Characterization of a novel polysaccharide with anti-colon cancer activity from Lactobacillus helveticus MB2-1

Article abstract of DOI:10.1016/j.carres.2014.12.014

The present study aimed at investigating the potential anti-colon cancer activity of three purified exopolysaccharides fractions (LHEPS-1, LHEPS-2 and LHEPS-3) from the Lactobacillus helveticus MB2-1. The experimental evidence showed that LHEPS-1 significantly inhibited cell proliferation of human colon cancer Caco-2 cells in both time- and concentration-dependent manners. In contrast, no significant improvements of the inhibitory effects of LHEPS-2 and LHEPS-3 on Caco-2 cells were observed with increasing sample concentrations or prolonged incubation time. Furthermore, the structure of LHEPS-1 was elucidated using methylated analysis, gas chromatography-mass spectroscopy (GC-MS) and nuclear magnetic resonance spectroscopy (NMR), including one- and two-dimensional nuclear magnetic resonance (1D and 2D NMR). Results indicated that the LHEPS-1 consisted of a decasaccharide repeating unit with the following structure (n≈122): Our results suggested that the LHEPS-1 produced by L. helveticus MB2-1 might be suitable for using as natural anti-colon cancer drugs and functional foods ingredients.

Full text of DOI:10.1016/j.carres.2014.12.014

Accepted Manuscript
Characterization of a novel polysaccharide with anti-colon cancer activity from
Lactobacillus helveticus MB2-1
Wei Li, Weizhi Tang, Juan Ji, Xiudong Xia, Xin Rui, Xiaohong Chen, Mei Jiang,
Jianzhong Zhou, Mingsheng Dong
PII:
S0008-6215(14)00466-2
10.1016/j.carres.2014.12.014
CAR 6916
DOI:
Reference:
To appear in: Carbohydrate Research
Received Date: 20 November 2014
Revised Date: 26 December 2014
Accepted Date: 28 December 2014
Please cite this article as: Li W, Tang W, Ji J, Xia X, Rui X, Chen X, Jiang M, Zhou J, Dong M,
Characterization of a novel polysaccharide with anti-colon cancer activity from Lactobacillus helveticus
MB2-1, Carbohydrate Research (2015), doi: 10.1016/j.carres.2014.12.014.
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ACCEPTED MANUSCRIPT
Characterization of a novel polysaccharide with anti-colon cancer activity from
Lactobacillus helveticus MB2-1
Wei Lia, Weizhi Tanga, Juan Jia, Xiudong Xiab, Xin Ruia, Xiaohong Chena, Mei Jianga, Jianzhong
Zhoub, Mingsheng Dong a,*
aCollege of Food Science and Technology, Nanjing Agricultural University, Nanjing, Jiangsu 210095,
P.R. China.
bInstitute of Agro-Product Processing, Jiangsu Academy of Agricultural Sciences, Nanjing, Jiangsu
210095, P.R. China.
*Corresponding author:
Dr Mingsheng Dong*
College of Food Science and Technology
Nanjing Agricultural University
1 Weigang Road, Nanjing, Jiangsu, PR China
Tel: +86 25 84396989
Fax: +86 25 84399090
E-mail address: dongms@njau.edu.cn
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ABSTRACT
The present study aimed at investigating the potential anti-conlon cancer activity of three purified
exopolysaccharides fractions (LHEPS-1, LHEPS-2 and LHEPS-3) from the Lactobacillus helveticus
MB2-1. The experimental evidence showed that LHEPS-1 significantly inhibited cell proliferation of
human colon cancer Caco-2 cells in both time- and concentration-dependent manners. In contrast, no
significant improvements of the inhibitory effects of LHEPS-2 and LHEPS-3 on Caco-2 cells were
observed with increasing sample concentrations or prolonged incubation time. Furthermore, the
structure of LHEPS-1 was elucidated using methylated analysis, gas chromatography–mass
spectroscopy (GC–MS) and nuclear magnetic resonance spectroscopy (NMR), including one- and two-
dimensional nuclear magnetic resonance (1D and 2D NMR). Results indicated that the LHEPS-1
consisted of a decasaccharide repeating unit with the following structure (n ≈ 122)：
Our results suggested that the LHEPS-1 produced by L. helveticus MB2-1 might be suitable for using
as natural anti-conlon cancer drugs and functional foods ingredients.
Keywords: Lactobacillus helveticus MB2-1; Exopolysaccharides; Anti-conlon cancer activity;
Structural characterization; Methylated analysis; Nuclear magnetic resonance spectroscopy.
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1. Introduction
Colon cancer is a major health problem of global concern and the third leading cause of
cancer-related mortality throughout the world.1 Usually, colon cancer is treated by surgical resection
and chemotherapy. Both 5-Fluorouracil (5-Fu) and oxaliplatin are the most effective cytotoxic agents
used in colon cancer treatment. However, some undesirable effects, including nausea, vomiting and
fatigue, declined the quality of patients’ lives.2,3 In addition, inherent and acquired resistances to the
drugs limited their applications in colon cancer chemotherapy.4 One obstacle ahead is the high risk of
locoregional relapse and distant metastasis.5 Therefore, it is important to search for new alternative
strategies for the prevention and treatment of colon cancer.
