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Biomedicine & Pharmacotherapy
journal homepage: www.elsevier.com/locate/biopha
Prebiotic oligosaccharides from dragon fruits alter gut motility in mice
Pissared Khuituan
a,⁎
, Sakena K-da
a,b
, Kanrawee Bannob
a,b
, Fittree Hayeeawaema
a
,
Saranya Peerakietkhajorn
b
, Chittipong Tipbunjong
c
, Santad Wichienchot
d
,
Narattaphol Charoenphandhu
e
a
Department of Physiology, Faculty of Science, Prince of Songkla University, Songkhla, Thailand
b
Department of Biology, Faculty of Science, Prince of Songkla University, Songkhla, Thailand
c
Department of Anatomy, Faculty of Science, Prince of Songkla University, Songkhla, Thailand
d
Interdisciplinary Graduate School of Nutraceutical and Functional Food, Prince of Songkla University, Songkhla, Thailand
e
Department of Physiology, Faculty of Science, Mahidol University, Bangkok, Thailand
ARTICLE INFO
Keywords:
Dragon fruit oligosaccharide
Colonic contractility
Colonic smooth muscle
Gut transit time
ABSTRACT
Dragon fruit oligosaccharide (DFO) has a prebiotic property which improves gut health by selectively stimu-
lating the colonic microbiota. Altering microbiota composition may affect intestinal motility. However, no study
has been done to understand the DFO effects on gut motor functions. This research thus aimed to investigate the
DFO effects on mice colons compared to the prebiotic fructo-oligosaccharide (FOS) and probiotic bifidobacteria.
The mice in this study received distilled water; 100, 500, and 1000 mg/kg DFO; 1000mg/kg FOS; or 10
9
CFU
Bifidobacterium animalis daily for 1 week and some treatments for 2 weeks. Gastrointestinal transits were ana-
lysed and motility patterns, smooth muscle (SM) contractions and morphological structures of the colons were
assessed. Administration of FOS, 500 and 1000 mg/kg DFO significantly increased fecal output when compared
to the control group. In mice treated with FOS and bifidobacteria, gut transit time was reduced, while upper gut
transit was increased in comparison to DFO groups. Spatiotemporal maps of colonic wall motions showed that
DFO increased the number of colonic non-propagation contractions and fecal pellet velocity, consistent with the
results from groups treated with FOS and bifidobacteria. DFO also increased the amplitude and duration of
colonic SM contractions. Histological stains showed normal epithelia, crypts, goblet cells, and SM thickness in all
groups. In conclusion, DFO increased colonic SM contractions without morphological change and acted as a
bulk-forming and stimulant laxative to increase fecal output and intestinal motility. Thus, DFO as a dietary
supplement may promote gut health and correct gastrointestinal motility disorders.
1. Introduction
The imbalance of enteric microbiota affects various gastrointestinal
(GI) functions including motility and may result in inflammatory bowel
disease (IBD), irritable bowel syndrome (IBS), diarrhea, or constipation
[1–3]. Microbial populations in the GI tract have been maintained in a
balanced state by probiotics and prebiotics [4,5]. Probiotics, such as
bifidobacteria and lactobacilli, are live microbes that are beneficial to
human and animal GI health. Probiotics modulate intestinal motility,
reducing both diarrhea and constipation [6–8]. Despite their benefits,
probiotics have limitations for patients who suffer from acute
pancreatitis or allergies. Heat and acid may destroy probiotics and they
are difficult to process in some foodstuffs[9,10]. An alternative ap-
proach to selective modification of the composition and activity of the
intestinal microbiota is dietary supplementation with prebiotics.
Prebiotics are non-digestible food ingredients that enter the colon
without being altered by digestion and absorption. They can serve as a
source of nutrition for beneficial bacteria in the colon. Prebiotics not
only promote specific changes to the composition and/or activities of
the GI microbiota; they also induce microbial competition and reduce
populations of undesirable bacteria [11,12]. The major products of
prebiotic fermentation in the colon are short chain fatty acids (SCFAs)
https://doi.org/10.1016/j.biopha.2019.108821
Received 28 December 2018; Received in revised form 20 March 2019; Accepted 26 March 2019
Abbreviations: BW, body weight; CFU, colony-forming unit; DFO, dragon fruit oligosaccharide; DP, degree of polymerization; DW, distilled water; FOS, fructo-
oligosaccharide; GI, gastrointestinal; GIMM, gastrointestinal Motility Monitor; GOS, galacto-oligosaccharides; HE, Hematoxylin-eosin; HPLC, high performance liquid
chromatography; IBD, inflammatory bowel disease; IBS, irritable bowel syndrome; MW, molecular weight; PAS, periodic acid-Schiff; SCFAs, short chain fatty acids;
SM, smooth muscle
⁎
Corresponding author at: Department of Physiology, Faculty of Science, Prince of Songkla University, 15 Karnjanavanich Rd., Hat Yai, Songkhla 90110 Thailand.
