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Phytochemical characterization and immunomodulatory effects of aqueous,
ethanolic extracts and essential oil of Syzygium aromaticum L. on human
neutrophils
Article in Scientific African · October 2022
DOI: 10.1016/j.sciaf.2022.e01395
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Scientific African 18 (2022) e01395
Contents lists available at ScienceDirect
Scientific African
journal homepage: www.elsevier.com/locate/sciaf
Phytochemical characterization and immunomodulatory
effects of aqueous, ethanolic extracts and essential oil of
Syzygium aromaticum L. on human neutrophils
Othman El Faqer a, Salma Bendiar a, Samira Rais a,c, Ismail Elkoraichi a,
Mohamed Dakir b, Anass Elouaddari b, Abdelaziz El Amrani b, Mounia Oudghiri a,
El Mostafa Mtairag a,∗
a
Département de Biologie, Faculté des Sciences Ain Chock, Laboratoire d’immunologie et Biodiversité, Université Hassan II, Casablanca,
Morocco
b
Département de Chimie, Faculté des Sciences Ain Chock, Laboratoire Synthèse Organique, Extraction et Valorisation, Université Hassan
II, Casablanca, Morocco
c
Département de Biologie, Faculté des Sciences Ben M’sik, Université Hassan II, Casablanca, Morocco
a r t i c l e
i n f o
Article history:
Received 31 December 2021
Revised 24 August 2022
Accepted 12 October 2022
Editor: DR B Gyampoh
Keywords:
Syzygium aromaticum L
Eugenol
HPLC
GC-MS
Human neutrophils
Immunomodulation
a b s t r a c t
In Morocco, the flower buds of Syzygium aromaticum L (Clove) from Myrtaceae are essential in traditional medicine; they are used in many forms (infusion, maceration, and essential oil) and are suggested to mitigate inflammatory conditions such as muscle and dental
pain as well as rheumatic diseases. This study aims to chemically characterize the aqueous
and ethanolic extracts as well as the essential oil from cloves; also, we aimed to evaluate their effects on the bactericidal activity of human neutrophils compared with eugenol.
The chemical composition of extracts was evaluated via qualitative phytochemical screening followed by quantitative screening using spectrophotometry and HPLC technique. The
essential oil was analyzed by the GC-MS technique. The PMNs bactericidal activity of extracts, essential oil, and eugenol was carried out by MTT assay. The screening of extracts
showed the presence of phenols, flavonoids, flavones aglycones, coumarins, and tannins.
HPLC analysis revealed the presence of numerous phenolic compounds such as gallic acid,
rutin, and quercetin while the GC-MS analysis of essential oil showed that the main components are eugenol (78.67%), eugenyl acetate (11.77%), and caryophyllene (6.85%). The
aqueous, ethanolic extracts and essential oil showed an immunomodulatory activity by
exerting a significant inhibition of neutrophil bactericidal activity in a dose dependentmanner reaching maximal inhibition at the concentration of 200 μg/ml with only 29.92%,
32.24%, and 48.15%, respectively (p < 0.001).
Abbreviations: AlCl3 , aluminum chloride; ANOVA, analysis of variance; DMSO, dimethylsulfoxide; EO, essential oil; FBS, fetal bovine serum; FeCl3 , iron
III chloride; FID, flame ionization detector; fMLP, N-formyl-methionyl-leucyl-phenylalanine; GC, gas chromatography; GC-MS, gas chromatography-mass
spectrometry; HCl, hydrochloric acid; HPLC, high-performance liquid chromatography; MRSA, methicillin-resistant staphylococcus aureus; MTT, 3-[4,5dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide; NaOH, sodium hydroxide; NIAID, national institute of allergy and infectious diseases; OD, optical
density; PBS, phosphate buffered saline; PMA, phorbol 12-myristate 13-acetate; PMNs, polymorphonuclear neutrophils; ROS, reactive oxygen species; RPMI,
roswell park memorial institute medium; S. aromaticum, Syzygium aromaticum L; TFC, total flavonoid compounds; TPC, total phenolic compounds.
∗
Corresponding author.
E-mail address: mtairag@hotmail.com (E.M. Mtairag).
https://doi.org/10.1016/j.sciaf.2022.e01395
2468-2276/© 2022 The Authors. Published by Elsevier B.V. on behalf of African Institute of Mathematical Sciences / Next Einstein Initiative. This is an
open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)
O.E. Faqer, S. Bendiar, S. Rais et al.