Polysaccharides are important components derived from plants, animals and microorganisms, and
have been attracted intensive attentions in the medical and food research fields due to their
immunmodulatory and anticancer activities.6-8 Among microbial polysaccharide, those
exopolysaccharides (EPSs) produced by lactic acid bacteria (LAB) are receiving increasing attention
because of the food-grade status of these microorganisms coupled with their long historical
application.9,10 EPSs, one of the most important secondary metabolites produced by LAB, have also
been correlated with multiple pharmacological activities, such as immunomodulatory, cholesterol
lowering, antioxidant and anticancer activities.11-13 Thus, EPSs derived from LAB might serve as ideal
candidate for prevention and therapeutics of colon cancer.
The anticancer activity of polysaccharides including EPSs is affected by many factors such as
monosaccharide composition, molecular weight (Mw), structure of the polymeric backbone and side
chains, and even number of branching points.14,15 The moderate size of polysaccharides has been
proved to be favorable for anticancer activity.16 Other structural properties such as the presences of
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mannose and glucose residues and existing of branching points in repeating unit are also conducive to
increase their anticancer activity.17-19 Recently, we reported that the production, purification,
characterization and antioxidant activities in vitro of EPS from Lactobacillus helveticus MB2-1. The
crude EPS obtained from the whey medium was purified by anion exchange and gel filtration
chromatography, affording three fractions of LHEPS-1, LHEPS-2 and LHEPS-3.20 The Mw of three
purified EPS fractions (LHEPS-1, LHEPS-2 and LHEPS-3) were estimated to be 2.08 × 105, 2.04 × 105
and 2.01 × 105 Da, respectively, and they were composed of galactose, glucose and mannose with a
molar ratio of 1.33:2.75:1.00, 1.00:1.43:9.34 and 1.17:1.00:2.96, respectively. In vitro antioxidant
activities assays demonstrated that LHEPS-1, LHEPS-2 and LHEPS-3 had strong scavenging activities
against free radicals.20 In addition, we found that all the LHEPS fractions, especially LHEPS-2 exerted
high inhibitory activity in vitro on human gastric cancer BGC-823 cells. Furthermore, we elucidated
the
fine
structure
of
the
purified
LHEPS-2,
which
was
a
→4,6-
D
-Manp-(1→6)-
D
-Manp-(1→4)-D-Galp-(1→
backbone
with
a
single
T-D
-Glcp-(1→4)-
D
-Manp-(1→6)-D-Glcp-(1→ side-branching in O-6 of →4,6-D
-Manp-(1→ residue.21
These results indicated that EPSs from L. helveticus MB2-1 had potential application as natural
anticancer drugs or functional food ingredients for gastric cancer therapies. However, little information
is presented regarding the anticancer activity of EPS fractions against human colon cancer Caco-2 cells.
Thus, in the present study, the in vitro anticancer effects were determined using Caco-2 cells for three
purified EPS fractions from L. helveticus MB2-1. In addition, for a better understanding and evaluation
of the structural characteristic and high anticancer activity of EPS fractions, the structure of neutral
LHEPS-1 was elucidated by using methylated analysis, gas chromatography–mass spectroscopy
(GC–MS), and nuclear magnetic resonance spectroscopy (NMR), including one- and two-dimensional
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nuclear magnetic resonance (1D and 2D NMR).
2. Results and discussion
2.1. Assay of inhibitory effects of LHEPS on the growth of human colon cancer Caco-2 cells
It has been reported that some polysaccharides exerted anticancer activity.22,23 However, only a few
studies investigated the anticancer activities of the polysaccharides of LAB.21,24 In the present study,
therefore, the anticancer activity of LHEPS in vitro was evaluated. The growth inhibitory effects on
colon cancer Caco-2 cells were measured using MTT assay after the cells were treated with increasing
concentrations (0, 50, 100, 200, 400 and 600 µg/mL) of the three purified LHEPS fractions and 5-Fu
(50 µg /mL) for 24, 48 and 72 h, respectively. As shown in Fig. 1, LHEPS-1 significantly inhibited the
growth of Caco-2 cells in both time- and dose-dependent manners within 72 h of incubation (p < 0.05).
