Fractionation and Characterization of Lycopene-Oxidation Products by LC-MS/MS (ESI)+: Elucidation of the Chemopreventative Potency of Oxidized Lycopene in Breast-Cancer Cell Lines

Article abstract of DOI:10.1021/acs.jafc.8b04850

Lycopene (LYC) has been correlated with the reduction of certain cancers and chronic diseases. However, the existence and biofunctionality of degraded, oxidized, and biotransformed LYC products in vivo have not been revealed. Therefore, this study aimed t

Full text of DOI:10.1021/acs.jafc.8b04850

Subscriber access provided by University of Sunderland
Bioactive Constituents, Metabolites, and Functions
Fractionation and characterization of lycopene oxidation
products by LC-MSMS (ESI)+: Elucidation of chemoprevention
potency of oxidized lycopene in breast cancer cell lines
Bangalore Prabhashankar Arathi, Poorigali Raghavendra-Rao Sowmya, Gini
Chempakathinal Kuriakose, Shivaprasad Shilpa, Hulikere Jagdish Shwetha, Sharath
Kumar, Marisiddaiah Raju, Baskaran Vallikannan, and Rangaswamy Lakshminarayana
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b04850 • Publication Date (Web): 27 Sep 2018
Downloaded from http://pubs.acs.org on September 28, 2018
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 47
Journal of Agricultural and Food Chemistry
1
2
Fractionation and characterization of lycopene oxidation products by LCꢀMSMS (ESI)+:
Elucidation of chemoprevention potency of oxidized lycopene in breast cancer cell lines
3
4
Bangalore Prabhashankar Arathia, Poorigali RaghavendraꢀRao Sowmyaa, Gini
5
Chempakathinal Kuriakoseb Shivaprasad Shilpaa, Hulikere Jagdish Shwethaa, Sharath
,
6
Kumarc, Marisiddaiah Rajud, Vallikannan Baskarane, Rangaswamy Lakshminarayanaa*.
7
8
aDepartment of Biotechnology, Jnana Bharathi Campus, Bangalore University, Bengaluru-
560 056, India.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
bDepartment of Biochemistry, Indian Institute of Science, Bengaluru-560 012, India.
cHimalaya drug company, Makali, Bengaluru- 562 162, India.
dDepartment of Botany, St. Joseph's College Autonomous, PB 27094, 36 Lalbagh Main
Road, Bangalore - 560 027, Karnataka, India.
eDepartment of Biochemistry, CSIR-Central Food Technological Research Institute, Mysuru
570 020, India.
*Correspondence address
Rangaswamy Lakshminarayana,
Assistant Professor, Department of Biotechnology,
Jnana Bharathi Campus, Bangalore University, Bengaluruꢀ 560 056, India.
Eꢀmail: rlnarn21@gmail.com; Phone: +91ꢀ080ꢀ22961461; Fax: +91ꢀ080ꢀ23211020.
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 2 of 47
26 ꢀ ABSTRACT
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
Lycopene (LYC) has been correlated with reduction of certain cancer and chronic diseases.
However, the existence of degraded/oxidized/biotransformed LYC products in vivo
questioned to reveal their bioꢀfunctionality. Therefore, this study aimed to screen and
elucidate the potential bioactive lycopene derived products in breast cancer and nonꢀ
cancerous cells. LYC oxidized/cleavage products were generated using KMnO4. These
oxidized products were separated as fraction’s (IꢀIII) by silica column chromatography using
gradient solvent systems. Further, LCꢀMSMS (ESI)+ technique was used to elucidate the
possible fragmentation pattern and the structure. The fraction II showed higher cytotoxicity
(IC50 value 64.5 ꢁM), cellular uptake and apoptosisꢀinducing activity in MCFꢀ7 cells. This
fraction consists of major peak 323 m/z, and identified as apoꢀ8, 6'ꢀcarotendial. The cytotoxic
inducing activity may be due to partial ROS generation with mitochondrial dysfunction.
Further, the role of apoꢀ8, 6'ꢀcarotendial in the induction of apoptosis is demonstrated for the
first time. These results illustrated that LYC oxidative derivatives or metabolites involve in
growth inhibition of cancer cells. Exploration of specific carotenoid oxidized products will
give further insight into the field of nutritional biochemistry.
44
Keywords:
45
46
47
48
49
Lycopene oxidized products, Breast cancer, apoptosis, reactive oxygen species,
mitochondrial membrane potential.
ACS Paragon Plus Environment

Page 3 of 47
Journal of Agricultural and Food Chemistry
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
ꢀ INTRODUCTION
Several epidemiological studies have shown an inverse relation between LYC consumption
with decreased risk of cancer and other chronic diseases.1ꢀ4 Studies have suggested that LYC
decreases the risk of certain types of cancers including breast.5ꢀ6 The effect of LYC is
attributed to its antioxidant and cell signaling properties.7 The unique chemical property of
LYC is very sensitive to oxidative modification/degradation.8 Stability studies have shown
isomerization and degradation of (allꢀE)ꢀLYC when exposed to light, oxygen, and high
temperature.9 In general, carotenoids are more prone to various modifications such as
hydrogenation, dehydrogenation, doubleꢀbond migration, chain shortening or extension,
rearrangement, isomerization, oxidation or combinations of these processes resulting in
numerous products.10 Apart from these, carotenoid oxidative metabolites may also be
formed due to the presence of monooxygenase, cyclooxygenase, and dioxygenase enzymes.11
Typically, the breakdown of lycopene occurs through isomerization followed by oxidation to
yield epoxides or undergoes eccentric cleavage by caroteneꢀ9', 10'ꢀmonooxygenase (CMOꢀII)
to form apoꢀlycopenoids.12ꢀ13 Although conversion and biological function of βꢀcarotene
metabolites are well documented in humans14, however bioactivity of specific LYC oxidative
derivative is poorly explained. Carotenoid metabolites/oxidized products are reported to be
involved in the chemoprevention of cancer.15ꢀ17 Previously, lycopene oxidative metabolites
have been identified in vitro and in vivo.18ꢀ24 Few notable studies support the concept that
biological activities of lycopene may be mediated by its oxidative cleavage products.25ꢀ28
Even though studies claim the possible role of carotenoids metabolites, the specific function
of oxidative derivatives/metabolite’s or its rich fraction’s is not explained as compared to its
parent compound. Earlier, we speculated the possible cytotoxic effect of LYC oxidized
mixture in inhibition of different cancer cells.29 Although enzymatic/oxidative degradation
products of LYC ascertain health benefits, however, the fate of these derivatives on
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 4 of 47
75
76
77
78
anticancer activity is still not well detailed. Furthermore, elucidation of such potential
component/oxidized products is considered to be important to understand their role against
chronic disease progression. Hence, the present study was aimed to isolate and identify the
major oxidative cleavage products of LYC and to elucidate their antiꢀcancer properties.
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
ꢀ MATERIALS AND METHODS
Chemicals. (allꢀE)ꢀLycopene (> 90%), potassium permanganate, dichloroꢀdihydroꢀ
fluorescein diacetate (DCHFꢀDA) dye, 4,6ꢀdiamidinoꢀ2ꢀphenylindole dilactate (DAPI) and
butylated hydroxytoluene (BHT), 5,5′,6,6′ꢀtetrachloroꢀ1,1′,3,3′ꢀtetraethylbenzimidazolocarboꢀ
cyanine iodide (JCꢀ1 dye), tetrahydrofuran (stabilized with BHT) were purchased from
SigmaꢀAldrich (St Louis, MO, USA). Methanol, acetonitrile, Dichloromethane (DCM),
isopropyl alcohol and tetrahydrofuran (THF) of HPLCꢀ grade solvents, PVDF syringe filters
(0.45 ꢁm) were procured from Merck (Mumbai, India). Minimum essential medium (MEM),
fetal bovine serum (FBS), 3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenyl tetrazolium bromide
(MTT), antibioticꢀantimycotic solution, calcium magnesium free phosphate buffer saline
(PBS), and cell culture consumables were purchased from HiꢀMedia Chemical Laboratories
(Mumbai, India). FITC AnnexinꢀV apoptosis detection kit was purchased from BD
pharmingen (BD Bioscience, San Diego, CA). All other chemicals and solvents of analytical
grades were purchased from Sisco Research Laboratories (Mumbai, India).
Isolation of LYC from the tomato. Ripened tomatoes were utilized for LYC extraction
(Indian hybrid) as per our earlier method.30 The peak identity, absorption maxima (λ max),
and characteristics UVꢀVisible spectra of isolated LYC confirmed by LCꢀMSMS analysis
using the reference standard.30 Extraction and preparation of LYC from sample and standard
were carried out under dim yellow light at 4 oC to prevent isomerization and degradation.
ACS Paragon Plus Environment

