Cytotoxic effect of crude and purified pectins from Campomanesia xanthocarpa Berg on human glioblastoma cells

Carbohydrate Polymers 224 (2019) 115140

Contents lists available at ScienceDirect

Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol

Cytotoxic effect of crude and purified pectins from Campomanesia
xanthocarpa Berg on human glioblastoma cells

T

Sarah da Costa Amarala,1, Shayla Fernanda Barbieria,1, Andrea Caroline Ruthesd,e,
⁎
Juliana Müller Barka, Sheila Maria Brochado Winnischofera,b,c, Joana Léa Meira Silveiraa,b,
a

Postgraduate Program in Biochemistry Sciences, Sector of Biological Sciences, Federal University of Paraná, Curitiba, PR, 81531-990, Brazil
Department of Biochemistry and Molecular Biology, Federal University of Paraná, CEP 81.531-980, Curitiba-PR, Brazil
c
Postgraduate Program in Cellular and Molecular Biology, Federal University of Paraná, CEP 81.531-980, Curitiba-PR, Brazil
d
Division of Glycoscience, Royal Institute of Technology - KTH, Sweden
e
Department of Entomology and Nematology, University of Florida, Gulf Coast Research and Education Center (GCREC-UF), Wimauma, USA
b

ARTICLE INFO

ABSTRACT

Keywords:
Gabiroba
Pectin
NMR analysis
Glioblastoma cells
Cytotoxicity
ROS

A new source of pectin with a cytotoxic effect on glioblastoma cells is presented. A homogeneous GWP-FP-S
fraction (Mw of 29,170 g mol−1) was obtained by fractionating the crude pectin extract (GW) from
Campomanesia xanthocarpa pulp. According to the monosaccharide composition, the GWP-FP-S was composed of
galacturonic acid (58.8%), arabinose (28.5%), galactose (11.3%) and rhamnose (1.1%), comprising 57.7% of
homogalacturonans (HG) and 42.0% of type I rhamnogalacturonans (RG-I). These structures were characterized
by chromatographic and spectroscopic methods; GW and GWP-FP-S fractions were evaluated by MTT and crystal
violet assays for their cytotoxic effects. Both fractions induced cytotoxicity (15.55–37.65%) with concomitant
increase in the cellular ROS levels in human glioblastoma cells at 25–400 μg mL−1, after 48 h of treatment,
whereas no cytotoxicity was observed for normal NIH 3T3 cells. This is the first report of in vitro bioactivity and
the first investigation of the antitumor potential of gabiroba pectins.

1. Introduction
Glioblastoma is one of the most frequent and most lethal malignant
primary brain tumors and is associated with poor prognosis (Bailey
et al., 2015). Current therapy includes surgical resection, followed by
radiotherapy and/or concomitant adjuvant temozolomide (TMZ) chemotherapy (Arbab et al., 2017). Despite modern advance in therapies,
over 90% of patients experience tumor recurrence, and the average
period of survival for patients diagnosed with glioblastoma is only
about 14 months (Bailey et al., 2015; Tanaka, Louis, Curry, Batchelor, &
Dietrich, 2013). Therefore, the search for additional therapeutic strategies remains a high priority.
Recently, polysaccharides (such as pectins) have been exploited for
their anticancer potential, due to their broad spectrum of therapeutic

properties and their low toxicity to healthy cells (Munarin et al., 2015;
Noreen et al., 2017). Although studies have shown that pectins are
effective against a range of tumor models, including prostate cancer
(Prado et al., 2017), breast cancer (Cobs-Rosas, Concha-Olmos,
Weinstein-Oppenheimer, & Zúñiga-Hansena, 2015) and melanoma

(Vayssade et al., 2010), most studies have focused on a colon cancer
model (Zhang, Xu, & Zhang, 2015). To our knowledge, no report has
been made on the antitumor potential of pectins against glioblastoma
cells.
Pectins are anionic polysaccharides composed of (1, 4)-linked-Dgalacturonic acid residues and a variety of neutral monosaccharides,
such as rhamnose, galactose, and arabinose. They can be classified into
three main types, according to common features: Homogalacturonans
(HG), type I Rhamnogalaturonans (RG-I), and type II
Rhamnogalaturonans (RG-II) (Caffal & Mohnen, 2009; Chan, Choo,
Young, & Loh, 2017; Yapo, 2011).
These polysaccharides are the main component of the peels and
pulps of several fruits, although most biological studies are performed
with commercial pectins that have been extracted from citrus peel and
apple pomace (Noreen et al., 2017; Zhang et al., 2015). As a result, a
large number of native fruits around the world continue to go underused, despite the abundance of the species. Brazil is believed to harbor
the earth's richest flora, including several native fruits from the Myrtaceae family (Donado-Pestana et al., 2018).

Corresponding author at: Department of Biochemistry and Molecular Biology, P.B.19046, Federal University of Paraná, CEP 81.531-980, Curitiba-PR, Brazil.
E-mail address: (J.L.M. Silveira).
1
These authors equally contributed to this work.
⁎

/>Received 16 April 2019; Received in revised form 26 July 2019; Accepted 27 July 2019

Available online 02 August 2019
0144-8617/ © 2019 Elsevier Ltd. All rights reserved.


Carbohydrate Polymers 224 (2019) 115140

S.d.C. Amaral, et al.

Biological properties have been reported of polysaccharides extracted from Myrtaceae family fruits. A pectic arabinogalactan from
edible jambo (Syzygium jambos) fruits showed immunomodulatory
properties (Tamiello, Nascimento, Iacomini, & Cordeiro, 2018), while
water-extracted polysaccharides from guava (Psidium guajava and Psidium littorale) fruits exhibited anti-glycated and α-glucosidase inhibitory activity (Yan, Lee, Kong, & Zhang, 2013; Zhang et al., 2016).
This work investigated a Brazilian Myrtaceae family species,
Campomanesia xanthocarpa Berg, popularly known as gabiroba (Barroso
& Barroso, 1978). This species is used in folk medicine to treat such
pathologies as inflammatory, renal, and digestive diseases; obesity; and
hypercholesterolemia (Alice et al., 1995). In addition, scientific studies
have confirmed that the leaf and fruit extracts of C. xanthocarpa demonstrate a broad spectrum of therapeutic effects, including: antioxidant properties (Pereira et al., 2012), antibacterial effects
(Czaikoski, Mesomo, Krüger, Queiroga, & Corazza, 2015), antiulcerogenic effects (Markman, Bacchi, & Kato, 2004), antidiabetic effects (Vinagre et al., 2010), reduction of cholesterol levels (Viecili et al.,
2014) and obesity (Biavatti et al., 2004). However, most previous studies focused only on low molar mass compounds, and the aforementioned health benefits were attributed to the presence of vitamins
(mainly ascorbic acid), flavonoids, volatile oils, carotenoids, and phenolic compounds.
In our previous works, polysaccharides extracted from gabiroba
pulp were analyzed (Barbieri et al., 2017), and the presence of pectins
was demonstrated (Barbieri et al., 2019). However, the bioactivities of
pectins extracted from gabiroba fruits, as well as their antitumor potential, have remained unexplored.
Thus, the aim of the present study was to purify the crude pectin
extract obtained from gabiroba pulp, elucidate its chemical structure,
and evaluate the antitumor potential of crude and purified pectin in a
human glioblastoma model.