However, the inhibitory effects of LHEPS-2 and LHEPS-3 on Caco-2 cells did not significantly
affected (p > 0.05) by the different sample concentrations (50−600 µg/mL) at 72 h of incubation, and
no significant differences were observed (p > 0.05) for the inhibitory effects of LHEPS-2 and LHEPS-3
at the treatments of 24, 48 and 72 h when the sample concentrations were higher than 400 µg/mL. At a
concentration of 600 µg/mL and 72 h incubation, the inhibitory effect of LHEPS-1 on Caco-2 cells
reached the highest (inhibition rate, 56.34%), although slightly lower than that of 5-Fu (79.98%) at 50
µg/mL. However, the level of LHEPS-1-mediated cytotoxicity in normal colon HCoEpiC cells was not
dramatic. Even at the high dose of 600 µg/mL, the cytotoxicity induced by a 72 h incubation of normal
colon cells with LHEPS-1 was only 4% (data not shown). Apparently, LHEPS-1 possessed a relative
higher anti-colon cancer activity in vitro than LHEPS-2 and LHEPS-3 whereas it was no cytotoxic
effect on normal colon cells. These results suggested that LHEPS-1 might be a potential natural drug or
health food for colon cancer therapeutics. Therefore, the fine structure characterization of LHEPS-1
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was further investigated in the present study, which was based on our previous data.20
Fig. 1
2.2. Methylation analysis of LHEPS-1
After methylation for four times, the hydroxyl group absorption at (3700–3100 cm−1) in FT-IR
disappeared, indicating the completeness of methylation. The individual peaks of the methylated
monosaccharide residues and fragmentation patterns were identified by their retention time in GC and
by comparison with literature MS patterns.25-29 The identification and the related linkage patterns are
shown in Table 1, and the molar ratio of monosaccharide residues was calculated according to the peak
areas and response factor of the total ion chromatogram (TIC) in Agilent GC–MS system. Methylation
analysis of the LHEPS-1 showed the presences of ten components, namely 2,4,6-Me3Glcp;
2,3,4-Me3Manp; 3,4,6-Me3Glcp; 2,3,6-Me3Glcp; 2,3,4,6-Me4-Glcp; 2,3,4,6-Me4-Galp; 2,3,6-Me3Galp;
2,3,4-Me3Glcp; 2,3-Me2Manp and 2,4-Me2Glcp. Based on the data available in Table 1, LHEPS-1 was
composed
of
(1→3)-linked-D-Glcp,
(1→6)-linked-
D
-Manp,
(1→2)-linked-
D
-Glcp,
(1→4)-linked-
D
D
-Glcp,
(1→)-linked-
D
-Glcp,
(1→)-linked-
D
-Galp,
(1→4)-linked-D
-Galp,
(1→6)-linked-
-Glcp, (1→4,6)-linked-D-Manp and (1→3,6)-linked-D-Glcp, in molar ratio of 1.25,
1.21, 1.07, 1.00, 1.31, 1.14, 1.19, 1.26, 1.16 and 1.33 (about 1:1:1:1:1:1:1:1:1:1), respectively.
Furthermore, methylation linkage analysis of LHEPS-1 indicated that the residues of branch structure
were linked to the oxygen of C-6 positions of (1→4)-linked-D-Manp and (1→3)-linked-D-Glcp,
respectively. In addition, the molar ratios of D-Glcp, D-Manp and D-Galp residues in fully methylated
analysis were respectively 3.10:1.02:1.00, which was in good agreement with the monosaccharide
composition of LHEPS-1 obtained from GC analysis.20
Table 1
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2.3. 1D and 2D NMR analysis of LHEPS-1
In order to get more insight into the structural information of the LHEPS-1 from L. helveticus
MB2-1, both detailed 1D and 2D NMR studies on the LHEPS-1 were performed. The general
representative peaks of sugar residue were ascribed as follows: anomeric proton (H-1) signals of
glycosides were assigned to δ 4.6–5.6 ppm, and H-2, H-3, H-4, H-5 and H-6 from glycosidic ring were
assigned to δ 3.5–4.5 ppm. The signal for residual D2O in the sample occurred at the anomeric region
in the spectrum at δ 4.7 ppm. All the signals were assigned based on component analysis, linkage
analysis and literature values.20,21,26-28,30 1D NMR spectra of LHEPS-1 are shown in Fig. 2A and B. Ten
obvious chemical shift signals of anomeric protons were found at δ 5.46, 5.33, 5.29, 5.26, 5.24, 5.23,
4.71, 4.70, 4.66 and 4.64 ppm in 1H NMR spectrum (Fig. 2A). The 13C NMR (Fig. 2B) chemical shifts
in the area of anomeric carbon atoms also suggested ten kinds of α- or β-linkages existed for the
D-Glcp, D-Manp and D-Galp residues. The C-1 signals of the residues were detectable at δ 105.72,
105.68, 105.38, 105.33, 104.58, 104.46, 104.29, 102.90, 100.73, 100.62 ppm, respectively. It was
evident from Fig. 2 that there were three regions of H-1/C-1 signals corresponding to α- anomers (H-1
5.2-5.6 ppm, C-1 100-105 ppm) and beta-anomers (H-1 4.6-4.8 ppm, C-1 105-107 ppm). Combined
with the result of monosaccharide analysis, the A–F residues might reflect six different α-type
glycosidic bonds of sugar residues, and G–J residues might indicate four different β-type glycosidic
bonds of sugar residues. Molar proportion of the ten residues was about
1.00:0.86:1.04:0.83:0.84:1.20:1.20:1.18:1.15:1.19, which estimated by the ratio of peak area of the
integration of the H-1 signal for A-J residues. However, the corresponding H chemical shifts of
residues A–J for other positions were packed in the range of δ 3.5–4.5 ppm and even difficult to
discriminate.
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Fig. 2
1
The complete assignments of the H and 13C chemical shifts of the LHEPS-1 were carried out by
means of the 2D HSQC, COSY, TOCSY, HMBC and NOESY experiments based on FTIR analysis
(data not shown), methylation analysis and the data from literatures.18,20,21,25–32 The single-bond
correlations between the protons and the corresponding carbons obtained from HSQC spectra (Fig. 2C)
of LHEPS-1 in D2O enabled all the 13C NMR to be assigned. The C-1 signals at δ105.72, 105.68,
105.38, 105.33, 104.58, 104.46, 104.29, 102.90, 100.73, 100.62 ppm could be assigned to the J, I, H,
G, B, E, F, A, C and D, respectively. These signals cross link to the proton signals at chemical shifts δ
4.64, 4.66, 4.70, 4.71, 5.33, 5.24, 5.23, 5.46, 5.29 and 5.26, respectively. The results from COSY (Fig.
