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OPEN Phytochemical analysis

and biological activities of essential
oils extracted from Origanum
grossii and Thymus pallidus: in vitro
and in silico analysis
Hind Zejli 1*, Aziza Fitat 1, Youssra Lefrioui 2, Farhan Siddique 3, Mohammed Bourhia 4,
Fatima Zahra Bousseraf 1, Ahmad Mohammad Salamatullah 5, Hiba‑Allah Nafidi 6,
Amare Bitew Mekonnen 7*, Abdelkader Gourch 1, Mustapha Taleb 1 & Abdelfattah Abdellaoui 1

The study aimed at investigating the phytochemical composition, antioxidant and antibacterial
activities of essential oils (EOs) of Origanum grossii and Thymus pallidus. The selection of these plants
for the study was driven by a comprehensive survey conducted in the Ribat Elkheir region of Morocco,
where these plants are widely utilized. The results reflect the valorization of these plants based
on the findings of the regional survey. The GC–MS phytochemical analysis revealed that the main
constituents of the essential oil were carvacrol and thymol for O. grossii and T. pallidus respectively.
Quantitative assays demonstrated that O. grossii exhibited higher levels of polyphenols (0.136 mg
AGE/mg EO) and flavonoids (0.207 mg QE/mg EO) compared to T. pallidus. The DPPH assay indicated
that O. grossii EOs possessed approximately twice the antiradical activity of T. pallidus, with ­IC50
values of approximately 0.073 mg/mL and 0.131 mg/mL, respectively. The antibacterial activity tests
showed that both essential oils exhibited significant inhibition zones ranging from 26 to 42 mm
against all tested bacterial strains. The MIC values varied among the bacteria, generally falling within
the range of 0.31 to 2.44 µg/mL, demonstrating the potency of the EOs to serve as antibacterial.
Molecular docking revealed that O. grossii and T. pallidus essential oils interact with antibacterial and
antioxidant proteins (1AJ6 and 6QME). Key compounds in O. grossii include p-cymene, eucalyptol,
and carvacrol, while T. pallidus contains potent chemicals like p-cymene, ɤ-maaliene, valencene,
α-terpinene, caryophyllene, himachalene, and thymol. Notably, the most potent chemicals in
Origanum grossii are p-cymene, eucalyptol, and carvacrol, while the most potent chemicals in Thymus
pallidus are p-cymene, α-terpinene, and thymol. These findings suggest that these plant EOs could be
used to develop new natural products with antibacterial and antioxidant activity.

Historically, naturally occurring substances have been utilized as food additives and have also been explored for
their therapeutic potential. The properties which are domiciled in different parts of the plant often draw wide
attention, with the essential oils (EO) of aromatic plants being a cynosure in this ­context1,2. These EOs often pos-
sess a surfeit of pharmacological properties including anticancer, anti-inflammatory, antioxidant, insecticidal, and
antimicrobial properties among many others. Concomitant with the presence of these properties is the presence

1
Laboratory of Engineering, Electrochemistry, Modeling and Environment, Faculty of Sciences Dhar El Mahraz,
Sidi Mohammed Ben Abdellah University, B. P. 1796, Fes‑Atlas, Morocco. 2Laboratory of Biotechnology, Health,
Agrofood and Environment, Faculty of Sciences Dhar El Mahraz, Sidi Mohammed Ben Abdellah University,
B. P. 1796, Fes‑Atlas, Morocco. 3Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Bahauddin
Zakariya University, Multan 60800, Pakistan. 4Department of Chemistry and Biochemistry, Faculty of Medicine and
Pharmacy, Ibn Zohr University, 70000 Laayoune, Morocco. 5Department of Food Science & Nutrition, College of
Food and Agricultural Sciences, King Saud University, 11, P.O. Box 2460, Riyadh 11451, Saudi Arabia. 6Department
of Food Science, Faculty of Agricultural and Food Sciences, Laval University, 2325, Quebec City, QC G1V 0A6,
Canada. 7Department of Biology, Bahir Dar University, P. O. Box 79, Bahir Dar, Ethiopia. *email: hind.zejli@
usmba.ac.ma; amarebitew@gmail.com

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of an array of phytochemicals in EOs which confers these potentials. However, the phytochemical composition of
EOs is affected by different factors including ecological (geographical origin, soil composition, climate condition),
genotype (species, clone, cultivar, ecotype), and technical (elements relating to agriculture, forms of collecting,
and crude material storage)3,4. Consequently, EO from similar plant species can express different features and
phytochemical composition which could be dependent on or influenced by the previously mentioned factors.
The Lamiaceae family, which consists of a wide variety of herbs, plays a significant role in the Mediterranean
region because of its well-known medicinal and aromatic qualities. Two notable members of this family, specifi-
cally Origanum grossii (O. grossii) and Thymus pallidus (T. pallidus), epitomize the importance of this family.
The Lamiaceae family is distinguished by a common physiological and morphological structure, with a notable
feature being the presence of essential oils (EOs). These oils have attracted considerable interest due to their
potential therapeutic p ­ roperties5. The significance of investigating the phytochemical constituents of these herbs
is highlighted by this shared characteristic.
The genus Origanum is widely recognized and utilized globally due to its numerous species that hold culinary
and medicinal ­value6. However, O. grossii is considered a distinct entity that has received less attention due to its
limited distribution within the Northwest (NW) region of ­Morocco6,7. The unique nature of its habitat is further
emphasized by its preference for high elevations and humid climates within the Western Rif Mountain r­ ange8.
The species’ specific ecological preferences have resulted in its unique physical characteristics, which include
compact stems covered in pubescent hair, branches of different lengths (~ 55 cm), noticeable bracts (3–5 mm),
and small leaves (11 mm-5-19)9. The distinctive attributes of O. grossii set it apart from other members of the
Lamiaceae family, making it an intriguing subject for further investigation into its bioactive properties.
The Thymus genus, found in the Moroccan landscape, is notable for its significant diversity, boasting around
nine endemic species. These plants possess a significant pharmacological value due to their ample presence of
bioactive compounds, including phenolic acids, tannins, flavonoids, and resins. These compounds, which are
present in both the aerial parts and essential oils, possess unique medicinal ­properties6,10. Notably, thymol and
carvacrol are prominent constituents found in thyme oils. These compounds are recognized for their antioxidant,
antiseptic, antibacterial, and antifungal ­properties11.
Within the domain of traditional healing, T. pallidus, a crucial member of the Thymus genus, assumes a
prominent role. The historical use of this substance has included a variety of applications, such as powders and
infusions, for the purpose of alleviating gastrointestinal disorders, whooping cough, bronchitis, influenza, and
oral infections. The ethnobotanical uses mentioned are in accordance with scientifically supported biological
activities, including antibacterial, antifungal, antioxidant, anti-inflammatory, analgesic, and spasmolytic e­ ffects12.
The distinctive characteristics of T. pallidus highlight its therapeutic potential and align with the well-established
reputation of the Lamiaceae family as a source of medicinal compounds.
Antimicrobial resistance (AMR) is a noteworthy issue and poses a significant threat to public health. While
several mechanisms including genetic mutations and the acquisition of resistance genes through horizontal gene
transfer contribute to the development of AMR, the misuse and overuse of antimicrobial drugs, both in human
healthcare and in agriculture, contribute to the development and spread of the ­resistance13. The effect of AMR has
hampered the treatment of infectious diseases, hence leading to increased morbidity, mortality, and healthcare
costs. Notably, AMR continues to rise globally, hence, the search for a newer class of antimicrobial drugs remains
­unabated14. The phytochemical constituents of aromatic and medicinal plants have been to possess antimicrobial
properties, with their EOs also possessing the ability to regulate pathogenic ­bacteria15,16. Within this particular
context, it is imperative to undertake a comprehensive examination of the intricate tapestry presented by the
various members of the Lamiaceae family, namely O. grossii and T. pallidus. This exploration aims to unravel
the unique bioactive characteristics exhibited by these species and their significance within the realms of both
traditional and contemporary medicinal practices.
This study has a dual objective: to showcase the value of O. grossii and T. pallidus plants and to explore their
potential in combating radicals and bacteria. Specifically, the research aims to evaluate the antimicrobial prop-
erties of essential oils (EOs) extracted from these plants. The primary focus involves testing the effectiveness of
these EOs against four bacterial strains—Salmonella sp, Streptococcus sp, S. aureus, and E. coli. These strains were
isolated from patients’ vomit or fecal samples collected at the emergency department of the University Hospital
of Fez in Morocco. Through this assessment, the researchers intend to determine the presence of significant
antimicrobial and antioxidant activity within these essential oils. The study’s findings could shed light on the
potential of these EOs as potent agents with antimicrobial and antioxidative properties, offering insights into
natural solutions for these challenges.

