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Carbohydrate Polymers 136 (2016) 923929

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

Modied sugar beet pectin induces apoptosis of colon cancer cells


via an interaction with the neutral sugar side-chains
Ellen G. Maxwell a, , Ian J. Colquhoun a , Hoa K. Chau b , Arland T. Hotchkiss b ,
Keith W. Waldron a , Victor J. Morris a , Nigel J. Belshaw a
a

Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, UK
United States Department of Agriculture, Agricultural Research Service, Eastern Regional Research Center, 600 E Mermaid Lane, Wyndmoor,
PA 19038-8598, USA
b

a r t i c l e

i n f o

Article history:
Received 14 July 2015
Received in revised form
15 September 2015
Accepted 21 September 2015
Available online 26 September 2015
Keywords:
Modied pectin
Polysaccharides
Sugar beet
Rhamnogalacturonan I
Galactan
Colon cancer

a b s t r a c t
Pectins extracted from a variety of sources and modied with heat and/or pH have previously been
shown to exhibit activity towards several cancer cell lines. However, the structural basis for the anticancer activity of modied pectin requires clarication. Sugar beet and citrus pectin extracts have been
compared. Pectin extracted from sugar beet pulp only weakly affected the viability of colon cancer cells.
Alkali treatment increased the anti-cancer effect of sugar beet pectin via an induction of apoptosis. Alkali
treatment decreased the degree of esterication (DE) and increased the ratio of rhamnogalacturonan
I (RGI) to homogalacturonan. Low DE per se did not play a signicant role in the anti-cancer activity.
However, the enzymatic removal of galactose and, to a lesser extent, arabinose from the pectin decreased
the effect on cancer cells indicating that the neutral sugar-containing RGI regions are important for pectin
bioactivity.
2015 Elsevier Ltd. All rights reserved.

1. Introduction
Colorectal cancer (CRC) is the third most common cancer worldwide and the most common diet-related cancer. Epidemiological
studies have shown that increased fruit and vegetable consumption is associated with a reduced risk of developing CRC (Aune
et al., 2011; Watson & Collins, 2011). Dietary bre, which includes
virtually all carbohydrates resistant to hydrolysis in the small intestine, is considered a signicant component in the modulation of
CRC risk by fruits and vegetables acting by various mechanisms.
Pectin, a family of complex polysaccharides, is a component of all
fruits and vegetables and is a signicant source of dietary bre.
It has an extremely complex structure made up of several structural elements but a basic model of pectin extracts comprises linear
regions of homogalacturonan (HG) interspersed with rhamnogalacturonan I (RGI) regions in which neutral sugars are present
as side-chains (Maxwell, Belshaw, Waldron, & Morris, 2012). These

Corresponding author.
E-mail addresses: ellen.maxwell@ifr.ac.uk (E.G. Maxwell),
ian.colquhoun@ifr.ac.uk (I.J. Colquhoun), rose.chau@ars.usda.gov (H.K. Chau),
arland.hotchkiss@ars.usda.gov (A.T. Hotchkiss), keith.waldron@ifr.ac.uk
(K.W. Waldron), vic.morris@ifr.ac.uk (V.J. Morris), nigel.belshaw@ifr.ac.uk
(N.J. Belshaw).
http://dx.doi.org/10.1016/j.carbpol.2015.09.063
0144-8617/ 2015 Elsevier Ltd. All rights reserved.

side-chains, consisting mainly of galactans, arabinans and arabinogalactans are more frequent in sugar beet pectin compared to citrus
pectin (Buchholt, Christensen, Fallesen, Ralet, & Thibault, 2004; Sun
& Hughes, 1999). Pectins from numerous sources such as citrus
(Guess et al., 2003; Jackson et al., 2007; Olano-Martin, Rimbach,
Gibson, & Rastall, 2003; Platt & Raz, 1992; Yan & Katz, 2010), apple
(Olano-Martin et al., 2003; Li et al., 2010), okra (Vayssade et al.,
2010), and ginseng (Cheng et al., 2011; Fan et al., 2010), extracted
and modied in various ways, have been investigated for their anticancer effects and have been shown to reduce cell proliferation,
migration, adhesion, and induce apoptosis in a variety of cancer
cell lines (Maxwell et al., 2012).
Pectin that has been treated with pH (low or high), heat or
enzymes is generally referred to as modied pectin (MP), although
this term remains ambiguous, as pectin is a highly heterogeneous
material. Modied pectin structure can vary widely depending
on the pectin source, extraction and method of modication. The
majority of research into the bioactive effects of modied pectin
has been carried out with citrus pectins, and several studies have
demonstrated the benets of modied citrus pectin (MCP) over
conventional, un-modied citrus pectin (CP) (Liu, Huang, Yang, Lu,
& Yu, 2008; Nangia-Makker et al., 2002; Pienta et al., 1995; Platt
& Raz, 1992). It is generally understood that modifying CP with
heat and pH will decrease MW and proportionally increase total

