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Phytochemical characterization and immunomodulatory effects of aqueous

and ethanolic extracts and essential oil of Moroccan Laurus nobilis L.
(Lauraceae) on human neutrophils

Article in Journal of Herbmed Pharmacology · January 2023

DOI: 10.34172/jhp.2023.08

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 J Herbmed Pharmacol. 2023; 12(1): 92-99.

http://www.herbmedpharmacol.com doi: 10.34172/jhp.2023.08

Journal of Herbmed Pharmacology

Phytochemical characterization and immunomodulatory

effects of aqueous and ethanolic extracts and essential oil of
Moroccan Laurus nobilis L. (Lauraceae) on human neutrophils
ID ID ID ID ID
Othman El Faqer1 , Samira Rais1,2 , Ismail Elkoraichi1 , Abdelaziz El Amrani3 , Mohamed Dakir3 , Younes
ID
Zaid1,4 , El Mostafa Mtairag1* ID
1
Immunology and Biodiversity Laboratory, Faculty of Sciences Ain Chock, Hassan II University, Casablanca, Morocco
2
Department of Biology, Faculty of Sciences Ben M’Sick, Hassan II University, Casablanca, Morocco
3
Laboratory of Organic Synthesis, Extraction, and Valorization, Faculty of Sciences Aïn Chock, Hassan II University, Casablanca, Morocco
4
Department of Biology, Faculty of Sciences, Mohammed V University, Rabat, Morocco

ARTICLE INFO ABSTRACT

Article Type: Introduction: The leaves of Moroccan bay laurel (Laurus nobilis L.) have been used in several
Original Article forms of extracts to cure rheumatic pain due to their anti-inflammatory properties. Our work
aimed to evaluate the effects of aqueous and ethanolic extracts, as well as the essential oil (EO)
Article History: from laurel, on the microbicidal activity of human neutrophils when compared to the effect
Received: 21 July 2022 of eucalyptol.
Accepted: 3 October 2022 Methods: The extracts (ethanolic and aqueous) were subject to phytochemical profiling
and high-performance liquid chromatography (HPLC) analyses. The EO obtained by
Keywords:
hydrodistillation from laurel was analyzed by gas chromatography-mass spectrometry (GC-
Bay laurel
MS). The immunomodulatory effects on neutrophil microbicidal activity of the extracts,
1,8-Cineole
EO, and eugenol were carried out by 3-(4,5-diméthylthiazol-2-yl)-2,5-diphényltétrazolium
Bioactive compounds
(MTT) assay.
Polymorphonuclear neutrophil
Results: The phytochemical analysis of the extracts revealed the presence of flavonoids,
Immunomodulatory compounds
coumarins, phenols, flavone aglycones, and tannins. HPLC analysis showed the presence
of numerous phenolic molecules such as syringic acid, ferulic acid, gallic acid, caffeine,
and quercetin. The chemical composition of EO revealed that the major components were
eucalyptol (44.14%), α-terpinyl acetate (11.11%), and β-phellandrene (6.74%). Aqueous
and ethanolic extracts and EO revealed a significant and dose-dependent ability to inhibit
neutrophils microbicidal activity with maximal inhibition at 200 µg/mL concentration with
30.42%, 24.7%, and 38.13%, respectively (P < 0.001).
Conclusion: The obtained results revealed the immunomodulatory properties of laurel as
a potential natural anti-inflammatory agent that would also allow the development of new
anti-inflammatory drugs.

Implication for health policy/practice/research/medical education:

The extracts and essential oil (EO) of bay laurel, as well as eucalyptol, revealed a good potential immunomodulatory effect
on human polymorphonuclear neutrophils (PMNs) microbicidal activity. In vivo assays are solicited to obtain more evidence
for the observed effect. Molecules present in bay laurel extracts and EO might be used as an alternative to drugs to attenuate
inflammatory problems.
Please cite this paper as: El Faqer O, Rais S, Elkoraichi I, El Amrani A, Dakir M, Zaid Y, et al. Phytochemical characterization
and immunomodulatory effects of aqueous and ethanolic extracts and essential oil of Moroccan Laurus nobilis L. (Lauraceae) on
human neutrophils. J Herbmed Pharmacol. 2023;12(1):92-99. doi: 10.34172/jhp.2023.08.

