FCT 7615 No.

of Pages 10, Model 5G

8 October 2013

Food and Chemical Toxicology xxx (2013) xxx–xxx

1

Contents lists available at ScienceDirect

Food and Chemical Toxicology

journal homepage: www.elsevier.com/locate/foodchemtox

5
6

3 Apolar Laurus nobilis leaf extracts induce cytotoxicity and apoptosis

4 towards three nervous system cell lines
7 Q1 Severina Pacifico a,1,⇑, Marialuisa Gallicchio a,1, Peter Lorenz b, Nicoletta Potenza a, Silvia Galasso a,
8 Sabina Marciano a, Antonio Fiorentino a, Florian C. Stintzing b, Pietro Monaco a
9 a
Department of Environmental, Biological and Pharmaceutical Sciences and Technologies, Second University of Naples, Via Vivaldi 43, I-81100 Caserta, Italy
10 b
WALA Heilmittel GmbH, BadBoll/Eckwälden, Germany

12
11
13
a r t i c l e i n f o a b s t r a c t
1
2 5
8
16 Article history: In the course of a bioactivity screening of Mediterranean plants, the assessment of neuroprotective prop- 29
17 Received 18 July 2013 erties of Laurus nobilis L. was of interest. Dried leaves were extracted by sonication using CHCl3 as solvent. 30
18 Accepted 24 September 2013 The CHCl3 parental extract (CHCl3-pe) was fractionated to yield CHCl3 (LnC-1), EtOAc (LnC-2), MeOH 31
19 Available online xxxx
(LnC-3) fractions. Each fraction underwent an extensive screening towards human neuroblastoma 32
(SK-N-BE(2)-C, and SH-SY5Y) and rat glioma (C6) cell lines. MTT and SRB cytotoxicity tests were per- 33
20 Keywords: formed. The effect on the plasma membrane integrity was evaluated by assessment of LDH release. 34
21 Laurus nobilis L.
The caspase-3 activation enzyme and DNA fragmentation were also evaluated. The oxidant/antioxidant 35
22 Neuroprotection
23 Assay guided fractionation
ability of all the extracts were evaluated using different methods. Furthermore, a metabolite profiling 36
24 Sesquiterpenes of the investigated extracts was carried out by GC–EI-MS. CHCl3-pe contained terpenes, allylphenols, 37
25 Apoptosis and a-tocopherol. Dehydrocostus lactone was the main constituent. As result of the fractionation tech- 38
26 Metabolic profiling nique, the LnC-1 extract was mainly composed of a-tocopherol, whereas the LnC-2 fraction was enriched 39
27 in guaiane and eudesmane terpenes. The most cytotoxic LnC-2 fraction induced apoptosis; it was ineffec- 40
tive in preventing in vitro free radicals production. Overall, the experimental results support a possible 41
role of LnC-2 preparation as a chemopreventive agent for neuronal cells or other cells of the CNS. 42
Ó 2013 Elsevier Ltd. All rights reserved. 43

44
45
46 1. Introduction recent improvements in diagnostic techniques, therapeutic 65
approaches remain disappointing and unsuccessful. Consequently, 66
47 The medicinal properties of plants are widely known to man, there is an urgent need for promising anticancer agents or lead 67
48 who, since ancient times, has learned about their beneficial and structures for the development of new drugs to raise the overall 68
49 curative effects. The cumulative action exerted by the different survival of patients with brain cancer. 69
50 natural constituents of a medicinal plant, or its part (plant drug), Several in vitro and in vivo studies showed the anti-brain tumor 70
51 is the basis of unique pharmacological properties (beneficial and/ properties of some plant extracts, both as therapeutics and as com- 71
52 or adverse). Plant complex (phytocomplex) is the biochemical ponents of the diet for chemoprevention (Pathak et al., 2003; 72
53 entity in which the bioactivity of the medicinal plant is expressed Huang et al., 2007; Lantto et al., 2009). The cytotoxic effects were 73
54 in its fullness. In fact, in it synergistic and additive effects, an im- related to their secondary metabolites content. It was reported, 74
55 proved bioavailability, or even a reduced toxicity are combined for instance, that a chloroform extract from Angelica sinensis, con- 75
56 with an enhanced pharmacological efficacy, often not attributable taining large amount of low-molecular weight phenolics as ferulic 76
57 to a single active substance (Bagetta et al., 2012). Although there acid, and butylidenephthalide, had the ability to inhibit growth and 77
58 are various molecular targets for which natural products have induce apoptosis of glioblastoma multiforme (GBM) tumor 78
59 proven their efficiency, biomedical research is now particularly through p53-dependent and p53-independent pathways (Tsai 79
60 interested in defining their role in the treatment of those disorders et al., 2005; Lin et al., 2012). Grape seed extract (GSE) treatment 80
61 and diseases that affect global human health, such as neurodegen- induced accumulation of intracellular reactive oxygen species 81
62 erative diseases and brain cancer (Iriti et al., 2010; Kannappan (ROS) in head and neck squamous cell carcinoma (HNSCC) cells 82
63 et al., 2011). For example, malignant brain tumors are particularly leading to DNA damage causing a cell cycle arrest in G2/M phase 83
64 aggressive in both children and adults (Li et al., 2013). Despite together with cell growth inhibition and apoptotic cell death 84
(Shrotriya et al., 2012). Moreover, chokeberry extract (Aronia 85
melanocarpa), which is rich in anthocyanins, and curcumin from 86
⇑ Corresponding author. Tel.: +39 0823 274572; fax: +39 0823 274571. Curcuma longa were reported to possess anticancer properties in 87
E-mail address: severina.pacifico@unina2.it (S. Pacifico). an established brain cancer cell line (Abdullah Thani et al., 2012). 88
1
These authors contributed equally to this research

0278-6915/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.fct.2013.09.029

Please cite this article in press as: Pacifico, S., et al. Apolar Laurus nobilis leaf extracts induce cytotoxicity and apoptosis towards three nervous system cell
lines. Food Chem. Toxicol. (2013), http://dx.doi.org/10.1016/j.fct.2013.09.029
 FCT 7615 No. of Pages 10, Model 5G
8 October 2013

2 S. Pacifico et al. / Food and Chemical Toxicology xxx (2013) xxx–xxx

89 A particular interest is focus on sesquiterpene-rich plant extracts. 2. Materials and methods 155
90 Sesquiterpenes are a class of naturally occurring molecules that have
2.1. Reagents and chemicals 156
91 demonstrated therapeutic potential in decreasing the progression of
92 cancer. These 15-carbon isoprenoid compounds are widespread in All of the solvents and reagents used for assessing antioxidant screening were 157
93 the plant kingdom (Modzelewska et al., 2005; Chen et al., 2011). A purchased from Sigma–Aldrich Chemie (Buchs, Switzerland) except ABTS [2,20 - 158
94 sesquiterpene enriched dichloromethane fraction from leaves of azinobis-(3-ethylbenzothiazolin-6-sulfonic acid)], which was from Roche Diagnos- 159
tics (Roche Diagnostics, Mannheim, Germany). Cell culture media and reagents for 160
95 Eremanthus crotonoides showed antiproliferative effects against
cytotoxicity testing were purchased from Invitrogen (Paisley, UK), MTT [3-(4,5-di- 161
96 U87-MG, and U251 glioblastoma multiforme cell lines (Lobo et al., methyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide], SRB (sulforhodamine 162
97 2012). Centratherin and goyazensolide were identified as the main B), INT [(2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride)], 163
98 constituents of the herbal extract through UPLC-PDA-ESI-MS/MS phenazine methosulfate, and lactic acid were from Sigma–Aldrich Chemie. 164
99 analysis. Different purified sesquiterpenoids were also successfully
100 tested for their cytotoxic activity against Central Nerve System
101 (CNS) tumor cell lines. b-Elemene, a volatile terpene found in 2.2. Plant collection and fractionation 165
102 botanicals such as celery, mint, and in many herbs used in traditional
Leaves from L. nobilis were collected in Caserta (Italy) in May 2011 and identi- 166
103 medicine, was shown to have cytotoxic efficacy towards different
fied by Dr. Assunta Esposito of the Second University of Naples. A voucher specimen 167
104 brain tumor cell lines (A172, CCF-STTG1, and U-87MG), effectively (CE000215) has been deposited at the Herbarium of the Department of Environ- 168
105 suppressing tumor cell survival. The inhibitory effect of b-elemene mental, Biological and Pharmaceutical Sciences and Technologies of the Second 169
106 was mediated by the induction of apoptosis (Li et al., 2013). Alanto- University of Naples. After drying in a thermo-ventilated oven at 40 °C for 7 days, 170
107 lactone, a sesquiterpene lactone compound, was found to effectively leaves were powdered in a mill and extracted by sonication (Dr. Hielscher UP 171
200S) for 1 h, using CHCl3 as extracting solvent. Then the samples were centrifuged 172
108 inhibit growth and triggering apoptosis in glioblastoma cells in a 173
at 3500 rpm for 10 min in a Beckman GS-15R centrifuge (Beckman Coulter, Milano,
109 time- and dose-dependent manner. The alantolactone-induced Italy) fitted with rotor S4180. The supernatant was dried under vacuum to yield a 174
110 apoptosis was found to be associated with glutathione (GSH) deple- crude extract (6.1 g, CHCl3-pe; pe = parental extract), which was afterwards 175
111 tion, and reactive oxygen species (ROS) generation (Khan et al., chromatographed by CC-SiO2 using three solvents with increasing polarity 176
(CHCl3, EtOAc, and MeOH) (Fig. 1). After removing the solvent in vacuo by rotary 177
112 2012). In order to assess the neuroprotective properties of plant ex-
evaporation, the resulting fractions were LnC-1 (46.9 mg), LnC-2 (471.0 mg) and 178
113 tracts, prepared, from herbal drugs commonly used in traditional LnC-3 (66.1 mg). 179
114 Mediterranean medicine, our attention has been paid to bay leaves.
115 The bay tree, also called bay laurel, sweet bay or laurel (Laurus nobilis
116 L.) is an evergreen shrub or small tree, belonging to the Lauraceae 180
2.3. GC/MS analyses
117 family (Pignatti, 1982; Kumar et al., 2004). Since ancient times bay
118 leaves were used in the Mediterranean cuisine as a condiment, but For GC/MS analysis the solid fractions were re-dissolved in CHCl3, EtOAc or 181
119 also applied as a mild bitter digestive (amarum). An essential oil MeOH, respectively (10 mg + 2 mL solvent, w/v). An aliquot of 1 lL each was 182
injected into the GC/MS injection port. GC/MS was performed with a PerkinElmer 183
120 obtained by steam distillation of laurel leaves is employed in per-
Clarus 500 gas chromatograph with split injection (split ratio 30:1) coupled to a 184
121 fumery and aroma industry. In traditional medicine, infusions from mass detector. The column used was a Zebron ZB-5 ms capillary column 185
122 the leaves were used for soothing of stomach troubles and therapy of (60 m  0.25 mm i.d.  0.25 lm film thickness, 5% phenylpolysiloxane and 95% 186
123 renal or bladder complaints (Speroni et al., 2011). The essential bay dimethylpolysiloxane coating; Phenomenex, Torrance, USA). Helium was the carrier 187
124 leaves oil was also administered for topical treatment of rheuma- gas at a flow rate of 1 mL/min. The injector employed was a PSS (programmed-tem- 188
perature split/splitless injector; temperature: 250 °C). The temperature program for 189
125 tism, pulled muscles or abrasions of the skin (Van Wyk and Wink, 190
the column oven was 100–320 °C at 4 °C/min with a final hold time of 30 min (total
126 2004). The chemical composition of bay leaves has been extensively run time: 85 min). The mass spectrometer was operated in the electron ionization 191
127 studied: essential oil constituents (Verdian-Rizi and Hadjiakhoondi, mode (70 eV) and molecular ions between m/z 60–610 Da were captured. The iden- 192
128 2008; Al-Kalaldeh et al., 2010), sesquiterpenes (De Marino et al., tification of the analytes was based on comparison of the mass spectra (see Table 1) 193
with the NIST database (NIST, Stein et al., 2008) as well as literature data. 194
129 2005; Julianti et al., 2012), aporphin-type alkaloids (Pech and Brune-
130 ton, 1982), flavonoids (Lee et al., 2012), phenolic acids and lignans
131 (De Marino et al., 2004; Dall’Acqua et al., 2006, 2009) have been
132 identified. Although the pharmacological and biological effects of 2.4. Assessment of the reducing capacity 195