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E.G. Maxwell et al. / Carbohydrate Polymers 136 (2016) 923929

neutral sugars, although in the majority of studies the extent of


structure modication was not examined. Generally it is thought
that pectins with a high neutral sugar content are more bioactive
due to the hypothesis that galactan side-chains on pectin can bind
to and inhibit the pro-metastatic protein galectin-3, resulting in the
suppression of cancer cell proliferation, aggregation, adhesion and
metastasis (Inohara & Raz, 1994; Nangia-Makker et al., 2002). However, conclusive evidence to support this hypothesis for all forms
of bioactivity remains to be presented.
In this study, the relationship between pectin structure and
anti-cancer activity for a range of sugar beet pectins, extracted
and modied in a variety of ways was explored. Modication by
alkali treatment increased the anti-cancer effect and this was associated with an increased ratio of RGI to HG. The importance of the
neutral sugar side-chains of RGI for bioactivity was conrmed by a
decreased anti-cancer effect following their removal.
2. Materials and methods
2.1. Pectin extraction and modication
Pectins were prepared at CP Kelco (Lille Skensved, Denmark).
50 kg of freshly dug sugar beet was chopped and washed in 30 C
water under agitation for 30 min, drained, then washed once more
for 30 min under agitation. Sugar beet pulp (12.5 kg) and citrus peel
(1 kg) were individually extracted with 50 L de-ionised water at
70 C for 4 h and adjusted to pH 1.7 with HNO3 . The sugar beet
extract was concentrated by vacuum evaporation at 60 C. After
ltering with Filtercel 450 (Advanced Minerals Corporation, CA,
USA), the liquid fraction was precipitated in three parts 80% IPA,
washed in 5 L 60% isopropanol (IPA), adjusted to pH 4 with HNO3 ,
then dried and milled to obtain conventional sugar beet (SBC) and
citrus (CP) pectins. The residue from the sugar beet pectin extract
was extracted again with 5 L de-ionised water at 98 C for 1 h at
pH 2 to obtain SBH. Half the SBH was mixed in 5 L of 60% IPA at
5 C and adjusted to pH 12.5 with NaOH for 1 h, then adjusted to
pH 4 with HNO3 . After precipitation, drying and milling, SBA was
dissolved in water at 10 mg/mL and centrifuged at 3700 g for 1 h
in order to obtain the soluble fraction (SBA), which was separated
from the pellet and lyophilised. In a separate extraction, sugar beet
pulp (12.5 kg) was extracted with 50 L de-ionised water at 70 C for
2 h at pH 3.5 with oxalic acid, and concentrated by vacuum evaporation at 60 C to obtain SBO. The residue was re-extracted in 5 L
de-ionised water with 15 mL Rohament PL polygalacturonase (AB
Enzymes, Darmstadt, Germany) in 100 mL water at pH 3.5 for 1 h
and vacuum evaporated at 60 C prior to precipitation, to obtain
SBOPG. Apple pectins were a gift from Danisco.
2.2. Monosaccharide, molar mass and protein analysis of pectins
Monosaccharide analysis was performed following methanolysis (Manderson et al., 2005) using high-performance anionexchange chromatography with pulsed amperometric detection
(HPAEC-PAD). A Dionex DX-500 system (Dionex Corp, Sunnyvale,
CA) was used, which included a CarboPac PA-20 column operated
at 0.5 mL/min. Neutral and acidic monosaccharides were separated in a single run using a mobile phase of 10 mM NaOH for
10 min, followed by a 060 mM CH3 COONa gradient in 100 mM
NaOH for 3 min and 60120 mM CH3 COONa in 100 mM NaOH for
17 min, as modied from that reported previously (Zhao et al.,
2008). Molecular weight (MW) was determined by High Pressure
Size Exclusion chromatography (HPSEC) as reported previously (Qi,
Chau, Fishman, Wickham, & Hotchkiss, 2014). MW values reported
are weight average molar mass values unless otherwise stated.
Polydispersity was determined by weight average molar mass