Introduction elimination and permitting the curative process (2).

In the innate immune system, inflammation is the Many cells are involved in this type of immune response.
response to damaging stimuli (pathogens, irradiation, Polymorphonuclear neutrophils (PMNs) represent 70% of
damaged cells, or toxic compounds) (1), leading to its the circulating leukocyte population in the bloodstream.

*Corresponding author: El Mostafa Mtairag,

Email: [email protected]
 Immunomodulatory effect of Laurus nobilis L. on human PMNs

They are considered one of the principal effectors of Material and Methods
the innate response; they are among the first cells to Preparation of plant material and extracts
be present at the inflammatory site and are capable of The plant L. nobilis was purchased from a local market
phagocyte and degrading a wide range of pathogens in Casablanca, Morocco. It was collected during the full
(3). The microbicidal function of PMNs is ensured by flowering period on May 3, 2019 in Taza – Morocco
the production of antibacterial oxidants, peptides, and (Latitude 33.938848 and Longitude -4.305174). The
proteases (4). Moreover, major dysfunction of the primary identification of the plant was realized as described in
function of these cells by prolonged activation, excessive our previous work (19). A specimen was deposited under
and unnecessary accumulation, over-activation, or certificate number LN 05062019 for Laurus nobilis L.
disruption can cause several health problems (3). Briefly, the leaves aqueous and ethanolic extracts were
Recently, many researchers focused their research on prepared respectively, by infusion and maceration of the
numerous studies on medicinal plants (MP), one of them powdered leaves of L. nobilis (50 g each) with 500 mL of
being an ethnopharmacological investigation permitting the appropriate solvents. The extraction procedure was
the identification of their chemical composition, toxicity, further continued according to the described scheme in
and biological properties leading them to discover novel our previous work (19).
therapeutic molecules (5). For ages, people have treated
inflammatory diseases using MP (6). Research suggests Extraction of essential oil
that it is linked to the presence of natural products like Extraction of EO was realized by hydrodistillation from
polyphenols, terpenes, and tannins (7). It has been laurel for 3 hours using a Clevenger-type apparatus. The
found that these biomolecules may regulate the immune EO was kept at -20°C in the dark until use.
response by inhibiting or stimulating cellular signaling
pathways of the inflammatory response (8). Phytochemical screening
Among the Mediterranean countries, Morocco has a To identify the different classes of natural products present
particular geographical position granting it a wide strip in L. nobilis extracts, a qualitative phytochemical screening
of very diverse vegetation, in particular MP. In Morocco, was realized according to the methods described in the
the use of MP is a very old practice, and this empirical literature (20,21).
knowledge has been handed over verbally to generations The presence of phenols was confirmed by a purple,
and has been enriched through the country’s strategic green, red, or blue coloration after adding Iron III chloride
approaches. Laurus nobilis L. (bay laurel) from the (FeCl3) to samples. A yellow coloration after adding 1%
Lauraceae family is an aromatic MP located in the Rif of aluminum chloride (AlCl3) to samples indicates the
Morocco known for its healing properties (9). It is used presence of flavonoids. The red-orange color after adding
in Mediterranean gastronomy (10) for seasoning and metallic magnesium and pure hydrochloric acid (HCl)
folk medicine, in addition to the use of its fruits to relieve to the heated samples confirms the presence of flavones
and treat many diseases, such as impaired digestion, aglycones. Saponins were detected by the formation of
viral infections, rheumatism, and other health problems foam persisting for at least 15 minutes after vigorously
(11). Many researchers reported the antimicrobial (12), mixing samples with distilled water. Green and dark
antioxidant, anticancer (13), anti-inflammatory (14,15), blue precipitation after the addition of 3 drops of 1%
and antifungal (16) effects of bay laurel extracts and FeCl3 solution confirm the presence of catechol tannins
essential oil (EO). These activities give bay laurel extracts and gallic tannins, respectively. A yellow coloration
and EO numerous utilization in various industries. after adding 10% NaOH to samples was an indicator of
The isolated components from the leaves showed great coumarins. Triterpenes and sterols were detected by
interest compared to those of the other parts of the dissolving dried samples in 0.5 mL of acetic anhydride
plant. The presence of eucalyptol, α-pinene, methyl 0.5 mL of chloroform then 0.5 mL of pure sulphuric acid
eugenol, α-terpineol, and β-pinene have been suggested was added. A red-brown coloration was the triterpenes
as major compounds in bay laurel EO responsible for indicator and green or violet coloration was the sterols
the antioxidant, antibacterial, and anti-inflammatory indicator.
effects (17,18). Despite several studies engaged in the
phytochemical analysis of EO and natural products from Determination of total phenolic and flavonoids content
the extracts of bay laurel collected from different countries To quantify total phenolic content (TPC) and total
and origins, their immunomodulatory effects on human flavonoid content (TFC), Folin-Ciocalteu and aluminum
PMNs are rare, and there are no Moroccan species colorimetric methods were used, respectively (22,23).
described in the literature. This study aims to evaluate Phenol and flavonoid contents were expressed in
the immunomodulatory effects of aqueous and ethanolic milligrams per gram of gallic acid (mg GAE/g extract) and
extracts and EO of Moroccan L. nobilis on human PMNs quercetin (mg of QuE/g extract) equivalents, respectively,
microbicidal activity in comparison to eucalyptol. from calibration curves.

