Parva Namiranian,Omid Sadatpour,Sadegh Azimzadeh Jamalkandi,Mohammad Hossein Ayati,Mehrdad Karimi

Parva Namiranian,Mohammad Hossein Ayati,Mehrdad Karimi,Department of Traditional Medicine,School of Persian Medicine,Tehran University of Medical Sciences,Tehran,Iran

Omid Sadatpour,Department of Immunology,School of Medicine,Tehran University of Medical Sciences,Tehran,Iran

Sadegh Azimzadeh Jamalkandi,Chemical Injuries Research Center,Systems Biology and Poisonings Institute,Baqiyatallah University of Medical Sciences,Tehran,Iran

Abstract OBJECTIVE:To investigate the in vitro and in vivo studies of natural compounds and medicinal plants with anti-coronavirus activity.METHODS:A systematic review was performed based on Preferred Reporting Items for Systematic Reviews and Meta-Analyses and Animal Research:Reporting of in vivo experiments guidelines to find data for medicinal plants and natural products effective against human coronaviruses in in vitro or in vivo studies.Studies published up to September 6,2020 were included.Studies (in vitro or in vivo) reporting the effect of medicinal plants and natural products or their derivatives on human coronavirus were included RESULTS:Promising anti-coronavirus effects are seen with different herbal compounds like some diterpenoids,sesquiterpenoids,and three compounds in tea with 3CLpro inhibiting effect of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV);Hirsutenone,Six cinnamic amides and bavachinin are PLpro inhibitors and Tanshinones are active on both 3CLpro and PLpro.Some flavonoid compounds of Citrus fruits act on Immunoregulation and target angiotensin-converting enzyme 2 which is used by SARS-COV for entry.Virus helicase is possibly inhibited by two compounds myricetin and scutellarein.CONCLUSION:This review shows that complementary medicine have the potential for new drug discovery against coronavirus.Further research is needed before definitive conclusions can be made concerning the safety and efficacy of the use of these medicinal plants.

Keywords:Coronavirus;medicine,Persian;medicine,Chinese traditional;plants,medicinal;new drug discovery

1.INTRODUCTION

Human coronaviruses (HCoVs) are the main viruses that cause upper respiratory tract illness in humans.1Seven types of coronaviruses infect humans,including four low-pathogenic members of HCoV-OC43,HCoV-229E,HCoV-HKU1,and HCoV-NL63whand three high pathogenic members of severe acute respiratory syndrome coronavirus (SARS-CoV),SARS-CoV-2,and Middle East respiratory syndrome coronavirus (MERSCoV).2Genetically,there is about 79% similarity between SARS-CoV-2 and SARS-CoV,and 50%similarity between SARS-CoV-2 and MERS-CoV.3Currently,COVID-19 infection has affected many people worldwide since its first emergence in late December 2019 in Wuhan,China.4The disease is spreading very fast,with a transmission rate among humans faster than both SARS-CoV and MERS-CoV.5,6There have been ongoing efforts to produce effective medication for treatment with prophylactic effects for the disease.The development of antiviral drugs and vaccines is time-consuming,and this necessitates the use of possible compounds for the prevention and treatment of COVID-19.Natural products are an essential source for new drug discovery,including a wide range of biologically relevant chemicals.7Herbal medicine and phytotherapy can be used as an adjuvant to conventional therapies.During past epidemics,there have been some clinical trials in which Traditional Chinese Medicine(TCM) medications were used along with modern medicine,8-13causing shorter hospitalization,improvement or disappearing of symptoms,decreasing the dosage of corticosteroids,and side effects of several drugs,and improving the quality of life of SARS patients.14,15In China,using TCM herbs in different medicinal forms has been considered for the current infection,too,16-22and even for preventing the disease.23It was reported that China had the lowest mortality rate during the SARS crisis.The mortality rate in mainland china was 7%,while the corresponding percentage was higher in other territories such as France with 14% or Canada with 17%.24This low rate and quick control of the outbreak were associated with integrating TCM with current medicine30,31Triterpenoids and their derivatives,also called the steroids,are considered a big family of natural compounds.Most of these compounds are present in nature as ferulic acid,esters such as fatty acids and free acids,or triterpenoid saponins linked with one or more sugar chains.They make the principle active components of various medicinal herbs,like glycyrrhizin inGlycyrrhiza uralensis,the ginsenosides inPanax ginseng,and saikosaponins inRadix bupleuri.25From 125 registered clinical trials as of February 18,2020,targeting COVID-19 treatment,33.3% were about herbs or TCM.26

Thus,it is imperative to gather and study natural compounds and medicinal plants with anti-coronavirus activity.In this systematic review,we have broadly investigated thein vitroandin vivostudies of medicinal plants and discussed their characteristics regarding different aspects of coronaviral infections.

2.METHODS

2.1.Search strategy

A systematic review was performed based on Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) and Animal Research:Reporting ofIn VivoExperiments (ARRIVE) guidelines to find data available for medicinal plants and natural products found to be effective against human coronaviruses inin vitroorin vivostudies.Online academic databases including PubMed,Web of Science,Wiley online library,Cochrane library,Google Scholar,and ScienceDirect were systematically searched based on the terms of[SARS OR MERS OR coronavirus OR COVID-19 OR 2019-nCoV OR "Severe Acute Respiratory Syndrome Coronavirus 2" OR SARS-CoV-2 OR "3-chymotrypsinlike protease" OR "papain-like protease" OR Furin OR TMPRSS2] AND [plant extract OR leaf OR root OR fruit OR petal OR peel OR seed OR bark OR rhizome OR medicinal plants OR herbal medicine OR Chinese medicine OR TCM OR and combinations].Then manuscripts texts were investigated forin vivoandin vitrostudies.Studies published up to September 6,2020 without language and time restrictions were included.Studies (in vitroorin vivo) reporting the effect of medicinal plants and natural products or their derivatives on human coronavirus were included.In silicoand docking studies,network pharmacology articles,randomized controlled trials (RCTs),manuscripts not peer-reviewed,studies on coronaviruses infecting other than humans,and non-peer-reviewed papers were excluded.