was deionized with cation exchange resin and dialyzed with Cellulose
6–8 kDa cut off membrane (Spectrum Labs™), where the precipitation of
some of the material could be observed inside the membrane. Thus, the
sample was removed from the membrane and centrifuged (3000 rpm,
20 min, 4 °C), resulting in an insoluble fraction (GWP-FP-P) and a soluble fraction (GWP-FP-S). The soluble fraction GWP-FP-S was freezedried, structurally characterized, and analyzed for the biological activity tests (Fig. 1).
2.2. Monosaccharide composition
Uronic acid content of the GWP-FP-S fraction was quantified by the
colorimetric m-hydroxybiphenyl method, using galacturonic acid as the
standard (Blumenkrantz & Asboe-Hansen, 1973). The identity of the
uronic acid was determined by anion exchange chromatography, with
pulse amperometric detection (HPAEC-PAD). Samples were hydrolyzed
with 2 mol L−1 TFA for 8 h at 100 °C, dried, and washed with methanol
(x 3) to remove the acid. Hydrolyzed samples (1 mg mL−1) were injected into a Thermo Scientific Dionex ICS-5000 chromatograph
(Thermo Fisher Scientific, USA) with CarboPac PA20 column
(3 × 150 mm), using a gradient of 0.5 mol L−1 NaOH and 1 mol L−1
NaOAc as eluent (Nagel, Sirisakulwat, Carle, & Neidhart, 2014) in an N2
atmosphere in a flow of 0.2 ml min−1 at 24 °C. The analysis was carried
out in triplicate, and the data were collected and analyzed using the
Chromeleon TM 7.2 Chromatography Data System software.
The neutral monosaccharides were evaluated through total acid
hydrolysis, with 2 mol L−1 TFA for 8 h at 100 °C. The hydrolysates were
converted to alditol acetates through treatment with NaBH4 (Wolfrom
& Thompson, 1963a), followed by acetylation with acetic anhydride
(Ac2O)-pyridine (1:1, v/v, 1 mL) at 100 °C for 30 min (Wolfrom &
Thompson, 1963b). The alditol acetates were extracted with CHCl3 and
were analyzed in a Thermo Scientific Trace GC Ultra gas chromatograph, with a mixture of He, N2, and compressed air as the carrier gas at
1 ml min−1. The chromatograph also used a DB-225-MS column
(0.32 mm internal diameter x 30 m x film thickness 0.25 μm) programmed from 100 °C to 230 °C at a heating rate of 60 °C min−1. The
alditol acetates were identified by their profile, and retention times
were compared with standards.

2. Materials and methods
2.1. Isolation of pectic polysaccharides
Gabiroba fruits were collected and prepared according to Barbieri
et al. (2017). Briefly, crude pectic fraction from gabiroba pulp was
obtained by hot water extraction (GW) as reported in our previous work
(Barbieri et al., 2019). In order to obtain a homogeneous fraction, GW
was submitted to the fractionation process (Fig. 1). During this process,
the resulted freezing-thawing precipitated fraction (GWP) was treated
with Fehling solution (Jones & Stoodley, 1965; Ruthes, Smiderle, &
Iacomini, 2015) obtaining the precipitated fraction (GWP-FP), which

2.3. High performance size exclusion chromatography coupled to
multidetectors (HPSEC-MALLS-RI)
The homogeneity and average molar mass (Mw) of soluble polysaccharides were evaluated by high performance size exclusion chromatography (HPSEC), coupled with multi-angle laser light scattering
(MALLS) (DSP-F, Wyatt Technology, Santa Barbara, CA, USA) and refractive index (RI) detectors (Waters 2410, Milford, MA, USA) (HPSECMALLS-RI). The log plot Mw versus elution time was calculated from the
Rayleigh-Debye-Gans equation (Wyatt, 1993; Zimm, 1948).
The chromatography was carried out on a Waters system containing
four gel permeation columns packed with Ultrahydrogel® 2000, 500,
250, and 120, connected in series, with exclusion limits of 7 × 106,
4 × 105, 8 × 104, and 5 × 103 g mol−1, respectively. The flow rate
used was 0.6 ml min−1, with 0.1 mol L−1 sodium nitrite as the mobile
phase and 0.2 g L−1 sodium azide as a preservative, at a temperature of
25 °C. The data was collected and processed by Wyatt Technology
ASTRA software, version 4.70.07.
2.4. Protein quantification
Protein content of the GWP-FP-S fraction was determined using the
Bradford method (Bradford, 1976). A calibration curve of bovine serum
albumin was built as a standard, and the results were expressed in g
protein/100 g of sample.

Fig. 1. Scheme of fractionation of water-extracted polysaccharides from gabiroba pulp (Campomanesia xanthocarpa). *GW and GWP-TEP was previously
characterized (Barbieri et al., 2019).
2


Carbohydrate Polymers 224 (2019) 115140

S.d.C. Amaral, et al.

2.5. Nuclear magnetic resonance (NMR) spectroscopy

permeable probe, which oxidizes intracellularly to the highly fluorescent DCF (2′,7′-dichlorofluorescein) (Kalyanaraman et al., 2012).
U251-MG and T98 G glioblastoma cells (4 × 103 cells/well) were
seeded on black 96-well plates for 24 h of incubation, and treated as
previously described for the cytotoxicity assay. Subsequently, the culture medium was removed, cells were washed with PBS, and then incubated with 5 mM of DCFH-DA at 37 °C for 30 min in the dark.
Fluorescence was measured on a spectrofluorometer (Infinite M200,
Tecan Trading AG, Switzerland) with a 485 nm excitation filter and a
520 nm emission. Hydrogen peroxide (H2O2, 400 μM) was used as a
positive control 30 min before the measurement. Values were normalized using crystal violet assay standardization and the intracellular
fluorescence levels of control cells (vehicle used to dissolve the extract milliQ water) were considered as 1.

Mono-dimensional (13C- and 1H-) and bi-dimensional (1H/13C
HSQC) NMR spectra were acquired at 70 °C on a Bruker AVANCE III
400 NMR spectrometer and equipped with a 5-mm multinuclear inverse
detection probe with z-gradient, operating at 9.5 T and observing 1H at
400.13 MHz and 13C at 100.61 MHz. The samples were acquired in D2O
with chemical shifts expressed as δ (ppm), using the resonances of
CH3e groups of acetone (1H at δ 2.22; 13C at δ 30.20) as internal references. The data was collected and processed on Topspin 3.2 (Bruker
BioSpin Corporation, Billerica, MA, USA).

2.6. Cell culture and treatment with pectic fractions
U251-MG and T98 G human glioblastoma cell lines and normal
murine fibroblast NIH 3T3 cell line were kindly provided by Dr. Mari
Cleide Sogayar, Cell and Molecular Therapy Center (NUCEL/NETCEM),
Faculty of Medicine, University of São Paulo (FMUSP). Cells were cultured in a DMEM high-glucose medium (Sigma-Aldrich), supplemented
with 10% fetal bovine serum (FBS, Gibco) and 50 μg mL−1 gentamicin
(Sigma-Aldrich), and maintained at 37 °C in a 5% CO2 atmosphere.
For the following experiments, GW and GWP-FP-S fractions obtained from gabiroba pulp were separately dissolved in ultrapure water
−
(5 mg mL 1) and stored at −20 °C until utilized.