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 El Faqer et al

Identification of phenolic compounds by HPLC samples to completely dissolve the residual formazan.
The quantitative high-performance liquid chromatography Produced formazan by viable bacteria was quantified at
(HPLC) assay of L. nobilis extracts was carried out 560 nm. The results were expressed as:
using the HPLC technique as described in our previous
work using the same equipment and a fully validated (OD sample) − (OD 90% killing )
% of killed bacteria =
1− × 90%
(OD 0% killing ) − (OD 90% killing )
chromatographic procedure (19).

Essential oil analysis Statistical analysis

The chemical analysis of EO was realized using GC/MS All experiments were realized in triplicate. Results were
technique as described in our previous work (19). presented as mean ± standard deviation. The comparison
test was carried out by a one-way ANOVA test followed by
PMNs microbicidal activity Tukey’s multiple for multiple groups. Statistical tests were
The microbicidal activity of PMNs was realized according performed by GraphPad Prism 8.0.2. Differences were
to methods established by Stevens et al (24). significant when P < 0.05.

Isolation of PMNs Results

To isolate PMNs, human heparinized venous blood Screening analysis
was obtained from healthy donors following a protocol We studied the chemical composition of L. nobilis extracts
approved by the Institutional Review Board for Human and the results are presented in Table 1. Both extracts
Subjects of the National Institute of Allergy and Infectious contained phenols, flavone aglycones, flavonoids, tannins,
Diseases (NIAID) (Bethesda, MD, USA). PMNs were and coumarins. The ethanolic extract revealed the
isolated as previously described (25). The viability of presence of saponins while being absent in the aqueous
PMNs was 95% determined by the trypan blue dye extract and no triterpenes were registered in both extracts.
method. PMNs were resuspended in RPMI 1640 and then
maintained on ice until use. Total phenolic and flavonoid content
The results of the quantification of the total phenolic and
In vitro treatment of PMNs flavonoid content of L. nobilis extracts are presented in
Fifteen microliters of extracts, and EO of L. nobilis (50, Table 2. The aqueous extract showed a higher level of TPC
100, 150, and 200 µg/mL) and eucalyptol (Elevations than the ethanolic extract with 13.32 ± 1.24 mg and 7.27
terpenes) at (5, 10, 15, and 20 µg/mL) were added to 50 µL ± 1.45 of GAE/g extract, respectively. For TFC level, there
of PMNs solution (107/mL) in RPMI 1640 supplemented was no difference between aqueous and ethanolic extract
with 5% of fetal bovine serum (FBS) and incubated for with 0.63 ± 0.69 mg, and 0.64 ± 0.28 mg of QE/g extract,
30 minutes at 37°C before use. The PMNs viability was respectively.
not affected by the range of concentration used; however,
PMNs viability decreased with concentrations above the HPLC analysis
aforementioned values. To dissolve EO and eucalyptol, The aim of this study was the analysis and determination
a 0.1% of dimethylsulfoxide (DMSO) solution was used, of biomolecules present in L. nobilis extracts using HPLC.
which did not affect the PMNs function (26). The identified molecules by HPLC in the extracts are
given in Table 3. In the ethanolic extract, we identified
Colorimetric microbicidal assay quercetin, cafein, rutin, ferulic, gallic, and syringic acids,
Bacteria (Methicillin-resistant Staphylococcus aureus
ATCC 43300) were opsonized with 10% of autologous
Table 1. Qualitative screening of Laurus nobilis extracts
inactivated human serum for 20 minutes at 37°C.
Briefly, 50 µL of bacteria (108 CFU/mL) were mixed with L. nobilis
Chemical constituents
untreated PMNs (10/1) and let incubated under agitation Ethanolic Aqueous
at 37°C for 1 hour. A control sample containing 50 µL Flavonoids + +
of bacteria and 50 µL of untreated PMNs was incubated Coumarins + +
in parallel. After incubation, 50 µL of Triton X-100 (0.2 Phenols + +
%) was added to lyse PMNs, 50 µL of MTT solution (2 Saponins + -
mg/mL) was added in each sample, and then incubated Flavones aglycones + +
at ambient temperature for 10 minutes. All samples were Triterpenes - -
centrifuged at 1600 g for 5 minutes. After removing the Sterols + +
supernatant, 150 µL of DMSO was added to the samples Catechic tannins + +
and left incubated at ambient temperature for 10 minutes Gallic tannins - -
under shaking. In the end, PBS (50 µL) was added to the − Absence, + Present.

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 Immunomodulatory effect of Laurus nobilis L. on human PMNs

Table 2. Total phenol and flavonoid content of Moroccan Laurus nobilis extracts

Sample Extraction yield (%) Phenolic content (mg GAE/gextract) Flavonoid content (mg QuE/gextract)

Ethanolic 17.44 7.27 ± 1.45* 0.64 ± 0.28ns

L. nobilis
Aqueous 18.22 13.32 ± 1.24* 0.63 ± 0.69ns

Results are presented as mean ± standard deviation (n = 3), * P < 0.001 Phenolic content of aqueous extract compared to phenolic content of ethanolic
extract; ns: P = 0.08 Flavonoid content of aqueous extract compared to the flavonoid content of the ethanolic extract. QE: Quercetin equivalent; GAE,
Gallic acid equivalent.

