Volume 17, Number 3,September 2024

JJBS ISSN 1995-6673

Pages 463 – 470
https://doi.org/10.54319/jjbs/170308
Jordan Journal of Biological Sciences

Antimicrobial, Antibiofilm and Antioxidant Properties of

Algerian Satureja graeca L. Against Human Pathogens
Mouna Menakh 1,*, Saber Boutellaa 2, Amar Zellagui3, Ozgur Ceylan 4, Mehmet
Öztürk 5, Chawki Bensouici 6
1
Department of Nature and Life Sciences, University Center Abdehafid Boussouf, Mila, Algeria; 2 Laboratory of Natural Sciences and
Materials (LSNM), Institute of Sciences and Technology, University Center AbdehafidBoussouf, Mila, Algeria ; 3 Laboratory of Bio-
molecules and Plant Breeding, Department of Nature and Life Sciences, Faculty of Exact Sciences, Oum El Bouaghi University, Oum EL
Bouaghi, Algeria; 4Apiculture Program, Ali Kocman Vocational School, Mugla Sitki Kocman University, 48640, Ula, Mugla, Turkey;
5
Department of Chemistry, Faculty of Sciences, Muğla Sıtkı Koçman University, 48000 Mugla, Turkey; 6 Biotechnology Research Center,
P.B E73/UV N°03 Ali Mendjeli Nouvelle Ville, 25000 Constantine, Algeria ;

Received: September 1, 2023; Revised: November 4, 2023; Accepted: November 29, 2023

Abstract

In this study, we evaluated the antimicrobial, antibiofilm and antioxidant properties, in addition to the chemical constituents
of Satureja graeca L. ethyl acetate and n-butanol fractions using HPLC-DAD analysis, phenolic compound detection was
established. Six in vitro assays (DPPH, ABTS, ß-carotene, O2•-, CUPRAC, and Reducing power) were employed to assess
the antioxidant capacities. In order to determine the minimum inhibitory concentrations (MIC) of both extracts against six
bacterial strains and two fungi, the serial micro-dilution method was used. According to analysis, the extracts' main phenolic
components were chlorogenic acid, rutin, ellagic acid, vanillin, and caffeic acid. Both fractions revealed high antioxidant
capacities at different levels in all assays and they showed a strong antimicrobial activity against all tested strains as MIC
came out to be 0.3–10 mg/ml. However, ethyl acetate fraction exerted important effect compared to that of n-butanol fraction
on Enterococcus faecalis ATCC19433 (1.2 and 2.5 mg/mL), Candida albicans ATCC10239 (2.5 and 10 mg/mL) and
Candida tropicalis RSKK 665 (2.5 and 5 mg/mL). Furthermore, ethyl acetate fraction showed a significant antibiofilm
activity against C. tropicalis RSKK 665 (41.9%) and C. albicans ATCC 10239 (38.6%) at 2.5 mg/mL. These findings
suggest that Satureja graeca L. can be used in food and may be a promising therapeutic agent for treating a wide range of
disorders.

Keywords: Antibiofilm Activity; Microbial Inhibition; Antioxidant Properties; Phenolic profile; Greek Savory.

becoming less effective (Danaei et al., 2021). The genus

1. Introduction Satureja, part of the Lamiaceae family, the Nepetoideae
subfamily, and the Menteae tribe, include around 200
The production of excessive free radicals in the human species of fragrant plants and herbs native to the
body, combined with the inadequacy of both endogenous Mediterranean, Europe, Middle East, and western Asia.
and exogenous antioxidant systems, results in oxidative Algeria is home to 16 species of Satureja, which can be
stress. This oxidative stress causes significant harm to used as both a culinary and medicinal herb that can also be
cellular DNA, proteins, carbohydrates, and lipids (Sharifi- planted as a decorative plant. (Bensouici et al., 2013;
Rad et al., 2020). Antibiotic resistance and the ability of Haouat et al., 2022). In Algeria and Morocco, Satureja is
certain microorganisms to form biofilms pose a major highly regarded for its ability to treat respiratory
threat to public health and are considered one of the most infections, coughs, and indigestion (Amiri, 2011). Recent
significant challenges of the 21st century (Júnior et al., studies have also shown that Satureja possesses
2018; Al-kafaween et al., 2020; Zullkiflee et al., 2023). vasodilatory, emmenagogue, antihyperlipidemic,
The emergence of new pathogens, the reemergence of bactericidal, fungicidal, antioxidant, antidiabetic
previously controlled ones, and the lack of effective characteristics (Seyedtaghiya et al., 2021).
treatments only compound the problem, making the This research is centered on a comprehensive analysis
discovery of new active molecules a continued necessity of the chemical profile and a thorough evaluation of
(Venkatesan, 2021). biological properties of the ethyl acetate and n-butanol
The plant world offers a diverse array of molecules extracts derived from the aerial parts of Satureja graeca L.
with a range of various pharmacological effects, especially This particular species of Satureja is of significant
phenolic substances, which have been investigated for medicinal and botanical interest in Algeria. The primary
their potential to combat diseases linked to oxidative stress aim of this study is to uncover the therapeutic potential of
and as alternatives to conventional antibiotics, which are these extracts, with a specific focus on their antioxidant