E-mail address: pissared.k@psu.ac.th (P. Khuituan).
Biomedicine & Pharmacotherapy 114 (2019) 108821
0753-3322/ © 2019 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/BY-NC-ND/4.0/).
T
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that include acetate, propionate, and butyrate. SCFAs are energy
sources for colonic epithelial cells and play roles in electrolyte trans-
port, cell differentiation, cell growth, and colonic motility [13,14]. The
best known prebiotics are fructo-oligosaccharides (FOS), galacto-oli-
gosaccharides (GOS), and inulin [15–18]. Other non-digestible oligo-
saccharides, such as some prebiotic-rich fruits and vegetables, have also
been tested for prebiotic properties.
Dragon fruit oligosaccharide (DFO) is extracted and purified from
dragon fruit or pitaya. This fruit is native to Central and South America
and has gained popularity worldwide. It is now grown across South East
Asia [19–21]. The fruit is rich in β-carotene, lycopene, vitamin E and
essential fatty acids and exhibits antioxidant and anti-inflammatory
activities [22–24]. Both red pitaya with white-flesh (Hylocereus undatus)
and red pitaya with red-flesh (Hylocereus polyrhizus) have been reported
as a source of DFO found in its flesh and peel [25,26]. In an artificial
colon, DFO was resistant to hydrolysis by artificial human gastric juice
and α-amylase and stimulated the growth of lactobacilli and bifido-
bacteria [25,27]. Although the prebiotic properties of DFO are quite
evident from in vitro studies, the prebiotic effects of DFO on GI func-
tions, especially intestinal motility, have not been confirmed in an in
vivo model.
When managing the balance of the intestinal microbiota to improve
intestinal motility disorders such as constipation and diarrhea, it is
useful to know the effective dose and duration of prebiotic intake.
Therefore, the present study aimed to investigate the in vivo effects of
DFO in male ICR mice. The mice were supplemented with DFO for 1
and 2 weeks. Responses of interest were fecal output, intestinal transit,
evacuation time, colonic motility patterns, colonic pellet propulsion
velocity, proximal and distal colonic circular and longitudinal SM
contractions, and colonic morphological changes.
2. Materials and methods
2.1. Animals
This study was approved and guided by the Animal Ethics
Committee of the Prince of Songkla University, Thailand (Project li-
cense number MOE0521.11/799). Adult male ICR/Mlac mice (Mus
musculus; 5 weeks old, weighing 20–25 g) were obtained from National
Laboratory Animal Center, Mahidol University and housed at Southern
Laboratory Animal Facility, Prince of Songkla University. The animals
were housed four per cage and kept under standard environmental
conditions at 23 to 27 °C, with 50 to 55% humidity under a 12-hour
light/12-hour dark cycle. They were fed standard commercial food
pellets (Perfect Companion Group Co., Ltd., Thailand) with filtered
water ad libitum.
2.2. Chemicals and equipment
Reference prebiotic and probiotic supplements were FOS (Sigma-
Aldrich, St. Louis, MO, USA) and Bifidobacterium animalis (FD-DVS nu-
trish
®
BB-12
®
) (Chr. Hansen Holding A/S, Hoersholm, Denmark). Krebs
solution was composed as follows (in mM): 119 NaCl, 2.5 CaCl
2
, 4.5
KCl, 2.5 MgSO
4
, 25 NaHCO
3
, 1.2 KH
2
PO
4
, and 11.1 D-glucose [28,29]
(all purchased from Merck, Co., Ltd., Darmstadt, Germany) and the
working solution was made fresh on the day of the experiment. The
Gastrointestinal Motility Monitor (GIMM) System for ex vivo study of
colonic propulsive motility was purchased from Catamount Research
and Development, St. Albans, VT.