while in the aqueous extract we detected rutin, cafein, Table 4. Chemical constituents of Moroccan Laurus nobilis essential oil
detected by gas chromatography/mass spectrometry
syringic, gallic and ferulic acids.
Kovats index
Components Area %
Essential oil analysis KI Expa KI Litb
GC/MS analysis permitted the identification of Benzene 654 655 0.36
34 compounds representing 92.9% of the total EO 2,4-Dimethylhexane 734 732 0.84
composition (Table 4). The major compounds were as Isovaleric acid 827 826 0.03
follows: eucalyptol (44.14%), α-terpinyl acetate (11.11%), 2-Heptanol 894 895 0.03
and β-phellandrene (6.74%). Isobutyl isobutyrate 908 905 0.08
α-Thujene 924 922 0.27
PMNs microbicidal assay α-Pinene 932 930 3.98
We studied the effect of L. nobilis extracts, EO, and Trans-1,2-bis-(1-methylethenyl)
934 933 2.43
eucalyptol on human PMNs microbicidal activity. As cyclobutane
shown in Figure 1, pretreatment with extracts, EO, and Camphene 946 944 0.44
eucalyptol caused a significant decrease in PMNs function β-Pinene 974 975 3.86
in a dose-dependently (P < 0.001). The highest decrease in β-Myrcene 988 989 0.45
this activity was obtained at 200 µg/mL with a reduction δ-Carene 1001 1000 0.32
of 38.13%, 30.42%, and 24.7% for the EO, the aqueous β-Phellandrene 1002 1002 6.74
extract and the ethanolic extract, respectively (Figure p-Cymene 1020 1018 2.51
1A, B, C). Meanwhile, the use of eucalyptol (20 µg/mL), Eucalyptol 1026 1025 44.14
revealed a higher inhibition than that obtained with EO β-cis-Ocimene 1032 1032 0.28
reaching 44.56% (Figure 1D). cis-Sabinene hydrate 1041 1040 0.19
γ-Terpinene 1054 1051 0.74
Discussion Linalool 1095 1096 3.56
According to the literature, degranulation and oxidative Terpinen-4-ol 1174 1175 2.31
burst are the main functions of PMNs to destroy α-Terpineol 1186 1184 1.89
pathogens (26), and also control cellular homeostasis (27). Anisole, p-allyl- 1198 1196 0.14
Nevertheless, the immoderate and exorbitant activation Nerol 1227 1227 0.18
of PMNs by a dysregulation of the immune system may 3,8-Dimethylundecane 1228 1228 0.08
Bornyl acetate 1284 1283 0.46
Table 3. HPLC profile of Moroccan Laurus nobilis extracts α-Terpinyl acetate 1346 1345 11.11
2-Dodecanone 1388 1389 0.11
Retention time L. nobilis
Components Caryophyllene 1417 1416 0.04
(min) Ethanolic Aqueous
(E) Methyl isoeugenol 1451 1450 2.77
Gallic acid 3.38 + +
cis-Aconitic anhydride 1524 1523 0.02
Caffeic acid 8.22 - - 10-Oxocyclodec-2-enecarboxylic acid,
1680 1679 0.03
Catechine 9.10 - - methyl ester
Cafein 9.60 + + 1,6-Octadien-3-ol, 3,7-dimethyl-,
2157 2155 0.16
Syringic acid 14.11 + + 2-aminobenzoate
3-Allyl guaiacol (CAS Number.: 1941-12-4) 2169 2170 0.61
Ferulic acid 19.63 + +
Coumarin 20.96 - - 1H-Inden-5-ol, 2,3-dihydro-3-(4-
2331 2330 1.72
Rutin 23.33 + + hydroxyphenyl)-1,1,3-trimethyl-

Vanillin 24.06 - - Total 92.9