(Mw)/number average molar mass (Mn). Protein analysis was as


described previously (Fishman, Chau, Cooke, Yadav, & Hotchkiss,
2009).
2.3. Degree of esterication (DE) and degree of acetylation (DAc)
analysis of pectin extracts
DE was determined by titration with 0.1 M NaOH. Samples were
analysed according to the procedure given by United Nations Food
and Agricultural Organisation (FAO, 2007) with two minor modications. The pectin powder was collected by centrifugation rather
than ltration after washing in acidied and neutral alcoholwater
mixtures. Acetic acid was determined with an enzyme kit according to the manufacturers instructions (R-Biopharm, Darmstadt,
Germany). The kit converts acetic acid to acetyl-CoA whereby
NADH is formed which is measured by the increase in light
absorbance at 340 nm.
2.4. NMR spectroscopy
NMR spectra were obtained on a Bruker Avance III spectrometer
operating at 600 MHz for 1 H and 151 MHz for 13 C; the software was
Topspin v3.2. The spectrometer was equipped with a TCI cryoprobe.
Samples were prepared as solutions (10 mg/mL) in D2 O and spectra
were recorded at 334 K. Details of 1D and 2D NMR pulse sequences
and parameters were as provided previously (Maxwell et al., 2015).
2.5. Cell culture
HT29 and DLD1 colon cancer cells were obtained from the American Type Culture Collection and maintained in Dulbeccos Modied
Eagle Medium: Nutrient Mixture F-12 (DMEM/F-12) (Invitrogen,
UK), supplemented with 10% heat inactivated foetal bovine serum
and 2% Penicillin/streptomycin (1000 U/mL) at 37 C in a humidied
atmosphere containing 5% CO2 .
The effect of pectin extracts on cell viability was determined
following the treatment of cells in 96-well plates at the doses
and times specied by replacing the medium with 100 L fresh
medium containing 10 L WST-1 reagent (Roche Diagnostics, UK)
and measuring the absorbance at 450 nm. Results were expressed
as percentage of viable cells remaining after treatment relative to
the untreated control.
2.6. Apoptosis detection by ow cytometry
HT29 cells in 6-well plates were incubated for 72 h in medium
with or without 0.5 or 1 mg/mL SBA or 0.01 g/mL staurosporine.
After incubation, the supernatant from each well was removed,
de-clumped with a CellTrics 50 M lter (Partec, UK) and transferred to a 96-well plate. Adherent cells were trypsinised to detach
all cells, then centrifuged and washed in PBS. Cell staining was
carried out using a FITC-Annexin V Apoptosis Detection Kit with
Propidium Iodide (PI) (Biolegend, UK). Cells were centrifuged and
washed twice with 1 mL cell staining buffer at 4 C, ltered and resuspended in 100 L Annexin V binding buffer, 2 L FITC-Annexin V
and 4 L PI. After incubation in the dark for 15 min, 100 L Annexin
V binding buffer was added and samples were transferred to a 96well plate along with the supernatants, controls of unstained cells,
and staining solution without cells. Data acquisition and analysis
were carried out using an EC800 Sony Eclipse ow cytometer with
EC800 version 1.3.6 software (Sony Biotechnology, UK).
2.7. Enzyme digestion of alkali-treated sugar beet pectin
The enzymes -galactosidase (3200 U/mL), endo-1,4-galactanase (1300 U/mL), -l-arabinofuranosidase (400 U/mL) and

E.G. Maxwell et al. / Carbohydrate Polymers 136 (2016) 923929

925

Table 1
Monosaccharide composition of the pectin extracts.
mol (%)

CP
SBC
SBH
SBA
SBO
SBOPG

Ratios of monosaccharides

GalA

Rha

Gal

Ara

Xyl

Glc

GlcA

Fuc

GalA:Rha

Gal:Rha

Ara:Rha

91
62
60.1
52.5
36.9
43.6

1.2
5.5
6.4
11.4
3.9
3.2

4.6
12.4
14.4
18.5
11.1
9.4

2.2
13
6.9
7.8
36
31

0.4
1.6
3.7
3
3.3
3.9

0.4
4.5
7.6
4.1
2.4
8

0.2
0.9
0.9
2.6
0.7
0.5

0.1
0.1
0.1
0.2
0.1
0.2

73.9
11.4
9.3
4.6
9.4
13.8

3.7
2.3
2.2
1.6
2.8
3

1.8
2.4
1.1
0.7
9.2
10

GalA: galacturonic acid, Rha: rhamnose, Gal: galactose, Ara: arabinose, Xyl: xylose, Glc: glucose, GlcA: glucuronic acid, Fuc: fucose.

endo-arabinase (200 U/mL) were purchased from Megazyme


(Wicklow, Ireland), diluted in MilliQ water to 5 U/mL
(-galactosidase and endo-1,4--galactanase) or 1 U/mL (-larabinofuranosidase and endo-arabinase) and ltered with a
0.2 m syringe lter. Enzymes were added to SBA at 10 mg/mL
and incubated at 40 C for 24 h under agitation. Samples were then
heated to 70 C for 10 min to halt digestion. 0.6 mL of each sample
was taken and lyophilised for NMR analysis. SBA only and enzymes
only were used as controls.