Page 5 of 47
Journal of Agricultural and Food Chemistry
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
Oxidation of LYC. LYC isolated from tomato was subjected to oxidation by using
KMnO4 as per the procedure of CarisꢀVeyrat et al.20 . In brief, LYC (30 mg) and
cetyltrimethylammonium bromide (6 mg) dissolved in 30 mL DCM/toluene (1:1 ratio). The
oxidation of LYC was initiated by adding 1.5 mL of aqueous solution of KMnO4 stock (135
o
mg in 9 mL water), and stirred on a magnetic stirrer for 30 min at 26 ± 2 C. Organic phase
containing LYC oxidized products were separated by repeated washing with double distilled
water (10 mL), then filtered through a PVDF filter (0.45 µm) and dried on sodium sulphate
under nitrogen environment. The residue of LYC oxidized mixture reꢀdissolved in petroleum
ether, and filtered with PVDF filter to remove the KMnO4 remnant. LCꢀMSMS ESI +ve mode
was applied to analyze crude LYC oxidized products and different fractions obtained by
gradient open column chromatography. An aliquot of oxidized fractions were used for cell
culture treatments or sealed under a nitrogen environment and stored at ꢀ80 oC in amber vials.
Fractionation of LYC oxidized products.
Open column chromatography was employed to separate LYC oxidized products (OCC, 20
cm × 1.5 cm) on silica G (particle size 70ꢀ230 mesh) using specific gradient solvent systems.
In brief, an aliquot of the LYC oxidized products was dried under a nitrogen environment and
reꢀdissolved in a known volume of hexane/acetone (50/50 v/v), and applied onto OCC. The
oxidized products were eluted using hexane/acetone in the following ratios (90:10, 80:20, and
70:30 v/v), and each fraction was collected separately in amber vials and subjected for HPLC
analyses. LCꢀMSMS analysis confirmed the peak identity and their respective spectra of each
fractions. Further, the bioactiveꢀ rich fraction subjected for MSMS analysis to understand its
fragmented pattern.
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 6 of 47
124
125
126
127
128
129
130
131
132
133
134
135
136
137
138
139
140
141
142
143
144
145
146
HPLC analysis of LYC oxidized products. Chromatographic separation of LYC oxidized
products was standardized using an AB SCIEX API 2000 system (Applied Biosystems,
USA), with Cꢀ30 column (5 ꢁm; 250 x 4.6 mm; Princeton, Cranbury, USA). The mobile
phase consisted of acetonitrile: methanol: isopropyl alcohol (40:40:20, v/v/v) under the
isocratic condition with a flow rate of 0.8 mL/min was monitored at 420 and 450 nm using
UV detector. An aliquot (20 µL) of LYC and its oxidized products injected on column
o
maintained at 40 C. Data acquisition and processing were carried out using Analyst 1.5
version software (Applied Biosystems, USA).
LCꢀMSMS conditions for analysis of LYC oxidized products. Analysis of LYC
oxidized products was performed using a AB SCIEX API 2000 mass spectrometer (Applied
Biosystems, USA) via an ESI source operated in positive ion mode. An aliquot (20 µL) of
LYC and its oxidized products (~20 µg/mL) was injected onto the column. The conditions
used are the same as mentioned in the HPLC analysis. The instrument was conditioned by
following parameters: ion source voltage (3,500 V), declustering potential (20 V), focusing
o
potential (400 V), entrance potential (10 V), source temperature (420 C), GSꢀI (50 psi), GSꢀ
II (60 psi), curtain gas (nitrogen) (30 psi). Mass spectra of LYC oxidized products were
recorded with an m/z 50ꢀ1000 scan range at 450 nm. Data were processed by using Analyst
1.5 version software (Applied Biosystems, USA). Further, collisionꢀinduced dissociation of
ions was recorded and compared for the molecular ions of LYC oxidized products. Collisionꢀ
induced dissociation energy 6 V was used to check the fragmentation pattern. Due to the
constraint in the sample size and unavailability of standards for LYC oxidative products, the
possible structures were elucidated using Chemsketch 8.0 software (ACD Labs, USA).
ACS Paragon Plus Environment

