Evaluation of the anti-SARS-CoV-2 properties of essential oils and aromatic extracts

Essential oils and aromatic extracts (oleoresins, absolutes, concretes, resinoids) are often used as food flavorings and constituents of fragrance compositions. The flavor and fragrance industry observed significant growth in the sales of some natural materials during the COVID-19 outbreak. Some companies worldwide are making false claims regarding the effectiveness of their essential oils or blends (or indirectly point toward this conclusion) against coronaviruses, even though the available data on the activity of plant materials against highly pathogenic human coronaviruses are very scarce. Our exploratory study aimed to develop pioneering knowledge and provide the first experimental results on the inhibitory properties of hundreds of flavor and fragrance materials against SARS-CoV-2 main and papain-like proteases and the antiviral potential of the most active protease inhibitors. As essential oils are volatile products, they could provide an interesting therapeutic strategy for subsidiary inhalation in the long term.

Inhibitor screening. Experimental conditions (concentration of enzyme and substrate, time) were tailored to obtain optimal enzyme activity during measurement like in our previous communications: PL pro10 and M pro38 . Control experiments were carried out in the absence of the inhibitor (100% activity), enzyme or substrate. SARS-CoV-2 M pro (75 nM) was preincubated in assay buffer containing 20 mM Tris, 150 mM NaCl, 1 mM EDTA, and 1 mM DTT, pH 7.3, for 10 min at 37 °C. Then, the enzyme was added to wells containing inhibitors (50 µg/mL essential oils or aromatic extracts), and the mixture was incubated for 30 min at 37 °C. After the incubation period, fluorogenic substrate (Ac-Abu-Tle-Leu-Gln-ACC) was added to the wells (final concentration 50 μM). ACC liberation was monitored for 30 min at 37 °C (λ ex = 355 nm, λ em = 460 nm) using a Molecular Devices Spectramax Gemini XPS spectrofluorometer. Each experiment was repeated twice (inhibition ≤ 50%) or five times (inhibition > 50%). The same experiments were performed for SARS-CoV-2 PL pro . SARS-CoV-2 PL pro (150 nM) was preincubated in assay buffer containing 50 mM Tris, 5 mM NaCl, 0.075% BSA, and 5 mM DTT, pH 7.5, for 10 min at 37 °C. The assay conditions were the same as described above (Ac-Leu-Arg-Gly-Gly-ACC was used as the substrate to measure SARS-CoV-2 PL pro residual activity). IC 50 determination. Experimental conditions (concentration of enzyme and substrate, time) were tailored to obtain optimal enzyme activity during measurement like in our previous communications: PL pro10 and M pro38 . Control experiments were carried out in the absence of the inhibitor (100% activity), enzyme or substrate. For selected inhibitors, the IC 50 value was determined. Serial dilutions of inhibitors in assay buffer were prepared on 96-well plates (20 µL of each dilution in wells). SARS-CoV-2 M pro (75 nM) or SARS-CoV-2 PL pro (100 nM) was preincubated in assay buffer (20 mM Tris, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, pH 7.3; 50 mM Tris, 5 mM NaCl, 0.075% BSA, 5 mM DTT, pH 7.5) for 10 min at 37 °C. Then, 60 µL of the enzyme was added to the wells containing serial dilutions of inhibitors (ranging from 1 µg/mL to 80 µg/mL), and the mixture was incubated for 30 min at 37 °C. After that time, 20 µL of the substrate (Ac-Abu-Tle-Leu-Gln-ACC for SARS-CoV-2 M pro or Ac-Leu-Arg-Gly-Gly-ACC for SARS-CoV-2 PL pro ) was added to each well. Measurements were carried out at 37 °C for 40 min (λ ex = 355 nm, λ em = 460 nm). The experiments were repeated three times. IC 50 values were determined in GraphPad Prism software using nonlinear regression (dose-response -inhibition equation) and presented as relative enzyme activity vs. inhibitor concentration.