Materials and methods

Plant material
Fresh leaves of T. pallidus and O. grossii were systematically collected in June 2017 from the region of Ribat EL
Kheir located approximately 75 km from the city of Fez, Morocco (33° 49′ north, 4° 25′ west). The identification
of the plant specimens was performed by Professor Amina Bari, a botanist affiliated with the Department of
Biological Sciences in the Faculty of Science at Sidi Mohammed Ben Abdellah University, Fez, Morocco. Nota-
bly, O. grossii is given voucher number A51/08/06/2018/SE, whilst T. pallidus is given A52/08/06/2018/SE. The
collected leaves were air-dried under suitable shade conditions to preserve their phytochemical composition.
Subsequently, the dried leaf samples were stored in a dry and cool environment at a temperature of 5 °C until
they were ready for further analysis or experimentation.

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Isolation of essential oils

Under the established protocols outlined in the European P ­ harmacopoeia17, the EOs were extracted from the
air-dried plant materials using a Clevenger-type apparatus for 3 h. The EOs were collected in a sealed vial and
subsequently stored at a temperature of 4 °C to ensure their preservation and maintain their quality. The yield
of essential oil was determined using the formula:
 
weight of obtained EO
Yield(%) = × 100, (1)
weight of the material plant
where the weight of the obtained essential oil refers to the mass of essential oil extracted from the plant material
and the weight of the plant material is the initial mass of the plant material used for extraction.

Phytochemical analysis
The analysis of the extracted essential oils was carried out using gas chromatography-mass spectrometry
(GC–MS) techniques. The analysis employed fused silica capillary columns, specifically SPB-1 and Supelco-
Wax-10, as described by ­reference18. These column types are commonly used for the separation and identification
of volatile compounds present in E­ O17.

Total flavonoids content (TFC) and the total phenolic content (TPC)
The aluminium chloride colorimetric assay was employed to assess the total flavonoid concentration of EOs as
described by r­ eference19. The solution was prepared, and the absorbance at 510 nm was measured using a Jasco
v-530 spectrophotometer, with a blank sample for comparison. Galic acid was used as the standard to create a
calibration curve. The overall quantity of flavonoid content in each sample was expressed in terms of gallic acid
equivalents per gram of mg of EO (mg GAE/mg EO).
For the determination of the phenolic content in the samples, the Folin-Ciocalteu method was employed, as
outlined ­by20. The reaction took place at room temperature in the dark for 2 h, and the absorbance at 760 nm
was measured using a Jasco v-530 spectrophotometer. Gallic acid was used as the standard for comparison. The
total phenol content was expressed in milligrams of gallic acid equivalents per gram of EO (mg GAE/mg EO).
Each specimen was evaluated in triplicate to ensure the accuracy and reproducibility of the results.

Antioxidant activity
DPPH radical scavenging activity
The DPPH radical-scavenging ability of the essential oils was assessed using the approach described b­ y21. Note-
worthy, this technique was originally developed by Blois in 1­ 95822. After a 30-min incubation period at room
temperature in the absence of light, the absorbance of the mixture was measured at 517 nm using a Jasco V-530
spectrophotometer. The percentage inhibition was calculated using the following formula:
 
As
I(%) = 1 − × 100, (2)
A0
where I (%): inhibition percentage, as sample absorbance, and A0: represents the absorbance of the blank control.
While BHT was utilized as a positive control in the experiment. The ­IC50 values were determined based on
the concentration of the essential oils that inhibited the DPPH radical by 50%23.

Reducing power capacity

The assessment of reduction power for EO was conducted in accordance with the methodology outlined b ­ y24.
Quercetin is utilized as the standard. The ­IC50 values, denoting the concentration at which the absorbance reached
0.5, were determined to quantitatively evaluate the reduction power. To achieve this, a graph was constructed
by plotting the absorbance against the corresponding concentration, allowing for the determination of the ­IC50
value in milligrams per milliliter (mg/mL).

Total antioxidant capacity (TAC test)

The determination of the total antioxidant capacity of the EOs was performed as described b ­ y25. The assay was
based on the conversion of Mo (VI) to Mo (V) and the subsequent formation of a green phosphate/Mo (V)
complex under acidic conditions. The absorbance of the resulting complex was measured at 695 nm using a
Jasco v-530 spectrophotometer. The antioxidant activity was expressed in terms of ascorbic acid equivalents (mg
AAE/g DW), providing a quantitative measure of the overall antioxidant capacity.

Antibacterial activity
Bacterial strains
In this study, the antibacterial activity of oregano and thyme EO against four bacterial strains, including Gram-
positive Staphylococcus aureus and Streptococcus faecalis D, as well as Gram-negative Escherichia coli and
Salmonella, provided by the Regional Laboratory of Epidemiological Diagnosis and Environmental Hygiene in
Fez, Morocco. The density was changed to correspond to a 0.5 McFarland Standard’s turbidity, equivalent to 1–5
108 CFU per milliliter. Agar disc diffusion a­ nalysis26.

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Agar well diffusion (AWD) assay

The AWD test was run in triplicate using a modified version of the Kirby-Bauer ­method27 Standardized suspen-
sions (108 CFU/mL) were used to inoculate Mueller Hinton agar plates before Whatman paper discs (3 mm)
were applied to the agar’s surface. After that, essential oils were infused into the discs. At 37 °C, all plates were
incubated for a day. The widths of the inhibitory zones were measured after incubation using a ruler. By assessing
the zones of inhibition against the examined bacterial strains, the antibacterial efficacy was evaluated.

