Anti-COVID-19 drug candidates: A review on potential biological activities of natural products in the management of new coronavirus infection

Abstract

Background and aim

The novel coronavirus disease (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is now become a worldwide pandemic bringing over 71 million confirmed cases, while the specific drugs and vaccines approved for this disease are still limited regarding their effectiveness and adverse events. Since virus incidences are still on rise, infectivity and mortality may also rise in the near future, natural products are highly considered to be valuable sources for the discovery of new antiviral drugs against SARS-CoV-2. This present review aims to comprehensively summarize the up-to-date scientific literatures on biological activities of plant- and mushroom-derived compounds relevant to mechanistic targets involved in SARS-CoV-2 infection and inflammatory-associated pathogenesis, including viral entry, replication and release, and the renin-angiotensin-aldosterone system (RAAS).

Experimental procedure

Data were retrieved from a literature search available on PubMed, Scopus and Google Scholar databases and collected until the end of May 2020. The findings from in vitro cell and non-cell based studies were considered, while the results of in silico studies were excluded.

Results and conclusion

Based on the previous findings in SARS-CoV studies, except in silico molecular docking analysis, herein, we provide a total of 150 natural compounds as potential candidates for development of new anti-COVID-19 drugs with higher efficacy and lower toxicity than the existing therapeutic agents. Several natural compounds have showed their promising actions on multiple therapeutic targets, which should be further explored. Among them, quercetin, one of the most abundant of plant flavonoids, is proposed as a lead candidate with its ability on the virus side to inhibit SARS-CoV spike protein-angiotensin-converting enzyme 2 (ACE2) interaction, viral protease and helicase activities, as well as on the host cell side to inhibit ACE activity and increase intracellular zinc level.

Graphical abstract

Highlights of the findings and novelties

• •Relevant and up-to-date publications in natural products with anti-COVID-19 potential.
• •Emphasis on the potential of anti-COVID-19 plant/mushroom-based medicine.
• •Twenty four proposed natural compounds for the anti-COVID-19 drug candidates.
• •Quercetin emerged as the most promising compound acting on multiple therapeutic targets.

List of abbreviations

3CL^pro

3-chymotrypsin-like main protease

ACE

Angiotensin-converting enzyme

ARB

Angiotensin-receptor blocker

ARDS

Acute respiratory distress syndrome

AT1R

Angiotensin II type 1 receptor

COVID-19

Coronavirus Disease 2019

MERS-CoV

Middle East Respiratory Syndrome Coronavirus

Nsp

Non-structural protein

PL^pro

Papain-like protease

RAAS

Renin–angiotensin–aldosterone system

RdRp

RNA-dependent RNA polymerase

RTC

Replication-transcription complex

SARS-CoV

Severe Acute Respiratory Syndrome Coronavirus

SARS-CoV-2

Severe Acute Respiratory Syndrome Coronavirus 2

TMPRSS2

Transmembrane protease serine 2

V-ATPase

Vacuolar-type H⁺-ATPase

1. Introduction

On 31 December 2019, several cases of pneumonia were reported in Wuhan, the epicenter of the outbreak in Hubei province of China.¹ The novel coronavirus was identified as Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) which causes Coronavirus Disease 2019 (COVID-19) pandemic.^2,3 From the time of emergence until present, COVID-19 has spread worldwide in which a total of over 71 million confirmed cases with over 1.6 million death tolls has been reported by the World Health Organization (WHO). The COVID-19 positive cases continue rising and is widely distributed throughout the world with the prevalence ranging from highest in America, followed by Europe and South-East Asia, and lowest in Western Pacific region. Asymptomatic patients and patients with mild symptoms can be recovered under home care and isolation while patients with severe complications including acute respiratory distress syndrome (ARDS) require intensive care unit (ICU) which involves oxygen therapy.^4,5 Currently, there is scant evidence from clinical trials for WHO to approve any standard drugs or vaccines as several trials have failed due to efficacy and safety concerns.^6,7 Natural compounds from plant and fungi sources have been recognized in their antiviral properties with numerous mechanisms to prevent infection and strengthen host immunity.^8,9 Herein, we reviewed potential antiviral compounds with multiple targets of action relating to coronaviruses including inhibiting of viral entry, replication and release, and compounds targeting renin–angiotensin–aldosterone system (RAAS) which exhibit promising effects against the disease. We also proposed future perspectives in adopting natural compounds to combat against the COVID-19.

2. Promising therapeutic strategies for the treatment of COVID-19 infection

Presently, there is no clinically approved therapeutics for treating COVID-19, while the rapid human-to-human transmission of this viral infection has expanded worldwide. As the efficacy and safety of natural products on the treatment of a number of viruses including SARS-CoV and Middle East Respiratory Syndrome Coronavirus (MERS-CoV), have been widely acknowledged for several years,¹⁰ the compounds derived from natural sources, e.g. plants and fungi, could have the potential to be a powerful anti-COVID-19 drug. In this review, we focused on four main categories of therapeutic strategies that aim to target the cellular machinery at each step of virus life cycle, starting from viral entry and replication to the release of viral progenies, as well as the RAAS which is a main target of the treatment of hypertension and has recently been proposed as another promising alternative in the treatment of COVID-19. The multiple potential therapeutic mechanisms, both specific and general, that could be capable of tackling COVID-19 infection are presented in Fig. 1.

Fig. 1.

Schematic illustration of potential therapeutic mechanisms in COVID-19 infection. The potential therapeutic strategies for SARS-CoV-2 infection proposed here fall into four main categories based on the cellular and molecular machinery required for the viral life cycle and its related pathogenic mechanisms: inhibition of virus entry, inhibition of virus replication, blocking the release of viral progenies, and modifying the RAAS. The selective blockade of the S protein-ACE2 binding (❶), TMPRSS2 activity (❷), and endocytic pathway-associated proteins such as clathrin, the vacuolar-type H⁺-ATPase (V-ATPase), and cathepsin L (❸), prevent the internalization of virus within the cell. Virus multiplication can be blocked through direct inhibition of proteolytic activity of two viral proteases, 3CL^pro and PL^pro (❹), and replicative activity of viral RTC components e.g., RdRp and helicase (❺), or indirect enzyme inhibition by increasing intracellular Zn²⁺ concentration (❻). Silencing the expression and ion channel activity of viroporin 3a suppresses the release of viral particles from infected cells (❼). Overactivation of Ang II/AT1R axis which contributes to excessive inflammation, can be suppressed by blockade of ACE (❽) and AT1R (❾). 3CL^pro, 3-chymotrypsin-like protease; ACE2, angiotensin-converting enzyme 2; Ang, angiotensin; AT1R, angiotensin II type 1 receptor; E, envelope; MasR, mitochondrial assembly receptor; M, membrane; N, nucleocapsid; PL^pro, papain-like protease; pp, polyprotein; RAAS, renin-angiotensin-aldosterone system; RdRp, RNA-dependent RNA polymerase; RTC, replication-transcription complex; S, spike; TMPRSS2, transmembrane protease serine 2.

The first therapeutic strategy targets on the mechanisms of virus entry in which the selective blockade of molecules that facilitates the internalization of virus into the host cells could be effective to prevent infection. Upon the binding of a virus surface spike (S) protein to a cellular receptor angiotensin-converting enzyme 2 (ACE2), the SARS-CoV-2 generally enters into target host cells via two primary routes; viral membrane fusion and the more common endocytic uptake.¹¹ The first entry mechanism is assisted by proteolytic activation of S protein by a host cell transmembrane protease serine 2 (TMPRSS2), which allows not only direct fusion of virus at the plasma membrane surface, but also release of viral genomic RNA into the cytoplasm. On the other hand, without the membrane bound protease TMPRSS2, the latter entry mechanism allows the whole viral particle to be uptaken via receptor-mediated endocytosis, before subsequently uncoated following the S protein cleavage by cathepsin L within the endosome, to unveil its RNA genome into the cell.

