''Evaluation of the volatile oil composition and antiproliferative activity of Laurus nobilis L. (Lauraceae) on breast cancer cell line models''

Abstract

Volatile oil composition and antiproliferative activity of Laurus nobilis L. (Lauraceae) fruits and leaves grown in Jordan were investigated. GC-MS analysis of the essential oil of the fruits resulted in the identification of 45 components representing 99.7 % of the total oil content, while the leaf essential oil yielded 37 compounds representing 93.7% of the total oil content. Oxygenated monoterpene 1,8-cineole was the main component in the fruit and leaf oils. Using sulphorhodamine B assay; the crude ethanol fraction, among other solvent extracts, showed strong antiproliferative activity for both leaves and fruits, nevertheless, the fruits were more potent against both breast cancer cell models (MCF7 and T47D). At IC50 values; the mechanism of apoptosis was nevertheless different: where L. nobilis fruit proapoptotic efficacy was not regulated by either p53 or p21, L. nobilis leaf extract components enhanced the p53 levels substantially. In both extracts, apoptosis was not caspase-8 or Fas Ligand and sFas (Fas/APO-1) dependent. Our studies highlight L. nobilis as a potential natural agent for breast cancer therapy. Compared with non induced basal cells, both L. nobilis fruits and leaves induced a significant enrichment in the cytoplasmic mono-and oligonucleosomes after assumed induction of programmed MCF7 cell death.

Full text

ORIGINAL ARTICLE
The article was published by Academy of Chemistry of Globe Publications
www.acgpubs.org/RNP © Published 03/19/2014 EISSN:
1307-6167
Rec. Nat. Prod. 8:2 (2014) 136-147
Evaluation of the Volatile Oil Composition and Antiproliferative
Activity of Laurus nobilis L. (Lauraceae) on Breast Cancer Cell
Line Models
Rana Abu-Dahab
∗
∗∗
∗
, Violet Kasabri and Fatma Ulku Afifi
Faculty of Pharmacy, The University of Jordan, Amman 11942, Queen Rania Street, Amman JORDAN
(Received April 15, 2013; Revised October 06, 2013; Accepted October 09, 2013)
Abstract: Volatile oil composition and antiproliferative activity of Laurus nobilis L. (Lauraceae) fruits and
leaves grown in Jordan were investigated. GC-MS analysis of the essential oil of the fruits resulted in the
identification of 45 components representing 99.7 % of the total oil content, while the leaf essential oil yielded
37 compounds representing 93.7% of the total oil content. Oxygenated monoterpene 1,8-cineole was the main
component in the fruit and leaf oils. Using sulphorhodamine B assay; the crude ethanol fraction, among other
solvent extracts, showed strong antiproliferative activity for both leaves and fruits, nevertheless, the fruits were
more potent against both breast cancer cell models (MCF7 and T47D). At IC
50
values; the mechanism of
apoptosis was nevertheless different: where L. nobilis fruit proapoptotic efficacy was not regulated by either p53
or p21, L. nobilis leaf extract components enhanced the p53 levels substantially. In both extracts, apoptosis was
not caspase-8 or Fas Ligand and sFas (Fas/APO-1) dependent. Our studies highlight L. nobilis as a potential
natural agent for breast cancer therapy. Compared with non induced basal cells, both L. nobilis fruits and leaves
induced a significant enrichment in the cytoplasmic mono- and oligonucleosomes after assumed induction of
programmed MCF7 cell death.
Keywords: Laurus nobilis; Volatile oils; Anticancer; Apoptosis; Jordan. © 2014 ACG Publications. All rights
reserved.
1. Introduction
Laurus nobilis L. (Lauraceae), commonly known as bay leaf, has been used as a spice
worldwide and a medicinal plant in Mediterranean countries, including Jordan. Leaves and fruits have
been reported to possess aromatic, stimulant and narcotic properties [1]. The anti-convulsive and
antiepileptic activities of L. nobilis extracts have been confirmed [2]. The leaves of L. nobilis are
traditionally used orally to treat the symptoms of gastrointestinal problems, such as epigastric bloating,
digestion, and flatulence [3]. Preliminary brine shrimp toxicity tests and few other studies related to its
cytotoxic properties were carried out on the leaf extract of Turkish L. nobilis [4]. While the majority of
the phytochemical and biological reports dealt with the leaves of L. nobilis, there has been very little
work on its fruits. The major setbacks of most available chemotherapies are the non-selective
∗
Corresponding author: E-mail: abudahab@ju.edu.jo; Phone: +96265355000; Fax:+ 96265300250

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Abu-Dahab et.al., Rec. Nat. Prod.(2014) 8:2 136-147
cytotoxicity, severe side effects, and chemo-resistance [5]. A cornerstone of newer cancer therapy is to
develop alternatives that have high specificity to induce molecular apoptosis in cancer cells.