2.2.Data Extraction and quality control

Data regarding the type of study being whetherin vitroorin vivo,phytochemical name/active ingredients,the scientific names of plants,plant parts,type of extracts,name of natural products,compounds,type of studied coronavirus,stage of inhibition/ function,half-maximal effective concentration in μg/mL (EC50),mean 50%cytotoxic concentration in μg/mL (CC50),half maximal inhibitory concentration (IC50),and Selectivity Index(SI) (equal to CC50/IC50) were collected independently from every manuscript and captured using a standardized Word document form.

2.3.Classification of studies

The SARS-CoV-2 genome encodes proteins,which are non-structural,structural,and accessory.Non-structural proteins include 3-chymotrypsin-like protease (3CLpro),RNA-dependent RNA polymerase,papain-like protease(PLpro),and helicase.It contains four structural proteins,including spike glycoprotein (S),membrane (M),envelope (E),and nucleocapsid (N) proteins.27Targeting specific proteins that show preservation in all SARSCoV-2 genomes can be useful in designing drugs.Studies have shown that 3CLpro is preserved,with 100%similarity among all SARS-CoV-2 genomes.It has 96.08%,87.00%,and 90.00% identical sequence with SARS-CoV,MERS-CoV,and Human-CoV homologs,respectively.28Plpro is another protein that is used for drug discovery purposes.We divided medicinal herbs into two subsidiary classes of in vitro and aminal studies based on the analyzed studies.Then thein vitrostudies were classified into six main classes based on their effects on SARS-CoV,including Class I) targeting viral entry,Class II) targeting viral replication,Class III)targeting viral pLpro protein,Class IV) targeting viral 3CLpro protein,Class V) targeting other proteins,and Class VI) targeting other involved mechanisms.

3.RESULTS

Full versions of potentially relevant articles were obtained to assess eligibility,and each was individually evaluated for inclusion (Figure 1).

Figure 1 PRISMA flow diagram for the systematic review detailing the database searches,the number of abstracts screened and the full texts retrieved

3.1.In vitro studies

3.1.1 Class I:targeting viral entry

There were fourteen eligible studies identified from our search activities related to compounds inhibiting the entry process of SARS-CoV into cells in different ways.SARS-S glycoprotein uses angiotensin-converting enzyme 2 (ACE2) as the receptor for entry29,30and utilizes TMPRSS2,which is a cellular serine protease,for S protein priming.29,31Radix et Rhizoma Rhei(the root tubers ofRheum officinaleBaill.),RadixPolygoni multiflori(the root tubers ofPolygonum multiflorumThunb.),and CaulisPolygoni multiflori(the vines ofPolygonum multiflorumThunb.) were inhibitors of the attachment of SARS-CoV S protein to ACE2.Emodin,an anthraquinone compound extracted from genusRheumandPolygonum,was also an effective inhibitor of the S protein and ACE2 fusion that acts in a dosedependent way.32Luteolin and tetra-O-galloyl-β-Dglucose (TGG) had apparent properties against SARSCoV.These two molecules exhibited vivid anti-SARSCoV effects with binding to the SARS S2 glycoprotein that interferes with the entry of SARS-CoV into Vero E6 cells.33

Griffithsin,a lectin derived from red-alga,attaches to SARS-CoV spike glycoprotein.34Therefore,it may be a potent spike inhibitor for SARS-CoV-2,too.Griffithsin was previously used in the form of a gel or as an enema to prevent HIV.35Its route of administration and efficacy on SARS-CoV-2 needs further studies.Griffithsin was found to inhibit the entry of MERS-CoV into host cells by inhibiting spike proteins' function.This inhibitory effect also happens within the early phases of the virus replication cycle.36

Some medicinal herbs such asScrophularia scorodonia,Heteromorpha spp.,Bupleurum spp.,andRadix bupleuricontain Saikosaponins.37,38Saikosaponins (A,B2,C,and D) had antiviral properties at different concentrations,and the most potent one on human coronavirus 229E was Saikosaponin B2.Saikosaponin B2 might exert its anticoronavirus characteristic by interfering with virus fusion to the cell surface (Figure 2).38Details are shown in supplementary Table 1.

