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J. Martínez de la Ossa, Food Funct., 2017, DOI: 10.1039/C7FO00877E.

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Food & Function Accepted Manuscript

Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
Selective Antitumoural Action of Pressurized Mango Leaf Extracts
against Minimally and Highly Invasive Breast Cancer
M.T. Fernández-Ponce*a,c, A. López-Biedmab,c, C. Sánchez-Quesadab,c,d, L. Casasa,c, C. Mantella,c, J.J.
Gaforiob,c,d,e and E.J. Martínez de la Ossaa,c
Mango leaf tea has been traditionally used by different cultures to reduce inflammation in the body. There is evidence that
chronic inflammation increases the risk of cancer. This study investigates the antitumoural effects of pressurized mango
leaf extracts on minimally (MCF7) and highly invasive (MDA-MB-231) breast cancer cells as well as on non-tumourigenic
cells (MCF10). Extracts showed protective properties against oxidation and cytotoxic effects in breast cancer cell lines,
causing minor damages in non-carcinogenic cells. Nontheless, some selective activity, depending on hormone receptor
status, was observed. This was possibly related to the presence of minor compounds. Extracts with high levels of
gallotannins showed cytotoxic action in MCF7 cells, while those who had methyl gallate and homomangiferin as common
components were more effective against MDA-MB-231 cells. Therefore, the cytotoxic effect of mango leaf extracts might
be attributed to the synergistic effects of different polyphenols and not just to mangiferin on its own, as the predominant
compound in mango leaves.

against different types of cancer. These polyphenols are mainly

1. Introduction
Breast cancer is presently one of the most common global threats
to women. Therapies used to treat breast cancer are mainly based
on the antitumoural effects of certain chemicals, which promote
the death of cancer and normal epithelial cells, with the subsequent
secondary effects. Recent studies indicate that the use of natural
bioactive compounds could promote tumour death without any
1-2
damage to epithelial cells. Indeed, authors have focused on the
discovery of natural chemopreventive agents that elude secondary
effects on non-tumourigenic cells. Soy isoflavonegenistein,
4-7
squalene, lignans or triterpenes from olive oil, and pomegranate ,
to mention some of them, have exhibited antitumoural properties
against breast cancer cells.
Mango (Mangifera indica L.) polyphenols, such as mangiferin,
quercetin, phenolic acids and gallotannins, thanks to their
9-18
antioxidant and anti-inflammatory effects, are also natural
bioactive compounds that have shown chemopreventive effects
19
present in M. indica leaves and bark. However, scientists have
mainly focused on the evaluation of the anticarcinogenic effects of
10,20-23
mango pulp, peel and bark. Vimang, a commercial extract
from mango bark, is used in Cuba as a preventive antioxidant
supplement and also to improve the quality of life of patients with
10
chronic diseases such as cancer or HIV.
Other cultures, however, have traditionally used M. indica leaf tea
to control different inflammatory processes such as asthma,
dysentery, periodontal diseases, neuropathic pain, and so forth. The
24
3 extracts from mango leaves have been reported as antioxidant,
25 26 27
8 antidiabetic, anti-inflammatory, antihypertensive, and so forth.
Ethanolic/methanolic extracts from mango leaves have been
assessed for favourable hypolipidemic and hepatoprotective
28-29
activities. Aqueous extract presented gastroprotective effects
30
against several ulcerogenic agents. In addition, a current study
demonstrated cytotoxic activity of mango leaf extract against
25
different types of cancer such as colon and gastric carcinoma, but
no reports have been published on its use to prevent breast cancer.
Mango leaf extracts are commonly obtained by conventional
extraction techniques, including decoction and maceration in water,
25,28-30
ethanol or methanol. However, novel extraction techniques,
such as supercritical fluid extraction (SFE), pressurized liquid
extraction (PLE) and enhanced solvent extraction (ESE), have been
proposed as valid alternatives to traditional extraction methods,
since they are highly efficient and environment-friendly, as well as
24,31-33
energy and solvent/time saving. All of these techniques
employ green solvents, including carbon dioxide, water or ethanol,
under high pressure and at moderate to high temperature. The