*
Corresponding author. e-mail: m.menakh@centre-univ-mila.dz.
464 © 2024 Jordan Journal of Biological Sciences. All rights reserved - Volume 17, Number 3

capabilities, antibacterial effects, and antibiofilm gram of dry weight, and their characterization was based
properties. on a comparison of the retention times (Tel-Çayan et al.,
2015).
2. Material and Methods 2.4. Antioxidant activity
Using a microplate reader (Perkin Elmer, Enspire), all
2.1. Plant material experiments were carried out in 96-well microplates. The
Satureja graeca L. aerial components were gathered in tests used to define the results were CUPRAC and
March 2020 in Mila, North Eastern Algeria Reducing power assays, where A0.5 refers to the dose
(36°30'25.55"N Latitude and 6°21'43.70"E Longitude, at a indicating 0.5 absorbance, and DPPH, ABTS, O2•- and ß-
height of 240 m), during the floral stage. At the Life Carotene-linoleic acid, where IC50 refers to sample dose
Science and Nature Department of the University of Larbi giving 50% activity.
Ben Mhidi Oum El Bouaghi in Algeria, a sample was kept 2.4.1. DPPH assay
in the herbarium of the Biomolecules and Plant Breeding
Laboratory. According to BLOIS (1958), diphenyl-picrylhydrazyl
(DPPH) free radicals were used to measure the antiradical
2.2. Preparation of extracts effect of ethyl acetate and n-butanol fractions. 160 µL of
The aerial portions of S. graeca L. (50 g) were 0.1mM DPPH methanolic solution was added with
pulverized and macerated for three cycles of 24 hours each triplicate samples (40 µL of 6.25-200 µg.mL-1). A blank
with 80% ethanol and water at room temperature. The (methanol) was used as a reference during a half-hour
resultant hydroalcoholic extract was concentrated under incubation period at 25°C to quantify the mixture's
decreased pressure (40°C) and twice filtered through absorbance. The following equation was employed to
ordinary filter paper to remove ethanol. After that, it was compute the proportion of DPPH scavenger activity:
fractionated using progressively polar solvents. Before %I= [(A1- A2)/A1] × 100
usage, the fractions were kept at 4°C.
Where; A1 and A2 are respectively absorbencies of negative
2.3. Phytochemical analysis control and sample.

2.3.1. Total phenolics and flavonoids quantification 2.4.2. ABTS Cation Assay
The content of total soluble phenolics in the ethyle To create the ABTS cation radical, A 7 mM ABTS
acetate (EtOAc) and butanolic extracts (n-but) from the solution in water received the addition of 2.45 mM
aerial portions of S. graeca L. was evaluated using the potassium persulfate, which was then kept for 12 hours at
Folin reagent by colorimetry (Singleton et al., 1965). 500 room temperature. Later, using H2O, the solution's
µL of each extract, diluted to a dose of 0.25 mg/mL, 2500 absorption was brought to 0.700±0.020 at 734 nm. 40 µL
µL of F.C. reagent, diluted a tenth in water, and 2000 µL of each sample and standard was diluted in triplicate with
of sodium carbonate (20 g/l) made up the reaction mixture. 160 µL of this solution in each well. As typical
The absorbance at 760 nm was determined after 90 antioxidants, BHT and BHA were utilized, while methanol
minutes. The procedure utilizing aluminum trichloride was served as a blank. The absorbance was measured at 734
used to determine the flavonoid content of both extracts nm after ten minutes of incubation at 25°C. Using the
(Miliauskas et al., 2004). A 2% methanolic solution of DPPH formula the inhibition % was then determined (Re
AlCl3 was combined with one milliliter of each extract et al., 1999).
(250 µg/mL) in methanol, and after incubating at room
2.4.3. β-Carotene assay
temperature for 10 minutes, the absorbance was measured
at 430 nm. The total phenolics and flavonoids were According to Marco (1968), 25 µL of linoleic acid were
expressed in µg of GA equivalent and µg of quercetin combined with 0.5 mg of β-carotene in 1 mL of
equivalent per milligram of extract, respectively, using chloroform, and 200 µL of Tween 40 were used to
gallic acid and quercetin as reference standards to generate emulsify the mixture. Chloroform was evaporated at 40°C,
calibration curves. and the rest was recovered using 100 mL of purified water
that had been infused with oxygen. With oxygenated
2.3.2. HPLC-DAD screening of phenolics
water, the absorbance was changed to (0.8 - 0.9) at 470
To analyze ethyle acetate and butanolic extracts as well nm. 40 µL of samples at concentrations ranging from 6.25
as 27 standard phenolics, we used a Shimadzu reverse to 200 µg/mL were added to 160 µL of this solution. β -
stationary phase HPLC system which is controlled by LC- carotene bleaching percentage was calculated after two
solution and includes a Shimadzu model LC-20AT solvent hours of incubation at 50 °C using an absorbance
supply unit. 35°C was chosen as the column temperature. measurement at 470 nm and the result was calculated as
Aqueous acetic acid 0.1% (A) and methanol served as the follows:
mobile phases for the chromatographic separation, which R = (ln a1/a2)/t
was carried out on an Inertsil ODS-3 guard column (4 µm, a1 and a2 were the relative absorbencies at the beginning of the
4.0 mm x 150 mm) column (B). Elution was done in reaction (t0) and 120 minutes afterward (t120). Antioxidant
gradients ranging from 2% to 100%. Sample stock capacity was computed using the following formula:
solutions were created in methanol at a concentration of
A% = [(Rcontrol – Rsample)/ Rcontrol] x 100
8mg.mL-1 and filtered through an Agilent 0.45 µm filter.
20 µL of fluid was injected. A diode array detector (DAD) 2.4.4. Superoxide anion assay
operating at a wavelength of 254 nm was used to find the
The alkaline DMSO technique, with minor
phenolics. The results were presented as micrograms per
modifications, was used to test O2•- scavenging activity
 © 2024 Jordan Journal of Biological Sciences. All rights reserved - Volume 17, Number 3 465