Table 2
Molecular weight (MW), polydispersity (Mw/Mn), degree of esterication (DE) and
degree of acetylation (DAc) of the pectin extracts.
MW (kDa)
CP
SBC
SBH
SBA
SBO
SBOPG

144
548
535
419
1381
594

2
11
10
7
19
15

Mw/Mn

DE (%)

DAc (%)

2.31
2.94
2.87
3.23
2.48
2.1

70.2
56.8
54.7
18
62.4
62.4

1.5
23.4
21.6
8.8
25.3
32

denotes standard deviation.

2.8. Statistical analysis


All experiments were performed with replication as indicated
and statistical analysis was performed using SPSS 16.0 software.
The statistical differences for the comparison of individual means
were determined by the students t-test and were considered signicant at P < 0.05.
3. Results and discussion
3.1. Structural features of pectin extracts
Various modied pectin extracts have been shown to have
anti-cancer activity, although the structural features of pectin
responsible for this activity have rarely been described. However,
it has previously been proposed that the components of pectin
responsible for anti-cancer activity reside in the neutral sugar-rich
RGI regions (Platt & Raz, 1992), implying that those pectins with
a higher neutral sugar content should provide greater bioactivity.
Sugar beet was chosen as a source of pectin due to its high neutral sugar content compared with citrus pectin. One citrus and ve
sugar beet pectin extracts were prepared to provide an array of
pectins with varying structural features in order to explore their
relationships with effects on cells.
All pectin extracts primarily contain galactose (Gal), arabinose
(Ara), rhamnose (Rha) and galacturonic acid (GalA) residues in
ratios shown in Table 1. Relatively high amounts of xylose and
glucose present in sugar beet pectin extracts may derive from additional hemicellulose components extracted with the pectin, such as
xyloglucan, or may possibly be sugars present in xylogalacturonan
or more complex structural features of pectin such as RGII regions
(Maxwell et al., 2012). Pectin extracted from sugar beet pulp differs
considerably from that extracted from citrus peel. SBC has a considerably higher neutral sugar and lower GalA content than CP, as
well as a lower degree of esterication (DE) and a higher degree of
acetylation (DAc) (Table 2). The GalA:Rha ratios in Table 1 show the
number of GalA residues per Rha residue, which gives an indication
of the RGI backbone to HG content, and the Gal:Rha and Ara:Rha
ratios indicate the number of neutral sugar residues attached to
the RGI backbone. These ratios indicate that SBC is almost 7-fold
richer in RGI regions than CP, although the number of neutral sugar
residues per Rha is similar, indicating similar average chain lengths.
SBH, acid-extracted at high temperature, has lower arabinose

content reduced from 13.1% to 6.9%. Further treatment with alkali


yielded SBA with a signicantly reduced DE and DAc from 55% to
18%, and 22% to 8.8%, respectively (Table 2). MW is also reduced,
coinciding with a decrease in GalA and an increase in the number of
RGI to HG regions, although Gal/Ara:Rha ratios indicate the neutral
sugar side-chains remained unchanged by this treatment.
SBO, which underwent a weak oxalic acid extraction, had more
than double the MW compared to its conventionally extracted
counterpart with a signicantly higher arabinose content of 36%
compared with the harsher, conventional extraction, which yielded
pectin with 13% arabinose. Galactose content was similar. Further
incubation of the residue from oxalic acid extraction with endopolygalacturonase (PG) to yield SBOPG considerably reduced the
MW, while the neutral sugar composition was unaffected. All pectin
samples had a similar degree of polydispersity (Table 2).
3.2. Effects of pectin extracts on colon cancer cell proliferation
The pectin extracts were assessed for effects on the proliferation of HT29 and DLD1 colon cancer cells following treatment with
each extract at 0.2, 0.5 or 1.0 mg/mL for 48 h. Only SBA signicantly
reduced HT29 cell proliferation in a dose-dependent manner with
SBA at 1.0 mg/mL reducing proliferation by 20.7 5.9% (P < 0.001).
None of the pectin extracts affected DLD1 cell proliferation after
48 h (data not shown). The effects of SBA on HT29 cell proliferation over time were investigated, together with the effects of CP
and SBC. Fig. 1A shows that HT29 cell proliferation decreased with
increased time of treatment with SBA. SBC, which did not signicantly affect cell proliferation after 48 h, also induced a signicant
decrease in cell proliferation after a longer treatment. Extending
the length of treatment with DLD1 cells also showed that SBA and,
to a lesser extent, SBC signicantly affected the proliferation of this
cell line (Fig. 1B). CP did not affect the proliferation of either cell
line.
The pectin extracts were analysed for neutral sugar and GalA
composition, DE, DAc and MW (Tables 1 and 2) in order to assess
any relationship between structure and bioactivity. Although the
unmodied acid-extracted sugar beet pectin, SBC, signicantly
affected cell proliferation, heat- and alkali-treatment of this pectin
to yield SBA signicantly increased its effect. The alkali treatment
decreased DE, suggesting that a lower DE is important for bioactivity. To investigate this further the effects of four pectin extracts