2.9. Statistical analysis
The statistical analyses are expressed as mean ± SD of at least
three independent experiments in quadruplicate. Analyses were performed on GraphPadPrism software version 5.01 for Windows
(GraphPad Software, San Diego, CA, USA), using a one-way analysis of
variance (ANOVA) followed by Tukey’s. A difference of p < 0.05 was
considered statistically significant.
3. Results and discussion

2.7. Cytotoxicity assay

3.1. Structural characterization of purified fraction extracted from gabiroba
pulp

First, 4 × 103 cells/well were seeded in 96-well culture plates for
24 h of incubation. Then, cells were treated either with vehicle control
(Ultrapure water – 18.2 MΩ cm−1, pH 6.9) or with different concentrations (10–400 μg mL−1) of GW and GWP-FP-S fractions.
The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide) method involves metabolically active cells reducing tetrazolium salt to formazan (Berridge, Herst, & Tan, 2005; Mosmann,
1983). For the MTT assay (Mosmann, 1983), cells were treated either

with vehicle control or with 10, 25, 50, 100, 200, 400 μg mL−1 of GW
and GWP-FP-S fractions. Then, the culture medium was removed after
48 h of treatment and replaced with fresh medium containing
0.5 mg mL−1 MTT (Sigma- Aldrich); this was protected from light for
3 h at 37 °C in a 5% CO2 atmosphere. The medium was removed, and
formazan production on viable cells was solubilized in dimethyl sulfoxide (DMSO). Absorbance was measured by spectrophotometry
(Epoch, BioTek, USA) at 545 nm, and results were expressed as a percentage of control (vehicle), assigned as 100% of metabolically active
cells.
Crystal violet is an alkaline dye with high affinity to nucleic acids
extensively used to determine the number of cells through DNA staining
of adhered and fixed cells (Kueng, Silber, & Eppenberger, 1989).
For the crystal violet assay (Kueng et al., 1989), cells were treated
either with vehicle control or with 25, 100, 400 μg mL−1 of GW and
GWP-FP-S fractions. After 48 h of treatment the culture medium was
removed and washed with PBS. Cells were then fixed in methanol for
10 min, stained with crystal violet for 3 min, and then washed with PBS.
Afterwards, the stain was diluted in sodium citrate and quantified in a
spectrophotometer (Epoch, BioTek, USA) at 545 nm. Absorbance was
proportional to cell number. Results were calculated by comparing
absorbance of treated-to-untreated cells (with control as the vehicle
used to dissolve the fractions – Ultrapure water), assigning control
absorbance as 100% of adhered cells.
Cell images were obtained using an inverted microscope (Axiovert
40, Carl Zeiss).

GW fraction (5.06% yield), recently characterized by Barbieri et al.
(2019), was submitted to fractionation by freeze-thawing and the
Fehling’s solution treatment, giving rise to a GWP-FP fraction (1.63%
yield). This fraction was purified to give the fraction of interest, GWPFP-S (0.64% yield) (Fig. 1).
Fig. 2A presents the HPSEC-RI elution profiles of the GWP-FP and

GWP-FP-S fractions. The GWP-FP fraction presented three peaks that
eluted at retention times between 42 and 60 min, showing a heterogeneous profile, such as the GW fraction analyzed by Barbieri et al.
(2019); meanwhile, the GWP-FP-S fraction presented a homogeneous
profile, with a single elution peak at 51 min, demonstrating that the
polysaccharide fractionation process was efficient. Assuming that
achieve the homogeneity is the primordial step for studies of polysaccharide structure, pharmacology, and its structure-activity relationships (Shi, 2016), the GWP-FP-S was suitable for the investigation
of its cytotoxic potential.
HPSEC-MALLS-RI chromatogram and molar mass distribution of the
GWP-FP-S fraction were performed (Fig. 2B). Through the light scattering signal (90°), a major peak can be observed between 35 and
45 min (with an invisible signal to the RI detector), corresponding to its
very low concentration. A minor peak between 45 and 55 min detected
by light scattering (90°) was demonstrated as a major peak by the RI
detector, showing a high concentration corresponding to the GWP-FP-S
fraction. The average molar mass (Mw) for GWP-FP-S was determined to
be 29,170 g mol−1 (dn/dc 0.113).
GWP-FP-S was predominantly composed of galacturonic acid (GalA,
58.8%), followed by arabinose (Ara, 28.5%), galactose (Gal, 11.3%),
and rhamnose (Rha, 1.1%), suggesting the presence of pectins
(Table 1).
In comparison to the crude fraction GW (54.5% Ara; 33.5% GalA;
7.6% Gal) (Barbieri et al., 2019), the purified fraction GWP-FP-S, exhibited an increase in GalA and Gal followed by a decrease in Ara
content (Table 1). Nevertheless, the content of GalA from the GWP-FP-S
fraction was similar to that found by Maxwell et al. (2016) for a
modified sugar beet pectin (52. 5% of GalA), which induced apoptosis
of colon cancer cells.
The relative amount of HG and RG-I domains of the pectins was

2.8. Detection of intracellular reactive oxygen species (ROS) levels
The intracellular levels of ROS were measured using DCFH-DA (2′,
7′-Dichlorofluorescin diacetate, Sigma-Aldrich) a non-fluorescent cell

3


Carbohydrate Polymers 224 (2019) 115140

S.d.C. Amaral, et al.

Fig. 2. HPSEC-MALLS-RI elution profile. (A) Elution profile by RI of the GWP-FP and GWP-FP-S fractions obtained from the gabiroba pulp. (B) GWP-FP-S Fraction.
Molar mass distribution (MwD), light scattering (LS-90°), and refractive index (RI).

estimated from the monosaccharide composition according to M’sakni
et al. (2006), using the equations:

signal was not observed for GWP-FP-S. A de-esterification may have
occurred due to the use of alkaline pH in the Fehling’s treatment. Deesterification was also observed by Nascimento et al. (2017) on sweet
pepper (Capsicum annum) pectin fraction after treatment with Fehling’s
solution.
The 13C-NMR spectrum also showed signals at δ 107.6, δ 107.2,
106.5, and 106.3 that are attributed to α-L-Araf (C-1). Signals for the →
4)-β-D-Galp-(1→units were observed at δ 104.3 (C-1), δ 72.0 (C-2), δ
73.4 (C-3), δ 77.6 (O-substituted C-4), δ 74.5 (C-5), and δ 60.8 (C-6).
All assignments obtained by 13C-NMR were confirmed by the analysis of the 1H/13C heterocorrelated HSQC-NMR spectrum (Table 2,
Fig. 4). The highest intensity peaks in the spectrum were attributed to
→4)-α-D-GalAp-(1→ units, confirming the presence of HG in the GWPFP-S fraction. Signals with lower intensity were also observed and were
attributed to unsubstituted →2)-α-L-Rhap-(1→ and substituted →2,4)α-L-Rhap-(1→ units from the backbone of type I rhamnogalacturonan.
Arabinose and galactose side chains were ascribed to signals of linear
backbone of →5)-α-L-Araf-(1→ units, as well as branched →3,5)-α-LAraf-(1→ units. In addition, the presence of galactans was observed at
correlations of →4)-β-D-Galp(1→ units (Colodel et al., 2018;
Klosterhoff et al., 2018; Tamiello et al., 2018). With the analysis of
1

H/13C heterocorrelated HSQC-NMR, it was possible to propose that the
purified GWP-FP-S fraction consists of a pectin formed predominantly
by HG regions and containing branched RG-I inserts with side chains of
arabinans, galactans, and possibly arabinogalactans.

