The molecular modeling studies were prepared with the protocol of our earlier studies 47. Crystal structures of the SARS-CoV-2 Mpro (PDB ID: 6XBH - 48) and SARS-CoV-2 PLpro (PDB ID: 6WX422) were obtained from the Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB-PDB). Waters, ligands, and ions were deleted, and the proteins were protonated at experimental pH and optimized 49. Before docking, the structures of the inhibitors were protonated in experimental pH typical for each enzyme and optimized by LigPrep 50. Induced Fit Docking protocol was used for molecular docking studies. The site of the enclosing box was set at 20 Å on the centroid of the catalytical cysteines 51. The VSGB (variable-dielectric generalized Born) model, which incorporates residue-dependent effects, was used. The solvent was water. The active center amino acids were optimized within 5.0 Å of ligand poses, and Glide redocking was carried out with the XP (extra precision) algorithm. The top three poses for ligand were saved. The last steps, rePrime refinement, and MM-GBSA (molecular mechanics-generalized Born surface area) calculations were performed to calculate the Gibbs free energies with protein flexibility, and the distance from the ligand was also set to 5.0 Å. The one with the lowest free binding energy was considered for publication. The protocol and all the parameters were the same for the second docking, but the ligands were forced to interact with the catalytical cysteine (Cys145 or Cys111) during the IFD.
Essential oils and related products
All samples of investigated plant volatile products were generously donated by members of the industry – manufacturers of the products at the best possible quality. Nomenclature of the products according to specifications.
Essential oils of bitter orange (fruit) distilled, petitgrain bigarade sur fleurs, cabbage rose, carrot seed, celery seed, celery leaf, chamomile blue, cinnamon basil CT methyl cinnamate, cinnamon basil CT methyl chavicol, cinnamon basil CT linalool, cinnamon basil CT citral, clary sage, coriander seed, coriander leaf, dill leaf, jasmine, anise seed, cumin, lantana, leek, fennel bitter, fennel sweet, neroli, neroli BdN, parsley seed, parsley leaf, petitgrain bigarade, petitgrain key lime, petitgrain mandarin, winter savory, summer savory, absolutes of artichoke, cabbage rose, cabbage rose leaves, calendula, carnation, carrot leaf, cassie, Rosa damascena, bitter orange blossom hydrolate, honeysuckle, jasmine, mango leaf, nasturtium, nettle, olive leaf, rocket, spinach, strawberry leaf, and tomato leaf, and concretes of cabbage rose and jasmine.
Essential oils of traditional and complete cistus, absolutes of cistus, cistus SEV, and tonka bean.
Amigo & Arditi
Essential oils of cabreuva red, guaiac wood, citronella organic, and petitgrain bitter orange.
Essential oils of Irish lace, spiked pepper, copal, and palo santo.
Essential oils of angelica root, angelica seed, anise (Pimpinella anisum), Artemisia afra, rosewood, buchu leaf betulina, buchu leaf crenulata, calamus, cascarilla bark, catnip, coffee, cognac white, cubeb, dill seed, Eucalyptus smithii, fir needle China, lavandin abrialis, lovage leaf, lovage root, mandarin red, milfoil, Ocotea cymbarum, parsley leaf, Rosa damascena Bulgaria, Rosa damascena Turkey, peppermint Yakima, Pinus pumilio, perilla, sassafras, savory, siam wood, tagetes, valerian root, and wormwood European, fir balsam, absolutes of spruce, Rosa damascena Morocco and Bulgaria, orris root concrete, gums of labdanum and storax.
Essential oils of labdacistus, rue, bitter fennel, clementine, cypriol, laurel, onion, tarragon, tangerine, petitgrain lemon, petitgrain mandarin, vetiver Java, ylang ylang 2nd, citronella Java type, orange CP Valencia, and thyme red, absolutes of labdanum, jasmine, spike lavender, thyme red, and thyme grey, oleoresins of cumin and turmeric.
Essential oils of black pine, maritime pine, and silver fir cones.
Citrus and Allied Essences Ltd.
Essential oils of fir needle Siberian, ginger China, lemon California type CP Extra, lemon Argentina Tucuman CP, orange Valencia CP, orange Brazil CP, orange Brazil 5-fold, corn mint India (partially dementholized, Mentha arvensis), tangerine Dancy, geranium China.
D.V. Deo Industries
Essential oils of palmarosa, cardamom, and gingergrass.
Dutjahn Sandalwood Oils.
Santalum spicatum essential oil.
Essential Oils and Herbs
Melissa and yarrow essential oils.
Essential oils of rose geranium, tea tree, lemon-scented tea tree, Eucalyptus smithii, Eucalyptus radiata, tagete.
