Nat Prod Res
Nat Prod Res
Nat Prod Res
SHORT COMMUNICATION
1. Introduction
Olea europaea, the olive tree, contains phytochemicals that possess various biological
properties like anti-inflammatory, anti-oxidant, anti-hypertensive, antitumoral, antibac-
terial, antifungal, anthelminthic agents, neuroprotective, hypoglycemic, and cytotoxic.
(Hashmi et al. 2015; Alesci et al. 2021). Apart from these, olives and olive oil contain
diacylglycerols, triacylglycerols, fatty acids, and hydrocarbons with cholesterol-lowering
and anti-diabetic properties due to their phytochemical abundance (Khaliq et al.
2020). These were attributed due to the presence of polyphenols, fatty acids, tannins,
terpenoids, glycosides, saponins, etc. (Bianco et al. 2004; Bianco and Ramunno 2006).
Several essential bioactive compounds from olive fruit like oleuropein, kaempferol, aro-
madendrine, nocellaralactone, vanillin, oleanolic acid, oleic acid, tyrosol, hydroxyl-tyro-
sol are proven anti-inflammatory agents (Serrilli et al. 2013; Venditti et al. 2013;
Rhouma et al. 2021). In addition, phenolic compounds from the flowers of O. europaea
also possess antiproliferative, anticancer, and apoptosis-inducing properties. A few
studies have reported the antiviral property of O. europaea, predominantly focusing
on the bioactive compounds from its leaves, however not from the fruit pulp (Micol
et al. 2005). It is essential to identify the best extraction technique to extract all the
components from the fruit without the loss of activity. COVID-19 has been a threat to
humanity since its outbreak in Wuhan, China, in December 2019, spreading infections
globally killing millions of people (Zheng et al. 2020). There is no antidote for this pan-
demic responsible virus; however, many vaccines have cleared their clinical trials and
are under the process of administration throughout the globe. Several FDA approved
drugs like hydroxychloroquine, remdesivir, etc., are currently repurposed and used as
an alternative drug of choice to solve this ongoing pandemic (Rathi et al. 2020). The
SARS-CoV-2 main-protease (PDB Id: 6W63) is a cysteine protease catalytically active at
CYS145 and HIS41 amino acid residues with a molecular weight of 33.79 kDa are pre-
dominantly responsible for the formation of functional proteins, are involved in viral
replication and transcription processes (Mandour et al. 2020). The viral replication is
stopped by inhibiting its catalytic center, where chemical drugs and biologically active
phytochemicals come. The importance of viral main-protease and the absence of
strictly related homologs in Homo sapiens make it an attractive target for inhibition
(Pillaiyar et al. 2016). The present research work thus throws light on the effective
extraction, characterization, and antiviral potential of ethanolic extract of O. europaea
against the SARS-CoV-2 as an effort towards developing plant-based cure through
computational approaches have not been reported as of our knowledge. The workflow
of the research work is shown in Figure S1.
hydrophobic nature, causing smooth water displacement from the ligand-binding site
of Mpro. Squalene is widely used as an adjuvant in the vaccine. The n-hexadecanoic
acid, a saturated fatty acid, showed binding energy of 4.6 kcal/mol (Ki ¼ 421 mM)
formed interactions with THR24, THR25, HIS41, CYS44, THR45, SER46, MET49, TYR54,
MET165, ASP187, ARG188, and GLN189 residues through conventional hydrogen
bonds, van der Waals, alkyl, and p-alkyl interactions. Cis-9-hexadecenoic acid ranked
third with a binding energy of 4.3 kcal/mol (Ki ¼ 699.1 mM), forming interactions
with THR25, HIS41, CYS44, THR45, MET49, HIS164, MET165, GLU166, ASP187, ARG188,
and GLN189 residues of Mpro with three hydrogen conventional bonds, van der Waals,
alkyl, and p-alkyl non-covalent interactions. (E)-9-octadecenoic acid ethyl ester (Ki ¼
980.3 mM) interacted with HIS41, CYS44, MET49, CYS145, HIS164, MET165, GLU166,
PRO168, ASP187, ARG188, GLN189, THR190, and GLN192 residues of the Mpro forming
van der Waals, alkyl, p-alkyl, and p-sigma interactions. The free binding energy of co-
crystallized inhibitor X77 was 8.3 kcal/mol, which interacted with THR25, HIS41,
MET49, TYR54, PHE140, LEU141, ASN142, SER144, CYS145, HIS163, MET165, GLU166,
PRO168, ASP187, ARG188, and GLN189 amino acids forming three conventional hydro-
gen bonds with Mpro. The re-docked X77-Mpro superimposed onto the actual co-crystal
structure without any stearic and atomic clashes with a low RMSD of 0.75 Å (Figure
S5), thus validating the protocol. Remdesivir was used as the control drug and pos-
sessed binding energy of 7.3 kcal/mol interacting with THR25, HIS41, CYS44, THR45,
SER46, MET49, TYR54, PHE140, LEU141, ASN142, SER144, CYS145, HIS163, HIS164,
MET165, GLU166, HIS172, ASP187, ARG188, and GLN189 with six hydrogen bonds
which is slightly lower than the binding energy of squalene. From the interactions, we
can notice all the compounds interacted with the amino acids in the catalytic site of
Mpro, thereby causing inhibition. These supramolecular non-covalent interactions
played a pivotal role by inhibiting viral replication and transcription. All the four bio-
active metabolites satisfied Lipinski’s drug-likeliness; these compounds have the poten-
tial to be used as an oral drug (Table S4). Only a few research have focussed on the
anti-COVID activity of O. europaea. Thangavel et al. 2020 performed molecular docking
of various polyphenolic flavonoids from O. europaea fruits against SARS-CoV-2 Mpro
(Thangavel et al. 2020) and reported the inhibition constant value as low as 11.58 mM;
the present study reported a minimum inhibition constant value of 28.2 mM, which is
slightly higher. Alhadrami et al. (2021) reported an IC50 ¼ 3.22–14.55 mM using O. euro-
paea leaves against SARS-CoV-2 Mpro, whose values are close to the present research
work (Alhadrami et al. 2021). Thus, the bioactive compounds of O. europaea exhibited
a good inhibition potential, with squalene been the most potent inhibitor.