(Kunchandy and Rao, 1990). This process depends on the formation ability of both bacteria and yeasts using
synthesis of alkaline DMSO while changing the yellow polystyrene microplates (Ceylan and Ugur, 2015). The
color of Nitroblue tetrazolium (NBT) into formazan (blue microorganisms were cultured overnight in 5 ml of
hue). Following the addition of 30 µL of NBT (1 mg/mL Tryptone-Soy Broth (TSB) supplemented with glucose
in distilled water), 40 µL of each extract was combined (1%). The microbial suspension was then diluted (5 x 105
with 130 µL of alkaline DMSO (created by dissolving 20 CFU ml-1), and each well received 100 µL along with 100
mg of NaOH in 100 mL of DMSO to create O2•-). Tannic µL of each extract at various concentrations (MIC, MIC/2,
acid and α-tocopherol served as benchmarks. Following 10 and MIC/4) or 100 μL of the control (culture medium).
min of incubation at ambient temperature, the absorbance Each well was rinsed with water after being incubated for
was recorded at 560 nm, and the ratio of O2•- blockage was 48 h at 37°C to get rid of any planktonic bacteria. The
calculated employing the DPPH formula. remaining bacteria were then dyed for 10 minutes at 25°C
2.4.5. Reducing power assay with a 0.1% crystal violet solution. To remove the crystal
violet solution (which had not particularly colored the
Using Oyaizu (1986) method of Fe3+ to Fe2+ reductive attached bacteria), the wells were washed once more. To
capability, the extracts' reductive capacities were remove any extra liquid, the microplates were flipped and
evaluated. 50 µL of potassium ferricyanide (1%) (1 g of firmly tapped on absorbent paper. They were then allowed
K3Fe (CN)6 in 100 mL H2O) and 40 µL of phosphate to air dry. The wells containing Gram-negative and Gram-
buffer (pH 6.6) were added to 10 µL of each sample positive bacteria, respectively, received 200 l each of 95%
(1.562-50 µg/mL), followed by a 20 min incubation period ethanol and 33% glacial acetic acid, and the plates were
at 50°C for the combination. Then, 10 µl of ferric chloride automatically shaken. Spots of the biofilm dissolved at
FeCl3 (0.1%), 40 µl of water, and 50 µl of trichloroacetic room temperature. Finally, a microplate reader was used to
acid (TCA) (10%) were added to the mixture. On a blank detect the absorbance at a wavelength of 550 nm. The
surface (methanol), at 700 nm, absorption was determined. biofilm formation inhibition percentages were calculated
2.4.6. Cupric reducing capacity (CUPRAC) using the next equation:
I% = [(Abcontrol – Absample) / Abcontrol] x 100
A mixture consisting of 60 µL of ammonium acetate
buffer (pH 7.0), 50 µL of 10 mM (Cu Cl2, 2H2O), and 50 2.7. Statistical analysis
µL of 7.5 mM neocuproin was prepared. To this mixture,
40 µL of sample solutions with 6.25-200 µg/mL All results and findings were obtained in triplicate and
concentrations were then supplemented (Apak et al., reported as mean ± S.D. The data were processed by SPSS
2008). After one hour of incubation, the intensity of V.16. One-way analysis of variance (ANOVA) and Tukey
absorption at 450 nm was obtained and contrasted with a Post Hoc test were performed to establish the differences
blank solution. BHT and BHA were common antioxidants among the means. P values < 0.05 were considered to be
employed in this assay. significant. The IC50 values (50% inhibition concentration)
for the antioxidant activity were calculated by the linear
2.5. Antimicrobial activity regression method from the curve [% inhibition = f
According to the micro-dilution method of Rota et al., (concentrations)].
(2004), minimal inhibitory concentration (MIC) and
minimal bactericide concentration (MBC) of the extracts, 3. Results
which reflect the lowest concentration that gave no
observable growth, were determined. E. foecalis 3.1. Total phenolics and flavonoid contents
ATCC19433, L. monocytogenes ATCC7644, S. aureus
ATCC 25923, and S. aureus MU40 have been chosen as Total phenolics and flavonoid contents of ethyl acetate
positive Gram bacteria. C. violaceum CV 026 and P. (EtOAc) and butanolic extracts (n-but) from aerial parts of
fluorescens RSKK240 have been chosen as negative Gram S.graeca L. are presented in Table 1. A great richness of
bacteria. All mentioned bacterial strains were put to grow both extracts in total phenols was noted. However, EtOAc
in nutrient broth and incubated at 37°C for 24 h excepted extract had the highest content significantly (268.4 ±1.9 μg
P. fluorescens RSKK240, L. monocytogenes ATCC7644 EAG/mg E) compared to that of n-but extract (P < 0.05).
and Fungi (30°C for 24-48 h). Afterwards, inocula 5×105 In contrast, the latter contained more flavonoids (37.6 ±
colony-forming units (CFU)/mL were prepared. The test 0.2 EQ/mg E) than EtOAc extract (20.9 ± 0.2 EQ/mg
medium was Mueller Hinton Broth (MHB) from Biolife in extract) (P < 0.05).
Milan, Italy. Each extract was produced in five final doses
(0.625–10) mg/mL in DMSO solution. 10 µL of cell
suspension were added to each well of a 96-well
microplate containing 170 uL of sterile MHB. Then, 20 uL
of sample were added in duplicate at various
concentrations. Three wells were reserved for negative
controls (media, media + bacteria and media + bacteria +
DMSO). For 24 hours, microplates with inoculums were
incubated at 37°C.
2.6. Antibiofilm activity
We tested the impact of extracts in different
concentrations ranging from the minimum inhibitory
concentration (MIC) to one-fourth MIC on the biofilm
466 © 2024 Jordan Journal of Biological Sciences. All rights reserved - Volume 17, Number 3
(a) Microgram of Gallic Acid Equivalent per milligram of
extract,(b) Microgram of Quercetin Equivalent per milligram of
extract. ***P<0.001, ** P <0.01 and * P <0.05 compared
between means.

