Extraction and characterization of metabolites from Olea europaea pulp

and their molecular docking against SARS-CoV-2 main-protease (Mpro)

Venkataraghavan Ragunathan1 and K. Chithra1, *

1
Nanomaterials and Environmental Research Laboratory, Department of Chemical
Engineering,

Alagappa College of Technology, Anna University, Chennai – 600025

*Corresponding author: chithraakumaran@gmail.com

Abstract

The present study is the first to extract the bioactive metabolites from Olea europaea fruit
using the Soxhlet-maceration extraction method. The preliminary phytochemical; Fourier
transform-infrared spectroscopy (FT-IR); gas chromatography-mass spectrometry (GC-MS)
analyses, and their potential against SARS-CoV-2 Mpro through molecular docking were
studied. The preliminary qualitative phytochemical analyses showed coumarin glycosides,
tannins, terpenoids, cholesterol, carbohydrates, and proteins. FT-IR spectroscopy revealed C-
H, C=O, O-H, C-N, C-O-C, C-O, CO-O-CO, C=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 (ΔG = -6.2 kcal/mol) posed the best inhibition
potential and was comparable with the control drug remdesivir. The compounds possessed
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.

Keywords: Phytochemical; gas chromatography-mass spectrometry; Olea europaea;

metabolites; SARS-CoV-2
 Fig S1: Workflow of research work

Experimental procedure

All chemicals used in the research work were of analytical grade.

Collection of Olea europaea fruit

Olea europaea were procured from the local markets in and around Chennai, Tamil Nadu,
India. The fruits were then soaked and washed with running tap water, followed by Milli-Q
distilled water to remove sand and other debris. The fruits were pitted to remove the endocarp
seed inside, and the mesocarps were shade dried for two days. The shade-dried pulps were
packed in an air-tight container and stored until use.

Extraction of bioactive metabolites from Olea europaea pulp

The bioactive constituents were extracted using the conventional continuous extraction
system using the Soxhlet apparatus (Borosil, India) followed by maceration extraction to
ensure complete extraction under static and dynamic conditions. Thirty grams of powdered
fruit was transferred to a Whatman cellulose thimble and loaded 250 mL of ethanol onto the
apparatus in the round bottom flask. The extraction was performed until the constituents
leached out, denoted by an endpoint colour change in the thimble. The extracted debris was
then transferred to a conical flask with 150 mL of ethanol and agitated at 150 rpm in an
orbital shaker (Scigenics (India) Pvt. Ltd., India) for 48 hours. The extracted constituents
from both the extraction were concentrated using a rotary evaporator (Buchi, Switzerland),
and the percentage yield was calculated using equation (1).
 (1)

The concentrated extracts were stored at -20ºC until further use. The extraction was
performed in duplicates to maintain concordance of the results.

Table S1: Extraction yield of bioactive compounds using Soxhlet-maceration method

Trial Soxhlet Maceration Soxhlet-maceration Total yield (%)

extraction (%) extraction (%) extraction (%)
1 29.88 4.50 34.23
30.53±5.23
2 22.73 4.10 26.83

Characterization of Olea europaea pulp extract

Preliminary qualitative phytochemical analyses for the presence of phytochemicals

flavonoids, terpenoids, phlobatannins, tannins, cardiac glycosides, alkaloids, saponins,
coumarin glycosides, carbohydrates, cholesterol, and proteins were performed according to
the standard procedures of (Harborne 1984; Gul et al. 2017), and the results were tabulated.
FT-IR spectroscopy was performed to detect the functional groups using JASCO FT/IR-4700
type Spectrometer (JASCO, Japan) in the mid-infrared region using the attenuated total
reflectance mode at 4 cm-1 resolution. The bioactive metabolites were detected using Perkin
Elmer Clarus 680 chromatography (Perkin Elmer, USA) using Elite-5MS fused silica
column, helium as a carrier gas, and 1 mL/min flow rate. The injection temperature was set at
260ºC for the complete evaporation of the sample, and oven conditions were as follows, i)
60°C for 2 minutes; an increase to 300°C at the rate of 10°C per minute; held for 6 minutes at
300°C. The mass detector conditions were as follows, transfer line temperature 240°C; ion
source temperature 240°C; ionization mode electron impact 70 eV; scan interval of 0.1
seconds. The compounds were detected between 40 to 600 Daltons. The spectra of
components were then compared with the standard database of GC-MS NIST (2008) library
of known compounds.

Molecular docking of bioactive compounds from O. europaea against SARS-CoV-2

main-protease using AutoDock Vina

The GC-MS detected bioactive compounds were downloaded from the PubChem database in
3-dimensional SDF format. The energy was minimized using ArgusLab 4.0.1 with QM/MM
force field and the Hartree-Fock algorithm for 200 iterations. It converted to PDBQT format
using Open Babel GUI software. The enzyme (PDB Id:6W63) was chosen based on the
literature references (Pant et al. 2020) and downloaded from the protein data bank website,
refined using PyMOL 2.3 to remove heteroatoms. The polar hydrogen, Kollman, and
Gasteiger charges were added using AutoDock 4.2 (Morris et al. 2009) and saved in PDBQT
format before docking. The ligand-binding site of the enzyme was determined using the
MetaPocket 2.0 webserver, and the grid boxes were set such that the box covers the amino
acids in the ligand-binding site completely in all dimensions. Molecular docking was
performed using AutoDock Vina software (Trott and Olson 2009) for 100 genetic algorithm
runs with an exhaustiveness value of 8. The response inhibition potentials were studied in
terms of free binding energy (ΔG) and inhibition constant (Ki) for each run based on the
entropy change (ΔS) and bond formations during the interaction of the compound with the
enzyme. The compound with the lowest binding energy is considered the best potential
inhibitor. The ligand X77 was re-docked against the Mopar and superimposed onto the actual
co-crystal structure, which we performed to validate the docking protocol. A typical
superimposed executive RMSD < 2Å between the complexes is considered valid. The docked
interactions and superimposed complexes were elucidated using Biovia’s Discovery studio
2020 and LigPlotv.2.2 software and tabulated. The Ki value was calculated using equation
(2).

