European Journal of Integrative Medicine 42 (2021) 101292

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European Journal of Integrative Medicine

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Research paper

Antioxidant, antibacterial, and cytotoxic activities of Cedrus atlantica

organic extracts and essential oil
Nassim Belkacem a,∗, Bachra Khettal a, Mohammad Hudaib b,c, Yasser Bustanji c,d,
Bashaer Abu-Irmaileh d, Chiraz Soumia M. Amrine e
a
Laboratoire de Biotechnologies Végétales et Ethnobotanique, Faculté des Sciences de la Nature et de la Vie, Université de Bejaia, 06000 Bejaia, Algeria
b
College of Pharmacy, Al Ain University of Science and Technology, Abu Dhabi 112612, UAE
c
Department of Pharmaceutical Sciences, School of Pharmacy, The University of Jordan, Amman 11942, Jordan
d
Hamdi Mango Center for Scientific Research, The University of Jordan, Amman 11942, Jordan
e
Department of Physical Sciences, Arkansas Tech University, Russellville AR 72801, USA

a r t i c l e i n f o a b s t r a c t

Keywords: Introduction: Cedrus atlantica is an endemic pine tree species well known for its wood oil. Its traditional medicinal
Cedrus atlantica usages are mainly anti-inflammatory and antibacterial. The current study aimed to explore both the essential oil
essential oil, chemical composition of the tree’s cones and the organic extracts of the branches, chemically and biologically.
antioxidant
antibacterial Methods: The essential oil was analyzed using gas chromatography coupled with mass spectrometry (GC-MS).
cytotoxic Phenolic contents of the extracts and fractions were determined following diverse methods described in the
literature. The bioactivity was assessed for their antioxidant (FRAP, DPPH• and ABTS•+ radical scavenging
activities), antibacterial (Broth microdilution method), and cytotoxic effects (MTT assay).
Results: 𝛼-pinene (81.49%) was found to be the major constituent of the essential oil. The extracts and frac-
tions were found to be rich in polyphenols. The antioxidant activity was better in the ethyl acetate fraction.
Staphylococcus aureus appeared to be the most susceptible strain to C. atlantica’s extracts and oil, with a minimum
inhibitory concentration and minimum bactericidal concentration values of 62.5 and 125 μg/mL for the ethyl ac-
etate fraction, respectively, and values of 0.25 and 0.5% for the essential oil, respectively. C. atlantica’s essential
oil exhibited a potent cytotoxic activity against MCF-7 breast cancer cell line with an IC50 value of 143.13±14.6
μg/mL.
Conclusion: C. atlantica’s essential oil and organic extracts showed antioxidant, antibacterial, and anticancer
activities. Thereby, the ethnobotanical use of C. atlantica in traditional preparations is worth investigating as the
plant appears to be a potential source of interesting metabolites.

1. Introduction chemotherapy resistance, adverse effects of commercial drugs, and the

economic stress on the growing interest into natural products chemistry
Medicinal plants continue to play an important role as a source research [5-7].
of significant secondary metabolites of therapeutic interest [1,2]. It Cedrus atlantica belongs to the family of Pinaceae. This endemic
has been reported that 69% of new antibacterial agents between 1981 species is well known, in part, for its Cedar wood essential oil and for its
and 2006 were of natural product origin [3]. Similarly, 49% of anti- high wood quality [8,9]. The Cedar wood oil has been investigated in
cancer agents on the market, between the period of 1940 and 2014 previous studies for several biological activities. It demonstrated promis-
were secondary metabolites isolated from nature or their derivatives ing antioxidant [10], antimicrobial [11], antifungal [12], antiviral [13],
[4]. anti-inflammatory [14], analgesic [15,16], anticancer [17], and anti-
The increasing need of finding new treatments for diverse health cholinesterase activities. Furthermore, the essential oil of C. atlantica
issues, as well as, the emergence of multidrug-resistant bacteria, cancer showed to have mollucidal [18], larvicidal [19], acaricidal [20], insecti-

Abbreviations: MIC, Minimum inhibitory concentration; MBC, Minimum bactericidal concentration; Chl, Chloroform; EtOAc, Ethyl acetate; But, n-butanol; Aq,
Aqueous fractions; TPC, Total phenolic content; FC, Flavonoid content; CTC, Condensed tannin content.
∗
Corresponding author.
E-mail address: [email protected] (N. Belkacem).