3.2. Compounds identified by HPLC-DAD

The results of HPLC analysis are given in Table 1.
Only five minor compounds were identified in EtOAc
fraction: chlorogenic acid (4.8µg/g), caffeic acid (1.4µg/g),
vanillin (1.3µg/g), protocatechuic acid (0.8µg/g) and 4-
hydroxybenzoic acid (0.2 µg/g). The majority of the
detected peaks were not identified due to the absence of
suitable standards (Figure 1). For n-but fraction, nine
compounds were identified: Rutin (41.6 µg/g), chlorogenic
Figure 1. Total phenolics and flavonoids contents in S. graeca L. acid (41.1 µg/g), ellagic acid (3.3 µg/g), 2,4-
extracts dihydroxybenzoic acid (0.3 µg/g), Coumarin (0.3 µg/g), p-
coumaric acid (0.2 µg/g), ferulic acid (0.1 µg/g), trans-
Results are expressed as means ± standard deviation of three
cinnamic acid (0.2 µg/g) Quercetin (0.2 µg/g) and
measurements.
Rosmarinic acid (0.1 µg/g).
Table 1. Bioactive phenolics obtained from of S. graeca L. extracts.

No Compounds Rt (min) EtOAc (g/g) n-but (g/g)

1 Protocatechuic acid 14.0 0.8 -
2 4-hydroxybenzoic acid 19.5 0.2 -
3 Caffeic acid 24.1 1.4 -
4 Vanillin 24.8 1.3 -
5 2,4,-dihydroxybenzoic acid 25.2 - 0.3
6 Chlorogenic acid 27.3 4.8 41.1
7 p-coumaric acid 29.8 - 0.2
8 Ferrulic acid 31.0 - 0.1
9 Coumarin 32.5 - 0.2
10 Rutin 34.2 - 41.6
11 Ellagic acid 38.1 - 3.3
12 trans-cinnamic acid 43.2 - 0.2
13 Quercetin 44.0 - 0.2
14 Rosmarinic acid 45.3 - 0.1
(-): not detected
mAU
254nm,4nm (1.00)
1250

1000

750
3,4-dihydroxybenzoic acid

4-hydroxy benzoic acid

500
Chlorojenic acid
Caffeic acid

250
Vanilin

0
0 10 20 30 40 50 60 70 80 min
Figure 2. Chromatogram of S. graeca L. EtOAc fraction at 254 nm.
 © 2024 Jordan Journal of Biological Sciences. All rights reserved - Volume 17, Number 3 467

mAU
254nm,4nm (1.00)

1750

1500

1250

Rutin hydrate
1000

Rosmarinic acid Quercetin

2,4-dihydroxy benzoic acid
Chlorojenic acid
750

trans-cinnamic acid
500

p-Coumaric acid

Ellagic acid
Ferrulc acid
Coumarin
250

0
0 10 20 30 40 50 60 70 80 min
Figure 3. Chromatogram of S. graeca L. n-but t fraction at 254 nm.

3.3. Antioxidant activity extracts exhibited a very significant activity in comparison

to standard antioxidants, as we have recorded CI50 values
Results of antioxidant activities are represented as very near to those of BHA in the ABTS and β-carotene
percentage inhibition for each concentration as well as tests (P <0.05), significantly better than those of BHT in
50% radical scavenging concentration (IC50) values for the DPPH (CI50: 10.6 ± 0.4 µg/mL and 11.4 ± 0.5 µg/mL) and
DPPH, ABTS, O2•- and β-Carotene tests, and A0.5 values, CUPRAC (A0.5: 4.4 ± 0.4 µg/mL and 6.2 ± 0.3 µg/mL)
which correspond to the concentrations indicating 0.5 tests (P <0.01) and significantly better than that of α-
absorbance for CUPRAC and reducing power (RP) tests. tocopherol in the RP test (A0.5: 6.9 ± 0.6 µg/mL and 11.5 ±
According to data mentioned in Tables 2 and 3, and based 0.7 µg/mL) for EtOAc and n-but, respectively (P<0.001).
on the values of CI50 and A0.5, we have found that both

Table 2. Antioxidant activity of S. graeca L. extracts by DPPH•, ABTS•+, O2•-, β-carotene, inhibition % at minimum concentration and IC50.
DPPH• assay ABTS•+ assay β-carotene-linoleic O2•- assay
acid assay
Inhibition IC50 µg/mL Inhibition IC50 µg/mL Inhibition IC50 µg/mL Inhibition IC50 µg/mL
(%) at (%) at (%) at (%) at
6.2µg/mL 6.2µg/mL 6.2µg/mL 6.2µg/mL
EtOAc 28.9±0.1* 10.6 ±0.4** 89.5±0.3* 3.3±0.1* 66.4±2.5* 2.4±0.1* 82.8±0.4 1.2±0.1*
n-but 26.5±1.3* 11.4 ±0.5** 65.2±1.8 4.6±0.3* 61.1±0.4* 2.6±0.1* 78.1±1.4 7.3±0.2***
BHA 54.3±1.6** 5.7 ±0.4*** 93.5±0.1* 1.8±0.1* 90.1±0.6** 0.9±0.1* nt nt
BHT 22.2±1.3* 13.0 ±0.4** 78.5±3.4* 1.2±0.3** 86.1±1.0** 1.0±0.1* nt nt
Tannic ac nt nt nt nt Nt nt 78.8±0.9 1.6±0.1*
α-Tocopherol nt nt nt nt Nt nt 64.9±1.0* 3.1±0.4*
Results are expressed as means ± standard deviation of three measurements
(-) : no inhibition; nt : not tested. ***P<0.001, ** P <0.01 and * P <0.05 compared between means.