In the last decades, newer anti-cancer drugs have been introduced, with about half of them
derived from natural sources [6-7]. In fact, natural compounds from flowering plants have played a
significant role in cancer chemotherapy. Some of these agents include vincristine and vinblastine from
Catharanthus roseus, paclitaxel and taxotere from species of yew (Taxus spp.), etoposide derived from
lignans of Podophyllum spp. and camptothecin analogues, such as topotecan, from Camptotheca
acuminata. These fundamentally cytotoxic components inhibit cell proliferation through different
mechanisms [8]. Apoptosis is a highly regulated mechanism by which cells undergo cell death in an
active way. Accordingly, one of the challenging tasks concerning cancer therapy is to induce and
augment apoptosis in malignant cells. Therefore, there is an escalating focus on natural products to
modulate apoptotic signaling pathways and their emerging molecular targets [9].
Though few reports were related to L. nobilis antiproliferative capacity [10-12]; presently in this
study, water, ethanol, butanol, ethylacetate and chloroform extracts of L. nobilis leaves and fruits as
well as their hydrodistilled volatile oils were tested for their antiproliferative activity. To shed light on
the possible anticancer mechanisms of L. nobilis leaves and fruits extracts, p53 and p21 levels, Fas
ligand and sFas and caspase-8 activities were determined, all of which are strongly associated with the
signal transduction pathway of apoptosis [13-15].
2. Materials and Methods
2.1. Plant Material and Preparation of Crude Extracts
Leaves and fruits of L. nobilis L., collected from the trees grown in the campus of The
University of Jordan in June 2010, were identified by Prof. Barakat Abu Irmaileh (Faculty of
Agriculture, The University of Jordan). Leaves and fruits were air dried at room temperature (RT) in
the shade until constant weight. Voucher specimens for L. nobilis fruits [1LAUR FMJ] and leaves
[2LAUR FMJ] have been deposited in the Department of Pharmaceutical Sciences, Faculty of
Pharmacy, the University of Jordan.
Each 10 g of the dried and coarsely powdered plant material was refluxed for 30 min using
different solvents, kept overnight, filtered and solvents were evaporated. The ethanol extracts of the
fruits and leaves were tested for the presence of different classes of secondary metabolites using Thin
Layer Chromatography (TLC) [16]. Coated analytical TLC plates were procured from Merck. For
biological activity tests, 100 mg of the extracts were dissolved in 10 mL DMSO (stock solution).
2.2. Distillation of Plant Material
Air dried plants were coarsely powdered and then hydro-distilled using a Clevenger apparatus
for 3 h. The distillation was repeated twice and the oils obtained were pooled separately, dried over
anhydrous sodium sulfate (Na
2
SO
4
) and stored at 4º C in amber glass vials until analysis.
2.3. GC-MS and GC-FID Analysis
About 1 µL aliquot of each oil sample, appropriately diluted to 10 µL in GC grade n-hexane,
was subjected to qualitative and quantitative GC-MS analysis. A hydrocarbon mixture of n-alkanes
(C
8
-C
20
) was analyzed separately by GC-MS under the same chromatographic conditions using the
same DP-5 column. Identification of compounds was based on the built-in libraries (NIST Co and
Wiley Co, USA) and by comparing their calculated retention indices (RI) relative to (C
8
-C
20
) n-
alkanes literature values, measured with columns of identical polarity, or in comparison with authentic
samples [17].
α- and β-pinenes, p-cymene, limonene, linalool (Fluka, Buchs, Switzerland), eugenol,
and sabinene hydrate (Sigma-Aldrich, Buchs, Switzerland) were used as reference substances in GC-
MS analysis. GC-grade hexane and analytical reagent grade anhydrous Na
2
SO
4
were from Scharlau
(Barcelona, Spain) and UCB (Bruxelles, Belgium), respectively. Each sample was analyzed twice.