Figure 2 Natural compounds inhibitory actions throughout coronavirus life cycle

Fifteen plant lectins were found to inhibit both SARSCoV and Feline infectious peritonitis (FIP) caused by feline coronavirus (FCoV)in vitro,with five lectinsactive only against SARS-CoV and two only on FIPV.The lectins with anti-corona characteristics were galactose-,mannose-,glucose-,N-acetyl glucosamineand N-acetyl galactosamine-specific agglutinins.Details are given in supplementary Table 1.Generally,the group that inhibited coronaviruses were mannose-specific plant lectins.The lectin that inhibited the SARS-CoV-induced cytopathy most remarkably was the mannose-specific plant lectin taken from leek.Indeed,the N-acetyl glucosamine-specific lectins purified from the stinging nettle (Urtica dioica agglutinin,UDA) and the tobacco plant (Nictaba) blocked SARS-CoV significantly.Two probable targets for antiviral activity were found;one in the early phase of virus replication (most likely fusion)and the other target in the final stages of the virus cycle.39As previously mentioned,coronaviruses are enveloped viruses.Their surface is covered with carbohydratecontaining proteins.Carbohydrate-binding agents (CBA)were identified to be coronavirus inhibitors.Two strains of coronaviruses,Feline Infectious Peritonitis virus,and Mouse Hepatitis Virus were exposed to CBA,including the plant lectinsGalanthus nivalisagglutinin,UDA,Hippeastrumhybrid agglutinin,and the non-peptidic mannose-binding antibiotic pradimicin A.CBA targeted the S and M protein,of both viruses.It was shown that CBA was not a virus-cell attachment inhibitor but acted on virus entry after the binding stage.The sensitivity of coronaviruses towards CBA is dependent on N-linked carbohydrates processing.40

The effect of ethanol extract produced from FormosanaNakai stem and numerous phenolic acid components on HCoV-NL63 was evaluated.Gallic acid,caffeic acid,and chlorogenic acid showed anti-HCoVNL63 characteristics,with caffeic acid being the most potent constituent,which inhibited attachment of virus strongly and blocked viral replication in a cell-typeindependent way.41

3.1.2 Class II:targeting viral replication

Thirteen studies from our search activities focused on compounds affecting the replication of SARS-CoV.The essential oil ofLaurus nobiliswas reported to have anti-SARS-CoV property.L.nobilis,T.orientalis,andJ.oxycedrusssp.oils inhibited SARS-CoV more potently.They had a great margin of safety compared to glycyrrhizin as positive control.T.orientalisandJ.oxycedrusssp.oxycedrusoils were found to inhibit SARS-CoV replication.A weak anti-SARS-CoV effect was seen whenC.sempervirensssp.pyramidalisandS.officinalisessential oils were added to virus culture.42TheLaurus nobilisessential oil is made up of 21.83%alpha-ocimeme;9.43% 1,8-cineole;3.65% small amounts of eremanthin,and 7,52% dehydro-costuslactone.43Twenty-six components madeSalviaofficinalis(94.39% of the total oil);Among them,1,8-cineole made up to 43.62%;alpha-thujone 12.99%;sabinene 6.97%;and camphor 5.71%.42Cimicifuga rhizoma,Meliae cortex,Coptidis rhizoma,Phellodendron cortex,andSophora subprostrata radixlower the mouse hepatitis virus A59 (MHV-A59)replication.The details are shown in supplementary Table 2.

After boiling,the crude extract is taken from the leaf ofToona sinensis to formTSL-1.When a nanometermanufacturing method was applied on TSL-1,the pieces were not more than a nanometer,and TSL-1nm was produced.44TSL-1 was shown to inhibit SARS-CoV replicationin vitro.45Toona sinensisRoem is also known asCedrela sinensis.

Cell-based cytopathogenic effect (CPE) assay in SARSCoV-infected Vero E6 cells showed that some herbal extracts were effective against SARS-CoV.Studying these plants using enzyme-linked immunosorbent assay,herbal extracts fromGentianae Radix,Dioscoreae Rhizoma,Cassiae Semen,andLoranthi Ramus(the dried stem,with the leaf ofTaxillus chinensis)Rhizoma Cibotii(the dried rhizome ofCibotium barometz) inhibited SARS-CoV significantly at different concentrations.46Details are given in supplementary Table 2.Pudilan Xiaoyan Oral Liquid (PDL) and Lianhuaqingwen (LH)were able to lower viral replication in Vero E6 cells infected with SARS-CoV-2.47LH is composed of 13 medicinal herbs,and it causes particle morphology related to virion to become abnormal after administration.48Resveratrol (trans-3,5,4′-trihydroxystilbene) inhibits MERS-CoV replication in Vero E6 cells.This natural stilbene derivative,which is available in various plants,such as cranberry (Vacciniummacrocarpon),Huzhang (Polygonum cuspidatum),and grape (Vitis vinifera),49does this by blocking nucleocapsid (N) protein expression of the virus which is fundamental for viral replication.Cellular apoptosis rate mediated by the virus was also reduced.Besides,the consequent application of resveratrol decreased virus activity in lower dosages.50Three prescriptions used in TCM known as Fu-Fang-Lian-Pu pellet,Lian-Hua-Qing-Wen capsule,and Di-Kang's injection on SARS-CoV in Vero-E6 cells showed viral replication inhibition.51Standardized extract ofPelargonium sidoides(EPs®7630) affects the replication of some respiratory viruses.This product decreased replication of human coronavirus at concentrations up to 100 μg/mL.52

UDA fromstinging nettle rhizomewas reported to block replication of SARS-CoV strains in Vero 76 cells in a dose-dependent way.Details are given in supplementary Table 3.Therefore,viral loads of the Urbani strain were decreased by UDA.Further studies of UDAin vitroshowed that the replication reduction of live SARS-CoV or SARS-CoV pseudotyped virus happened when added just before adsorption (early phases of replication).Thus,adsorption or penetration may be the target of UDA to show inhibitory actions.UDA may also bind to the SARS-CoV spike glycoprotein to neutralize the virus infectivity.UDA was exposed to N-acetylglucosamine,and it was added to cells just before adsorption.UDA did not affect virus infection inhibition.According to these data,it may be concluded that by binding to Nacetylglucosamine-like residues of envelope glycolproteins,UDA inhibits virus attachment to cells.53