133 bay leaves have been thoroughly investigated in the literature and 196
Five methods were employed for assessing the antioxidant properties of ex-
134 anti-oxidant, anti-bacterial, anti-fungal, anti-convulsive, anti-leu- tracts from L. nobilis leaves. CHCl3-pe extract and the fractions derived therefrom, 197
135 kemia, sedative, neuromuscular as well as wound-healing effects dissolved in DMSO as stock solutions of 125 mg/mL, were evaluated at different 198
136 have been published (Matsuda et al., 2000; Kang et al., 2002; Dada- levels. Tests were carried out performing three replicate measurements for three 199
samples (n = 3) of each extract (in total, 3  3 measurements). Recorded activities 200
137 lioglu and Evrendilek, 2004; Barla et al., 2007; Ramos et al., 2012;
were compared to a blank arranged in parallel to the samples. Radical Scavenging 201
138 Saab et al., 2012), their neuroprotective potential and anti-cancer Capacity Percentage (RCS%) was calculated using the following formula: 202
139 activity have not been addressed with few exceptions. Cho et al. RSC% = [(Absblank  Abssample)/Absblank]  100. 203
140 (2010) observed that L. nobilis chloroform fraction was able to
141 protect against cerebral ischemia neuronal damage. The inhibitory
142 effects of spirafolide (Ham et al., 2010), and 3a-acetoxyeudesma-
143 1,4(15),11(13)-trien-12,6a-olide (Koo et al., 2011) from laurel
144 leaves on DA-induced apoptosis in dopaminergic SH-SY5Y cells
145 were also observed.
146 In the current study bay leaves were extracted with CHCl3 to ob-
147 tain a lipophilic parental extract (CHCl3-pe), which underwent an as-
148 say guided fractionation. Thus, each extract obtained with solvents
149 of increasing polarity, was screened in order to assess its specific
150 cytotoxic and apoptotic properties towards human neuroblastoma
151 (SK-N-BE(2)-C, and SH-SY5Y) and rat glioma (C6) cell lines. Because
152 of the pivotal role of ROS in triggering apoptosis, the oxidant/antiox-
UE = Ultrasound Extraction
153 idant ability of parental extract and the partially purified fractions
154 derived therefrom were also thoroughly evaluated. Fig. 1. CHCl3-pe fractionation scheme.

Please cite this article in press as: Pacifico, S., et al. Apolar Laurus nobilis leaf extracts induce cytotoxicity and apoptosis towards three nervous system cell
lines. Food Chem. Toxicol. (2013), http://dx.doi.org/10.1016/j.fct.2013.09.029
 FCT 7615 No. of Pages 10, Model 5G
8 October 2013

S. Pacifico et al. / Food and Chemical Toxicology xxx (2013) xxx–xxx 3

Table 1
L. nobilis extracts investigated by GC–EI-MS profiling.

Compound tR EI MS m/z (rel. int.) Match CHCl3- CHCl3 (LnC- EtOAc (LnC-
(min) NISTa peb 1)b 2)b
Eucalyptol 7.4 154 [M]+, 136 [M-H2O]+(13), 126 (17), 125 (15), 121 (10), 112 (6), 111 (82), 669 3.25 2.12 0.75
109 (9), 108 (100), 97 (19), 96 (60), 95 (36), 93 (57), 85 (8), 84 (98), 83 (46)
(Gören et al., 2002)
Limonene 8.5 136 [M]+ (25), 121 (24), 94 (30), 93 (60), 91 (23), 79 (40), 68 (100), 66 (25), 536 2.49 0.08 6.19
54 (42), 39 (58)
a-Terpinylacetate 15.2 196 [M, not observed]+, 137 (22), 136(68), 122 (10), 121 (100), 107 (18), 94 754 8.25 11.47 n.d.
(15), 93 (81), 90 (24), 81 (20), 78 (26), 76 (20), 68 (12), 67 (27), 66 (28), 58
(12), 54 (10), 52 (11)
Eugenol 15.4 164 [M]+ (100), 149 (33), 137 (19), 131 (26), 121 (13), 107 (5), 103 (29), 91 735 2.04 1.52 n.d.
(24), 77 (22), 65 (8) (Miele et al., 2001)
Methyleugenol 16.6 178 [M]+ (100), 163 (25), 152 (10), 147 (24), 135 (9), 115 (11), 107 (18), 103 791 9.51 11.73 n.d.
(23), 91 (26), 77 (11) (Miele et al., 2001)
Elemicin 21.0 208 [M]+ (100), 193 (56), 177 (13), 133 (15), 91 (12), 77 (15) (Zouari et al., 726 1.19 n.d. 1.25
2013)
Spatulenol 22.3 220 [M, not observed]+, 205 [M-CH3]+ (60), 202 [M-OH]+ (10), 187 (13), 177 590 1.75 n.d. 1.92
(8), 159 (37), 145 (12), 131 (27), 119 (70), 105 (55), 90 (100), 80 (25), 78
(52) (Werner et al., 2003)
b-Eudesmol 24.5 222 [M]+ (4), 204 (12), 189 (13), 164 (24), 161 (22), 149 (60), 135 (10), 122 593 1.27 n.d. 2.02
(24), 109 (27), 105 (25), 95 (24), 92 (37), 80 (27), 78 (26), 66 (24), 58 (100),
54 (22)
Dehydrocostus 33.3 230 [M]+ (27), 215 (20), 201 (45), 150 (90), 91 (66), 90 (100), 78 (75), 52(54) 776c 51.69 31.16 76.83
lactone (Kim et al., 2008)
Eremanthin 33.6 230 [M]+ (20), 187 (10), 173 (25), 172 (49), 150 (100), 145 (12), 129 (13), 765 3.39 0.82 4.31
123 (20)
Santamarine 37.7 248 [M]+ (49), 152 (40), 133 (20), 119 (20), 107 (100), 90 (60), 80 (35), 78 – n.d. n.d. 0.63
(38) (Fang et al., 2005)
a-Tocopherol 55.3 430 [M]+ (100), 205 (12), 165 (93), 43 (25) (Fiorentino et al., 2009) 617 13.39 41.09 n.d.
b-Sitosterol 59.1 416 [M]+ (18), 415 (100), 330 (50), 304 (40), 213 (45), 145 (70), 105 (80), 54 551 1.77 0 6.10
(77)
a
Match factors obtained by searching the NIST database (1000 = 100%);
b
percentage of each identified compound based on its peak area;
c
identification based on literature MS data and an isomer found in the NIST database.