HG (%) = GalA (%) – Rha (%) and,
RG-I (%) = [GalA (%) – HG(%)] + Rha + Ara + Gal
Thus, the GWP-FP-S chain is represented by 42.0% of RG-I and
57.7% of HG. These results showed a decrease in the RG-I proportion
and an increase in the HG proportion of GWP-FP-S in relation to the GW
fraction, which presented 65.3% RG-I and 31.9% HG (Barbieri et al.,
2019). Structural differences in the main chain can be attributed to the
process of purification. According to the literature, HG-rich chains form
a complex with Cu2+ and precipitate after being treated with Fehling’s
solution (Nascimento, Iacomini, & Cordeiro, 2017).
In addition, in order to estimate the extension of neutral side chains
attached to the RG-I (Houben, Jolie, Fraeye, Van Loey, & Hendrickx,
2011), the ratio (Gal + Ara)/Rha was calculated. The value obtained
for GWP-FP-S was 37.6 (Table 1). Despite the differences in the proportions of the HG and RG-I domains of GWP-FP-S and GW, it can be
observed that the values obtained to the neutral side chains attached to
the RG-I (37.6 for GWP-FP-S and 38.8 for GW) was similar for both
fractions, demonstrating that the side chain extension was not affected
by the fractionation process adopted.
Regarding the protein content, GWP-FP-S showed no significant
amount when evaluated by the Bradford method.
The GWP-FP-S fraction was investigated by 13C-NMR. The 13C-NMR
spectrum (Fig. 3) showed a typical chemical shift from HG observed at δ
99.7 and 70.3, which correspond to C-1 and C-5, respectively, of unesterified α-D-GalAp units. The remaining assignments of D-GalAp ring
carbons were seen at δ 78.1 (O-substituted C-4/H-4), δ 68.3 (C-3), and δ
68.0 (C-2). Signals of C-6 from unesterified α-D-GalAp units could be

assigned as δ 171.9. In the literature, the signal of methyl groups linked
to α-D-GalAp units can usually be observed at δ 52.8 ppm (Colodel,
Vriesmann, & Petkowicz, 2018; Nascimento et al., 2017); however, this

3.2. Cytotoxic effect of crude and purified pectin fractions extracted from
gabiroba pulp
Fig. 5 shows the cytotoxic effect of GW and GWP-FP-S pectins on
glioblastoma cell lines. U251-MG and T98 G cells were exposed to different concentrations of pectins (25–400 μg mL−1) for 48 h, and cell
cytotoxicity was determined by crystal violet assay.
Both of these fractions were able to reduce the number of adherent

Table 1
Monosaccharide composition of crude and purified pectin fractions extracted from gabiroba pulp.
Monosaccharide composition (%)a

Fraction

GWd
GWP-FP-S
a
b
c
d

GalAb

Rha

Ara

Xyl

Man

Gal

Glc

HG (%)c

RG-I (%)c

33.5
58.8

1.6
1.1

54.5
28.5

1.0
0.2

tr
tr

7.6
11.3

0.9
tr

31.9
57.7

65.3
42.0

% of peak area of monosaccharide composition relative to the total peak area, determined by GLC.
Uronic acids, determined using the m-hydroxybiphenyl method (Blumenkrantz & Asboe-Hansen, 1973), and identified by HPAEC-PAD.
HG = GalA – Rha ; RG-I = 2(Rha) + Ara + Gal (M’sakni et al., 2006); tr = trace.
GW from Barbieri at al. (2019).
4

(Gal+Ara)
Rha

38.8
37.6


Carbohydrate Polymers 224 (2019) 115140

S.d.C. Amaral, et al.

Fig. 3.

13

C NMR spectrum of the GWP-FP-S fraction from the pulp of gabiroba fruits, obtained at 70 °C in D2O (chemical shifts are expressed in δ, ppm).

glioblastoma cells (stained by crystal violet assay) (Fig.5). These effects
were also evidenced by optical microscopy, which showed that both
GW (Fig. 5D) and GWP-FP-S (Fig. 5E) treatments promote significant
changes in U251-MG cell morphology beyond altering cell number, as
compared to a vehicle-treated control group (Fig. 5C).
All tested concentrations of both GW and GWP-FP-S fractions were
able to reduce the percentage of adherent U251-MG and T98 G cells,
reaching a decrease of 18.04–33.74% and 20.05–37.65% of adherent
U251-MG cells after GW and GWP-FP-S treatment, respectively, at
different concentrations. Similar effects were also achieved for T98 G
glioblastoma cells, where GW and GWP-FP-S treatments were able to
inhibit 15.78–27.32% and 25.48–35.55% of adherent T98 G cells, respectively, indicating that the cytotoxic effect of these pectins was not
selective, and is independent of cell-line-specific characteristics. In view
of this, the next investigations were continued with only one glioblastoma cell line (U251-MG).
The cytotoxic effects were also confirmed by MTT assay (Fig. 6).
When U251-MG cells were treated with GW and GWP-FP-S at high
concentrations (100–400 μg mL−1), the percentage of metabolicallyactive U251-MG cells were reduced in an equal manner. However,
U251-MG cells seemed to respond differently when pectin fractions
were used at low concentrations. A decrease in metabolic U251-MG cell

Table 2
1
H and 13C NMR chemical shifts (δ, ppm) of GWP-FP-S fraction from the pulp of
gabiroba.
Glycosil residues

Nucleus

→4)-α -D-GalAp-(1→

13

→2)-α-L-Rhap-(1→

13

→2,4)-α-L-Rhap-(1→
→5)-α-L-Araf-(1→
→3,5)-α-L-Araf-(1→
t-α-L-Araf-(1→
→4)-β-D-Galp-(1→

C
H
C
1
H
13
C
1
H
13
C
1
H
13
C
1

H
13
C
1
H
13
C
1
H
1

Chemical shifts, δ (ppm)
1

2

3

4

5

99.7
5.10
99.1
5.22
99.1
5.22
107.7
5.08

106.6
5.23
107.6
5.17
104.3
4.61

68.0
3.78
76.7
4.08
–

68.5
4.01
–

78.1
4.49
–

70.3
5.01
–

–

–

–

81.4
4.13
–

76.8
4.01
–

79.8
4.27
72.0
3.68

76.7
3.97
73.4
3.75

82.8
4.19
82.3
3.90
84.1
4.04
77.6
4.15

66.8
3.88

66.6
3.91
61.3
3.73
74.5
3.70

6

16.5
1.25
16.8
1.32

60.8
3.80

Acetone was used as internal standard (δ2.22/30.2), and the analysis was
carried out at 70 °C.

Fig. 4. 1H/13C HSQC- NMR spectrum of the GWP-FP-S fraction from the pulp of gabiroba fruits, obtained at 70 °C in D2O (chemical shifts are expressed in δ, ppm).

5


Carbohydrate Polymers 224 (2019) 115140

S.d.C. Amaral, et al.

Fig. 5. Cytotoxic effect of GW and GWP-FP-S pectins. Glioblastoma cell lines: (A) U251-MG and (B) T98 G cells were treated with GW or GWP-FP-S at different

concentrations (25–400 μg mL−1) for 48 h. The percentage of adherent cells were evaluated by crystal violet assay. The values represent the means ± SD of
percentage of cells compared with the control (data represent at least three independent experiments, each in quadruplicate). *p < 0.05; **p < 0.01;
***p < 0.001. Morphology of U251-MG cells: (C) Control group (cells treated with vehicle). (D) Cells treated with GW at 400 μg mL−1. (E) Cells treated with GWPFP-S at 400 μg mL−1. Magnification 100 × .