Anti HCoV-OC43 property of cepharanthine (CEP),tetrandrine (TET),and fangchinoline (FAN) was evaluated using MRC-5 human embryonic lung fibroblast.The central origin of these bisbenzylisoquinoline alkaloids isStephania tetrandra andother related species of Menispermaceae.The results revealed that viral replication was significantly reduced,and N and S proteins' expression was blocked.Thus,it can be inferred that these compounds can be effective for prophylaxis and HCoV-OC43 treatment.54

3.1.3 Class III:targeting viral pLpro protein

Five studies related to compounds with corona virus'Papain-like protease down regulatory activities were found based on our search activities.Total ethanol extract related to the root ofBroussonetia papyriferaand its numerous fractionsincluding broussochalcone A and B,broussoflavan A,4-hydroxyisolonchocarpin,papyriflavonol A,kazinol A,B,F and J,3′-(3-methylbut-2-enyl)-3′,4,7-trihydroxyflavane's effects on proteases of MERS-CoV and SARS Co-V showed that all of ten polyphenols inhibited SARS-CoV PLpro in a dosedependent manner.They blocked it more significantly than the other cysteine proteases,with Papyriflavonol A having the most potent effect on PLpro.55Ethanol extract ofPsoralea corylifoliaseeds is another potent PLpro inhibitor of SARS CoV.Fractionation of them yielded in six aromatic components:bavachinin (1),neobavaisoflavone (2),isobavachalcone (3),4'-O-methylbavachalcone (4),psoralidin (5),and corylifol A (6).These components can also significantly inhibit PLpro,too (55) Methanol extract,on the other hand,ofTribulus terrestrisfruits were shown to inhibit SARS CoV PLpro potently.Six cinnamic amides (1-6) and ferulic acid (7)resulted from this extract fractionation.Of them,compounds (1-6) were potent PLpro inhibitors.Test results showed that these six compounds were highly available in native fruits.56

Five new geranylated flavonoids,including tomentin A,B,C,D,E from fruits ofPaulownia tomentosatree,were investigated to search for the PLpro inhibitory effect.Some known extracts including 3′-O-methyldiplacone,3′-O-methyldiplacol,4′-O-methyldiplacol,diplacone,4′-O-methyldiplacone,mimulone,and 6-geranyl-4′,5,7-trihydroxy-3′,5′-dimethoxyflavanone were also studied.Most compounds inhibited PLpro in a dose-dependent way.All new compounds had a 3,4-dihydro-2H-pyran group and a better antiviral effect than their parent compounds.57Another compound with anti-PLpro activity is diarylheptanoids 1-9 fromAlnus japonica.Thus,it is the most potent inhibitor of Plpro.Catechol and α,β-unsaturated carbonyl moiety in the molecule were essential for PLpro inhibition.58The half-maximal inhibitory concentration (IC50) of compounds on PLpro is given in supplementary Table 4.

Tryptanthrin blocked HCoV-NL63 replication in early and late stages,mainly by suppressing the synthesis of the viral RNA genome and blocking PLpro.in addition to tryptanthrin,indigodole B revealed significant viricidal characteristics against HCoV-NL63,too.59

3.1.4 Class IV:targeting viral 3CLpro protein

Fifteen studies related to compounds with corona virus'3-chymotrypsin-like protease down regulatory activities were found based on our search activities.Natural polyphenols of tea,including 3-isotheaflavin-3-gallate(TF2B) and Tannic acid inhibit 3CLpro activity.In addition to these two compounds,theaflavin-3,3'-digallate (TF3) is also a 3CLpro inhibitor.3CLpro inhibitory activity was observed in Pure and black tea extracts compared to green or oolong teas.60There are two gallate groups attached to the 3 and 3' position in theaflavin-3-3'-digallate (in contrast to one gallate group in theaflavin 3-gallate or nil in theaflavin).Since catechins are less active than theaflavins,the gallate group attached to 3' position might be necessary for inhibiting the 3CLpro.It is noteworthy that black tea's theaflavins can neutralize coronavirus because tea is a very routine drink in many countries.61Furthermore,aqueous extract of Haritaki (Terminalia chebula) and Tea (Camellia sinensis)block SARS-COV-2 3CLpro,too.62

Eight out of nine phlorotannins derived from the edible brown algaeEcklonia cavainhibited SARS-CoV 3CLpro dose-dependently.Among these compounds,two eckol groups with a diphenyl ether-linked dieckol (8)exhibited the most potent SARS-CoV 3CLpro inhibitor activity63IC50 of compounds on 3CLpro (supplementary Table 5).