204 2.4.1. Determination of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging the samples. The antioxidant activity (ORAC value) was calculated by using the 239
205 capacity TroloxÒ calibration curve. The ORAC values were expressed as lmol TroloxÒ 240
206 In order to estimate the DPPH scavenging capability, investigated extracts (2.5, equivalents. 241
207 5.0, 10.0, 25.0, 50.0, and 100.0 lg/mL, final concentration levels) were dissolved in a
208 DPPH methanol solution (9.4  105 M; 1.0 mL) at room temperature. After 30 min,
209 the absorption at 515 nm was measured by a Shimadzu UV-1700 spectrophotome- 2.4.4. Determination of Superoxide Anion Radical (O2 ) Scavenging Capacity
242
210 ter in reference to a blank. The results were expressed in terms of the percentage The inhibition of nitroblue tetrazolium (NBT) reduction by photochemically 243
211 decrease of the initial DPPH radical absorption by the test samples.

generated O2 was used to determine the superoxide anion radical scavenging activ- 244
ity of the extracts according to the literature (Fiorentino et al., 2008). L. nobilis 245
extracts (2.5, 5.0, 10.0, 25.0, 50.0, and 100.0 lg/mL, final concentration levels) were 246
dissolved at room temperature in 3.0 mL of a reaction mixture containing sodium 247
212 2.4.2. Determination of ABTS [2,20 -azinobis-(3-ethylbenzothiazolin-6-sulfonic acid)] phosphate buffer (50.0 mM, pH 7.8), methionine (13.0 mM), riboflavin (2.0 lM), 248
213 radical cation scavenging capacity EDTA (100.0 lM) and NBT (75.0 lM). The O 249
2 production was followed by monitor-
214 The determination of ABTS+ solution scavenging capacity was estimated as ing the increase in absorbance at 560 nm after 10 min illumination with a fluores- 250
215 previously reported (Di Maro et al., 2013). ABTS radical cation was generated by cent lamp. 251
216 reacting ABTS (7.0 mM) and potassium persulfate (2.45 mM). The mixture was
217 allowed to stand in the dark at room temperature for 12–16 h. Thus, the ABTS+
218 solution was diluted with PBS (pH 7.4) in order to reach an absorbance of 0.70 at 2.4.5. Determination of hydroxyl radical (OH) scavenging capacity 252
219 734 nm. L. nobilis extracts (2.5, 5.0, 10.0, 25.0, 50.0, and 100.0 lg/mL, final concen- Fe(III)-EDTA-H2O2 solutions were used as hydroxyl radical generating system 253
220 tration levels) were dissolved in 1.0 mL of diluted ABTS+ solution. After 6 min of (Fenton reaction), and the radical scavenging capabilities of L. nobilis extracts were 254
221 incubation, the absorption at 734 nm was measured by a Shimadzu UV-1700 evaluated from the determination of malondialdehyde (MDA) from the oxidative 255
222 spectrophotometer in reference to a blank. The results were expressed in terms degradation of 2-deoxyribose. The reagent mixture, consisting of H2O2 (100.0 lM) 256
223 of the percentage decrease of the initial ABTS+ absorption by the test samples. aqueous solution, EDTA (100.0 lM), FeCl3 (100.0 lM), and ascorbic acid 257
(100.0 lM) in KH2PO4/KOH buffer (10 mM, pH 7.4), was incubated at 37 °C for 258
1 h. 2-Deoxyribose (28 mM) was added. The reaction mixture was allowed to stand 259
at 37 °C for 5 min. Thus, 1 mL of diluted reagent mixture was added into tubes con- 260
224 2.4.3. Determination of oxygen radical absorbance capacity (ORAC) taining test samples, which were incubated at 37 °C for 1 h. To each tube 1 mL of a 261
225 The ORAC assay was performed as described by Prior et al. (2003). Each extract 1% TBA (thiobarbituric acid) in 50 mM NaOH and 1 mL of a solution at 2.8% of TCA 262
226 (20 lL; 1.0, 2.5, and 5.0 lg/mL, final concentration levels) and fluorescein (120.0 lL; (trichloroacetic acid) was added and placed in a water bath at 90 °C for 30 min, 263
227 70 nM, final concentration) were preincubated for 15 min at 37 °C in 75 mM phos- before taking the reading at 532 nm. 264
228 phate buffer (pH 7.4). Then 2,20 -azobis-(2-amidinopropane)-dihydrochloride
229 (AAPH) solution (60.0 lL; 12 mM, final concentration) was rapidly added. In
230 parallel with the test samples, a blank (FL + AAPH) and solutions of the reference 2.5. Cytotoxicity assessment 265
231 antioxidant TroloxÒ (1–8 lM, final concentration levels) were properly prepared
232 in PBS. The fluorescence decay (kex = 485 nm, kem = 525 nm) was recorded every Cytotoxicity assays which use different parameters associated with cell death 266
233 minute for 80 min using a Tecan SpectraFluor fluorescence and absorbance reader. and proliferation were performed. Tests were carried out performing 12 replicate 267
234 Antioxidant curves (fluorescence vs. time) were first normalized to the curve of the (n = 12) measurements for three samples of each extract (in total: 12  3 measure- 268
235 blank by multiplying original data by the fluorescence blank factor (t = 0/fluores- ments). Samples of each extract were prepared as a stock solutions of 125 mg/mL in 269
236 cence sample, t = 0). From the normalized curves, the area under the fluorescence DMSO. They were further diluted in cell culture medium to appropriate final dose 270
237 decay curve (AUC) was calculated. Linear regression equations between net AUC levels (DMSO final concentration was equal to 0.1% (v/v)). Recorded activities were 271
238 (AUCantioxidant  AUCblank) and antioxidant concentration were calculated for all compared to an untreated blank arranged in parallel to the samples. 272

Please cite this article in press as: Pacifico, S., et al. Apolar Laurus nobilis leaf extracts induce cytotoxicity and apoptosis towards three nervous system cell
lines. Food Chem. Toxicol. (2013), http://dx.doi.org/10.1016/j.fct.2013.09.029
 FCT 7615 No. of Pages 10, Model 5G
8 October 2013

4 S. Pacifico et al. / Food and Chemical Toxicology xxx (2013) xxx–xxx

273 2.5.1. Cell cultures 2.6. Assessment of apoptosis-inducing activities 345

274 C6 rat glioma cell line and, SH-SY5Y human neuroblastoma cells were pur-
275 chased from ATCC (American Type Culture Collection); SK-N-BE(2)-C human bone 2.6.1. Caspase-3 activation assay 346
276 marrow neuroblastoma cells were purchased from ICLC (Interlab Cell Line Collec- Caspases are crucial mediators of programmed cell death. Among them, cas- 347
277 tion) at Istituto Nazionale per la Ricerca sul Cancro, Genoa (Italy). C6 and SH- pase-3 (also known as CPP32, Yama or apopain) is a frequently activated death pro- 348
278 SY5Y cell lines were grown in DMEM high glucose medium supplemented with tease, catalyzing the specific cleavage of many key cellular proteins. Caspase-3 349
279 10% fetal bovine serum, 50.0 U/mL penicillin, and 100.0 lg/mL streptomycin, at activation was detected through the Caspase-3/CPP32 Colorimetric Kit (Invitrogen), 350
280 37 °C in a humidified atmosphere containing 5% CO2. The SK-N-BE(2)-C cell line following the protocol described by the manufacturer. The Caspase-3 colorimetric 351
281 was plated and grown under the same conditions, except that RPMI 1640 was used protease assay provides a simple and convenient means for quantification of casp- 352
282 instead of DMEM. ases that recognize the amino acid sequence, DEVD. The substrate, DEVD-pNA, is 353
composed of the chromophore, p-nitroanilide (pNA), and a synthetic tetrapeptide, 354
DEVD (Asp-Glu-Val-Asp), which is the upstream amino acid sequence of the cas- 355
pase-3 cleavage site in PARP (Poly ADP ribose polymerase). Upon cleavage of the 356
283 2.5.2. MTT cell viability test substrate by caspase-3, free pNA light absorbance can be spectrophotometrically 357
284 The MTT test allows the assessment of cell viability by determining the levels of quantified. Comparison of the absorbance of pNA from apoptotic sample with a con- 358
285 activity of mitochondrial dehydrogenases towards 3-(4,5-dimethyl-2-thiazolyl)- trol allows determination of the fold increase in caspase-3 activity. Briefly, cells 359
286 2,5-diphenyl-2H-tetrazolium bromide (MTT). were plated in 6-multiwell plates at a density of 1.6  105 cells/well. Twenty-four 360
287 C6, SH-SY5Y, and SK-N-BE(2)-C cell lines were seeded in 96-multiwell plates at hours after seeding, the cells were treated with extracts (100.0 lg/mL) from L. nobi- 361
288 a density of 1.0  104 cells/well. After 24 h of incubation, cells were treated with ex- lis and incubated for another 24 h. Cells were resuspended in 50 lL of chilled cell 362
289 tracts from L. nobilis at six dose levels (2.5, 10.0, 25.0, 50.0, 100.0, and 250.0 lg/mL). lysis buffer and incubated on ice for 10 min. After centrifugation at 10,000g and 363
290 At 24, 48, and 72 h of incubation, cells were treated with 150 lL of MTT (0.50 mg/ 4 °C for 1 min, the supernatant (cytosol extract) was transferred to a fresh tube 364
291 mL), dissolved in the culture medium, for 1 h at 37 °C in a 5% CO2 humidified and put on ice. Protein in supernatant was quantified by the Lowry method with 365
292 atmosphere. The MTT solution was then removed and 100 lL of DMSO were added bovine serum albumin as standard. Each cytosol extract was diluted to a concentra- 366
293 to dissolve the formazan originated. Finally, the absorbance at 570 nm of each well tion of 100 lg protein per 50 lL cell lysis buffer (2 mg/mL). Thus, 50 lL of 2 367
294 was determined using a Tecan SpectraFluor fluorescence and absorbance reader. reaction buffer (containing 10 mM dithiothreitol) and 5 lL of the 4 mM 368
295 Cell viability was expressed as percentage of mitochondrial redox activity of the DEVD-pNA caspase 3 substrate (200 lM final concentration of each) were added. 369
296 cells treated with the extracts compared to the untreated control (Pacifico et al., After 2 h of incubation at 37 °C, formation of p-nitroanilide was measured at 370
297 2012). Cell viability inhibition (CVI,%) was calculated using the following formula: 405 nm by use of a Tecan SpectraFluor fluorescence and absorbance reader. 371
298 [(Absuntreated cells)  (Abstreated cells)/(Absuntreated cells)]  100. Caspase-3 activation percentage was calculated using the following formula: [(Abs- 372
treated cells  Absblank)/Absblank]  100. 373