Antiviral activity of essential oils and aromatic extracts toward a SARS-CoV-2 strain in
VeroE6-GFP cell culture. VeroE6-GFP cells were seeded at a density of 25,000 cells/well in 96-well plates (Greiner Bio One, catalog no. 655090) and pretreated with threefold serial dilutions of the compounds overnight. On the next day (Day 0), cells were infected with the SARS-CoV-2 inoculum at a multiplicity of infection (MOI) of 0.001 tissue culture infectious dose (TCID 50 ) per cell. The number of fluorescent pixels of the GFP signal determined by high-content imaging (HCI) on Day 4 postinfection (p. i.) was used as a read-out. The percent inhibition was calculated by subtracting the background (untreated-infected control wells) and normalizing to the untreated-uninfected control wells (also background subtracted). Experiments were carried out twice. The 50% effective concentration (EC 50 ) was determined using logarithmic interpolation. The potential toxicity of compounds was assessed in a similar setup in treated-uninfected cultures, where metabolic activity was quanti- www.nature.com/scientificreports/ fied at Day 5 using the MTS assay as described earlier 39 . The 50% cytotoxic concentration (CC 50 ) was calculated by logarithmic interpolation.
Gas chromatography (FID). GC analyses were performed using a Shimadzu GC-2010 Plus gas chromatograph equipped with an FID detector and DB-5 (0.25 mm i.d. × 30 m, 0.25 μm film thickness, Agilent, Santa Clara, USA) capillary column. The injection port was maintained at 250 °C. The split ratio was set as 25:1, and 1 μL of the sample was injected. The oven temperature was set at 40 °C and increased to 300 °C at a rate of 2 °C/ min, with a constant nitrogen carrier gas flow of 1.5 mL/min. The linear retention indices (RIs) of the compounds were calculated using the retention times of n-alkanes from C 8 to C 26 .
Gas chromatography-mass spectrometry (GC-MS). GC-MS analyses were performed using an Agilent 7890A gas chromatograph equipped with an HP-5-MS capillary column (0.25 mm i.d. × 30 m, 0.25 μm film thickness, Agilent, Santa Clara, USA) combined with a WATERS GCT high-resolution mass spectrometer (TOF, EI +). The injection port was maintained at 250 °C. One microliter of the sample was injected in splitless mode. The oven temperature was held at 40 °C and raised to 300 °C at a rate of 2 °C/min, with a constant helium carrier gas flow of 1.5 mL/min. Mass spectra in electron impact (EI) mode were recorded at 70 eV ionization energy.
Essential oil fractionation. Distillation of the petitgrain mandarin essential oil was carried out using a Buchi B-585 oven equipped with a Kugelrohr accessory. Eleven grams of the essential oil was placed in a 40 mL round-bottom flask and fractionated under a controlled distillation setup (2 mbar, 40-260 °C). Three fractions (3.22 g, 2.09 g, 3.91 g) and a residue (1.78 g) were analyzed using GC-FID and GC-MS.

Results and discussion
Natural flavorants and fragrances exhibit different inhibitory activities on both SARS-CoV-2 cysteine proteases.. A total of 538 samples of essential oils, aromatic extracts, and F&F raw materials of various origins were screened for their inhibitory properties on SARS-CoV-2 M pro and PL pro . Screening (spectrofluorimetric enzymatic assay) was carried out with fluorogenic substrates designed by our group in previous works on SARS-CoV-2 proteases 10,37 . Detailed results for each material are presented in the supplementary data (Table S1). No or the lowest inhibitory activities were observed for all materials with high monoterpene and monoterpenoid contents. The most active natural flavorants and fragrances (inhibitory activity > 70%, Table 1) constitute mostly materials with both volatile and nonvolatile fractions, such as resinoids (storax, benzoin Sumatra and Siam, galbanum, and tolu), gums (elemi, storax, and labdanum), and absolutes (rocket, spinach, and oakmoss). The inhibitory potential of complex mixtures such as natural F&F materials is directly related to their composition, which depends on factors such as source plant species, country of origin, part of the plants used, and method of isolation. Clear differences can be observed, for example, between lovage root and leaf essential oils, where the former exhibits higher inhibitory activity on both SARS-CoV-2 proteases (77% and 39% for M pro and PL pro , respectively, Table 1) than the latter (16% and 0% for M pro and PL pro , respectively, Table S1, entry 52). Flavorants and fragrances derived from Cistus ladanifer are very good examples showing the distribution of activity within materials derived from one plant species. Cistus ladanifer is a pyrophoric plant and a source of highly prized labdanum gum. When the plant's flowers begin to fade, the shrub develops leafy twigs. Its branches are covered with secretory hairs, which release abundant quantities of gum with an amber-like fragrance. The gum was historically harvested by combing goats, whose coats were covered with the exudate as they crisscrossed the hillsides 40 . Currently, cistus branches are gathered and processed industrially. Hydrodistillation or steam diffusion of cistus branches results in cistus