Quercetin 24.18 + -
a
Experimental linear retention index. b
Relative retention index in
literature.

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 El Faqer et al

Figure 1. In vitro effect of L. nobilis on human PMNs microbicidal activity. (A) aqueous extract, (B) ethanolic extract, (C) essential oil, and (D) eucalyptol.
Data are expressed as means ± standard deviation (n = 3) level of the percentage of killed bacteria. * P < 0.001 compared to the control group (untreated
PMNs) by ordinary one-way ANOVA test.

cause inflammatory diseases and lead to damage to the of pyrogallol, resorcinol, gallic, and coumaric acids in
human biological system (28). ethanolic extract (32). The fact that quercetin is present
In the current work, we studied the chemical profile in the ethanol extract could be related to the solvent and
of Moroccan L. nobilis extracts (aqueous and ethanolic) the extraction technique. One can explain its presence in
and we found that they contained flavonoid and phenolic the ethanolic extract by the fact of its solubility in ethanol
molecules. Previous studies have revealed the attendance (33).
of these biomolecules in these extraction forms. Silva et Additionally, we were able to fully characterize the
al analyzed the aqueous extract of L. nobilis and showed components of L. nobilis EO with the gas chromatography-
the presence of flavonoid and phenolic molecules (29). mass spectrometry (GC-MS). Thirty-four compounds
Pugazhenti et al identified the composition of Indian were identified with eucalyptol (44.14%), α-terpinyl
laurel and found that both ethanolic and aqueous extracts acetate (11.11%), and β-phellandrene (6.74%) as major
contained flavonoid and phenolic compounds (30). compounds. Our EO was characterized by a high content
This is in concordance with our results obtained on the of eucalyptol, which was much higher than what was
Moroccan specie. previously mentioned by Fiorini et al (34), who found
To identify the bioactive molecules of Moroccan L. an amount of 39% and lower than Yalçini et al (35)
nobilis extracts, an HPLC analysis was done. The results and Chericoni et al (36), who noticed the presence of
showed the presence of six components in the ethanolic eucalyptol at 58.59% and 50%, respectively. For α-terpinyl
extract and five in the aqueous extract. Many studies have acetate, Yalçini et al and Chericoni et al reported a low
shown the existence of other phenolic molecules. A study content of 8.8% and 6%, respectively. Fiorini et al found
realized by Muchuweti et al identified ferulic, caffeic, 18% of α-terpinyl acetate, which is higher compared to our
and vanillic acids in Zimbabwean laurel extract (31). An findings. Many factors can influence the composition of L.
HPLC analysis of Mexican laurel showed the presence nobilis extracts and EO. Solvents, extraction techniques,