299 2.5.3. SRB cell viability test

300 The sulforhodamine B (SRB) assay is used for cell density determination, based 2.6.2. Genomic DNA fragmentation assay 374
301 on the measurement of cellular protein content (Skehan et al., 1990). C6, SH-SY5Y, Oligonucleosomal fragmentation of genomic DNA is one of the hallmarks of 375
302 and SK-N-BE(2)-C cell lines were seeded in 96-multiwell plates at a density of apoptosis. It can be detected as a ladder pattern on agarose gel electrophoresis. Cells 376
303 1.0  104 cells/well. After 24 h of incubation, cells were treated with extracts from were seeded in 6-multiwell plates at a density of 1.6  105 cells/well. Twenty-four 377
304 L. nobilis at six dose levels (2.5, 10.0, 25.0, 50.0, 100.0, and 250.0 lg/mL). At 24, 48, hours after seeding, the cells were treated with extracts (100.0 lg/mL) from L. nobi- 378
305 and 72 h of incubation, cells were fixed with ice-cold TCA (10% w/v, 40 lL) for 1 h at lis, and incubated for another 24 h. In parallel, an untreated control was set up. Cells 379
306 4 °C. The plates were washed five times in distilled water and allowed to dry. Then, were washed with PBS, harvested by centrifugation (6000 rpm, 5 min) and lysed by 380
307 50 lL of sulphorhodamine B (SBR, 0.4% w/v in 1% aqueous acetic acid) solution was incubating at 55 °C for 2 h in lysis buffer (50 mM Tris–HCl pH 8.0, 10.0 mM EDTA, 381
308 added to each well of the dried 96-well plates and incubated at room temperature 0.5% SDS) with proteinase K (0.5 mg/mL). Then, genomic DNA was isolated by eth- 382
309 for 30 min. In order to remove unbound dye, the plates were quickly washed with anol precipitation. In brief, DNA solution was mixed with 2.5 volumes of cold abso- 383
310 1% aqueous acetic acid and dried subsequently. The bound SRB was solubilized by lute ethanol, in the presence of sodium acetate (0.1 volumes, 3 M, pH 5.2), and 384
311 adding 100 lL of 10 mM unbuffered Tris Base (pH 10.5) to each well and shaking for incubated at 80 °C; DNA was pelletted by centrifugation (13,000 rpm, 15 min), 385
312 5 min on a shaker platform. Finally, the absorbance at 570 nm of each well was washed by 70% ethanol. Dried pellets were dissolved in distilled water and incu- 386
313 measured using a Tecan SpectraFluor fluorescence and absorbance reader. bated at 37 °C for 30 min with Rnase A to a final concentration of 100 lg/mL. 387
314 Cell viability inhibition (CVI,%) was calculated using the following formula: DNA samples were quantified using a NanoDrop 2000/2000c Spectrophotometers 388
315 [(Absuntreated cells)  (Abstreated cells)/(Absuntreated cells)]  100.

316 2.5.4. Lactate dehydrogenase (LDH) leakage assay

317 One parameter for cell death is the integrity of the cell membrane, which can be
318 measured by the cytoplasmic enzyme released by damaged cells. Lactate dehydro-
319 genase (LDH) is a stable cytoplasmic enzyme abundant in the cytosol of all mamma-
320 lian cells. It is rapidly released into the cell culture supernatant upon damage of the
321 plasma membrane (Weyermann et al., 2005).
322 The lactate dehydrogenase (LDH) leakage assay is a colorimetric assay for the
323 quantification of cell death and cell lysis based on the measurement of LDH released
324 from the cytosol of damaged cells into the supernatant. LDH activity can be
325 determined by a coupled enzymatic reaction: LDH oxidizes lactate to pyruvate
326 which then reacts with tetrazolium salt 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phe-
327 nyltetrazolium chloride (INT) to form a water-soluble formazan dye. The increase in
328 the amount of formazan produced in culture supernatant directly correlates to the
329 increase in the number of lysed cells.
330 C6, SH-SY5Y, and SK-N-BE(2)-C cell lines were seeded in 24-multiwell plates at
331 a density of 4.0  104 cells/well. Twenty-four hours after seeding, the cells were
332 treated with extracts (100.0 lg/mL) from L. nobilis, and incubated for other 24, 48
333 and, 72 h. Then, each supernatant (100 lL) was treated with 100 lL of the reaction
334 mixture (0.7 mM INT; 54.0 mM lactic acid; 0.3 mM phenazine methosulfate;
335 0.8 mM NAD+ in 0.2 M Tris–HCl pH 8.0) in a 96-wells plate. The reaction was carried
336 out for 45 min in the dark under gentle stirring at 37 °C and stopped by addition of
337 1 M HCl (50 lL).
338 The absorbance was read at 492 nm by a Tecan SpectraFluor fluorescence and
339 absorbance reader. Data obtained were expressed in reference to a positive blank
340 prepared using a Triton X-100 (1%) solution. The amount of enzyme activity
341 detected in the culture supernatant reflects the membrane integrity and correlates Fig. 2. CHCl3-pe Cell Viability Inhibition (CVI,%) towards SK-N-BE(2)-C, SH-SY5Y,
342 with the proportion of lysed cells. Hence, cell vitality is inversely proportional to the and C6 cell lines at 24 h, 48 h, and 72 h exposure times. A. MTT test results; B. SRB
343 LDH released. LDH release percentage was calculated using the following formula: test results. Values, reported as percentage vs. an untreated control, are the
344 [(Abstreated cells  Absuntreated cells)/(AbsTriton-X100 treated cells  Absuntreated cells)]  100. mean ± SD of measurements carried out on 3 samples (n = 3) analyzed 12 times.

Please cite this article in press as: Pacifico, S., et al. Apolar Laurus nobilis leaf extracts induce cytotoxicity and apoptosis towards three nervous system cell
lines. Food Chem. Toxicol. (2013), http://dx.doi.org/10.1016/j.fct.2013.09.029
 FCT 7615 No. of Pages 10, Model 5G
8 October 2013

S. Pacifico et al. / Food and Chemical Toxicology xxx (2013) xxx–xxx 5

O O O O
O O
OH O O
O

α -terpinyl acetate limonene eucalyptol eugenol methyleugenol elemicin

OH
HO H H H H H

H H H H OH
H O O O
O
O O
spatulenol dehydrocostus lactone eremathine santamarine β -eudesmol

HO H
H
H
O H H
3
HO
α-tocopherol β -sitosterol

Fig. 3. Main metabolites identified in L. nobilis investigated extracts.

389 (ThermoScientific, Wilmington, DE, USA) and stored at 4 °C. 3.0 lg of each DNA of cell proliferation and an induction of cell death. Cell viability did 402
390 sample, were loaded on 2.0% agarose gel (containing 10 lg/mL ethidium bromide)
not seem to be affected by exposure time. The C6 murine cells were 403
391 in 1 TAE buffer, along with a molecular weight marker.
shown to be less sensitive to the addition of the toxic agent; cell 404
viability inhibition was weak at all the three exposure times 405
392 3. Results considered. It was only at the highest tested doses (100.0 and 406
250.0 lg/mL) that the extract exerted a significant inhibition on 407
393 3.1. CHCl3 parental extract cytotoxicity and GC–EI-MS metabolic the growth of the C6 murine cells. C6 cells as a key modulator of 408
394 profiling neuroprotection, are generally less susceptible for cytotoxic or 409
ROS generating substances, because of high glutathione (GSH) 410
395 The CHCl3 parental extract (CHCl3-pe) from L. nobilis leaves content (Kleinkauf-Rocha et al., 2013). 411
396 underwent an extensive screening to assess its cytotoxic proper- In order to obtain qualitative information about the chemical 412
397 ties. In order to assess cell growth, MTT and SRB assays, widely de- composition and to pinpoint the substances responsible for the 413
398 scribed for in vitro chemosensitivity testing of tumor cell lines, activity detected, a GC/MS profiling was carried out. Full scan anal- 414
399 were performed. The two colorimetric methods afforded compara- ysis was used to determine the chromatographic and MS traits of 415
400 ble results (Fig. 2): SH-SY5Y was the most sensitive cell line, in the different compounds (Table 1, Figs. 3 and 4). The extract 416
401 which CHCl3-pe treatment resulted in a dose-dependent inhibition contained highly volatile monoterpenes as eucalyptol, limonene 417

Fig. 4. GC/MS profile section of a CHCl3 fraction obtained from Laurus nobilis.

Please cite this article in press as: Pacifico, S., et al. Apolar Laurus nobilis leaf extracts induce cytotoxicity and apoptosis towards three nervous system cell
lines. Food Chem. Toxicol. (2013), http://dx.doi.org/10.1016/j.fct.2013.09.029
 FCT 7615 No. of Pages 10, Model 5G
8 October 2013

6 S. Pacifico et al. / Food and Chemical Toxicology xxx (2013) xxx–xxx

Fig. 5. LnC-1fraction Cell Viability Inhibition (CVI,%) towards SK-N-BE(2)-C, SH-
SY5Y, and C6 cell lines at 24 h, 48 h, and 72 h exposure times. A. MTT test results; B.
SRB test results. Values, reported as percentage vs. an untreated control, are the
mean ± SD of measurements carried out on 3 samples (n = 3) analyzed 12 times.
Fig. 7. LnC-3 fraction Cell Viability Inhibition (CVI,%) towards SK-N-BE(2)-C, SH-
SY5Y, and C6 cell lines at 24 h, 48 h, and 72 h exposure times. A. MTT test results; B.
SRB test results. Values, reported as percentage vs. an untreated control, are the
mean ± SD of measurements carried out on 3 samples (n = 3) analyzed 12 times.