3A), based on stepwise magnetization transfers from the anomeric protons, could help to assign the
chemical shifts of other protons. The TOCSY spectrum provided all intraresidue connectivity of all
protons of the A–J, respectively, as shown in Fig. 3C. From the HMBC spectrum, the linkage of
residue was obtained. Inspection of the NOESY spectrum showed on the F and I H-1 track NOE
connectivity with D and J H-6 in agreement with the F-(1→6)-D and I-(1→6)-J linkage, respectively.
In addition, inter-residue NOE cross-peaks between D H-1 and A H-6 supported the D-(1ꢀ6)-A
linkage, and between J H-1 and B H-6 supported the J-(1ꢀ4)-B linkage, which indicated that the
existing two branching points and the branching position of residue D and J, respectively (data not
shown). Thus a summarized of the information from the 1D and 2D NMR spectra gave a complete
assignment of all the linkage patterns which was shown in Table 2. According to the results, it has been
considered that residues A, B, E, F, G and I were substituted at C-6, C-4, C-3, C-6, C-4 and C-2
position, respectively. Thus, residues A, B, E, F, G and I belonged to (1→6)-linked-D-Manp,
(1→4)-linked-
D
-Galp, (1→3)-linked-
D
-Glcp, (1→6)-linked-D-Glcp, (1→4)-linked-D-Glcp and
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(1→2)-linked-
D
-Glcp, respectively. Residues D and J were assigned to (1→4,6)-linked-D-Manp and
(1→3,6)-linked-D-Glcp containing branching point, respectively. In addition, residues C and H were
determined as α-terminal-D-Glcp and β-terminal-D-Galp, respectively. On the basis of above
mentioned results, the suggested repeat unit of LHEPS-1 was concluded (Fig. 4). In this study, the
value of ‘n’ that represented the repeated structural unit of LHEPS-1 was calculated using the following
formula. The value of ‘n’ = (Mw of LHEPS-1) / [(Mw of monosaccharide × the number of
monosaccharide residues in the repeating unit) – (Mw of water molecule × the number of glycosidic
bonds in the repeating unit)]. In our study, Mw of LHEPS-1, Mw of monosaccharide, the number of
monosaccharide residues in the repeating unit, Mw of water molecule and the number of glycosidic
bonds in the repeating unit were 2.08 × 105 Da, 180 Da, 10, 18 Da and 11, respectively.20 Therefore,
the value of ‘n’ that repeat structural unit of LHEPS-1 was approximately equal to 122. In the current
study, a neutral EPS fraction (LHEPS-1) was derived from L. helveticus MB2-1, and its structure was
identified as heteropolysaccharide.
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Table 2
Fig. 3
Fig. 4
In general, EPS from Lactobacillus spp. are divided into heteropolysaccharide and
homopolysaccharide as other bacterial EPS.33,34 Homopolysaccharides are composed of one kind of
monosaccharide (ie. glucose or galactose), whereas several different types of monosaccharides
constitute heteropolysaccharides. Heteropolysaccharides have a great variability in structures. EPS
backbones of Lactobacillus spp. (L. helveticus, L. rhamnosus and L. delbrueckii bulgaricus) have
repeated units composed of seven monosaccharides, where glucose, galactose and mannose are the
main sugar residues.30,35 Heteropolysaccharides are often linear or branched with different types of
linkages. Their structures are also complex depending on the principal monosaccharide, for example,
glucogalactan or galactoglucan and depending on the ratios of each sugar they are named as
glucomannogalactan or galactoglucomannan. Obviously, LHEPS-1 was a kind of moderate branching
glucomannogalactan.
2.4. Structure-anticancer activity correlation
It has been reported that the anticancer effects of polysaccharides were influenced by their chemical
composition and structure of the polymeric backbone.19 According to our previously study, although
the Mw of LHEPS-1 was similar with that of LHEPS-2 and LHEPS-3, the chemical composition and
structure of LHEPS-1 was greatly differed from that of LHEPS-2 and LHEPS-3. LHEPS-1 exhibited
more average molar ratios between the composed monosaccharide than LHEPS-2 and LHEPS-3.20
Moreover, differed from LHEPS-2 and LHEPS-3, LHEPS-1 possessed more side chains which
probably facilitated the exposure of active groups and/or its spatial structure. In our study, the degrees
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of branching of the different LHEPS fractions were calculated using the following formula. The degree
of branching = the number of branching point / the number of monosaccharide residues in the repeating
unit. In our study, the number of branching points of LHEPS-1, LHEPS-2 and LHEPS-3 were 2, 1 and
1, respectively. The number of monosaccharide residues in the repeating unit of LHEPS-1, LHEPS-2
and LHEPS-3 were 10, 6 and 7, respectively20, 21. Thus, the degree of branching was 20% for LHEPS-1
which was higher than LHEPS-2 (17%) and LHEPS-3 (14%), according to the molar ratios of branched
to linear sugar residues. Therefore, structural characteristics such as the average molar ratios of
monosaccharide composition, moderate Mw, configuration and high degree of branching might
attribute LHEPS-1 a high anti-colon cancer activity. However, the mechanism for the inhibitory
activity of cancer cell growth of polysaccharide is complex. Ma et al.3 found a water-soluble
polysaccharide named SFPSA could inhibit the proliferation and growth of human colon cancer cells in
a time- and dose-dependent manner within 48 h, induce the apoptosis and increase the accumulation in
G2/M phase of colon cancer cells. Furthermore, it was found that SFPSA could decrease Bcl-2 mRNA
level, and enhance the mRNA expressions of Bax and p53, as well as the activity of caspase-3. It could
induce apoptosis of colon cancer cells through regulation of apoptosis-associated gene expressions and
the activation of caspase-3. Therefore, further study was needed to shed light on the mechanism for the
inhibitory activity of cancer cell growth of LHEPS-1 from L. helveticus MB2-1 .