Laurus nobilis antiproliferative activity
138
2.4. In vitro Assay for Antiproliferative Activity
Cell lines under investigation were human breast adenocarcinoma (MCF7, ATCC no. HTB-22)
and human ductal carcinoma (T47D, ATCC no. HTB-133). Cells were cultured in RPMI media
fortified with 10% heat inactivated bovine serum, 1% of 2 mmol/L L-glutamine, 50 IU/mL penicillin,
and 50 µg/mL streptomycin. Human periodontal fibroblasts (PDL), which are a primary cell culture,
were kindly provided by Dr. Suhad Al-Jundi and Dr. Nizar Mhaidat from Jordan University of Science
and Technology, Irbid, Jordan.
Cells were seeded with a density of 5000 cell/well (15 000 cell/cm
2
) and incubated at 37° C in a
humidified atmosphere containing 5% CO
2
. After 24 h, the cells were treated with the extracts, volatile
oils, and controls. Test compounds were incubated with the cells for 72 h at 37º C in humidified
conditions containing 5% CO
2
. At the end of the exposure time, cell growth was measured using the
sulphorhodamine B (SRB) assay as described earlier [18-19]. As positive controls, cisplatin and
doxorubicin were used [19].
2.5. Tests for Apoptosis
Quantum Protein (Bicinchoninic Protein assay kit; EuroClone S.p.A, Siziano, Italy),
Nucleosome ELISA (Roche Diagnostics GmbH; Mannheim, Germany), human total p53 ELISA,
human total p21 ELISA, human Fas Ligand, human sFas immunoassay ELISA, caspase-8
colorimetric assay kits, wash buffer and substrate and stop solutions (R&D Systems Europe, Ltd;
Abingdon, UK) were used.
2.6. Measurement of Apoptosis by ELISA
In our preliminary investigations, none of T47D selected extrinsic apoptosis biomarkers, namely
FasL, sFas or caspase 8, were differentially or significantly up-regulated/activated in comparison to
MCF7’s. Thus, the decision was formulated to preferentially select for MCF7 over T47D in the
antiproliferative action mechanism studies of L. nobilis aerial parts. In addition, it has been reported
that proteins implicated in apoptosis regulation are more strongly expressed in MCF7 as compared to
T47D [20]. The induction of apoptosis was assayed using the nucleosome ELISA kit. As described in
the manufacturer's protocol, this kit is a photometric enzyme-immunoassay for the determination of
cytoplasmic histone-associated-DNA-fragments after induced cell death. MCF7 cells were incubated
with vehicle alone (0.1% DMSO) and with the obtained IC
50
’s of the extracts under investigation for
72 h compared to the untreated (non-induced) basal cell incubations.
2.7. Assays of the Levels of p53, p21/WAF1, FasL (Fas Ligand) and sFas (Fas/APO-1)
MCF7 cells were treated as above and as described in the manufacturer’s protocol. The samples
of cell lysate were placed in 96-wellplates coated with monoclonal antibodies, and incubated for 2 h
(sFas, p53, p21, or FasL) at RT. After removing the unbound material by washing buffer, horseradish
peroxidase conjugated streptavidin was added. The absorbance was measured at 450 nm, and
concentrations of p53, p21, FasL and sFas were directly determined by interpolating from standard
curves. Results are presented as the percentage of the change of respective non-induced control
determinations.
2.8. Assay for Caspase-8 Activity
As per the manufacturer’s instructions, cell lysates were incubated with peptide substrate in
assay buffer (100 mM NaCl, 50 mM HEPES, 10 mM dithiothreitol, 1 mM EDTA, 10% glycerol, 0.1%
CHAPS, pH 7.4) for 2 h at 37
o
C. The release of p-nitroaniline was monitored at 405 nm. Results are
represented as the percentage of the change of the activity compared to the untreated control cells.
2.9. Statistical Analysis
Results were expressed means (as %control) ± S.E.M (standard error of the mean). Statistical
comparisons of the results were determined by ANOVA followed by Dunnett’s post-test whenever
appropriate using Graphpad Prism (version 3.02 for windows; GraphPad Software, San Diego, CA,

139
Abu-Dahab et.al., Rec. Nat. Prod.(2014) 8:2 136-147
USA). Values of the means of untreated control and treated cells were considered significantly
different if P<0.05 and highly significantly different if P<0.01 and P<0.001.