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differences between them are based on the solvent system. SFE
uses carbon dioxide as the extraction solvent, and both pressure
and temperature are above its critical point. It also proposes the
addition of small quantities of organic co-solvents (<10%) in order
24
to enhance the recovery of polar compounds such as polyphenols.
PLE, also known as hot-pressure extraction, uses liquid solvents
(mainly water and ethanol) at high pressure and high temperature
in order to increase the extraction efficiency of the polar
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substances. On the other hand, ESE combines the advantages of SFE
and PLE by using mixtures of CO2 together with rather high
percentages of organic solvents (over 10%). These latter mixtures
31-33
are considered as subcritical solvents.
Previous studies have evaluated the efficiency of these techniques
to obtain extracts from mango leaves. The extracts obtained by PLE
and ESE contained high levels of polyphenols and their antioxidant
action was superior to the well-known antioxidant α-
tocopherol.24,31-33 Recent studies have also demonstrated the anti-
inflammatory, anti-diabetic, antioxidant and neuroprotective
effects of pressurized mango leaf extracts.24,32-35
Furthermore, the valorization of this by-product would be of great
interest, since huge amounts of prune residues are generated each
year.
Therefore, bearing in mind the information outlined above, this
study intends to find a potential application of mango leaf extracts
as a chemopreventive agent against human breast cancer. The
antitumoural activity of pressurized mango leaf extracts was
evaluated by means of different in vitro assays. The influence that
the extraction process (PLE or ESE), the solvent system and the
phenolic content of the extracts had on the chemopreventive effect
of the extracts was evaluated. In addition, a fraction obtained by
concurrent extraction with SC-CO2 and hydroalcoholic mixtures was
also analyzed. Both, intracellular reactive oxygen species (ROS) and
cell cytotoxicity levels in MDA-MB-231 and MCF7 human breast
cancer cells as well as in MCF10A non-tumourigenic human breast
epithelial cells treated with pressurized mango leaf extracts were
determined and statistically analyzed.
2. Materials and methods
2.1 Materials
Mango (Mangifera indica, cv. Kent) leaves were provided by the
Institute for Mediterranean and Subtropical Horticulture ‘La
Mayora’ (IHSM) property of the Spanish National Research Council
(CSIC) (Malaga, Spain). The leaves were collected in September
2013. All the leaves were dried at ambient conditions to constant
weight and the samples were stored at room temperature in the
absence of light.
2.2 Chemicals and reagents
The following materials were purchased from Sigma-Aldrich Co. (St
Louis, MO): Hepes buffer; Sodium pyruvate; non-essential amino
acids mixture 100% (NEAA); 2',7'-dichlorofluorescein diacetate
(DCFH-DA); dimethyl sulfoxide (DMSO). Minimum essential medium
with Eagle’s salts (MEM) and fetal bovine serum (FBS) were
obtained from PAA Laboratories GmbH (Pasching, Austria). HuMEC
2 | J. Name., 2012, 00, 1-3
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DOI: 10.1039/C7FO00877E
Journal Name
Ready Medium kit was obtained from Invitrogen (Eugene, OR).
Culture plates and cell culture flasks were obtained from NUNC A/S
(Roskilde, Denmark). The CellTiter Blue reagent was obtained from
Promega Biotech Ibérica, SL (Madrid, Spain). Carbon dioxide
(99.99%) was obtained from Abello-Linde S.A. (Barcelona, Spain).
K2S2O8 (CAS 7727-21-1) and the organic solvents HPLC grade
ethanol, acetonitrile and formic acid were supplied by Panreac
(Barcelona, Spain). The standard compounds gallic acid, methyl
gallate (98%), 3,4-dihydroxybenzoic acid (≥97%), mangiferin (≥98%),
quercetin 3-D-galactoside (≥97%), quercetin 3-β-D-glucoside
(≥90%), quercetin 3-O-α-L-arabinopyranoside (≥95%), penta-O-
Food & Function Accepted Manuscript
galloyl-β-D-glucose hydrate (≥96%) and quercetin (≥98%) were
supplied by Sigma-Aldrich (Steinheim, Germany). Ultrapure water
(Milli-Q) was used.
2.3 Cell culture
Minimally invasive MCF7 human breast cancer cells (estrogen and
progesterone receptor-positive), highly invasive MDA-MB-231
human breast cancer cells (estrogen and progesterone receptor-
negative), and non-tumorigenic human breast epithelial cells
(MCF10A) were obtained from the American Type Culture
Collection (ATCC, Manassas, VA, USA). Human mammary epithelial
cells (MCF10A) were grown in HuMEC Ready Medium. Breast
tumour cells (MCF7 and MDA-MB-231) were cultivated in Minimum
Essential Medium (MEM) with Eagle's salts with L-glutamine
supplemented with 1% non-essential amino acids (NEAA), 1% Hepes
buffer, 1% sodium pyruvate and 10% FBS. All the cells were
maintained at 37 °C in a humidified atmosphere with a 5% CO2
content. Cells in the exponential growth phase with approximately
90–95% of confluence were used in all the experiments.
2.4 Production of mango leaf extracts by high pressure techniques
Mango leaf extracts were obtained by means of high pressure
extraction techniques: pressurized liquid extraction (PLE) and
enhanced solvent extraction (ESE). All the extraction tests were
carried out in a high pressure system (Thar Technology, model
SF100, Pittsburgh, PA, USA) fitted with a thermostatted vessel
(capacity 100 mL), two pumps with a maximum flow rate of 50
g/min (one for carbon dioxide and the other one for co-solvent), a
back pressure regulator valve (BPR) and a cyclonic separator to
allow periodic discharges of the extracted material. A schematic
diagram of the equipment is shown in Fig. 1.
Condenser BPR
CO2
Pump
CO2
Vessel Extractor
Heater
Cyclonic
Separator
Co-solvent Pump
Co-solvent
Fig. 1 Schematic diagram of high pressure equipment
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The solvent system was changed according to each extraction
technique. For PLE, pure ethanol (PET) and a hydroalcoholic mixture
ethanol-water 50:50 (PEW) were used as extraction solvents. For
ESE, the solvent used was a mixture of CO2-ethanol-water 50:25:25
(CEW). In addition, a fractionation (FEW) was carried out by
sequential pressurized extraction, first using supercritical-CO2 (SC-
CO2) and secondly ethanol-water 50:50.
For the different extraction techniques, 100°C of temperature, 12
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MPa of pressure, 10 g/min of flow rate and 3 h of extraction time
were set up as extraction conditions. With regards to the
fractionation process, the first step with pure CO2 was carried out at
40 MPa, 55 °C, 20 g/min and 3 h. Extraction conditions were
stablished both by economic aspects and by the results from
24,32
previous studies. After completing the extraction procedure, the
solvent was evaporated in a rotary evaporator to constant weight.
The dried extracts were stored at -4 °C prior to analysis.
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DOI: 10.1039/C7FO00877E
ARTICLE
10,20-
actions of extracts obtained from mango leaf, pulp and peel.
23,25
2.6.1 Cellular cytotoxicity assay
CellTiter Blue® reagent was used for the cytotoxicity assays
following a slightly modified manufacturer’s protocol. Cells in the
3
exponential phase were cultured in 96-well plates (5 × 10 cells/well
3
for MDA-MB-231 and MCF7 or 2.5 × 10 cells/well for MCF10A).
After overnight incubation to allow cell attachment, the medium
was removed and replaced with fresh medium which contained
mango leaf extracts at the concentration levels mentioned in
Food & Function Accepted Manuscript
section 2.6, and plates were incubated for a further 24 h. Finally,
CellTiter Blue was added. Fluorescence (Ex. λ485/Em. λ595) was
measured by means of a TECAN GENios Plus microplate reader
(Tecan Group Ltd.; Switzerland) after 3 h of incubation in darkness
at 5% CO2 and 37°C. Viability was calculated by equation (1):
%  
= 
  ⁄
  × 100 (Eq. 1)
2.5 Identification and quantification of phenolic compounds
The phenolic compounds present in pressurized mango leaf extracts
were quantified by high performance liquid chromatography
(HPLC). Analyses were performed using an Agilent HPLC series 1100
system (Agilent, Germany) fitted with a quaternary pump, an
autosampler, and a UV/vis detector connected to an HP
ChemStation®. 20 µL of extract was filtered and injected into the
HPLC system. Phenolic compounds were separated on a Synergi
Hydro-RP C18 column (150 mm × 3 mm i.d.; 4 μm) (Phenomenex,
USA) with a 4.0 mm × 2.0 mm i.d. C18 ODS guard column, and
eluted at 0.6 mL/min with a mobile phase consisting on water/0.1%
formic acid (solvent A) and acetonitrile/0.1% formic acid (solvent B).
A linear gradient profile with the following proportions of solvent B
[t (min), %B] was applied: (0, 0), (0.2, 0), (0.3, 7), (14.7, 8.5), (40,
19), (45, 33), (48, 50), (50, 95), (57, 0), (63, 0). Phenolic compounds
were detected at 278 nm and identified according to the retention
Where A stands for the relative fluorescence units in each sample.
All measurements were performed in quadruplicate in three
independent replicates.
2.6.2 Detection of intracellular reactive oxygen species (ROS)
7
As previously mentioned, ROS levels were detected using DCFH-DA.
3
In brief, MCF10A (5.5 × 10 cells/well), MDA-MB-231 and MCF7 cells
3
(7 × 10 cells/well) were cultured in 96 well plates for 24 h. After
adding the extracts at the tested concentration levels, the cells
were incubated for an additional 24 h period. DCFH-DA (100 μM)
was then added and the plates were incubated for another 30 min.
After this time, fluorimetric measurements (Ex. λ485/Em. λ535)
were carried out by means of a TECAN GENios Plus microplate
reader.
The intracellular ROS percentage was calculated according to
equation 2:
time and the elution behaviour determined by a previous
characterization study of mango leaf extracts by high pressure
liquid chromatography-electrospray ionization-mass spectrometry
32
(HPLC-ESI-MS). The quantification was carried out on the basis of
calibration curves generated from the standard compounds: gallic
acid, mangiferin, quercetin 3-β-D-glucoside, quercetin, 3-D-
galactoside, quercetin 3-O-α-L-arabinopyranoside and penta-O-
galloyl glucose. Compounds without commercial standards were
quantified as follows: iriflophenones according to the calibration
curve of mangiferin, quercetin 3-O-xyloside according to that of
quercetin glucoside, and tetragalloylglucose according to that of
pentagalloylglucose.
2.6 Evaluation of chemopreventive action by mango leaf extracts
The antitumor or chemopreventive action of mango leaf extracts
against breast cancer and mammary epithelial cells were evaluated
through a cellular cytotoxicity assay and the detection of
intracellular reactive oxygen species using 2',7'-dichlorofluorescein
diacetate (DCFH-DA). All the extracts were dissolved in DMSO at
concentrations ranging from 0.01 to 100.0 µg/mL and then filtered.
The final concentrations at 0.01, 0.1, 1.0, 10.0 and 100.0 µg/mL
were used for cytotoxicity and DCFH-DA assays, which were
determined by previous studies that evaluated the anticarcinogenic
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 =  ! − ! ⁄! # × 100 (Eq.2)
Where Ft0 is the fluorescence at t = 0 min and Ft30 is the
fluorescence at t = 30 min.
It has been reported that the addition of H2O2 increases oxidative
36
stress in cultured cells. Therefore H2O2 was added at 500 µM 30
minutes before fluorescence quantification. This would allow us to
evaluate the protective properties of mango leaf extracts against
induced oxidative stress. Both sets of experimental conditions were
run in triplicate in three independent replicates.
2.7 Statistical analyses
Results are presented as mean (± SEM), as a percentage relative to
the control solvent (DMSO), which was considered as 100%.
Statistical analysis was performed using analysis of variance
(ANOVA) followed by Dunnett test. Statistical analysis was carried
® ®
out with IBM SPSS Statistics Version 20 (IBM Corporation, USA). P-
values of less than 0.05 were considered statistically significant for a
confidence level of 95%, and p<0.01 for a confidence level of 99%.
3. Results and discussion
3.1. Chemical characterization of extracts obtained by high
pressure techniques
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DOI: 10.1039/C7FO00877E
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Pressurized liquid extraction (PLE) and enhanced solvent extraction