https://doi.org/10.1016/j.eujim.2021.101292
Received 17 August 2020; Received in revised form 9 January 2021; Accepted 16 January 2021
1876-3820/© 2021 Elsevier GmbH. All rights reserved.
N. Belkacem, B. Khettal, M. Hudaib et al. European Journal of Integrative Medicine 42 (2021) 101292

Fig. 1. Flow chart for the partitioning proto-

col.

cidal [21,22] and repellent properties [23], as well as an activity against 2.2.2. Preparation of the organic extracts
phytopathogenic agents. An amount of 21 g of the dry branches powder was subjected to or-
To the best of our knowledge, there is no study published about phe- ganic extraction with 200 mL of different solvents (methanol, ethanol,
nolic compounds extracted from C. atlantica branches. Moreover, liter- and acetone) separately. Extraction was performed using soxhlet appara-
ature shows a lack of data on phenolic compounds from C. atlantica tus (Reihenheizgerät 4, Germany). The obtained extract solutions were
[24,25]. The present study aims to contribute into investigating for the filtered then, evaporated using a rotavapor (Heidolph, Germany).
first time, the chemical composition of the essential oil of the Algerian C. The methanol extract showing the best yield was subjected to par-
atlantica cones. We also profiled the phenolic contents of the organic ex- tition following the protocol described by Rostagno and Prado (2013)
tracts from the branches. Furthermore, we evaluated the antioxidant and [27], (Fig. 1). The amount of 10 g of the methanol extract was dissolved
antibacterial activities in vitro, as well as their cytotoxic effects against in 200 mL of 5% methanol solvent. This later reconstituted solution was
MCF-7 breast cancer cell line. partitioned against a volume of 150 mL of solvents of increased polarity
(H: hexane, Chl: chloroform, EtOAc: ethyl acetate and But: n-butanol).
2. Materials and Methods The obtained solutions were filtered and evaporated separately. The
dried organic extracts and the fractions of the methanol extract were
2.1. Plant material conserved in darkness and stored at 4°C.

C. atlantica branches and cones were harvested in Akfadou forest 2.3. Gas chromatography-mass spectrometry analysis
(Adekar, Algeria) in May 2018. Botanical identification was carried out
in the Ecology and Environment Laboratory at Bejaia University by Dr. A volume of 1 μL of C. atlantica’s essential oil diluted in 0.1 mL of
F. Bekdouche using Quezel and Santa flora [26]. A voucher specimen GC grade n-hexane was analyzed using gas chromatography-mass spec-
(Cea-2018-5-42) was deposited in the School of Pharmacy at the Univer- trometry (Varian Chrompack CP-3800 GC-MS/MS-200. Saturn, Nether-
sity of Jordan. Branches were dried at room temperature in the shade. lands). The GC-MS was equipped with an automatic injector in the split
Then, they were subsequently grounded into powder of less than 250 mode and a flame ionization detector. A DB-5 GC capillary column was
μm in diameter. C. atlantica cones were dried at room temperature. used (30 m × 0.25 mm; 0.25 μm) consisting of 95% dimethylpolysilox-
ane and 5% diphenyl. Helium was used as the carrier gas at a flow rate
2.2. Extraction procedure of 1 mL/min. A linear temperature program was applied starting from
the initial column temperature of 60°C (hold time: 1 min) that increased
2.2.1. Essential oil extraction to 250°C (hold time 3 min) at a heating rate of 3°C/min.
The dried cones of C. atlantica were cut into small pieces and imme- The identification of the chemical constituents of the essential oil
diately hydro-distilled for 2.5 h using Clevenger-type apparatus. Anhy- was carried out by comparing their arithmetic retention indices (Kovat’s
drous sodium sulphate was added to the transparent distilled essential Index) with the reported values in the literature (Adam’s library) [28].
oil in order to eliminate any residual water. The final essential oil was A mixture of n-alkane hydrocarbons (C8-C20) was subjected to GC-MS
stored in a hermetically sealed amber vial at 4°C. analysis under the same chromatographic conditions described above