Cell-based assay to evaluate the cytopathogenic effect of SARS-CoV on Vero E6 cells showed that some compounds were potent 3CLpro inhibitors,including lignoids,sesquiterpenoids,diterpenoids,triterpenoids,baicalin,Shuanghuanglian preparation,and curcumin.Shuanghuanglian preparation is a potent TCM medication composed ofForsythia suspense(Thunb.),Scutellaria baicalensisGeorgi,andLonicera japonicaThunb which is used for acute infections of the respiratory system.The most effective compounds belonged to the following groups:four diterpenoids with numbers 1,5,6,8;a triterpenoid with number 14;and a lignoid numbered 16.Betulinic acid and savinin appeared to be competitive inhibitors of SARS-CoV 3CLpro.Specific abietane-type diterpenoids and lignoids have anti-SARS-CoV properties.Possible mechanisms of their action are protease inhibition and preventing SARS-CoV entry at the post binding stage during the fusion phase.64,65It is concluded that lignoids and some abietane-type diterpenoids could inhibit SARS-CoV in a significant way.Curcumin,on the other hand,inhibited SARS-CoV replication and 3CLpro mildly.65Shuanghuanglian preparation and baicalin with the plant source ofS.baicalensis,had not only a potent 3CLpro inhibitory effect but also blocked virus replication.66

Cell-free and cell-based cleavage assays for five major compounds ofI.indigoticaroot,Isatis indigoticaroot extract,and seven plant-derived phenolic compounds in order to study their interactions with SARS-CoV 3CLpro showed IC50 values of μM ranges by cleavage assays with the 3CLpro forI.indigoticaroot extract,hesperetin,aloe-emodin,sinigrin,and indigo.Sinigrin exhibited to be a more potent 3CLpro inhibitor in the cell-based test than indigo and beta-sitosterol.Dose-dependent inhibition of cleavage activity of the 3CLpro in the cellbased assay was seen for two phenolic compounds aloeemodin and hesperetin.67

The Methanol extract of the dried rhizome ofCibotium barometzand Methanol extract ofDioscoreae Rhizoma(the tuber ofDioscorea batatas) were shown to inhibit SARS-CoV 3CLpro remarkably.46Torreya nuciferaleaves and ethanol extract inhibited SARS-CoV 3CLpro.After Bioactivity-guided fractionation,anti-3CLpro activity of eight diterpenoids (1-8) and four biflavonoids(9-12) were studied using fluorescence resonance energy transfer (FRET) analysis.Biflavone amentoflavone (9)blocked 3CLpro most significantly.Authentic flavones(quercetin,luteolin,and apigenin) were also 3CLpro inhibitors.The results were supported by a molecular docking study.68Houttuynia cordatawater extract was a potent SARS-CoV 3CLpro inhibitor.This species applies its inhibitory effects in a dose-dependent way.69Four quinone-methide triterpenes,numbered 67-70,are isolated fromTriterygium regelii.By hydrogenation under palladium-carbon catalyst,compound 67 is converted to its derivative 71.All five compounds were evaluated for SARS-CoV 3CLpro inhibitory effect.Compounds 67-70 had IC50 values better than compound number 71,which had a phenol moiety.Therefore,it may be concluded that the relatively marked effect in inhibition is caused by the quinone-methide moiety.25Investigation of Quinone-methide tingenone,iguesterin,pristimerin,triterpenes,and celastrol taken fromTriterygium regelii,and also,dihydrocelastrol,which is synthesized by hydrogenation under palladium catalyst was found to be significant inhibitors of 3CLpro.Dihydrocelastrol,which had phenol moiety,was not very effective.Thus,quinone-methide moiety in A-ring and more hydrophobic E-ring helps the compound be more functional.70

Rheumspp.has anthraquinone derivatives,including emodin,aloe-emodin,and rhein,as main active principles.71It was found thatRheum palmatum L.components were significant SARS-CoV 3CLpro inhibitors.The inhibition rate was up to 96%,and it can be inferred that extracts fromRheum palmatum L.might be an effective inhibitor for SARS-CoV-2,too.72

3.1.5 Class V:targeting other proteins

Coronaviruses use a 5′ cap-dependent way to initiate viral mRNA translation.Silvestrol was found to significantly block cap-dependent viral mRNA translation of HCoV-229E and MERS-CoV in human embryonic lung fibroblast cells.Peripheral blood mononuclear cells (PBMCs) and Huh-7 cells were also used for further study of HCoV-229E,which showed that Silvestrol blocks the expression of structural and nonstructural proteins (N,nsp8) in coronavirus significantly and also prevents viral replication/transcription complexes73(supplementary Table 6).

Myricetin and Scutellarein are potent inhibitors of the nsP13 (SARS-CoV helicase protein) that affect ATPase activity.Besides,these two compounds do not have cytotoxic effects on normal breast epithelial MCF10A cells.74

SARS-CoV's RNA-dependent RNA polymerase (RdRp),an enzyme that plays an essential role in replication and gene transcription of the virus,is inhibited by the extracts of Kwan Du Bu Fei Dang (KDBFD) in a dose-dependent manner.Kwan Du Bu Fei Dang is a Chinese formula of several plants,includingRhizoma Phragmitis,Semen Armeniacae,Amarum Folium Mori,Radix Astragali,Fructus Forsythiae,Radix Glycyrrhizae,Radix Scutellariae,Flos Chrysanthemi,Herba Menthae,Radix Platycodonis,Folium Isatidis,and Radix Saposhnikovia.Other SARS-CoV RdRp inhibitors areGanoderma lucidum,Sinomenium acutum,andCoriolus versicolor,and Houttuynia cordata.75

