Beruflich Dokumente
Kultur Dokumente
Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
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
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a r t i c l e
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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
924
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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.
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
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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.
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.
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928
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.
Table 3
Neutral sugar side-chain content of enzyme-digested SBA.
Sample
Treatment
% 1,4-galactan contenta
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).
929
Buchholt, H. C., Christensen, T. M. I. E., Fallesen, B., Ralet, M. C., & Thibault, J. F.
(2004). Preparation and properties of enzymatically and chemically modied
sugar beet pectins. Carbohydrate Polymers, 58, 149161.
Cheng, H., Li, S., Fan, Y., Gao, X., Hao, M., Wang, J., et al. (2011). Comparative studies
of the antiproliferative effects of ginseng polysaccharides on HT-29 human
colon cancer cells. Medical Oncology, 28(1), 175181.
Fan, Y. Y., Cheng, H. R., Li, S. S., Wang, J., Liu, D., Hao, M. A., et al. (2010).
Relationship of the inhibition of cell migration with the structure of ginseng
pectic polysaccharides. Carbohydrate Polymers, 81(2), 340347.
FAO. (2007). Compendium of food additive specications: Joint FAO/WHO Expert
Committee on Food Additives: 68th meeting 2007. Rome: FAO.
Fishman, M. L., Chau, H. K., Cooke, P. H., Yadav, M. P., & Hotchkiss, A. T. (2009).
Physico-chemical characterization of alkaline soluble polysaccharides from
sugar beet pulp. Food Hydrocolloids, 23(6).
Gao, X., Zhi, Y., Sun, L., Peng, X., Zhang, T., Xue, H., et al. (2013). The inhibitory
effects of a rhamnogalacturonan I (RG-I) domain from ginseng pectin on
galectin-3 and its structureactivity relationship. Journal of Biological
Chemistry, 288(47), 3395333965.
Guess, B. W., Scholz, M. C., Strum, S. B., Lam, R. Y., Johnson, H. J., & Jenrich, R. J.
(2003). Modied citrus pectin (MCP) increases the prostate-specic antigen
doubling time in men with prostate cancer: A phase II pilot study. Prostate
Cancer and Prostatic Diseases, 6, 301304.
Inohara, H., & Raz, A. (1994). Effects of natural complex carbohydrate (citrus
pectin) on murine melanoma cell properties related to galectin-3 functions.
Glycoconjugate Journal, 11(6), 527532.
Jackson, C. L., Dreaden, T. M., Theobald, L. K., Tran, N. M., Beal, T. L., Eid, M., et al.
(2007). Pectin induces apoptosis in human prostate cancer cells: Correlation of
apoptotic function with pectin structure. Glycobiology, 17(8), 805819.
Li, Y. H., Niu, Y. B., Wu, H. J., Sun, Y., Li, Q. A., Kong, X. H., et al. (2010). Modied
apple polysaccharides could induce apoptosis in colorectal cancer cells. Journal
of Food Science, 75(8), H224H229.
Liu, H. Y., Huang, Z. L., Yang, G. H., Lu, W. Q., & Yu, N. R. (2008). Inhibitory effect of
modied citrus pectin on liver metastases in a mouse colon cancer model.
World Journal of Gastroenterology, 14(48), 73867391.
Liu, L., Li, Y. H., Niu, Y. B., Sun, Y., Guo, Z. J., Li, Q. A., et al. (2010). An apple
oligogalactan prevents against inammation and carcinogenesis by targeting
LPS/TLR4/NF-kappa B pathway in a mouse model of colitis-associated colon
cancer. Carcinogenesis, 31(10), 18221832.
Manderson, K., Pinart, M., Tuohy, K. M., Grace, W. E., Hotchkiss, A. T., Widmer, W.,
et al. (2005). In vitro determination of prebiotic properties of oligosaccharides
derived from an orange juice manufacturing by-product stream. Applied and
Environmental Microbiology, 71(12), 83838389.
Maxwell, E. G., Belshaw, N. J., Waldron, K. W., & Morris, V. J. (2012). Pectin An
emerging new bioactive food polysaccharide. Trends in Food Science &
Technology, 24(2), 6473.
Maxwell, E. G., Colquhoun, I. J., Chau, H. K., Hotchkiss, A. T., Waldron, K. W., Morris,
V. J., et al. (2015). Rhamnogalacturonan I containing homogalacturonan
inhibits colon cancer cell proliferation by decreasing ICAM1 expression.
Carbohydrate Polymers, 132, 546553.
Nangia-Makker, P., Hogan, V., Honjo, Y., Baccarini, S., Tait, L., Bresalier, R., et al.
(2002). Inhibition of human cancer cell growth and metastasis in nude mice by
oral intake of modied citrus pectin. Journal of the National Cancer Institute,
94(24), 18541862.
Olano-Martin, E., Rimbach, G. H., Gibson, G. R., & Rastall, R. A. (2003). Pectin and
pectic-oligosaccharides induce apoptosis in in vitro human colonic
adenocarcinoma cells. Anticancer Research, 23, 341346.
Pienta, K. J., Naik, H., Akhtar, A., Yamazaki, K., Replogle, T. S., Lehr, J., et al. (1995).
Inhibition of spontaneous metastasis in a rat prostate-cancer model by
oral-administration of modied citrus pectin. Journal of the National Cancer
Institute, 87(5), 348353.
Platt, D., & Raz, A. (1992). Modulation of the lung colonization of B16-F1
melanoma-cells by citrus pectin. Journal of the National Cancer Institute, 84(6),
438442.
Qi, P., Chau, H. K., Fishman, M. L., Wickham, E. D., & Hotchkiss, A. T. (2014).
Investigation of molecular interactions between b-lactoglobulin and sugar
beet pectin by multi-detection HPSEC. Carbohydrate Polymers, 107, 198
208.
Sun, R. C., & Hughes, S. (1999). Fractional isolation and physico-chemical
characterization of alkali-soluble polysaccharides from sugar beet pulp.
Carbohydrate Polymers, 38, 273281.
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. Phytotherapy Research, 24(7), 982989.
Watson, A. J., & Collins, P. D. (2011). Colon cancer: A civilization disorder. Digestive
Diseases, 29(2), 222228.
Yan, J., & Katz, A. (2010). PectaSol-C modied citrus pectin induces apoptosis and
inhibition of proliferation in human and mouse androgen-dependent and
-independent prostate cancer cells. Integrative Cancer Therapies, 9(2),
197203.
Zhao, Z. Y., Liang, L., Fan, X., Yu, Z., Hotchkiss, A. T., Wilk, B. J., et al. (2008). The role
of modied citrus pectin as an effective chelator of lead in children
hospitalized with toxic lead levels. Alternative Therapies in Health and Medicine,
14(4), 3438.