2.3. Extraction and purification method of DFO (briefly)
White-fleshed dragon fruits (Hylocereus undatus (Haw) Britt. and
Rose) were grown on and purchased from a certified organic, GAP
(good agricultural practice) contract fruit farm in Hat Yai, Songkhla,
Thailand. The fruits were selected from one batch to ensure consistency
of chemical composition, particularly oligosaccharide content. The
fruits were washed and separated into flesh and peel parts. The flesh
and peel parts were chopped into small pieces, finely ground and ex-
tracted using pectinase enzyme in a 50 L reactor [30]. We analyzed the
sugar content of white-fleshed dragon fruit - mostly glucose, fructose
and some oligosaccharides - by high performance liquid chromato-
graphy (HPLC) in a previous study [25]. The low molecular weight
(MW) fraction, glucose and fructose, which does not have prebiotic
properties, was removed biologically by two-step yeast (Saccharomyces
cerevisiae) cultivation. Yeast cells were then removed by filtration and
centrifugation. The purified DFO extract was concentrated by eva-
poration and spray dried to obtain DFO powder. The DFO powder was
stored at −20 °C to ensure stability and was used for all experiments.
The MW distribution of the mixed oligosaccharides was confirmed by
mass spectrometry. There were four components of 716, 700, 490 and
474 Da with relative percentages of 100, 68, 45 and 21, respectively
[25]. Therefore, the degree of polymerization (DP) of mixed DFO is 3–4,
which is in the same range as some FOSs. The dietary composition of
DFO is listed in Supplemental Table S1.
2.4. Experimental design, surgical procedure and tissue preparation
After a week of acclimatization, groups of animals had their diet
variously supplemented by gavage once per day. Six groups were sup-
plemented for a week with 0.2 mL distilled water (DW) (vehicle con-
trol) or with DFO at 100, 500, or 1000 mg/kg, or with FOS at 1000 mg/
kg or with bifidobacteria at 10
9
CFU. Three groups were supplemented
for two weeks with DW or with 500 mg/kg DFO or with 1000 mg/kg
FOS. An overview of the experimental procedure over 21 days is pro-
vided in Supplemental Fig. S1. Body weight (BW), food and water in-
takes, and fecal pellet outputs of all mice were recorded every day. The
colonic propulsive motility functions of the animals were analyzed ex
vivo. Animals were anesthetized by intraperitoneal injection of 70 mg/
kg Thiopental sodium (Anesthal
®
) so the abdominal cavity could be
rapidly dissected. The colon was sectioned whole from the cecum to the
rectum and placed with contents in oxygenated (5% CO
2
and 95% O
2
)
ice-cold Krebs solution (pH 7.4 with an osmolality of 289–292 mmol/kg
H
2
O). To study the effects of DFO on colonic propulsive motility, the
whole colon was mounted horizontally in a 50 mL GIMM organ bath
containing oxygenated Krebs solution at 37 °C. To study SM con-
tractility, the colon was divided into two sections. The proximal colon
was sectioned 3 cm distal to the cecum and the distal colon 3 cm
proximal to the rectum. The two segments were transversely sectioned
into lengths of 1 cm. To study longitudinal SM contraction, 1 cm seg-
ments were suspended in the organ bath longitudinally. To study cir-
cular SM contraction, 1 cm segments were opened along the mesenteric
border and the muscular layer was separated. The muscular layer was
opened out to form a strip and then suspended so that contractions
occurred vertically in a 20 mL organ bath containing Krebs solution.
2.5. Fecal pellet output, gut transit and evacuation time assay
During treatments, fecal pellets evacuated over a six-hour period
were counted and hourly averages calculated. To determine fecal water
content, feces were weighed, dried at 100 °C for 30 min and weighed
again. The fecal water content (%) was calculated by ((wet weight –dry
weight)/wet weight) × 100%. To measure total gut transit time, the
mice were fed an Evan-blue marker meal (5% Evan-blue in 1.5% me-
thyl cellulose; 0.1 mL (i.g.)) and were observed every 10 min until the
first blue pellet was expelled. Evacuation time was measured by using a
bead expulsion test. A 3-mm glass bead was inserted through the anus
2 cm into the colon using a petroleum jelly-lubricated plastic tip and
time was measured until the bead was expelled. For upper gut transit
measurement, the mice received a charcoal meal and 60 min later
charcoal transit (%) was calculated by (the distance of charcoal meal/
total length of the small intestine) × 100%. These methodologies were
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performed and modified according to previously described methods
[31,32].
2.6. Measurement of ex vivo colonic motility
A GIMM organ bath, lined with Sylgard, was placed on top of lu-
minance plates to silhouette colonic segments and continuously per-
fused with Krebs solution at 10 mL/min. The segment was pinned in situ
at both oral and caudal ends of its length. Before recording, the segment
was allowed to equilibrate in Krebs solution for 30 min without flushing
out fecal pellets. Pellet movement was recorded for 30 min using a
video camera connected to a computer running GIMM software (2
times/individual). Spatiotemporal maps of motility were constructed
from recordings acquired from individual runs. In each video frame, the
image of the colonic segment was converted to a silhouette. The dia-
meter at each point along the entire length was calculated and con-
verted into a grey-scale. The contracted diameters (intestinal contrac-
tion) were coded as white and the relaxed diameters (intestinal
dilation) as black. The total number of spontaneous contractions was
defined as the sum of the number of non-propagation contractions and
the number of propagation contractions over 30 min. Non-propagation
contractions were defined as those contractions that failed to move the
pellet forward. The velocity of fecal pellet propulsion through the
whole colon was determined only in the propagation contraction pat-
tern and was performed using the fecal pellet tracking method in the
GIMM software, in which the pellet is digitally darkened and tracked
from the oral end to the caudal end. Fecal pellet velocity was calculated
and displayed in mm/second.