GW fraction did not alter their cellular metabolic activity, likely in an
attempt to maintain survival. This differential effect was no longer
observed when the treatment reached higher concentrations of either
fraction.
It is important to note that, from a pharmacological point of view,
cytotoxicity at lower concentrations is one of the most valuable potentials for clinical application (Cobs-Rosas et al., 2015). In the literature, modified sugar beet pectin reduced the number of viable HT29
colon cancer cells by 20.7% in 48 h at 1000 μg mL−1 (Maxwell et al.,
2016). Low-molecular-weight citrus pectin decreased cell viability of
AGS (gastric cancer) and SW-480 (colorectal cancer) cells by 24% and
28%, respectively, at a concentration of 5.0 mg mL−1 for 24 h (Wang,
Li, Lu, & Ling, 2016). However, until now, only one study has been
reported for glioblastoma model, when Fan et al. (2012) evaluated the
effect of Panax ginseng (ginseng) pectin on U87 human glioblastoma
cells, and no cytotoxicity was observed at any tested concentrations
(10–500 μg mL−1).
Considering that one of the pivotal pathways to activate cell death is
by excessive reactive oxygen species (ROS) levels (Kalyanaraman et al.,
2018), the impact of GW and GWP-FP-S on intracellular ROS levels was
measured using DCFH-DA probe. As illustrated in Fig. 7, the ROS levels
were significantly increased after treatment with 100–400 μg mL−1 of
GW, and 25–400 μg mL−1 of GWP-FP-S, in comparison to untreated
U251-MG cells, which suggest that cytotoxic effect of pectin fractions
could be related to increased intracellular ROS levels.
Citrus pectin (CP) and apple pectin (AP) have also been reported to
suppress viability in MDA-MB-231, MCF-7 and T47D human breast
cancer cells by increasing ROS content, which led to the caspase-dependent apoptosis (Salehi, Behboudi, Kavoosi, & Ardestani, 2018). The

Fig. 6. Cytotoxic effect of GW and GWP-FP-S pectins. U251-MG cells were
treated with GW or GWP-FP-S at different concentrations (10–400 μg mL−1) for
48 h. The percentage of metabolically active cells were evaluated by MTT assay.
The values represent the means ± SD of percentage of cells compared with the
control (data represent at least three independent experiments, each in quadruplicate). *p < 0.05; **p < 0.01; ***p < 0.001.

activity (19.55%) was observed using 50 μg mL−1 of GW (similar to
obtained with high concentrations), while half that amount (25 μg
mL−1) of GWP-FP-S was necessary to achieve the same effect (inhibition of 20.96% of metabolic human glioblastoma cell activity).
GW and GWP-FP-S treatments at 25 μg mL−1 decreased comparable
numbers of adherent cells; nevertheless, U251-MG cells treated with

6


Carbohydrate Polymers 224 (2019) 115140

S.d.C. Amaral, et al.

was observed in high concentrations (the cytotoxic effect was not statistically different for treatments using more than 25 μg mL−1).
However, is it possible to suggest that the major component responsible
for the biological activity of the crude GW is purified GWP-FP-S
structure, since after purification process, lower concentrations of this
homogeneous fraction were sufficient to exert the cytotoxic effect.
The crude GW fraction was revealed to be a heterogeneous fraction,
with a wide range of molar mass distribution and a degree of methylesterification (DM) of 60%. It was composed mostly of RG-I portion
(65.3%) and presented high extension of the neutral side chains and
high amounts of arabinose (54.5%) and galactose (7.6%) (Barbieri
et al., 2019). Evidence from the literature suggests that the antitumor

activity may be related to the RG-I domain, possibly by adopting a
conformation that maximizes the availability of neutral sugar side
chains for cellular interaction (Maxwell et al., 2016; Prado et al., 2017;
Vayssade et al., 2010). In addition, the importance of arabinose’s presence in the structure has recently been recognized due to its partial
influence on the pectin’s effect on cancer cell lines. Maxwell et al.
(2016) observed a reduction of colon cancer cell proliferation—reaching 7–15% of inhibition—after selective removal of
arabinose in modified sugar beet pectin.
In contrast, GWP-FP-S structure, represented by 57.7% HG and
lower proportions (42.0%) of branched RG-I (containing 28.5% of Ara
and 11.3% of Gal) and de-esterified (due to the employed alkaline pH in
the Fehling’s treatment). By the HPSEC-MALLS-RI analysis, it showed as
a homogeneous fraction, with low molar mass (29,170 g mol−1). The
molar mass of GWP-FP-S was similar to a modified citrus pectin (MCP,
30,000 g mol−1) (Gao et al., 2012). Modified pectins with lower molar
mass have been reported as producing more profound antitumor activity than naturally large pectins (Naqash, Masoodi, Rather, Wani, &
Gani, 2017; Zhang et al., 2015). These authors showed the MCP as a
galectin-3 (gal3) inhibitor (a carbohydrate recognition domain with the
ability to modulate important functions for cell survival migration and
metastasis) that represents an attractive target for cancer therapy
(Vladoiu, Labrie, & St-Pierre, 2014).
Another structural characteristic of pectins attributed to the anticancer effect by inhibiting gal3 function is the presence of →4)-β-DGalp-(1→ units on the branched chain in the RG-I (Gunning, Bongaerts,
& Morris, 2009; Zhang et al., 2015). This may explain the difference of
biological effect of GW and GWP-FP-S observed in lower concentrations
(25 μg mL−1 by MTT and ROS assay), where only GWP-FP-S (which
contains 1.5 times more galactose than GW) is capable to induce cytotoxicity.
In order to evaluate the cytotoxic effect of GW and GWP-FP-S
fractions on normal cells, NIH 3T3 murine fibroblast cells were exposed
to the same treatment conditions as GW and GWP-FP-S (10–400 μg
mL−1 for 48 h), and cytotoxic effect was measured by MTT assay. Both
pectin fractions showed no cytotoxicity for NIH 3T3 cells after 48 h of

treatment at all tested concentrations (Fig. 8). By contrast, traditional
anticancer agents are generally toxic to normal cells, producing severe
side effects (Huettemann & Sakka, 2005; Wang, Huang, Sun, & Pan,
2015). Indeed, pectins have been described as a biocompatible and nontoxic biopolymer (Chan et al., 2017; Munarin et al., 2015).
In addition, 400 μg mL−1 (or 13.7 μmol L-1) of GWP-FP-S, the
highest concentration tested on the human glioblastoma model, inhibited 26.5% of metabolically active glioblastoma cells; in contrast,
temozolomide (TMZ), the standard chemotherapy for glioma patients
(Bailey et al., 2015), is known to inhibit 24.7% of U251-MG viable cells
(also evaluated by MTT assay, on 48 h of treatment) at 100 μmol L−1
(Shen, Hu, & Zheng, 2014). Interestingly, GWP-FP-S concentration is
approximately 7 times lower than the necessary TMZ concentration for
a similar effect.
The antitumor activities of polysaccharides can be mediated
through three main approaches: direct cytotoxicity, immunoenhancement, and synergistic effects combined with conventional antitumor
drugs (Yang et al., 2013). In this context, GW and GWP-FP-S pectin

Fig. 7. Effect of GW and GWP-FP-S pectins on intracellular ROS levels. U251MG cells were treated with GW or GWP-FP-S at different concentrations
(25–400 μg mL−1) for 48 h. The intracellular ROS levels were evaluated spectrofluorometrically using a DCFH-DA probe. U251-MG cells treated with hydrogen peroxide (H2O2) were used as a positive control (400 μM, 30 min). The
values represent the means ± SD of percentage of cells compared with the
control (data represent at least three independent experiments, each in quadruplicate). *p < 0.05; **p < 0.01; ***p < 0.001.