The second and third therapeutic strategies focus on the inhibition of progeny virus production and release from infected cells. As far as the viral replication process is concerned,¹² it begins with the translation of released genome of SARS-CoV-2, a single-stranded (positive-sense) RNA of approximately 30 kb in length, into two precursor polyproteins, pp1a and pp1ab. Both are further cleaved by virus-encoded proteases into several non-structural proteins (nsps) including two key replicative enzymes: the nsp12-RNA-dependent RNA polymerase (RdRp) and the nsp13-helicase, to form the replication-transcription complex (RTC) for synthesizing a full-length genomic RNA (replication) or a nested set of subgenomic mRNA (transcription). These mRNAs are translated into all relevant structural proteins, which together with the viral genome are subsequently assembled into new virions and finally released outside the cell through viroporin-mediated viral budding.¹³

The last therapeutic strategy involves modulating the immune system with the RAAS which regulates blood pressure, fibrosis, and inflammation. In this system, angiotensin-converting enzyme (ACE) converts angiotensin I to angiotensin II which is then converted to lung-protective angiotensin-(1–7) by ACE2. The angiotensin-(1–7) is further recognized by its receptor, the G-protein coupled receptor Mas, to reduce blood pressure, fibrosis, and inflammation.¹⁴ However, as SARS-CoV-2 enters the cells by binding to ACE2, the normal functions of ACE2 are then suppressed. Therefore, instead of converting to angiotensin-(1–7), the angiotensin II is largely bound to type 1 angiotensin II type 1 receptor (AT1R) which causing increased inflammation and other deleterious effects, particularly in the renal and cardiovascular systems.¹⁵

3. Potential natural products as drug candidates against COVID-19

The data presented in this review were obtained from PubMed, Scopus and Google Scholar database up to May 2020. The terms of natural compound, natural product, plant and mushroom were individually searched along with the terms corresponding to each target molecule. Here, we summarize plant- and mushroom-derived compounds that have been reported of antiviral activity with known therapeutic mechanisms specifically against SARS-CoV infection, performed by in vitro cell or non-cell based experiments but not in silico method, as potential candidates to be further researched. We also propose certain promising natural compounds targeting general mechanisms involved in coronavirus infection (see Fig. 1). Additionally, the reports on natural compounds against SARS-CoV with unidentified mechanism of action were included in this review.

3.1. Natural bioactive compounds targeting viral entry

3.1.1. The S protein-ACE2 interaction

The S protein plays a pivotal role in the entry of coronaviruses into host cells by recognizing and binding to the ACE2 via multivalent bonds.¹⁶ The attachment of S protein to ACE2 receptor leads to the fusion between the viral envelope and host cell membrane resulting in successful transfer of viral genome into infected cells.^17,18 S protein is composed of two functional subunits, S1 and S2. The S1 is responsible for binding to the host cell receptor through the receptor binding domain (RDB), while the S2 causes fusion of the viral and cellular membranes.^19,20 Sequence alignment results showed that the homology of the S protein RBD sequence between the beta coronaviruses SARS-CoV and SARS-CoV-2 is 76%.²¹ A number of evidence revealed human ACE2 (hACE2) molecule as an entry receptor for both SARS-CoV and SARS-CoV-2 S proteins.22, 23, 24, 25 Notably, S protein of SARS-CoV-2 was found to exhibit greater affinity to the ACE2 receptor than that of SARS-CoV.²⁴ In addition, expression of ACE2 is ubiquitous with diverse functions, however its specific functions are demonstrated in several organs including lung, tongue, heart, kidney, gastrointestinal tract, pancreas and brain.^26,27 Accordingly, multiple symptoms could be observed in COVID-19 patients.²⁷ Several observations have been reported that the use of hydroxychloroquine, an ACE2 FDA-approved antagonist, was able to reduce mortality rate in hospitalized COVID-19 patients.²⁸ Therefore, it is apparent that the S protein-hACE2 interaction complex is the most crucial target for searching appropriate inhibitors to inhibit entry of the virus in the host cell.

Several natural compounds have been demonstrated their activity to inhibit SARS-CoV entry to the host cell as shown in Table 1. According to the literature, an anthraquinone compound, emodin, showed the potency to inhibit viral infection by blocking the binding of SARS-CoV S protein to ACE2 in a dose-dependent manner.²⁹ The plant sources which are likely to contain emodin as their active constituent were also found effective in blocking SARS-CoV S protein and ACE2 interaction, with showing IC₅₀ values for aqueous extracts from the root of Rheum palmatum, the root and stem of Polygonum multiflorum, ranged from 1 to 10 μg/ml.²⁹ Another previous study using the high-throughput screening technique revealed more promising natural antiviral compounds consisted in the extracts from Chinese herbs. Those small herbal molecules could strongly bind to the SARS-CoV S2 protein and inhibited the pseudovirus entry, possibly by interfering with the function of the S protein.³⁰

Table 1.

List of bioactive compounds from natural sources as potential anti-COVID-19 drug candidates and their mechanisms of action.