traditional essential oil, which has a lemony, fresh, and amberlike olfactory profile. The addition of an extract from distillation water to the traditional oil gives ciste (full) essential oil with predominant amber-like, warm, and gourmand notes. Extraction of cistus twigs and branches with hydrocarbon-based solvent leads to a cistus concrete, which can be processed by ethanolic extraction that gives traditional cistus absolute with dry wood and sweet notes or by molecular distillation resulting in cistus SEV absolute with balsamic, smoky, and hot notes. Young cistus branches can also be dipped in a hot solution of sodium carbonate. This solution was acidified with sulfuric acid, and raw labdanum gum was obtained. Its hydrodistillation results in labdanum oil with intensive amber-like, woody notes. Extraction of gum with a hydrocarbon solvent leads to labdanum concrete, which after ethanolic extraction gives labdanum absolute, which possesses alcoholic, balsamic, and mild notes. Ethanolic extraction of raw gum leads to labdanum resinoids with balsamic, sweet, and amber-like olfactory profiles. The acid fraction of a mixture of labdanum gum and resinoid processed by molecular distillation leads to a labdasur-aroma material with animalic and cheese-like notes. It should be noted that despite having the same plant origin, cistus isolates present different olfactory properties. This means that some constituents are absent in one isolate but present in another, which may result in different biological activities. In this case, the inhibitory activities of various cistus isolates on SARS-CoV-2 M pro are different, e.g., labdanum gum-74%, labdanum resinoid-33%, ciste EO-34%, cistus absolute-0%, and cistus SEV absolute-42%.
In some materials, the nonvolatile fraction is mostly responsible for the inhibitory activity. This is evident in the case of elemi (Canarium luzonicum) gum (97% M pro inhibition vs. 33% in the case of elemi essential oil) and galbanum (Ferula galbaniflua) resinoid (78% vs. 18% (EO) M pro inhibition). Very illustrative examples of the influence of the method of isolation are turmeric-derived flavorants and fragrances, where the most likely polar volatile part is responsible for the M pro inhibitory activity (essential oil-64%; isolation method: water/ steam distillation vs. CO 2 extract-33% inhibition) and the polar nonvolatile part for PL pro inhibitory activity (essential oil-6% and CO 2 extract-5% vs. oleoresin 64% inhibition). In the case of turmeric oleoresin, its polar nonvolatile fraction also enhances the M pro inhibitory activity (89%). The Cuprassaceae family constitutes a more abundant example of active essential oils. Cade crude ex-sabine EO was the most active (94%) against M pro , together with blue cypress and Siam wood essential oils. These activities were also retained for Chinese cedarwood (Cupressus funebris) and Virginian cedarwood (Juniperus virginiana) EOs (Table S1, 57% and 60% inhibition, respectively).
Most of the activity of the tested samples was attributed to the inhibition of the SARS-CoV-2 main protease. M pro is the primary protease of the virus. The papain-like protease has a regulatory role, and both are essential for the processing of the polyprotein. In addition, PL pro has an important role in counteracting the innate immunity of the host cell 9 . Ultimately, the best inhibitor would be the one that has high activities against one or both enzymes. There were only two examples (out of 400 tested) of such high activities-turmeric oleoresin (89% M pro and 64% PL pro inhibition) and petitgrain mandarin essential oil (85% M pro and 100% PL pro inhibition).
All the main compounds of oils, gums, and resinoids listed in Table 1 have been studied to determine whether they can be considered potent inhibitors of SARS-CoV-2 M pro and SARS-CoV-2 PL pro . They were docked in the active center of the enzymes in the S1 cavity but with large binding freedom (20 Å) due to their large shape. Mostly they bind at the S1-S1′ cavity but are also shifted toward further cavities on the substrates and the products on both sides ( Fig. S1A and B).
The structural similarity of the main compounds outside families is noted, and they can be divided into six main groups: benzoates and cinnamates, aliphatic acids, aromatic oxygenated compounds, low-molecular cyclic oxygenated compounds, terpene hydrocarbons, and terpenoid alcohols. The first group of compounds includes derivatives of organic acids based on the skeleton of benzoic or cinnamic acid. The extension of the ester or skeleton length could have a positive effect, increasing the inhibitory activity. Cinnamic acid esters could be more active than their shorter benzoate counterparts. Complex matrices of the remaining materials do not allow a direct comparison of the structure-inhibition relationship. The additional oxygen, hydroxy, or methoxy moieties or heteroatoms in other main groups of major constituents of the most active natural materials could not directly increase the inhibition of the enzyme. It is difficult to say whether it is certain, especially when a given natural product has an equal amount of individual ingredients.