Minimum inhibitory concentration (MIC)

With some modifications, the National Committee for Clinical Laboratory Standards’ e­ xperiment28 was used to
determine the MIC and 96-well plates were used for the test. The different concentrations of oregano EOs and
antibiotics were prepared in a suspension containing 0.2% agar in sterile distilled ­water29, they were carried out
using successive 1/2 dilutions of the EOs, ranging from 5000 to 9 µg/mL, and the antibiotics, ranging from 200
to 0.4 µg/mL. The corresponding well of the plate was filled with varying amounts of antibiotics or oregano EO.
The concentrations obtained in the wells ranged from 1250 to 2 μg/mL. The presence of bacteria was determined
by adding 20 μl of a 10% aqueous solution of 2.3.5-triphenyl tetrazolium chloride to each well. The lowest con-
­ IC26,30.
centration that doesn’t result in a red hue was designated as M

Molecular docking
The molecular docking methodology utilized a virtual screening process to analyze the crystal structures of dif-
ferent antibacterial and antioxidant proteins from the RCSB protein data bank, specifically PDB IDs ­1AJ631 and
­6QME32. The decision to select the crystal structures of these proteins for molecular docking methodology was
likely driven by their alignment with the study’s focus on assessing the antimicrobial and antioxidant activities of
essential oils. These protein targets have been chosen based on their relevance to the study’s objectives, potential
roles in a­ ntibacterial33–35 and ­antioxidant36–38 processes as indicated by prior research or literature, structural
availability in the RCSB Protein Data Bank, potential for validation and benchmarking, and the feasibility of
computational ­analysis39–44. Such protein selection allows for investigating interactions with known proteins that
are pertinent to the study’s goals, contributing to a comprehensive understanding of the essential oils’ poten-
tial effects. To perform this analysis, various software tools were employed, including MGLtools, Autodock4,
­Autogrid445, BIOVIA Discovery Studio V ­ isualizer46, Chemdraw U ­ ltra47, and Chemdraw 3D ­Pro48.
Initially, the protein structures were processed using BIOVIA’s Discovery Studio Visualizer, wherein heter-
oatoms, co-crystal ligands, and solvent molecules were eliminated. To optimize the protein structure for docking,
Autodock tools were utilized, assigning appropriate polar hydrogen and Kollman charges. The resulting optimized
protein structure was saved as a pdbqt ­file49. For the ligands, ChemDraw Ultra was used to draw their structures,
followed by energy minimization in Chem 3D Pro. The ligands were then converted to the pdbqt file format using
the OpenBabel GUI ­program50. The structure-based virtual screening was conducted using Autodock4, with
each ligand drug docked independently into the active site of each protein. The interactions between the ligands
and proteins were visualized using BIOVIA Discovery Studio Visualizer. To validate the results, the root-mean-
square deviation (RMSD) value was calculated, and the co-crystal ligand was re-docked. Poses were accepted if
both the docking and experimental ligand RMSD values were less than 2.051,52.

Statistical analysis
To analyze the obtained data, the means of the three-way analyses were calculated, and the results were presented
as mean ± standard deviation (SD). The IBM SPSS Statistics software version 20.0 was utilized to perform the
statistical analysis. The Fisher’s least significant difference (LSD) test and one-way analysis of variance (ANOVA)
were employed to assess the statistical significance between different groups at a significance level of P ≤ 0.05.

Plant collection approval

No approval is needed to collect Origanum grossii and Thymus pallidus in Morocco for research purposes.

IUCN policy statement

The collection of plant material complies with relevant institutional, national, and international guidelines and
legislation.

Results
Chemical composition of EOs
The extraction of essential oils (EOs) from the leaves of O. grossii and T. pallidus resulted in yields of 3% and 1%
(v/w), respectively, on a dry weight basis. The extracted EOs were then subjected to gas chromatography-mass
spectrometry (GC–MS) analysis, and the results are presented in Tables 1 and 2.
The GC–MS analysis provided in Table 1 showing detailed insights into the chemical constituents of the Eos
from T. pallidus. Key compounds, including p-cymene, γ-terpinene, Sabinene hydrate, linalool oxide, Santolina
triene, Borneol, Terpinen-4-ol, Thymol, Caryophyllene, Himachalene, Caryophyllene oxide, and Trans-cadinol,
were identified and quantified based on their retention indices, retention times, and respective areas in the chro-
matogram. Thymol, in particular, dominated the composition of the T. pallidus EO, constituting a significant
proportion (86.354%) of the total.
Table 2 outlines the comprehensive profile of phytochemical constituents within the O. grossii EOs. Through
GC–MS analysis, specific compounds such as p-cymene, Eucalyptol, γ-terpinene, Cis-sabinene, α-campholenol,
Isoborneol, Borneol, Isopulegone, Myrtenyl acetate, Carvacrol, Caryophyllene, Valencene, and γ-Mamliene are

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RI
RT Chemical names Obs Lit Chemical formula Chemical class Area (%)
15.58 p-cymene 1022 1024 C10H14 Monoterpene hydrocarbons (MT.H) 0.563
17.96 Santolina triene 906 908 C10H16 MT.H 2.036
16.64 ɤ-terpinene 1055 1059 C10H16 MT.H 0.632
17 Sabinene hydrate 1067 1070 C10H18O Monoterpene oxygenated (MT.O) 0.231
17.64 Linalool oxide 1070 1072 C10H18O MT.O 0.146
20.23 Borneol 1168 1169 C10H18O MT.O 0.986
20.90 Terpinen-4-ol 1174 1177 C10H18O MT.O 0.741
24.03 Thymol 1288 1290 C10H14O MT.O 86.354
27.31 Caryophyllene 1418 1419 C15H24 Sesquiterpene hydrocarbons (ST.H) 0.324
29.23 Himachalene 1450 1451 C15H24 ST.H 1.631
31.56 Caryophyllene oxide 1563 1667 C15H24O Sesquiterpene oxygenated (ST.O) 0.463
32.22 Trans-cadinol 1639 1640 C15H26O ST.O 0.128
Monoterpene hydrocarbons (MT.H) 3.231
Monoterpene oxygenated (MT.O) 88.458
Sesquiterpene hydrocarbons (ST.H) 1.955
Sesquiterpene oxygenated (ST.O) 0.591
Total (%) 94.235

Table 1.  The phytochemical constituents of the EOs from T. pallidus. RI retention indices, RT retention time
in minutes, Obs retention indices calculate, Lit literature.

RI
RT Chemical names Obs Lit Chemical formula Chemical class Area (%)
15.56 p-cymene 1020 1024 C10H14 Monoterpene hydrocarbons (MT.H) 0.364
16.62 ɤ-terpinene 1054 1059 C10H16 MT.H 0.423
18.59 Cis-sabinene 1070 1070 C10H16 MT.H 1.063
15.78 Eucalyptol 1029 1031 C10H18O Monoterpene oxygenated (MT.O) 0.125
19.56 α-campholenal 1124 1126 C10H16O MT.O 1.157
20.22 Isoborneol 1160 1160 C10H18O MT.O 0.983
20.47 Borneol 1168 1169 C10H18O MT.O 0.897
21.34 isopulegone 1592 1596 C10H16O MT.O 0.143
23.05 Myrtenyl acetate 1321 1326 C12H18O2 Other (O) 0.684
24.56 Carvacrol 1296 1299 C10H14O MT.O 70.963
27.33 Caryophyllene 1418 1419 C15H24 Sesquiterpene hydrocarbons (ST.H) 2.376
29.23 ɤ- Maaliene 1472 1477 C15H24 ST.H 0.637
27.82 Valencene 1494 1494 C15H24 ST.H 0.218
31.55 Caryophyllene oxide 164 1667 C15H24O Sesquiterpene oxygenated (ST.O) 0.921
Monoterpene hydrocarbons (MT.H) 1.850
Monoterpene oxygenated (MT.O) 74.268
Sesquiterpene hydrocarbons (ST.H) 3.231
Sesquiterpene oxygenated (ST.O) 0.921
Other (O) 0.684
Total (%) 80.954

Table 2.  The phytochemical constituents of the EOs from O. grossii. RI retention indices, RT retention time in
minutes, Obs retention indices calculate, Lit. literature.

identified and quantified. Notably, Carvacrol stands out as the predominant component, constituting a substantial
proportion (70.963%) of the total composition. These details enrich our understanding of the chemical composi-
tion and potential bioactivity of the O. grossii EO.
The exploration of the phytochemical constituents within the essential oils (EOs) of T. pallidus, as conducted
in this study, unveiled a total of 12 compounds, collectively encompassing 94.235% of the total composition. A
noteworthy revelation is the prevalence of thymol, which emerged as the principal compound, constituting a
substantial majority at 86% (Table 1).