Compound	Class	Source	Biological action/Efficacy	Experiment	Reference
Inhibiting the SARS-CoV S protein-ACE2 interaction
Emodin	Anthraquinone	Rheum palmatuma	IC50 = 200 μM	Cell-free assay (Competitive biotinylated ELISA)	29
			94% inhibition at EC of 50 μM	Cell-based assay (IFA)
Luteolin	Flavonoid	Rhodiola kirilowiia	IC50 = 4.5 μM	Cell-free and cell-based assay (FAC/MS and Luciferase assay)	30
Quercetin	Flavonoid	Allium cepaa	IC50 = 83.4 μM	Cell-based assay (Luciferase assay)	30
Tetra-O-galloyl-$\mathrm{\beta }$-d-glucose (TGG)	Tannin	Galla chinensisa	IC50 = 10.6 μM	Cell-free and cell-based assay (FAC/MS and Luciferase assay)	30
Inhibiting the endocytic machinery
1-cinnamoyl-3,11-dihydroxy meliacarpin	Terpenoid	Melia azedarach	increased endolysosomal pH (EC of 7.5 μM)	Cell-based assay (AO staining)	38
25-O-acetyl-7,8-didehydrocimigenol 3-O-beta-d-xylopyranoside (ADCX)	Terpenoid	Cimicifugae rhizoma	inhibited degradation activity by decreasing cathepsin expression, but not endolysosomal acidity (EC of 24 μM)	Cell-based assay (AO staining, DQ-BSA staining and WB)	39
Alantolactone	Sesquiterpene lactone	Inula heleniuma	neutralized endo-lysosomal pH and reducing the expression and activity of cathepsins (EC of 10 μM)	Cell-based assay (LysoTracker Red and AO staining, WB and Cathepsin activity assay)	76
Cleistanthin A	Lignan glycoside	Cleistanthus collinua	inhibited the activity of V-type ATPase and elevated endolysosomal pH (EC of 0.1 μM)	Cell-based assay (pH sensitive fluorescent probe/LysoTracker Red staining and V-type ATPase activity assay)	77,78
Cleistanthoside A tetraacetate	Lignan glycoside	Phyllanthus taxodiifolius Beille a	neutralized endolysosomal acidity and decreased the activity of V-type ATPase (EC of 50 nM)	Cell-based assay (LysoTracker Red staining and V-type ATPase activity assay)	78
Dauricine	Alkaloid	Rhizoma Menispermia	elevated endolysosomal pH, decreased the levels of active cathepsins and inhibited the activity of V-type ATPase (EC of 10 μM)	Cell-based assay (LysoSensor Yellow/Blue staining, WB and V-type ATPase activity assay)	42
Daurisoline	Alkaloid	Rhizoma Menispermia	elevated endolysosomal pH, decreased the levels of active cathepsins and inhibited the activity of V-type ATPase (EC of 10 μM)	Cell-based assay (LysoSensor Yellow/Blue staining, WB and V-type ATPase activity assay)	42
Diphyllin	Lignan lactone	Cleistanthus collinusa	inhibited the activity of V-type ATPase (EC of 0.3 μM)	Cell-based assay (V-type ATPase activity assay)	79
Ginsenoside Ro	Triterpenoid saponin	Panax ginseng	raised endolysosomal pH and downregulating the expression and activity of cathepsins (EC of 50 μM)	Cell-based assay (AO staining, WB and Cathepsin activity assay)	80
Icariside II	Flavonoid	Epimedium koreanum Nakai	decreased endolysosomal acidity (EC of 25 μM)	Cell-based assay (AO staining)	81
Leelamine	Terpene	Pinus sylvestrisa	decreased endolysosomal acidity and inhibited cellular endocytosis (EC of 3 μM)	Cell-based assay (LysoTracker Red staining and Internalization of fluorescent transferrin-A488)	40
Matrine	Alkaloid	Sophora flavescens Ait	inhibited endolysosomal acidification and reduced the expression and activity of cathepsins (EC of 2 mM)	Cell-based assay (LysoSensor Yellow/Blue, WB and Cathepsin activity assay)	43
Myrtenal	Terpene	Elettaria cardamomuma	inhibited the activity of V-type ATPase and reduced endolysosomal acidification (EC of 100 μM)	Cell-based assay (AO staining and V-type ATPase activity assay)	41
Oblongifolin C	Benzophenone	Garcinia yunnanensis Hu	inhibited endolysosomal acidification and downregulated the expression and activity of cathepsins (EC of 15 μM)	Cell-based assay (AO staining, WB and Cathepsin activity assay)	82
Pulsatilla saponin D	Triterpenoid saponin	Pulsatilla chinensis (Bunge) Regel	elevated endolysosomal pH and downregulated cathepsins (EC of 1.25 μM)	Cell-based assay (LysoSensor Yellow/Blue, WB and Cathepsin activity assay)	83
Tetrandrine	Alkaloid	Stephania tetrandra S. Moore a	elevated endolysosomal pH in a concentration-dependent manner (EC of 1–10 μM)	Cell-based assay (LysoSensor Yellow/Blue staining)	44
Inhibiting the SARS-CoV 3CLproactivity
3’-(3-Methylbut-2-enyl)-3′,4,7-trihydroxyflavane	Flavonoid	Broussonetia papyrifera	IC50 = 30.2 μM	Cell-free assay (FRET)	84
4-Hydroxyderricin	Chalcone	Angelica keiskei	IC50 = 81.4 μM	Cell-free assay (FRET)	35
			IC50 = 50.8 μM	Cell-based assay (Luciferase reporter assay)
Betulinic acid	Terpenoid	Breynia fruticosea	IC50 = 10 μM	Cell-free assay (FRET)	49,50
Broussochalcone A	Chalcone	Broussonetia papyrifera	IC50 = 88.1 μM	Cell-free assay (FRET)	84
Broussochalcone B	Chalcone	Broussonetia papyrifera	IC50 = 57.8 μM	Cell-free assay (FRET)	84
Broussoflavan A	Flavonoid	Broussonetia papyrifera	IC50 = 92.4 μM	Cell-free assay (FRET)	84
Dihydrotanshinone I	Tanshinone	Salvia miltiorrhiza	IC50 = 14.4 μM	Cell-free assay (FRET)	51
Hesperetin	Flavonoid	Isatis indigotica	IC50 = 60 μM	Cell-free assay (ELISA)	48
			IC50 = 8.3 μM	Cell-based assay (Luciferase reporter assay)
Hirsutenone	Diarylheptanoid	Alnus japonica	IC50 = 36.2 μM	Cell-free assay (FRET)	85
Isobavachalcone	Chalcone	Angelica keiskei	IC50 = 39.4 μM	Cell-free assay (FRET)	35
			IC50 = 11.9 μM	Cell-based assay (Luciferase reporter assay)
Isoliquiritigenin	Chalcone	Glycyrrhiza glabraa	IC50 = 61.9 μM	Cell-free assay (FRET)	84,86
Kazinol A	Flavonoid	Broussonetia papyrifera	IC50 = 84.8 μM	Cell-free assay (FRET)	84
Kazinol F	Biphenyl propanoids	Broussonetia papyrifera	IC50 = 43.3 μM	Cell-free assay (FRET)	84
Kazinol J	Biphenyl propanoids	Broussonetia papyrifera	IC50 = 64.2 μM	Cell-free assay (FRET)	84
Methyl tanshinonate	Tanshinone	Salvia miltiorrhiza	IC50 = 21.1 μM	Cell-free assay (FRET)	51
Quercetin	Flavonoid	Allium cepaa	IC50 = 52.7 μM	Cell-free assay (FRET)	84,87
Quercetin-3-b-galactoside	Flavonoid	Machilus zuihoensisa	IC50 = 42.8 μM	Cell-free assay (FRET)	87,88
Rosmariquinone	Tanshinone	Salvia miltiorrhiza	IC50 = 21.1 μM	Cell-free assay (FRET)	51
Savinin	Lignoid	Chamaecyparis obtuse var. formosana	IC50 = 25 μM	Cell-free assay (FRET)	49
Tanshinone I	Tanshinone	Salvia miltiorrhiza	IC50 = 38.7 μM	Cell-free assay (FRET)	51
Tanshinone IIA	Tanshinone	Salvia miltiorrhiza	IC50 = 89.1 μM	Cell-free assay (FRET)	51
Tanshinone IIB	Tanshinone	Salvia miltiorrhiza	IC50 = 24.8 μM	Cell-free assay (FRET)	51
Xanthoangelol	Chalcone	Angelica keiskei	IC50 = 38.4 μM	Cell-free assay (FRET)	35
			IC50 = 5.8 μM	Cell-based assay (Luciferase reporter assay)
Xanthoangelol B	Chalcone	Angelica keiskei	IC50 = 22.2 μM	Cell-free assay (FRET)	35
			IC50 = 8.6 μM	Cell-based assay (Luciferase reporter assay)
Xanthoangelol D	Chalcone	Angelica keiskei	IC50 = 26.6 μM	Cell-free assay (FRET)	35
			IC50 = 9.3 μM	Cell-based assay (Luciferase reporter assay)
Xanthoangelol E	Chalcone	Angelica keiskei	IC50 = 11.4 μM	Cell-free assay (FRET)	35
			IC50 = 7.1 μM	Cell-based assay (Luciferase reporter assay)
Xanthoangelol F	Chalcone	Angelica keiskei	IC50 = 34.1 μM	Cell-free assay (FRET)	35
			IC50 = 32.6 μM	Cell-based assay (Luciferase reporter assay)
Xanthokeistal A	Chalcone	Angelica keiskei	IC50 = 44.1 μM	Cell-free assay (FRET)	35
			IC50 = 9.8 μM	Cell-based assay (Luciferase reporter assay)
Inhibiting the SARS-CoV PLproactivity
3′-O-Methyldiplacol	Flavonoid	Paulownia tomentosa	IC50 = 9.5 μM	Cell-free assay (Fluorescence-based deubiquitination)	89
3′-O-Methyldiplacone	Flavonoid	Paulownia tomentosa	IC50 = 13.2 μM	Cell-free assay (Fluorescence-based deubiquitination)	89
4′-O-Methylbavachalcone	Chalcone	Psoralea corylifolia	IC50 = 10.1 μM	Cell-free assay (Fluorescence-based deubiquitination)	90
4′-O-Methyldiplacol	Flavonoid	Paulownia tomentosa	IC50 = 9.2 μM	Cell-free assay (Fluorescence-based deubiquitination)	89
4′-O-Methyldiplacone	Flavonoid	Paulownia tomentosa	IC50 = 12.7 μM	Cell-free assay (Fluorescence-based deubiquitination)	89
6-Geranyl-4′,5,7-trihydroxy-3′,5′-dimethoxyflavanone	Flavonoid	Paulownia tomentosa	IC50 = 13.9 μM	Cell-free assay (Fluorescence-based deubiquitination)	89