However, the main constituents can be important for inhibitory activity; for example, (+)-bulnesol and (−)-guaiol prevail in blue cypress and guaiac wood essential oils. The results of inhibition for M pro are almost the same, but for PL pro , the additional ingredients of guaiac wood oil increase its activity against this enzyme. The variation in the inhibition could be related to the different ratios of the two major compounds in blue cypress and guaiac wood essential oils.
One compound contributes to the overall inhibitory activity of the petitgrain mandarin essential oil.. The essential oil isolated from mandarin leaves was the most potent in the preliminary screening studies. It comprises 6 main constituents ( Table 2): α-pinene, β-pinene, p-cymene, (+)-limonene, γ-terpinene, and dimethyl anthranilate (DMA), with the last constituent being the most abundant.
Petitgrain mandarin essential oil was fractionated on Kugelrohr, three fractions and distillation residue were analyzed chromatographically, and their activities on both SARS-CoV-2 proteases were assessed ( Table 3, the activity of fractions determined at the same concentration as the raw material during the screening phase). Before fractionation, the hypothesis was that the anthranilate part of the oil is responsible for the inhibitory activity (other materials with high monoterpene content exhibited low or no activity). The hypothesis was correct, as the inhibition rate increases with increasing dimethyl anthranilate content. The same can be observed for two fractions of PM essential oil (during the production of the oil, two fractions emerge-a fraction lighter than water, and a fraction heavier than water-the combination of both gives the commercial PM oil). This was further confirmed with the evaluation of pure natural dimethyl anthranilate, which fully inhibits both SARS-CoV-2 proteases under the same experimental conditions.
The detailed inhibition properties of the petitgrain mandarin essential oil, natural dimethyl anthranilate, and other most active essential oils and aromatic extracts were assessed ( Table 4). As suspected from the fractionation www.nature.com/scientificreports/ studies, PM oil and DMA were significantly stronger inhibitors of SARS-CoV-2 papain-like protease. The best inhibitors of M pro were benzoin Siam and Sumatra resinoids and rocket extracts (absolute and concrete) with IC 50 values in range of 3.41 to 5.24 µg/mL. Our exploratory study aimed to develop pioneering knowledge and provide the first experimental results on the inhibitory properties of hundreds of flavor and fragrance materials against SARS-CoV-2 main and papain-like proteases. The majority of available information is related to SARS-CoV. Only several plant extracts have been evaluated [47][48][49] for their activity against the key proteases, and they exhibited moderate to low inhibitory potential (Table S2, entries 3-5) compared to two natural plant constituents, 3-isotheaflavin-3-gallate and tannic acid 50 , which had relatively good inhibitory activities against the SARS-CoV main protease (Table S2, entries 1

and 2).
Molecular docking studies reveal the detailed binding method of DMA in the active sites of both enzymes.. Dimethyl anthranilate was docked in the active centers of the enzymes to investigate ligand-enzyme interactions. Two types of binding were considered. The ligand was allowed to bind freely 20 Å from the catalytic cysteine (Cys145 for M pro and Cys111 for PL pro ) or was forced to interact with the mentioned amino acid. In the first step of docking, DMA interacted with the catalytic groups of SARS-CoV-2 M pro in both docking pathways. The S1-S1′ pocket is not filled tightly by the ligand (Fig. 1A), which is directed with the methyl group of the ester to Cys145. The placement is stabilized by the hydrophobic interactions of Cys145--Me (3.28 Å) and His163--Me (3.27 Å). The carbonyl oxygen atom also interacts with histidine (3.33 Å). Its position is determined by a strong hydrogen bond with Ser144 (2.47 Å). Another strong hydrogen bond is noticeable for the amino group of the inhibitor with His164 (2.57 Å). The position of the phenyl ring and the N-methyl group seems to be rather constrained, and the interactions of the amino acids with them are weaker compared to the Table 3. Discovery of the most active constituents of the petitgrain mandarin (Citrus reticulata blanco var. Mandarin) oil against SARS-CoV-2 M pro and PL pro . a Area percentages of the main components of the petitgrain mandarin oil and its fractions: Fr. distillation fraction number, Res. distillation residue, EO essential oil, LFr light fraction of the oil, HFr heavy fraction of the oil.  www.nature.com/scientificreports/ ester of the carboxyl group. In addition, their positions might be influenced by a strong intermolecular interaction of neighboring COOMe and NHMe groups (1.92 Å). The optimization of the ligand-enzyme complex moved the dimethyl anthranilate toward the S2-S1 pocket (Fig. 1A). The phenyl ring finds itself in the middle of a hydrophobic area of a pocket built by His41 (4.05 Å), Cys44 (5.48 Å), and Met49 (3.98 Å). Intermolecular interactions are not formed because the side chain of Gln189 creates a strong hydrogen bond (1.90 Å) with the carbonyl oxygen atom, while the methyl ester group interacts with His41 (3.60 Å) and Met165 (4.03 Å). Such positioning of the ligand seems more natural and comparable to the enzyme-substrate complex. This is also confirmed by the large difference in the binding energy ΔG S1-S1′ = -16.70 kcal/mol vs. ΔG S2-S1 = -37.08 kcal/mol.