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In contrast, the investigation into the phytochemical makeup of O. grossii EO disclosed the presence of 14
distinct compounds that set it apart from T. pallidus. Notably, Carvacrol stood out as the predominant constitu-
ent, accounting for a significant proportion of 70% within the EOs (Table 2).

Antioxidant activity
DPPH scavenging activity
The antioxidant activity of the EOs was evaluated using the DPPH scavenging activity assay which is a frequently
utilized method for antioxidant activity evaluation. The results presented in Table 3 elucidate the DPPH radical-
scavenging potential of O. grossii and T. pallidus EOs. Notably, the EOs of O. grossii exhibited the highest radical-
scavenging ability, as evident by its I­ C50 value of 0.073 ± 0.001 mg/mL, followed by T. pallidus EO with an ­IC50
value of 0.131 ± 0.002 mg/mL. Interestingly, statistical analysis revealed that O. grossii EO displayed significantly
stronger antioxidant activity (P ≤ 0.05) in comparison to the pure reference antioxidant BHT, with an ­IC50 value
of 0.120 ± 0.001 mg/mL.

Ferric reducing power (FRAP)

As depicted in Table 3, the EOs of O. grossii exhibited superior reducing power against ferric ions compared to
the EOs of T. pallidus, yielding values of 56.333 ± 1.778mg/mL and 79.333 ± 1.556mg/mL.

Total antioxidant activity (TAC)

The results, presented in Fig. 1, were quantified in terms of BHT equivalents (mg BHT E/mg EO). Significantly
higher TAC values were observed for the EOs pf O. grossii compared to T. pallidus, with recorded values of
0.185 ± 0.005 mg BHT E/mg EO and 0.13 ± 0.004 mg BHT E/mg EO, respectively.

Quantification of total flavonoid content (TFC) and total phenolic content (TPC)
The determination of TFC and TPC for each test sample is elaborated in Table 4. The TFC values were standard-
ized with respect to the reference quercetin, as detailed in the same table. In a similar manner, the TPC outcomes
were aligned with the benchmark gallic acid and were expressed as milligrams of gallic acid equivalents per
gram of the sample (mg GAE/g of the sample). Noteworthy is the observation that among the samples, O. grossii

O. grossii T. pallidus BHT Quercetin

IC50 (mg/mL) 0.073 ± 0.001*** 0.131 ± 0.002 0.120 ± 0.001 –
EC50 (mg/mL) 56.333 ± 1.778*** 79.333 ± 1.556*** – 33.450 ± 0.027

Table 3.  DPPH radical scavenging activity and Ferric reducing/antioxidant power (FRAP) capacity. IC50:
half maximal inhibitory concentration, ­EC50: half maximal effective concentration, data are reported as mean
values ± SD of three measurements. Means were significantly different when ***P ≤ 0.001.

Figure 1.  The total antioxidant capacity of O. grossii and T. pallidus Eos.

O. grossii T. pallidus
Flavonoids in mg QE/mg EO 0.207 ± 0.007a 0.109 ± 0.006b
a
Polyphenols in mg GAE/mg EO 0.136 ± 0.003 0.125 ± 0.002b

Table 4.  Total phenolic (TPC) and total flavonoids (TF) contents. Data are reported as mean values ± SD of
three measurements. Means were significantly different when P < 0.05; values followed by different letters are
significantly different.

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demonstrated the most substantial TFC and TPC values, exhibiting measurements of 0.207 ± 0.007 mg QE/g EO
and 136.66 ± 0.003 mg GAE/g EO, respectively.

Pearson’s correlation analysis

In order to explore the interdependence between the total phenolic and flavonoid contents and the observed
antioxidant activity, Pearson’s correlation coefficient analysis was employed in this study, as delineated in Table 5.
The outcome of this analysis unveiled noteworthy and statistically significant correlations among these variables.

Antibacterial activity
The outcomes of the agar disc diffusion antibacterial activity test are detailed in Table 6 and illustrated in Fig. 2.
The results indicate that both essential oils (EOs) demonstrated significant antibacterial effects against all bac-
terial strains tested. Notably, the EOs exhibited heightened efficacy against a spectrum encompassing both
Gram-positive and Gram-negative bacteria. Specifically, noteworthy antibacterial activity was observed against
Salmonella sp., with O. grossii and T. pallidus EOs yielding the most substantial inhibition zone diameters of

Pearson correlation
IC50 EC50 TAC​ Flavonoids Polyphenols
IC50 1
EC50 0.183 1
TAC​ − 0.981** − 0.969** 1
Flavonoids − 0.981** − 0.997** 0.977** 1
Polyphenols − 0.852* − 0.780 0.871* 0.814* 1

Table 5.  Pearson correlation between antioxidant activity parameters. Correlations are: *significant at p < 0.01,
**significant at p < 0.001.

Essential oils Antibiotics

O grossii T pallidus OX5 S25 CRO30 ZOX30 OFX5 CN15 P10 CEC30
Bacteria IZ CMI IZ CMI IZ IZ IZ IZ IZ IZ IZ IZ
Staph. aureus 30.67 ± 0.89 0.61 26.67 ± 0.45 2.44 0 12 20 11 14 20 0 0
Strep. fecalus 32.33 ± 0.45 0.61 30.00 ± 0.67 0.61 0 14 0 0 27 14 0 0
Salmonella sp 42.67 ± 0.88 0.31 35.33 ± 0.45 0.31 0 16 18 12 20 20 0 0
E. coli 33.00 ± 2.00 0.61 28.33 ± 1.11 0.61 0 14 22 13 14 21 0 0

Table 6.  Antibacterial activity of Origanum grossii, Thymus pallidus, and some antibiotics. IZ inhibition
zone(mm), CMI minimum inhibitory concentration(ug/mL), OX5 Oxacillin, S25 streptomycin, CRO30
ceftriaxone, ZOX30 ceftizoxime, OFX5 ofloxacin, CN15 gentamicin, P10 penicillin G, CEC30 cefaclor.

Figure 2.  Antibacterial activity of O. grossii and T. pallidus EOs.

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42.6 ± 0.88 mm and 36 ± 0.5 mm, respectively. It is worth noting that the efficacy of O. grossii EO and T. pallidus
EO against E. coli was comparatively lower.
When contrasting the antibacterial activity of the essential oils (EOs) with that of conventional antibiotics
employed as positive controls, it becomes evident that the EOs displayed heightened potency. Through the
microdilution technique, the efficacious concentrations of O. grossii and T. pallidus EOs were ascertained to fall
within the range of 0.31 to 2.44 µg/mL, as outlined in Table 6. Particularly noteworthy is the concentration of
0.31 µg/mL, which emerged as the most efficacious against Salmonella sp. for both EOs.