Broussochalcone A	Chalcone	Broussonetia papyrifera	IC50 = 9.2 μM	Cell-free assay (Fluorescence-based deubiquitination)	84
Broussochalcone B	Chalcone	Broussonetia papyrifera	IC50 = 11.6 μM	Cell-free assay (Fluorescence-based deubiquitination)	84
Cryptotanshinone	Tanshinone	Salvia miltiorrhiza	IC50 = 0.8 μM	Cell-free assay (Fluorescence-based deubiquitination)	51
Curcumin	Polyphenol	Curcuma longaa	IC50 = 5.7 μM	Cell-free assay (Fluorescence-based deubiquitination)	85,91
Dihydrotanshinone I	Tanshinone	Salvia miltiorrhiza	IC50 = 4.9 μM	Cell-free assay (Fluorescence-based deubiquitination)	51
Diplacone	Flavonoid	Paulownia tomentosa	IC50 = 10.4 μM	Cell-free assay (Fluorescence-based deubiquitination)	89
Hirsutanonol	Diarylheptanoid	Alnus japonica	IC50 = 7.8 μM	Cell-free assay (Fluorescence-based deubiquitination)	85
Hirsutenone	Diarylheptanoid	Alnus japonica	IC50 = 4.1 μM	Cell-free assay (Fluorescence-based deubiquitination)	85
Isobavachalcone	Chalcone	Psoralea corylifolia	IC50 = 7.3 μM	Cell-free assay (Fluorescence-based deubiquitination)	90
		Angelica keiskei	IC50 = 13.0 μM	Cell-free assay (Fluorescence-based deubiquitination)	35
Isoliquiritigenin	Chalcone	Glycyrrhiza glabraa	IC50 = 24.6 μM	Cell-free assay (Fluorescence-based deubiquitination)	84,86
Kaempferol	Flavonoid	Zingiber officinalea	IC50 = 16.3 μM	Cell-free assay (Fluorescence-based deubiquitination)	84,92
Kazinol J	Biphenyl propanoids	Broussonetia papyrifera	IC50 = 15.2 μM	Cell-free assay (Fluorescence-based deubiquitination)	84
Methyl tanshinonate	Tanshinone	Salvia miltiorrhiza	IC50 = 9.2 μM	Cell-free assay (Fluorescence-based deubiquitination)	51
Mimulone	Flavonoid	Paulownia tomentosa	IC50 = 14.4 μM	Cell-free assay (Fluorescence-based deubiquitination)	89
Neobavaisoflavone	Flavonoid	Psoralea corylifolia	IC50 = 18.3 μM	Cell-free assay (Fluorescence-based deubiquitination)	90
Papyriflavonol A	Favonoid	Broussonetia papyrifera	IC50 = 3.7 μM	Cell-free assay (Fluorescence-based deubiquitination)	84
Psoralidin	Flavonoid	Psoralea corylifolia	IC50 = 4.2 μM	Cell-free assay (Fluorescence-based deubiquitination)	90
Quercetin	Flavonoid	Allium cepaa	IC50 = 8.6 μM	Cell-free assay (Fluorescence-based deubiquitination)	84,87
Rubranol	Diarylheptanoid	Alnus japonica	IC50 = 12.3 μM	Cell-free assay (Fluorescence-based deubiquitination)	85
Rubranoside A	Diarylheptanoid	Alnus japonica	IC50 = 9.1 μM	Cell-free assay (Fluorescence-based deubiquitination)	85
Rubranoside B	Diarylheptanoid	Alnus japonica	IC50 = 8.0 μM	Cell-free assay (Fluorescence-based deubiquitination)	85
Tanshinone I	Tanshinone	Salvia miltiorrhiza	IC50 = 8.8 μM	Cell-free assay (Fluorescence-based deubiquitination)	51
Tanshinone IIA	Tanshinone	Salvia miltiorrhiza	IC50 = 1.6 μM	Cell-free assay (Fluorescence-based deubiquitination)	51
Tanshinone IIB	Tanshinone	Salvia miltiorrhiza	IC50 = 10.7 μM	Cell-free assay (Fluorescence-based deubiquitination)	51
Terrestrimine	Cinnamic amide	Tribulus terrestris	IC50 = 15.8 μM	Cell-free assay (Fluorescence-based deubiquitination)	93
Tomentin A	Flavonoid	Paulownia tomentosa	IC50 = 6.2 μM	Cell-free assay (Fluorescence-based deubiquitination)	89
Tomentin B	Flavonoid	Paulownia tomentosa	IC50 = 6.1 μM	Cell-free assay (Fluorescence-based deubiquitination)	89
Tomentin C	Flavonoid	Paulownia tomentosa	IC50 = 11.6 μM	Cell-free assay (Fluorescence-based deubiquitination)	89
Tomentin D	Flavonoid	Paulownia tomentosa	IC50 = 12.5 μM	Cell-free assay (Fluorescence-based deubiquitination)	89
Tomentin E	Flavonoid	Paulownia tomentosa	IC50 = 5.0 μM	Cell-free assay (Fluorescence-based deubiquitination)	89
Xanthoangelol	Chalcone	Angelica keiskei	IC50 = 11.7 μM	Cell-free assay (Fluorescence-based deubiquitination)	35
Xanthoangelol B	Chalcone	Angelica keiskei	IC50 = 11.7 μM	Cell-free assay (Fluorescence-based deubiquitination)	35
Xanthoangelol D	Chalcone	Angelica keiskei	IC50 = 19.3 μM	Cell-free assay (Fluorescence-based deubiquitination)	35
Xanthoangelol E	Chalcone	Angelica keiskei	IC50 = 1.2 μM	Cell-free assay (Fluorescence-based deubiquitination)	35
Xanthoangelol F	Chalcone	Angelica keiskei	IC50 = 5.6 μM	Cell-free assay (Fluorescence-based deubiquitination)	35
Inhibiting the SARS-CoV helicase activity
Myricetin	Flavonoid	Camellia sinensisa	inhibited ATPase activity of SARS-CoV helicase with IC50 of 2.71 μM	Cell-free assay (Colorimetry-based ATP hydrolysis assay)	94
Quercetin	Flavonoid	Allium cepaa	inhibited duplex DNA-unwinding activity of SARS-CoV NTPase/helicase with IC50 of 8.1 μM	Cell-free assay (FRET-based dsDNA unwinding assay)	95
Scutellarein	Flavonoid glycoside	Scutellaria baicalensis	inhibited ATPase activity of SARS-CoV helicase with IC50 of 0.86 μM	Cell-free assay (Colorimetry-based ATP hydrolysis assay)	94
Increasing intracellular Zn2+
Caffeic acid	Phenolic acid	Ocimum basilicuma	increased intracellular Zn2+ level (3-fold increase at EC of 50 μM)	Cell-free assay (using liposome model)	60
Catechin	Flavonoid	Camellia sinensisa	increased intracellular Zn2+ level (2-fold increase at EC of 50 μM)	Cell-free assay (using liposome model)	60
Catechol	Phenol	Allium cepaa	increased intracellular Zn2+ level (2-fold increase at EC of 50 μM)	Cell-free assay (using liposome model)	60
Epigallocatechin-3-gallate (EGCG)	Flavonoid	Camellia sinensisa	increased intracellular Zn2+ level (36-fold increase at EC of 50 μM)	Cell-free assay (using liposome model)	60
			increased the uptake of Zn2+ in both cell (4-fold increase at EC of 100 μM) and liposome model (16-fold increase at EC of 10 μM)	Cell-based assay (Fluorescent Zn2+ indicator) and cell-free assay (using liposome model)	62
Gallic acid	Phenolic acid	Syzygium aromaticuma	increased intracellular Zn2+ level (8-fold increase at EC of 50 μM)	Cell-free assay (using liposome model)	60
Genistein	Flavonoid	Glycine maxa	increased intracellular Zn2+ level (2-fold increase at EC of 50 μM)	Cell-free assay (using liposome model)	60
Luteolin	Flavonoid	Rhodiola kirilowiia	increased intracellular Zn2+ level (12-fold increase at EC of 50 μM)	Cell-free assay (using liposome model)	60
Pyrithione	Organic sulfur compound	Allium stipitatuma	increased intracellular Zn2+ level (3-fold increase at EC of 10 μM)	Cell-based assay (Radioactive Zn2+ uptake)	96
Quercetin	Flavonoid	Allium cepaa	increased intracellular Zn2+ level (18-fold increase at EC of 50 μM)	Cell-free assay (using liposome model)	60
			increased the uptake of Zn2+ in both cell (2-fold increase at EC of 100 μM) and liposome model (8-fold increase at EC of 10 μM)	Cell-based assay (Fluorescent Zn2+ indicator) and cell-free assay (using liposome model)	[62]
Resveratrol	Polyphenol	Vitis viniferaa	increased intracellular Zn2+ level (7.5-fold increase at EC of 10 μM)	Cell-based assay (AAS)	61
Rutin	Flavonoid glycoside	Morus albaa	increased intracellular Zn2+ level (4-fold increase at EC of 50 μM)	Cell-free assay (using liposome model)	60
Tannic acid	Phenolic acid	Camellia sinensisa	increased intracellular Zn2+ level (12-fold increase at EC of 50 μM)	Cell-free assay (using liposome model)	60
Taxifolin	Flavonoid	Silybum marianuma	increased intracellular Zn2+ level (4-fold increase at EC of 50 μM)	Cell-free assay (using liposome model)	60
β-thujaplicin (Hinokitiol)	Terpene	Chamaecyparis obtusea	increased intracellular Zn2+ level (3-fold increase at EC of 125 μM)	Cell-based assay (Radioactive Zn2+ uptake)	96
Inhibiting the viroporin 3a activity
Afzelin	Flavonoid glycoside	Houttuynia cordataa	inhibited the ion channel activity of SARS-CoV 3a protein (17% inhibition at EC of 10 μM)	Cell-based assay (Voltage-clamp method in Xenopus oocyte model)	65
Emodin	Anthraquinone	Rheum tanguticum	inhibited the ion channel activity of SARS-CoV 3a protein with IC50 of 20 μM	Cell-based assay (Voltage-clamp method in Xenopus oocyte model)	66,97
Juglanine	Flavonoid glycoside	Polygonum avicularea	inhibited the ion channel activity of SARS-CoV 3a protein with IC50 of 2.3 μM	Cell-based assay (Voltage-clamp method in Xenopus oocyte model)	65