3.2. Cytotoxicity and metabolic profiling analysis of derived plant 432

extracts 433

The CHCl3-pe fractionation, carried out through SiO2 prepara- 434

tive column chromatography eluting with organic solvents with 435
increasing polarity (CHCl3, EtOAc and MeOH) allowed us to obtain 436
LnC-1, LnC-2, and LnC-3 extracts. Analogous to the CHCl3-pe, each 437
extract was subjected to cytotoxicity evaluation on the two neuro- 438
nal and one glial cell lines (Figs. 5–7) and to determination of the 439
metabolic profile by GC–EI-MS. 440
The LnC-1 fraction (Fig. 5), which, as highlighted by GC–MS 441
analysis, lacked the entire pool of sesquiterpene metabolites char- 442
acterizing the parental extract, exhibited a far less pronounced 443
activity. Data obtained from human neuroblastoma cell lines 444
showed that both cell lines were poorly affected when treated with 445
low dose levels of the extract (2.5, 10.0, 25.0, and 50.0 lg/mL). 446
After treatment with the highest tested dose (250.0 lg/mL), how- 447
ever, a large increase in cytotoxicity was detected. 448
The LnC-2 fraction, enriched in guaiane and eudesmane 449
terpenes and almost completely devoid of monoterpene and allyl- 450
phenol components, revealed a remarkable cytotoxic effect (Fig. 6). 451
Fig. 6. LnC-2 fraction Cell Viability Inhibition (CVI,%) towards SK-N-BE(2)-C, SH- MTT analyses revealed that the growth of human SK-N-BE(2)-C 452
SY5Y, and C6 cell lines at 24 h, 48 h, and 72 h exposure times. A. MTT test results; B. and SH-SY5Y cell lines was significantly inhibited after 24 h expo- 453
SRB test results. Values, reported as percentage vs. an untreated control, are the
mean ± SD of measurements carried out on 3 samples (n = 3) analyzed 12 times.
sure time at a dose level of 50.0 lg/mL. In the course of cytotoxicity 454
testing toward SH-SY5Y cells, the SRB assay was exhibiting broadly 455
418 and a-terpinylacetate. Allylphenols such as eugenol, known as an similar results to the MTT assay. ID50 values (Table 2), acquired on 456
419 antiseptic constituent of different essential oils from medicinal the basis of dose–response curves allowed us to estimate that the 457
420 plants, and its methyl derivative methyleugenol were also present. EtOAc extract (LnC-2) exhibited a higher cytotoxic activity than the 458
421 All these substances have been earlier described as components of parental extract (CHCl3-pe). Cytotoxic effects on C6 glioma cells 459
422 essential oils obtained from the plant leaves (Özcan and Chalchat, were not observed, even at an extract concentration of 100.0 lg/ 460
423 2005; Yalçın et al., 2007; Marzouki et al., 2009). Guaianolides, mL. A massive decrease in cell viability was recorded only when 461
424 including dehydrocostus lactone and its isomer eremanthin (Con- the 250.0 lg/mL dose level was applied. These findings suggest 462
425 forti et al., 2006) were the main sesquiterpene constituents. The that this cell line has a relatively high resistance against constitu- 463
426 extract also contained the sesquiterpene b-eudesmol (Verdian-Rizi ents of the L. nobilis extracts. 464
427 and Hadjiakhoondi, 2008) and represents a source particularly rich The MeOH (LnC-3) fraction, which solubility and polarity did 465
428 in a-tocopherol (Gómez-Coronado and Barbas, 2003; Ouchikh not allow GC–MS analysis, only showed a weak cytotoxic effect. 466
429 et al., 2011) and b-sitosterol. As minor constituents were analysed Only at 250.0 lg/mL the SK-N-BE(2)-C cell viability was inhibited 467
430 the hydrocarbon hentriacontane, and the aliphatic alcohol by 20% and 50% in MTT test and SRB test, respectively. A mild 468
431 pentatriacontanol. inhibition (equal to an average value of 25%) was registered for 469

Please cite this article in press as: Pacifico, S., et al. Apolar Laurus nobilis leaf extracts induce cytotoxicity and apoptosis towards three nervous system cell
lines. Food Chem. Toxicol. (2013), http://dx.doi.org/10.1016/j.fct.2013.09.029
 FCT 7615 No. of Pages 10, Model 5G
8 October 2013

S. Pacifico et al. / Food and Chemical Toxicology xxx (2013) xxx–xxx 7

Table 2
L. nobilis investigated extracts ID50 (50% inhibiting dose; lg/mL) values. ID50 values were calculated through the plotted dose–response curves from MTT and SRB tests, on SK-N-
BE(2)-C, SH-SY5Y, and C6 cell lines, at 24, 48, and 72 h exposure times. pe = parental extract.

MTT IC50 (lg/mL) SRB IC50 (lg/mL)

24 h 48 h 72 h 24 h 48 h 72 h
SK-N-BE(2) CHCl3-pe 18.6 18.3 15.2 43.5 49.0 45.9
LnC-1 127.5 153.4 135.4 >250 96.8 101.6
LnC-2 32.3 28.0 29.9 >250 17.9 20.3
LnC-3 >250 >250 >250 >250 >250 >250
SH-SY5Y CHCl3-pe 53.1 47.6 33.5 31.1 29.1 24.6
LnC-1 47.6 48.9 35.1 101.8 75.0 61.2
LnC-2 17.6 15.5 16.6 33.9 23.6 18.8
LnC-3 >250 >250 >250 >250 >250 >250
C6 CHCl3-pe 208.4 119.3 89.3 >250 183.7 105.5
LnC-1 >250 >250 >250 >250 >250 >250
LnC-2 215.8 173.4 127.4 >250 210.7 165.6
LnC-3 34.8 24.5 23.2 40.1 40.5 37.0

structures. Analysis and a detailed knowledge of the processes 481

inducing cytotoxicity play a crucial role from the health point of 482
view as possible inhibition of malignant cells could lead to reduc- 483
tion or even elimination of their harmful effects. 484
Apoptosis and necrosis are the two major forms of cell death 485
(Chan et al., 2013). In order to investigate the mechanism that 486
underlie cell viability inhibition exerted by the parental extract, 487
and its fractions therefrom, the effect on the plasma membrane 488
integrity was evaluated. Although there are many assays for apop- 489
tosis detection relatively few assays are available for measuring 490
necrosis. A key signature for necrotic cells is an increasing perme- 491
abilization of the plasma membrane, which can be quantified by 492
release of the intracellular enzyme lactate dehydrogenase (LDH). 493
The activation of the caspase-3 enzyme was also evaluated. Both 494
tests (Fig. 8) were carried out only on the neuroblastoma cell lines 495
using a dose of 100.0 lg/mL, at which all the extracts exhibited a 496
clear cytotoxic effect. The results clearly showed that the inhibition 497
of proliferation observed through MTT and SRB assays was not due 498
to necrosis. 499
In order to investigate the apoptosis inducing activity of the 500
extracts, the activation of caspase-3 was measured using the 501
tetrapeptide DEVD labelled with the chromophore p-nitroaniline 502
(pNA) as substrate. In the presence of active caspase-3, cleavage 503
and release of pNA from the substrate occurs. Following 24 h 504
treatment of human neuronal cell lines, caspase-3 activities were 505

Fig. 8. LDH release (±SD) and caspase-3 activation (±SD) of A. SK-N-BE(2)-C cell measured and compared with control cells. As shown in Fig. 8, 506
line, B. SH-SY5Y cell line. The evaluation of LDH release was carried out treating CHCl3-pe extract and, particularly, its LnC-2 fraction showed a 507
cells with 100.0 lg/mL extracts dose at 24 h, 48 h, and 72 h exposure times. The noteworthy increase of caspase-3 activity. Both extracts exhibited 508
evaluation of caspase-3 activation was carried out treating cells with 100.0 lg/mL
high levels of sesquiterpene metabolites. CHCl3-pe extract was also 509
extracts dose at 24 h exposure time. Values, reported as percentage vs. an untreated
control, are the mean ± SD of measurements carried out on 3 samples (n = 3) rich in a-tocopherol. The presence of the latter allowed us to 510
analyzed 12 times. explain the lower apoptosis activity of the parental extract. 511
Moreover DNA fragmentation assays also confirmed that CHCl3- 512
pe extract and LnC-2 induced apoptosis in human cell lines (Fig. 9). 513

470 exposure of the SH-SY5Y cells to the same dose in both tests used.
471 Again a dissimilar response from the murine cell line was ob- 3.4. L. nobilis extracts exhibit antioxidant capacity 514

472 served. In both cytotoxicity tests the MeOH fraction induced a cell
473 viability decrease by approximately 75% at a dose level of 50.0 lg/ In order to define the contribution of the intracellular produc- 515

474 mL (Fig. 7). tion of reactive radicals to apoptotic events, the oxidant/antioxi- 516
dant capacity of both parental extract and its partially purified 517
fractions was evaluated using five different tests. In particular, 518
475 3.3. L. nobilis extracts induce apoptosis the ability of the extracts to act as chain breaking compounds by 519
transferring hydrogen atom (Hydrogen Atom Transfer: HAT) or a 520
476 Cytotoxicity is a cellular stress condition that can be caused by single electron (Single Electron Transfer: SET) to radical species 521
477 many biotic or abiotic factors, e.g. toxins, radiation, reactive oxygen was evaluated. The extracts were tested for their radical scaveng- 522
478 or nitrogen species (ROS or RNS). It often results in cell death. The ing feature by measuring their capacity to scavenge DPPH radical, 523
479 molecular mechanisms that characterize cytotoxicity are ABTS cation, anion superoxide (O 
2 ), hydroxyl (OH ), and hydroper-
524

480 particularly complex and involve many components and cellular oxyl radicals (HOO ). The latter was estimated through the ORAC 525

Please cite this article in press as: Pacifico, S., et al. Apolar Laurus nobilis leaf extracts induce cytotoxicity and apoptosis towards three nervous system cell
lines. Food Chem. Toxicol. (2013), http://dx.doi.org/10.1016/j.fct.2013.09.029
 FCT 7615 No. of Pages 10, Model 5G
8 October 2013

8 S. Pacifico et al. / Food and Chemical Toxicology xxx (2013) xxx–xxx

Fig. 9. DNA fragmentation of SK-N-BE(2)-C, and SH-SY5Y cells induced by L. nobilis extracts (CHCl3-pe, LnC-1, LnC-2, and LnC-3). Genomic DNA was isolated from all cell
samples as described in Methods and analyzed by eletrophoresis on 2% agarose gel stained with ethidium bromide. A comparison with molecular weight marker indicated
that bay leaf extracts cause a ‘‘ladder’’ pattern of DNA in treated cells and absence of DNA fragmentation in untreated cells.