Page 7 of 47
Journal of Agricultural and Food Chemistry
147
148
149
150
151
152
153
154
155
156
157
158
159
160
161
162
163
Furthermore, the samples were subjected for highꢀresolution mass spectrometer Bruker
impact HD (QꢀTOF) to confirm the LYC oxidised products with higher accuracy.
(Supplementary figure 1).
Culture conditions and viability assay. MCFꢀ7 cells were obtained from the National
Centre for Cell Science (Pune, India) and cultured in standardized conditions.29 (. Cells were
grown in MEM medium supplemented with 10% heatꢀinactivated fetal bovine serum, 4 mM
glutamine, 40 g/mL penicillin, and 400 U/mL streptomycin. Similarly, normal breast
epithelial cells (MCFꢀ10A, ATCC, USA) cultured and maintained as per the previous
report.31 Preliminarily, we screened different concentration of lycopene (1ꢀ200 ꢁM) and its
oxidized products [fractions (I to III)] for cytotoxic assay. This study demonstrated that rich
fractions of LYC oxidized products showed cytotoxicity at higher concentrations (50, 100
and 200 µM equivalent to LYC concentration). Hence, in the current study, we used above
optimal level (100 ꢁM) of LYC concentration to compare its activity with different fractions
of LYC oxidized products for cellꢀbased assays. The LYC and different fractions of LYC
oxidized products obtained by OCC were dissolved in cell culture grade THF and added to
the cells in the medium at different doses [quantified based on the equivalent concentration of
164
165
166
167
168
169
170
171
standard LYC as per the Zhang et al.21 method]. The final concentration of THF in the culture
medium was 0.5 % (v/v). THF at this concentration did not induce cytotoxicity.ꢀ. The cells
were seeded into 96 well microtiter plates (5000 cells/well). After 24 h of cultivation, cell
viability was measured using MTT method as described previously.16
Apoptosis detection. Apoptosis detection was made with a FITC AnnexinꢀV apoptosis
detection kit according to the manufacturer’s instructions. Briefly, control and treated cells (1
X 105 cells/well in 12 well plates) incubated for 24 h were collected, then washed with iceꢀ
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 8 of 47
172
173
174
175
176
177
178
179
180
181
182
183
184
185
186
187
188
189
190
191
192
193
194
195
196
cold PBS and centrifuged at 2500 rpm for 5 min. The cell pellet was reꢀsuspended in the iceꢀ
cold 1X binding buffer, incubated with FITCꢀconjugated annexin V and propidium iodide
(PI) for 15 minutes at room temperature in the dark. Before the analysis, the instrument
calibrated with internal controls, such as unstained cells (negative control) and stained cells
(annexinꢀFITC, PI, and annexinꢀFITC with PI control cells). The samples were immediately
analyzed on FACS verse flow cytometer using the Diva analysis software.
Confocal microscopy. Untreated and treated cells with LYC oxidized products (50 and100
ꢁM) were harvested after 24 h incubation and used for annexin VꢀFITC+PI and DAPI
staining. Then, cells were observed under an inverted confocal laser scanning microscope
(Ziess LSM 510 MK4, Germany) and images captured.
ROS detection by flow cytometry. Control and treated cells (2 × 105) were harvested
after 24 h of incubation, washed with PBS, and suspended in 1 mL PBS. Followed by,
o
DCFHꢀDA (10 ꢁM) added and incubated for 15 minutes in 5% CO2 at 37 C. After
incubation, cells were washed and reꢀsuspended in PBS and analyzed within 1 h on FACS
Verse flow cytometry after calibration with negative (unstained) and positive control (H2O2)
treated cells. The results were expressed as fluorescence intensity of dichlorofluorescein
compared with control, treatment, and cells with H2O2 (positive control).
Detection of loss of mitochondrial transmembrane potential (Dym). Modification in
mitochondrial membrane potential was analyzed by flow cytometry using JCꢀ1 dye as
described earlier.32 Briefly, MCFꢀ7 cells treated either with LYC or oxidized products (50ꢀ
200 µM concentration) for 24 h. Then, cells were incubated with JCꢀ1 (0.5 mM) for 15 min at
37 oC and subjected for flow cytometric analysis. The ratios of red to green fluorescence was
ACS Paragon Plus Environment

Page 9 of 47
Journal of Agricultural and Food Chemistry
197
198
199
200
201
202
203
204
205
206
207
208
209
210
211
212
measured for each treatment and plotted. Cells treated with 2, 4ꢀDinitrophenol (2, 4ꢀDNP)
considered as positive control.
Cellular uptake of LYC vs. its oxidized products. MCFꢀ7 cells (106) in 75ꢀcm2 flasks
(n = 3) treated with 100 ꢁM purified LYC and its bioactive oxidized products (fraction II)
separately. Upon incubation for 24 h, cells were harvested, washed using MEM, and
extracted with suitable solvent systems. LYC and oxidized LYC content of all the treated
cells were quantified by HPLC and expressed after subtracting the content of carotenoids
adherent to the cell surface determined at 4 °C.33
Statistical analysis. Data tested for homogeneity of variances by the Bartlett test. When
homogenous variances were confirmed, the data further analyzed using ANOVA (GraphPad
software prism 5.1.). Tukey's test evaluated the differences between treated and control
groups. The significant differences between the experimental samples were considered at p <
0.05.
213
214
215
216
217
218
219
220
221
ꢀ RESULTS
Lycopenals formed with potassium permanganate. LCꢀMSMS analysis of KMnO4
induced LYC oxidized products shown the presence of single oxidative cleavage, and double
oxidative cleavage components of LYC. The single oxidative cleavage compounds contain
one end an aldehyde or a ketone group, and were identified as apoꢀlycopenal and apoꢀ
lycopenone, respectively. The second category of oxidized products contains two aldehyde
end groups and identified as apoꢀcarotendials. The chemical structures of the apoꢀ
lycopenals/ones and apoꢀcarotendials are shown in Table 1. Treatment with KMnO4 resulted
in cleaving the carbonꢀcarbon double bonds of LYC, yields a series of apoꢀlycopenals and
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 10 of 47
222
223
224
225
226
227
228
229
230
231
232
233
234
235
236
237
238
239
240
241
242
243
244
245
246
apoꢀcarotendials. The separation of these LYC cleaved products was achieved using C30
column (Figure 1). The lycopenals and apoꢀcarotendials eluted on C30 column in the
following order and were identified as: (2E, 4E, 6E, 8E, 10E, 12E, 14E, 16E, 18E)ꢀ
2,6,11,15,19ꢀpentamethyldocosa2,4,6,8,10,12,14,16,18ꢀnonaenedial (A,19.73%), (4E,6E,8E,
10E,12E,14E,16E,18E,20E,22E)ꢀ2,6,10,15,19,23,27ꢀheptamethyloctacosaꢀ2,4,6,8,10,12,14,
16,18,20,22,26ꢀdodecaene (B,7.5%), (4E,6E,8E,10E,12E,14E,16E)ꢀ4,8,12,17ꢀtetramethylꢀ18ꢀ
oxononadecaꢀ4,6,8,10,12,14,16ꢀheptaenal (C,13.5%) ,(2E,4E,6E,8E,10E,12E,14E,16E)ꢀ4,9
,13,17,21ꢀpentamethyldocosaꢀ2,4,6,8,10,12,14,16,20ꢀnonaenal (D, 3.9%), (2E, 4E, 6E,
8E,10E, 12E, 14E, 16E, 18E, 20E)ꢀ4, 8, 13, 17, 21ꢀ pentamethyltetracosa ꢀ2, 4, 6, 8, 10, 12,
14, 16, 18, 20ꢀdecaenedioic acid (E, 4.2%), (2E, 4E, 6E, 8E, 10E,12E,14E,16E)ꢀ2, 6, 11, 15ꢀ
tetram ethyloctadecaꢀ2, 4, 6, 8, 10, 12, 14, 16ꢀoctaenedial (F, 23.2%), 2E,4E,6E,8E,10E,12E,
14E)ꢀ2,7,11,15,19ꢀpentamethylicosaꢀ 2,4,6,8,10,12,14,18ꢀoctaenal (apoꢀ12'ꢀlycopenal) (G, 10
.8%), apoꢀ5,6'ꢀcarotendial (H, 4.9%), (2E,4E 6E,8E,10E,12E,14E,16E,18E)ꢀ2,6,11,15,19,23ꢀ
hexamethyl tetracosa 2,4,6,8,10,12,14, 16,18,22ꢀdecaenal (apoꢀ8'ꢀlycopenal) (I, 0.29%),
(2E,4E,6E,8E,10E,12E,14E,16E)ꢀ4,9,13,17,21ꢀpentamethyldocosaꢀ2,4,6,8,10,12,14,16,20ꢀ
nonaenal (apoꢀ10'ꢀlycopenal) (J, 0.15%) (Figure 1). The elution of rich fraction of LYC
oxidized products were obtained by OCC using hexane/acetone gradient solvent system in the
following ratios,90:10, 80:20 and 70:30 v/v. Fraction 1 identified and designated as (G) apoꢀ
12'ꢀlycopenal as a major peak Fraction 2 (F) consisted of apoꢀ8,6'ꢀcarotendial
[(2E,4E,6E,8E,10E,12E,14E,16E)ꢀ2,6,11,15ꢀtetramethyloctadecaꢀ2,4,6,8,10,12,14,16ꢀoctaen
edial] and Fraction 3 (A) consisted of (2E,4E,6E,8E,10E ,12E,14E,16E,18E)ꢀ2,6,11,15,19ꢀ
pentamethyldocosa 2,4,6,8,10,12,14,16,18ꢀnonaenedial, as major peaks. Fractions of LYC
cleavage products (Fraction IꢀIII) were further subjected to evaluate the bioactive role in
cancer and normal cell lines. Further, the MSMS analysis of the bioactive LYC cleavage
products was done to understand their fragmentation pattern (Figure 3).
ACS Paragon Plus Environment