Scientific African 18 (2022) e01395
Our study showed the immunomodulatory virtues of cloves as a natural antiinflammatory agent. The strength of this effect is related to the presence of eugenol and
the extraction forms used.
© 2022 The Authors. Published by Elsevier B.V. on behalf of African Institute of
Mathematical Sciences / Next Einstein Initiative.
This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/)
Introduction
Inflammation is a physiological process in the body’s defense against the endogenous or exogenous agent. The primary
function of the inflammatory response is to detect those agents, then eliminate or isolate them from the rest of the body,
and to allow the repair of damaged tissues as quickly as possible [1]. This reaction is controlled by specific cells of the
immune system such as Polymorphonuclear neutrophils (PMNs), who are one of the main cells of the innate immune system
fighting against pathogens [2]. The bactericidal function of PMNs is related to exclusive events, including the release of
enzymatic protein contents in the granules or the generation of reactive oxygen species through the respiratory burst [3].
The excessive and exaggerated activation of these types of cells may cause inflammatory diseases thereby partially damaging
the human biological systems [4]. The use of synthetic drugs has worked as a treatment for inflammatory diseases, but it
seems that it causes undesirable side effects following its long use and disrupts the functions of the body [5], hence the
renewed interest in medicinal plants and their therapeutic effects.
In the last decades, the interest in the field of medicinal plants has been steadily increasing. Researchers are deeply
involved in the ethnopharmacological investigation and characterization of the properties, composition, and toxicity of many
medicinal plants to present them as a successful remedy for many health diseases [6]. In this context, medicinal plants are
widely used to cure inflammatory diseases [7] and this effect is mainly due to the presence of secondary metabolites such
as polyphenols, flavonoids, and terpenes [8]. Polyphenols can modulate the response of the innate and adaptive immune
systems, by inhibiting or stimulating several metabolic pathways involved in the inflammatory response [9].
In Moroccan traditional medicine, Syzygium aromaticum L. (S. aromaticum) is considered one of the most important
medicinal plants for its healing properties [10]. The phytochemical analysis of various types of the extract revealed the
presence of different chemical groups such as phenolics, sesquiterpenes, and monoterpenes compounds. According to the
literature, eugenol, eugenol acetate and caryophyllene are the major compounds found in clove essential oil (EO) [11]. The
presence of these types of molecules gives S. aromaticum a large array of biological activities such as anti-inflammatory,
antioxidant, and antibacterial [12].
In the present study, we chemically characterized the aqueous, ethanolic extracts and EO of S. aromaticum, and we also
aimed to evaluate their immunomodulatory effects on the bactericidal activity of human PMNs compared to eugenol, known
as a major compound in S. aromaticum and for its immunomodulatory and anti-inflammatory effect [13,14].
Material and methods
Plant material
S. aromaticum was purchased and obtained from a local market in Casablanca. The plant was identified by Professor
Khyati Najat, from the department of biology, Faculty of Sciences, University Hassan II of Casablanca, Morocco, according
to the flora of Morocco [14]. Certified voucher specimens of the plant of air-dried leaves were deposited at the biology
department in the same faculty under voucher number SA 12032019 for Syzygium aromaticum L. After air-drying in the
shade for a week, the air-dried buds were powdered and used for the extraction.
Preparation of plant extracts
The aqueous extract was prepared by infusion. To 500 ml of distilled water which was previously brought to 70 °C,
50 g of dry powder were added and then allowed to cool down with continual stirring. The ethanolic extract was prepared
by maceration, adding 500 ml of absolute ethanol to 50 g dry powder for 48 h at room temperature in the dark. The
mixtures were centrifuged for 10 min at 2500 rpm, filtered through 3 MM Whatman paper, and then concentrated in a
rotary evaporator under vacuum (water bath set at 50–60 °C). The extracts were recovered and stored in sterile Phosphatebuffered saline (PBS) at pH = 7.4 and stored at −20 °C and in the dark until used.
Isolation of essential oil
EO was isolated by hydrodistillation for 3 h from 100 g of dry powder, using a Clevenger-type apparatus according to the
European Pharmacopoeia [15]. The obtained EO was stored at −20 °C in the dark until analysis.