3. Conclusion
In the present study, the inhibitory effects of three purified LHEPS fractions (LHEPS-1, LHEPS-2
and LHEPS-3) from the L. helveticus MB2-1 to the proliferation potential of human colon cancer
Caco-2 cells were studied. The results suggested the potential prospects of LHEPS-1 significantly
inhibited cell proliferation in Caco-2 cells in both time- and concentration-dependent manners. Further
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study revealed LHEPS-1 was a moderate branching glucomannogalactan that possessed a backbone
consisting
of
the
repeating
unit
→4)-α-D-Manp-(1→6)-α-D-Manp-α-(1→4)-β-D-Glcp-(1→3)-β-D-Glcp-(1→4)-α-D-Galp-(1→,
branching at O-6 of →4)-α-D-Manp-(1→ and →3)-β-D-Glcp-(1→, respectively, and terminated with
D-Glcp and D-Galp. This structure differed from the previously reported EPS from other Lactobacillus
spp. These results suggested the potential prospects of LHEPS-1 as foods and natural anticancer drugs
to prevent human colon cancer.
4. Experimental
4.1. Materials
The strain of L. helveticus MB2-1 was isolated from traditional Sayram ropy fermented milk which
was collected from Xinjiang, China.36 Human colon cancer Caco-2 cells were obtained from the Cell
Bank of Shanghai Institute of Cell Biology (Shanghai, China). Dulbecco’s modified Eagle’s medium
(DMEM), fetal bovine serum (FBS) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium
bromide (MTT) were purchased from Gibco/Invitrogen (Gibco BRL, Grand Island, NY, USA).
Dimethyl sulfoxide (DMSO), 5-Fu, penicillin and streptomycin were obtained from Sigma Chemical
Co. (St. Louis, MO, USA). All other reagents were of analytical grade.
4.2. Preparation of purified LHEPS fractions
The LHEPS was isolated and purified according to our previously method.20,21 Briefly, after 24 h of
incubation period, the cell was separated by centrifugation at 12,000 × g for 15 min at 4 °C. Then
trichloroacetic acid (TCA) solution was added to the culture to give a final concentration of 4% (w/v),
and the precipitated proteins were removed by centrifugation (12,000 × g for 30 min at 4 °C). The
supernatant was filtered through a 0.45 µm membrane filter, mixed with three volumes ice cold ethanol,
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stirred vigorously and kept at 4 °C for overnight. Crude LHEPS was collected by centrifugation at
15,000 × g for 15 min. The LHEPS pellet was dissolved in distilled water and dialyzed (Mw cut-off
8,000– 14,000 Da, Solarbio Co., Ltd, Beijing, China) against distilled water for 48 h at 4 °C and then
lyophilized. The freeze-dried sample was fractionated with a DEAE-cellulose column (2.6 × 30 cm),
followed by stepwise elution with distilled water, 0.1 and 0.3 M NaCl solutions at a flow rate of 60
mL/h. The fractions were assayed for carbohydrate content by the phenol–sulfuric acid method.36 Three
fractions (LHEPS-1, LHEPS-2 and LHEPS-3) eluted with 0, 0.1 and 0.3 M sodium chloride solutions
were collected, concentrated, dialyzed and lyophilized, respectively. These three fractions were then
purified by a Sephadex G-100 column (2.6 cm × 100 cm) with distilled water at a flow rate of 12 mL/h.
Finally, the three purified fractions were collected, concentrated, dialyzed and lyophilized for further
study.
4.3. Anticancer activity of three purified LHEPS fractions
To investigate the inhibition effects of three purified LHEPS fractions, human colon cancer Caco-2
cells and human colonic epithelial HCoEpiC cells were cultured in DMEM medium supplemented with
10% FBS, penicillin (100 U/mL) and streptomycin (100 mg/L) under 5% CO2 atmosphere at 37 °C,
respectively. The inhibition effects of different LHEPS fractions on Caco-2 and HCoEpiC cells in vitro
was evaluated using MTT-based colorimetric method according to our previously reported method.24
Briefly, 100 µL of cells were incubated on a 96-well plate at a concentration of 2 × 105 cells/well. After
inoculation under 5% CO2 at 37 °C for 24 h, the cells were then incubated with DMEM medium
containing various concentrations of purified LHEPS fractions (0, 50, 100, 200, 400 and 600 µg/mL)
and 5-Fu (50 µg /mL) for 24, 48 and 72 h, respectively. At the end of each treatment, 10 µL (5 mg/mL)
of MTT was dissolved in DMEM medium and was added and the cells were inoculated for another 4 h.
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The liquid was then removed and 100 µL DMSO was added to the well. After dissolving of the formed
crystal formazan, the absorbance was measured by a Synergy™-2 microplate reader (BioTek
Instruments, Inc., Burlington, VT) at 570 nm. The inhibitory rate was expressed as follows:
Inhibitory rate (%) = [1 – (Asample – Ablank) / (Acontrol – Ablank)] × 100
where Acontrol and Ablank were the absorbance of the system without the addition of samples and cells,
respectively.