Page 11 of 47
Journal of Agricultural and Food Chemistry
247
Effect of LYC oxidized products on cell viability. The influence of fractions (I to III) of
LYC oxidized products on cytotoxicity of MCFꢀ7 cells were shown in Figure 2. Among
fractions evaluated, fraction II rich Apoꢀ8ꢀ6' carotendial strongly inhibited the cell viability.
The % cell viability of MCFꢀ7 cells was decreased by 57.6 % (fraction 1), 77.5 % (fraction
II) and 54.5 % (fraction III) as compared to control. The IC50 concentration of fraction II was
found to be 64.5 ꢁM equivalent to LYC concentration in MCFꢀ7 cells.
248
249
250
251
252
253
254
255
256
257
258
259
260
261
262
263
264
265
266
267
268
269
270
271
Apoptosis induction of LYC oxidized products. An increase in apoptosis was observed
in MCFꢀ7 cells treated with fraction II (Figure 4). The influence of fraction II on apoptosis
was further confirmed by annexin FITCꢀPI staining as shown in Figure 4. Results of
confocal microscopic observation showed an early and late apoptosis, and nuclear
condensations in the treated cells as compared to control (Figure 5).
Intracellular ROS generation. ROS generation is considered as one of the intermediate
steps during activation of apoptosis. The levels of ROS were examined based on the
fluorescence of DCFDA. Influence of LYC and its oxidized fraction II on intracellular ROS
levels in MCFꢀ7 cells are shown in Figure 6. Fraction II increased the ROS levels at an initial
concentration (50 ꢁM) and decreased at higher concentration (100 ꢁM). Flow cytometric
evaluation showed no detectable levels of ROS production upon the treatment of fraction II.
The levels of ROS in control and LYC were lower by 13.1, and 10.1% than fraction II treated
cells in the initial concentration and not detected at higher concentration. The % of ROS in
positive control was found to be 81.3%.
LYC oxidized fraction II induces loss of mitochondrial transmembrane potential
(Dym). Loss of mitochondrial transmembrane potential (Dym) is well known to be an early
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 12 of 47
272
273
274
275
276
277
278
279
280
281
282
283
284
285
event during apoptosis. We measured the loss of mitochondrial membrane potential of LYC
and its oxidized fraction II treated MCFꢀ7 cells (24 h) by flow cytometry using JCꢀ1 dye.
Dym was measured from the shift in the ratio of red to green fluorescence emitting cells after
treatment. Results showed an increase in green fluorescence in the fraction II treated cells in a
concentrationꢀdependent manner, indicating a loss of mitochondrial transmembrane potential
in treated cells (Figure 7). Thus, data suggest that oxidized product (fraction II) induce
depolarization and mitochondrial transmembrane potential which collapse the cells leading to
activation of apoptosis.
Cellular uptake of LYC vs. its oxidized products. The cellular uptake of LYC and its
oxidized products (fraction IꢀIII) was in the order of fraction II > fraction I > fraction III>
LYC (Figure 8). The oxidized products showed higher cellular uptake compared to the intact
LYC.
286
287
288
289
290
291
292
293
294
295
296
ꢀ DISCUSSION
Studies have reported that consumption of carotenoids rich diet is inversely associated with
reduced risk of certain cancer. Among carotenoids, LYC has gained special attention due to
the worldwide consumption of tomato and its products. Further, higher levels of LYC in
plasma and tissues are positively correlated with reduction of chronic diseases including
cancer.34ꢀ35 In this regard, many studies attempted to elucidate the possible role of
carotenoids, majorly βꢀcarotene, then LYC and lutein on modulation of cancer progression.
In contrast, few reports have shown the nonꢀsignificant effect of carotenoids on cancers in
both in vitro and in vivo.36ꢀ39 Subsequently, studies were undertaken on βꢀcarotene and LYC
metabolites to test the hypothesis that metabolites render the higher antiꢀproliferative effect of
cancer cells compared to parent molecules. However, there is no detailed study even after two
ACS Paragon Plus Environment