Table 3. Antioxidant activity of S. graeca L. extracts py CUPRAC, Ferric reducing power, inhibition % at minimum concentration and A0.5.
CUPRAC Ferric reducing power
Inhibition (%) at 6.2µg/mL A0.50 µg/mL Inhibition (%) at 1.5µg/mL A0.50 µg/mL
EtOAc 0.6±0.1** 4.4±0.4** 0.3±0.00*** 6.9±0.6***
n-but 0.5±0.1** 6.1±0.3** 0.3±0.01*** 11.4±0.7***
BHA 0.8±0.1** 3.6±0.1** 0.1±0.01*** 8.4±0.6***
BHT 0.3±0.2** 9.6±0.8** 0.1±0.00*** >50***
Tannic ac nt nt Nt 5.3±0.9***
α-Tocopherol nt nt Nt 34.9±2.3***
***
Ascorbic ac nt nt 0.1±0.00 6.7±1.1***
Results are expressed as means ± standard deviation of three measurements
(-) : no inhibition; nt : not tested. ***P<0.001, ** P <0.01 and * P <0.05 compared between means.

3.4. Antimicrobial and Antibiofilm activities Table 4. Our data showed that EtOAc fraction of S. graeca
L. possesses a strong effect and a wide range of action
Antimicrobial activity of both S. graeca L. extracts covering Gram positive, Gram negative bacteria and
against 9 reference microbial strains tested in this study Candida genus, as MIC came out to be 0.3–10 mg/ml
was qualitatively evaluated by the minimum inhibitory (mean range). n-but fraction had the same antibacterial
concentrations (MICs) and results were represented in
468 © 2024 Jordan Journal of Biological Sciences. All rights reserved - Volume 17, Number 3

effect (MIC) against S.aureus ATCC25923 (0.6 mg/ml), L. Results of antibiofilm activity presented in Table 4 as
monocytogenes ATCC7644 (1.2 mg/ml) and P. fluorescens inhibition percentages showed that EtOAc extract exerted
RSKK240 (0.3 mg/ml). However, EtOAc fraction exerted a moderate effect of inhibition of the biofilm formed by
a significant effect compared to that of n-but on E. faecalis only three strains: L. monocytogenes (23.4%), C. albicans
ATCC19433 (1.2 and 2.5 mg/mL), C. albicans (38.6%) and C. tropicalis (42.1%). However, there was no
ATCC10239 (2.5 and 10 mg/mL) and C. tropicalis RSKK effect with the n-but fraction against the biofilm produced
665 (2.5 and 5 mg/mL). by the major strains examined.
Table 4. Antimicrobial and antibiofilm activities of S. graeca L. extracts values.

Strains n-but EtOAc

MIC % inhibition on biofilm formation MIC % inhibition on biofilm formation
mg/mL MIC MIC/2 MIC/4 Mg/mL MIC MIC/2 MIC/4
Gram- S.aureus MU40 0.62 - - - 0.6 - - -
Positive E. foecalis ATCC19433 2.5 - - - 1.2 - - -
bacteria L. monocytogenes 1.2 - - - 1.2 23.4±0.4 18.7±0.8 12.4±1.2
ATCC7644
B. subtilis ATCC6633 10 nt nt nt 5 nt Nt nt