Table 3
DPPH and ABTS ID50 (lg/mL) values. DAUC values and TroloxÒ (lM) Equivalents from ORAC test. pe = parental extract.

ID50 DPPH ID50 ABTS DAUC* ORAC TroloxÒ lM Eq

1.0 lg/mL 2.5 lg/mL 5.0 lg/mL 1.0 lg/mL 2.5 lg/mL 5.0 lg/mL
CHCl3-pe >100 >100 13.3 ± 0.2 23.3 ± 0.4 24.9 ± 0.7 3.16 6.48 7.00
LnC-1 >100 35.57 10.5 ± 0.7 19.5 ± 0.1 21.4 ± 1.2 2.24 5.24 5.88
LnC-2 >100 >100 12.0 ± 1.0 13.5 ± 0.9 19.0 ± 0.8 2.73 3.25 5.07
LnC-3 >100 55.87 18.7 ± 0.8 29.4 ± 1.1 33.2 ± 1.2 4.96 8.51 9.80
*
Values, reported as percentage vs. an untreated control, are the mean ± SD of measurements carried out on three samples (n = 3) analyzed three times.

526 method, which is considered to be most relevant because it utilizes 4. Discussion 557
527 a biologically relevant radical source. This method measures the
528 oxidative degradation of fluorescein in the presence of peroxyl The process by which a normal cell transforms and develops 558
529 radicals through the formation of a non-fluorescent product, which into a malignant tumour requires several cellular alterations. 559
530 can be easily quantified by spectrofluorimetric measurements. The Evasion of apoptosis is a hallmark of most, if not all cancers, 560
531 inhibition of peroxyl radical induced oxidations by antioxidants re-
532 flects the classical radical chain breaking antioxidant activity by
533 hydrogen atom transfer. Calculation of the antioxidant power of
534 the extracts investigated, was performed from their specific net
535 integrated areas under the fluorescence decay curves (AUC). Trolox
536 equivalents of each sample was also calculated (Table 3). The
537 MeOH extract seemed to be able to exert protective effects. The
538 results are depicted in Fig. 10 and in Table 3.
539 The CHCl3-pe extract showed a bland ABTS radical cation
540 scavenging efficacy, whereas highly dose-dependent. The pres-
541 ence of numerous terpene molecules seems to mask the poten-
542 tial antiradical capacity that takes shape in the evaluation of
543 LnC-1 extract, in which a-tocopherol is the main constituent.
544 In fact, the LnC-1 fraction was able to completely reduce the
545 metastable radical cation at 25.0 lg/mL dose (94% inhibition).
546 Further, the LnC-3 fraction was responsible for a scavenging effi-
547 cacy similar to that exerted from LnC-1. In fact, it was observed
548 that the most polar fraction, at the highest dose tested, was
549 capable of reducing by 79% and 23% ABTS+ radical and DPPH
550 radical, respectively (Fig. 10A). The LnC-2 fraction was ineffec-
551 tive. When the superoxide anion radical scavenging capability
552 was evaluated, it was observed that MeOH (LnC-3) extract was
553 the only one able to act as a scavenger. All other extracts in-
Fig. 10. Radical Scavenging Capacity (RSC,%) of L. nobilis investigated extracts. A.
554 creased the radical production in a dose-dependent manner. All RSC towards DPPH radical and ABTS+ radical. B. RSC towards OH radical and O 2
555 investigated extracts exhibited weak scavenging activity towards radical. Values, reported as percentage vs. a blank, are the mean ± SD of measure-
556 hydroxyl radicals within the test concentration (Fig. 10B). ments carried out on 3 samples (n = 3) analyzed three times.

Please cite this article in press as: Pacifico, S., et al. Apolar Laurus nobilis leaf extracts induce cytotoxicity and apoptosis towards three nervous system cell
lines. Food Chem. Toxicol. (2013), http://dx.doi.org/10.1016/j.fct.2013.09.029
 FCT 7615 No. of Pages 10, Model 5G
8 October 2013