4.4. Methylation and GC–MS analysis for LHEPS-1
Methylation and GC–MS analysis of LHEPS-1 were performed by using our previously reported
method.37 Complete methylation was confirmed by the disappearance of O–H absorption band (3700–
3100 cm−1) in fourier-transform infrared (FT-IR) spectrum. Briefly, the permethylated LHEPS-1 was
hydrolyzed with 2 M trifluoroacetic acid at 120 °C for 2 h. Then the methylated sugars were reduced
with NaBH4 for 2 h and acetylated with pyridine-acetic anhydride (1:1, v/v) at 100 °C for 1 h. The
resulting methylated alditol acetate derivatives were analyzed by GC–MS. GC–MS analysis was
performed on a Agilent 5975MSD-6890 GC–MS (Agilent Technologies, CA, USA), equipped with a
HP-5 capillary column was used with He as carrier gas. The oven temperature was initially set at
100 °C, programmed at 6 °C/min to 250 °C and held at 250 °C for 5 min.
4.5. 1D and 2D NMR analysis
1
The structure of LHEPS-1 (50 mg/mL) was identified by NMR. H NMR, 13C NMR and 2D NMR
spectra were recorded in D2O as solvent at 323 K with a Bruker AVANCE AV-500 spectrometer
(Bruker Group, Fällanden, Switzerland) using the residual solvent signal as internal standard. The
chemical shifts (δ) are given in parts per million (ppm), and coupling constants (J) are given in Hz. The
1
1
1
2D H–1H correlated spectroscopy (COSY), H–1H total correlation spectroscopy (TOCSY), H–13C
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1
heteronuclear single quantum coherence (HSQC), H–13C heteronuclear multiple quantum coherence
(HMBC) and nuclear Overhauser effect spectroscopy (NOESY) measurements were used to assign
signals and to determine the sequence of sugar residues.
4.6. Statistical analysis
Statistical analysis was performed using SPSS for windows, version 16.0. One-way analysis of
variance (ANOVA) and the Fisher's least significant difference (LSD) test were used to determine
significant differences for different samples. Differences were considered to be significant when P <
0.05. Data were obtained from three independent experiments and each sample was analyzed in
triplicate.
Acknowledgements
This work was co-financed by National Natural Science Foundation of China (No. 31371807 and
31201422 ), Fundamental Research Funds for the Central Universities (KYZ201419), Key Projects in
the National Science & Technology Pillar Program (No. 2013BAD18B01-4), High-Tech Research and
Development Program of China (No.2011AA100903) and was also supported by the Project Funded by
the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).
References
1. Siegel, R.; Ma, J.; Zou, Z.; Jemal, A. CA–Cancer J. Clin. 2014, 64, 9–29.
2. Adamsen, L.; Quist, M.; Midtgaard, J.; Andersen, C.; Moller, T.; Knutsen, L.; Tveterås, A.; Rorth, M.
Support. Care. Cancer. 2006, 14, 116–127.
3. Ma, L.; Qin, C.; Wang, M.; Gan, D.; Cao, L.; Ye, H.; Zeng, X. Food Chem. Toxicol. 2013, 60,
269–276.
15

ACCEPTED MANUSCRIPT
4. Ricci-Vitiani, L.; Lombardi, D. G.; Pilozzi, E.; Biffoni, M.; Todaro, M.; Peschle, C.; De Maria, R.
Nature. 2006, 445, 111–115.
5. Gunnarsson, H.; Ekholm, A.; Olsson, L. I. Eur. J. Surg. Oncol. 2013, 39, 831–836.
6. Liu, C. F.; Tseng, K. C.; Chiang, S. S.; Lee, B. H.; Hsu, W. H.; Pan, T. M. J. Sci. Food. Agr. 2011, 91,
2284–2291.
7. Goodarzi, N.; Varshochian, R.; Kamalinia, G.; Atyabi, F.; Dinarvand, R. Carbohyd. Polym. 2013, 92,
1280–1293.
8. Qiao, D.; Liu, J.; Ke, C.; Sun, Y.; Ye, H.; Zeng, X. Carbohyd. Polym. 2010, 82, 1184–1190.
9. Mazzoli, R; Bosco, F.; Mizrahi, I.; Bayer, E. A.; Pessione, E. Biotechnol. Adv. 2014, 32, 1216–1236.
10. Lin, T. Y.; Chien, M. F. C. Food Chem. 2007, 100, 1419–1423.
11. Pan, D.; Mei, X. Carbohyd. Polym. 2010, 80, 908–914.
12. Suresh, V.; Senthilkumar, N.; Thangam, R.; Rajkumar, M.; Anbazhagan, C.; Rengasamy, R.;
Gunasekaranc, P.; Kannan, S.; Palani, P. Process Biochem. 2013, 48, 364–373.
13. Yang, W.; Pei, F.; Shi, Y.; Zhao, L.; Fang, Y.; Hu, Q. Carbohyd. Polym. 2012, 88, 474–480.
14. Cui, F.; Tao, W.; Xu, Z.; Guo, W.; Xu, H.; Ao, Z.; Jin, J.; Wei, Y. Bioresource Technol. 2007, 98,
395–401.
15. Jiang, X.; Zhang, Z.; Chen, Y.; Cui, Z.; Shi, L. Carbohyd. Polym. 2014, 103, 434–441.
16. Zhao, Q.; Xie, B.; Yan, J.; Zhao, F.; Xiao, J.; Yao, L.; Zhao, B.; Huang, Y. Carbohyd. Polym. 2012,
87, 392–396.