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E.G. Maxwell et al. / Carbohydrate Polymers 136 (2016) 923929

studies. Contrary to our observations with sugar beet pectin, Jackson and co-workers showed that alkali treatment of heat-treated
citrus pectin abolished its ability to induce apoptosis in LNCaP
prostate cancer cells, leading them to suggest that the ester linkages
in pectin are essential for bioactivity (Jackson et al., 2007). However,
Bergman and co-workers showed that citrus pectins with DE of 30%
and 60% reduced HT29 cell proliferation by 45% and 57%, respectively, indicating that DE had little impact on bioactivity (Bergman,
Djaldetti, Salman, & Bessler, 2010). The alkali treatment also led to
a decrease in DAc. As DAc imparts some hydrophobic character, it
may provide steric barriers and inuence how the molecules orientate themselves relative to the surroundings. A reduction of DAc
may, therefore, increase bioactivity by potentially exposing functional pectin groups to cells. However, CP also has a low DAc but
did not affect cell proliferation.
A further consequence of alkali treatment is to decrease MW by
hydrolysis of the HG backbone, generating pectin fragments with a
lower GalA:Rha ratio, indicating an increased RGI content. Therefore it is possible that lowering the MW of pectin could increase its
bioactivity. However, a lower MW per se is insufcient for bioactivity as CP has a lower MW than SBA, but had no effect on cells.
Taken together this suggests that the increased RGI content may
be responsible for the enhanced effects of SBA on cell proliferation.
This is supported by the GalA:Rha ratios shown in Table 1, which
indicate that the RGI content of SBA is twice that of SBC and SBH, and
approximately 16-fold higher than in non-bioactive CP. The Gal:Rha
and Ara:Rha ratios indicate that the neutral sugar side-chains are
on average shorter in SBA than in SBH, SBC and CP (Table 1), suggesting that a higher RGI content rather than the length of their
neutral sugar side-chains is more important for bioactivity.
To gain an understanding of how SBA affects cell proliferation
the effect of SBA on HT29 cell growth was investigated by counting the number of cells every 24 h during treatment. Fig. 1C shows
that untreated and CP-treated cells increased in number by 2- to
3-fold every 24 h, while cells treated with SBA showed a signicantly reduced growth rate. However, an increase in cell number
was still detectable, indicating that treated cells continue to proliferate, but at a signicantly reduced rate compared with untreated
cells.

3.3. Effect of SBA on HT29 cell apoptosis and the cell cycle

Fig. 1. Effects of 1 mg/mL pectin extracts on (A) HT29 cell proliferation; (B) DLD1
cell proliferation and (C) HT29 cell number. Results are mean SEM (n = 5). *, **
and *** indicates signicantly different from untreated cells at P < 0.05, P < 0.01 and
P < 0.001, respectively.

from citrus peel or apple pomace with a low DE (1040%) on HT29


cell proliferation were evaluated. None of these pectin extracts
had a signicant effect on cell proliferation (data not shown)
indicating that the low DE per se may not be sufcient for bioactivity. The effect of pectin DE has been investigated in previous

