide derivatives 112 and 113, resulting in low SI values (2 to 5). Compounds 113 and 114, containing L-Cys(SBn) and Gly-Leu, respectively, were active against SARS-CoV. Compound 113 was 10-fold more active against SARS-CoV than 111, with an EC50 = 35 µM (SI = 41), whereas 114 exhibited an EC50 = 139 µM (SI = 2). On the contrary, the glycopeptide analogs 115–119 did not exhibit any activity against SARS-CoV in concentrations up to 1 mM. Therefore, it was concluded that a free carboxylic acid group at C-30 of the triterpene scaffold was vital for the anti-SARS-CoV activity of glycyrrhizin glycopeptide analogs since 113 and 114 contain a free carboxylic acid group at C-30 whereas 115–119 contain three amino acids or amino acid alkyl ester residues. Interestingly, 125 and 126, where the two glucuronic acid units were replaced by ortho-hydroxybenzoic acid and succinic acid, respectively, no anti-SARS-CoV activity was observed, indicating that the sugar moiety is essential for the anti-SARS-CoV activity (Table2)[63]. Molecules 2021, 26, x FOR PEER REVIEW 19 of 29

scaffold was vital for the anti-SARS-CoV activity of glycyrrhizin glycopeptide analogs since 113 and 114 contain a free carboxylic acid group at C-30 whereas 115–119 contain three amino acids or amino acid alkyl ester residues. Interestingly, 125 and 126, where the Molecules 2021, 26, 1754two glucuronic acid units were replaced by ortho-hydroxybenzoic acid and succinic acid, 19 of 28 respectively, no anti-SARS-CoV activity was observed, indicating that the sugar moiety is essential for the anti-SARS-CoV activity (Table 2) [63].

Figure 18.Figure Structures 18. Structures of glycyrrhizin of glycyrrhizin (111) and (111 its) andsynthetic its synthetic analogs analogs 112–126112–126. .

Another group of oleanane triterpene glycosides is saikosaponins. These compounds were isolated from medicinal plants such as Bupleurum spp., Heteromorpha spp. and Scrophularia scorodonia, and have been reported to possess a myriad of biological activities including antiviral activities against HIV [41], influenza virus [42], varicella-zoster [43], measles and herpes simplex [44]. Cheng et al. have investigated the in vitro antiviral activity and mode of action of saikosaponins A (127), D (128), B2 (129) and C (130) (Figure 19) against HCoV-229E, which has been recognized as an important cause of nosocomial respiratory viral infections in high-risk infants. It was found that 127-130, at concentrations of 25 μM or less, significantly inhibited HCoV-229E infection, being 129 the strongest inhibitor (Table 2). Mechanism study revealed that 129 decreased infection by HCoV-229E in a dose- and time-dependent manner, and the inhibition of viral infection was more effective when it was added before viral adsorption than after viral adsorption. This observation suggests that 129 may disturb the early stages of HCV-229E infection, including viral attachment and penetration. Experimental results revealed that 129 significantly inhibited HCoV-229E attachment in a dose-dependent manner, suggesting that 129 prevents the attachment of HCoV-229E into host cells. Compound 129 also inhibited penetration of HCoV-229E into host cells in a time-dependent manner, suggesting that it also prevents

Molecules 2021, 26, 1754 20 of 28

Table 2. Anti-Human Coronavirus (Anti-HCoV) activities of natural compounds.

No. Mode of Action IC50 (µM) CC50 (µM) EC50 (µM) SI Concentration (µM) Positive Control Ref. Alkaloids