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Scientific African 18 (2022) e01395
Phytochemical analysis
The aqueous and ethanolic extracts were subjected to qualitative phytochemical screening for the identification of various
classes of active chemical constituents such as phenols, flavonoids, flavones aglycones, coumarins, saponins, tannins, and
sterol-triterpene, using the method described in the literature [16,17].
To detect phenols, a small amount of plant extract was added to 1 ml of water in a test tube and 1 to 2 drops of Iron
III chloride (FeCl3 ) were added, with blue, green, red, or purple colorations being indicative of a positive test. For flavonoid
detection, 3 ml of plant extracts were mixed with 2 ml of 1% aluminum chloride (AlCl3 ) where yellow coloration confirmed
the presence of flavonoids.
As for flavones aglycones, 2 ml of the extract was heated and 5 drops of metallic magnesium was added with concentrated hydrochloric acid (HCl). A red-orange color indicates the presence of flavones aglycones. Regarding saponins, 5 ml of
plant extract were vigorously mixed with 10 ml of distilled water for 2 min. The presence of saponins was then confirmed
by the appearance of foam that persists for at least 15 min, or by the formation of an emulsion when adding olive oil. To
reveal the presence of tannins, 1 ml of plant extracts was mixed with 10 ml of distilled water and then filtered. Three drops
of FeCl3 were added to the filtrate. A blue-black precipitate confirmed the presence of gallic tannins whilst a green precipitate confirmed the presence of catechol tannins. Coumarins were detected by adding 3 ml of 10% NaOH to 2 ml of the
plant extract with yellow coloration indicating the presence of coumarins. Finally, to detect sterols and triterpenes, 10 ml of
the aqueous and ethanolic extracts were put in a beaker for evaporation. Acetic anhydride (0.5 ml) and chloroform (0.5 ml)
were added to the residue. All were transferred into a dry tube with 0.5 ml of added pure sulphuric acid. The presence of
triterpenes was indicated by an intense red-brown coloration whilst the presence of sterols was denoted by green or violet
coloration.
Total phenolic compounds
To determine total phenolic content (TPC), Folin–ciocalteu colorimetric assay was used [16]. 0.5 ml of sample (100 μg/ml)
and 2 ml of sodium carbonate solution (75 g/l) were added to 2.5 ml of 10% Folin-ciocalteu reagent. After 30 min of incubation at room temperature, the absorbance was measured at 765 nm. A standard of gallic acid was used as the calibration
curve. The results are expressed in mg gallic acid equivalents per gram extract (mg GAE/g extract ).
Flavonoids compounds
Total flavonoid compounds (TFC) were measured according to methods described by Ahn et al. [18]. 0.5 ml of 2% AlCl3
ethanol solution was added to 0.5 ml of sample (100 μg/ml) and incubated for 10 min at room temperature. The absorbance
was measured at 420 nm. Quercetin was used as a standard and the results are expressed in mg quercetin equivalents per
gram extract (mg of QuE/g extract ).
HPLC analysis
The determination of bioactive compounds present in the aqueous and ethanolic extracts of S. aromaticum was performed
by high-performance liquid chromatography (HPLC) type Shimadzu equipped with an SPD-20A UV/Vis detector and the LCSolution data processing station.
The extracts were eluted from an RP-C18 column with an isocratic elution of mobile phase water, acetonitrile (88, 12,
respectively), and monitored at 285 nm. The injection volume of all samples and the mixture of polyphenol standards was
20 μl. The flow rate was 1 ml/min.
Polyphenol standards used were: gallic acid, caffeic acid, catechin, caffein, syringic acid, ferulic acid, coumarin, rutin,
vanillin, and quercetin. The standards were procured from Sigma Chemical Company, USA. Chromatograms were registered
at 285 nm. The identification of bioactive compounds present in the extracts was accomplished by comparison of their
retention times with those of pure standards.
Oil analysis
S. aromaticum EO was analyzed by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS).
Gas chromatography (GC)
The analysis was carried out using Shimadzu GC-2010 Plus gas chromatograph equipped with BP-5 capillary column
(30 m x 0.25 mm i.d., film thickness 0.25 μm SGE Ltd) and a flame ionization detector (FID). The temperature was programmed from, 60 to 200 °C, at 3 °C.min−1 , then held isothermal for 5 min; the injector and detector temperatures were
280 and 300 °C, respectively; the carrier gas, nitrogen, adjusted to a linear velocity of 30 cm.s−1 . The samples were injected
using split sampling technique, ratio 1:50. The volume injection was 0.2 μl of a pentane-volatile solution (1:1).