(2)

Where ΔG denotes intermolecular free binding energy; R denotes gas constant; T denotes
temperature (298 K). Lipinski’s properties of the bioactive compounds were studied from the
PubChem database to assess the oral drug-likeliness.

Pharmacokinetic and toxicity properties of bioactive compounds O. europaea

Pharmacokinetic and toxicity properties of bioactive compounds were predicted using

admetSAR 1 & 2 (Yang et al. 2019) and SwissADME webservers (Daina et al. 2017).
Fig S2: FT-IR spectrum of ethanolic extract of Olea europaea pulp showing various
functional groups

(3)

(1)
(4)

(2)

Fig S3: GC-MS spectrum of ethanolic extract of Olea europaea pulp showing various
metabolites
 Table S2: List of bioactive metabolites detected using GC-MS

S. Compound RT Scan Height Area Area Norm

No % %

1 n- 18.035 3046 12,144,768,000 1,173,028,096.0 10.796 13.17

hexadecanoic
acid

2 (E)-9- 19.245 3288 3,812,718,592 138,604,192.0 1.276 1.56

octadecenoic
acid ethyl
ester

3 Cis-9- 19.755 3390 35,203,506,176 8,904,620,032.0 81.951 100.00

hexadecenoic
acid

4 Squalene 24.172 4273 8,048,815,104 649,593,664.0 5.978 7.30

Table S3: Binding energy and interaction of compounds with Mpro (PDB Id: 6W63)

S. No Compound Binding Interacting amino No. of Ki value

Energy acid residues hydrogen
(kcal/mol) bonds
1 n-hexadecanoic -4.6 THR24, THR25, 2 421 µM
acid HIS41, CYS44,
THR45, SER46,
MET49, TYR54,
MET165, ASP187,
ARG188, GLN189
2 (E)-9- -4.1 HIS41, CYS44, - 980.3 µM
octadecenoic MET49, CYS145,
acid ethyl ester HIS164, MET165,
GLU166, PRO168,
ASP187, ARG188,
GLN189, THR190,
GLN192
3 Cis-9- -4.3 THR25, HIS41, 3 699.1 µM
hexadecenoic CYS44, THR45,
acid MET49, HIS164,
MET165, GLU166,
ASP187, ARG188,
GLN189
4 Squalene -6.2 THR25, HIS41, - 28.2 µM
CYS44, MET49,
TYR54, PHE140,
LEU141, ASN142,
GLY143, SER144,
CYS145, HIS163,
MET165, GLU166,
ASP187, ARG188,
GLN189,
5 X77 -8.3 THR25, HIS41, 3 811.5 nM
MET49, TYR54,
PHE140, LEU141,
ASN142, SER144,
CYS145, HIS163,
MET165, GLU166,
PRO168, ASP187,
ARG188, GLN189
6 Remdesivir -7.3 THR25, HIS41, 6 4.39 µM
CYS44, THR45,
SER46, MET49,
TYR54, PHE140,
LEU141, ASN142,
SER144, CYS145,
HIS163, HIS164,
MET165, GLU166,
HIS172, ASP187,
ARG188, GLN189
Fig S4: Amino acid interactions of Mpro with various compounds: (1) n-hexadecanoic acid;
(2) (E)-9-octadecenoic acid ethyl ester; (3) cis-9-hexadecenoic acid; (4) squalene; (5) X77;
(6) Remdesivir
 Fig S5: Re-docked X77-Mpro (Blue) superimposed onto actual co-crystal structure (Green)

Table S4: Lipinski’s oral drug-likeliness properties of bioactive metabolites from O.

europaea
S. No Compound Molecular Log P H-bond H-bond Molar
weight in (<5) donor (<5) acceptor refractivity
g/mol (<10) (<130)
(<500 Da)
1 n-hexadecanoic acid 256 5.55 1 2 77.94
2 (E)-9-octadecenoic 310 6.58 0 2 96.08
acid ethyl ester
3 cis-9-hexadecenoic 254 5.32 1 2 77.85
acid
4 Squalene 410 10.60 0 0 140.06
 Table S5: Predicted pharmacokinetic properties of bioactive metabolites from O.
europaea
S. Compound GI BBB Caco-2 P- P- CYP1A2 CYP2C19 CYP2C9 CYP2D6 CYP3A4 Log Kp Skin Total
No absorption permeation permeability glycoprotein glycoprotein inhibitor inhibitor inhibitor inhibitor inhibitor permeation polar
substrate inhibitor (cm/s) surface
area
(Å2)

1 n- Yes Yes Yes No No Yes No No No No -2.77 37.30

hexadecanoic
acid

2 (E)-9- Yes Yes Yes No No Yes No No No No -4.52 54.37

octadecenoic
acid ethyl
ester

3 cis-9- Yes Yes Yes No No No No No No No -1.95 26.30

hexadecenoic
acid

4 Squalene Yes Yes Yes No Yes No No No No No -0.58 0.00

Table S6: Predicted toxicity properties of bioactive metabolites from O. europaea

S. Compound AMES Carcinogens Hepatotoxicity Biodegradation Acute oral

No toxicity toxicity
(kg/mol)
1 n- No No No Yes 1.376
hexadecanoic
acid
2 (E)-9- No No No Yes 1.742
octadecenoic
acid ethyl
ester
3 cis-9- No No No Yes 1.706
hexadecenoic
acid
4 Squalene No Yes No No 1.842

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