Interferon alpha, CC50 > a a 7 Inhibition of SARS-CoV (BJ-001 strain) - 14,980.0 ± 912.0 15.7 ± 1.2 954 - 100,000 ± 710.1 µM, EC50 = [23] 660.3 ± 119.1 µM, SI > 151 8 0.33 ± 0.03 13.41 ± 0.36 - 40.19 Inhibition of HCoV-OC43 infected MRC-5 9 1.01 ± 0.07 11.54 ± 0.46 - 11.46 - [30] human lung cells 2–20 10 0.83 ± 0.07 11.26 ± 0.69 - 13.63 11 Inhibition of SARS-CoV replication - 25 3.4 7.3 - - [31] Anthraquinones Blocks the interaction of SARS spike 18 200 - - - 0.1–400 Promazine [32] protein to ACE-2 Flavonoids and ﬂavonoid glycosides 19 Inhibition of SARS-CoV 3CL Protease 8.3 (2.5 ± 0.8 µg/mL) 2718 (820 ± 15 µg/mL) - - - - [47] Glycyrrhizin (111), Inhibition of wild-type 22 - 155 10.6 (9.2–12.2) 14.62 0.1–10,000 EC > 607.6 µM; [44,50] SARS-CoV infection 50 Ribavirin, not effect Inhibition of entry of HIV-luc/SARS 23 - 3320 83.4 39.81 0.1–10,000 - [60] pseudotypeed virus into Vero E6 cells 24 2.71 ± 0.19 - - - Inhibition of SARS-CoV helicase, nsP13 0.01–10 [51] 25 0.86 ± 0.48 - - - 31 33.17 - - - 32 Inhibition of SARS-CoV 3CL Protease 27.45 - - - 2–320 - [57] 33 37.78 - - - 36 42.79 ± 4.97 - - - 38 24.14 ± 4.32 - - - 39 Inhibition of SARS-CoV 3CL Protease 31.62 ± 2.43 - - - 9.4–80 - [58] 40 48.85 ± 8.15 - - - 41 61.46 ± 9.13 - - - Inhibition of SARS-CoV 3CL 36 127.89 ± 10.06 - - - 16.5–200 Protease Q189A 45 6.2 ± 0.04 - - - 46 6.1 ± 0.02 - - - 47 11.6 ± 0.13 - - - 48 12.5 ± 0.22 - - - 49 5.0 ± 0.06 - - - 50 Inhibition of SARS-CoV 9.5 ± 0.10 - - - 0.1–100 - [34] 51 papain-like protease 9.2 ± 0.13 - - - 52 13.2 ± 0.14 - - - Molecules 2021, 26, 1754 21 of 28

Table 2. Cont.

No. Mode of Action IC50 (µM) CC50 (µM) EC50 (µM) SI Concentration (µM) Positive Control Ref.

12.7 ± 0.19 - - - 53 54 14.4 ± 0.27 - - - 55 10.4 ± 0.16 - - - 56 13.9 ± 0.18 - - - 64 280.8 ± 21.4 - - - Luteolin (22), IC = 65 8.3 ± 1.2 - - - 50 20.0 ± 2.2 µM; 66 Inhibition of SARS-CoV 3CL Protease 72.3 ± 4.5 - - - 1–1000 [35] Quercetin (23), IC = 67 32.0 ± 1.7 - - - 50 23.8 ± 1.9 µM 68 34.8 ± 0.2 - - - Lignans and neolignans b 69 - >750 >10 N.C. Niclosamide, CC50 = 22.1 Inhibition of Vero E6 cell 70 - >750 1.13 >667 µM, EC50 < 0.1 µM, SI > 221; proliferation and 0.01–10 72 - 88.9 6.50 13.7 Valinomycin, CC50 = SARS-CoV replication [36] 73 - 68.3 3.80 18.0 67.5 µM, EC50 = 1.63 µM, 69 >100 - - - SI = 41.4 Inhibition of SARS-CoV 3CL Protease 8–80 Niclosamide, IC = 40 µM 70 25 - - - 50 Gallic acid derivatives Glycyrrhizin (111), EC > Inhibition of wild-type 50 74 - 1.08 4.5 (1.96–5.8) 240 0.1–10,000 607.6 µM; [60] SARS-CoV infection Ribavirin, not effect 75 3 - - - 76 Inhibition of SARS-CoV 3CL Protease 43 - - - 4–20 N-Ethylmaleimide [61] 77 9.5 - - - Sesquiterpenoids 84 - >750 >10 b Niclosamide, CC = 22.1 Inhibition of Vero E6 cell N.C. 50 0.01–10 µM, EC < 0.1 µM, SI > 221; [36] proliferation and 50 Valinomycin, CC = 85 SARS-CoV replication - 76.8 4.44 17.3 50 67.5 µM, EC50 = 1.63 µM, SI = 41.4 Molecules 2021, 26, 1754 22 of 28

Table 2. Cont.