Fig. 8. Cytotoxic effect of GW and GWP-FP-S pectins on normal fibroblast cells.
NIH 3T3 cells were treated with GW or GWP-FP-S at different concentrations
(10–400 μg mL−1) for 48 h, and MTT assay was performed. The values represent the means ± SD of percentage of metabolically active cells compared
with the control (data represent at least three independent experiments, each in
quadruplicate). *p < 0.05; **p < 0.01; ***p < 0.001.

elevated levels of ROS are reported to increase the vulnerability of
cancer cells to oxidative damage of various macromolecules, including
proteins, lipids, and DNA, which in turn results in cell death (Wang

et al., 2019).
Furthermore, it is well established in the literature (Galadari,
Rahman, Pallichankandy, & Thayyullathil, 2017; Panieri & Santoro,
2016) that cancer cells have an inherent elevated ROS level compared
to normal cells. This effect suggests that therapeutic strategies that increase ROS generation may push cancer cells beyond the breaking
point, leading to a toxic level, thereby activating ROS-induced cell
death pathways. This may explain why GW and GWP-FP-S were only
cytotoxic on glioblastoma cells (Figs. 5 and 6) and showed no cytotoxicity for NIH 3T3 cells (Fig. 8).
Interesting, only the purified pectin (GWP-FP-S) at 25 μg mL−1 was
capable to increase significantly (1.3 fold higher than control) the levels
of ROS, corroborating to cytotoxic effects observed by MTT assay
(Fig. 6). In addition, the effect observed for 400 μg mL−1 of GW (1.71
fold) is achieved in a 4-fold lower concentration of GWP-FP-S (100 μg
mL−1).
These results indicate that, despite differing chemical structural
characteristics between GW and GWP-FP-S, a similar cytotoxic effect
7


Carbohydrate Polymers 224 (2019) 115140

S.d.C. Amaral, et al.

fractions from gabiroba pulp could prove to be effective antitumor
candidates. The purification and chemical structural characterization
are important factors in establishing structure-function relationships.
According to the literature, the cytotoxic effects of pectins on cancer
cell lines may be related to different factors involving their structure, as
conformation of the molecule, degree of esterification, ratio of HG / RGI and side chain of these polysaccharides. In this work, by comparing
the data obtained about the chemical structure and response to cytotoxicity in glioblastoma cells, it may suggests that the antitumor activity of the pectic fractions extracted from gabiroba pulp (GW and

GWP-FP-S) is probably related to the presence of →4)-β-D-Galp-(1→
units on the branched chain in the RG-I (Gunning et al., 2009; Zhang
et al., 2015). The amount of galactose on the GWP-FP-S structure (1.5
times higher than GW fraction) may explain the difference of cytotoxic
effect of in lower concentrations (25 μg mL−1 by MTT and ROS assay),
where only GWP-FP-S is capable to induce cytotoxicity.
Else more, for the first time, a crude (GW) and purified (GWP-FP-S)
pectins from gabiroba pulp have demonstrated cytotoxicity in glioblastoma cell lines. In terms of purely cytotoxic purposes, crude pectin
GW presented similar effects, when compared to the purified GWP-FPS, with the advantages of higher yield and fewer steps in the extraction
process, which in turn mean lower cost and time of acquisition.
Nevertheless, GWP-FP-S requires less concentration to exert this effect
and is more suitable to continue further studies on the potential antitumor effect, since it is a homogeneous purified pectin.

References
Alice, C. B., Siqueira, N. C. S., Mentz, L. A., Brasil, E., Silva, G. A. A., & Jose, K. F. D.
(1995). Plantas medicinais de uso popular: Atlas Farmacognóstico. Canoas:
ULBRA59–61.
Arbab, A. S., Rashid, M. H., Angara, K., Borin, T. F., Lin, P. C., Jain, M., et al. (2017).
Major challenges and potential microenvironment-targeted therapies in glioblastoma.
International Journal of Molecular Sciences, 18, 2732–2750.
Barbieri, S. F., Ruthes, A. C., Petkowicz, C. L. O., Godoy, R. C. B., Sassaki, G. L., SantanaFilho, A., et al. (2017). Extraction, purification and structural characterization of a
galactoglucomannan from the gabiroba fruit (Campomanesia xanthocarpa Berg),
Myrtaceae family. Carbohydrate Polymers, 174, 887–895.
Barbieri, S. F., Amaral, S. C., Ruthes, A. C., Petkowicz, C. L. O., Kerkhoven, N. C., Silva, E.
R. A., et al. (2019). Pectins from the pulp of gabiroba (Campomanesia xanthocarpa
Berg): Structural characterization and rheological behavior. Carbohydrate Polymers,
214, 250–258.
Barroso, G. M. (1978). Sistemática das magnoliophytas. In G. M. Barroso (Ed.). Sistemática
de angiospermas do Brasil-parte II (pp. 114–126). São Paulo: Ed. Universidade de São
Paulo.

Bailey, L. A., Jamshidi-Parsian, A., Patel, T., Koonce, N. A., Diekman, A. B., Cifarelli, C. P.,
et al. (2015). Combined temozolomide and ionizing radiation induces galectin-1 and
galectin-3 expression in a model of human glioma. Tumor Microenvironment and
Therapy, 2(1), 19–31.
Berridge, M. V., Herst, P. M., & Tan, A. S. (2005). Tetrazolium dyes as tools in cell
biology: New insights into their cellular reduction. Biotechnology Annual Review, 11,
127–152.
Biavatti, M. W., Farias, C., Curtius, F., Brasil, L. M., Hort, S., Schuster, L., et al. (2004).
Preliminary studies on Campomanesia xanthocarpa (Berg.) and Cuphea carthagenensis
(Jacq.) J.F. Macbr. aqueous extract: weight control and biochemical parameters.
Journal of Etnopharmacology, 93, 385–389.
Blumenkrantz, N., & Asboe-Hansen, G. (1973). New method for quantitative determination of uronic acids. Analytical Biochemistry, 54(2), 484–489.
Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram
quantities of protein utilizing the principle of protein-dye binding. Analytical
Biochemistry, 72, 248–254.
Caffal, K. H., & Mohnen, D. (2009). The structure, function, and biosynthesis of plant cell
wall pectic polysaccharides. Carbohydrate Research, 344, 1879–1900.
Chan, S. Y., Choo, W. S., Young, D. J., & Loh, X. J. (2017). Pectin as a rheology modifier:
Origin, structure, commercial production and rheology. Carbohydrate Polymers, 161,
118–139.
Cobs-Rosas, M., Concha-Olmos, J., Weinstein-Oppenheimer, C., & Zúñiga-Hansena, M. E.
(2015). Assessment of antiproliferative activity of pectic substances obtained by
different extraction methods from rapeseed cake on cancer cell lines. Carbohydrate
Polymers, 117, 923–932.
Colodel, C., Vriesmann, L. C., & Petkowicz, C. L. O. (2018). Cell wall polysaccharides from
ponkan mandarin (Citrus reticulata Blanco cv. Ponkan) peel. Carbohydrate Polymers,
195, 120–127.
Czaikoski, K., Mesomo, M. C., Krüger, R. L., Queiroga, C. L., & Corazza, M. L. (2015).
Extraction of Campomanesia xanthocarpa fruit using supercritical CO2 and bioactivity
assessments. The Journal of Supercritical Fluids, 98, 79–85.