3. Results and Discussion
3.1. Volatile Oil Composition and Phytochemical Screening of Crude Extracts by TLC
After hydrodistillation, leaves and fruits of L. nobilis afforded yellowish oils with a yield of
moisture-free 4.3 and 2.8 % (v/w), respectively. The retention indices and relative percentages of the
identified compounds are presented in Table 1.
GC-MS analysis of the hydrodistilled essential oil of the fruits resulted in the identification of
45 components representing 99.7 % of the total oil content. Hydrocarbon and oxygenated
monoterpenes were found to have the highest contribution to the hydro-distilled essential oil
comprising 84.9 % of the total oil content. 1,8-cineole (eucalyptol) was the main oxygenated
monoterpene detected, accounting for 29.8 % of the total oil content. With the exemption of α-
terpinylacetate (5.6 %), α-terpineol (1.2 %) and γ-terpineol (1.5 %), other detected oxygenated
monoterpenes were found to occur in concentrations less than 1%. Sesquiterpenes were found only in
low concentration (12.7 %) with ß-elemene being the main constituent (6.2 %). GC-MS analysis of the
leaf essential oil resulted in the identification of 37 compounds representing 93.7 % of the total oil
content. 1,8-cineole (36.8%), α-terpinylacetate (14.6 %) and terpinene-4-ol (6.4 %) were the major
components of the monoterpenoid fraction (83.3 %). Sesquiterpenoids were detected only at a
concentration of 1.4 %.
Crude ethanolic extracts of L. nobilis leaves and fruits were rich in flavonoids and terpenoids. In
TLC experiments, the occurrence of the well known flavonoids quercetin, luteolin, kaempferol and
apigenin were identified in both the leaf and fruit extracts.
Volatile oil composition of the fruits and leaves of L. nobilis has been studied by several
researchers. A comparison of these reports showed differences in the concentration of the major
components. In addition to the genetic factors, determining the chemotype of the species, and the
season during which the plants are collected, the environmental conditions, soil characteristics,
methods of drying, extraction and analytical conditions do contribute to the differences in oil
composition [21]. Consistent with our findings, 1,8-cineole is the main component of the volatile
fraction as in all previous studies. The leaves of L. nobilis collected from Lebanon yielded 35.15%
1,8- cineole while the oil obtained from young and old leaves collected from North Black Sea region
of Turkey yielded 24.2 % and 32.1 %, respectively [22] . Two other studies were carried out with the
leaf oil from different location in South Turkey contained higher percentages for 1,8-cineole (46.6 %,
47.6 % , 59.9 % and 44.7 %, respectively) [23-24]. The findings of the current study are quite similar
to those of Loizzo et al. [25], and stands at 36.8 %, although earlier in our laboratories, using the fresh
leaves, higher percentage was detected (40.9 %) [18]. The lowest concentration for 1,8-cineole was
reported from Portugal (27.2%) [26]. On the other hand, the concentration of 1,8- cineole in the hydro-
distilled oil from the fruits of L. nobilis in the present study is higher (29.8 %) compared to the
studies from Lebanon (9.4 %) and Turkey (9.5 % and 18.1 %, 20. 5 % , 17.4 %, respectively) [22-23].
Fluctuations were recognized with other constituents of the volatile fractions of the leaves and
fruits, all studied L. nobilis oils from the Mediterranean countries were rich in monoterpenoids while
sesquiterpenoids were detected in very low concentrations. In the present study, monoterpene
hydrocarbons and oxygenated monoterpenoids had nearly equal share in the oil of the fruits (43.6 %
and 41.3 %, respectively) while the leaf oil contained predominantly oxygenated monoterpenoids
(66.2 %). Still oxygenated sesquiterpenes were absent in L. nobilis fruit oil. The main detected
sesquiterpene in the bay fruit oil was ß-elemene (6.2 %). The latter compound found in the leaf oil
only at a concentration of 0.3 %. Moreover, β-caryophyllene and caryophyllene oxide were detected in
both oils in concentrations below 2 %.