Table 2. Phenolic content of mango leaf extracts obtained on using ethanol (PEW), ethanol-water (PEW), ethanol-water-CO2 (CEW) and the hydroalcoholic fraction previously extracted with
(ESE) were explored in this work as techniques to obtain mango leaf

13.26 ± 0.01
0.66 ± 0.01
extracts with high contents of potential antioxidants and

7.22 ± 0.01
8.28 ± 0.01

4.36 ± 0.01
0.39 ± 0.01
1.40 ± 0.01
2.12 ± 0.01
1.34 ± 0.01
4.94 ± 0.01
FEW
anticarcinogenic compounds. In addition, the production of a

nd
nd

nd

nd
id

id
fraction from mango leaves obtained by sequential extraction with
SC-CO2, followed by pressurized liquid extraction with ethanol-
water 50:50 from the residue of the first stage was also studied for

Identity characterized by HPLC-MS and confirmed with standards. b Identity characterized by negative ion [M-H]- previously described in the literature32. c Retention time
the first time.
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The global yield and phenolic content of the different pressurized

3.02 ± 0.05

5.31 ± 0.03

3.46 ± 0.17
7.20 ± 0.07

1.58 ± 0.43
0.49 ± 0.04
0.87 ± 0.03
1.49 ± 0.22
0.66 ± 0.09
0.51 ± 0.12
0.98 ± 0.01
mango leaf extracts are shown in Tables 1 and 2.