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Scientific African 18 (2022) e01395
GC-MS
The GC-MS unit consisted of a Shimadzu GC-2010 gas chromatograph, equipped with BP-5 capillary column (30 m x
0.25 mm i.d., film thickness 0.25 μm; SGE, Ltd.), and interfaced with a Shimadzu QP2010 Plus mass spectrometer (software version 2.50 SU1). The oven temperature was programmed as described for GC analysis; transfer line temperature,
300 °C; ion source temperature,200 °C; carrier gas, helium, adjusted to a linear velocity of 36.5 cm.s−1 ; split ratio, 1:40;
ionization energy, 70 eV; scan range, 40 400 u; scan time, 1 s. Component identification was carried out by comparison
of their retention indices relative to C9 -C20 n alkanes on the BP-5 column [19], confirmed by comparison of recorded mass
spectra with those of a computer library (Shimadzu corporation library and NIST05 database/ ChemStation data system)
and from a homemade library, constructed based on the analyses of reference oils, laboratory-synthesized components and
commercially available standards and other literature data [20].
PMNs bactericidal assay
The PMNs bactericidal assay was carried out using methods previously described by Stevens et al. [21] with slight modifications.
Isolation of human PMNs
Human heparinized venous blood was obtained from healthy donors following a protocol approved by the National Institute of Allergy and Infectious Diseases (NIAID) Institutional Review Board for Human Subjects (Bethesda, MD, USA). Informed
written consent was acquired from each participant. PMNs were obtained by 2% dextran sedimentation followed by FicollPaque centrifugation, the erythrocytes were eliminated by hypo-osmotic lysis and isolated PMNs were maintained in RPMI
1640 and then kept on ice until used. The viability was assessed by trypan blue dye exclusion and the percentage of the
final preparation’s viability was 95%.
Preparation of opsonized bacteria
108 CFU of Methicillin-resistant Staphylococcus aureus (MRSA) ATCC 43,300 was suspended in 1 ml of RPMI 1640 containing 10% of inactivated autologous serum, then, rotated and incubated at 37 °C for 20 min. The opsonized bacteria were used
immediately after preparation.
In vitro treatment of PMNs
In sterile 96-well microtiter plates, using triplicate dispatching, 50 μl of 107 PMNs/ml in RPMI 1640 containing 5% of Fetal
Bovine Serum (FBS) were pre-treated with 15 μl of the aqueous and ethanolic extracts, EO at the following final concentrations: 50, 100, 150 and 200 μg/ml and eugenol (Sigma Aldrich, reagent plus 99%) at 5, 10, 15 and 20 μg/ml. The viability
of PMNs was not affected by the range of concentrations used. Any concentration beyond was observed to compromise
cell integrity. The EO and eugenol were dissolved in a 0.1% Dimethylsulfoxide (DMSO) solution which had no effect on the
bactericidal activity of PMNs [21]. The PMNs were incubated for 30 min at 37 °C before being used.
Colorimetric bactericidal assay
50 μl of opsonized MRSA were added to the treated PMNs resulting in a ratio of 10 bacteria per PMN. Plates were
then incubated for 1 h at 37 °C under agitation to permit the killing of bacteria by PMNs. A positive control comprised of
untreated PMNs and 50 μl of opsonized MRSA was incubated. A standard curve of bactericidal activity was established by
diluting opsonized bacteria in RPMI 1640 containing 5% FBS to correspond to 0, 30, 60, and 90 reduction in cell number.
PMNs were lysed by adding 50 μl of 0.2% Triton X-100, and 50 μl of 2 mg/ml of MTT were added to each well, then the
plates were incubated for 10 min at room temperature, followed by centrifugation at 1600 g for 5 min. Once the supernatant
was removed, 150 μl of DMSO were added to the wells as they were left to incubate for 10 min at room temperature. The
plates were then carefully shaken to facilitate the dissolution of formed formazan by DMSO. Finally, 50 μl of PBS pH = 7.4
were added to properly solubilize the remaining formazan.
To quantify the formazan produced by bacteria, a measure of absorbance was performed at 560 nm. An Optical density
(OD) corresponding to 0% and 90% of viable bacteria was established by linear regression analysis using a standard curve.