No. Mode of Action IC50 (µM) CC50 (µM) EC50 (µM) SI Concentration (µM) Positive Control Ref. Diterpenoids 87 - 80.4 1.39 58.0 88 - 305.1 4.00 76.3 Niclosamide, CC 90 - 78.5 >10 <7.9 50 = 22.1 µM, EC < 0.1 µM, 91 Inhibition of Vero E6 cell - >750 1.47 >510 50 SI > 221; 92 proliferation and - 127 1.15 111 0.01–10 [36] Valinomycin, CC = 93 SARS-CoV replication - 89.7 5.55 16.2 50 67.5 µM, EC = 1.63 µM, 94 - 303.3 1.57 193 50 SI = 41.4 95 - >750 4.71 >159 96 - 674 7.5 89.8 87 49.6 ± 1.5 - - - 97 220.8 ± 10.4 - - - 98 233.4 ± 22.2 - - - 99 163.2 ± 13.8 - - - Abietic acid (104), Inhibition of SARS-CoV 3CL Protease 0.1–1000 [35] 100 128.9 ± 25.2 - - - IC50 = 189.1 ± 15.5 µM 101 207.0 ± 14.3 - - - 102 283.5 ± 18.4 - - - 103 137.7 ± 12.5 - - - Triterpenoids 105 Inhibition of Vero E6 cell - 150 >10 <15 Niclosamide, CC = 22.1 - 50 106 proliferation and - 112 0.63 180 µM, EC50 < 0.1 µM, SI > 221; [36] SARS-CoV replication Valinomycin, CC50 = 67.5 µM, EC50 = 1.63 µM, 105 10 - - - SI = 41.4 Inhibition of SARS-CoV 3CL Protease 8–80 Niclosamide, IC = 40 µM 106 >100 - - - 50 Saponins 111 - >20,000 * 300 (51) * >67 - - [40] 112 - >3000 40 ± 13 >75 113 - 1462 ± 50 35 ± 7 41 114 - 215 ± 18 139 ± 20 2 Glycyrrhizin (111), CC50 > 120 Inhibition of SARS-CoV replication - 44 ± 6 8 ± 2 6 0.1–1000 24,000 µM, EC50 = [63] 121 - 250 ± 19 50 ± 10 5 365. ± 12 µM, SI > 65 122 - 15 ± 3 5 ± 3 3 123 - 66 ± 8 16 ± 1 4 Molecules 2021, 26, 1754 23 of 28

Table 2. Cont.

No. Mode of Action IC50 (µM) CC50 (µM) EC50 (µM) SI Concentration (µM) Positive Control Ref. 127 - 228.1 ± 3.8 8.6 ± 0.3 26.6 Actinomycin D, CC = 128 Inhibition of HCoV-OC43 infected - 383.3 ± 0.2 1.7 ± 0.1 ** 221.9 50 0.25–25 2.8 ± 0.3 µM, EC = [64] 129 MRC-5 human lung cells - 121.5 ± 0.1 19.9 ± 0.1 *† 19.2 50 0.02 ± 0.0 µM, SI = 140 130 - 176.2 ± 0.2 13.2 ± 0.3 *†‡ 13.3 131 Inhibition of SARS-CoV replication - 15.0 6.0 2.5 - - [30] a b † †‡ nm; not calculable; * mg/L; ** p < 0.05 compared with saikosaponin A; * p < 0.05 compared with saikosaponin B2;* p < 0.05 compared with saikosaponin C (student’s test). Molecules 2021, 26, 1754 24 of 28

Another group of oleanane triterpene glycosides is saikosaponins. These compounds were isolated from medicinal plants such as Bupleurum spp., Heteromorpha spp. and Scrophu- laria scorodonia, and have been reported to possess a myriad of biological activities including antiviral activities against HIV [41], influenza virus [42], varicella-zoster [43], measles and herpes simplex [44]. Cheng et al. have investigated the in vitro antiviral activity and mode of action of saikosaponins A (127), D (128), B2 (129) and C (130) (Figure 19) against HCoV-229E, which has been recognized as an important cause of nosocomial respiratory viral infections in high-risk infants. It was found that 127-130, at concentrations of 25 µM or less, significantly inhibited HCoV-229E infection, being 129 the strongest inhibitor (Table2) . Mechanism study revealed that 129 decreased infection by HCoV-229E in a dose- and time-dependent manner, and the inhibition of viral infection was more effective when it was added before viral adsorption than after viral adsorption. This observation suggests that 129 may disturb the early stages of HCV-229E infection, including viral attachment and penetration. Experimental results revealed that 129 significantly inhibited HCoV-229E attachment in a dose-dependent manner, suggesting that 129 prevents the attachment of HCoV-229E into host cells. Compound 129 also inhibited penetration of HCoV-229E into Molecules 2021, 26, x FOR PEER REVIEW 20 of 29 host cells in a time-dependent manner, suggesting that it also prevents the penetration of HCoV-229Ethe penetration into of host HCoV-229E cells, probably into host through cells, detachment probably through of virus detachment that has already of virus bound that tohas the already cell by bound disturbing to the viral cell glycoproteinsby disturbing [viral64]. glycoproteins [64].

FigureFigure 19.19. StructuresStructures ofof saikosaponinssaikosaponins127 127––130130..