A reduction in cell proliferation is often associated with an


effect on the regulation of the cell cycle and/or an induction
of apoptosis. Therefore, the effect of SBA on these mechanisms
in HT29 cells was examined using ow cytometry. To investigate apoptosis, HT29 cells treated with SBA (0.5 or 1 mg/mL) or
the known apoptosis-inducing agent staurosporine (ST) (Bertrand,
Solary, Oconnor, Kohn, & Pommier, 1994) for 72 h were stained with
Annexin V and PI. Fig. 2 shows the effects of treatment on the percentage of cells classied as either live, undergoing early or late
apoptosis or dead. SBA treatment led to a signicant decrease in
the proportion of live cells and a signicantly higher percentage
of cells in both early and late apoptosis. Examination of the supernatant from cells incubated with SBA can also give an indication of
cell death as cells undergoing apoptosis will often detach from the
plate. Fig. 3 shows a signicant increase in the number of detached
cells following treatment with SBA. Apoptosis is often induced following an arrest of the cell cycle, therefore the effect of SBA on
the cell cycle of HT29 cells was determined. There was no effect of
SBA on the proportion of cells in each phase of the cell cycle (data
not shown). Together these results show that the observed reduction in HT29 cell proliferation with SBA treatment results from an
induction of apoptosis without cell cycle arrest and ultimately cell
detachment.

E.G. Maxwell et al. / Carbohydrate Polymers 136 (2016) 923929

Fig. 2. The proportion of HT29 cells classied as live, undergoing early or late apoptosis, or dead following treatment with SBA (0.5 and 1 mg/mL) or staurosporine (ST,
0.01 g/mL) for 72 h. Results are mean SEM (n = 5). *, ** and *** indicates signicantly different from untreated cells at P < 0.05, P < 0.01 and P < 0.001, respectively.

Fig. 3. Detached HT29 cells present in the culture medium following treatment
with SBA (0.5 and 1 mg/mL) or staurosporine (ST, 0.01 g/mL) for 72 h. Results are
mean SEM (n = 5). * and *** indicates signicantly different from untreated cells at
P < 0.05 and P < 0.001, respectively.

3.4. Characterisation of SBA following the removal of the neutral


sugar side-chains by digestion with specic enzymes
In a parallel investigation of two pectic polysaccharides from
potato, RGI and galactan, were obtained from Megazyme (Wicklow,
Ireland). It was found that 1 H and 1 H/13 C HSQC NMR experiments
could distinguish three types of Rha unit within the rhamnogalacturonan backbone (Maxwell et al., 2015). These were unsubstituted
at O-4 (no Gal), substituted at O-4 with a single Gal residue (t-Gal) or
with a longer -(1,4)-linked Gal chain (dp 2). The ratios (t-Gal):
(dp 2): (no Gal) were [2.4:0.6:1] and [1:1:1] in RGI and galactan, respectively. Furthermore, the average lengths of the (dp 2)
chains were 3 and 25 in RGI and galactan, respectively. The
same method was used to analyse the side-chain distribution in
SBA with the result (t-Gal): (dp 2): (no Gal) was [0.4:0.6:1] and
the average (dp 2) chain length was 3.5, i.e. comparable with
the chain length to potato RGI but with a higher proportion of
longer chains to single galactose stubs. The precise linkage of Ara
side-chains in SBA (to backbone rhamnose or galactan side-chain)
could not be determined but using cross-sections of the HSQC NMR
spectrum it was found that the intensities of the C1 peaks for tAra (1 H/13 C = 5.14/109.8 ppm) and -(1,5)-linked Ara (5.07/110.1)
were practically equal. The high proportion of t-Ara implies that Ara
side-chains were mostly either single stubs or else very short.