Donado-Pestana, C. M., Moura, M. H. C., Araujo, R. L., Santiago, G. L., Barros, H. R. M., &
Genovese, M. I. (2018). Polyphenols from Brazilian native Myrtaceae fruits and their
potential health benefits against obesity and its associated complications. Current
Opinion in Food Science, 19, 42–49.
Fan, Y., Sun, C., Gao, X., Wang, F., Li, X., Kassim, et al. (2012). Neuroprotective effects of
ginseng pectin through the activation of ERK/MAPK and Akt survival signaling
pathways. Molecular Medicine Reports, 5, 1185–1190.
Galadari, S., Rahman, A., Pallichankandy, S., & Thayyullathil, F. (2017). Reactive oxygen
species and cancer paradox: To promote or to suppress? Free Radical Biology &
Medicine, 104, 144–164.
Gao, X., Zhi, Y., Zhang, T., Xue, H., Wang, X., Foday, A. D., et al. (2012). Analysis of the
neutral polysaccharide fraction of MCP and its inhibitory activity on galectin-3.
Glycoconjugate Journal, 29, 159–165.
Gunning, A. P., Bongaerts, R. J. M., & Morris, V. J. (2009). Recognition of galactan
components of pectin by galectin-3. The FASEB Journal, 23, 415–424.
Houben, K., Jolie, R. P., Fraeye, I., Van Loey, A. M., & Hendrickx, M. E. (2011).
Comparative study of the cell wall composition of broccoli, carrot, and tomato:
Structural characterization of the extractable pectins and hemicelluloses.
Carbohydrate Research, 346(9), 1105–1111.
Huettemann, E., & Sakka, S. G. (2005). Anaesthesia and anticancer chemotherapeutic
drugs. Current Opinion in Anaesthesiology, 18(3), 307–314.
Jones, J. K. N., & Stoodley, R. J. (1965). Fractionation using copper complexes. In R. L.
Whistler, M. L. Wolfrom, & J. N. BeMiller (Eds.). Methods in carbohydrate chemistry
(pp. 36–38). New York and London: Academic Press.
Kalyanaraman, B., Darley-Usmar, V., Davies, K. J. A., Dennery, P. A., Forman, H. J.,
Grisham, M. B., et al. (2012). Measuring reactive oxygen and nitrogen species with
fluorescent probes: Challenges and limitations. Free Radical Biology & Medicine,
52(1), 1–6.
Kalyanaraman, B., Cheng, G., Hardy, M., Ouari, O., Bennett, B., & Zielonka, J. (2018).
Teaching the basics of reactive oxygen species and their relevance to cancer biology:

Mitochondrial reactive oxygen species detection, redox signaling, and targeted
therapies. Redox Biology, 15, 347–362.
Klosterhoff, R. R., Bark, J. M., Glänzel, N. M., Iacomini, M., Martinez, G. R., Winnischofer,
S. M. B., et al. (2018). Structure and intracellular antioxidant activity of pectic
polysaccharide from acerola (Malpighia emarginata). International Journal of Biological

4. Conclusion
This paper presents a new source of pectin with a cytotoxic effect on
glioblastoma cells. GWP-FP-S pectin with low molar mass
(29,170 g mol−1) was purified from a crude (GW) pectin fraction from
gabiroba pulp and analyzed through monosaccharide composition,
homogeneity, and 1D and 2D nuclear magnetic resonance. The results
indicate the presence of HG, RG-I, and long side chains composed by
arabinose and galactose in the RG-I backbone. Crude GW and purified
GWP-FP-S pectins selectively exhibited a cytotoxic effect, even in low
concentrations, against human glioblastoma cells (U251-MG and T98 G
cell lines) and a concomitant increase in the cellular ROS levels, suggesting that these pectins could mediate cytotoxicity by altering the
cellular redox status. Also, no cytotoxicity was observed in normal fibroblast cells (NIH-3T3). The hypothesis that the antitumor effect of
pectins is associated with the Gal and Ara content of side chains on the
RG-I/HG backbone is corroborated by these results.
Acknowledgements
The authors gratefully acknowledge the following Brazilian agencies for financial support: the National Council for Scientific and
Technological Development – CNPq, the Coordination for the
Improvement of Higher Education Personnel - CAPES; the Araucaria
Foundation, Nanoglicobiotec and Ministry of Science and Technology/
CNPq, and the Federal University of Parana – Brazil. J.L.M.S. is a research member of the CNPq Foundation (nº 476950/2013-9; 308296/
2015-0; 309225/2018-3); S.M.B.W. is a research member of the CNPq
and Araucaria Foundation (nº 479356/2010-6; 307066/2012-6; 219/
2010-17497); S.C.A is the beneficiary of a post-graduation scholarship
(nº 141692/2018-9) provided by CNPq, and S.F.B. is the beneficiary of

a post-doctoral scholarship from Coordination of Superior Level Staff
Improvement - CAPES, nº 88887.335103/2019-00. The authors would
like to thank the NMR Center of UFPR for recording the NMR spectra,
the Brazilian Agricultural Research Corporation/Embrapa Forestry,
Rossana Catie Bueno de Godoy and Maria Cristina Medeiros Mazza for
provide the gabiroba pulp.
8


Carbohydrate Polymers 224 (2019) 115140

S.d.C. Amaral, et al.
Macromolecules, 106, 473–480.
Kueng, W., Silber, E., & Eppenberger, U. (1989). Quantification of cells cultured on 96well plates. Analytical Biochemistry, 182(1), 16–19.
Markman, B. E. O., Bacchi, E. M., & Kato, E. T. M. (2004). Antiulcerogenic effects of
Campomanesia xanthocarpa. Journal of Etnopharmacology, 94, 55–57.
Maxwell, E. G., Colquhoun, I. J., Chau, H. K., Hotchkiss, A. T., Waldron, K. W., Morris, V.
J., et al. (2016). Modified sugar beet pectin induces apoptosis of colon cancer cells via
an interaction with the neutral sugar side-chains. Carbohydrate Polymers, 136,
923–929.
Mosmann, T. (1983). Rapid colorimetric assay for cellular growth and survival:
Application to proliferation and cytotoxicity assays. Journal of lmmunological Methods,
63, 55–63.
Munarin, F., Petrini, M. P., Gentilini, R., Pillai, R. S., Dirè, S., & Sglavo, V. M. (2015).
Micro- and nano-hydroxyapatite as active reinforcement for soft biocomposites.
International Journal of Biological Macromolecules, 72, 199–209.
M’sakni, N. H., Majdoub, H., Roudesli, S., Picton, L., Cerf, D. L., Rihouey, C., et al. (2006).
Composition, structure and solution properties of polysaccharides extracted from
leaves of Mesembryanthenum crystallinum. European Polymer Journal, 42, 786–795.
Nagel, A., Sirisakulwat, S., Carle, R., & Neidhart, S. (2014). An acetate hydroxidegradient