Wu et al. have found that the oleanane type saponin aescin (131) (Figure 20), a major active principle of Aesculus hippocastanum L. (Sapindaceae), inhibited viral replication in SARS-CoV-infected Vero E6 cells with an EC50 of 6.0 μM and CC50 of 15 μM (SI = 2.5) [31]. In an attempt to decipher the mechanism of action of N. sativa L. for its usefulness against SARS-CoV-2, Mani et al. have used a network generated around ACE2, a target site for SARS-CoV-2, to depict a disease network and N. sativa L. constituents as a treatment network. Network analysis revealed various hub nodes, i.e., the proteins that control the disperse of signaling in the network including ACE2, which has various functions related to blood-vessel dilation, controlling blood pressure, cytokine production, inflammatory response, and protein transport. The symptoms of breathlessness, dysregulated blood pressure and inflammation are those that were initially reported in COVID-19 diagnosis. Glide Standard-Precision (SP) docking was performed and the best binding lig- and to ACE2 was found to be α-hederin (132) (Figure 20). Compound 132 (−6.265 Kcal/mol) was observed to be involved in a regulation of blood pressure, cell communication, vascular processes, negative regulation of cell death, response to stress and immune effector processes. The genes identified for regulating the immune response via 132 are ACE2, F2, SRC, etc. Since 132 has ACE as its predicted receptor in human system, this compound can be further explored for targeted therapies [65].

Molecules 2021, 26, 1754 25 of 28

FigureFigure 20. 20.Structures Structures of of triterpene triterpene glycosides glycosides aescin aescin ( 131(131)) and andα α-hederin-hederin ( 132(132).).

In an attempt to decipher the mechanism of action of N. sativa L. for its usefulness against SARS-CoV-2, Mani et al. have used a network generated around ACE2, a target site for SARS-CoV-2, to depict a disease network and N. sativa L. constituents as a treatment network. Network analysis revealed various hub nodes, i.e., the proteins that control the disperse of signaling in the network including ACE2, which has various functions related to blood-vessel dilation, controlling blood pressure, cytokine production, inflammatory response, and protein transport. The symptoms of breathlessness, dysregulated blood pressure and inflammation are those that were initially reported in COVID-19 diagnosis. Glide Standard-Precision (SP) docking was performed and the best binding ligand to ACE2 was found to be α-hederin (132) (Figure 20). Compound 132 (−6.265 Kcal/mol) was observed to be involved in a regulation of blood pressure, cell communication, vascular processes, negative regulation of cell death, response to stress and immune effector processes. The genes identified for regulating the immune response via 132 are ACE2, F2, SRC, etc. Since 132 has ACE as its predicted receptor in human system, this compound can be further explored for targeted therapies [65].

4. Future Perspectives and Conclusions This article presents an overview of the current state of knowledge on the effects of various classes of phytochemicals and their mode of action on SARS-CoV, which can be used as models for drug leads to combat the COVID-19 pandemic. In an attempt to ﬁnd new anti-HCoV drugs, this review can guide researchers to target medicinal plants with antiviral properties because of their valuable naturally occurring metabolites. We have shown that some structural classes of natural products such as the abietane diterpenes, friedelane and oleanane triterpenes and saponins along with geranylated and glycosylated ﬂavonoids could be considered as unique scaffolds for the development of anti-HCoV-229E drugs. On the other hand, the phenolic compounds such as those containing galloyl moiety can exert antiviral activity through the interaction with the enzyme active sites. Emerging from this review are the glycosylated secondary metabolites, in particular saponins, which have an

Molecules 2021, 26, 1754 26 of 28

intrinsic capacity to be used as anti-SARS-CoV diseases. Due to the novel profile of COVID- 19 pneumonia related to the viral binding site of ACE2, some secondary metabolites are more likely to represent potential antiviral drugs. Finally, yet importantly, a combination of IFNs, in particular IFN-β, with other antiviral and anti-inflammatory natural products might be of great interest in treatment of SARS-CoV. Further investigations and chemical modifications of anti-SARS-CoV naturally occurring compounds to search for parameters that increase efficacy and decrease toxicity and side effects are needed.

Author Contributions: Conceptualization: S.H.G. and A.K.; software: S.H.G. and A.K.; validation: M.E.-S., N.S. and A.K.; investigation: S.H.G. and M.E.-S.; writing—original draft preparation: S.H.G.; resources: N.S. and M.E.-S.; writing—review and editing: A.K.; supervision: A.K. All authors have read and approved the final version of the manuscript. Funding: This work is partially supported by national funds through FCT- Foundation for Science and Technology with the scope of UIDB/04423/2020 and UIDP/04423/2020. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Data sharing is not applicable to this article. Acknowledgments: The authors thank Golestan University and Instituto de Ciências Biomédicas Abel Salazar of the University of Porto for logistical supports. Conflicts of Interest: The authors declare no conflict of interest.

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