CEW

nd

nd
id
id

id
Mango leaf extracts

Food & Function Accepted Manuscript

Table 1. Global yield of mango leaf extracts obtained by different
extraction techniques and expressed as g/100g dry extract and
represented as the mean ± SD
Global yield Total Phenolic

2.57 ± 0.01

7.07 ± 0.01

3.60 ± 0.03
9.25 ± 0.04

2.20 ± 0.54
0.20 ± 0.01
0.77 ± 0.01
1.17 ± 0.04
0.44 ± 0.01
0.29 ± 0.01
0.85 ± 0.01
Extraction Technique (g/100g sample) Content

PEW
(g/100g extract)

nd
nd

nd
id

id
PLE
EtOH (PET) 36.37 ± 1.3 45.41 ± 0.02

EtOH-H2O 50:50 (PEW) 45.3 ± 1.29 28.41 ± 0.01

10.13 ± 0.01

12.75 ± 0.01

3.86 ± 0.80
4.42 ± 0.02

5.86 ± 0.01

0.67 ± 0.01
1.03 ± 0.04
2.80 ± 0.01
1.27 ± 0.01
0.88 ± 0.01
1.35 ± 0.01
ESE

PET
46.73 ± 1.78 25.57 ± 0.01

nd

nd

nd
nd
id
CO2-EtOH-H2O 50:25:25 (CEW)
Sequential Fractionation
1º CO2 1.22 ± 0.15 n.d

2º EtOH-H2O 50:50 (FEW) 43.08 ± 0.13 43.97 ± 0.03

(m/z,

287.1 (14), 407.2 (100), 816.3 (17), 815.3

527.1 (100), 528.2 (27), 590.2 (10), 641.2

711.2 (100), 712.12 (35), 713.1 (105)
Legend n.d: not detected; SD: Standard deviation obtained from triplicate
109.2 (55), 112.9 (22), 153.1 (100)

559.2 (100), 560.2 (30), 561.1 (7)

128 (94), 435.1 (100), 436.2 (45)

HPLC analyses.
303.1 (15, 423 (100), 424.1 (22)

300 (50), 301 (25), 463 (100)

300 (50), 301 (25), 463 (100)

617 (17), 769 (75), 939 (100)

300 (45, 301 (15). 433 (100)
It can be seen from Table 1 that the global yield obtained from
Major Fragments ions
%)

pressurized ethanol as extraction solvent (PET) was less than that

125 (45), 169 (100)

124 (40), 183 (100)

obtained by means of the other solvents (PEW, CEW, and the 301 (35), 421 (100)
SC-CO2 (FEW), expressed as g/100g dry extract and represented as the mean ± SD

Legend nd: not detected, id: identified by HPLC-MS, I: iriflophenone, Q: quercetin

sequential fractionation FEW). In contrast to this, the phenolic

787.1 (100)
content of PET was significantly superior to that obtained from PEW

433 (100)
and CEW. Such results indicate that the use of pure ethanol led to a
(35)

(12)
more selective extraction of phenolic compounds than
hydroalcoholic mixtures such as PEW and CEW. The addition of
water in the extraction solvent possibly decreased the selectivity of
[M-H]-

169.1
153.1
423.1
183.4
407.2

559.1

421.1
435.1
711.2
527.1

787.1
463.1
463.1
433.0
433.0
939.0
the extraction process thus reducing the content of polyphenols in
the extracts.
The fractionation process carried out by sequential extraction; first
10.32
12.30

22.57

26.16
28.90
32.80
37.39

38.12
38.84
39.67
41.60
42.40
43.44
(min)
4.09
6.25
7.24

using SC-CO2 and secondly using a pressurized hydroalcoholic

tRc

mixture, also favoured an increment of total phenolic content in

mango leaf extract (See Table 1). In the first fraction obtained with
SC-CO2, polyphenols could not be identified. Previous studies had
I 3-C-(2-6-di-O-galloyl)-β-D-glucosideb

reported that SC-CO2, a low polar solvent, did not have the capacity
I 3-C-(2-O-p-hydroxybenzoyl)-β-D-

to extract phenolic compounds from mango leaves and that it

I 3-C-(2-O-galloyl)-β-D-glucosideb
Phenolic compounds

Q 3-O-α-L arabinopyranosidea

would be necessary to increase the polarity of the solvent system

by adding ethanol/methanol.24 Therefore, in the second fraction
3,4-dihydroxybenzoic acida