for the quantitation of the neutral sugar and uronic acid profile ofpectins by HPAECPAD without postcolumn pH adjustment. Journal of Agricultural and Food Chemistry,
62, 2037–2048.
Naqash, F., Masoodi, F. A., Rather, S. A., Wani, S. M., & Gani, A. (2017). Emerging
concepts in the nutraceutical and functional properties of pectin-A Review.
Carbohydrate Polymers, 168, 227–239.
Nascimento, G. E., Iacomini, M., & Cordeiro, L. M. C. (2017). New findings on green sweet
pepper (Capsicum annum) pectins: Rhamnogalacturonan and type I and II arabinogalactans. Carbohydrate Polymers, 171, 292–299.
Noreen, A., Nazlic, Z.-I.-U., Akrama, J., Rasulb, I., Manshaa, N. Y., Iqbald, R., et al.
(2017). Pectins functionalized biomaterials, a new viable approach for biomedical
applications: A review. International Journal of Biological Macromolecules, 101,
254–272.
Panieri, E., & Santoro, M. M. (2016). ROS homeostasis and metabolism: a dangerous
liason in cancer cells. Cell Death & Disease, 7, 1–12 e2253.
Pereira, M. C., Steffens, R. S., Jablonski, A., Hertz, P. F., Rios, A. O., Vizzotto, M., et al.
(2012). Characterization and antioxidant potential of Brazilian fruits from the
Myrtaceae family. Journal of Agricultural and Food Chemistry, 60, 3061–3067.
Prado, S. B. R., Ferreira, G. F., Harazono, Y., Shiga, T. M., Raz, A., Carpita, N. C., et al.
(2017). Ripening-induced chemical modifications of papaya pectin inhibit cancer cell
proliferation. Scientific Reports, 7, 1–17.
Ruthes, A. C., Smiderle, F. R., & Iacomini, M. (2015). D-Glucans from edible mushrooms:
A review on the extraction, purification and chemical characterization approaches.
Carbohydrate Polymers, 117, 753–761.
Salehi, F., Behboudi, H., Kavoosi, G., & Ardestani, S. K. (2018). Oxidative DNA damage
induced by ROS-modulating agents with the ability to target DNA: A comparison of
the biological characteristics of citrus pectin and apple pectin. Scientific Reports,
8(13902-), 13918.
Shen, W., Hu, J. A., & Zheng, J. S. (2014). Mechanism of temozolomide-induced antitumor effects on glioma cells. The Journal of International Medical Research, 42(1),
164–172.
Shi, L. (2016). Bioactivities, isolation and purification methods of polysaccharides from
natural products: A review. International Journal of Biological Macromolecules, 92,

37–48.
Tamiello, C. S., Nascimento, G. E., Iacomini, M., & Cordeiro, L. M. C. (2018).

Arabinogalactan from edible jambo fruit induces different responses on cytokine
secretion by THP-1 macrophages in the absence and presence of proinflammatory
stimulus. International Journal of Biological Macromolecules, 107, 35–41.
Tanaka, S., Louis, D. N., Curry, W. T., Batchelor, T. T., & Dietrich, J. (2013). Diagnostic
and therapeutic avenues for glioblastoma: no longer a dead end? Nature Reviews
Clinical Oncology, 10, 14–26.
Vayssade, M., Sengkhamparn, N., Verhoef, R., Delaigue, C., Goundiam, O., Vigneron, P.,
et al. (2010). Antiproliferative and proapoptotic actions of Okra pectin on B16F10
melanoma cells. Phytoteraphy Research, 24, 982–989.
Viecili, P. R. N., Borges, D. O., Kirsten, K., Malheiros, J., Viecili, E., Melo, R. D., et al.
(2014). Effects of Campomanesia xanthocarpa on inflammatory processes, oxidative
stress, endothelial dysfunction and lipid biomarkers in hypercholesterolemic individuals. Atherosclerosis, 234, 85–92.
Vinagre, A. S., Rönnau, A. D. R. O., Pereira, S. F., Silveira, L. U., Wiilland, E. F., &
Suyenaga, E. S. (2010). Anti-diabetic effects of Campomanesia xanthocarpa (Berg) leaf
decoction. Brazilian Journal of Pharmaceutical Science, 46, 169–177.
Vladoiu, M. C., Labrie, M., & St-Pierre, Y. (2014). Intracellular galectins in cancer cells:
Potential new targets for therapy (Review). International Journal of Oncology, 44,
1001–1014.
Wang, Y., Huang, M., Sun, R., & Pan, L. (2015). Extraction, characterization of a Ginseng
fruits polysaccharide and its immune modulating activities in rats with Lewis lung
carcinoma. Carbohydrate Polymers, 127, 215–221.
Wang, S., Li, P., Lu, S. M., & Ling, Z. Q. (2016). Chemoprevention of low-molecularweight citrus pectin (LCP) in gastrointestinal cancer cells. International Journal of
Biological Sciences, 12(6), 746–756.
Wang, Y., Zhang, J., Yang, Y., Liu, Q., Xu, G., Zhang, R., et al. (2019). ROS generation and
autophagosome accumulation contribute to the DMAMCL-induced inhibition of
glioma cell proliferation by regulating the ROS/MAPK signaling pathway and suppressing the Akt/mTOR signaling pathway. OncoTargets and Therapy, 12, 1867–1880.
Wolfrom, M. L., & Thompson, A. (1963a). Reduction with sodium borohydride. In R. L.

Whistler, M. L. Wolfrom, & J. N. BeMiller (Eds.). Methods in carbohydrate chemistry
(pp. 65–68). New York and London: Academic Press Inc.
Wolfrom, M. L., & Thompson, A. (1963b). Acetylation. In R. L. Whistler, M. L. Wolfrom, &
J. N. BeMiller (Eds.). Methods in carbohydrate chemistry (pp. 211–215). New York and
London: Academic Press Inc.
Wyatt, P. J. (1993). Light scattering and the absolute characterization ofmacromolecules.
Analytica Chimica Acta, 272, l-40.
Yan, C., Lee, J., Kong, F., & Zhang, D. (2013). Anti-glycated activity prediction of polysaccharides from two guava fruits using artificial neural networks. Carbohydrate
Polymers, 98, 116–121.
Yang, C., Gou, Y., Chen, J., An, J., Chen, W., & Hu, F. (2013). Structural characterization
and antitumor activity of a pectic polysaccharide from Codonopsis pilosula.
Carbohydrate Polymers, 98, 886–895.
Yapo, B. M. (2011). Pectic substances: from simple pectic polysaccharides to complex
pectins - A new hypothetical model. Carbohydrate Polymers, 86, 373–385.
Zhang, W., Xu, P., & Zhang, H. (2015). Pectin in cancer therapy: A review. Trends in Food
Science & Technology, 44, 258–271.
Zhang, Z., Kong, F., Ni, H., Mo, Z., Wan, J. B., Hua, D., et al. (2016). Structural characterization, α-glucosidase inhibitory and DPPH scavenging activities of polysaccharides from guava. Carbohydrate Polymers, 144, 106–114.
Zimm, B. H. (1948). Apparatus and methods for measurement and interpretation of the
angular variations of light scattering; preliminary results on polystyrene solutions.
The Journal of Chemical Physics, 16(12), 1099–1116.

9