Laurus nobilis antiproliferative activity
140
Table 1. Comparison of the chemical composition of the essential oils hydrodistilled from the leaves
and fruits of Laurus nobilis. Results present the average of 2 independent trials
RI* Compound Dry leaves
Percentage
**
Fruits
Percentage
**
852 (E)-3-hexanol 1.0 -
864 (Z)-3-hexanol 3.8 -
926
a
/931
b
α-thujene 0.1 0.5
934 α-pinene 4.6 10.9
937 citronellene tetrahydro 0.1 -
944
β
-citronellene 0.2 -
951 camphene 0.5 1.3
974
a
/973
b
sabinene 3.1 4.4
980 β-pinene 3.6 8.4
985 trans-m-mentha-2,8-diene, 0.1 -
989 myrecene 0.4 -
992 dehydo-1,8-cineole 0.2 -
1009 α- phellandrene - 9.0
1010
δ
-2 -carene 0.4 -
1019
a
/1018
b
α- terpinene 0.4 0.4
1026 o-cymene 1.0 -
1033
a
/1027
b
p-cymene 0.3 2.5
1031
a
/1030
b
limonene 1.6 2.5
1035
a
/1034
b
1,8-cineole 36.8 29.8
1046 (E)-
β
-ocimene - 3.2
1059 γ-terpinene 0.6
0.4
1073
a
/1072
b
cis-sabinene hydrate 0.6 0.2
1088
a
/1087
b
terpinolene 0.1 0.1
1102
a
/1101
b
linalool 2.6 0.4
1128 1-terpineol 0.2 0.1
1146 cis-sabinol 0.3 0.2
1150 camphor - 0.2
1173 p-mentha-1,5-dien-8-ol 0.6 -
1175
a
/1173
b
terpinene-4-ol 6.4 0.2
1177 borneol - 0.4
1185
α
-terpineol - 1.2
1201
a
/1198
b
γ-terpineol 1.8 1.5
1208 p-cymene 9-ol - 0.2
1271 iso-3-thujyl acetate 0.5 -
1287
a
/1286
b
isobornylacetate 1.1 0.8
1316
a
/1315
b
cis-dihydro-α-terpinylacetate 0.3 0.3
1340 δ-elemene 0.4 -
1350
a
/1348
b
α-terpinylacetate 14.6 5.6
1360 nerylacetate - 0.2
1372 α-ylangene - 0.3
1385
β
-cubebene - 0.1
1392
a
/1390
b
β
-elemene 0.3 6.2
1409
a
/1405
b
methyleugenol 4.2 0.2
1421 2,5-dimethoxy-p-cymene 0.2 -
1423
β
-caryophyllene - 1.1
1438 α-guaiene - 0.5
1444 aromadendrene - 0.3
1452 cis-muurola 3,5-diene - 0.2

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Abu-Dahab et.al., Rec. Nat. Prod.(2014) 8:2 136-147
1459 α-humulene - 0.3
1484 γ-muurolene - 0.7
1491
β
-selinene 0.4 -
1493 germacrene D - 0.5
1500
δ
- selinene - 0.6
1505 α-selinene - 1.0
1511 germacrene A - 0.2
1517 γ-cadinene - 0.5
1521 δ-cadinene - 0.2
1525 undecenoic acid-10-methyl,
methyl ester - 0.8
1586 caryophyllene oxide 0.3 -
1596 dodecenoic acid ethyl ester - 1.1
Total identified 93.7 99.7
Monoterpenoids 83.3 84.9
Monoterpenoid hydrocarbons 17.1 43.6
Oxygenated monoterpenoids 66.2 41.3
Sesquiterpenoids 1.4 12.7
Sesquiterpene hydrocarbons 1.1 12.7
Oxygenated sesquiterpenes 0.3 -
Phenylpropanoids 4.2 0.2
Miscellaneous 4.8 1.9
Unidentified 6.3 0.3
* Retention indices (RI) calculated on (DB-5MS) column, ** Percentage is given as the average of two
independent measurements. Compounds are listed based on their elution order on the corresponding column.
a
refers to RI of the leaves,
b
refers to RI of the fruits.
3.2. Antiproliferative Activity
The in vitro antiproliferative activity of the different fractions is shown in Table 2. Results
present the IC
50
values of the fractions (concentration of the extract needed to reduce proliferation by
50% after 72 h of incubation compared to control wells). IC
50
values were determined by plotting dose
response curves for the fractions under investigation in the range of 0.1 to 100 µg/mL). The IC
50
values of reference drugs; cisplatin and doxorubicin were 7.3 ± 1. 9 µM and 0.16 ± 0.0 µM for MCF7
cells, 21.3 ± 9.7 µM and 0.2 ± 0.0 µM for T47D cells respectively.
Table 2. The antiproliferative activity of Laurus nobilis leaves- and fruits-extracts on MCF7
and T47D cell lines.