927

In order to investigate the role of the neutral sugar side-chains


of SBA in mediating the activity towards HT29 cells, Gal and/or
Ara side-chains were removed using combinations of commercial enzymes and the effects of treatment conrmed by NMR
analysis. Galactan side-chains were cleaved by treatment of SBA
with a combination of -galactosidase and endo-galactanase to
generate SBA-gal. Fig. 4 shows that the characteristic H1 (a)
and H4 (b) signals of a -(1,4)-linked galactan chain evident in
SBA that was incubated under the same conditions but without enzymes (SBA-ne, Fig. 4A) have been signicantly reduced
in SBA-gal (Fig. 4B). The neutral sugar side-chain content of the
enzyme-digested SBAs was determined from the NMR analysis
and is shown in Table 3. In addition to removing approximately
90% of the t-Gal-(1 4)-Gal and (1,4)-Gal-(1 4)-Gal content,
the endo-galactanase/-galactosidase treatment also removed
approximately 70% of the (1,5)-Ara-(1 5) and 64% t-Ara-(1 3)
content. However, a number of new signals are evident in the
anomeric region of SBA-gal. These include - and -GalA reducing
end signals, from both free GalA (d, h) and GalA that is 4-linked
to another GalA (c, g) representing the reducing end of an oligogalacturonide. Another new signal (i) corresponds to H4 of a
non-reducing end GalA unit. Free galactose was produced (signals
H-1 (e) and H-1 (h)) and the H-1 signal (g) of t-Gal (linked to
O-4 of Rha) could also be seen. It should be noted that the signals
labelled (g) and (h) have both been assigned to two different sugar
units as both are correlated to two different 13 C shifts in the HSQC
spectrum.
Although t-Gal units linked to Rha were also present in SBA-ne,
the Rha Me signals show that the ratio of substituted to unsubstituted units did not change in SBA-gal (or indeed with any of
the enzyme treatments). It is therefore concluded that the endogalactanase/-galactosidase treatment leaves at least a Gal stub
at every position which was occupied in the SBA RGI regions
by either a stub or a longer galactan chain. The observation of
free GalA and oligo-GalAs in SBA-gal shows that there must be
an unexpected polygalacturonase activity present in the commercial enzymes used (separate experiments showed that the
impurity was associated with the galactanase only). Therefore the
galactanase/galactosidase treatment induced a removal of galactan
side-chains in the RGI regions to leave mainly Gal stubs and, unexpectedly, a partial depolymerisation of the GalA HG regions. This
depolymerisation was also present in SBA incubated with all four
enzymes (SBA-all).
The arabinan side-chains were largely removed using a combination of -l-arabinofuranosidase and endo-arabinase, yielding
SBA-ara. The only new signals evident in SBA-Ara (Fig. 4C) were (j)
and (k) corresponding to H1 -Arap and H1 -Arap, respectively,
which are from released free arabinose existing in the pyranose
form rather than the furanose form found in arabinan side-chains.
The 1 H spectrum of SBA-ara (Fig. 4C) shows that an approximately
equivalent amount of Ara monosaccharide was released as from
SBA treated with all four enzymes (SBA-all, Fig. 4D) leading to
the assumption that the enzymes removed 100% (1,5)-Ara-(1 5)
units and 64% of t-Ara-(1 3) units (Table 2). However, there is
no information from the NMR spectra on how the remaining Ara is
linked to the polysaccharide so it is unknown whether Ara is linked
to Rha or Gal residues. The 1 H spectrum of SBA-all (Fig. 4D) appears
to be a simple summation of SBA-gal and SBA-ara without any evidence of major additional changes arising from a synergistic effect
of the enzyme combination.
3.5. Effects of enzyme-digested SBA on HT29 cell proliferation
The effect of the removal of the neutral sugar side-chains from
SBA on the proliferation of HT29 cells was investigated. Fig. 5
shows that treatment with SBA-ne signicantly reduced HT29 cell

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E.G. Maxwell et al. / Carbohydrate Polymers 136 (2016) 923929

Fig. 4. 600 MHz 1 H NMR spectra of enzyme-digested SBAs in D2 O at 334 K. (A) SBA-ne (control); (B) SBA-gal; (C) SBA-ara; (D) SBA-all. Signal assignments (residue involved
is shown in bold): (a) H1 -Gal(1 4)--Gal; (b) H4 -(1,4)-Gal; (c) H1 GalA(1 4)--GalARE ; (d) H1 -GalA; (e) H1 -Gal; (f) H1 -Rha; (g) H1 GalA(1 4)--GalARE
and H1 t--Gal-(1 4)--Rha; (h) H1 -GalA and H1 -Gal; (i) H4 -GalANR ; (j) H1 -Arap; (k) H1 -Arap.

Fig. 5. Effects of enzyme-digested SBAs on HT29 cell proliferation. Results are


mean SEM (n = 5). *, ** and *** indicates signicantly different from untreated cells
at P < 0.05, P < 0.01 and P < 0.001, respectively.

proliferation in a time-dependent manner to the same extent as


untreated SBA (data not shown). SBA-ara treatment also reduced
HT29 cell proliferation but the effect was signicantly decreased
by approximately 715% (P < 0.01) compared with SBA-ne