Molecular docking
The two macromolecules i.e. antibacterial protein PDB ID: 1AJ6 and antioxidant proteins 6QME were docked
with the various types of phytochemical constituents of essential oils obtained from the plants T. pallidus and O.
grossii and the results of docking were tabulated in the form of tables.
The phytochemical constituents p-cymene, santolina triene, ɤ-terpinene, sabinene hydrate, linalool oxide,
borneol, terpinen-4-ol, thymol, caryophyllene, himachalene, caryophyllene oxide, trans-cadinol was extracted
from T. pallidus plants virtually docked with antibacterial 1AJ6 proteins and showed binding affinity of − 5.7,
− 4.9, − 5.7, − 5.5, − 5.0, − 4.8, − 5.5, − 4.8, − 5.5, − 5.7, − 5.9, − 6.7, − 6.0, − 5.5 in (Kcal/mol) respectively tabu-
lated in Table 7. The p-cymene showed hydrophobic interaction with amino acids VAL43, VAL120, VAL167,
and ILE78 with distances 4.169, 3.982, 4.336, 5.287 (Å), santolina triene was bounded hydrophobically with
distances 4.092, 5.232, 5.279 (Å) to amino acid residues ALA47, ILE78, VAL43, ɤ-terpinene showed two types
of interaction with 1AJ6, one was electrostatic interaction to amino acid residues ASP49 with bond angle 4.092
(Å) and the other was hydrophobic interaction to amino acid ILE78 with bond distance 5.272 (Å). Sabinene

Interaction of EOs from T. pallidus with 1AJ6 Residues Types of interaction Bond distance (Å) Binding affinity (Kcal/mol)
VAL43 Hydrophobic 4.169
VAL120 Hydrophobic 3.982
p-cymene − 5.7
VAL167 Hydrophobic 4.336
ILE78 Hydrophobic 5.287
ALA47 Hydrophobic 4.092
Santolina triene ILE78 Hydrophobic 5.232 − 4.9
VAL43 Hydrophobic 5.279
ASP49 Electrostatic 4.974
ɤ-terpinene − 5.7
ILE78 Hydrophobic 5.272
ALA47 Hydrophobic 4.092
Sabinene hydrate ILE78 Hydrophobic 5.231 − 5.5
VAL43 Hydrophobic 5.279
GLU50 Hydrogen bond 2.668
Linalool oxide ILE78 Hydrophobic 3.745 − 5.0
ILE94 Hydrophobic 3.634
GLU50 Hydrogen bond 2.048
Borneol ARG76 Hydrogen bond 2.756 − 4.8
ILE78 Hydrophobic 5.026
ILE78 Hydrophobic 5.156
Terpinen-4-ol VAL120 Hydrophobic 3.772 − 5.5
VAL167 Hydrophobic 4.467
ASP73 Hydrogen bond 2.003
ALA47 Hydrogen bond 3.473
Thymol − 5.7
VAL167 Hydrophobic 4.702
ILE78 Hydrophobic 5.038
ARG190 Hydrophobic 5.293
Caryophyllene − 5.9
PHE41 Hydrophobic 4.599
ILE78 Hydrophobic 4.977
Himachalene − 6.7
ILE78 Hydrophobic 4.849
Caryophyllene oxide ILE78 Hydrophobic 5.150 − 6.0
ARG190 Hydrophobic 4.997
LYS189 Hydrophobic 3.991
Trans-cadinol − 5.5
ARG190 Hydrophobic 4.076
PHE41 Hydrophobic 5.137

Table 7.  Active site interactions (type of interactions, bond distance in Å) of phytochemical constituents of T.
pallidus plant with antibacterial protein 1AJ6 along with docking score (Kcal/mol).

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hydrate hydrophobically interacted with amino acid residues ALA47, ILE78, and VAL43 with bond distances
4.092, 5.231, 5.279 (Å), linalool oxide showed two types of interactions, one with GLU50 was bounded through
a hydrogen bond with bond distance 2.668 (Å) and ILE78, ILE94 interacted hydrophobically with bond dis-
tance 3.745, 3.634. In the Borneol-1AJ6 docking results, amino acid ILE78 was bounded through hydrophobic
interaction with bond distance 5.026 (Å) and amino acids GLU50, and ARG76 were bounded through a hydro-
gen bond with bond distance 2.048, 2.7565 (Å) mentioned in Table 7. The Terpinen-4-ol showed hydrophobic
interaction with distances 5.156, 3.772, 4.467(Å) to amino acid residues ILE78, VAL120, and VAL167 showed
in. Thymol interacted hydrophobically with amino acids VAL167, ILE78 with bond angles 4.702, 5.038 (Å) and
interacted through hydrogen bond to amino acids residues ASP73, ALA47 with bond distances 2.003, 3.473 (Å)
tabulated in Table 7. Caryophyllene interacted hydrophobically with residues ARG190, and PHE41 of 1AJ6 with
bond distances 5.293, 4.599 (Å); Trans-cadinol interacted hydrophobically with amino acids residues ARG190,
LYS189, and ARG190, PHE41 with bond distance 4.997, 3.991, 4.076, 5.137 (Å) tabulated in Table 7. The high-
est top-ranked docked essential oil himachalene concerning its binding affinity -6.7 (Kcal/mol) has interacted
through hydrophobic interaction with amino acids ILE78 with bond distances 4.977, 4.849 (Å) represented in
Fig. 3 and Table 7. The second highest essential oil caryophyllene oxide with binding energy − 6.0 (Kcal/mole)
bounded hydrophobically to ILE78 with a bond distance of 5.150 (Å) represented in Fig. 4 and Table 7. The two
compounds himachalene and caryophyllene oxide have the highest binding score with antibacterial protein 1AJ6
thus they were considered good antibacterial targets.
In this study Table 8 represents interaction of antibacterial protein 1AJ6 phytochemical constituents
p-cymene, ɤ-terpinene, Cis-sabinene, Eucalyptol, α-campholenal, isoborneol, borneol, isopulegone, myrtenyl
acetate, carvacrol, caryophyllene, ɤ-maaliene, valencene, caryophyllene oxide of essential oils obtained from
O. grossii and co-crystallized ligand novobiocin with their binding score − 5.7, − 5.7, − 4.9, − 4.9, − 4.7, − 4.7,
− 4.8, − 5.3, − 5.6, − 6.1, − 5.9, − 6.4, − 7.3, − 6.0, − 7.6 (Kcal/mol) respectively showed in Table 8. Some of the
constituents of O. grossii such as p-cymeme, ɤ-terpinene, borneol, caryophyllene, caryophyllene oxide were
the same as with the constituents of T. pallidus already described above and tabulated in Tables 7 and 8. The
remaining constituents like cis-sabinene were hydrophobically bound to amino acid residues of 1AJ6 ILE78,
ILE78, and ILE94 with distances 4.349, 3.872, 3.776 (Å); the eucalyptol was bounded hydrophobically to ILE94,
ALA100 with bond distance 5.397, 4.991 (Å); α-campholenal, isoborneol and caryophyllene oxide were inter-
acted hydrophobically to ILE78 with bond distance 4.529, 4.878, 5.150 (Å) respectively tabulated in Table 8. The
isopulegone has interacted hydrophobically with amino acids VAL120, and VAL167 with distances 4.112, and

Figure 3.  3D, hydrogen surface and 2D interaction of himachalene with 1AJ6.

Figure 4.  3D, hydrogen surface and 2D interaction of caryophyllene oxide with 1AJ6.

