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Natural Product Research

Formerly Natural Product Letters

ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/gnpl20

Extraction and characterization of metabolites


from Olea europaea pulp and their molecular
pro
docking against SARS-CoV-2 main-protease (M )

Venkataraghavan Ragunathan & K. Chithra

To cite this article: Venkataraghavan Ragunathan & K. Chithra (2021): Extraction


and characterization of metabolites from Olea�europaea pulp and their molecular
pro
docking against SARS-CoV-2 main-protease (M ), Natural Product Research, DOI:
10.1080/14786419.2021.1983813

To link to this article: https://doi.org/10.1080/14786419.2021.1983813

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Published online: 28 Sep 2021.

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NATURAL PRODUCT RESEARCH
https://doi.org/10.1080/14786419.2021.1983813

SHORT COMMUNICATION

Extraction and characterization of metabolites from Olea


europaea pulp and their molecular docking against SARS-
CoV-2 main-protease (Mpro)
Venkataraghavan Ragunathan and K. Chithra
Nanomaterials and Environmental Research Laboratory, Department of Chemical Engineering,
Alagappa College of Technology, Anna University, Chennai, India

ABSTRACT ARTICLE HISTORY


The present study is the first to extract the bioactive metabolites Received 28 May 2021
from Olea europaea fruit using the Soxhlet-maceration extraction Accepted 15 September 2021
method. The preliminary phytochemical; Fourier transform-infra-
red spectroscopy (FT-IR); gas chromatography-mass spectrometry KEYWORDS
(GC-MS) analyses, and their potential against SARS-CoV-2 Mpro Phytochemical; gas
chromatography-mass spec-
through molecular docking were studied. The preliminary qualita- trometry; Olea europaea;
tive phytochemical analyses showed coumarin glycosides, tannins, metabolites; SARS-CoV-2
terpenoids, cholesterol, carbohydrates, and proteins. FT-IR spec-
troscopy revealed C-H, C ¼ O, O-H, C-N, C-O-C, C-O, CO-O-CO,
C ¼ C, and C-Br functional groups in the extract. GC-MS analysis
was done and the compounds detected were docked against
SARS-CoV-2 Mpro using AutoDock Vina.The squalene (DG ¼
6.2 kcal/mol) posed the best inhibition potential and was com-
parable with the control drug remdesivir. The compounds pos-
sessed excellent pharmacokinetic and toxicity properties and are
safe and reliable. Thus, the present research unveiled the valuable
metabolites from O. europaea and their antiviral potential against
the SARS-CoV-2.

CONTACT K. Chithra [email protected]


Supplemental data for this article can be accessed online at https://doi.org/10.1080/14786419.2021.1983813.
ß 2021 Informa UK Limited, trading as Taylor & Francis Group
2 V. RAGUNATHAN AND K. CHITHRA

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.

2. Results and discussion


2.1. Extraction yield and preliminary phytochemical analyses
The novel present study obtained a significant yield of 30.53 ± 5.23% from the etha-
nolic fruit extract, which is more effective when compared to (Hiedayati et al. 2017),
who reported 26.07% using the maceration method alone from the ethanolic leaf
extract. In the present study, the residual phytoconstituents remaining in the pulp
NATURAL PRODUCT RESEARCH 3

after Soxhlet extraction was extracted entirely by the maceration method to


30.53 ± 5.23%, which is noteworthy. Thus, the yield obtained by combining the Soxhlet
and maceration method produced is more significant when compared to using one
mode of extraction alone. It is clear from Table S1 that the overall extraction yields
increased upto 4% using the sequential combination of Soxhlet and maceration
methods. The extracts from Soxhlet and maceration methods were then combined
and used for the rest of the research. Virot et al. 2007 obtained a yield of 35-40%
from the olive fruit using Soxhlet-microwave assisted extraction, which is slightly
higher than the yield of the current research work (Virot et al. 2007). Cho et al. 2020
reported a maximum yield of 17.55 ± 0.88% from O. europaea leaf extract using 50%
ethanol (Cho et al. 2020) which is very low compared to the yield reported in the pre-
sent study. The yield of Soxhlet-maceration extraction was entirely satisfactory com-
pared to the previously reported studies. The qualitative phytochemical analyses
revealed six phytochemicals, viz., terpenoids, tannins, coumarin glycosides, carbohy-
drates, cholesterol, and proteins.

2.2. Characterization of bioactive metabolites from O. europaea pulp


The FT-IR (Figure S2) showed the following peaks viz., medium peaks at 3005.52,
2920.66, and 2852.2 cm1 denoted the vibrations of C-H, a strong peak at
1743.33 cm1 denoted C ¼ O, a medium peak at 1376.93 cm1 depicted the vibration
of O-H. A peak at 1277.61 and 1240.97 cm1 denoted C-N group, a medium peak at
1162.87 cm1 showed the vibration of C-O-C, 1091.51 cm1showed the vibration of C-
O, a medium peak at 1048.12 cm1 showed the presence of CO-O-CO, a stretching at
721.247 cm1 denoted C ¼ C group, and a tiny peak at 641.251 cm1 showed the pres-
ence of C-Br functional groups. From the characteristic peaks of FT-IR, we confirm the
presence of aromatic compounds, phenolics, carbonyl, esters, and triglycerides in the
ethanolic extract of O. europaea pulp. The GC-MS analysis (Figure S3) detected four
volatile bioactive compounds at various retention times (Table S2) viz., (1) n-hexadeca-
noic acid (C16H32O2) at retention time 18.035 min with a peak area of 10.796%; (2) (E)-
9-octadecenoic acid ethyl ester (C20H38O2) at retention time 19.245 min with a peak
area of 1.276%; (3) cis-9-hexadecenoic acid (C16H30O2) at retention time19.755 min
with a peak area of 81.951%; (4) squalene (C30H50) at retention time 24.172 min with a
peak area of 5.978% were detected. Similar components in olives were also reported
by (Irmak and Tokuşoglu 2017; Pacetti et al. 2019).

2.3. Molecular docking of bioactive compounds against mpro using


AutoDock vina
Out of the four compounds, the polyunsaturated triterpene squalene showed the low-
est binding energy of 6.2 kcal/mol (Ki ¼ 28.2 mM) interacting with THR25, HIS41,
CYS44, MET49, TYR54, PHE140, LEU141, ASN142, GLY143, SER144, CYS145, HIS163,
MET165, GLU166, ASP187, ARG188, and GLN189 residues in the active site forming
non-covalent bonding like van der Waals, alkyl, and p-alkyl interactions (Table S3 ad
Figure S4). This low DG was attributed due to the low entropy change because of its
4 V. RAGUNATHAN AND K. CHITHRA

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.

2.4. Predicted pharmacokinetic and toxicity properties of bioactive metabolites


from O. europaea
Table S5 shows that all the bioactive metabolites had high gastrointestinal absorption,
penetrate the blood-brain barrier, and Caco-2 permeability due to their low polar sur-
face area (Matsson and Kihlberg 2017). All the compounds were not the substrate for
P-glycoprotein substrate. Except for the triterpene squalene, all other compounds
were not inhibitors of P-glycoprotein. The metabolic properties of compounds
NATURAL PRODUCT RESEARCH 5

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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