Kaempferol	Flavonoid	Zingiber officinalea	inhibited the ion channel activity of SARS-CoV 3a protein (18% inhibition at EC of 20 μM)	Cell-based assay (Voltage-clamp method in Xenopus oocyte model)	65,66
Kaempferol-3-O-α-rhamnopyranosyl (1 → 2) [α-rhamno pyranosyl(1 → 6)]-β-glucopyranoside	Flavonoid glycoside	Clitoria ternateaa	inhibited the ion channel activity of SARS-CoV 3a protein (32% inhibition at EC of 20 μM)	Cell-based assay (Voltage-clamp method in Xenopus oocyte model)	65
Tiliroside	Flavonoid glycoside	Althaea officinalisa	inhibited the ion channel activity of SARS-CoV 3a protein (52% inhibition at EC of 20 μM)	Cell-based assay (Voltage-clamp method in Xenopus oocyte model)	65,66
Inhibiting the ACE activity
25-O-methylalisol F	Triterpenoid	Alisma orientale	Reduced ACE and AT1R protein expression (∼30% and ∼10% inhibition at EC of 10 μM)	Cell-based assay (WB analysis)	98
3,5-dihydroxy-4- methoxybenzoic acid	Phenolic acid	Tamarix hohenackeri	46.2% inhibition at EC of 20 mg/mL	Cell-free assay (HHL degradation assay)	99
4′-hydroxy Pd-C-III	Coumarin	Angelica decursiva	IC50 = 9.4 μM	Cell-free assay (FAPGG degradation assay)	100
4′-methoxy Pd–C–I	Coumarin	Angelica decursiva	IC50 = 16 μM	Cell-free assay (FAPGG degradation assay)	100
Ampleopsin C	Stilbenoid	Vitis thunbergii var. Taiwanian	IC50 = 18.2 μM	Cell-free assay (FAPGG degradation assay)	101
Apigenin	Flavonoid	Adinandra nitidaa	30.3% inhibition at EC of 500 μg/mL	Cell-free assay (HHL degradation assay)	102
Asparaptine	Organic sulfur compound	Asparagus officinalis	IC50 = 113 μM	Cell-free assay (3HB-GGG hydrolysis assay)	103
Caffeic acid	Phenolic acid	Echinacea purpureaa	IC50 = 0.1 μM	Cell-free assay (HHL degradation assay)	71
Camellianin A	Flavonoid	Adinandra nitida	30.2% inhibition at EC of 500 μg/mL	Cell-free assay (HHL degradation assay)	102
Camellianin B	Flavonoid	Adinandra nitida	40.7% inhibition at EC of 500 μg/mL	Cell-free assay (HHL degradation assay)	102
Carlinoside	Flavonoid glycoside	Desmodium styracifolium	IC50 = 33.6 μM	Cell-free assay (HHL degradation assay)	104
Catechin	Flavonoid	Malus domestica(a)	IC50 = 109 μM	Cell-free assay (HHL degradation assay)	105
Chlorogenic acid	Phenolic acid	Echinacea purpurea(a)	IC50 = 0.1 μM	Cell-free assay (HHL degradation assay)	71
Chrysin	Flavonoid	Malus domestica(a)	IC50 = 146 μM	Cell-free assay (HHL degradation assay)	105
Chrysoeriol	Flavonoid	Tamarix hohenackeri	57.6% inhibition at EC of 20 mg/mL	Cell-free assay (HHL degradation assay)	99
Coretincone	Phenolic glycoside	Coreopsis tinctoria	IC50 = 228 μM	Cell-free assay (HHL degradation assay)	106
Curcumin	Polyphenol	Curcuma longa(a)	76.9% inhibition at EC of 10 μM	Cell-free assay (HHL degradation assay)	107
Cyanidin-3-O-glucoside	Flavonoid glycoside	Malus domestica(a)	IC50 = 174 μM	Cell-free assay (HHL degradation assay)	105
Cyanidin-3-O-galactoside	Flavonoid glycoside	Malus domestica(a)	IC50 = 206 μM	Cell-free assay (HHL degradation assay)	105
Cyanidin-3-O-rhamnosdie	Flavonoid glycoside	Malus domestica(a)	IC50 = 114 μM	Cell-free assay (HHL degradation assay)	105
Cyanidin-3-O-sambubioside	Flavonoid glycoside	Hibiscus sabdariffa	IC50 = 117.7 μM	Cell-free assay (FAPGG degradation assay)	108
Cyanidin-3-O-β-glucoside	Flavonoid glycoside	Rosa damascena	IC50 = 138.8 μM	Cell-free assay (HHL degradation assay)	109
Decursidin	Coumarin	Angelica decursiva	IC50 = 20 μM	Cell-free assay (FAPGG degradation assay)	100
(+)-trans-Decursidinol	Coumarin	Angelica decursiva	IC50 = 4.7 μM	Cell-free assay (FAPGG degradation assay)	100
Decursinol	Coumarin	Angelica decursiva	IC50 = 18.3 μM	Cell-free assay (FAPGG degradation assay)	100
Delphinidin-3-O-sambubioside	Flavonoid glycoside	Hibiscus sabdariffa	IC50 = 141.6 μM	Cell-free assay (FAPGG degradation assay)	108
Epicatechin	Flavonoid	Malus domestica(a)	IC50 = 73 μM	Cell-free assay (HHL degradation assay)	105
Gallic acid	Phenolic acid	Tamarix hohenackeri	43.1% inhibition at EC of 20 mg/mL	Cell-free assay (HHL degradation assay)	99
Gluco-aurantioobtusin	Anthraquinone glycoside	Cassia tora	IC50 = 30.2 μM	Cell-free assay (FAPGG degradation assay)	110
(+)-Hopeaphenol	Stilbenoid	Ampelopsis brevipedunculata var. hancei	IC50 = 1.6 μM	Cell-free assay (HHL degradation assay)	72
Isoferulic acid	Phenolic acid	Tamarix hohenackeri	30.6% inhibition at EC of 20 mg/mL	Cell-free assay (HHL degradation assay)	99
Isoquercetrin	Flavonoid	Tropaeolum majus(a)	Reduced plasmatic ACE activity in SHR rats (43% inhibition at EC of 10 mg/kg)	Cell-free assay (HHL degradation assay)	111
Isorutarine	Coumarin	Angelica decursiva	IC50 = 68.4 μM	Cell-free assay (FAPGG degradation assay)	100
Junipediol A-8-O-β-d-glucoside	Phenylpropa-noid glycoside	Apium graveolens	IC50 = 210 μM	Cell-free assay (HHL degradation assay)	112
(S)-Malic acid 1′-O-β-gentiobioside	Organic acid glycoside	Lactuca sativa	IC50 = 27.8 μM	Cell-free assay (HHL degradation assay)	113
Mangiferin	Xanthone glycoside	Swertia chirayita(a)	31.5% inhibition at EC of 500 μM	Cell-free assay (HHL degradation assay)	114
Miquelianin	Flavonoid glycoside	Cuphea glutinosa	32.1% inhibition at EC of 100 ng/mL	Cell-free assay (FAPGG degradation assay)	115
N1,N4,N8-tris (dihydrocaffeoyl)spermidine	Polyamine	Solanum quitoense	IC50 = 9.6 ppm	Cell-free assay (3HB-GGG hydrolysis assay)	116
Methyl gallate	Phenolic acid	Tamarix hohenackeri	35.7% inhibition at EC of 20 mg/mL	Cell-free assay (HHL degradation assay)	99
Naringenin	Flavonoid	Malus domestica(a)	IC50 = 78 μM	Cell-free assay (HHL degradation assay)	105
Onopordia	Polyphenol	Onopordum acanthium L.	IC50 = 300 μM	Cell-free assay (HHL degradation assay)	117,118
Orotic acid	Organic acid	Daucus carota(a)	40.3% inhibition at EC of 5 μg/mL	Cell-free assay (HHL degradation assay)	119
Pd–C–I	Coumarin	Angelica decursiva	IC50 = 6.8 μM	Cell-free assay (FAPGG degradation assay)	100
Pd-C-II	Coumarin	Angelica decursiva	IC50 = 12.4 μM	Cell-free assay (FAPGG degradation assay)	100
Pd-C-III	Coumarin	Angelica decursiva	IC50 = 15.3 μM	Cell-free assay (FAPGG degradation assay)	100
Quercetin	Flavonoid	Malus domestica(a)	IC50 = 151 μM	Cell-free assay (HHL degradation assay)	105
		Tamarix hohenackeri	48.6% inhibition at EC of 20 mg/mL	Cell-free assay (HHL degradation assay)	99
Quercetin-3-O-galactoside	Flavonoid glycoside	Malus domestica(a)	IC50 = 180 μM	Cell-free assay (HHL degradation assay)	105
Quercetin-3-O-glucoside	Flavonoid glycoside	Malus domestica(a)	IC50 = 71 μM	Cell-free assay (HHL degradation assay)	105
Quercetin-3-O-glucuronic acid	Flavonoid conjugate	Malus domestica(a)	IC50 = 27 μM	Cell-free assay (HHL degradation assay)	105
Quercetin-3-O-rhamnoside	Flavonoid glycoside	Malus domestica(a)	IC50 = 100 μM	Cell-free assay (HHL degradation assay)	105
Quercetin-3-O-rutinoside	Flavonoid glycoside	Malus domestica(a)	IC50 = 90 μM	Cell-free assay (HHL degradation assay)	105
Quercetin-3-O-sulfate	Flavonoid conjugate	Malus domestica(a)	IC50 = 131 μM	Cell-free assay (HHL degradation assay)	105
Quercetin-4′-O-glucoside	Flavonoid glycoside	Malus domestica(a)	IC50 = 211 μM	Cell-free assay (HHL degradation assay)	105
Schaftoside	Flavonoid glycoside	Desmodium styracifolium	IC50 = 58.4 μM	Cell-free assay (HHL degradation assay)	104
Tannic acid	Phenolic acid	Camellia sinensis(a)	IC50 = 230 μM	Cell-free assay (HHL degradation assay)	120
Taxifolin	Flavonoid	Coreopsis tinctoria	IC50 = 145.7 μM	Cell-free assay (HHL degradation assay)	106
Vicenin 1	Flavonoid glycoside	Desmodium styracifolium	IC50a52.5 μM	Cell-free assay (HHL degradation assay)	104
Vicenin 2	Flavonoid glycoside	Desmodium styracifolium	IC50 = 43.8 μM	Cell-free assay (HHL degradation assay)	104
Vicenin 3	Flavonoid glycoside	Desmodium styracifolium	IC50 = 46.9 μM	Cell-free assay (HHL degradation assay)	104
(+)-ε-Viniferin	Stilbenoid	Vitis thunbergii var. taiwanian	IC50 = 35.5 μM	Cell-free assay (FAPGG degradation assay)	101
(+)-Vitisin A	Stilbenoid	Vitis thunbergii var. taiwanian	IC50 = 3.3 μM	Cell-free assay (FAPGG degradation assay)	101
		Ampelopsis brevipedunculata var. hancei	IC50 = 1.5 μM	Cell-free assay (HHL degradation assay)	72