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Interaction of EOs from O. grossii with 1AJ6 Residues Types of interaction Bond distance (Å) Binding affinity (Kcal/mole)
VAL43 Hydrophobic 4.169
VAL120 Hydrophobic 3.982
p-cymene − 5.7
VAL167 Hydrophobic 4.337
ILE78 Hydrophobic 5.287
ASP49 Electrostatic 4.974
ɤ -terpinene − 5.7
ILE78 Hydrophobic 5.272
ILE78 Hydrophobic 4.349
Cis-sabinene ILE78 Hydrophobic 3.872 − 4.9
ILE94 Hydrophobic 3.776
ILE94 Hydrophobic 5.397
Eucalyptol − 4.9
ALA100 Hydrophobic 4.991
α-campholenal ILE78 Hydrophobic 4.529 − 4.7
Isoborneol ILE78 Hydrophobic 4.847 − 4.6
GLU50 Hydrogen bond 2.048
Borneol ARG76 Hydrogen bond 2.756 − 4.8
ILE78 Hydrophobic 5.026
VAL120 Hydrophobic 4.112
isopulegone − 5.3
VAL167 Hydrophobic 4.401
THR165 Hydrogen bond 2.251
Myrtenyl acetate − 5.6
ILE78 Hydrophobic 5.302
ASP73 Hydrogen bond 2.126
Carvacrol ALA47 Hydrogen bond 3.562 − 6.1
ILE78 Hydrophobic 5.203
ARG190 Hydrophobic 5.293
Caryophyllene − 5.9
PHE41 Hydrophobic 4.599
ILE78 Hydrophobic 4.594
ɤ -Maaliene − 6.4
ILE78 Hydrophobic 4.522
ILE78 Hydrophobic 4.648
ILE78 Hydrophobic 3.989
Valencene − 7.3
VAL43 Hydrophobic 4.628
VAL167 Hydrophobic 4.579
Caryophyllene oxide ILE78 Hydrophobic 5.150 − 6.0
THR165 Hydrogen bond 2.167
LYS103 Hydrogen bond 3.419
ASN46 Hydrogen bond 4.016
ILE78 Hydrophobic 3.937
co-crystallized ligand novobiocin − 7.6
GLY77 Hydrophobic 3.879
ILE78 Hydrophobic 4.324
ILE94 Hydrophobic 5.034
PRO79 Hydrophobic 4.994

Table 8.  Active site interactions (type of interactions, bond distance in Å) of phytochemical constituents of O.
grossii plant with antibacterial protein 1AJ6 along with docking score (Kcal/mol).

4.401 (Å); myrtenyl acetate showed two types of interaction one was hydrophobic to ILE78 with a distance of
2.251 (Å), and one was hydrogen bond interaction to THR165 with distance 5.302 (Å); Carvacrol has interacted
through a hydrogen bond to ASP73, ALA47 and hydrophobic bond to ILE78 with bond distance 2.126, 3.562,
5.203 respectively in Table 8.
The highest top ranked constitute valencene with a docking score of − 7.3 (Kcal/mol) has interacted through
hydrophobic interaction with amino acids residues ILE78, ILE78, VAL43, and VAL167 with bond distances 4.648,
3.989, 4.628, 4.579 (Å) respectively represented in Fig. 5 and Table 8. The second highest top ranked ɤ- maaliene
with a docking score of -6.4 (Kcal/mol) hydrophobically interacted with ILE78 with bond distances 4.594, 4.522
(Å) represented in Fig. 6 and Table 8.
The co-crystallized ligand novobiocin has greater binding affinity 0f. − 7.6 (Kcal/mol) with respect to all the
phytochemical constituents of T. pallidus and O. grossii plants have interacted with two types of interactions; one
was hydrogen bond interaction to residues THR165, LYS103, ASN46 with bond distance 2.167, 3.419, 4.016 (Å)
and the second was hydrophobic interaction to ILE78, GLY77, ILE78, ILE94, PRO79 with bond distance 3.937,
3.879, 4.324, 5.034, 4.994 (Å) respectively represented in Fig. 7 and Table 8.

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Figure 5.  3D, hydrogen surface and 2D interaction of ɤ -Maaliene with 1AJ6.

Figure 6.  3D, hydrogen surface and 2D interaction of Valencene with 1AJ6.

Figure 7.  3D, hydrogen surface and 2D interaction of co-crystallized ligand novobiocin with 1AJ6.

Table 9 represented the interactions of phytochemical constituents p-cymene, santolina triene, ɤ-terpinene,
sabinene hydrate, linalool oxide, borneol, terpinen-4-ol, thymol, caryophyllene, himachalene, caryophyllene
oxide, trans-cadinol were extracted from T. pallidus plants virtually docked with antioxidant 6QME proteins
and showed binding score of − 5.2, − 4.7, − 5.2, − 5.8, − 6.2, − 6.1, − 5.7, − 5.9, − 7.5, − 7.1, − 7.9, − 7.0 (Kcal/
mol). As we have discussed earlier some of the constituents are same for the both plants such as p-cymeme,
ɤ-terpinene, borneol, caryophyllene, caryophyllene oxide, the type of interaction with residues and binding
affinity were the same after docked with the protein 6QME tabulated in Tables 9 and 10. The p-cymene hydro-
phobically interacted with ALA366 with a distance of 3.586 (Å); ɤ-terpinene interacted hydrophobically with
TYR572, TYR334, TYR334, ALA556 with a distance of 3.625, 4.655, 4.338, 5.320 (Å); Borneol was bounded
hydrophobically to ALA366 with bond length 4.791 (Å) while bounded through hydrogen bond interaction to
ILE559 with distance 2.177 (Å) in Tables 8 and 9. The highest top-ranked compound Caryophyllene oxide with
a binding affinity of − 7.9 and the 2nd highest top-ranked compound Caryophyllene with a binding affinity of

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Interaction of EOs from T. pallidus with 6QME Residues Types of interaction Bond distance (Å) Binding affinity (Kcal/mole)
p-cymene ALA366 Hydrophobic 3.586 − 5.2
ALA366 Hydrophobic 3.915
Santolina triene − 4.7
VAL465 Hydrophobic 4.314
TYR572 Hydrophobic 3.625
TYR334 Hydrophobic 4.655
ɤ-terpinene − 5.2
TYR334 Hydrophobic 4.338
ALA556 Hydrophobic 5.320
ALA366 Hydrophobic 3.915
Sabinene hydrate − 5.8
VAL465 Hydrophobic 4.314
Linalool oxide ILE416 Hydrogen bond 1.959 − 6.2
ILE559 Hydrogen bond 2.177
Borneol − 6.1
ALA366 Hydrophobic 4.791
ALA366 Hydrophobic 5.141
Terpinen-4-ol − 5.7
ILE559 Hydrophobic 5.35
LEU365 Hydrogen bond 2.603
ILE416 Hydrogen bond 2.495
ALA556 Hydrophobic 3.774
Thymol − 5.9
ARG415 Hydrophobic 3.968
ARG415 Hydrophobic 5.144
ALA556 Hydrophobic 5.218
Caryophyllene ALA366 Hydrophobic 4.179 − 7.5
Himachalene ALA366 Hydrophobic 4.797 − 7.1
Caryophyllene oxide ALA366 Hydrophobic 4.941 − 7.9
ILE559 Hydrogen bond 2.013
CYS368 Hydrophobic 5.128
ALA466 Hydrophobic 4.839
VAL467 Hydrophobic 5.439
Trans-cadinol − 7.0
ALA607 Hydrophobic 4.947
CYS368 Hydrophobic 4.679
VAL369 Hydrophobic 4.952
VAL420 Hydrophobic 4.333

Table 9.  Active site interacting residues, distance (Å) and binding affinity (Kcal/mol) of phytochemical
constituents of T. pallidus with 6QME.