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N. Belkacem, B. Khettal, M. Hudaib et al. European Journal of Integrative Medicine 42 (2021) 101292

(Supp.Fig.1). The corresponding retention times were used to calculate 2.6. Antibacterial activity evaluation
the arithmetic Kovat’s Index for each component present in the essential
oil according to Van den Dool and Kratz equations [28]. Moreover, the 2.6.1. Preparation of bacterial inocula
obtained mass spectra were compared with those of the data bank of the Bacterial suspensions of each strain (Gram-positive strains: Staphylo-
TerpeneThermoQuest, General purpose and NIST libraries. The percent coccus aureus ATCC 25923 and Bacillus cereus ATCC 11778); and (Gram-
content of each identified compound of the essential oil was obtained negative strain: Escherichia coli ATCC 25921) were prepared by diluting
by integrating the corresponding peak area [28,29]. a few colonies in sterile aqueous saline solution (NaCl, 0.85%) scraped
from an overnight culture in agar plates incubated at 37°C. The turbidity
was adjusted to 0.5 McFarland corresponding to 108 CFU/mL [38] and
2.4. Phenolic compounds analysis
compared to a suspension having an optical density between 0.08 and
0.13 at 625 nm. This solution consists of 50 μL of anhydrous barium
2.4.1. Total polyphenol content
chloride (BaCl2 , 1.175%) added to 9.95 mL of sulphuric acid (H2 SO4 ,
The determination of total polyphenol content (TPC) was performed
1%) [38,39]. Bacterial suspensions were diluted in Muller Hinton Broth
using the Folin-Ciocalteu method [30,31]. The TPC was calculated using
(MHB) to yield approximately 5 × 106 CFU/mL. These later suspensions
a Gallic acid calibration curve (Abs=0.011[GA]+0.035, R2 =0.996) and
were used within 15 min following their preparation.
the results expressed in milligram equivalent Gallic acid per gram of dry
extract or fraction (mg EqGA/g).
2.6.2. Determination of minimum inhibitory and minimum bactericidal
concentrations
2.4.2. Flavonoid content Minimum inhibitory concentrations (MIC) were determined using a
The flavonoid content (FC) was determined using the aluminium broth micro-dilution method [39]. Stock solutions of each extract (es-
chloride method [32]. The FC was calculated using a Quercetin calibra- sential oil and fractions: EtOAc, But and Aq) were dissolved in dimethyl
tion curve (Abs=0.029[Q]-0.025, R2 =0.997) and the results expressed sulfoxide (DMSO) and filtered through a 0.22 μm sterile syringe filter.
in milligram equivalent Quercetin per gram of dry extract or fraction A volume of 100 μL of diluted solutions was transferred separately into
(mg EqQ/g). a 96-well microplate then subjected to serial twofold dilution to get a
concentration range from 31.25 to 1000 μg/mL of the fractions and from
0.03 to 1% of the essential oil. Subsequently, 100 μL of bacterial suspen-
2.4.3. Condensed tannin content
sions were added separately to each well. The microplates were then in-
The amount of condensed tannin (CTC) was estimated using the
cubated at 37°C for 18-24 h. Gentamicin was used as a positive control.
vanillin method [33]. The CTC was calculated using a Catechin calibra-
A mixture of MHB and DMSO was used as a negative control. The low-
tion curve (Abs=0.002[C]+0.038, R2 =0.992) and the results expressed
est concentration with no visible bacterial growth was recorded as the
in milligram equivalent Catechin per gram of dry extract or fraction (mg
minimum inhibitory concentration (MIC). To get the minimum bacteri-
EqC/g).
cidal concentration (MBC), 50 μL from the wells with no visible growth
were sub-cultured on agar plates and incubated at 37°C for 18-24 h. The