The amino acids building the S1 and S1′ active pockets of SARS-CoV-2 PL pro are close, and the catalytic Cys111 is involved in the construction of both. Dimethyl anthranilate is placed very well in the S1 area (Fig. 1B). It creates strong hydrogen bonds between its carbonyl oxygen atom and the catalytic amino acids Trp106 (1.90 Å) and Cys111 (3.25 Å). The methyl ester group plays the role of a dovetail interlocking the compound in its position with hydrophobic interactions with Asn109 (3.25 Å), Tyr112 (5.13 Å), and Leu162 (3.97 Å). The leucine interacts hydrophobically with the N-methyl group (4.17 Å) likewise. A small role in the binding of the inhibitor is played by Cys270, which arranges the aromatic part of DMA (5.45 Å). Intramolecular interaction of COOMe and NHMe (1.96 Å) also occurs.
Benzoin resinoids (Siam and Sumatra) demonstrated very promising inhibitory activity against M pro . They are comprised of derivatives of benzoic and cinnamic acids. Four main constituents were evaluated in silico on SARS-CoV-2 M pro (Fig. 2): benzyl benzoate ( Fig. 2A), benzyl cinnamate (Fig. 2B), cinnamyl cinnamate (Fig. 2C), and cinnamic acid (Fig. 2D). For the general nature of the inhibition, we assumed that the unsaturated parts of the compounds are trans-isomers.
Benzyl derivatives ( Fig. 2A and B) are directed by the phenyl ring of the acid part toward the deep part of the S2 pocket of the active center. Arenes are stabilized by multiple hydrophobic interactions with lipophilic amino acids, such as His41 and Met49. However, none of the amino acids create hydrogen bonds between the ligand and protein. Hydrogen interaction occurs when the backbone is elongated. Carbonyl oxygen interacts with the amino group's hydrogen atom from Glu166. The attracted ligands are too distant to form a weak phenyl-Cys145 The binding for each enzyme is shown for the S1-S1′ and S2-S1 pockets. The surface of the enzymes is colored light blue (M pro ) or light pink (PL pro ). The surface of methyl N-methylanthranilate is colored dark brown for the S1-S1′ pockets and green for the S2-S1 pockets. The ligands and the side chains of the amino acids are shown as sticks, and the bond order is not shown. Benzoin Sumatra resinoid has the most prominent antiviral activity.. The most active protease inhibitors were also tested for their antiviral activity using a fully replication-competent SARS-CoV-2 strain in VeroE6-GFP cell culture (Table S3). The benzoin Sumatra resinoid showed selective inhibition (halfmaximal effective concentration (EC 50 ) = 31.5 ± 2.4 µg/mL) with limited toxicity (50% cytotoxic concentration (CC 50 ) = 85.5 ± 1.9 µg/mL). The best PL pro inhibitor, petitgrain mandarin essential oil, showed lower antiviral activity (> 100 µg/mL). Available data on the activity of plant materials against highly pathogenic human coronaviruses are scarce. A few studies have been conducted on the anti-SARS-CoV activities of plant extracts and essential oils (Table S4). Evaluation of cinnamon bark and clove bud aqueous extracts (Table S4, entries [1][2][3][4][5] showed that the hydroalcoholic extract of cinnamon bark and its butanol fraction possess moderate inhibitory activity against SARS-CoV infection 51 . Screening of 121 Chinese herbs resulted in the identification of two compounds (Table S4, entries 6 and 7), tetra-O-galloyl-β-D-glucose from Galla chinensis and luteolin from Veronica linariifolia, which could inhibit SARS-CoV infection in a dose-dependent manner 52 . Only one reference addresses the evaluation of anti-SARS-CoV activities of essential oils 53 (Table S4, entries 10-16), with laurel EO being the most active (modestly).