− 7.5 hydrophobically interacted with ALA366 with a distance of 4.179, 4.941 (Å) tabulated in Tables 9 and 10
and interactions represented in Figs. 8 and 9
The 3rd highest compound from T. pallidus was Himachalene with binding affinity − 7.1 (Kcal/mole) and
showed hydrophobic interaction to ALA366 with bond distance 4.797 (Å) represented in Fig. 10 and Table 9.
The sabinene hydrate interacted hydrophobically with amino acids ALA366, and VAL465 with distances
3.915, and 4.314 (Å); linalool oxide was hydrophically bounded to residue ILE416 with a distance of 1.959 (Å);
Terpinen-4-ol also interacted through the hydrophobic bond to ALA366, ILE559 with bond distance 5.141, 5.35
(Å); thymol was interacted by a hydrogen bond to LEU365, ILE416 with distance 2.603, 2.495 while thymol
interacted by hydrophobic interaction to residues ALA556, ARG415, ARG415, ALA556 with distance 3.774,
3.968, 5.144, 5.218 (Å); Trans-cadinol was interacted by hydrophobic bond CYS368, ALA466, VAL467, ALA607,
CYS368, VAL369, VAL420 with bond distance 5.128, 4.839, 5.439, 4.947, 4.679, 4.952, 4.333 (Å) shown in Table 9.
Table 10 showed the interaction between the phytochemical constituents p-cymene, ɤ-terpinene, cis-sabi-
nene, eucalyptol, α-campholenal, isoborneol, borneol, isopulegone, myrtenyl acetate, carvacrol, caryophyllene,
ɤ-maaliene, valencene, caryophyllene oxide of essential oils obtained from O. grossii and co-crystallized ligand
J6Q to antioxidant protein 6QME with docking score − 5.2, − 5.2, − 5.1, − 5.8, – 5.5, − 6.1, − 6.1, − 5.7, − 6.6,
5.9, − 7.5, − 7.4, − 7.9, − 7.2, − 8.3 respectively tabulated in Table 10. The cis-sabinene and eucalyptol, myrtenyl
acetate, and valencene were bounded by hydrophobic bonds to ALA366 with bond distances 3.586, 5.327, 4.030,
3.831 (Å) respectively shown in Table 10.
The α-campholenal was bounded through hydrogen bond interaction to GLY464 and bounded hydrophobi-
cally to ALA366 with distances 2.564, and 4.372 (Å) respectively shown in Table 10. The Isoborneol represented
two types of interactions, one was by hydrogen bond to residues ILE416, VAL463, GLY417 with bond distance
2.753, 2.592, 3.561 (Å) and interacted hydrophobically to residues ALA366 with bond distance 5.239 (Å) tabu-
lated in Table 10.
The isopulegone showed the hydrophobic interaction to residues ALA556, and ARG415 with bond distances
3.987, 3.951 (Å); carvacrol was bounded through hydrogen bonds to residues VAL418, and VAL465 with bond

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Interaction of EOs from O. grossii with 6QME Residues Types of interaction Bond distance (Å) Binding affinity (Kcal/mole)
p-cymene ALA366 Hydrophobic 3.586 − 5.2
TYR572 Hydrophobic 3.625
TYR334 Hydrophobic 4.655
ɤ-terpinene − 5.2
TYR334 Hydrophobic 4.338
ALA556 Hydrophobic 5.320
Cis-sabinene ALA366 Hydrophobic 4.748 − 5.1
Eucalyptol ALA366 Hydrophobic 5.327 − 5.8
GLY464 Hydrogen bond 2.564
α-campholenal − 5.5
ALA366 Hydrophobic 4.372
ILE416 Hydrogen bond 2.753
VAL463 Hydrogen bond 2.592
Isoborneol − 6.1
GLY417 Hydrogen bond 3.561
ALA366 Hydrophobic 5.239
ILE559 Hydrogen Bond 2.177
Borneol − 6.1
ALA366 Hydrophobic 4.791
ALA556 Hydrophobic 3.987
isopulegone − 5.7
ARG415 Hydrophobic 3.951
Myrtenyl acetate ALA366 Hydrophobic 4.030 − 6.6
VAL418 Hydrogen bond 3.170
Carvacrol VAL465 Hydrogen bond 2.389 − 5.9
ALA366 Hydrophobic 3.759
Caryophyllene ALA366 Hydrophobic 4.179 − 7.5
ALA366 Hydrophobic 4.556
ɤ- Maaliene − 7.4
ALA366 Hydrophobic 4.727
Caryophyllene oxide ALA366 Hydrophobic 4.9411 − 7.9
Valencene ALA366 Hydrophobic 3.831 − 7.2
SER508 Hydrogen bond 2.560
SER602 Hydrogen bond 2.152
ALA556 Hydrophobic 3.779
co-crystallized ligand J6Q TYR525 Hydrophobic 3.769 − 8.3
TYR525 Hydrophobic 4.388
TYR572 Hydrophobic 5.408
ALA556 Hydrophobic 4.137

Table 10.  Active site interacting residues, distance (Å) and binding affinity (Kcal/mol) of phytochemical
constituents of O. grossii with 6QME.

Figure 8.  3D, hydrogen surface and 2D interaction of Caryophyllene with 6QME.

distances 3.170, 2.389 (Å) and showed hydrophobic interaction to residues ALA366 with bond distance 3.759
(Å) shown in Table 10.
The first two highest top-ranked constituents of O. grossii with 6QME were already discussed above and
represented in Figs. 6 and 8; while the third highest top-ranked compound ɤ-Maaliene with docking score

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Figure 9.  3D, hydrogen surface and 2D interaction of Caryophyllene oxide with 6QME.

Figure 10.  3D, hydrogen surface and 2D interaction of Himachalene with 6QME.

− 7.4 (Kcal/mole) showed hydrophobic interaction to residues ALA366 with bond distance 4.556, 4.727 (Å)
represented in Fig. 11 and Table 10.
The co-crystallized ligand J6Q showed two types of interactions, one was hydrogen bond to residues SER508,
and SER602 with bond lengths 2.560, 2.152 (Å) while the other was hydrophobic interactions to residues ALA556,
TYR525, and TYR525, TYR572, ALA556 with bond distance 3.779, 3.769, 4.388, 5.408, 4.137 with docking score
− 8.3 (Kcal/mole) which was closely related to the highest 3 top ranked constituents of O. grossii and T. pallidus
plants Fig. 12 and Table 10.

Discussion
The yields of both plants were higher compared to previous studies on similar species, which reported yields of
2% and 0.26% for O. grossii and T. pallidus, ­respectively9,12. The determination of the chemical constituents and
flavonoid concentration of EOs holds considerable significance in the taxonomic classification and differentiation

Figure 11.  3D, hydrogen surface and 2D interaction of ɤ-Maaliene with 6QME.

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Figure 12.  3D, hydrogen surface and 2D interaction of co-crystallized ligand J6Q with 6QME.