2.5. Antioxidant activity evaluation lowest concentration that exhibited no bacterial growth was recorded as
MBC. All measurements were carried out in triplicate.
2.5.1. DPPH• radical scavenging
Diphenylpicrylhydrazyl radical (DPPH• ) scavenging activity was 2.7. Anticancer activity evaluation
carried out using the protocol described by Shirwaikar et al (2006) [34].
The DPPH• radical scavenging activity (%) and the IC50 values were de- 2.7.1. Cell culture
termined. Butylhydroxyanisol (BHA) and Vitamin C (VitC) were used as Breast cancer cell lines (MCF-7) obtained from the American type
positive controls at the same concentrations and conditions. culture collection were cultured in RPMI 1640 with L-Glutamine (Eu-
roClone, Italy) complemented with 10% fetal bovine serum (FBS) (GE
Healthcare, USA), 10 mM HEPES buffer (pH 7.3) (Caisson, USA), 100
2.5.2. ABTS•+ radical scavenging
U/mL of penicillin (EuroClone, Italy), and 100 U/ml of Streptomycin
ABTS•+ radical scavenging activity was carried out using the proto-
(EuroClone, Italy). The cells were incubated at 37°C in a humidified at-
col described by Le et al (2007) [35]. The radical ABTS•+ was obtained
mosphere of 5% CO2 . The medium was changed every 48 to 72 h. When
by mixing an aqueous ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-
cells reached confluence, the medium was withdrawn from the 75 cm2
sulphonic acid) solution (7 mM) with an aqueous potassium persulfate
flask, and then they were washed with phosphate-buffered saline (PBS)
solution (2.45 mM), with a ratio of 2:1. The mixture was then incu-
(EuroClone, Italy). Subsequently, 1.5 mL of Trypsin-EDTA without phe-
bated for 16 h in darkness at room temperature. BHA and VitC were
nol red, calcium, and magnesium (EuroClone, Italy) was added to the
used as positive controls at the same concentrations and conditions. The
flask. The cells were then incubated at 37°C in a humidified atmosphere
results were expressed in mmol equivalent Trolox per gram of dry sam-
of 5% CO2 for 5 min to detach the cells. To stop the trypsin effect, 3
ple (mmolEqT/g) obtained from a standard curve plotted using Trolox
mL of the medium was added. The cells were sub-cultured by pipetting
(Abs=206.8[T]+1.73, R2 =0.992). Also, the ABTS•+ radical scavenging
them into new flasks. Cell count after trypsinization was performed via
activity (%) was calculated and the IC50 determined from the plot.
a haemocytometer using trypan blue dye (Promega Corporation, USA)
exclusion assay [40].
2.5.3. Ferric reducing antioxidant power (FRAP)
The ferric reducing antioxidant power was assessed using the proto- 2.7.2. Cytotoxicity and MTT assays
col described by Thaipong et al (2006) [36]. FRAP solution was made by Cytotoxicity of each plant sample was assessed by MTT (3-[4,5-
mixing sodium acetate buffer (300 mM, pH 3.6), 2,4,6-Tris(2-pyridyl)- dimethylthiazol-2-yl]-2,5diphenyl tetrazolium bromide) assay [40]. A
s-triazine (TPTZ, 10 mM in 40 mM HCl), and ferric chloride (20 mM) in volume of 100 μL of MCF-7 cells was seeded at a density of 7000
darkness at 37°C with a ratio of 10:1:1 [37]. The absorbance of the final cells/well in a 96-well microplate and allowed to attach overnight. A
solution was measured at 593 nm after 15 min incubation in darkness similar volume of each plant extract (essential oil and fractions: EtOAc,
at 37°C. The results were expressed in milligram equivalent vitamin C But and Aq) dissolved in DMSO was tested in triplicate at different con-
per gram of dry sample (mg EqVitC/g) obtained from a standard curve centrations diluted with the prepared RPMI medium. The final concen-
plotted using VitC (Abs=0.014[VitC]-0.031, R2 =0.995). tration of DMSO in all wells was 1%. RPMI medium with 1% DMSO