S. Pacifico et al. / Food and Chemical Toxicology xxx (2013) xxx–xxx 9

561 because defects in its regulators invariably accompany tumorigen- further studies aimed to prove its fighting properties against 624
562 esis and sustain malignant progression. Cellular oxidants, deriva- neuronal cancer in vivo. 625
563 tives of oxygen, are essential mediators of apoptosis. In general,
564 ROS are generated in the cell as metabolic by-products and their
565 role have been described in several diseases including cancer. Conflict of Interest 626
566 However, ROS are like a double-edged sword and when their
567 intracellular levels overwhelm the cellular antioxidant capacity, The authors declare that there are no conflicts of interest. 627
568 this may be detrimental to cell survival (Circu and Aw, 2010). Con-
569 sidering the potential role of ROS in inducing DNA damage and
References 628
570 apoptosis in cancer cells, the proposed strategy relevant for cancer
571 treatment would be to increase intracellular ROS in cancer cells to Abdullah Thani, N.A., Sallis, B., Nuttall, R., Schubert, F.R., Ahsan, M., Davies, D., 629
572 toxic level that causes oxidative stress, inactivates redox-signaling Purewal, S., Cooper, A., Rooprai, H.K., 2012. Induction of apoptosis and reduction 630
of MMP gene expression in the U373 cell line by polyphenolics in Aronia 631
573 molecules and induce cell death (Schumacker, 2006). 632
melanocarpa and by curcumin. Oncol. Rep. 28, 1435–1442.
574 Our study clearly demonstrated the ability of bay leaves CHCl3 Al-Kalaldeh, J.Z., Abu-Dahab, R., Afifi, F.U., 2010. Volatile oil composition and 633
575 parental extract (CHCl3-pe) to cause cytotoxicity and apoptotic cell antiproliferative activity of Laurus nobilis, Origanum syriacum, Origanum vulgare, 634
and Salvia triloba against human breast adenocarcinoma cells. Nutr. Res. 30, 635
576 death. The efficacy of the CHCl3-pe could be further enhanced by 636
271–278.
577 fractionation. In fact, the adopted purification approach allowed Bagetta, G., Cosentino, M., Corasaniti, M.T., Sakurada, S., 2012. Herbal Medicines: 637
578 us to obtain three partially purified fractions of which the LnC-2 Development and Validation of Plant-Derived Medicines for Human Health. CRC 638
Press, Taylor & Francis Group, Roca Baton, Florida. 639
579 (EtOAc) fraction was estimated as a promising anti-cancer agent.
Barla, A., Topçua, G., Öksüza, S., Tümenb, G., Kingston, D.G.I., 2007. Identification of 640
580 The metabolic fingerprinting of this fraction highlighted its sesqui- cytotoxic sesquiterpenes from Laurus nobilis L. Food Chem. 104, 1478–1484. 641
581 terpene constitution: its main component, dehydrocostus lactone, Chan, F.K., Moriwaki, K., De Rosa, M.J., 2013. Detection of necrosis by release of 642
582 was observed to be a promising lead candidate for the develop- lactate dehydrogenase activity. Methods Mol. Biol. 979, 65–70. 643
Chen, Q.F., Liu, Z.P., Wang, F.P., 2011. Natural sesquiterpenoids as cytotoxic 644
583 ment of therapeutic agents against drug-resistant sarcoma tumors anticancer agents. Mini Rev. Med. Chem. 11, 1153–1164. 645
584 (Kretschmer et al., 2012). Oh et al. (2004) demonstrated that Cho, E.Y., Lee, S.J., Nam, K.W., Shin, J., Oh, K.B., Kim, K.H., Mar, W., 2010. 646
585 dehydrocostus lactone was able to inhibit NF-jB activation by Amelioration of oxygen and glucose deprivation-induced neuronal death by 647
chloroform fraction of bay leaves (Laurus nobilis). Biosci. Biotechnol. Biochem. 648
586 preventing TNF-a-induced degradation and phosphorylation of 74, 2029–2035. 649
587 its inhibitory protein I-jBa in human leukemia HL-60 cells. Phar- Circu, M.L., Aw, T.Y., 2010. Reactive oxygen species, cellular redox systems and 650
588 macological inhibition of NF-jB offers the promise of enhancing apoptosis. Free Rad. Biol. Med. 48, 749–762. 651
Conforti, F., Statti, G., Uzunov, D., Menichini, F., 2006. Comparative chemical 652
589 the efficacy of anticancer therapies. Moreover, the anti-angiogenic 653
composition and antioxidant activities of wild and cultivated Laurus nobilis L.
590 mechanism of action of dehydrocostus lactone was also reported leaves and Foeniculum vulgare subsp. piperitum (Ucria) coutinho seeds. Biol. 654
591 (Wang et al., 2012). Pharm. Bull. 29, 2056–2064. 655
Dadalioglu, I., Evrendilek, G.A., 2004. Chemical compositions and antibacterial 656
592 The peculiar effect of LnC-2 fraction could also be due to the loss 657
effects of essential oils of Turkish oregano (Origanum minutiflorum), bay laurel
593 of a-tocopherol from the parental extract. The powerful antioxi- (Laurus nobilis), Spanish lavender (Lavandula stoechas L.), and fennel (Foeniculum 658
594 dant a-tocopherol inhibits ROS generation and interferes with vulgare) on common foodborne pathogens. J. Agric. Food Chem. 52, 8255–8260. 659
Dall’Acqua, S., Viola, G., Giorgetti, M., Loi, M.C., Innocenti, G., 2006. Two new 660
595 the therapeutic activity of anticancer drugs that kill cancer cells
sesquiterpene lactones from the leaves of Laurus nobilis. Chem. Pharm. Bull. 54, 661
596 by apoptosis (Salganik, 2001). In analogy to this finding, it was 1187–1189. 662
597 demonstrated previously that cisplatin, a widely applied Dall’Acqua, S., Cervellati, R., Speroni, E., Costa, S., Guerra, M.C., Stella, L., Greco, E., 663
598 anticancer drug, induced apoptosis in human breast cancer cells Innocenti, G., 2009. Phytochemical composition and antioxidant activity of 664
Laurus nobilis L. leaf infusion. J. Med. Food 12, 869–876. 665
599 by an increase in ROS generation, whereas the co-treatment De Marino, S., Borbone, N., Zollo, F., Ianaro, A., Di Meglio, P., Iorizzi, M., 2004. 666
600 cisplatin/a-tocopherol suppressed the cell death protective Megastigmane and phenolic components from Laurus nobilis L. leaves and their 667
601 mechanism by ROS reduction. If depletion of ROS by antioxidants inhibitory effects on nitric oxide production. J. Agric. Food Chem. 52, 7525– 668
7531. 669
602 downregulates apoptosis, then a rise in ROS concentration could De Marino, S., Borbone, N., Zollo, F., Ianaro, A., Di Meglio, P., Iorizzi, M., 2005. New 670
603 enhance the apoptotic death of cancer cells. The concentration of sesquiterpene lactones from Laurus nobilis leaves as inhibitors of nitric oxide 671
604 ROS can be increased by enhancing ROS generation or by depleting production. Planta Med. 71, 706–710. 672
Di Maro, A., Pacifico, S., Fiorentino, A., Galasso, S., Gallicchio, M., Guida, V., Severino, 673
605 antioxidants. Experiments to verify this reasoning were performed 674
V., Monaco, P., Parente, A., 2013. Raviscanina wild asparagus (Asparagus
606 using transgenic mice developing brain tumors (Salganik et al., acutifolius L.): a nutritionally valuable crop with antioxidant and 675
607 2000). Mice were fed a diet depleted of antioxidants, while control antiproliferative properties. Food Res. Int. 53, 180–188. 676
Fang, F., Shengmin, S., Chen, K.Y., Gosslau, A., Ho, C.-T., Rosen, R.T., 2005. Isolation 677
608 mice were fed a standard diet. The antioxidant-depleted diet sig- 678
and identification of cytotoxic compounds from bay leaf Laurus nobilis. Food
609 nificantly increased ROS concentration in brain tumors that, in Chem. 93, 497–501. 679
610 turn, led to a dramatic increase in apoptotic death of brain tumor Fiorentino, A., D’Abrosca, B., Pacifico, S., Mastellone, C., Piscopo, V., Caputo, R., 680
Monaco, P., 2008. Isolation and structure elucidation of antioxidant polyphenols 681
611 cells. Consequently a sharp decrease in tumor volume resulted.
from quince (Cydonia vulgaris) peels. J. Agric. Food Chem. 56, 2660–2667. 682
612 An enhancement of apoptosis was not observed in normal tissues Fiorentino, A., Mastellone, C., D’Abrosca, B., Pacifico, S., Scognamiglio, M., Cefarelli, 683
613 of animals fed the antioxidant-depleted diet. Thus, the use of an G., Caputo, R., Monaco, P., 2009. D-Tocomonoenol: a new vitamin E from kiwi 684
614 herbal formula able to enhance oxidant production in tumor cells (Actinidia chinensis) fruits. Food Chem. 115, 187–192. 685
Gómez-Coronado, D.J., Barbas, C., 2003. Optimized and validated HPLC method for 686
615 could represent a valuable strategy for counteracting brain tumor alpha- and gamma-tocopherol measurement in Laurus nobilis leaves: new data 687
616 development. on tocopherol content. J. Agric. Food Chem. 51, 5196–5201. 688
Gören, A., Topçu, G., Bilsel, G., Bilsel, M., Aydoğmusß, Z., Pezzuto, J.M., 2002. The 689
chemical constituents and biological activity of essential oil of Lavandula 690
stoechas ssp. stoechas. Z. Naturforsch. C 57, 797–800. 691
617 5. Conclusions Ham, A., Kim, B., Koo, U., Nam, K.W., Lee, S.J., Kim, K.H., Shin, J., Mar, W., 2010. 692
Spirafolide from bay leaf (Laurus nobilis) prevents dopamine-induced apoptosis 693
by decreasing reactive oxygen species production in human neuroblastoma SH- 694
618 The CHCl3 parental extract (CHCl3-pe) from bay leaves was able 695
SY5Y cells. Arch. Pharmacal Res. 33, 1953–1958.
619 to exert a strong cytotoxicity and apoptosis inducing activity. Huang, W.J., Huang, C.H., Wu, C.L., Lin, J.K., Chen, Y.W., Lin, C.L., Chuang, S.E., Huang, 696
620 These activities of the phytocomplex (CHCl3-pe) were enhanced C.Y., Chen, C.N., 2007. Propolin G, a prenylflavanone, isolated from Taiwanese 697
propolis, induces caspase-dependent apoptosis in brain cancer cells. J. Agric. 698
621 when the extract underwent an assay guided fractionation. In fact,
Food Chem. 55, 7366–7376. 699
622 one of the fractions obtained, LnC-2, exhibited considerably high Iriti, M., Vitalini, S., Fico, G., Faoro, F., 2010. Neuroprotective herbs and foods from 700
623 and promising cytotoxic and apoptotic effects, which encourage different traditional medicines and diets. Molecules 15, 3517–3555. 701

Please cite this article in press as: Pacifico, S., et al. Apolar Laurus nobilis leaf extracts induce cytotoxicity and apoptosis towards three nervous system cell
lines. Food Chem. Toxicol. (2013), http://dx.doi.org/10.1016/j.fct.2013.09.029
 FCT 7615 No. of Pages 10, Model 5G
8 October 2013