of various thyme ­species53. Notably, in T. pallidus, thymol emerged as the predominant compound, constituting
a substantial proportion of 86% (Table 1). However, an alternative study reported a distinctive composition,
with borneol representing the major constituent at 41.67%54. Furthermore, another study on the EOs reported
the presence of 15 compounds, with γ-Terpinene emerging as the principal component, comprising 29.6% of
the total c­ omposition55. For O. grossii, it is worth noting that although the primary component remained con-
sistent, Figueredo56 observed structural variations in the chemical profile of O. grossii EO, suggesting inherent
­heterogeneity56. This heterogeneity in EO composition within the same species can be attributed to diverse fac-
tors, including environmental conditions, geographical location, timing of harvest, and variations in distillation
techniques. Notably, climatic factors have been identified as particularly influential c­ ontributors57.
Antioxidant activity was evaluated through DPPH scavenging ability, Ion reducing ability and Total Antioxi-
dant Activity. For DPPH activity, noteworthy, limited literature is available concerning O. grossii EO. Conversely,
the EOs of T. pallidus exhibited a higher antioxidant activity compared to the findings reported by Laila El
Bouzidi ­(IC50 = 345.11 ± 7.46 μg/mL)55, as well as Thymus vulgaris ­(IC50 = 0.259 ± 0.476 μg/mL) as reported ­in58.
As regards the ferric-reducing power capacity assay was employed to investigate the redox-modulating poten-
tial of EOs, as well as their ability to neutralize reactive s­ pecies59. The underlying principle of this assay involves
the reduction of hydroxyl radicals generated by the interaction of F ­ e2 + and ­H2O2, facilitated by the antioxidants
present in the EOs, which subsequently chelate the resulting ­Fe2 + hydroxyl ­radicals60. Interestingly, the values
of our test surpassed those reported for the EOs of other Origanum and Thymus species, including Origanum
Vulgaris, T. satureioides, T. maroccanus, and T. broussonetii EOs, as documented b ­ y55,61. However, it is pertinent to
note that the antioxidant activity of the tested EOs was notably lower than that of the pure reference antioxidant
Quercetin (0.03 g/mL)62–64.
The total antioxidant capacity (TAC) of the investigated EOs, reference antioxidants, and BHT was assessed
using the phosphomolybdenum method, as detailed by Pilar Prieto et al.25. Noteworthy, the methodology involves
the conversion of Mo (VI) to Mo (V) in the presence of an antioxidant compound. The determination of TAC
serves as a crucial parameter to evaluate the capacity of a substance to counteract unwanted oxidation processes.
It holds biological significance by providing insights into the antioxidative strength exhibited by the studied
substance. The antioxidant potential of phenolic compounds is attributed to the presence of hydroxyl groups,
which confer their scavenging ability towards free radicals. Notably, a higher content of hydroxyl groups within
these compounds corresponds to an enhanced capability to neutralize reactive s­ pecies21,65.
The antioxidant property of phenolic compounds is closely linked to their structural features, which often
include functional groups capable of binding and neutralizing free r­ adicals66. Comparing the TPC and TFC
results of T. pallidus in our study to those reported in the literature for Thymus species like T. vulgaris (19.2 ± 0.3
mg GAE/g)66, we observed that T. pallidus exhibited higher values. Moreover, T. daenensis subsp. and T. kotschy-
anus demonstrated TPC values of 295.93 ± 34.07 mg GAE/g and 337.00 ± 8.31 mg GAE/g, ­respectively67. Regard-
ing TFC, T. capitatus displayed a flavonoid concentration of 10.62 ± 0.24 mg QE/g68.
Given that phenolic compounds often contain molecules that effectively bind and counteract free radicals,
the antioxidative characteristic is inherently linked to its phenolic s­ tructure66. Consequently, to evaluate the
antioxidant activity of O. grossii and T. pallidus essential oils, we examined the total flavonoid content (TFC)
and total phenolic content (TPC) of each test sample. The results of this evaluation are presented in Table 4.
TFC results were standardized against quercetin as a reference, and the outcomes were expressed in terms of mg
Q E (milligrams of quercetin equivalents) per gram of essential oil, as shown in Table 4. Similarly, the findings
of the comparison of TPC against the standard gallic acid were reported as mg GAE (milligrams of gallic acid
equivalents) per gram of the sample. Notably, the essential oil from O. grossii exhibited the highest total flavonoid
and phenolic content, measuring 0.207 ± 0.007 mg Q E/g EO and 0.136 ± 0.13 mg GAE/g EO, r­ espectively69–71.
The analysis of Pearson’s Correlation revealed significant correlations between these variables. The total
flavonoid content (TFC) exhibited strong positive correlations with the total antioxidant capacity (TAC) assay
(r = 0.977), indicating that the flavonoids present in the samples contribute significantly to their antioxidative
effects. Conversely, TFC showed negative linear correlations with the DPPH assay and the FRAP assay (r = − 0.997
and − 0.981, respectively), suggesting an inverse relationship between TFC and the scavenging of DPPH radicals
and the reducing power of the samples. Furthermore, the DPPH assay demonstrated the highest correlation
with the total phenolic content (TPC) among the antioxidant tests (r = − 0.852), indicating that polyphenols

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play a crucial role in the observed antioxidant activity measured by the DPPH assay. It is important to note
that different phenolic compounds may exhibit varying responses to different antioxidant mechanisms, lead-
ing to variations in correlation strengths depending on the specific antioxidant assay employed. These findings
highlight the significance of flavonoids and polyphenols in the antioxidant properties of the O. grossii and T.
pallidus samples. The correlations observed between the total phenolic and flavonoid contents and the different
antioxidant assays provide valuable insights into the specific mechanisms and compounds responsible for the
observed antioxidant activity.
The antibacterial activity of O. grossii and T. pallidus EOs was evaluated against four bacterial strains: Sal-
monella sp, Streptococcus sp, S. aureus, and E. coli, which are known to be responsible for foodborne infections.
These bacterial strains were obtained from the Center of the Hospital University of Fez, Morocco, and stored
in the regional laboratory of epidemiological diagnosis and hygiene in Fez. The potent antibacterial activity of
O. grossii and T. pallidus EOs can be attributed to their rich compositions of carvacrol and thymol, respectively.
Previous studies investigating the antibacterial properties of Lippia sidoides EO and its main components, thymol,
and carvacrol, have demonstrated their strong inhibitory effects against bacteria and f­ ungi72,73. These findings
highlight the potential of O. grossii and T. pallidus EOs as natural antibacterial agents due to their chemical
compositions and strong inhibitory effects against both Gram-positive and Gram-negative ­bacteria74,75. Since
Eos’ mechanism of action and its constituents (thymol and carvacrol) are hydrophobic, they may interact with
the lipid bilayer of bacterial cytoplasmic membranes, causing a loss of integrity, increasing the fluidity and
permeability of the membrane, and allowing cellular components like ions, ATP, and nucleic acids to leak ­out75.

Conclusions
Our study focused on the antioxidant capacity based on DPPH radical scavenging activity, CAT, FRAP, TPC, and
TFC of that O. grossi and T. pallidus, and the antibacterial effect against bacteria species causing food poisoning.
The results of this study suggest that the chemicals in O. grossii and T. pallidus plant EOs have antibacterial and
antioxidant properties. These findings could lead to the development of new natural products with antibacterial
and antioxidant activity. The chemicals in O. grossii that were found to have antibacterial activity were p-cymene,
eucalyptol, and carvacrol. These chemicals are known to work by disrupting the cell membranes of bacteria,
which can lead to cell death. The chemicals in T. pallidus that were found to have antibacterial activity were
p-cymene, α-terpinene, and thymol. These chemicals are also known to work by disrupting the cell membranes
of bacteria. The chemicals in both plants that were found to have antioxidant activity were p-cymene, eucalyptus,
carvacrol, α-terpinene, and thymol. These chemicals are known to work by scavenging free radicals, which can
damage cells. The findings of this study suggest that the chemicals in the studied EOs could be used to develop
new natural products with antibacterial and antioxidant activity. These products could be used to treat a variety
of conditions, including infections and inflammation.

Data availability
All data generated or analyzed during this study are included in this published article.

Received: 12 June 2023; Accepted: 10 November 2023

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Author contributions
Conceptualization, Z.H.; Methodology and software, Z.H., F.S.; validation, L.A., B.F.Z. and L.Y.; formal analysis,
A.A.; writing—original draft preparation, Z.H.; writing—review and editing, Z.H. and L.Y.; supervision, T.M.
and G.A.; project administration, A.A. Writing—original draft preparation, reviewing and editing, data valida-
tion, and data curation: F.S., M.B., A.M.S., H.-A.N. All authors have read and agreed to the published version
of the manuscript.

Funding
This work is financially supported by the Researchers Supporting Project number (RSP-2023R437), King Saud
University, Riyadh, Saudi Arabia.

Competing interests
The authors declare no competing interests.

Additional information
Correspondence and requests for materials should be addressed to H.Z. or A.B.M.
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