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N. Belkacem, B. Khettal, M. Hudaib et al. European Journal of Integrative Medicine 42 (2021) 101292

was used as a negative control, while doxorubicin was chosen to be the Table 1
positive control. After 24 h incubation, the medium was removed from Essential oil composition of Cedrus atlantica cones.
each well and replaced with 100 μL of fresh RPMI medium. Then, 15 μL Peak No RT (min) RI Component Area Percentage %
of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
1 5.63 926 Alpha-pinene 6191000 81.49
(Promega Corporation, USA) was added in the shade to each well, fol-
2 6.80 969 Sabinene 243959 3.21
lowed by an incubation for 3 h at 37°C in a humidified atmosphere of 3 7.14 982 Beta-pinene 148445 1.95
5% CO2 . To solubilize the blue formazan crystals, 100 μL solubilization 4 8.47 1024 Beta-phellandrene 192346 2.53
stop solution mix (Promega Corporation, USA) was added to each well 5 18.76 1283 Bornyl acetate 225345 2.96
6 25.82 1454 Beta-farnesene 73505 0.96
and left for 24 h. Optical densities (OD) were measured using an ELIZA
7 45.41 - ND∗ 59307 0.78
microplate reader (model Elx 808, BioTek Instruments, USA) at 570 nm. 8 47.49 - ND∗ 160155 2.10
Cytotoxicity effect (C(%)) was calculated by the following formula: 9 54.05 - ND∗ 55854 0.73
[( ) ] 10 57.54 - ND∗ 152775 2.01
𝐶 (%) = 𝑂𝐷𝐶 − 𝑂𝐷𝑇 ∕𝑂𝐷𝐶 × 100 11 57.64 - ND∗ 93850 1.23
Total 7596541 93.13
Where, ODT and ODC are the optical density of treated cells and of the
negative control, respectively. IC50 values were calculated as the aver- ND∗ : Not determined: 6.85%
age of three replicates. Monoterpene hydrocarbons 89.18%
Sesquiterpene hydrocarbons 0.96%
2.8. Statistical analysis Miscellaneous 2.96%
Total identified 93.13%
All samples were analyzed in triplicates. The results were expressed
as mean ± standard deviation (SD). The data were subjected to t-test
and 36.46±1.91% (P>0.05), respectively. Approximately, 70% of the
statistical analysis using GraphPadPrism statistical software (version 5).
constituents were isolated in the polar fractions (Butanol and aqueous).
Statistical differences of P≤0.05 were considered significant.
Several studies have shown that the solvent’s nature influences the ex-
traction yield, the phenolic content, and consequently the biological ac-
3. Results and discussion
tivities of the organic extracts [42-44].
3.1. Extract yield
3.2. GC-MS analysis
The extraction yield of the essential oil of C. atlantica was 0.41%
v/w. The yield is lower than the one obtained from the cones harvested The constituents of the essential oil of C. atlantica identified by GC-
in the region of Ifrane (Morocco) (0.62% v/w) [41]. MS analysis and the quantitative data are summarized in Fig. 2 and
On the other hand, the different yields of the branch extracts were Table 1. Six compounds, representing 93% of the total composition,
found to be dependant of the nature of the solvent used in the extrac- were successfully identified using the retention index and mass spectra
tion process via soxhlet (P<0.05). Methanol exhibited the highest yield matching methods (Fig. 2). C. atlantica’s essential oil was characterized
with 19.70±2.68%, followed by ethanol with 11.54±1.17%, whereas, by dominant levels of monoterpene hydrocarbons (89.18%). However, a
acetone gave a yield of 9.69±1.07% (P<0.05, Supp.Table1). This is cer- lower content of sesquiterpene hydrocarbons was detected. This later is
tainly due to the presence of components with various polarities and sol- represented by traces of 𝛽-farnesene (0.96%). Alpha-pinene was found to
ubilities. The methanol extract showing the highest yield was subjected be the major monoterpene hydrocarbon component (81.49%), followed
to further fractionation using solvents of increasing polarities. Butanol by sabinene (3.21%), 𝛽-phellandrene (2.53%) and 𝛽-pinene (1.95%).
and aqueous fractions exhibited the highest yields with 35.39±1.22% Furthermore, the essential oil contained also a low amount of bornyl

Fig. 2. GC-MS chromatogram of the essential

oil of Cedrus atlantica cones.