3HB-GGG = 3-hydryoxybutyryl-Gly-Gly-Gly; AAS = Atomic absorption spectrophotometry; AO = Acridine orange; ATP = Adenosine triphosphate; DQ-BSA = Dye quenched-bovine serum albumin; EC = The effective test concentration; ELISA = Enzyme Linked Immunosorbent Assay; FAC/MS = Frontal affinity chromatography-Mass spectrometry; FAPGG = furylacryloyl-phenylalanyl-glycyl-glycine; FRET = Fluorescence resonance energy transfer; HHL = hippuryl-L-histidyl-l-leucine; IC₅₀ = The half maximal inhibitory concentration; IFA = Immunofluorescence assay; SHR = spontaneously hypertensive rat; WB = WesternBlot.

^aThe study used commercial products. Here provides a natural source of compound as an example.

3.1.2. The plasma membrane protease TMPRSS2

Recognized as a host trypsin-like serine protease, TMPRSS2 highly expressed in alveolar cells has been demonstrated to facilitate viral entry by priming of viral S protein. Inhibition of TMPRSS2 activity could prevent infection of coronaviruses including MERS-CoV, SARS-CoV and SARS-CoV-2.³¹ Now, several synthetic drugs like camostat mesylate, nafamostat mesylate and bromhexine which are serine protease inhibitors showed potential to inhibit SARS-CoV-2 infection.32, 33, 34 However, on the side of natural products, only few compounds were reported. Xanthoangelol G isolated from Angelica keiskei was reported to inhibit a trypsin-like serine protease with its IC₅₀ value of 51.6 μM.³⁵ Notably, in vitro cell-based and in vivo experiments are needed to be done for the development of anti-SARS-CoV-2 drugs.