penta-O-galloyl-glucosea
b
tetra-O-galloyl-glucose

FEW, obtained by using pressurized ethanol-water, a high content

I 3-C-β-D-glucosideb
maclurin glucosideb

Q 3-β-D-glucosidea

of phenolic compounds (43.97 ± 0.03 g/100g dried extract) was

Q 3-D-galactosidea
homomangiferin

Q 3-O-xylosideb
methyl gallateb

detected (see Table 1). This phenolic content was higher than that
mangiferina

observed in the non-fractionated extracts obtained by using similar

gallic acida

glucosideb

hydroalcoholic mixtures, PEW and CEW (25.57–28.41 g/100g dried

extract). The extraction efficiency of mango polyphenols, which are
a

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& Function
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medium to high polar compounds, was increased by means of a
previous extraction with SC-CO2. This result could be explained by
the fact that when a pre-treatment with supercritical CO2 was
applied, nonpolar and lipophilic substances should be removed due
to their high solubility in SC-CO2, thus modifying the solute-matrix
diffusion process of these compounds, which liberates active sites
in the matrix. This fact promotes changes in the interactions
between the residual matrix and the solutes that could not be
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extracted in the first stage with SC-CO2, as they become more
available and easily extractable in a second extraction step, where
37
polar solvents are used. García-Mendoza et al. also reported
positive effects related to the use of sequential extraction with CO2
and pressurized ethanol for the recovery of phenolic compounds
37
from mango peel.
According to the phenolic profile, it can be seen from Table 2 that
all the extracts -PEW, CEW and PET- and the fraction FEW contained
similar phenolic compounds, such as phenolic acids (gallic acid),
xanthones (mangiferin), galloylated benzophenones (iriflophenone
3-C-β-D-glucoside, iriflophenone 3-C-(2-O-p-hydroxybenzoyl)-β-D-
glucoside and iriflophenone 3-C-(2-O-galloyl)-β-D-glucoside),
flavonols (quercetin 3-D-galactoside, quercetin 3-β-D-glucoside,
quercetin 3-O-xyloside and quercetin 3-O-α-L arabinopyranoside),
and gallotannins (tetra- and penta-O-galloyl-glucose). Mangiferin
was the predominant compound, followed by iriflophenone-
glucoside from PET, PEW and CEW, whereas it was the second most
abundant phenolic compound in FEW fraction.
According to other phenolic compounds detected in the extracts by
HPLC-MS (Table 2) other differences were observed.
Homomangiferin and methyl gallate were identified in FEW and
PEW, although FEW did not contain penta-O-galloyl-glucose. In
contrast, PET and CEW contained maclurin 3-C-β-D-glucoside as a
common component. Furthermore, iriflophenone 3-C-(2,6-di-O-
galloyl)-β-D-glucoside and 3,4-dihydroxybenzoic acid were only
detected in the CEW extract.
The phenolic compounds identified in FEW were similar to those
detected in the non-fractioned extract obtained with the same
hydroalcoholic mixture (PEW). However, in the case of FEW, an
increment in the content of iriflophenone derivatives and
quercetin-arabinopyranoside was observed. Also, at the same time,
the content of gallic acid and pentagalloylglucose decreased. The
removal of nonpolar compounds by means of CO2 pre-treatment
may have enhanced the recovery of some polyphenols (such as
iriflophenone derivatives and quercetin-arabinopyranoside) due to
the modification of active sites, which may have increased the
37
solubilisation of these compounds from the matrix.
3.2. Chemopreventive action in breast cancer cells
The presence of different polyphenols with antitumoural activity in
pressurized mango leaf extracts (see Table 2) suggests that they
have a potential as chemopreventive agents against breast cancer.
Therefore, the anticarcinogenic activity of pressurized mango leaf
extracts against MCF7 and MDA-MB-231 breast cancer cells as well
as non-tumorigenic mammary epithelial cells (MCF10A) was
explored in terms of cytotoxic activity, intracellular ROS generation
and protection against oxidative damage. Mango leaf extracts at a
concentration range of 0.01–100 µg/mL were evaluated, and the
effect of the extraction solvents (ethanol, ethanol-water 50:50, CO2-
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ethanol-water 50:25:25 and the hydroalcoholic fraction previously
extracted with SC-CO2) on the antitumor activity was also analyzed.
3.2.1 Effect on cellular cytotoxicity
Uncontrolled cell division, together with suppressed apoptosis, is a
primary key in the progression of human tumours. Therefore, the
cytotoxicity of pressurized mango leaf extracts in human breast
cancer cells and non-tumorigenic human mammary epithelial cells
at a concentration range of 0.01–100 µg/mL was evaluated and the
results are shown in Fig. 2. It can be seen from this figure that
breast cancer cells survival was inhibited after being exposed to
Food & Function Accepted Manuscript
pressurized mango leaf extracts and that it was dependent on each
extraction solvent.
For metastatic breast cancer cell line (MDA-MB-231), PEW and FEW
showed significant cytotoxic action at concentration levels between
0.01 and 10 µg/mL. PEW showed a significant reduction of viable
cells when a concentration treatment of 10 µg/mL was applied
(p<0.05). This effect was more noticeable when using low
concentrations of PEW and FEW (0.01 and 0.1 µg/mL) which could
reduce MDA-MB-231 cell survival to values just over 40% with a
confidence level of 99% (Fig. 2A). It is important to point out (as
shown in Fig. 2C) that at 0.1 μg/mL treatment, PEW extract was not
cytotoxic for non-tumorigenic human mammary epithelial cells
(MCF10), and FEW extract caused a slight decrease of viable cells
(below 10%, p<0.05), which was much lower than that observed in
MDA-MB-231 breast cancer cells (~30%).
In contrast to the above, the treatment with PET and CEW extracts
caused low cytotoxicity in MDA-MD-231 cells. Only CEW extract
slightly inhibited the growth of the MDA-MB-231 breast cancer cell
line (~10%) at the lowest concentration applied (0.01 µg/mL) and
with a confidence level of 99%. On the other hand, after MCF7 cells
were exposed to PET and CEW extracts at concentration levels
ranging between 0.01 and 0.1 µg/mL, a significant cytotoxic effect
was observed (p<0.01), together with a reduction of the number of
viable cells by ~20%. This effect was also achieved by the CEW
extract at 1.0 µg/mL concentration level (p<0.01) (Fig. 2B). The
other extracts analyzed (PEW and FEW) did not significantly modify
the viability of MCF7 breast cancer cells. It is also remarkable that
these extracts (PET and CEW) besides its anti-proliferative effect on
MCF7 cells also protect the non-carcinogenic mammary cells, since
a non-cytotoxic effect was observed at the mentioned
concentration level (See Fig. 2C). These results suggest that the
extraction solvent play a key role in the production of mango leaf
extracts, since the extracts produced present different functional
properties and antitumoural effects.
With regards to the effect of mango leaf extracts in normal
mammary epithelial cells (MCF10), only a slight decrease (<20%) in
the number of viable cells was observed when the lowest
concentration level of CEW (p<0.01) and PEW (p<0.05) was applied
(0.01 µg/mL), and this effect was also observed at 0.1 µg/mL when
using FEW (p<0.05). These results suggest that mango leaf extracts
have potential properties as chemopreventive agent, with the
added advantage of causing a minor damage to healthy mammary
tissue. However, when the highest concentration level was applied
(100 µg/mL), all of the extracts showed cytotoxic action in MCF10
cells with a confidence level of 99% (Fig. 2C).
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Food & Function Accepted Manuscript