Page 13 of 47
Journal of Agricultural and Food Chemistry
297
decades of research on the bioactivity of carotenoid metabolites or oxidized products due to
non availability of standards, lesser concentration, stability, and challenging issues in
isolation and characterization from biological samples. In the present study, we demonstrated
LYC oxidized products and anticancer property of fractionꢀII rich in apoꢀ8, 6'ꢀcarotendial
purified by column chromatography using suitable solvent ratios and optimized condition.
The apoptotic property of fraction II indicates an opportunity for its potential application. We
also attempted for the first time to compare the anticancer effect of LYC with its oxidized
fraction II. Several reports have proposed the possible cleavage pattern of LYC to obtain its
metabolites in vitro and in vivo.17,18,20,40
298
299
300
301
302
303
304
305
306
307
308
309
310
311
312
313
314
315
316
317
318
319
320
321
Studies have suggested that lycopene decreases the risk of breast cancer.6,41ꢀ43
Further, Sharoni et al.27 suggested that some of the cellular effects of carotenoids are
mediated through their derivatives formed either by chemical oxidation or by enzymatic
cleavage inside the cells. Also, Ford et al.44 demonstrated that LYC and its metabolite apoꢀ
12′ꢀlycopenal significantly suppressed the proliferation of cells through alteration in cell
cycle regulation. However, our results are contradictory, showing a nonꢀsignificant difference
in intact LYC treatment, compared to LYC oxidized products (fraction II) on apoptosis
induction in MCFꢀ7 cells. Apoptosisꢀinducing activity of LYC oxidized product (fraction II
with major peak apoꢀ8ꢀ6'ꢀcarotendial) was confirmed by annexin FITCꢀPI and JCꢀ1 staining
(Figure 4, 5, 6 and 7)which may be due to change in polarity and its cellular uptake (Figure
8) when compared to LYC and other fractions. It is evident from Figure 8 that fraction II
cellular uptake is comparatively higher than LYC. Studies demonstrated that uptake rate and
intracellular concentration in a different stage of cells might play important roles in the
chemopreventive effect of lycopene.45 Cellular uptake of fraction II is higher than other
fractions and LYC, which may be due to the difference in their structure, polarity, and
solubility.46 Further, in the present study, a strong relationship between fraction II
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 14 of 47
322
323
324
325
326
327
328
329
330
331
332
333
334
335
336
337
338
339
340
341
342
343
344
345
346
accumulation, cytotoxicity, and cell antiꢀproliferation is evident. It is possible that cell growth
modality, metabolic status, and membrane composition, may influence carotenoid
accumulation depending on cell type.47 Further, ROS generation is considered as one of the
intermediate steps during activation of apoptosis. In our study, we observed slightly higher
ROS levels at 50 µM and minimal levels of ROS production upon fraction II treated at higher
concentration (Figure 7). We determined that ROS was generated with fraction II at 50 µM,
as it suppressed ROS levels when treated in combination with Nꢀacetylcysteine (NAC), a
ROS scavenger (Supplementary Figure 2). The higher oxidative stress by fraction II at 50 µM
treatment may be either due to an imbalance in oxidant and antioxidant in the cells. In case of
100 and 200 µM concentration, a decreased ROS levels were evident as compared to 50 µM
concentration of fraction (II) treated cells (Figure 7). These contrast results were clarified
with repeated experiments. The higher ROS levels at 50 µM may trigger the apoptosis either
through the extrinsic or intrinsic pathway within a short period of exposure. This could be the
reason for minimal levels of ROS at 100 and 200 µM of fraction (II) as similar to control
showing ROS independent pathway. Interestingly, the percent apoptosis ratio and loss of
mitochondrial potential are higher significantly in a doseꢀdependent manner. The induction of
the cell death mechanism in fraction II treated cells may be either through ROS dependent or
independent pathway, still needs to be detailed. The cell viability results confirmed that the
LYC and its oxidized fractions did not affect the normal cells (MCFꢀ10A) under the
experimental conditions, suggesting that LYC oxidized products or metabolites play an
important functionality against cancer cell proliferation. Based on the current study, we
propose that LYC derived products especially apoꢀ8ꢀ6' carotendial may involve in biological
functions. The overall results indicated the potentiality of LYC derived products as a chemoꢀ
preventive molecule. This observation gives a further platform to address the active role of
carotenoids derived products. Although the role of specific carotenoid metabolites needs to
ACS Paragon Plus Environment

Page 15 of 47
Journal of Agricultural and Food Chemistry
347
be explored, it is challenging due to following limitations such as unavailability of standards,
coꢀelution, lesser concentration and circulation in a biological system. Further, studies related
to anticancer properties of specific oxidized product on molecular mechanisms such as
cyclins, the inhibitor of apoptosis proteins and caspases activities needs to be addressed
(under progress). The in vivo studies related to the role of these specific metabolites also
necessitate addressing. Apart from these, the lifespan/halfꢀlife of these oxidized
products/metabolites needs to be compared with isotopically labelled studies to show further
potentiality.
348
349
350
351
352
353
354
355
356
357
358
359
360
361
362
363
364
365
ꢀ ABBREVIATIONS
LYC, Lycopene; KMnO4, Potassium permanganate; ROS, Reactive oxygen Species; RAR,
Retinoic acid receptor; HPLC, High performance liquid chromatography; THF,
Tetrahydrofuran; MTT, 3ꢀ(4, 5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2, 5ꢀdiphenyl tetrazolium bromide;
DCM, Dichloromethane; FBS, Fetal bovine serum; MEM, Minimum essential medium;
PVDF, Polyvinylidene difluoride; FITC, Fluorescein isothiocyanate; DCHFꢀDA, dichloroꢀ
dihydroꢀfluorescein diacetate; DAPI, 4',6ꢀdiamidinoꢀ2ꢀphenylindole; JCꢀ1, 5, 5′, 6, 6′ꢀ
tetrachloroꢀ1,1′,3,3′ꢀtetraethylbenzimidazolocarboꢀcyanine iodide; LCꢀMSMS, Liquid
chromatography–mass spectrometry; MCPBA, Metaꢀchloroperoxybenzoic acid, NAC, Nꢀ
acetylcysteine.
366
367
368
369
370
ꢀ ACKNOWLEDGMENTS
Arathi B.P. acknowledges DST (Govt. of India) for grant of Women Scientist Fellowship
(F.NO.SR/WOSꢀA/LSꢀ35/2012). Authors acknowledge DSTꢀSERB (NO.SB/EMEQꢀ
233/2013) for the project grant, Department of Microbiology and Biotechnology (Bangalore
University), UGCꢀSAP, DSTꢀFIST. Authors also acknowledge Dr. Ravi Sundaresan,
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 16 of 47
371
372
373
374
375
376
377
378
379
380
381
382
383
384
385
386
387
388
389
390
391
392
393
394
395
Department of Microbiology and Cell biology, Indian Institute of Science for permitting to
use FACS and central analytical spectroscopy facilities.
ACS Paragon Plus Environment