17. Lavi, I.; Friesem, D.; Geresh, S.; Hadar, Y.; Schwartz, B. Cancer Lett. 2006, 44, 61–70.
18. Nie, S.; Cui, S.; Phillips, A.; Xie, M.; Phillips, G.; Al-Assaf, S.; Zhang, X. Carbohyd. Polym. 2011,
84, 894–899.
16

ACCEPTED MANUSCRIPT
19. Xu, R.; Ye, H.; Sun, Y.; Tu, Y.; Zeng, X. Food Chem. Toxicol. 2012, 50, 2473–2480.
20. Li, W.; Ji, J.; Chen, X.; Jiang, M.; Rui, X.; Dong, M. Carbohyd. Polym. 2014, 102, 351–359.
21. Li, W.; Ji, J.; Tang, W.; Rui, X.; Chen, X.; Jiang, M.; Dong, M. Carbohyd. Polym. 2014, 105,
334–340.
22. Li, W.; Mutuvulla, M.; Chen, X.; Jiang, M.; Dong, M. Eur. Food Res. Technol. 2012, 235, 497–505.
23. Wang, K.; Li, W.; Rui, X.; Chen, X.; Jiang, M.; Dong, M. Int. J. Biol. Macromol. 2014, 63,
133–139.
24. Wang, K.; Li, W.; Rui, X.; Chen, X.; Jiang, M.; Dong, M. Int. J. Biol. Macromol. 2014, 67,
6771–78.
25. Persin, Z.; Stana-Kleinschek, K.; Foster, T. J.; Van Dam, J. E. J. E. Boeriu, C. G.; Navard, P.
Carbohyd. Polym. 2011, 84, 22–32.
26. Warrand, J. Food Technol. Biotech. 2006, 44, 355–370.
27. Charchoghlyan, H.; Park, H. D. Food Hydrocolloid. 2012, 30, 512–518.
28. Górska, S.; Jachymek, W.; Rybka, J.; Strus, M.; Heczko, P. B.; Gamian, A. Carbohyd. Res. 2010,
345, 108–114.
29. Notararigo, S.; Nácher-Vázquez, M.; Ibarburu, I.; Werning, M. L.; de Palencia, P. F.; Dueñas, M.
T.; Aznar, R.; López, P.; Prieto, A. Carbohyd. Polym. 2013, 93, 57–64.
30. Rodríguez-Carvajal, M. A.; Ignacio Sánchez, J.; Campelo, A. B.; Martínez, B.; Rodríguez, A.;
Gil-Serrano, A. M. Carbohyd. Res. 2008, 343, 3066–3070.
31. Yin, J.; Lin, H.; Li, J.; Wang, Y.; Cui, S.; Nie, S.; Xie, M. Carbohyd. Polym. 2012, 87, 2416–2424.
32. Lebeer, S.; Verhoeven, T. L.; Francius, G.; Schoofs, G.; Lambrichts, I.; Dufrêne, Y.; Vanderleyden,
J.; De Keersmaecker, S. C. J. Appl. Environ. Microb. 2009, 75, 3554–3563.
17

ACCEPTED MANUSCRIPT
33. Li, B.; Dobruchowska, J. M.; Gerwig, G. J.; Dijkhuizen, L.; Kamerling, J. P. Carbohyd. Polym.
2013, 91, 314–321.
34. Zhou, S.; Liu, Y.; Yang, Y.; Jia, W.; Tang, Q.; Tang, C.; Feng, N.; Zhang, J. Bioact. Carbohyd.
Dietary Fibre. 2013, 1, 99–104.
35. Badel, S.; Bernardi, T.; Michaud, P. Biotechnol Adv. 2011, 29, 54–66.
36. Welman, A. D.; Maddox, I. S. Trends Biotechnol. 2003, 21, 269–274.
37. Torino, M. I.; Mozzi, F.; de Valdez, G. F. Appl. Microbiol. Biot. 2005, 68, 259–265.
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Table 1. Methylation analysis of LHEPS-1 from L. helveticus MB2-1
Retention
time (min)
15.459
Methylated
Molar
ratio
1.25
1.21
1.07
1.00
1.31
1.14
1.19
1.26
1.16
1.33
Primary mass fragments (m/z)
Deduced linkage
sugar
2,4,6-Me3Glcp
2,3,4-Me3Manp
3,4,6-Me3Glcp
2,3,6-Me3Glcp
43.1, 71.1, 101.1, 129, 161.1, 205.1, 253.1, 282.1
43.1, 65, 85, 117, 139, 159.1, 201.1, 231, 261.1, 281.9
43.1, 87, 129, 161.1, 189.1, 281.1, 326.9
→3-
→6-
→2-
→4-
D
D
D
D
-Glcp-(1→
-Manp-(1→
-Glcp-(1→
-Glcp-(1→
15.812
16.985
17.095
43.1, 87, 117, 145, 173, 207, 234.1, 281.1, 401
17.222
2,3,4,6-Me4-Glcp 43.1, 71, 101.1, 129, 161.1, 205.1, 249, 355.2
T
-
-
D
-Glcp-(1→
-Galp-(1→
17.536
2,3,4,6-Me4-Galp 43.1, 71.1, 101.1, 129, 161.1, 189.1, 233, 281.1, 341.1
T
D
18.048
2,3,6-Me3Galp
2,3,4-Me3Glcp
2,3-Me2Manp
2,4-Me2Glcp
43.1, 71, 101.1, 125, 145.1, 173.1, 205.1, 245.2, 281.1
43.1, 87, 129, 159.1, 189.1, 233.1, 261, 355.1
→4-
D
D
-Galp-(1→
-Glcp-(1→
18.996
→6-
19.073
43.1, 68, 87.1, 117, 142.1, 161.1, 189.1, 208.9, 233.1, 281.1
43.1, 85, 115, 145, 187, 217.1, 259.1, 289.1, 361.1
→4,6-
D
-Manp-(1→
-Glcp-(1→
20.996
→3,6-
D
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Table 2. Chemical shifts (ppm) of 1H and 13CNMR signals for the LHEPS-1, recorded in D2O at 323 K.