MCF7 T47D
Leaves Fruits Leaves Fruits
Ethanol 48.2±5.2 28±1.7 19.8±4.3 12.3±4.0
Chloroform 48.8±10.3 31.2±5.24 58.2±14.1 35. 6 ± 5.6
Butanol Non toxic 72. 5±5.8 Non toxic 75.4±6.4
Ethyl acetate 61.0±18.4 20.6 ± 4.5 27.9±1.3 33.7±13.9
Aqueous Non toxic Non toxic Non toxic Non toxic
Volatile oil 93.1±4.7 41.9±3.3 64.3±3.1 142±43.6
Results present the IC
50
in µg/mL (concentration needed to reduce cell proliferation by 50%). Results
present the average of at least two determinations on two cell line passages and each is an average with
standard deviation of four wells.
For both cell lines, L. nobilis fruit extracts showed higher antiproliferative activity compared to
the leaf extracts. In both parts, aqueous and butanol fractions demonstrated weak or no reduction in

Laurus nobilis antiproliferative activity
142
cellular viability at the concentration range under investigation. The criteria of cytotoxic activity for
the crude extracts, as established by the American National Cancer Institute (NCI) is an IC
50
less than
30 µg/mL in the preliminary assay [27]. The ethanol extract of the fruits exhibited prominent
antiproliferative activity with an IC
50
of 12.3 µg/mL for T47D cell line and 28 µg/mL for MCF7 cells,
while the IC
50
values of the leaves were higher (19.8 µg/mL for T47D cells and 48.2 µg/mL for
MCF7).
Similar results were observed for L. nobilis grown in Turkey, where methanol extracts of leaves,
fruits and seeds were evaluated for their ovarian cytotoxic activity and DNA damaging properties
against three types of yeast. In this study, the most cytotoxic extract was that of the fruit and further
identification led to cytotoxic sesquiterpenes isolated from the fruits rather than from the leaves or
seeds [28]. In another study, L. nobilis leaves from Palestine were also tested for their anticancer
activity against MCF7 as well as L929sA (murine fibrosarcoma cell line) and MDA-MB 231(breast
cancer cell line). Dichloromethane: methanol extracts were used and here the leaves were reported as
not cytotoxic against the breast cancer cell line models used and had an IC
50
of 174 µg/mL against the
murine fibrosarcoma [29]. The concentration range of the crude extracts under investigation was
nevertheless not mentioned.
Cytotoxic activity has been reported for the crude bay leaf extract and for sesquiterpene lactones
isolated from the leaves and fruits of L. nobilis. The cytotoxicity was evaluated in vitro using human
tumor cell lines such as Jurkat, HL-60, LoVo, SH-SY5Y, MCF7 and A2780 and promising results
were obtained, especially with the isolated sesquiterpene lactones [28-31].
The antiproliferative activity of the volatile oils
and pure compounds
was investigated. Firstly, the
crude oils were tested for their antiproliferative activity and here, the activity was less than that for
both the ethanol and ethylacetate fractions. Secondly, the antitumor propensities of selected pure
volatile compounds of the volatile oil fraction were tested, this included, 1,8-cineole, α-pinene, β-
pinene, limonene, linalool, borneol, β-caryophyllene and caryophyllene oxide. From those, with the
exception of β-caryophyllene and caryophyllene oxide, no/or minimal antiproliferative activity was
detected from 0.1 up to 100 µg/mL. β-Caryophyllene and caryophyllene oxide exhibited potent
biological activity; 14.4 ± 1.6 µg/mL for β-caryophyllene and 8.1 ± 0.7 µg/mL for caryophyllene
oxide against MCF7 cells and 14.4 ±1.3 µg/mL for β-caryophyllene and 6.1 ± 0.8 µg/mL for
caryophyllene oxide against T47D cells.
Although present in low concentrations; β-caryophyllene and caryophyllene oxide, could have
contributed to the antiproliferative activities observed with the volatile oil fractions and crude extracts
of the tested plants. Moreover, the well known flavonoids quercetin, luteolin, kaempferol and apigenin
were identified in the ethanol extracts. In earlier publications [19] the IC
50
of luteolin was determined
(5.3 ± 0.4 µg/mL for MCF7 cells and 4.3 ± 0.1 µg/mL for T47D) and it was recognized as a potent
antiproliferative agent in Eminium spiculatum. It can be concluded that the observed antiproliferative
activity of the extracts are attributed to the flavonoids in addition to the mentioned pure volatile
compounds. It was postulated by Kwom et al. [32] that induction of apoptosis by kaempferol and
quercetin was caspase-3 dependent. Similar caspase-3 (and caspase-7) dependent apoptotic pathways
were described for luteolin [33].