Fig. 2 Cell survival of: A) MDA-MB-231 B) MCF7 and C) MCF10A cells measured by means of CellTiter Blue after 24 hours exposure to PET, CEW, PEW and
FEW extracts. Data are represented as the treatment average (±SEM) with respect to the control solvent (DMSO). Statistically significant differences relative
to the control solvent by Dunnet test were indicated as * and ** PET, ¥ and ¥¥ CEW, ϕ and ϕϕ PEW, ϐ and ϐϐ, FEW at p< 0.05 and p< 0.01 respectively.

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Previous studies have attributed M. indica extract’s antitumoural
properties mainly to mangiferin, which is the predominant active
12
compound in mango leaf extracts. Mangiferin exerts anti-
metastatic and anti-invasive actions by selective gene expression
regulation of metalloproteinases (MMP), which plays a key role in
cell proliferation and has the capacity to inhibit epithelial
mesenchymal transition, a process linked to metastatic propensity
that is characterized by the loss of cell adhesion.
10-12
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However, different phenolic compounds detected in mango leaf
extracts have also demonstrated to exert antitumoural action and
could interfere in the cytotoxic action and the specificity of mango
leaf extracts for a particular breast cancer cell type (estrogen and
progesterone receptor-negative or -positive) (see Table 2).
Quercetin, gallic acid and pentagalloylglucose have exhibited
antitumoural activity by modulation of gene expression, induction
of apoptosis, and reduction of MMP and kinase activity.10,13-18 In
addition, benzophenones have exhibited a regulatory capacity on
pro-apoptotic genes in breast cancer.20
This study has identified a certain correlation between the
selectivity of mango leaf extracts for a particular type of breast
cancer cells (hormone receptor-positive or –negative) and the
presence of other phenolic compounds in the extracts, other than
mangiferin. PEW and FEW extracts which share as common
compounds homomangiferin and methyl gallate, showed cytotoxic
action against estrogen receptor-negative MDA-MD-231 cell lines.
In contrast to this, PET and CEW extracts, which contained high
levels of gallotannins (tetragalloylglucose and pentagalloylglucose),
were cytotoxic for estrogen receptor-positive MCF7 breast cancer
cells. Previous studies have revealed that pentagalloylglucose
inhibits breast cancer MCF-7 cell growth by suppression of the
estrogen receptor function.17 The way gallotannins act may have
enhanced the cytotoxic action of PET and PEW extracts in the MCF7
cell line. Furthermore, pentagalloylglucose was not present in the
fraction FEW extract, which had the lowest activity level in the
MCF7 cell line.
In addition, a correlation between the total phenolic content and
the cytotoxic action of pressurized mango leaf extracts was not
observed. For example, CEW extract showed similar cytotoxic
action in MCF7 cells to that corresponding to PET extract; although
this latter solvent presented a greater total phenolic content than
CEW’s (see Table 1). This negative correlation between total
phenolic content and antiproliferative action of mango peel extracts
have been also observed in previous studies.38 These results lead us
to think that the antitumoural activity of pressurized mango leaf
extracts cannot be attributed to a specific compound. The cytotoxic
action and the specificity of the pressurized mango leaf extracts on
a particular type of breast cancer cell line (hormone receptor-
positive or -negative) might be due to the combined action of the
different bioactive compounds that have been identified in the
extracts. Nonetheless, further studies will be necessary to
determine the synergistic effects of the different mango
polyphenols involved in the selective antitumoural effect on
aggressive or minimally invasive breast tumour cells.
On the other hand, it is worth highlighting that treatments with a
low concentration level of pressurized mango leaf extract (0.01 and
0.1 µg/mL) achieved the highest breast cancer cell growth inhibition
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in both tumorigenic cell lines (Fig. 2A and 2B). In contrast to the
above said, higher doses of conventional mango extracts have been
10,22
required to inhibit cell proliferation of tumour cells. The
cytotoxic action of mango bark extract against the metastatic breast
cancer MDA-MB-231 cell line, for example, was observed at a
10
concentration that ranged between 25 and 800 µg/mL. Mango
peel extract with an IC50 of 10 µg/mL presented cytotoxicity against
22
the MDA-MB-231 and MCF7 cell lines, as well as mango leaf
extract with an IC50>200 µg/mL in the treatment of liver, colon and
24
gastric carcinoma. Usually, novel extraction techniques, such as
high pressure techniques, resulted in extracts with superior
Food & Function Accepted Manuscript
biological properties than those observed in extracts obtained by
conventional extraction techniques. Extraction processes carried
out at high pressure (100–600 bar) increase mass transfer rates due
to enhancement of cell permeability and secondary metabolite
diffusion. In addition, the extraction procedure is carried out in
absence of light and oxygen and at mild temperature, and this
conditions are favourable to avoid the degradation of the active
32,37
compounds and to preserve their functional properties.
Furthermore, pure phenolic compounds have also shown cytotoxic
activity at doses greater than those obtained from pressurized
mango leaf extract. Mangiferin, for example, has demonstrated to
reduce cell proliferation and to induce apoptosis in MCF7 and MDA-
MB-231 breast cancer cells at a concentration range of 75‒300 µM
11
(equal to 32–127 μg/mL). Gallic acid has shown dose-dependent
cytotoxicity in MDA-MB-231 breast cancer cells at a concentration
10
level range of 1.25–80.0 µg/mL with an IC50 of 10 µg/mL.
Quercetin has shown anti-proliferative effect in breast cancer cells
regardless of estrogen signalling at a concentration level range of 1–
14,16,39-41
300 µM (equal to 0.3–90 µg/mL). The fact that lower
concentration levels of pressurized mango leaf extracts are
necessary to obtain an anti-proliferative action in comparison to
those required when using pure compounds, may be due to the
enhancement of the anticarcinogenic activity enabled by the
synergistic effects of mango polyphenols. Pre-clinical and clinical
trials have provided solid scientific evidence that mango extracts
present similar or higher activity levels than the predominant active
42
compound mangiferin. Therefore, a hypothesis that a
combination of various phenolic compounds can be more effective
than purified compounds on their own can be set up. Such results
could be of interest because of the possibility of using mango leaf
extracts at low concentration levels as chemopreventive agent.
On the other hand, with regards to extract concentration, no dose-
dependent cytotoxic action has been observed. In fact, a reduction
of cytotoxicity was noticed when mango leaf extract doses were
increased (see Fig. 2A and 2B). Furthermore, an increment in the
number of viable cells with respect to the control cells was
observed at some concentration levels. Particularly in the MDA-MB-
231 cell line with a PET treatments at 1.0 µg/mL (p<0.01) and 100.0
µg/mL (p<0.05) concentration levels (Fig. 2A). This effect has also
been observed during the treatment of breast cancer cells with high
doses of quercetin. An increment in the number of viable cells
compared to that in the control cells was observed when the