Page 17 of 47
Journal of Agricultural and Food Chemistry
396
ꢀ REFERENCES
397
398
399
400
401
402
403
404
405
406
407
408
409
410
411
412
413
414
415
416
417
418
419
(1) Rao, A.V.; Agarwal, S. Role of antioxidant lycopene in cancer and heart disease. J. Am.
Coll. Nutr. 2000, 19, 563ꢀ569.
(2) Hwang, E.S.; Bowen, P.E. Cell cycle arrest and induction of apoptosis by lycopene in
LNCaP human prostate cancer cells. J. Med. Food. 2004, 7, 284ꢀ289.
(3) Beilby, J.; Ambrosini, G.L.; Rossi, E.; de Klerk, N.H.; Musk, A.W. Serum levels of
folate, lycopene, βꢀcarotene, retinol and vitamin E and prostate cancer risk. Eur. J. Clin. Nutr.
2010, 64, 1235ꢀ1238.
(4) Riccioni, G.; Scotti, L.; Di Ilio, E.; Bucciarelli, V.; Ballone, E.; De Girolamo, M.D.;
Orazio, N.; Martini, F.; Aceto, A.; Bucciarelli, T. Lycopene and preclinical carotid
atherosclerosis. J. Biol. Regul. Homeost. Agents. 2011, 25,435ꢀ441.
(5) Levy, J.; Bosin, E.; Feldman, B.; Giat, Y.; Miinster, A.; Danilenko, M.; Sharoni, Y.
Lycopene is a more potent inhibitor of human cancer cell proliferation than either αꢀcarotene
or βꢀcarotene. Nutr. Cancer. 1995, 24, 257ꢀ266.
(6) Nahum, A.; Hirsch, K.; Danilenko, M.; Watts, C.K.; Prall, O.W.; Levy, J.; Sharoni, Y.
Lycopene inhibition of cell cycle progression in breast and endometrial cancer cells is
associated with reduction in cyclin D levels and retention of p27 Kip1 in the cyclin Eꢀcdk2
complexes. Oncogene. 2001, 20, 3428ꢀ3436.
(7) Cristina, T.S.; Jose, P.C.; Mónica, T.R.; Dolores, J.F.; Arturo, C.S.; Norma, S.G.; Laura,
O.R.; Julio, S. Multiple molecular and cellular mechanisms of action of lycopene in cancer
inhibition. J. Evid. Based Complementary Altern. Med. 2013, 705121,
http://dx.doi.org/10.1155/2013/705121.
(8) Britton, G. Structure and properties of carotenoids in relation to function. FASEB J.
1995, 9, 1551ꢀ1558.
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 18 of 47
420
421
422
423
424
425
426
427
428
429
430
431
432
433
434
435
436
437
438
439
440
441
442
443
444
(9) Anguelova ,T.; Warthesen, J. Lycopene stability in tomato powders. J. Food Sci. 2000,
65, 67ꢀ70.
(10) RodriguezꢀAmaya, D.B.; Kimura, M. Harvestplus handbook of carotenoid analysis.
2004, Washington, DC.
(11) Yeum, K.J.; Leekim, Y.C.; Yoon, S.; Lee, K.Y.; Park, I.S.; Lee, K.S.; Kim, B.S.; Tang,
G.W.; Russell, R.M.; Krinsky, N.I. Similar metabolites formed from βꢀcarotene by human
gastricmucosal homogenates, lipoxygenase, or linoleicꢀacid hydroperoxide. Arch. Biochem.
Biophys. 1995, 321, 167ꢀ174.
(12) Hu, K.Q.; Liu, C.; Ernst, H.; Krinsky, N.I.; Russel, R.M.; Wang, XꢀD. The biochemical
characterization of ferret caroteneꢀ9′, 10′ꢀmonooxygenase catalyzing cleavage of carotenoids
in vitro and in vivo. J. Biol. Chem. 2006, 281, 19327ꢀ19338.
(13) Wang, X.D. Lycopene metabolism and its biological significance. Am. J. Clin. Nutr.
2012, 96, 1214ꢀ1222.
(14) Nagao, A. Oxidative conversion of carotenoids to retinoids and other products. J. Nutr.
2004, 134, 237ꢀ240.
(15) King, T.J.; Khachik, F.; Bortkiewicz, H.; Fukushima, L.H.; Morioka, S.; Bertram, J.S.
Metabolites of dietary carotenoids as potential cancer preventive agents. Pure Appl. Chem.
1997, 69, 2135ꢀ2140.
(16) Lakshminarayana, R.; Aruna, G.; Sathisha, U.V.; Dharmesh, S.M.; Baskaran, V.
Structural elucidation of possible lutein oxidation products mediated through peroxyl radical
inducer 2, 2′ꢀAzobis (2ꢀmethylpropionamidine) dihydrochloride: Antioxidant and cytotoxic
influence of oxidized lutein in HeLa cells. Chem. Biol. Interact. 2013, 203, 448ꢀ455.
(17) Arathi, B.P.; Sowmya, P.R.; Vijay, K.; Baskaran, V.; Lakshminarayana, R.
Metabolomics of carotenoids: The challenges and prospects ꢀ A review. Trends Food Sci.
Technol. 2015a, 45, 105ꢀ117.
ACS Paragon Plus Environment