β-Elemene (found in L. nobilis fruits at a percentage of 6.9 but only 0.3 % in its leaves) may
contribute to the differences in cytotoxic activity of the crude volatile oils as well as the ethanol,
chloroform and ethyl acetate extracts from the two different aerial parts. β-Elemene has been reported
to have anticancer activity against cancers of brain, breast, lung and other tissues after different in vivo
and in vitro experiments. The mechanism of its anticancer effect is still unclear; nevertheless, it could
be due to direct cytotoxic activity, and inhibition of free radical formation. Several reports attribute
this activity to induction of apoptosis and suppression of tolemerase activity [34].
In an attempt to study the safety of these extracts, the antiproliferative activity of the crude
ethanol extract was tested against normal freshly excised human periodontal cells. In those, and after
72 h incubation, the IC
50
values for both parts were higher than those determined against the selected
cancer cell lines; (81.3 µg/mL for the fruits and 41.8 µg/mL for the leaves). This suggests its
preferential selectivity for cancer tissues over normal ones.

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Abu-Dahab et.al., Rec. Nat. Prod.(2014) 8:2 136-147
3.3. Effects of Fruit and Leaf Extracts on Induction of Apoptosis as Detected and Quantified
by Nucleosome ELISA
Apoptosis is governed by a complex network of effector molecules. It is characterized by DNA
fragmentation, cell shrinkage and nuclear condensation, and phosphatidylserine translocation from the
inner to the outer leaflet of the plasma membrane bilayer [5]. Cisplatin's potent cytotoxicity is
primarily mediated by its ability to cause DNA damage and subsequent apoptotic cell death. However,
it was found that its anti-tumerogenic efficacy and proapoptotic features in many cancer cell lines
involves the activation of different forms of cell death, i.e. the receptor mediated apoptotic extrinsic
pathway, the intrinsic pathway as in alterations in mitochondrial membrane permeability and
cytochrome c release and a death process mediated by endoplasmic reticulum stress [35].
Compared with basal control wells, 28 µg/mL of L. nobilis fruit extract induced a significantly
detectable enrichment in the cytoplasmic mono- and oligonucleosomes after assumed induction of
programmed MCF7 cell death (227.3 ± 23.3%, p<0.05, n=4, Figure 1). More importantly, 48.2 µg/mL
of L. nobilis leaf extract augmented highly significantly cytoplasmic enrichment following
polynucleosomes disintegration and DNA fragmentation in treated MCF7 wells over 72 h ( 1295 ±
37.2 %, n=4, p<0.001 vs. basal (untreated) controls, the same figure).
3.4. Effect of L. nobilis Fruit and Leaf Extracts on Receptor-mediated Apoptosis-target
Molecules
Previous reports have indicated that MCF7 cells have a normal (non-mutated) tumor suppression
gene, p53. Cisplatin is a chemotherapeutic drug used for several human malignancies. Exceptionally,
cisplatin 72 hours incubation induced a highly significant augmentation of p53 levels (233.8 ± 10.4%,
n=4, p<0.001 vs. basal control MCF7 wells).
In examining the effects of L. nobilis fruit and leaf extracts on cell cycle regulatory molecules,
including p53 and its downstream molecule p21, Figure 2 demonstrates that L. nobilis fruit (28
µg/mL) enhanced the expression of neither protein at the examined incubation time. Thus, L. nobilis
fruit extract proapoptotic efficacy might not be regulated by either p53 or p21. Interestingly, our
results indicate that L. nobilis leaf extract components enhanced the p53 levels highly substantially
(140.1 ± 4.4%, n=4, p<0.001 vs. basal control MCF7 wells, Figure 2) less effectively than cisplatin,
although mostly unlikely, L. nobilis leaf extract proapoptotic effect was p21-independent. To establish
the sequence of events involved in L. nobilis induction of apoptosis, the recruitment of sFas/FasL-
mediated execution of apoptosis was investigated. Over 72 h, sFas/FasL system was not selectively
upregulated in either L. nobilis-parts mediated inhibition of proliferation in MCF7 treatments (Figure
3). We next examined the downstream caspase of sFas/FasL system, and consistent with our findings
on the lack of sFas/FasL system modulation, neither of L. nobilis parts increased caspase 8 activity at
72 h compared to basal control wells (Figure 4).