The percentage of killed bacteria was determined using the following formula:
1−
(ODsample ) − (OD 90% kil l ing)
× 90%
(OD 0% kil l ing) − (OD 90% kil l ing)
Statistical analysis
Data are presented as mean ± SD. One-way ANOVA test followed by Tukey’s multiple comparisons test was applied for
multiple groups. Statistical analyses were conducted by GraphPad Prism 8.0.2 (GraphPad Software Inc, San Diego, CA, USA).
Differences were considered significant at p < 0.05.
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Scientific African 18 (2022) e01395
Table 1
Phytochemical screening of aqueous and ethanolic extracts of S. aromaticum.
Chemical
constituents
S. aromaticum
Aqueous
Ethanolic
Phenols
Flavonoids
Flavones aglycones
Coumarins
Saponins
Gallic tannins
Catechic tannins
Sterol
Triterpene
+++
++
++
+++
–
++
–
+++
–
+++
+++
++
++
+
++
–
+++
–
− Negative result, + Positive result, ++ Present in high concentration
and +++ Present in very high concentration.
Table 2
The yield of crude extracts and total phenols and flavonoid content of aqueous and ethanolic extracts of S. aromaticum.
S. aromaticum
∗
Extract
Yield (%)
Total phenolic compounds
(TPC) (mg GAE/gextract )
Total flavonoid compounds
(TFC) (mg QE/gextract )
Aqueous
Ethanol
30.00
26.00
10.53 ± 0.32∗
62.42 ± 0.92∗
1.47 ± 0.24ns
1.13 ± 0.11ns
p < 0.001 TPC clove: aqueous vs ethanolic; ns p = 0.08 TFC clove: aqueous vs ethanolic, with n = 3.
Table 3
Phenolic compounds identified by HPLC, in aqueous and ethanolic extracts of S. aromaticum.
Compounds
Retention time
(min)
S. aromaticum
Peak N°
Aqueous
Ethanolic
1
2
3
4
5
6
7
8
9
10
Gallic acid
Caffeic acid
Catechine
Cafein
Syringic acid
Ferulic acid
Coumarin
Rutin
Vanillin
Quercetin
3.38
8.22
9.10
9.60
14.11
19.63
20.96
23.33
24.06
24.18
+
–
+
–
+
–
+
–
–
–
+
–
+
–
+
–
–
–
–
+
Results
Phytochemical screening
The results of phytochemical screening of the aqueous and ethanolic extracts of S. aromaticum are listed in Table 1. All
extracts showed the presence of phenols, flavonoids, flavones aglycones, coumarins, and tannins. An absence of saponins
was observed in the aqueous while they are present in the ethanolic extract. There was no presence of triterpenes in both
extracts.
Total phenolic and flavonoid composition
The results obtained from TPC and TFC composites of the aqueous and ethanolic extracts of S. aromaticum are presented
in Table 2. These results show that the TPC of the ethanolic and aqueous extracts of S. aromaticum is 62.42 ± 0.92 mg
of GAE/g extract and 10.53 ± 0.32 mg of GAE/g extract , respectively. Concerning the TFC, we obtained concentrations of
1.47 ± 0.24 mg of QE/g extract and 1.13 ± 0.11 mg of QE/g extract of the aqueous and ethanolic extracts, respectively.
Qualitative analysis of extracts by HPLC
The focus of this study was to determine and analyze the presence of bioactive compounds present in the ethanolic
and aqueous extracts of S. aromaticum using HPLC. The identified compounds by matching retention times of phenolic standards and the studied extracts are presented in Table 3. The results obtained revealed the presence of gallic, syringic acids,
catechin, and coumarin for the aqueous extract, and gallic, syringic acids, catechin, and quercetin for the ethanolic extract.
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Scientific African 18 (2022) e01395
Table 4
Chemical composition of S. aromaticum. EO.
Kovats index
Components
Cyclopropyl carbinol
Octane
Carvacrol
Eugenol
(Z) Caryophyllene
(E) Caryophyllene
γ -cadinene
Eugenyl acetate
Caryophyllene oxide
Total
KI Exp
a
Percentage (%)
KI Lit
648
800
1298
1356
1408
1417
1513
1521
1582
647
802
1296
1355
1406
1418
1510
1518
1579
b
0.12
0.05
0.16
78.67
6.85
0.27
0.10
11.77
0.45
98.4
a
Linear retention index on BP-5 column, experimentally determined by using homologous series
of C9 –C20 alkanes.
b
Relative linear retention index literature taken from Adams for BP-5 capillary column.