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N. Belkacem, B. Khettal, M. Hudaib et al. European Journal of Integrative Medicine 42 (2021) 101292

acetate (2.96%) with a miscellaneous structure. Diterpenes, oxygenated Table 2

monoterpenes, and sesquiterpenes could not be identified. Antioxidant activity of extracts and fractions of Cedrus atlantica branches
To our best knowledge, this is the first time where the composition Samples DPPH IC50 (μg/mL) ABTS IC50 (μg/mL) FRAP (mg Eq Vit C/g)∗
of the essential oil from the Algerian C. atlantica cones via GC-MS is
Extracts:
reported. There was no previous study in the literature.
A 13.27±0.53d 147.46±3.91e 388.80±9.09d
Compared to our results, the essential oil of C. atlantica cones har- E 14.04±1.51d 160.9±8.02e 236.66±18.64f
vested in the region of Ifrane (Morocco) showed to have a different M 13.05±0.31d 158.4±8.57e 325.95±5.05e
composition. The major components were 𝛽-himachalène (29.4%), 𝛼- Fractions:
Chl 30.78±1.03e 263.2±26.12f 190.71±10.54g
longipinene (20.75%), 𝛽-chamigrene (14.39%), and longifolene (V4)
EtOAc 6.16±0.01b 45.34±1.53c 692.38±37.96b
(11.61%). Scientists reported the variations in the essential oil compo- But 8.23±0.36c 113.53±10.17d 459.52±17.04c
sition to be related to different factors such as plant parts, harvesting Aq 35.51±4.78e 232.8±27.97f 142.14±1.52h
period, geographical origin and genetic parameters [45,46]. BHA 6.03±0.13b 30.23±0.54b -
On the other hand, the study carried out by Boudarene et al (2004) Vit C 5.47±0.13a 24.92±1.32a 1000a

[47] on the essential oil of C. atlantica seeds collected in two different A: Acetone, E: Ethanol, M: Methanol, H: Hexane, Chl: Chloroform, EtOAc: Ethyl
areas of Algeria (Ould Yakoub and Tala Guilef) revealed that the oils acetate, But: n-Butanol, Aq: Aqueous.
contained relatively comparable constituents to our findings. The main For each column, values followed by the same letter were not significantly dif-
components found in the previous studies were 𝛼-pinene (37.1-5.5%), ferent (P<0.05).
𝛽-pinene (8.6-1.9%), myrcene (3.6-0.6%), limonene (2.5-0.6%), bornyl Values were expressed as Mean ± SD (n=3). .
∗
acetate (5.4-4%), 𝛽-farnesene (6.8-1.9%), and manool (8.3-20.7%). per gram of extract or fraction.-: not determined