3.1.3. The endocytic machinery

Clathrin-mediated endocytosis has been recognized as the primary cell entry route for multiple coronaviruses, including new emerging SARS-CoV-2, by utilizing the binding of viral S protein to host receptor ACE2 molecule.³⁶ After endocytosis into the target cell, the viral particle undergoes the cleavage of S protein mediated by a pH-dependent cysteine protease cathepsin L at an acidic endolysosomal pH (∼3.0–6.5), which finally triggers membrane fusion between virus and endosome, followed by release of viral genetic material into the cytoplasm. Hence, targeting endocytic pathway-associated proteins are considered to be one of promising strategies for inhibiting SARS-CoV-2 entry.³⁷ Following this idea, Table 1 summarizes natural compounds that have been reported with inhibitory effects on the vacuolar-type H⁺-ATPase (V-ATPase) activity, the expression and activity of cathepsins, or an increasing effect on lysosomal pH, which lead to impaired acidification and protein degradation of intracellular vesicles like endolysosome. Our search revealed that terpenes/terpenoids38, 39, 40, 41 and alkaloids42, 43, 44 are two major classes of compounds acting through this strategy. In addition to individual compounds, some crude plant extracts have shown their potential for being developed as an anti-COVID-19 drug. The traditional Japanese herbal formulation named Maoto, prepared from a mixture of four plants (Ephedrae herba, Armeniacae semen, Cinnamomi cortex and Glycyrrhizae radix), was recently shown to inhibit endolysosomal acidification.⁴⁵ Zhuang et al. also demonstrated that butanol crude fraction from C. cortex was able to inhibit the clathrin-dependent endocytosis pathway as well as the infection of SARS-CoV using cell-based assays.⁴⁶

3.2. Natural bioactive compounds targeting viral replication

3.2.1. The 3-chymotrypsin-like main protease (3CL^pro)

The 3CL^pro is an enzyme that plays important role in replication of coronaviruses. It is responsible for the cleavage of polyproteins to functional proteins. Base on the protein structures, 3CL^pro of SARS-CoV and SARS-CoV-2 show similarity of amino acid sequence at 96%, and both enzymes exhibit high conservation of active residues.⁴⁷ Therefore, small molecules with SAR-CoV 3CL^pro inhibitory activity may also inhibit 3CL^pro of SARS-CoV-2. Numerous studies have revealed for plant and mushroom derived natural compounds that could suppress SARS-CoV replication by blocking 3CL^pro activity with IC₅₀ range from 8.3 to 92.4 μM in either cell-free or cell-based assays. Among them, hesperetin, a phenolic compound isolated from Isatis indigotica root exhibited the greatest inhibitory activity against SARS-CoV 3CL^pro (IC₅₀ = 8.3 μM) in an African green monkey kidney (Vero) cell line and this effective dose did not toxic to the cells (CC₅₀ = 2.7 mM).⁴⁸ Other phytochemical classes that have shown promise in the inhibition of this enzyme are lignoid, terpenoid, tanshinone and chalcone with IC₅₀ less than 25 μM.^35,49, 50, 51 Interestingly, the lignoid savinin was able to reduce both viral replication (Selective index > 667) and cytopathic effect on SARS-CoV-infected Vero E6 cells.⁴⁹ The summary of bioactive compounds against SARS-CoV 3CL^pro inhibitory activity is tabulated in Table 1. Regarding to the similarity between 3CL^pro of SARS-CoV and SARS-CoV-2, these natural compounds are interesting substances to screen as inhibitors of SARS-CoV-2 3CL^pro activity furthermore.

3.2.2. The papain-like protease (PL^pro)

Similar to 3CL^pro, the function of PL^pro is essential for coronavirus replication by generating RTC through proteolytic processing of viral polyprotein. Hence, PL^pro could be served as another attractive target of drug discovery for treatment of coronavirus infection, especially SARS-CoV-2. At present, there is no FDA approved PL^pro inhibitor available, therefore identification of bioactive compounds from medicinal plants that specifically inhibit PL^pro has been focused to develop a new class of anti-coronavirus drug. According to high similarity of protein sequences and active residues between SARS-CoV and SARS-CoV-2 PL^pro (83%),⁴⁷ the compounds that have been reported as inhibitors of SARS-CoV PL^pro may also be effective against SARS-CoV-2. Table 1 lists many interesting compounds from natural sources that exhibited SARS-CoV PL^pro inhibitory activity. The IC₅₀ values of the compounds ranged from 0.8 to 19.3 μM, demonstrating their strong inhibitory potential. Among them, the cryptotanshinone and tanshinone IIA were regarded as two most excellent inhibitors.⁵¹

3.2.3. The replication/transcription complex (RTC)

The replication of full-length genomic RNA and the discontinuous transcription of subgenomic RNA transcripts are crucial for the production of new coronavirus particles inside the host cell. Both processes are mediated by the coronavirus RTC composed of multiple viral nsps including two key replicative enzymes like the RdRp (nsp12) and helicase (nsp13),⁵² which are now considered as potential targets for COVID-19 therapy. Considering a strikingly high homology of nucleotide sequence, amino acid sequence and protein structure between SARS-CoV and SARS-CoV-2 RdRp,⁵³ the natural compounds with previous reports of inhibitory activities towards RdRp of SARS-CoV could also have the potential to suppress the activities of those enzymes of the SARS-CoV-2. It was shown that the water extract from Houttuynia cordata exhibited a dose-dependent inhibition on SARS-CoV RdRp activity with the highest decrease by 74% in the treatment of 800 μg/mL.⁵⁴ That activity of H. cordata was confirmed in another study by Fung et al., along with Sinomenium acutum, Coriolus versicolor, Ganoderma lucidum and a traditional Chinese herbal formula Kwan Du Bu Fei Dang. Their IC₅₀ values were 251.1, 198.6, 108.4, 41.9 and 471.3 μg/mL, respectively.⁵⁵ The inhibitors of SARS-CoV helicase also serve as a potential drug candidate since this enzyme has a highly conserved sequence among coronaviruses and shares the similar structure to that of SARS-CoV-2.⁵² Herein, three plant-derived bioactive compounds that could be natural inhibitors of SARS-CoV-2 helicase are listed in Table 1.

3.2.4. The zinc ion

Zinc is an essential micronutrient that is required for various cellular metabolic processes, not only in human immunity but also in the replication of many viruses.⁵⁶ Although Zinc ion (Zn²⁺) acts as a cofactor for several important viral enzymes such as RdRp, 3CL^pro and PL^pro, it is interesting that its high intracellular concentration was found to inhibit those enzyme activities of a variety of RNA viruses including SARS-CoV,56, 57, 58 thus leading to subsequent decrease in the production of new virions. Therefore, Zn²⁺ possesses antiviral properties through generating host immune responses and inhibiting viral replication. As of now, several researchers have suggested the use of Zn²⁺ ionophore, a compound that stimulates cellular import of Zn²⁺ (e.g., chloroquine and its derivatives), as a possible option for the treatment of COVID-19.⁵⁹ In Table 1, we summarized some natural compounds with Zn²⁺ ionophore activity. The most promising compound is epigallocatechin-3-gallate (EGCG), followed by quercetin, luteolin, tannic acid and resveratrol.60, 61, 62