3.1.6 Class VI:targeting other involved mechanisms

It is known that the cytokine storm has an essential role in COVID-19.Severe cases have high amounts of cytokines,like IL-1β,IFNγ,TNF-α,MCP-1,and IL-10.4Flavonoid compounds of three species of citrus fruits,including mandarin (Citrus reticulata),pummelo (Citrus maxima),and sweet orange (Citrus sinensis) were studied.The flavonoids were naringin,naringeninm,hesperetin,hesperidin,nobiletin,and neohesperidin.76In vitroandin vivoexperiments on naringin and naringenin showed that naringin might prevent cytokine storm.Naringin suppressed some proinflammatory cytokines,including IL-6,COX-2,IL-1β,and iNOS in the Raw macrophage cell line.Naringin content was higher inCitrusmaxima.This mentioned cytokine restrain may happenviablocking HMGB1 expression in a mouse model.HMGB1 is a ubiquitous DNA-binding nuclear protein released actively by immune cells,such as macrophages and monocytes,following inflammatory stimulation.It also acts as a proinflammatory cytokine and regulates cytokine storm.The amount of hesperidin and hesperetin were high in sweet orange and mandarin.76Hesperetin was found to inhibit SARS-CoV 3CLpro significantly.67Due to the potential inhibitory effects of flavonoids on coronavirus and antiinflammatory property of flavonoids,the citrus fruit or its extracted phytochemicals have been proposed as probably effective for prevention and treatment of COVID-19.76

The efficacy of extracts from peels ofCitrus sinensis(Cs)on coronavirus was evaluated.MHV-A59 inoculated cells were used.Expression of some genes,including TRPM8,TRPC4,TRPV4,TRPM7,TRPA1,and TRPM6 changed remarkably after using Cs extract,and the virus load diminished.77Citrus sinensis(Cs),Anthemis hyalina(Ah),andNigella Sativa(Ns) extracts have been shown to decrease Coronavirus load after application to infected cells.The Ah extract was found to have the most potent anti-coronavirus effect.TRP gene expression levels were generally downregulated,and extracts affected these TRP channels:TRPV4,TRPC4,TRPM8,TRPM7,TRPM6,and TRPA1 and downregulated each one of them During CoV infection.78

IL-6 also plays an important role in COVID-19 and its complications.79The effect ofNigella sativaL.seeds oil on inflammation was evaluated using Simpson-Golabi-Behmel syndrome human pre-adipocytes.Two types of this product were used according to their storage time.The fresh extracted oil (FEO),which had a higher amount of thymoquinone,caused a significant decrease in IL-6 levels.The stored extracted oil (SEO),which had a higher anti-oxidant level,inhibited IL-1beta.80Parthenolide (PTN) decreased the amount of IL-6 in cultured lipopolysaccharide (LPS)-stimulated BV-2 microglia.This sesquiterpene lactonic derivative constitutes the main part of Feverfew (Tanacetum parthenium);81The effect was dose-dependent,and at 5 µm,IL-6 was 98% reduced,and TNF-α showed a significant reduction,too.82In vitroandin vivoexperiments of parthenolide showed promising antiinflammatory effects.83Propolis blocks not only IL-6,but also TGF-β mediated Th17 differentiationin vitro.This inhibitory activity may be used in the control of unbalanced cytokine networks.84Although propolis has not been studied separately on SARS-COVin vitro,many components have been found to be effective against SARS-CoV in previous in vitro studies;so it may be effective.85

Butanol fraction of Cinnamomi Cortex is a moderate inhibitor of wild-type SARS coronavirus.Butanol fraction of Cinnamomi Cortex has unknown inhibiting substances on the virus that possibly interfere with endocytosis,and it also contains procyanidins that do not inhibit the internalization but inhibit the infection.Procyanidin A2 and procyanidin B1 are two fractionated compounds purified from the Cinnamomi Cortex that showed moderate activity against wild-type SARSCoV.86

The cytopathic effect in Vero 76 cells induced by human SARS coronavirus is decreased significantly by tylophorine compounds.The compounds are derived from the methanol extracts ofT.indicaandT.ovata.These compounds are effective in the reduction of viral replication.87Another compound called myricetin,a bioflavonoid,has had an excellent antiviral effect against coronavirus.88Lycoris radiataand it is active components lycorine,Lindera aggregata,Artemisia annua,andPyrrosia linguablocked SARS-CoV moderately to potently.The most influential wasLycoris radiata.Details are given in supplementary Table 2.More fractionation,purification,and CPE/MTS evaluations onL.radiataextract to determine the active component recognized a single substance lycorine as a SARS-CoV inhibitor.89

3a channel of SARS coronavirus is necessary to be active before virus release occurs;Thus,if a medication blocks the channel,that drug inhibits the virus release.90Flavonols kaempferol,kaempferol glycosides,and acylated kaempferol glucoside derivatives were evaluated to inhibit the 3a channel's efficacy.Xenopus oocyte was used for this purpose.The glycoside juglanin was the most prominent drug which carried an arabinose residue.It seems that Kaempferol derivatives with rhamnose residue are effective on coronaviruses' 3a channel proteins.90,91Emodin also inhibited the 3amediated current.90The 3a ion channel of HCoV-OC43 and SARS-CoV are inhibited by emodin.Emodin also inhibits virus release from HCoV-OC43.92,93

Fifteen compounds structurally related to glycyrrhizin and Aescin,and six compounds related to Reserpine were studied94and of them,Four glycyrrhizin and Aescin derivatives,along with all six analogs of Reserpine,blocked SARS-CoV at ＜100 μM.One of the active compounds inPanax ginsengis Ginsenoside-Rb1.95,96This compound is antiviral at 100 µM,although the compound's toxicity is needed to be clarified.33Forty unsymmetrical aromatic disulfides are synthesized chemically that can inhibit SARS-CoV Main protease.97Twenty-three compounds from the ethanolic extract ofEuphorbianeriifolialeaves that included 22 triterpenoids and one flavonoid glycoside,were studied to evaluate the inhibitory activity of the separated triterpenoids on human coronavirus (HCoV).With actinomycin D used as the positive control,3β-Friedelanol was a more significant antiviral compound.Cell survival percent related to 3β-Friedelanol was 132.4,while it is 69.5 percent in actinomycin D.Epitaraxero,friedelin,and 3β-acetoxy friedelane was also related to 111,109,and 80.9 % Cell survival,respectively.98