quercetin concentration level was increased from 300 to 600 µM
(90–181 µg/mL) in MCF7 cells and also with the increment of the
treatment time (48 h) of the MDA-MB-231 cells. According to the
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author, other cellular mechanisms, possibly those involved in cell
43
survival, might come into play in treatments with high doses.
3.2.2. Effect on intracellular ROS levels
ROS generation seems to be involved in cancer initiation, since
cancer cells use ROS-sensitive signalling pathways to launch
proliferation, cell survival, glucose metabolism, invasion and other
mechanisms of tumour progression. The inhibition of ROS
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generation is therefore considered to be an antitumoural effect of
natural antioxidants, as this may help to slow down tumour
44-45
progression.
In a previous work by the same authors, the in vitro antioxidant
activity of pressurized mango leaf extracts was evaluated by DPPH
assay. The results showed that mango leaf extracts presented a
very powerful antioxidant activity that was even higher than that of
24,32
the renowned antioxidant α-tocopherol. Therefore, the
antioxidant activity of mango leaf extracts in breast cancer cells and
non-tumorigenic cells was explored by intracellular ROS detection
under basal conditions after the cells were exposed to mango leaf
extracts at concentrations in the range of 0.01–100 µg/mL. The
results for MDA-MB-231, MCF7 and MCF10A cells are shown in Fig.
3.
As can be seen in Fig. 3B, the treatment of MCF7 cells on applying
PET, PEW and FEW extracts at concentration levels of 1.0 and 10.0
µg/mL led to significant reductions in ROS levels (~30%) with a
confidence level of 99%, and this effect was maintained by PEW
extract at 0.1 µ/mL (p<0.01) and 0.01 µg/mL concentration levels
(p<0.05). In contrast, the MDA-MB-231 cell line only showed
antioxidant reactions at basal conditions in cells treated with PEW
extract at 1.0 and 10 µg/mL concentration levels. This extract
reduced ROS levels by ~20% with a confidence level of 95% (Fig.
3A).
As far as non-tumorigenic cells are concerned, when the MCF10A
cells were treated with PEW and FEW extracts at 10.0 µg/mL
concentration level, a statistical significant reduction of intracellular
ROS levels at basal conditions was observed. The same effect was
also observed with PEW extract at 1.0 µg/mL concentration level
(p<0.01). According to the results, PEW extract presented a higher
antioxidant protective effect on breast cancer cell lines and non-
tumorigenic cell lines than the other extracts that were analyzed.
Such antioxidant effect could be beneficial in breast cancer
prevention, since inhibition of ROS generation may contribute to
11
decelerate tumour progression.
However, at the highest mango leaf extract concentration level
applied (100 µg/mL), all the extracts seemed to be significantly pro-
oxidants in non-tumorigenic MCF10A cells and also in MCF7 breast
cancer cells (see Fig. 3B and 3C). Therefore, the cytotoxicity that
mango leaf extracts previously exhibited for MCF10A cells at a
concentration of 100 µg/mL (see Fig. 2C) could be promoted by the
severe oxidative stress induced by mango polyphenols at this high
concentration level, which may lead to cell death. This pro-oxidant
effect at high concentrations has also been observed in some
flavonoids. Quercetin has exhibited hormesis; a biphasic dose-
dependent response, in which this compound acts as an antioxidant
when it is applied at low concentration levels (<40 µM), while it
becomes a pro-oxidant at concentration levels greater than 40 µM.
In the case of breast cancer cells, a pro-oxidant effect could be
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useful to reduce tumour progression on the basis of an increased
basal oxidative stress above a toxic threshold level, which would
34
induce apoptosis and would kill tumour cells.
3.2.3 Effect on protection after induced oxidative damage
Reactive oxygen species causes DNA damage and promotes tumour
progression. Therefore, the potential of pressurized mango leaf
extracts for the protection of breast cancer cells and non-
tumorigenic mammary epithelial against H2O2 oxidative injury was
investigated. Intracellular ROS levels were measured in cells
previously treated with mango leaf extracts at increasing
Food & Function Accepted Manuscript
concentration levels from 0.01 to 100 µg/mL and the results are
shown in Fig. 4. In terms of oxidative stress stimulus, at the highest
concentration level applied (100 µg/mL), all the pressurized extracts
were able to reduce oxidative injury in both breast cancer cell types
(MCF7 and MDA-MB-231) as well as in non-tumorigenic mammary
epithelial cells (MCF10A) by a significant reduction of ROS levels
(p<0.01). This effect was more noticeable in MDA-MB-231 and
MCF10A cells, with a reduction of around 60% in ROS levels relative
to the control solvent (p<0.01). MCF7 cells also presented a
reduction of H2O2-induced ROS levels by a substantial ~50%
(p<0.01) after treatment with the different mango leaf extracts at
100 µg/mL concentration level.
With regards to MDA-MB-231 cells, the protective effect of mango
leaf extracts against oxidative injury was also observed at
concentration levels below 100 µg/mL. PEW extracts presented a
significant antioxidant action at all the concentration levels applied
(0.01‒100 µg/mL). ROS levels were significantly reduced by more
than 20% in a dose-dependent manner (p<0.01). Besides, PET and
FEW extracts significantly reduced intracellular ROS levels by ~20%
(p<0.01) at 10.0 µg/mL while FEW extract also showed the same
effect at 1.0 µg/mL (p<0.05). (Fig. 4.A).
However, as far as minimally invasive cells are concerned, a
significant decrease of intracellular ROS levels by ~50% was only
observed when MCF7 cells were pre-treated with extracts at 100
µg/mL (p<0.01) concentration level. FEW extracts presented a slight
reduction by ~15% also at 10 µg/mL (p<0.05). The extracts at
concentration levels below 10 µg/mL did not reduce ROS levels
after oxidative injury (Fig. 4.B).
In contrast to the above said, a statistically significant pro-oxidant
effect (p<0.01) was observed in MCF7 cell lines when tumour cells
were treated with CEW extract at concentration levels between
0.01 and 0.1 µg/mL. This pro-oxidant role in breast cancer cells
could be quite relevant if we bear in mind that high enough levels
of ROS may induce apoptosis in tumour cells, while non-
tumorigenic cells can tolerate such levels. Indeed, the treatment
with CEW extracts at low concentration levels (0.01‒0.1 µg/mL)
presented cytotoxicity in MCF7 cells, which could be favoured by
this pro-oxidant effect. The climb in ROS levels after H2O2-induced
oxidative shock may promote the death of MCF7 cells without any
damage to epithelial cells. PET extract was also cytotoxic for MCF7
cells at low concentrations, and presented a slightly pro-oxidant
effect on MCF7 cells but such effect was not statistically significant
with respect to the control solvent.
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Fig. 3 ROS levels under basal conditions in (A) MDA-MB-231, (B) MCF7 and (C) MCF10A after 24 hours of exposure to the PET, CEW, PEW and FEW extracts at
different concentration levels (0.01‒100 µg/mL) or the control solvent (DMSO). Data are represented as mean ± SEM with respect to the control solvent in
four independent assays carried out in triplicate. *p < 0.05 and **p < 0.01 vs. control solvent by Dunnett test.