Page 19 of 47
Journal of Agricultural and Food Chemistry
445
(18) Khachik, F.; Spangler, C.J.; Cecil Smith, J. Identification, quantification, and relative
concentrations of carotenoids and their metabolites in human milk and serum. Anal. Chem.
1997, 69, 1873ꢀ1881.
446
447
448
449
450
451
452
453
454
455
456
457
458
459
460
461
462
463
464
465
466
467
468
469
(19) Kim, S.J.; Nara, E.; Kobayashi, H.; Terao, J.; Nagao, A. Formation of cleavage products
by autoxidation of lycopene. Lipids. 2001, 36, 191ꢀ199.
(20) CarisꢀVeyrat, C.; Schmid, A.; Carail, M.; & Böhm, V. Cleavage products of lycopene
produced by in vitro oxidations: characterization and mechanisms of formation. J. Agric.
Food. Chem. 2003, 51, 7318ꢀ7325.
(21) Zhang, H.; KotakeꢀNara, E.; Ono, H.; Nagao, A. A novel cleavage product formed by
autoxidation of lycopene induces apoptosis in HLꢀ60 cells. Free Radic. Biol. Med. 2003, 35,
1653ꢀ1663.
(22) Gajic, M.; Zaripheh, S.; Sun, F.R.; Erdman Jr, J.W. Apoꢀ8′ꢀlycopenal and apoꢀ12′ꢀ
lycopenal are metabolic products of lycopene in rat liver. J. Nutr. 2006, 136, 1552ꢀ1557.
(23) Rodriguez, E.B.; RodriguezꢀAmaya, D.B. Lycopene epoxides and apoꢀlycopenals
formed by chemical reactions and autoxidation in model systems and processed foods. J.
Food Sci, 2009, 74, 674ꢀ682.
(24) Kopec, R.E.; Ried, K.M.; Harrison, E.H.; Curley, Jr R.W.; Hruszkewycz, D.P.; Clinton,
S.K. Identification and quantification of apoꢀlycopenals in fruits, vegetables and human
plasma. J. Agric. Food Chem. 2010, 58, 3290ꢀ3296.
(25) Reynaud, E.; Aydemir, G.; Rühl, R.; Dangles. O.; CarisꢀVeyrat C. Organic synthesis of
new putative lycopene metabolites and preliminary investigation of their cellꢀsignaling
effects. J. Agric. Food. Chem. 2011, 59, 1457ꢀ63.
(26) Pascale, G.; Eric, R.; Olivier, D.; CarisꢀVeyrat C. Antioxidant activity of (allꢀE)ꢀ
lycopene and synthetic apoꢀlycopenoids in a chemical model of oxidative stress in the gastroꢀ
intestinal tract. New J. Chem. 2012, 36, 575–587.
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 20 of 47
470
471
472
473
474
475
476
477
478
479
480
481
482
483
484
485
486
487
488
489
490
491
492
493
494
(27) Sharoni Y1, LinnewielꢀHermoni K, Zango G, Khanin M, Salman H, Veprik
A, Danilenko M, Levy J. The role of lycopene and its derivatives in the regulation of
transcription systems: implications for cancer prevention. Am. J. Clin. Nutr. 2012, 96, 1173ꢀ
1178.
(28) Narayanasamy, S.; Sun, J.; Pavlovicz, R.E.; Eroglu, A.; Rush, C.E.; Sunkel, B.D.; Li,
C.; Harrison, E.H.; Curley, R.W. Jr. Synthesis of apoꢀ13ꢀ, and apoꢀ15ꢀlycopenoids, cleavage
products of lycopene that are retinoic acid antagonists. J. Lipid Res. 2017, 58,1021ꢀ1029.
(29) Arathi, B.P.; Sowmya, P.R.; Kuriakose, G.C.; Vijay, K.; Baskaran, V.; Jayabaskaran, C.;
Lakshminarayana, R. Enhanced cytotoxic and apoptosis inducing activity of lycopene
oxidation products in different cancer cell lines. Food Chem. Toxicol. 2016, 97, 265ꢀ276.
(30) Arathi, B.P.; Sowmya, P.R.; Vijay, K.; Dilshad, P.; Saikat, B.; Gopal, V.;
Lakshminarayana, R. An improved method of UPLCꢀPDAꢀMS/MS analysis of lycopene
isomers. Food Anal. Method. 2015b, 8, 1962ꢀ1969.
(31) Sowmya, P.R.; Arathi, B.P.; Vijay, K.; Baskaran, V.; Lakshminarayana, R. Astaxanthin
from shrimp efficiently modulates oxidative stress and allied cell death progression in MCFꢀ7
cells treated synergistically with βꢀcarotene and lutein from greens. Food Chem. Toxicol.
2017, 106, 58ꢀ69.
(32) Srivastava, M.; Hegde, M.; Chiruvella, K.K.; Koroth, J.; Bhattacharya, S.; Choudhary,
B.; Raghavan, S.C. Sapodilla plum (Achras sapota) induces apoptosis in cancer cell lines and
inhibits tumor progression in mice. Sci. Rep. 2014, 21, 6147.
(33) Lin, C.; Huang, C.; Hu, M. The use of fetal bovine serum as delivery vehicle to improve
the uptake and stability of lycopene in cell culture studies. Br. J. Nutr. 2007, 98, 226ꢀ232.
(34) Re, R.; Mishra, G.D.; Thane, C.W.; Bates, C.J. Tomato consumption and plasma
lycopene concentration in people aged 65 y and over in a British national survey. Eur. J. Clin.
Nutr. 2003, 57, 1545ꢀ54.
ACS Paragon Plus Environment

Page 21 of 47
Journal of Agricultural and Food Chemistry
495
(35) Fielding, J.M.; Rowley, K.G.; Cooper, P.; Dea, K. Increases in plasma lycopene
concentration after consumption of tomatoes cooked with olive oil. Asia Pac. J. Clin. Nutr.
2005, 14, 131ꢀ136.
496
497
498
499
500
501
502
503
504
505
506
507
508
509
510
511
512
513
514
515
516
517
(36) Forbes, K.; Gillette, K.; Sehgal, I. Lycopene increases urokinase receptor and fails to
inhibit growth or connexin expression in a metastatically passaged prostate cancer cell line: a
brief communication. Exp. Biol. Med, 2003, 228, 967ꢀ971.
(37) Wu, K.; Erdman, J.W., Schwartz, S.; Platz, E.A.; Leitzmann, M.; Clinton, S.K.;
DeGroff, V.; Willett, W.C., Giovannucci, E. Plasma and dietary carotenoids, and the risk of
prostate cancer: A nested caseꢀcontrol study. Cancer Epidemiol. Biomarkers Prev. 2004, 13,
260ꢀ269.
(38) Sesso, H.D.; Buring, J.E.; Zhang, S.M.; Norkus, E.P.; & Gaziano, J.M. Dietary and
plasma lycopene and the risk of breast cancer. Cancer Epidemiol Biomarkers Prev. 2005, 14,
1074ꢀ1081.
(39) Burgess, L.C.; Rice, E.; Fischer, T.; Seekins, J.R.; Burgess, T.P.; Sticka, S.J.; Klatt, K.
Lycopene has limited effect on cell proliferation in only two of seven human cell lines (both
cancerous and noncancerous) in an in vitro system with doses across the physiological range.
Toxicol. In Vitro. 2008, 22, 1297ꢀ1300.
(40) Pennathur, S.; Maitra, D.; Byun, J.; Sliskovic, I.; Abdul Hamid, I.; Saed, G.M.;
Diamond, M.P.; AbuꢀSoud, H.M. Potent antioxidative activity of lycopene: A potential role
in scavenging hypochlorous acid. Free Radic. Biol. Med. 2010, 49, 205ꢀ213.
(41) Teodoro, A.J.; Oliveira, F.L.; Martins, N.B.; Maia, G.D.A.; Martucci, R.B.; Borojevic,
R. Effect of lycopene on cell viability and cell cycle progression in human cancer cell lines.
Cancer Cell Int. 2012, 12, 36.
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 22 of 47
518
519
520
521
522
523
524
525
526
527
528
529
530
531
532
533
534
535
536
(42) Takeshima, M.; Ono, M.; Higuchi, T.; Chen, C.; Hara, T.; Nakano, S. Antiꢀproliferative
and apoptosis inducing activity of lycopene against three subtypes of human breast cancer
cell lines. Cancer Sci. 2014, 105, 252ꢀ257.
(43) Dos Santos, R.C.; Ombredane, A.S.; Souza, J.M.T.; Vasconcelos, A.G.; Plácido, A.;
Amorim, A.D.G.N; Barbosa, E.A.; Lima, F.C.D.A.; Ropke, C.D.; Alves, M.M.M.; Arcanjo,
D.D.R.; Carvalho, F.A.A.; DelerueꢀMatos, C.; Joanitti, G.A.; Leite, J.R.S.A. Lycopeneꢀrich
extract from red guava (Psidium guajava L.) displays cytotoxic effect against human breast
adenocarcinoma cell line MCFꢀ7 via an apoptoticꢀlike pathway. Food Res. Int. 2018, 105,
184ꢀ196.
(44) Ford, N.A.; Elsen, A.C.; Zuniga, K.; Lindshield, B.L. Lycopene and apoꢀ12'ꢀlycopenal
reduce cell proliferation and alter cell cycle progression in human prostate cancer cells. Nutr.
Cancer. 2011, 63, 256ꢀ263.
(45) Liu, A.; Pajkovic, N.; Pang, Y.; Zhu, D.; Calamini, B.; Mesecar, A.L.; van Breemen,
R.B. Absorption and subcellular localization of lycopene in human prostate cancer cells. Mol.
Cancer Ther. 2006, 5, 2879ꢀ85.
(46) van den Berg, H. Carotenoid interactions. Nutr. Rev. 1999, 57, 1ꢀ10.
(47) Palozza, P., Serini, S., Di Nicuolo, F., Calviello, G. Modulation of apoptotic signalling
by carotenoids in cancer cells. Arch. Biochem. Biophyics. 2004, 430, 104ꢀ109.
537
538
539
540
ACS Paragon Plus Environment