Essential oils of citronella China, garlic China, geranium China, geranium Egypt, ginger China, neroli Morocco, peppermint arvensis China, petitgrain bigarade, sandalwood India, shiu (Ho wood) China, vetivert Bourbon, and ylang-ylang, absolutes of coffee, fenugreek, jasmine India, orris, and orange flower, ginger China and pink pepper CO2 extracts.
Mane Kancor Ingredients Private Limited
Essential oils of carrot seed, ajowan, black pepper CO2, capsicum CO2, cumin seed, black cumin, celery seed, fennel, mace, tuberose absolute and concrete.
Essential oils of allspice, blood orange, catnip, cedarwood Himalayan, Eucalyptus polybractea, goldenrod, grapefruit red, Lavandula stoechas, mandarin red, Peru balsam, Rosa damascena, black spruce, tea tree, ylang-ylang I, ylang-ylang II, and neem oil.
Les Arômes du Maroc
Mastic essential oil, orange flower water absolute, orange flower, and bran concretes.
Essential oils of cedarleaf, cedarwood Texas, citronella Java type, geranium Egypt, ginger China, ginger Nigeria, gurjun balsam, lavender fresh Bulgarian, lemon Spain, sweet orange CP Valencia, sweet orange CP Brazil, pennyroyal, peppermint Arvensis, thuja, thyme red, star aniseed, cassie, citrate, cognac green, coriander seed, cumin “ex-distilled”, dill leaf, Eucalyptus radiata, lemongrass, key lime distilled type Mexico, key lime expressed type Mexico, mandarin green, myrtle, nutmeg, parsley seed, patchouli molecular distillation, patchouli super dark, pimento berry, pimento leaf, pine needle, scotch pine, ravintsara, sweet basil, tropical basil, verbena, ylang-ylang I, ylang ylang III, oleoresins of basil, capsicum 1000000 SHU, paprika 40000 SHU, paprika 60000 SHU, paprika 80000 SHU, pepper black 40/20, and thyme red, copaiba balsam, and gurjun balsam rectified (copaene).
New Zealand Mānuka Group
Essential oils of mānuka (MBTK 5+), mānuka (MBTK 20+), mānuka (MBTK 25+), and kanuka.
Clove stem essential oil and cocoa butter CO2 extract.
Bran, blackcurrant buds, and broom absolutes.
Essential oils of angelica root, artemisia, Artemisia taurica, blood orange, blue cypress, buchu leaf, buddha wood, coriander herb, Eucalyptus kochii, hinoki, kumquat, kunzea, lemon myrtle, Rosalina, CO2 extracts of juniper berry, star anise, turmeric, vetiver, black seed, cardamom green, coffee arabica, coffee robusta, and ginger.
The remaining samples of essential oils and other aromatic materials were bought from PerfumersWorld Ltd. Essential oils were named according to the ISO 4720:2018 norm - Essential oils — Nomenclature where possible.
The expression of recombinant enzymes - SARS-CoV-2 Mpro and PLpro is described in our previous works 15,22.
The synthesis of ACC-labeled substrate for Mpro (Ac-Abu-Tle-Leu-Gln-ACC) and PLpro (Ac-Leu-Arg-Gly-Gly-ACC) is described in our previous works 21,22.
SARS-CoV-2 Mpro (75 nM) was preincubated in assay buffer 20 mM Tris, 150 mM NaCl, 1 mM EDTA, 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 (λex = 355 nm, λem = 460 nm) using Molecular Devices Spectramax Gemini XPS spectrofluorometer. Negative control experiments were carried out in the absence of the enzyme. Each experiment was repeated twice (inhibition ≤ 50%) or five times (inhibition > 50%). The same experiments were performed for SARS-CoV-2 PLpro. SARS-CoV-2 PLpro (150 nM) was preincubated in assay buffer 50 mM Tris, 5 mM NaCl, 0.075% BSA, 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 PLpro residual activity).
For selected inhibitors, the IC50 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 Mpro (75 nM) or SARS-CoV-2 PLpro (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 respectively) 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 Mpro or Ac-Leu-Arg-Gly-Gly-ACC for SARS-CoV-2 PLpro) was added to each well. Measurements were carried out at 37oC for 40 min (λex = 355 nm, λem = 460 nm). The experiments were repeated three times. IC50 values were determined in GraphPad Prism software using non-linear regression (dose-response – Inhibition equation) and presented as relative enzyme activity vs. inhibitor concentration.
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 C8 to C26.
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. 1 μL of the sample was injected in the 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 Buchi B-585 oven equipped with the kugelrohr accessory. 11 g of the essential oil was placed in a 40 mL round-bottom flask and was fractionated under controlled distillation set-up (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.