S.aureus ATCC25923 ˃ 10 - - - 5 - - -
Gram- C. violaceum CV 026 1.2 - - - 1.2 - - -
Negative P. fluorescens 0.3 - - - 0.3 - - -
bacteria RSKK240
Fungi C. albicans ATCC 10 19.3±3.2* - - 2.5 38.6±2.2* 16.9±2.7* -
10239
C. tropicalis RSKK 5 - - - 2.5 42.1.±0.2 - -
665
Results are expressed as means ± standard deviation of three measurements.
MIC: minimal inhibitory concentration (mg. mL-1)
(- ): no inhibition; nt : not tested. * P <0.05 compared between means.
(n-but) fractions of S. graeca L. based on different
4. Discussion mechanisms of action such as radical scavenging
properties (DPPH•, ABTS•+ and O2•-), ability to prevent
Infectious diseases brought on by bacteria, viruses, and lipid peroxidation (β-carotene) and ability to reduce copper
fungi continue to pose a significant threat to public health ( and ferric ions (CUPRAC and RP). Our findings
Al-kafaween et al., 2020; Shidiki and Vyas, 2022). consistently demonstrated that both the AcOEt and n-but
Finding alternate methods to combat bacterial infection extracts exhibited similar antioxidant activities across most
has become urgently necessary due to the rise in of these tests. Notably, the AcOEt extract displayed a
multidrug-resistant pathogenic microorganisms ( Zullkiflee slightly higher level of activity compared to the n-but
et al., 2023). extract. This observation underscores the robust
In the current study, rutin, ellagic acid, vanillin, caffeic antioxidant potential of both extracts.
and chlorogenic acids, were shown to be the major Additionally, our analysis of the extracts' composition
phenolic components in Satureja graeca L. extracts. revealed high and closely matched contents of total
According to several studies, phenolic chemicals are phenolic compounds and flavonoids in both fractions.
commonly found in species of Satureja. For example, p- These results suggest that the extracts' antioxidant
coumaric, ferulic, caffeic and protocatechuic acids have activities can be attributed to their rich content of these
been detected in ethyle acetate and n-butanol extracts of S. bioactive compounds, further supporting their potential
montana L. which are analyzed qualitatively by HPLC- utility in medicinal and therapeutic applications.
DAD, as well as in the methanolic extract of S. montana Our antioxidant properties investigation of S.graeca L.
subsp. Kitaibelii of Serbia (López-Cobo et al., 2015). extracts are consistent with those reported by previous
HPLC analysis of S. hortensis (Georgia) ethanol extract studies for aqueous and ethanolic extracts of S. hispidula
confirmed the presence of several phenolic compounds (Algeria), for wild and cultivated S. bachtiarica (Iran), for
such as caffeic, p-coumaric acids, rutin, hesperidin and 7- methanolic extract of S. rechingeri (Iran) depending on the
glucoside (Boroja et al., 2018). phenological stage and for S. montana (Serbia) who have
Antioxidant activity can be determined using a variety all found similar relationships between these species' total
of techniques. Depending on the test employed, the phenolic compound concentration and antioxidant capacity
chemical structure of extracts, which sometimes consists (Alizadeh, 2015; Veličković et al., 2018; Haouat et al.,
of several of compounds mixed together with different 2022). Thus, HPLC analyses have proven a diversity of
functional groups, polarity, and chemical behaviors, may phenolic compounds in both extracts. The presence of
produce dispersed results (Boutellaa et al., 2019; Menakh Chlorogenic acid and Rutin at high concentrations and
et al., 2020). Therefore, it would be more beneficial and Ellagic acid in n-but extract, on one hand, and the presence
even required to utilize a method that involves many tests of Caffeic acid, Vanillin and Protocatechuic acids in
to estimate the antioxidant capability of extracts. In this AcOEt extract could be the reason for their most powerful
investigation, six methods were used to evaluate in vitro antioxidant potential. Moreover, the proportion of
antioxidant activity of ethyl acetate (AcOEt) and n-butanol
 © 2024 Jordan Journal of Biological Sciences. All rights reserved - Volume 17, Number 3 469