The complement system's role in viral infections,including SARS,is crucial.The classical pathway (CP)and the alternative pathway (AP) can activate this system.The complement inhibitory effects of a Chinese multiherb remedy which is made up of HerbaArtemisiae Scopariae,Herba Eupatorii,Herba Houttuyniae,FlosChrysanthemi Indici,andFructus Tsaokowas utilized for prophylaxis and treatment of SARS;and was examined by hemolytic assays through the complement systemin vitro.Thisin vitrostudy was done by testing compounds for complement-inhibiting activities on the CP and AP.Fifteen compounds were isolated.Inhibition of the CP and AP was seen in nine flavonoids.The most potent one was luteolin (CH50 and AP50 values of 0.19 and 0.17 mM).99Another compound with prophylactic capacity is Echinaforce (comprising ethanol extracts ofEchinacea purpurea,95% aerial parts plus 5% roots)(94),which showed a protective effect used on respiratory epithelial cells before getting exposed to HCoV-229E.Therefore,it is concluded thatEchinacea purpureapreparations could be effective as a prophylactic treatment for CoVs,including SARS-CoV-2.100

Calendula officinalis has various phytochemicals,including quinones,coumarins,carotenoids,terpenoids,flavonoids,and other constituents.α-cadinol,an active component inC.officinalisflower,shows antiviral activity against SARS coronavirus.101

Cell culture has revealed that plant lectins are significant inhibitors of coronavirus replication.The GlcNAcspecific agglutinin Nictaba,the mannose-specific HHA(Hippeastrumhybrid lectin;amaryllis bulbs),and the(GlcNAc) n-specific agglutinin UDA were included in cultures.UDA blocked a mouse-adapted Urbani strain and the SARS-CoV Urbani strain in Vero cells.UDA inhibited SARS-CoV replication significantly,and there was no cytotoxicity for the lectins in allin vitrostudies.102

Benzoxazinones,which areDianthranthramidederiveatives,showed significant activities against human coronavirus in MRC-5 cells (human fibroblasts cells,in 3% FBS-DMEM medium),HCoV (strain 229E)cultures.Dianthusspecies of Caryophyllaceae were the source of isolation ofDianthramidesas phytoalexin.103The activity of some plant extracts has been studied on other coronaviruses except for SARS.Human coronavirus NL63 (HCoV-NL63) infection is an example that is frequently found among children,immuneocompromised individuals,and older adults.The methanol extract ofS.cusialeaf and its major components inhibit the cytopathic effects of HCoVNL63 on infected cells in a concentration-dependent way.Also,some compounds in the leaves of this plant,like indigodole B (5aR-ethyltryptanthrin) and tryptanthrin inhibited the virus104(supplementary Table 7).The different parts of coronavirus and the inhibitory effects of various natural compounds are illustrated in Figure 3.

Figure 3 Coronavirus genome and natural compounds inhibitory effects against its products

3.2.Animal studies

There are fewin vivostudies on animal coronaviruses and medicinal plants.Although animal models such as mice,Syrian hamster,non-human primate models,and ferrets have been used to study SARS and MERS,most studies have been in the field of vaccine development or chemical-based therapeutics.105There are relatively fewin vivostudies about natural products and medicinal plants.

The expression of recombinant nucleocapsid protein (rN)of SARS-CoV in tobacco (Nicotiana benthamiana) as a plant-based vaccine and its immunogenicity was surveyed in BALB/c mice.After three times immunization intraperitoneally,the specific IgG for rN protein reached 1 ∶1800 in the sera of mice that recommended B-cell maturation and differentiation in mice.Besides,splenocytes exhibited upregulation of IL-10 and IFN-γ.Thus,it is concluded that potent cellular and humoral responses may be mediated by rN of SARSCoV in mice.106

A new strain of SARS-CoV (strain v2163),which is fatal in five to six-week-old BALB/c mice,has been studied.This virus increased IL-1α,MCP-1,RANTES,MIP-1α,and IL-6 in mice.Increased expression of IL-6 correlated with mortality.The infection was very similar to human disease,except for a lack of hyaline membrane formation.Known inhibitors of SARS-CoV replication showedin vitroefficacy against v2163.In these infected mice,stinging nettle lectin (UDA) showed partial protection.UDA decreased IL-6 expression and inhibited SARSCoV replication.107In another study,BALB/c mice infected with mouse-adapted Urbani SARS-CoV (MA15 virus) were given intranasal griffithsin.They found that this substance can significantly inhibit the virus by attaching to the virus's spike protein and preventing virus entry.Also,all mice survived,and there was a reduction of virus amount in the lungs.108The intraperitoneal UDA lead to the essential protection of the fatal SARS-CoVinfected BALB/c mice.However,this did not cause a significant reduction of virus load in the lung.Altogether,it is proposed that using UDA in SARS infected mice can help mice survive.53