Laurus nobilis antiproliferative activity
144
0
250
500
750
1000
1250
1500
Control Laurus nobilis fruit Laurus nobilis leaf
DNA Fragmentation and nu cleosomes
levels (As %Control)
***
*
Figure 1. Effects of antiproliferative Laurus nobilis extracts on proapotosis DNA fragmentation.Cell lysates
containing cytoplasmic oligonucleosomes of apoptotic cells were analyzed by means of nucleosome ELISA kit
to investigate the induction of apoptosis in MCF7 cells by antiproliferative Laurus spp. Results expressed as
%control are mean ± SEM (n=4 independent determinations). *P<0.05 and ***P<0.001 indicates that bioactive
plants extracts had highly significant statistical differences compared to control (non-induced) wells and, as
analyzed by ANOVA followed by Dunnett's test.
0
50
100
150
Control Laurus nobili s fruit Laurus nobilis leaf
p53/p 21 Levels (As %Control)
p53 p21
***
Figure 2. Effects of antiproliferative Laurus nobilis on protein expression of p53/p21 in Human breast
adenocarcinoma MCF7 cells, as determined by p53/p21 ELISA kits. Results expressed as % control are mean ±
SEM (n=4 independent replicates). ***P<0.001 indicate that plants' incubations had highly significant statistical
differences vs. control (basal non- induced) wells, as analyzed by ANOVA followed by Dunnett's test.

145
Abu-Dahab et.al., Rec. Nat. Prod.(2014) 8:2 136-147
0
50
100
150
Control Laurus nobilis fruit Laurus nobilis leaf
sFas/ FasL (As %Control)
sFas FasL
Figure 3. The lack of Fas/Fas ligand apoptotic system involvement in Laurus nobilis induction of apoptosis in
MCF7 cells. Results expressed as %control are mean ± SEM (n=4 independent replicates). None of the
bioactive plants’-treatment wells had a statistically significant difference vs. control (non-induced) incubations,
as analyzed by ANOVA followed by Dunnett's test.
0
50
100
150
Control Laurus nobilis fruit Laurus nobilis l eaf
Caspas e 8 Activity (As %Control)
Figure 4. The lack of activation of caspase -8 in MCF7 cells by antiprolifertaive Laurus nobilis extracts. Results
are expressed as %control are mean ± SEM (n=4 independent determinations). None of the bioactive plants’-
treatment wells had a statistically significant difference vs. control (non-induced) incubations, as analyzed by
ANOVA followed by Dunnett's test
.
The
findings of the present study coincide with those of Jiang et al. [36], where p53 activation
was an early signal for apoptosis during cisplatin treatment [36]. Unequivocally, only L. nobilis leaf
extract evoked a highly marked increase in p53 without a concomitant increase in the downstream
effector molecule p21. The pro-apoptotic efficacy of fruits and leaf extracts was further verified and
substantiated, at least in part, by DNA-fragmenting executions. Both extracts, on the other hand, were
unable to augment the assembly of the MCF7 death-inducing signaling complex responsible for the
activation of caspase-8 and thus unable to enhance MCF7 cells’ extrinsic apoptosis cascade. This
signified the caspase-8 independent action mechanism of these extracts. Importantly, caspases are
very well known to act as key intermediates of apoptosis and to contribute to the apoptotic
morphology through the cleavage of various cellular substrates. Consequently, it can be hypothesized
that L. nobilis antiproliferative effectiveness may invoke other possible pathways and further studies
are necessary to investigate the precise mechanisms responsible. Fruits and leaves did not seem to
target succinctly the same signal transduction effectors. Hence, more elaborative investigation may
verify the plant’s promising multi-targeted malignancy therapeutics.
Taken together, the present data highlight L. nobilis as a potential natural agent for breast cancer
therapy. They also present L. nobilis important implication as a safe and effective alternative to
conventional breast cancer intervention.

Laurus nobilis antiproliferative activity
146
Acknowledgment
This work has been supported by an SRTD grant (contract JO/2008/RGS/034). The technical
assistance of Miss Lara Majdalawi and Mr. Ismail Abaza is appreciated.
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