3.3. Phenolic compounds analysis A similar trend was observed with the ABTS assay. According to the
solvent’s nature, the capacity of the extracts to scavenge the free radical
C. atlantica extracts exhibited high levels of TPC and CTC ABTS• + measured as IC50 varied significantly (P<0.05). The acetone ex-
(Supp.Table1). Few amounts of flavonoids were detected. The effect tract showed the lowest IC50 value with 147.46±3.91 μg/mL (P<0.05),
of solvent influences significantly the total phenolic and tannin content followed by the methanol and the ethanol extracts with 158.4±8.57
(P<0.05). In fact, the highest TPC was displayed by the acetonic extrac- μg/mL and 160.9±8.02 μg/mL (P>0.05), respectively. The IC50 val-
tion (233.21±18.27 mg EqGA/g extract), whereas the methanolic ex- ues exhibited by Chl, EtOAc, But and Aq fractions were as follows:
traction had the best CTC (143.5±12.72 mg EqC/g extract). It should be 263.2±26.12 μg/mL, 45.34±1.53 μg/mL, 113.53±10.17 μg/mL, and
noted that methanol exhibited the best extraction yield and dissolved 232.8±27.97 μg/mL (P<0.05), respectively. The EtOAc fraction showed
the highest levels of phenolic compounds from C. atlantica branches’ a potent antioxidant capacity against the radical ABTS•+ with an IC50
powder with 39.43±1.7 mg EqGA/g dry powder, versus 22.59±1.77 mg value comparable to those of the positive controls BHA and VitC with
EqGA/g dry powder for acetone (P<0.05). Indeed, methanol was found 30.23±0.54 μg/mL and 24.92±1.32 μg/mL (P<0.05), respectively.
in the literature to have high abilities to extract phenolic compounds
from other plant samples [44,48,49]. 3.4.2. Ferric reducing antioxidant power (FRAP)
Ethyl acetate demonstrated the highest TPC, CTC and FC with FRAP assay is an electron transfer based-assay. The pH was main-
474.39±13.34 mg EqGA/g, 504.83±18.9 mg EqC/g, and 18.78±0.43 tained at 3.6 to enhance the solubility of iron and to allow important
mg EqQ/g, followed by the butanolic fraction with 337.57±8.86 mg drive electron transfer [51]. The highest reducing capacity was obtained
EqGA/g, 366±18.75 mg EqC/g, and 3.73±0.28 mg EqQ/g. by the acetone extract with 388.80±9.09 mg EqVitC/g extract followed
A positive and high correlation (r=0.989) between TPC and CTC was by the methanol extract with 325.95±5.05 mg EqVitC/g extract, then
recorded suggesting that the phenolic components extracted from C. at- the ethanol extract with 236.66±18.64 mg EqVitC/g extract (P<0.05).
lantica branches were mainly tannins. However, to the best of our knowl- Among the fractions of the methanol extract, EtOAc and But showed
edge, no data have been reported on the phytochemical composition of the highest ferric reducing antioxidant power with 692.38±37.96 mg
C. atlantica branches. EqVitC/g fraction and 459.52±17.04 mg EqVitC/g fraction (P<0.05),
respectively.
3.4. Antioxidant activity evaluation A positive and high correlation was observed between the three an-
tioxidant assays (0.894≤r≤0.967). The relationship between phenolic
There is no standardized method for the evaluation of the antioxidant compounds (TPC, FC and CTC) and antioxidant activities (DPPH, ABTS
activity of plant extracts. Therefore, the use of more than one method and FRAP) is reported in the supplementary document (Supp.Table2).
is recommended to provide more comprehensive information [50]. In CTC exhibited a highly significant correlation with the antioxidant as-
the present study, different extracts and fractions from C. atlantica were says (0.827≤r≤0.962) compared to FC (0.472≤r≤0.801). The later re-
investigated for their antioxidant activity in vitro by three methods: Two sults demonstrate that tannins could be considered as the main contrib-
antiradical scavenging capacity (DPPH and ABTS) assays and a ferric utors to the total antioxidant activity of C. atlantica extracts. However,
reducing antioxidant power (FRAP) assay. The results are presented in several reports showed that the antioxidant activity is not only related
Table 2. to the amount of phenolic compounds but is strongly linked to their
chemical structures [52]. It was reported that organic extracts may act
3.4.1. DPPH and ABTS radical scavenging capacity on the cell surface by breaking the radical chain reaction. Inside the
DPPH• radical scavenging ability was evaluated in terms of inhibi- cell, the extracts work by scavenging intracellular oxidants owing to
tion percentage of free radical DPPH• by antioxidants in the different their highest ability of hydrogen donating, electron transfer and metal
samples and the respective IC50 values were recorded. The lowest IC50 scavenging capacity [53]. Polyphenol-intake plays a key role in the Re-
value reflects the best antioxidant activity. Accordingly, all tested ex- active Oxygen Species (ROS) inhibition and oxidative stress, leading to
tracts exhibited high radical scavenging ability. Among fractions, EtOAc degenerative disease prevention [54,55].
and But showed the highest antioxidant activity with IC50 values of
6.16±0.01 μg/mL and 8.23±0.36 μg/mL (P<0.05), respectively. More- 3.5. Antibacterial activity evaluation
over, the potent antioxidant power recorded for the EtOAc fraction was
close to those exhibited by the positive controls BHA and VitC with The essential oil exhibited antibacterial effect against all tested
6.03±0.13 μg/mL and 5.47±0.13 μg/mL (P<0.05), respectively. Gram-positive and negative bacteria, showing the better potency against

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N. Belkacem, B. Khettal, M. Hudaib et al. European Journal of Integrative Medicine 42 (2021) 101292