assessed on the liver microsomal enzymes were responsible for catalysing the drug
metabolism. Bioactive compounds n-hexadecanoic acid and (E)-9-octadecenoic acid
ethyl ester inhibited the CYP1A2 enzyme; however, all the other compounds were
non-inhibitors of the other microsomal enzymes as expected. Table S6 shows that the
compounds were non-hepatotoxic, non-AMES toxic properties, and biodegradable.
Squalene showed slightly type III (slightly toxic) oral toxicity properties; (E)-9-octadece-
noic acid ethyl ester showed type III oral toxicity. However, several studies reveal that
squalene is effective as a chemopreventive medicine in various cancers (Smith 2000).
Lead optimization could be done by altering the toxic moieties from type III to non-
carcinogenic and type IV (least toxic) toxicity (Abdul-Hammed et al. 2021). Moreover,
metabolites from O. europaea are safe and non-toxic even in high concentrations
(Acar-Tek and Ag agu
€ndu
€z 2020). Thus, all the compounds possessed good pharmaco-
kinetic properties and high potential as inhibitors as therapeutic agents after in-vitro
and in-vivo studies.
3. Conclusion
Thus, the present research work involved extracting bioactive metabolites from Olea
europaea fruits using the novel Soxhlet-maceration sequential mode with a yield of
30.53 ± 5.23%. The characterized fruit extract using FT-IR and GC-MS analytical techni-
ques revealed the identity of various bioactive compounds. The GC-MS revealed four
compounds: n-hexadecanoic acid, (E)-9-octadecenoic acid ethyl ester, cis-9-hexadece-
noic acid, and squalene. The binding energies of the bioactive compounds subjected
to molecular docking against SARS-CoV-2 Mpro were squalene (-6.2 kcal/mol); n-hexa-
decanoic acid (-4.6 kcal/mol); cis-9-hexadecenoic acid (-4.3 kcal/mol); (E)-9-octadecenoic
acid ethyl ester (-4.1 kcal/mol). Out of the four compounds, squalene possessed the
lowest binding energy interacting with amino acids in the active site forming non-
covalent supramolecular interactions. Re-docking studies using the co-crystallized lig-
and validated the docking protocol. Moreover, all the compounds satisfied Lipinski’s
rule of five for oral bioavailability and drug-likeliness and possessed excellent pharma-
cokinetic and toxicity properties for the possibility of use as drugs. On the whole, the
bioactive compounds from O. europaea showed inhibition against Mpro with the sig-
nificant antiviral property. The present research work will pave the way for developing
a plant-based regimen for COVID-19. Furthermore, it will help scientists developing
anti-COVID drugs to investigate further and optimize many other properties to be
used as a drug. Our future studies aim to study the molecular dynamics simulations
and energy studies of docked complexes followed by in-vitro studies of crude and
purified extracts against SARS-CoV-2 infections.
Acknowledgement
The authors extend their gratitude to the Centre for Analysis, Testing, Evaluation & Reporting
Services, CSIR-CLRI, Chennai for performing FT-IR spectroscopy, and Sophisticated
Instrumentation Facility, School of Advanced Sciences, VIT Vellore, Tamil Nadu, for performing
GC-MS analysis.
6 V. RAGUNATHAN AND K. CHITHRA
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
The author(s) reported there is no funding associated with the work featured in this article.
ORCID
Venkataraghavan Ragunathan http://orcid.org/0000-0003-2487-9972
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