Scientific Reports
In addition to the in vitro evaluations, several randomized controlled trials showed some promise of Chinese herbal medicine combined with conventional medicine for the treatment of SARS, but the evidence was insufficient due to the low methodological quality of the trials 54 .
Relevant data on the activity of plant materials against MERS-CoV and SARS-CoV-2 are even more scarce, as most of the references are focused on in silico evaluations of natural products (including essential oil constituents) 55 or perspectives in this field 56 . Two of the few meaningful references describe the antiviral value of the hydroalcoholic extract of Echinacea purpurea herb and roots (Echinaforce ® ) against MERS-CoV and SARS-CoV-2 57 . Fifty micrograms/ml Echinaforce ® (the final concentration of ethanol was 0.2%, and it was www.nature.com/scientificreports/ proven that the tested coronaviruses were not sensitive to this residual concentration) fully blocked the infectivity of MERS-CoV and SARS-CoV-2. Most recently, one study described the anti-SARS-CoV-2 activity of some Lamiaceae EOs and their monoterpene constituents 58 . The most active essential oil (Mentha vilosa) exhibited moderate inhibitory activity (127.00 ± 4.63 ppm), as did carvacrol (80.23 ± 6.07 µM). The main omission of the abovementioned study is the lack of stereochemical descriptors of the evaluated optically active monoterpenes. In addition, toxicological aspects of pennyroyal (Mentha pulegium) essential oil, menthofuran, and pulegone are not discussed in enough detail. The results clearly show no or limited toxicity of the abovementioned materials to Vero 76 cells, but they are known to be highly toxic to humans. Despite the lack of experimental in vitro data on the potential of flavor and fragrance materials against SARS-CoV-2, two clinical trials evaluated the potential of a commercial product containing essential oil constituents (Listerine ® -originally EO-based, now comprising a mixture of eucalyptol, (−)-menthol, thymol, and methyl salicylate) to eliminate SARS-CoV-2 in the throat and nasopharynx of COVID-19 patients. The first trial ( 59 , NCT04410159) was completed and showed high viral clearance [two negative RT-PCR (of swabs from the oropharynx and nasopharynx) results at least 24 h apart] rates for 1% iodine-povidone (Betadine ® ) and "essential oils" (Listerine ® ) gargles (100% and 80%, respectively) 4 days after intervention (median time without intervention-14 days). These results should be treated with caution, as the testing groups were very small (5 patients in each gargle group). The second "gargle" clinical trial (NCT04584684), "Antiviral Efficacy and Acceptability of Therapeutic Antiseptic Mouth Rinses for Inactivation of COVID SARS-2 Virus", is ongoing. Its goal is to test the efficacy of antiseptic mouth rinses (e.g., Listerine ® ) to inactivate SARS-CoV-2 in the saliva of COVID-19-positive patients (480 patients). Listerine ® is a hydroalcoholic mixture of essential oil constituents. The ethanol concentration is at the level of 21%, which is significant, but it has been proven that the concentration of ethyl alcohol for the complete inactivation of SARS-CoV-2 must be ≥ 30% ( 60 , 20% EtOH concentration resulted in a reduction factor of only 1.1).

Conclusion
F&F materials are important for society because they are commonly used in various areas of human interest, have established toxicological profiles and are relatively safe. Until recently, the tools to assess the inhibitory potential of chemicals against the key SARS-CoV-2 proteases were limited. Our recent developments 10,37 provided a framework for the discovery of new materials/molecules with potential therapeutic value. The results of this study give the scientific community the selection criteria for further in vitro and in vivo testing of the most promising EOs, AEs, and isolated natural compounds. They can also be used for the rational design of SARS-CoV-2 protease inhibitors. Even though important countermeasures for the spread of SARS-CoV-2 are in motion (i.e., the introduction of vaccines), these results will be important to the field because though coronaviruses can change, the coronaviral proteases are conserved (broad application of results in potential future outbreaks).

Data availability
The data that support the findings of this study are available from the corresponding author upon reasonable request.