10 S. Pacifico et al. / Food and Chemical Toxicology xxx (2013) xxx–xxx

702 Julianti, E., Jang, K.H., Lee, S., Lee, D., Woongchon, M., Oh, K.-B., Shin, J., 2012. Pacifico, S., Gallicchio, M., Fiorentino, A., Fischer, A., Meyer, U., Stintzing, F.C., 2012. 770
703 Sesquiterpenes from leaves of Laurus nobilis L. Phytochemistry 80, 70–76. Antioxidant properties and cytotoxic effects on human cancer cell lines of 771
704 Kang, H.W., Yu, K.W., Jun, W.J., Chang, I.S., Han, S.B., Kim, H.Y., Cho, H.Y., 2002. aqueous fermented and lipophilic quince (Cydonia oblonga Mill.) preparations. 772
705 Isolation and characterization of alkyl peroxy radical scavenging compound Food Chem. Toxicol. 50, 4130–4135. 773
706 from leaves of Laurus nobilis. Biol. Pharm. Bull. 25, 102–108. Pathak, S., Multani, A.S., Banerji, P., Banerji, P., 2003. Ruta 6 selectively induces cell 774
707 Kannappan, R., Gupta, S.C., Kim, J.H., Reuter, S., Aggarwal, B.B., 2011. death in brain cancer cells but proliferation in normal peripheral blood 775
708 Neuroprotection by spice-derived nutraceuticals: you are what you eat! Mol. lymphocytes: a novel treatment for human brain cancer. Int. J. Oncol. 23, 776
709 Neurobiol. 44, 142–159. 975–982. 777
710 Khan, M., Yi, F., Rasul, A., Li, T., Wang, N., Gao, H., Gao, R.M., 2012. Alantolactone Pech, B., Bruneton, J., 1982. Alkaloids from Laurus nobilis. J. Nat. Prod. 45, 560–563. 778
711 induces apoptosis in glioblastoma cells via GSH depletion, ROS generation, and Pignatti, S., 1982. Flora d’Italia. Edagricole, Bologna, Italy. 779
712 mitochondrial dysfunction. IUBMB Life 64, 783–794. Prior, R.L., Hoang, H., Gu, L., Wu, X., Bacchiocca, M., Howard, L., Hampsch-Woodill, 780
713 Kim, E.J., Lim, S.S., Park, S.Y., Shin, H.H., Kim, J.S., Park, J.H.Y., 2008. Apoptosis of M., Huang, D., Ou, B., Jacob, R., 2003. Assays for hydrophilic and lipophilic 781
714 DU145 human prostate cancer cells induced by dehydrocostus lactone isolated antioxidant capacity (oxygen radical absorbance capacity (ORACFL)) of plasma 782
715 from the root of Saussurea lappa. Food Chem. Toxicol. 46, 3651–3658. and other biological and food samples. J. Agric. Food Chem. 51, 3273–3279. 783
716 Kleinkauf-Rocha, J., Bobermin, L.D., Machado Pde, M., Gonçalves, C.A., Gottfried, C., Ramos, C., Teixeira, B., Batista, I., Matos, O., Serrano, C., Neng, N.R., Nogueira, J.M., 784
717 Quincozes-Santos, A., 2013. Lipoic acid increases glutamate uptake, glutamine Nunes, M.L., Marques, A., 2012. Antioxidant and antibacterial activity of 785
718 synthetase activity and glutathione content in C6 astrocyte cell line. Int. J. Dev. essential oil and extracts of bay laurel Laurus nobilis Linnaeus (Lauraceae) 786
719 Neurosci. 31, 165–170. from Portugal. Nat. Prod. Res. 26, 518–529. 787
720 Koo, U., Nam, K.W., Ham, A., Lyu, D., Kim, B., Lee, S.J., Kim, K.H., Oh, K.B., Mar, W., Saab, A.M., Tundis, R., Loizzo, M.R., Lampronti, I., Borgatti, M., Gambari, R., 788
721 Shin, J., 2011. Neuroprotective effects of 3a-acetoxyeudesma-1,4(15),11(13)- Menichini, F., Esseily, F., Menichini, F., 2012. Antioxidant and antiproliferative 789
722 trien-12,6a-olide against dopamine-induced apoptosis in the human activity of Laurus nobilis L. (Lauraceae) leaves and seeds essential oils against 790
723 neuroblastoma SH-SY5Y cell line. Neurochem. Res. 36, 1991–2001. K562 human chronic myelogenous leukaemia cells. Nat. Prod. Res. 26, 1741– 791
724 Kumar, S., Singh, J., Sharma, A., 2004. Bay leaves. In: Peter, K.V. (Ed.), Handbook of 1745. 792
725 Herbs and Spices. Woodhead Publishing Limited, Cambridge, England, pp. 52– Salganik, R.I., Albright, C.D., Rodgers, J., Kim, J., Zeisel, S.H., Sivashinskiy, M.S., Van 793
726 61. Dyke, T.A., 2000. Dietary antioxidant depletion: enhancement of tumor 794
727 Kretschmer, N., Rinner, B., Stuendl, N., Kaltenegger, H., Wolf, E., Kunert, O., apoptosis and inhibition of brain tumor growth in transgenic mice. 795
728 Boechzelt, H., Leithner, A., Bauer, R., Lohberger, B., 2012. Effect of costunolide Carcinogenesis 21, 909–914. 796
729 and dehydrocostus Lactone on cell cycle, apoptosis, and ABC transporter Salganik, R.I., 2001. The benefits and hazards of antioxidants: controlling apoptosis 797
730 expression in human soft tissue sarcoma cells. Planta Med. 78, 1749–1756. and other protective mechanisms in cancer patients and the human population. 798
731 Lantto, T.A., Colucci, M., Závadová, V., Hiltunen, R., Raasmaja, A., 2009. Cytotoxicity J. Am. Coll. Nutr. 20, 464S–472S. 799
732 of curcumin, resveratrol and plant extracts from basil, juniper, laurel and Schumacker, P.T., 2006. Reactive oxygen species in cancer cells: live by the sword, 800
733 parsley in SH-SY5Y and CV1-P cells. Food Chem. 117, 405–411. die by the sword. Cancer Cell 10, 175–176. 801
734 Lee, S., Chung, S.-C., Lee, S.-H., Park, W., Oh, I., Mar, W., Shin, J., Oh, K.-B., 2012. Shrotriya, S., Deep, G., Gu, M., Kaur, M., Jain, A.K., Inturi, S., Agarwal, R., Agarwal, C., 802
735 Acylated kaempferol glycosides from Laurus nobilis leaves and their inhibitory 2012. Generation of reactive oxygen species by grape seed extract causes 803
736 effects on Na+/K+-adenosine triphosphatase. Biol. Pharm. Bull. 35, 428–432. irreparable DNA damage leading to G2/M arrest and apoptosis selectively in 804
737 Li, Q.Q., Lee, R.X., Liang, H., Zhong, Y., 2013. Anticancer activity of b-Elemene and its head and neck squamous cell carcinoma cells. Carcinogenesis 33, 848–858. 805
738 synthetic analogs in human malignant brain tumor cells. Anticancer Res. 33, Skehan, P., Storeng, R., Scudiero, D., Monks, A., McMahon, J., Vistica, D., Warren, J.T., 806
739 65–76. Bokesch, H., Kenney, S., Boyd, M.R., 1990. New colorimetric cytotoxicity assay 807
740 Lin, P.-C., Liuc, P.-Y., Lin, S.-Z., Harne, H.-J., 2012. Angelica sinensis: a Chinese herb for for anticancer-drug screening. J. Natl. Cancer Inst. 82, 1107–1112. 808
741 brain cancer therapy. BioMedicine 2, 30–35. Speroni, E., Cervellati, R., Dall’Acqua, S., Guerra, M.C., Greco, E., Govoni, P., Innocenti, 809
742 Lobo, J.F.R., Castro, E.S., Gouvea, D.R., Fernandes, C.P., de Almeida, F.B., de Amorim, G., 2011. Gastroprotective effect and antioxidant properties of different Laurus 810
743 L.M.F., Burth, P., Rocha, L., Santos, M.G., Harmerski, L., Lopes, N.P., Pinto, A.C., nobilis L. leaf extracts. J. Med. Food 14, 499–504. 811
744 2012. Antiproliferative activity of Eremanthus crotonoides extracts and Stein, S., Mirokhin Y., Tchekhovskoi, D., Mallard, G., 2008. The NIST Mass Spectral 812
745 centratherin demonstrated in brain tumor cell lines. Rev. bras. Farmacogn. 22, Search Program, NIST/EPA/NIH Spectral Library, Vers. 2.0, distributed by 813
746 1295–1300. National Institute of Standard and Technology (USA). 814
747 Marzouki, H., Elaissib, A., Khaldic, A., Bouzidd, S., Falconierie, D., Marongiu, B., Tsai, N.-M., Lin, S.-Z., Lee, C.-C., Chen, S.-P., Su, H.-C., Chang, W.-L., Harn, H.-J., 2005. 815
748 Pirasa, A., Porcedda, S., 2009. Seasonal and geographical variation of Laurus The Antitumor Effects of Angelica sinensis on malignant brain tumors in vitro 816
749 nobilis L. essential oil from Tunisia. TONPJ 2, 86–91. and in vivo. Clin. Cancer Res. 11, 3475–3484. 817
750 Matsuda, H., Kagerura, T., Toguchida, I., Ueda, H., Morikawa, T., Yoshikawa, M., 2000. Van Wyk, B.-E., Wink, M., 2004. Medicinal Plants of the World and Their Uses. 818
751 Inhibitory effects of sesquiterpenes from bay leaf on nitric oxide production in Timber Press, Portland, Oregon (USA). 819
752 lipopolysaccharide-activated macrophages: structure requirement and role of Verdian-Rizi, M., Hadjiakhoondi, A., 2008. Essential oil composition of Laurus nobilis 820
753 heat shock protein induction. Life Sci. 66, 2151–2157. L. of different growth stages growing in Iran. Z. Naturforsch. C. 63, 785–788. 821
754 Miele, M., Dondero, R., Ciarallo, G., Mazzei, M., 2001. Methyleugenol in Ocimum Wang, C.-Y., Tsai, A.-C., Peng, C.-Y., Chang, Y.-L., Lee, K.-H., Teng, C.-M., Pan, S.-L., 822
755 basilicum L. cv. genovese gigante. J. Agric. Food Chem. 49, 517–521. 2012. Dehydrocostuslactone suppresses angiogenesis in vitro and in vivo 823
756 Modzelewska, A., Sur, S., Kumar, S.K., Khan, S.R., 2005. Sesquiterpenes: natural through inhibition of Akt/GSK-3b and mTOR signaling pathways. PLoS ONE 7, 824
757 products that decrease cancer growth. Curr. Med. Chem. Anticancer Agents 5, e31195. 825
758 477–499. Werner, I., Glasl, S., Presser, A., Haslinger, E., Jurenitsch, J., 2003. Sesquiterpenes 826
759 Oh, G.S., Pae, H.O., Chung, H.T., Kwon, J.W., Lee, J.H., Kwon, T.O., Kwon, S.Y., Chon, from Achillea pannonica Scheele. Z. Naturforsch. C. 58, 303–307. 827
760 B.H., Yun, Y.G., 2004. Dehydrocostus lactone enhances tumor necrosis factor- Weyermann, J., Lochmanna, D., Zimmerb, A., 2005. A practical note on the use of 828
761 alpha-induced apoptosis of human leukemia HL-60 cells. Immunopharmacol. cytotoxicity assays. Int. J. Pharm. 288, 369–376. 829
762 Immunotoxicol. 26, 163–175. Yalçın, H., Anık, M., S ß anda, M.A., Çakır, A., 2007. Gas Chromatography/Mass 830
763 Ouchikh, O., Chahed, T., Ksouri, R., Taarit, M.B., Faleh, H., Abdelly, C., Kchouk, E.M., Spectrometry analysis of Laurus nobilis essential oil composition of Northern 831
764 Marzouk, B., 2011. The effects of extraction method on the measured tocopherol Cyprus. J. Med. Food 10, 715–719. 832
765 level and antioxidant activity of L. nobilis vegetative organs. J. Food Comp. Anal. Zouari, S., Ketata, M., Boudhrioua, N., Ammar, E., 2013. Allium roseum L. volatile 833
766 24, 103–110. compounds profile and antioxydant activity for chemotype discrimination – 834
767 Özcan, M., Chalchat, J.-C., 2005. Effect of different locations on the chemical case study of the wild plant of Sfax (Tunisia). Ind. Crop. Prod. 41, 172–178. 835
768 composition of essential oils of laurel (Laurus nobilis L.) leaves growing wild in 836
769 Turkey. J. Med. Food 8, 408–411.

Please cite this article in press as: Pacifico, S., et al. Apolar Laurus nobilis leaf extracts induce cytotoxicity and apoptosis towards three nervous system cell
lines. Food Chem. Toxicol. (2013), http://dx.doi.org/10.1016/j.fct.2013.09.029