Chemical composition of EO
The results of the identified compounds and their respective percentages within the EO of S. aromaticum are summarized in Table 4. A total of 9 compounds representing 98.4% were identified and characterized by high amounts of eugenol
(78.67%), eugenol acetate (11.77%), and caryophyllene (6.85%).
Neutrophil bactericidal assay
The effect of the aqueous, ethanolic extracts, EO of S. aromaticum, and eugenol on the bactericidal activity of PMNs
was evaluated by colorimetric assay. The pre-treatment of PMNs for 30 min with 100, 150, 200 μg/ml of S. aromaticum
aqueous and ethanolic extracts, EO, and 5, 10, 15, 20 μg/ml of eugenol revealed inhibition of PMNs bactericidal activity in
a dose-dependent manner (p < 0.001). Maximal inhibition of PMNs bacterial activity was obtained at the concentration of
200 μg/ml with only 29.92%, 32.24%, and 48.15% for the aqueous, ethanolic extracts, and EO, respectively (Fig. 1A–C). Eugenol
at the concentration of 20 μg/ml, showed the same inhibition of this activity as that obtained with EO reaching only 49.75%
(Fig. 1D).
Discussion
Medicinal plants play a major role in the treatment and prevention of many health disorders and diseases. The established presence of bioactive compounds in plants suggests different potential biological activities that could play an important role in immunomodulation [22]. It is known that PMNs have an important role in eliminating microorganisms via their
major function, i.e. degranulation and oxidative burst, and play a role in cellular homeostasis [23]. Therefore, if the production level of reactive oxygen species (ROS) is increased by deregulation of the immune system, it may cause severe damage
to healthy cells, leading to auto-immune diseases [24].
The phytochemical screening of the aqueous and ethanolic extracts of S. aromaticum indicates the presence of an interesting level of phenolic and flavonoid compounds. According to the literature, many studies showed the presence of these
molecules in these extraction types. Gowri and Manimegalai [25] and Gupta et al. [26] reported the same result as us in the
phytochemical screening for the ethanolic and aqueous extracts. Jimoh et al. showed the presence of phenolic and flavonoid
compounds [27]. This is in accordance with our results. It seems interesting that for S. aromaticum extracts, the ethanolic
extract contains more TPC than the aqueous extracts. In agreement with our results, El Ghallab et al. reported the amount
of TPC in clove and found that the ethanolic extract has more TPC than the aqueous extract with 351.83 mg GAE/gextract and
45.57 mg GAE/gextract , respectively [28]. On the other hand, the amount of TFC seems almost identical in the two extracts
and we can conclude that for TFC there is no difference between the solvents.
HPLC analysis allowed us to identify the bioactive compounds present in both the aqueous and ethanolic extracts of
S. aromaticum. Results showed the presence of four compounds in S. aromaticum extracts. Adefegha et al. analyzed the
HPLC phenolic profile of clove buds from Nigeria. The raw aqueous extract revealed the presence of gallic, caffeic, ellagic,
chlorogenic acids, rutin, catechin quercitrin, quercetin, kaempferol, and luteolin [29]. Hina et al. investigated the chemical
composition of cloves ethanolic extract from Pakistan and they found six major phenolic compounds, namely gallic, ferulic,
vanillic, sinapic, p-coumaric acids, and quercetin [30]. The presence of quercetin in the ethanolic extract could be explained
through the extraction method and the used solvent because it is known for its solubility in ethanol which explains its
presence in the ethanolic extract [31].
In this study, GC-MS allowed the characterization of the plant’s EO components. Nine molecules were identified for clove
and the major components were eugenol (78.67%), eugenol acetate (11.77%), and caryophyllene (6.85%). It was reported by
6
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Scientific African 18 (2022) e01395
Fig. 1. In vitro effect of aqueous, ethanolic extracts, EO of S. aromaticum, and eugenol on human PMNs bacterial activity. Results are represented as the
mean (n = 3) ± SD level of the percentage of killed bacteria. ∗ p < 0.001, aqueous (A), ethanolic (B) extracts, EO (C), eugenol (D) vs control by Ordinary
one-way ANOVA test.