Page 23 of 47
Journal of Agricultural and Food Chemistry
541
Figure captions
542
543
544
Figure 1. LCꢀMSMS analysis and elucidation of possible LYC oxidized products (AꢀB
fraction III, CꢀF fraction II, GꢀJ fraction I). MSMS scan 391>331, 463>377, 338>321,
429>359, 449>312, 323>251, 351>281, 349>331, 417>347, 377>307
545
546
547
548
549
Figure 2. (a) Effect of LYC and oxidised fractions (IꢀIII) (1ꢀ200 ꢁM) of on viability of MCFꢀ
7 cancer cell lines (b) Effect of LYC and oxidised fractions (IꢀIII) (1ꢀ200 ꢁM) on viability of
MCFꢀ10A cell lines. The asterisk indicates values are significantly different from the control
(*P < 0.05, **P < 0.001, ***P < 0.0001). Statistical comparisons were made by one way
ANOVA followed by Tukey’s test. Values are mean ± SD (n=3).
550
Figure 3. LCꢀMS MS analysis of LYC oxidized products (fraction II major peak, 323 m/z)
551
552
553
Figure 4. Detection of apoptosis induced by LYC (100 ꢁM) and oxidized LYC (fraction ꢀII,
50ꢀ200ꢁM) in MCFꢀ7 cell line. Values are mean ± SD (n=3), Ctrl=control, EA=early
apoptosis, LA=late apoptosis.
554
555
556
Figure 5. Apoptotic cells stained with annexin V FITC/PI and examined under microscopy
(100 X) after treatment with fraction II (50 and 100 ꢁM) in MCFꢀ7 cells. DAPI staining was
used as nuclear marker.
557
558
559
560
Figure 6. Effect of LYC (100 ꢁM) and oxidized LYC (fraction ꢀII, 50ꢀ200ꢁM) on production
of reactive oxygen species in MCFꢀ7 cell line. The asterisk indicates values are significantly
different from the control (*P < 0.05, ***P < 0.0001). Statistical comparisons were made by
one way ANOVA followed by Tukey’s test. Values are mean ± SD (n=3).
561
562
Figure 7. Effect of LYC (100 ꢁM) and oxidized LYC (fraction ꢀII, 50ꢀ200 ꢁM) on
mitochondrial integrity in MCFꢀ7 cell line. The asterisk indicates values are significantly
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 24 of 47
563
564
different from the control (**P < 0.001, ***P < 0.0001). Statistical comparisons were made
by one way ANOVA followed by Tukey’s test. Values are mean ± SD (n=3).
565
566
567
568
Figure 8. The cellular uptake of LYC and oxidized products at 100 ꢁM equivalent LYC
concentration. The asterisk indicates values are significantly different from the LYC (**P <
0.001, ***P < 0.0001). Statistical comparisons were made by one way ANOVA followed by
Tukey’s test. Values are mean ± SD (n=3).
569
570
571
572
ACS Paragon Plus Environment

Page 25 of 47
Journal of Agricultural and Food Chemistry
Figure 1.
Fraction II
Fraction III
Fraction I
(A)
(B)
(C)
(D)
(E)
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 26 of 47
Continued…
ACS Paragon Plus Environment

Page 27 of 47
Journal of Agricultural and Food Chemistry
Figure 2.
120
100
80
60
40
20
0
MCFꢀ7
LYC
fraction I
fraction II
fraction III
**
*
**
*
**
**
***
***
***
***
***
***
***
***
***
***
0
1
10
25
50
100
200
Carotenoids (µM)
120
100
80
60
40
20
0
MCFꢀ10A
LYC
fraction I
fraction II
fraction III
*
*
*
**
**
*
**
0
1
10
25
50
100
200
Carotenoids (µM)
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 28 of 47
Figure 3.
(Lycopene)
(Apoꢀ8,6’ carotendial)
C18H21O•
253.159
C5H6O••
82.0419
O
O
C4H5O•
69.034
••
C13H16
172.125
ACS Paragon Plus Environment

Page 29 of 47
Journal of Agricultural and Food Chemistry
Figure 4.
0.8 5 ±
0.8 %
1.2 ± 1.1 %
3.8 ± 0.1 %
0.1 ±
0.04 %
0.4 ± 0.1 %
0.1 ± 0.03 %
1.5 ±
1.0 %
3.6 ± 0.8 %
26.2 ± 3.8 %
1.0 ± 0.5 %
0.9 ±
2.3 ±
1.9 %
0.2 %
16.1 ± 2.0%
58.8 ± 1.5 %
0.4 ±
0.4 %
0.4 ± 0.05 %
1.3 ± 0.9 %
4.6 ±
1.8 %
27.2 ± 2.5 %
40.9 ± 2.3 %
100
EA
LA
80
60
40
20
0
Necrosis
Treatments
ACS Paragon Plus Environment