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level. These results indicate the capacity of pressurized mango leaf

extracts to protect normal cells from oxidative damage.

Conclusions
High-pressure extraction techniques (PLE and ESE) proved to
be efficient to obtain mango leaf extracts with a potential
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capacity as chemopreventive agents against different types of

breast cancer while causing no damage to normal mammary
epithelial cells. Such extracts, at low concentration levels,

Food & Function Accepted Manuscript

presented cytotoxic action against breast cancer cells while
favoured the reduction of ROS generation levels. Additionally,
some protective effect in non-tumorigenic cells against
oxidative damage was observed. Results also suggest that the
selective action of the extracts depending on breast tumour
cells hormone receptor status could also be exploited. A
specific activity against a type of breast cancer (-positive or –
negative hormone receptor) was observed when the
extraction solvent was changed and it was possibly correlated
with each extract’s phenolic profile. The presence of other
polyphenols different to mangiferin – the predominant
compound of M. indica extracts – exerted a likely influence on
the selective antitumoural activity of mango leaf extracts.
Extracts with a high content of gallotannins showed cytotoxic
activity against MCF7 cells, while those which shared methyl
gallate and homomangiferin as minor compounds were more
effective in MDA-MB-231 cells. However, such antitumoural
properties cannot be attributed to any specific compound on
its own, but to the combined action of the different phenolic
compounds found in the extracts. The data obtained from the
present study represent valuable information for the
subsequent development of nutraceutical or natural
chemopreventive agents to be used for the prevention of
breast cancer with different hormone receptor status. Further
studies are, however, required in order to identify the
mechanisms of action and the synergistic effects of mango
polyphenols involved in selective antitumoural activity against
aggressive or minimally invasive breast tumour cells.

Fig. 4 ROS levels in (A) MDA-MB-231, (B) MCF7 and (C) MCF10A cells after
Conflict of interest
the addition of H2O2 measured with DCFH-DA after 24 hours exposure to
the extracts PET, CEW, PEW and FEW at different concentrations (0.01‒100 None of the authors declared a conflict of interest.
µg/mL) or the control solvent (DMSO). Data are represented as mean ± SEM
with respect to the control solvent in four independent assays carried out in
triplicate. *p < 0.05 and **p < 0.01 vs. control solvent by Dunnett test. Acknowledgements
The authors would like to thank the Secretariat of State for
Research, Development and Innovation, the European
On the other hand, mango leaf extracts showed a protective effect Regional Development Fund for its financial support (Project
in non-tumorigenic (MFC10A) cell lines against H2O2-induced CTQ2011- 22974) and the Institute for Mediterranean and
oxidative stress at the highest concentration applied (100 µg/mL), Subtropical Horticulture ‘La Mayora’ (IHSM) (Malaga, Spain)
since intracellular ROS levels were greatly reduced (more than 60%) for the provision of the mango leaves for the study.
with a confidence level of 99% (Fig. 4.C). PET extract has also
showed a significant protective effect close to 20% at a
concentration level range of 0.01–10 µg/mL, while CEW extract at References
10 µg/mL (p<0.05) led to a significant reduction in intracellular ROS

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