unidentified compounds in both fractions is generally non- antibiofilm effects of the plant extract may be caused by a
negligible. reduction in structure development, such as a decrease in
The creation of bioactive secondary metabolites, which exopolysaccharide production, or an alteration in the
leads to the discovery of antibiotics, has attracted the transcription of genes linked to biofilms (Swamy et al.,
attention of researchers who have been mining the 2016).
understudied sources to find novel secondary metabolites In summary, our research not only highlights the
that are of considerable interest in the present promising antibiofilm properties of S. graeca L. extracts
(Abdolhosseini et al., 2019; Abdel-Mawgoud et al., 2019). but also underscores the need for more extensive studies to
Our funding demonstrated that S. graeca L. extracts elucidate the mechanisms underlying these properties and
possess a strong antimicrobial activity because they are their suitability for practical applications in areas such as
abundant in phenolic and flavonoid chemicals, which can medicine, agriculture, and biotechnology.
be used as an alternative to antibiotics. Besides,
chlorogenic acid and rutin, known for their antimicrobial 5. Conclusion
potential, were detected as major phenolic compounds,
which could explain the observed effect (Kabir et al., In conclusion, our study has revealed that Satureja
2014; Stojković et al., 2013). According to the literature graeca extracts, characterized by their rich composition of
review, there was no work on the antimicrobial activity of phenolic compounds and flavonoids, have the potential to
S. graeca L. extracts. Previous studies have examined the serve as a valuable source of bioactive phenolic
antimicrobial properties of extracts from other species compounds. These compounds hold promise in the fields
within the Satureja genus. For instance, research on S. of medicine and therapy due to their demonstrated
kitaibelii explored the antimicrobial potential of antioxidant and antimicrobial properties. Furthermore, this
chloroform, ethyl acetate, and n-butanol extracts. The research has contributed to a deeper understanding of the
results indicated that the ethyl acetate extract exhibited the antibiofilm properties of Satureja graeca L. extracts,
highest activity, with a minimum inhibitory concentration showing their effectiveness in inhibiting biofilm formation
(MIC) of 10 mg/ml against P. aeruginosa and S. aureus, by bacterial and yeast species. This discovery is
while the chloroform extract was particularly effective particularly significant, as biofilm inhibition has wide-
against P. aeruginosa. However, the n-butanol extract ranging implications in various industries, including
showed weaker activity with an MIC of 50 mg/ml against healthcare and agriculture. While our findings are
S. aureus (López-Cobo et al., 2015). In another study, promising, it is important to note that further research is
aqueous and EtOH extracts of S. boissieri (Turkey), rich in necessary to validate the applicability of these extracts,
hesperidin and ac rosmarinique, showed weak activity both as feed additives and in clinical settings. Future in
against S. aureus and P. aeruginosa, and were not vivo studies are warranted to confirm the efficacy and
effective against C. albicans (Aras et al., 2018). Our safety of these extracts for potential use in practical
results are similar with those of previous funding who applications.
reported a strong antimicrobial activity of S. kitaibelii
MeOH extract from Serbia against S. aureus (0.6 mg/mL), Disclosure statement
L. monocytogenes (1.25 mg/mL), P. aeruginosa (1.2
mg/mL) and C. albicans (0.6 mg/mL) (Stanojković et al., The authors report no conflicts of interest.
2013) . Moreover, with MICs varying from 0.02 to 0.4
mg/mL, the antibacterial activity of S. hortensis (Turkey) Acknowledgements
extracts in EtOH, MeOH, and dichloromethane was
significant against S. aureus, E. faecalis, P. aeruginosa, This study was supported by the Ministry of Higher
and C. albicans (Sharifi et al., 2018). Education and Scientific Research of Algeria.
Bacterial survival in challenging environments is
supported by biofilms, which are essential cell colonies References
that bind to biotic or abiotic environments and produce an
extracellular matrix. Different degrees of biofilm Abdel-Mawgoud M, Khedr F G and Mohammed E I.
formation are capable of occurring in both Gram-positive 2019.Phenolic Compounds, Antioxidant and Antibacterial
and Gram-negative bacteria ( Al-kafaween et al., 2020; Activities of Rhus flexicaulis Baker. Jordan J Biol Sci., 12(1): 17–
Seyedtaghiya et al., 2021). 21.
The results of the current study's evaluation of the Abdolhosseini M, Zamani HA and Salehzadeh A. 2019.
antibiofilm qualities of S. graeca L. extracts revealed that Synergistic antimicrobial potential of ciprofloxacin with silver
AcOEt extract reduced the development of biofilms at nanoparticles conjugated to thiosemicarbazide against
concentrations lower than MIC. This finding holds ciprofloxacin resistant Pseudomonas aeruginosa by attenuation
of MexA-B efflux pump genes. Biologia., 74 : 1191–1196.
significant promise for the potential application of S.
graeca L. extracts in combating biofilm-related issues, a Alizadeh A. 2015. Essential oil composition, phenolic content,
subject for which data have been notably scarce in the antioxidant, and antimicrobial activity of cultivated Satureja
existing literature. Currently, there is a dearth of data on rechingeri Jamzad at different phenological stages. Z
Naturforsch., 70(1): 51–58.
the antibiofilm activity of S. graeca L. extracts, making our
study one of the pioneering works in this regard. While Al-kafaween MA, Hilmi ABM, Jaffar N, Al-Jamal HAN, Zahri
previous research has focused on related species like S. MK and Jibril FI. 2020. Antibacterial and Antibiofilm activities
hortensis and its essential oil (Seyedtaghiya et al., 2021; of Malaysian Trigona honey against Pseudomonas aeruginosa
ATCC 10145 and Streptococcus pyogenes ATCC 19615. Jordan J
Sharifi et al., 2018), few studies have specifically delved
Biol Sci., 13(1): 69–76.
into the antibiofilm qualities of S. graeca L. extracts. The
470 © 2024 Jordan Journal of Biological Sciences. All rights reserved - Volume 17, Number 3
Amiri H. 2011. The in vitro antioxidative properties of the Miliauskas G, Venskutonis P R and Van Beek TA .2004.