indicating that, although not essential, the arabinan in the neutral


sugar side-chains plays a role in the bioactivity of SBA.
SBA-gal treatment did not signicantly affect HT29 cell proliferation after 72 h but after 96 and 120 h cell proliferation was
reduced. However, this reduction in proliferation was still signicantly less than that produced by SBA-ne (P < 0.001). This indicates
that the galactan side-chains play a signicant role in mediating
the effects of SBA on cell proliferation. However, it should be noted
that the galactanase/galactosidase treatment of SBA also induced
a partial depolymerisation of the GalA HG regions. This could suggest that the HG backbone may also play a role in the bioactivity
of SBA; however, this is unlikely since a signicant amount of HG
remains in SBA-gal. It is possible that the reduction in cell proliferation with prolonged treatment with SBA-gal may be due to
the remaining 413% galactan chains. However, it is also possible
that the HG or RG backbone of SBA could be a secondary bioactive component with less signicant effects that take longer to
detect. The two distinct phases observed in the effects of SBA-gal
on HT29 cell proliferation strongly suggest that there may be more
than one mechanism of action. Previous studies have suggested
distinct bioactive roles for HG pectin structures. In a recent study
the importance of HG in mediating the anti-proliferative effects
of RGI on DLD1 colon cancer cells was demonstrated (Maxwell
et al., 2015). Polygalacturonic acid has also been reported to induce
apoptosis in rat pituitary tumour cells (Attari, Sepehri, Delphi,
& Goliaei, 2009) and Liu and co-workers showed pentamers of
GalA to be active against inammation and carcinogenesis in a

Table 3
Neutral sugar side-chain content of enzyme-digested SBA.
Sample

Treatment

% 1,4-galactan contenta

% 1,5-linked arabinan contentb

% Terminal Ara contentc

SBA-ne
SBA-ara
SBA-gal
SBA-all

Control
-l-Arabinofuranosidase + endo-arabinase
-Galactosidase + endo-galactanase
All four enzymes

100
100
413
420

100
0
30
0

100
36
36
36

a
Measured by comparison of C1 Gal-(1 4)-Gal signal intensities from HSQC cross-peak at ( 4.61/106.9) and C4 signal intensities in 4-linked Gal units, cross-peak (
4.15/80.15).
b
Measured by comparison of C1 (1,5)-Ara-(1 5) signal intensities from HSQC cross-peak at ( 5.07/110.1).
c
Measured by comparison of C1 t-Ara-(1 3) signal intensities from HSQC cross-peak at ( 5.14/109.8).

E.G. Maxwell et al. / Carbohydrate Polymers 136 (2016) 923929

mouse model of colitis-associated CRC (Liu et al., 2010). Gao and


co-workers showed that the backbone of ginseng RGI, depleted
of all neutral sugar side-chains, inhibited haemagglutination,
albeit this required higher concentrations than for the intact RGI
(Gao et al., 2013).
The removal of the arabinan side-chains from SBA-gal to yield
SBA-all did not signicantly alter the effect on cell proliferation
compared with SBA-gal indicating that the arabinan side-chains
alone do not mediate the effects of SBA on cells. However, since
the selective removal of arabinan to generate SBA-ara moderately
impacted the effects of SBA on cell proliferation, this suggests that
the arabinan may assist in the presentation of galactan to receptors,
as has been noted previously (Gao et al., 2013). Taken together these
data indicate that the conformation adopted by the neutral sugar
side-chains in cooperation with the HG/RG backbone is required
for optimal bioactivity.
4. Conclusion
Sugar beet pectin extract demonstrated weak but signicant
anti-proliferative activity towards colon cancer cells highlighting
a potential novel exploitation of the by-product from sugar beet
pulp. Alkali treatment of the sugar beet pectin extract signicantly
increased its effects on cells by inducing apoptosis without an effect
on the cell cycle. Alkali treatment increased the ratio of RGI to HG
content suggesting that the neutral sugar side-chains in the RGI
regions are important for bioactivity. This was conrmed following their removal with commercial enzymes: this showed that the
galactan side-chains play a prominent role in bioactivity, which
is weakly enhanced by the presence of arabinan side-chains. The
almost complete removal of the neutral sugar side-chains did not
abolish the effects on cells suggesting that the RGI/HG backbone
may also be bioactive, perhaps by an alternative mechanism. In conclusion, galactan and arabinan side-chains together with an RGI/HG
backbone cooperate to exert optimal bioactivity, possibly by adopting a conformation that maximises the availability of the galactan
chains for cellular interaction. By providing greater insight into the
structural requirements for pectin bioactivity, this study highlights
the need for detailed mechanistic investigations at the molecular
and cellular level for understanding the bioactive roles of MPs that
have been extensively characterised.
Acknowledgements
This work was supported by the International Pectin Producers
Association and by the UK Biotechnology and Biological Sciences
Research Councils core strategic grant to the Institute of Food
Researchs Gut Health and Food Safety (BB/J004529/1) and Food
and Health (BB/J004545/1) programmes. We are also grateful to
Roy Bongaerts for his assistance with the ow cytometry analyses
and to Claus Rolin from CPKelco for his advice in the selection and
preparation of sugar beet pectin extracts.
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