Moreover,KDBFD and herbal extracts were applied to Splenic lymphocytes related to Balb/c mice.Mouse splenic lymphocytes proliferated significantly whenCoriolus versicolorextract was applied.Besides,this extract increased the CD4+and CD8+T cell proportion.75

Pudilan Xiaoyan Oral Liquid (PDL),which is a TCM formula including Scutellaria Amoena (ScutellariaBaicalensis),Mongolian Dandelion (TaraxacumMongolicum),Bunge Corydalis (Corydalis Bungeana),and Indigowoad Root (Isatis Indigotica) inhibits SARSCoV-2in vivosignificantly.Applying PDLviathe intragastric route to HACE2 transgenic mice infected with SARS-CoV-2,resulted in body weight increase post-infection while the control group experienced weight loss.Besides,hACE2 mice treated with PDL had significantly lower viral RNA copies in lung post-SARSCoV-2-infection.47

4.DISCUSSION

Different types of investigations have been done on coronaviruses,includingin vitrostudies,in vivostudies,RCTs,case reports or case series,molecular docking,andin silicostudies.Iranian Traditional Medicine is one of the branches of complementary medicine with a precious treasure of plants,many of which have antiviral properties.109,110Among these antiviral herbs,there are specific herbs with specific studies on various types of viruses,including coronavirus.

Some diterpenoids,sesquiterpenoids,lignoids triterpenoids,curcumin,and quercetin,inhibit 3CLpro of SARS-CoV virus.Three compounds in tea have the same action:TF3,TF2B,and Tannic acid.Quercetin inhibits the same enzyme.dieckol,quinone-methide triterpenes,hesperetin,luteolin,amentoflavone,and apigenin are also among the compounds within vitrostudies supporting their inhibiting effect on 3CLpro of SARSCoV.Some flavonoid compounds of Citrus fruits act on Immunoregulation and also target ACE2 probably.

While Saikosaponins are more potent in inhibiting viral attachment and entry,TGG and Luteolin of tea inhibit the S2 subunit of spike glycoprotein.Reports show that Griffithsin and emodin are effective in preventing virus entry.Emodin is seen to be a 3a ion channel inhibitor as well as inhibiting virus release.Flavonols kaempferol glycosides,kaempferol,and acylated kaempferol glucoside derivatives block 3a ion channel,too.Moreover,glycyrrhizin is an inhibitor of virus replication,according to various studies.Urtica dioicaagglutinin and tylophorine compounds act on virus replication,too.Tanshinones are active on both 3CLpro and PLpro.Mannose-specific agglutinins,GlcNAc-specific agglutinins,are the group that inhibits viral attachment and put an end to the virus cycle.The same is true about Gal-specific agglutinins,(GlcNAc) n-specific agglutinins,Man/Glc-specific agglutinins,Gal/GalNAc specific agglutinins,GalNAc (＞ Gal) specific agglutinins,GalNAcα (1,3) Gal ＞ GalNAc ＞ Gal-specific agglutinins,Man/GalNAc-specific agglutinins.

Hirsutenone,six cinnamic amides,and bavachinin,4'-Omethylbavachalcone,neobavaisoflavone,isobavachalcone,psoralidin,corylifol A are PLpro inhibitors.Procyanidin A2,B1,and Butanol fraction of Cinnamomi Cortex probably block virus endocytosis.Virus helicase is possibly inhibited by myricetin and scutellarein.Some other natural compounds are acting as SARS-CoV inhibitors,that their mechanism of action is still not clear.Considering these positive effects,it is concluded that complementary medicines and especially herbal medicine can potentially discover new drug discovery.Furthermore,the whole extract of some plants or special parts of plants like roots,flowers or leaves has various properties (supplementary Table 7) that are sometimes different from small molecules.This reminds the significance of a holistic viewpoint and highlights ancient medical systems like Iranian Traditional Medicine or Chinese Traditional Medicine.

The main inquiry to be dealt with is whether or not these plant products will have toxicity in animal models so that they may be used for the next stages of trials and use as the work of Fung et al75in which they noticed Kwan Du Bu Fei Dang (KDBFD) formula,Coriolus versicolor,andHouttuynia cordatato be principally nontoxic on laboratory Balb/c mice.However,mortality was seen withGanoderma lucidumandSinomenium acutum.Any compound which is chosen for treatment should be nontoxic (＞50% cell viability) at concentrations of more than 300 µM in order to be developed as a useful lead drug.39Drug safety is a critical issue,after which the efficacy of drugs should be taken into account.

This review included mostlyin vitroandin vivostudies of medicinal plants which provide a basis for clinical studies.Thus,the effect and efficacy of these compounds in clinical setting and human being needs to be studied yet.Although some studies and clinical trials have been done on patients infected with SARS-CoV-2,their results are still to be published.

The other note is about probable interaction between conventional medicine medications used for COVID-19 patients and medicinal herbs and natural compounds.When there is simultaneous use of herbs and modern medicine drugs,their possible interactions must be taken into account with especial attention to the age of patients,potential polypharmacy and medications taken for underlying diseases.

In conclusion,medicinal herbs are a good source for fighting against bacterial and viral infections.In previous reports,these plants have shown favourable effects on viral diseases.Considering the promising effect of some herbs and natural products used in phytotherapy on coronavirus,it is concluded that these compounds have the potential to be used as medicines.Thus,future search for therapeutic effects of herbal medicine against COVID-19 will be of value.Reframing prevention and treatment strategies to integrate herbal medicine and Western medicine will result in a more robust approach to control and eradicate COVID-19.

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