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 Immunomodulatory effect of Laurus nobilis L. on human PMNs

species, regions, and the climatic conditions of plant suggesting their immunomodulatory effects and their
culture can play a role in this observed composition (37). potential use as anti-inflammatory agents. This begs the
In this work, we also studied the immunomodulation question with regards to the signaling pathways involved
effects of extracts and EO of L. nobilis compared to in EO and eucalyptol effects on the main PMNs functions,
eucalyptol on human PMNs microbicidal activity. Our which can be studied in vitro. In this context, more
data revealed that all extracts, EO, and eucalyptol were insightful studies are still required to evaluate the effect of
significantly able to inhibit the microbicidal function EO and eucalyptol on degranulation and oxidative burst
of PMNs (P < 0.001). According to these results, we of N-formylmethionyl-leucyl-phenylalanine or phorbol
can explain this effect with the presence of secondary 12-myristate 13-acetate (PMA)-stimulated PMNs.
metabolites that could exert an effect on the main PMNs
functions, degranulation, and oxidative burst by interfering Acknowledgments
with their signaling pathways. During these twenty years, The authors would like to thank Faculté des Sciences,
researchers have concentrated their attention on exploring Université Hassan II-Ain Chock for facilitating the
the potential therapeutic and health-promoting effects accomplishment of the current study.
of secondary metabolites (38). Numerous studies have
demonstrated the biological properties of polyphenols Author contributions
as immunomodulatory, anti-inflammatory, antioxidant, All authors contributed to the study design, experiments,
and antimicrobial agents (39). In the same context, a data analysis, and interpretation. OEF: Conceptualization,
higher level of TPC was obtained in the aqueous extract writing original draft; SR: Conceptualization, validation;
than in the ethanolic, which could explain the difference IEK: Conceptualization, formal analysis; AEA:
in the observed immunomodulatory effects on PMNs. Conceptualization, methodology, data analysis; MD:
According to the literature, plant extracts can inhibit PMNs Conceptualization, methodology; YZ: Conceptualization,
functions. Bedouhene et al studied the effect of Algerian visualization; EMM: Conceptualization, supervision.
olive mill wastewater polyphenol extract on PMNs. They
reported that the polyphenols exert an inhibition of Conflict of interests
reactive oxygen species production by PMNs proposing The authors have no conflicts of interest to declare.
their use as possible anti-inflammatory drugs (40). Denev
et al studied the neutrophil-modulating effect of Bulgarian Ethical considerations
MP: raspberry (Rubus idaeus) leaves, meadowsweet All authors have inspected the ethical issues of plagiarism,
(Filipendula ulmaria) aerial parts, chokeberry (Aronia misconduct, data fabrication, falsification, double
melanocarpa) leaves, hawthorn (Crataegus monogyna) publication, or redundancy related to the manuscript.
leaves, and blackberry (Rubus fruticosus) leaves and found
that all six studied extracts blocked ROS production by Funding/Support
neutrophils (41). This research did not receive any specific grant from
For EO, we could attribute the effect obtained to the funding agencies in the public, commercial, or not-for-
presence of eucalyptol (44.14%). Regardless of using profit sectors.
low concentrations of eucalyptol, our study showed an
inhibitory effect slightly higher compared to EO which References
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