Table 3 motic control dysfunction [60,61]. Moreover, the aromatic ring and
MIC and MBC values of the different plant samples and gentamicin phenolic hydroxyl groups may interfere with hydrogen bonds of the en-
towards the tested bacteria. zymes active sites [58].
S.aureus B. cereus E.coli MIC and MBC values of the tested fractions (EtOAc, But and Aq)
Samples obtained from the methanol extract varied from 31.25 to 1000 μg/mL
MIC MBC MIC MBC MIC MBC
and 62.5 to 2000 μg/mL, respectively. Wherein, the most active frac-
a a a a a
EtOAc 62.5 125 125 250 250 1000a tions were EtOAc and But. Gram-negative bacteria were shown less sus-
But 62.5a 125a 250a 125a 250a 1000a
ceptible to antibacterial agents as their outer membrane is imperme-
Aq 500a 1000a 1000a 2000a 1000a 2000a
EO 0.25b 0.5b 0.25b 0.5b 0.5b 1b able to most of them [62]. Furthermore, the preliminary tests assessed
G 6.25a 6.25a 6.25a 12.5a 12.5a 25a with the agar diffusion method, showed that Chl fraction did not exhibit
any antibacterial effect. The most potent fractions showed high levels
EtOAc: Ethyl acetate, But: n-Butanol, Aq: Aqueous, EO: Essential
of polyphenols (mainly tannins). These secondary metabolites isolated
oil, G: Gentamicin
a
Values in μg/ml
from different plants were always known for their antimicrobial activity
b
Values in % (v/v)n=3 [63]. Scalbert (1991) [64] reported tannins’ antimicrobial mechanism
to be due to their role in enzyme inhibition and substrate deprivation.
Other mechanisms are related to their action on membranes by oxida-
tive phosphorylation and electron transport system inhibition, or metal
Staphylococcus aureus and Bacillus cereus with MIC and MBC values of
ion deprivation by the formation of tannins-metal precipitates. To the
0.25% (v/v) and 0.5% (v/v), respectively (Table 3). It seems reason-
best of our knowledge, this is the first study that reports the antibacte-
able to hypothesize the activity attributed to monoterpene hydrocarbons
rial activity of extracts from C. atlantica branches. However, other stud-
found as the major components in the oil, mainly 𝛼-pinene. However,
ies have been conducted on the antimicrobial potential of extracts from
minor components were found essential in a couple of studies, due to a
other Cedrus species, namely C. libani [65,66] and C. deodara [67,68].
synergetic effect with the main active compounds [56,57]. The essential
Therefore, the above-mentioned results demonstrated that Cedrus may
oil derived from the Moroccan C. atlantica cones showed no inhibition
serve as an important natural antibacterial alternative.
of the two strains Staphylococcus aureus and Escherichia coli. In addition,
some antimicrobial studies were performed on C. atlantica essential oils
derived from wood [11,12] and leaves [58]. However, our current find- 3.6. Cytotoxicity and MTT assays
ings are in agreement with a previous study conducted by Derwich et
al (2010) [58] on the essential oils obtained from C. atlantica’s leaves In the cytotoxicity assay, mitochondrial dehydrogenase enzymes of
grown in Morocco, where the major component was 𝛼-pinene. active cells reduce the MTT to blue formazan reflecting cell viability
Similarly, several studies of essential oils rich in 𝛼-pinene revealed [69]. The MTT assay results showed that cancer cell lines were sensitive
potential antibacterial activities [58,59]. It has been reported that ter- to doxorubicin (positive control) with an IC50 value of 0.59±0.05 μg/mL
penes may attack the cell membranes and cause toxicity by chemios- (Fig. 3 and Supp.Fig.2). However, there was no significant cytotoxic

Fig. 3. Cytotoxic effect on MCF-7 treated with (A) EtOAc fraction; (B) But fraction; (C) Aq fraction; and (D) EO (Cell viability percentage). Doxorubicin was tested
on MCF-7 as positive control. Three experiments conducted in triplicate.

6
N. Belkacem, B. Khettal, M. Hudaib et al. European Journal of Integrative Medicine 42 (2021) 101292

activity on the MCF-7 cell lines treated with the fractions (EtOAc, But, Data availability
and Aq). On the other hand, an IC50 value of 143.13±14.6 μg/mL was
obtained with the essential oil (Fig. 3). This suggested that C. atlantica All data associated with this study are included within this
EO had a promising cytotoxic activity against MCF-7 breast cancer cell manuscript and can be obtained from the corresponding author upon
lines that is worth further investigation. request.
No data was found in the literature about the anticancer activity of C.
atlantica’s essential oil on the MCF-7 breast cancer cell lines. However, References
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