essential oils and methanol extracts of Satureja macrosiphonia Screening of radical scavenging activity of some medicinal and
Bornm. Nat Prod Res., 25(3): 232–243. aromatic plant extracts. Food Chem., 85(2): 231–237.
Apak R, Güçlü K, Özyürek M and Çelik SE. 2008. Mechanism of Oyaizu M .1986. Studies on Products of Browning Reaction. Jap
antioxidant capacity assays and the CUPRAC (cupric ion reducing J of Nutr., 44(6): 307–315.
antioxidant capacity) assay. Microchim Acta .,160(4):413–419.
Re R, Pellegrini N, Proteggente A, Pannala A, Yang M and Rice-
Aras A, Bursal E, Alan Y, Turkan F, Alkan H and Kılıç Ö. 2018. Evans C. 1999. Antioxidant activity aplying an improved ABTS
Polyphenolic Content, Antioxidant Potential and Antimicrobial radical cation decolorization assay. Free Radic Biol Med.,
Activity of Satureja boissieri. Iran J Chem Chem Eng., 37(6): 26(9):1231–1237.
209–219.
Rota C, Carraminana JJ, Burillo J and Herrera A .2004. In vitro
Bensouici C, Benmerache A, Chibani S, Kabouche CS, antimicrobial activity of essential oils from aromatic plants
Abuhamdah S and Semra Z. 2013. Antibacterial activity and against selected foodborne pathogens. J of Food Prot.,
chemical composition of the essential oil of satureja calamintha 67(6):1252-1256.
ssp. sylvatica from Jijel, Algeria. Der Pharm Lett., 5(2): 224–227.
Seyedtaghiya MH, Fasaei BN and Peighambari SM .2021.
BLOIS MS .1958. Antioxidant Determinations by the Use of a Antimicrobial and antibiofilm effects of Satureja hortensis
Stable Free Radical. Nature., 181(4617):1199–1200. essential oil against Escherichia coli and Salmonella isolated from
poultry. Iran J Microbiolo., 13(1): 5485–5495.
Boroja T, Katanić J, Rosić G, Selaković D, Joksimović J, Mišić D
and Mihailović V .2018. Summer savory (Satureja hortensis L.) Sharifi-Rad M, Anil Kumar NV, Zucca P, Varoni EM, Dini L,
extract: Phytochemical profile and modulation of cisplatin- Panzarini E and Sharifi-Rad J .2020. Lifestyle, Oxidative Stress,
induced liver, renal and testicular toxicity. Food Chem Toxicol., and Antioxidants: Back and Forth in the Pathophysiology of
118: 252–263. Chronic Diseases. Front Physiol., 11: 694.
Boutellaa S, Zellagui A, Öztürk M, Bensouici C, Ölmez ÖT, Sharifi A, Mohammadzadeh A, Zahraei Salehi T and Mahmoodi P
Menakh M and Duru M E .2019. HPLC-DAD profiling and .2018. Antibacterial, antibiofilm and antiquorum sensing effects
antioxidant activity of the butanol extract from aerial parts of of Thymus daenensis and Satureja hortensis essential oils against
Algerian Crithmum maritimum L. Acta Sci Nat., 6(1): 8–16. Staphylococcus aureus isolates. J App Microbiol., 124(2): 379–
388.
Ceylan O and Ugur A .2015. Chemical composition and anti-
biofilm activity of Thymus sipyleus BOISS. subsp. sipyleus Boiss. Shidiki A and Vyas A .2022. Evaluation of Antibacterial Potential
var. davisianus Ronniger essential oil. Arch Pharm Res., 38(6): of Syzygium cumini against Methicillin-Resistant Staphylococcus
957–965. aureus and Macrolide-Lincosamide-Streptogramin B Strains of
Danaei M, Motaghi MM, Naghmachi M, Frazane A and Moravej Staphylococcus aureus and Its Liquid Chromatography Mass
Spectroscopy Analysis. Biomed Biotechnol Res J., 6(1): 66-72.
R. 2021. Green synthesis of silver nanoparticles (AgNPs) by
filamentous algae extract: comprehensive evaluation of Singleton VL, Joseph A and Rossi J .1965. Colorimetry of Total
antimicrobial and anti-biofilm effects against nosocomial Phenolics with Phosphomolybdic-Phosphotungstic Acid Reagents.
pathogens. Biologia., 76(10): 3057–3069. Amer J Enol Viticult., 16(3): 144–158.
Haouat A, Rechek H, Pinto DCGA, Cardoso SM, Válega MSGA, Stanojković T, Kolundžija B, Ćirić A, Soković M, nikolić D and
Boudjerda M and Mekkiou R. 2022. Metabolite Profiling, Kundaković T .2013. Cytotoxicity and antimicrobial activity of
Antioxidant and Key Enzymes Linked to Hyperglycemia Satureja kitaibelii wierzb. Ex heuff (Lamiaceae). Dig J
Inhibitory Activities of Satureja hispidula: An Underexplored Nanomater Biostruct., 8(2): 845–854.
Species from Algeria. Molecules.,27(24):8657.
Stojković D, Petrović J, Soković M, Glamočlija J, Kukić-
Júnior S D da C, Santos JVde O, Campos LA de A, Pereira MA, Marković J and Petrović S .2013. In situ antioxidant and
Magalhães NSS and Cavalcanti IMF. 2018. Antibacterial and antimicrobial activities of naturally occurring caffeic acid, p-
antibiofilm activities of quercetin against clinical isolates of coumaric acid and rutin, using food systems. J Sci Food Agricul.,
Staphyloccocus aureus and Staphylococcus saprophyticus with 93(13): 3205–3208.
resistance profile. Inter J Env Agr Biotech., 3(5): 1948–1958.
Swamy MK, Akhtar MS and Sinniah UR .2016. Antimicrobial
Kabir F, Katayama S, Tanji N and Nakamura S. 2014. Properties of Plant Essential Oils against Human Pathogens and
Antimicrobial effects of chlorogenic acid and related compounds. Their Mode of Action: An Updated Review. Evid Based
J Korean Soc App Biol Chem., 57(3): 359–365. Complement Alternat Med., 2016, 3012462.
Kunchandy E and Rao MNA. 1990. Oxygen radical scavenging Tel-Çayan G, Öztürk M, Duru ME, Rehman MU, Adhikari A,
activity of curcumin. Inter J Pharm., 58(3): 237–240. Türkoglu A and Choudhary MI .2015. Phytochemical
investigation, antioxidant and anticholinesterase activities of
López-Cobo A, Gómez-Caravaca AM, Švarc-Gajić J, Segura-
Ganoderma adspersum. Indus Crops and Prod., 76: 749–754.
Carretero A and Fernández-Gutiérrez A .2015. Determination of
phenolic compounds and antioxidant activity of a Mediterranean Veličković V, Mašković JM and Mašković PZ .2018. Analytical
plant: The case of Satureja montana subsp. kitaibelii. J Func Control and Antioxidative Activity of Different Teas in Serbia.
Foods., 18: 1167–1178. Der Chem Sin., 9(1): 599–604.
Marco G J .1968. A rapid method for evaluation of antioxidants. J Venkatesan P .2021. Re-emergence of infectious diseases
Am Oil Chem Soc., 45(9): 594–598. associated with the past. The Lancet Microbe., 2(4): e140.
Menakh M, Mahdi D, Boutellaa S, Zellagui A, Lahouel M and Zullkiflee N, Hashim F, Taha H and Usman A. 2023. Antifungal
Bensouici C .2020. antioxidant activity and protective effect of and Antiamoebic Activities, Cytotoxicity, and Toxicity of
Hertia cheiriefolia L. n-butanol extract against liver and heart Aqueous and Ethanolic Extracts of Propolis Produced by Brunei
mitochondrial oxidative stress in rat. Acta Sci Nat., 7(1): 33–45. Stingless Bees. Jordan J Biol Sci., 16(2): 259–266.