3.3. Natural bioactive compounds targeting viral release

3.3.1. The viroporin 3a

The viroporins are small, pore-forming, viral-encoded accessory proteins with ion channel activity that have been known to play an essential role in mediating several processes in the life cycle of many viruses, including coronaviruses.⁶³ Viroporin 3a functions are strongly involved in the regulation of viral budding and release from infected cells.¹³ Interestingly, this protein was found unique to SARS-CoV and SARS-CoV-2 and not present in other known coronaviruses,⁶⁴ thus the viroporin 3a protein can be an important potential therapeutic target for COVID-19. Summary of natural compounds with inhibitory effect on viroporin 3a activity is presented in Table 1. Schwarz et al. revealed that flavonoid compounds like kaempferol and its derivatives were capable of blocking the ion channel activity of SARS-CoV viroporin 3a protein. Among them, the most potent one is the glycoside juglanine, kaempferol 3-O-α-l-arabinopyranoside, exhibiting IC₅₀ of 2.3 μM.⁶⁵ Another kaempferol glycoside tiliroside and the anthraquinone emodin also showed good inhibitory activity with and IC₅₀ of 20 μM.⁶⁶

3.4. Natural bioactive compounds targeting inflammation-related pathogenesis

Upon binding to SARS-CoV-2 S protein, the ACE2 function is downregulated which leads to increased angiotensin II level and overactivation of the AT1R signaling, causing the deleterious effects associated with excessive inflammation on several tissues.⁶⁷ Therefore, suppressing angiotensin II production by ACE inhibitors and blocking of AT1R by angiotensin-receptor blockers (ARBs) may be of benefit to ameliorate Ang II/AT1R-mediated inflammation in COVID-19 patients. Moreover, it was shown that an ARB could not only reduce AT1R activation, but also activate the AT2R, thus resulting in a production of vasodilation benefit.⁶⁸

Currently, ACE inhibitors and ARBs are commonly prescribed in COVID-19 patients with severe symptoms. Even though risks of the use of hypertensive drugs were concerned, accumulating evidence has not suggested the association between the drugs and worse clinical outcomes.^69,70 Interestingly, a great number of natural compounds have been identified as potent ACE inhibitors and ARBs. Given that there are minimal side effects of using drugs from natural sources, those compounds with potential activity should be considered and investigated. Bioactive compounds derived from natural sources which possess ACE inhibitory activity are summarized in Table 1. Among them, the excellent inhibitory properties against ACE were exerted by the phenolic caffeic acid and chlorogenic acid, and the stilbenoid hopeaphenol and vitisin A, with IC₅₀ less than 2 μM.^71,72 These two stilbenoids were also found to be resveratrol tetramers exhibiting multifaceted properties including anti-inflammation⁷³ and antiviral infection as a potent inhibitor of hepatitis C virus helicase.⁷⁴ However, only few compounds have shown the ability to block AT1R which one of them is [6]-gingerol, the major bioactive compounds present in Zingiber officinale. According to the report by Liu and colleagues, it could inhibit AT1R activity with IC₅₀ of 8.2 μM as detected by cell-based calcium mobilization assay.⁷⁵

3.5. Anti-SARS-CoV natural compounds with unidentified mechanism of action

Some natural occurring compounds have been reported their beneficial effect to inhibit SARS-CoV, even though their mechanisms of action have not yet been identified (Table 2). Accordingly, the compounds from those previous studies might also have a potency to inhibit COVID-19 infection. Using HIV/SARS-CoV S pseudovirus and wild-type SARS-CoV, three anthocyanins derived from Cinnamomi cortex, cinnamtannin B1, procyanidin A2 and procyanidin B1, were reported their inhibitory activities against the infection of both viruses, but at least not through the inhibition of clathrin-mediated endocytosis.⁴⁶ This study also investigated the effects of some crude plant extracts and found that aqueous extract of Caryophylli Flos exhibited moderate inhibition to pseudovirus (IC₅₀ = 58.8 μM) and wild-type virus (IC₅₀ = 50.1 μM).⁴⁶ In addition, the natural alkaloid lycorine, isolated from Lycoris radiate, has been suggested as an anti-SARS-CoV compound with an IC₅₀ value of 15.7 nM.¹²¹

Table 2.

List of anti-SARS-CoV compounds from natural sources with unidentified mechanism of action.

Compound	Class	Source	Biological action/Efficacy	Experiment	Reference
Cinnamtannin B1	Flavonoid	Cinnamomi cortex	IC50 = 32.9 μM (HIV/SARS-CoV S pseudovirus)	Cell-based assay (Luciferase reporter assay)	46
			IC50 = 32.9 μM (Wild-type SARS-CoV)	Cell-based assay (Plaque reduction assay)
Lycorine	Crystalline alkaloid	Lycoris radiata	IC50 = 15.7 nM	Cell-based assay (CPE/MTS assay)	121
Procyanidin A2	Flavonoid	Cinnamomi cortex	IC50 = 120.7 μM (HIV/SARS-CoV S pseudovirus)	Cell-based assay (Luciferase reporter assay)	46
			IC50 = 29.9 μM (Wild-type SARS-CoV)	Cell-based assay (Plaque reduction assay)
Procyanidin B1	Flavonoid	Cinnamomi cortex	IC50 = 161.1 μM (HIV/SARS-CoV S pseudovirus)	Cell-based assay (Luciferase reporter assay)	46
			IC50 = 41.3 μM (Wild-type SARS-CoV)	Cell-based assay (Plaque reduction assay)

CPE/MTS = cytopathic effect-based MTS reduction; IC₅₀ = the half maximal inhibitory concentration.

^(a) The study used commercial products. Here provides a natural source of compound as an example.

4. Conclusion and further prospects

Emerged as the most devastating viral infection in this era for the human race, the COVID-19 pandemic has introduced “new normal” for changing life as we recognize it. As numbers of new COVID-19 infected cases are rising globally, disruption of the transmission chain to minimize this spread is seriously unavoidable. This rise in COVID-19 infection is hardly disrupted unless its infective mechanisms including entry, replication and release, and modification of RAAS can be properly eliminated by humans. Certainly, we are waiting for effective strategies including drugs and vaccines to fight against COVID-19. Due to the unavailability of drugs to treat this infection, natural compounds are a main area of anti-COVID-19 research discovery. Our review suggests that 24 natural compounds have showed their potential actions on multiple therapeutic targets, which should be further explored for anti-COVID-19 plant/mushroom-based medicines (Fig. 2). The classes of these phytochemical compounds include chalcones (n = 7), flavonoids (n = 5), tanshinones (n = 5), phenolic acids (n = 3), polyphenol (n = 1), anthraquinone (n = 1), diarylheptanoid (n = 1) and biphenylpropanoid (n = 1). Among them, a natural flavonoid quercetin is found as a lead candidate with its ability on the virus side to inhibit SARS-CoV S protein-ACE2 interaction, viral protease and helicase activities, as well as on the host cell side to inhibit ACE activity and increase intracellular zinc level, thus making it very promising to reduce the disease burden. Although it is previously speculated that certain ACE inhibitors with an increased activity of ACE2 receptor may indeed enhance viral infectivity, recent studies revealed no substantial association between increased risks of infection and ACE inhibitor medications.^69,70 Therefore, it is worth noting that many potential mechanisms of anti-COVID-19 natural agents are required to carefully and substantially investigated. Together with proper proactive investments, it is our great hope that qualified natural compound-based medicines from promising leads described here will be developed as anti-COVID-19 soon to benefit the human race in this “new normal” era.

Fig. 2.

Chemical structures of natural compounds with potential antiviral properties against multiple therapeutic targets for COVID-19.

Taxonomy (classification by EVISE)

Emerging Infectious Disease, Viral Infection of Respiratory System, Severe Acute Respiratory Syndrome Coronavirus, Cell culture, Molecular Biology, Traditional herbal medicine, Natural Product Analysis.

Declaration of competing interest

The authors declare that they have no conflict of interest.