Residue
H-1
5.46
5.33
5.29
H-2
4.29
4.25
4.19
4.19
4.29
4.39
3.51
3.50
3.66
3.68
C-2
H-3
4.12
4.14
3.98
3.99
4.02
4.25
3.72
3.71
4.13
4.07
C-3
H-4
4.08
4.11
3.95
3.96
4.22
4.15
3.81
3.80
4.11
4.10
C-4
H-5
3.94
3.85
3.86
3.95
3.94
3.96
3.64
3.65
3.85
3.84
C-5
H-6
4.20
3.95
4.09
4.08
4.09
4.08
3.69
4.04
4.11
3.89
C-6
A
B
C
D
E
F
G
H
I
→6-α-
→4-α-
α-
→4,6-α-
D
-Manp-(1→
D
-Galp-(1→
D
-Glcp-(1→
D
-Manp-(1→ 5.26
5.24
5.23
4.71
4.70
4.66
4.64
C-1
→3-
→6-
→4-
D
D
D
-α-Glcp-(1→
-α-Glcp-(1→
-β-Glcp-(1→
β-
→2-
→3,6-
D
-Galp-(1→
-β-Glcp-(1→
-β-Glcp-(1→
D
J
D
A
B
C
D
E
F
G
H
I
→6-α-
D
-Manp-(1→
-Galp-(1→
102.90 80.52 80.63 71.00 71.42 80.83
104.58 72.40 80.25 77.25 75.08 63.38
100.73 80.75 75.51 75.13 69.22 63.38
→4-α-
D
α-D-Glcp-(1→
→4,6-α-
D
-Manp-(1→ 100.62 80.83 75.73 75.01 69.18 69.18
104.46 71.31 81.00 71.22 73.90 63.44
104.29 71.93 71.20 71.32 69.29 69.19
105.33 75.45 75.10 73.18 77.10 62.88
105.38 75.32 75.27 73.09 77.15 62.90
105.68 78.00 77.98 75.41 71.95 63.21
105.72 78.02 80.98 71.90 69.25 69.15
→3-
→6-
→4-
D
D
D
-α-Glcp-(1→
-α-Glcp-(1→
-β-Glcp-(1→
β-
→2-
→3,6-
D
-Galp-(1→
-β-Glcp-(1→
-β-Glcp-(1→
D
J
D
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Figure captions
Figure 1. Inhibitory effect in vitro of LHEPS-1, LHEPS-2 and LHEPS-3 against human colon cancer
Caco-2 cells at 24 h (A), 48 h (B) and 72 h (C) treatment. Data are means ± SD of triplicate.
1
Figure 2. The 500-MHz H, 13C NMR and HSQC spectra of LHEPS-1 from L. helveticus MB2-1
recorded on Bruker Avance DRX-500 spectrometer in D2O. 1H NMR spectrum (A); 13C NMR spectrum
(B); HSQC spectrum (C). The assignments of sugar residues in anomeric region (D) and non-anomeric
region for HSQC spectrum (E, F and G).
Figure 3. COSY spectrum of LHEPS-1 from L. helveticus MB2-1 recorded on Bruker Avance
DRX-500 spectrometer in D2O (A). The assignments of sugar residues in non-anomeric region for
COSY spectrum (B). TOCSY spectrum of LHEPS-1 recorded on Bruker Avance DRX-500
spectrometer (C). The assignments of sugar residues in anomeric region for TOCSY spectrum (D).
Figure 4. Proposed structure of LHEPS-1 from L. helveticus MB2-1.
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30
25
20
15
10
5
LHEPS-1
LHEPS-2
LHEPS-3
(A)
0
50
100
200
400
600
Concentrations of LHEPS (µg/mL)
60
LHEPS-1
LHEPS-2
LHEPS-3
(B)
50
40
30
20
10
0
50
100
200
400
600
Concentrations of LHEPS (µg/mL)
70
LHEPS-1
LHEPS-2
LHEPS-3
(C)
60
50
40
30
20
10
0
50
100
200
400
600
Concentrations of LHEPS (µg/mL)
Fig. 1
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(A)
(B)
(C)
23

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(D)
(E)
(F)
(G)
Fig. 2
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(A)
(B)
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(C)
(D)
Fig. 3
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Fig. 4
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Highlights:
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
LHEPS-1 could significantly inhibit the growth of human colon cancer Caco-2 cells;
Structural feature of LHEPS-1 was elucidated by methylation, GC-MS and 1D/2D NMR;
LHEPS-1 was a kind of moderate branching glucomannogalactan;
A possible structure of LHEPS-1 was proposed;
Anti-colon cancer activity was correlated with structure of LHEPS-1.