Chaieb et al. that 36 components were found in cloves’ oil with a higher proportion of eugenol (88.58%) whilst eugenol
acetate was the second most abundant component similar to our results [32]. However, 13 molecules were identified by El
Ghallab et al. where eugenol was the most abundant at a percentage of 55.28%, and the second most abundant molecule
was β -caryophyllene [28]. It is suggested that this level of difference in the composition of the aqueous, ethanolic extracts
and EO of cloves might be due to numerous factors such as geographic zones, genetic species, climatic conditions, as well
as extraction methods, and the solvent selectivity [33].
We investigated the immunomodulatory effect of extracts and EO of S. aromaticum and eugenol on the bactericidal function of human PMNs. The results of this in vitro study showed that all extracts, EO, and eugenol exert inhibition of the
bactericidal function of PMNs in a dose dependent-manner (p < 0.001). With these results, we suggest that the presence of
polyphenols compounds could affect the PMNs functions by suppressing Myeloperoxidase in degranulation, the production
of ROS in the oxidative burst, and also the phagocytic function. The observed inhibition effect obtained with EO could be
attributed to the presence of eugenol (78.4%). Despite the low concentrations of eugenol used, we obtained almost the same
percentage of inhibition as EO, which proves that eugenol could be the main actor in the suppressed antibacterial activity
of PMNs. Eugenol, a major phenolic compound in S. aromaticum, represents between 45 and 90% of the EO and possesses
a large scale of pharmacological activities such as analgesic, antioxidant, and anti-inflammatory [34]. In agreement with
our results, Chen et al. found that eugenol exerts a suppression effect on the antimicrobial functions of PMNs towards oral
pathogens Streptococcus mutans and Actinobacillus actinomycetemcomitans [3].
Over the past two decades, the amount of research into the beneficial health and therapeutic effects of polyphenols has
increased [22,35]. Polyphenols have shown anti-inflammatory, antioxidant, immunomodulatory, antimicrobial, and anticar7
O.E. Faqer, S. Bendiar, S. Rais et al.
Scientific African 18 (2022) e01395
cinogenic properties [36]. In the same approach, we found that our ethanolic extract contains more TPC than the aqueous
one, and could explain the difference in inhibition level between them.
Many studies demonstrated that plant extracts inhibit neutrophil functions, in effect, Kenny et al. found in an in vitro
study that the presence of flavonoids in cocoa moderates the signaling pathways derived from LPS stimulation on neutrophil
oxidative burst [37]. The presence of flavonoids in the aqueous extract of corn (Zea mays) silk was observed to exert a decrease in the bactericidal function of PMNs in a dose-dependent manner [38]. Pincemail et al. demonstrated the inhibitory
effect of the aqueous extract of ginkgo (Ginkgo biloba) on PMNs’ oxidative burst when stimulated by Phorbol 12-myristate
13-acetate (PMA). This effect is essentially related to the presence of flavonoid compounds in this type of extract [39]. Furthermore, Hung et al. investigated the in vitro effect of Areca catechu nut aqueous extract, which is rich with polyphenols
compounds, on the PMNs functions and demonstrated that it could reduce bactericidal activity and superoxide anion production [40]. Thus, the extracts may interact with PMNs by directly or indirectly inhibiting the signaling pathways involved
in the main functions.
Conclusion
Investigation of the immunomodulatory effect of S. aromaticum extracts, EO, and eugenol on the bactericidal function
of PMNs, revealed their potential use as anti-inflammatory agents. Interestingly, eugenol can be used as an active principle
to study in vitro, the signaling pathways involved in this immunomodulatory effect on PMNs stimulated with N-formylMethionyl-Leucyl-Phenylalanine (fMLP) or PMA. Thus, further studies are currently underway to evaluate the effect of EO
and eugenol on the principle PMNs functions: oxidative burst and degranulation and also the signaling pathways of their
effects.
Funding
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
CRediT authorship contribution statement
Othman El Faqer: Conceptualization, Writing – original draft. Salma Bendiar: Conceptualization, Formal analysis. Samira
Rais: Conceptualization, Validation. Ismail Elkoraichi: Conceptualization, Formal analysis. Mohamed Dakir: Conceptualization, Visualization. Anass Elouaddari: Conceptualization, Investigation. Abdelaziz El Amrani: Conceptualization, Methodology, Data curation. Mounia Oudghiri: Conceptualization, Methodology. El Mostafa Mtairag: Conceptualization, Supervision.
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