2.7. Measurement of in vitro smooth muscle contractility
The distal end of the colonic segment was tied to an organ holder
while the proximal end was secured with a silk thread to an isometric
force transducer (Model FT03, Grass, USA) and stretched passively
under a load of 500 mg. The signal output of mechanical activity, am-
plified and digitized via a Bridge Amp and PowerLab
®
System (AD
Instruments, Australia), was stored on a computer for subsequent
analysis using LabChart7 program software. Signals were acquired and
analyzed twice per individual. Following the 30 min equilibration
period, the spontaneous contractions representing basal activity in the
colonic SM were recorded for 5 min. To stimulate contraction, carba-
chol (Tocris Bioscience, Bristol, UK) was added to the Krebs solution in
the organ bath in a cumulative fashion. The concentrations progressed
from 0.1 to 1 to 10 μM without washing between concentrations. The
amplitude, duration, and frequency of contractions of each colonic
segment were calculated. The mean amplitude (in mg) was calculated
as the average of peak to peak differences over 5 min and was expressed
as a percentage of the values recorded in the presence of 1 μM CCh
(maximal contraction). The frequency and duration were respectively
expressed as the number of contractions per minute (times/min) and
the mean of contraction times (in seconds) recorded in a 5-min period.
Fig. 1. Effects of one-week and two-week dietary supplementation with DFO on the number of fecal pellets and time of evacuation in mice. Mice were treated with (A
and B) 0.2 mL DW, DFO (100, 500, and 1000 mg/kg, p.o.), FOS (1000 mg/kg, p.o.), and bifidobacteria (10
9
CFU, p.o.) for a week, then with (C and D)0.2 mL DW,
DFO (500 mg/kg, p.o.) and FOS (1000 mg/kg, p.o.) for two weeks. Each bar of the data represents means ± SEM (n = 4–6).
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2.8. Histological study
Colonic segments were fixed in 10% formalin solution, embedded in
paraffin, and sectioned every 7 μm. The sections were deparaffinized
with xylene and rehydrated in serial graded ethanol. Hematoxylin-eosin
(HE) and Periodic acid-Schiff(PAS) staining were performed according
to the standard protocols, and tissues were observed by light micro-
scopy. SM thickness was measured with image J software.
2.9. Statistical analysis
Results obtained from this study were expressed as means ± SEM
with n in parentheses denoting the number of animals. Data were
analyzed using the statistical program GraphPad Prism 5.0 (GraphPad
Software Inc., San Diego, California, USA). Comparison among multiple
groups was made using one-way analysis of variance test followed by
the Bonferroni post hoc test. The determination of sample size was
computed using the MINITAB Statistical Analysis Package (Minitab 16,
Minitab Inc., Pennsylvania, USA). The level of significance for all sta-
tistical tests was P< 0.05.
3. Results
3.1. Effects of DFO on body weight, food and water intakes, and fecal pellet
output
There was no significant change in BW, food intake, or water intake
in mice whose diet was supplemented by the vehicle control or all doses
of DFO for one and two weeks (Supplemental Figs. S2 and S3). The
mean fecal pellet number for all the DFO-treated groups did not sig-
nificantly differ from that for the control group (Fig. 1A and C). How-
ever, significant differences were found in mean fecal pellet wet weight.
Among the group supplemented with 500 and 1000 mg/kg DFO for a
week, mean fecal pellet wet weight increased by 2.3 times compared to
the vehicle control and among the group that received 500 mg/kg DFO
for two weeks, the weight increase was 2 times. These increases in
mean fecal pellet wet weight were similar to the effect of supple-
mentation with the reference prebiotic, 1000 mg/kg FOS (Fig. 2A and
C). The increases in fecal mass occurred despite the similarity in food
intakes between the experimental groups and the control group. We
concluded that the change was induced by DFO. The changes in fecal
mass among two DFO-treated groups support the supposed prebiotic
effects of DFO put forward in our previous study [25]. However, fecal
water content was not significantly different in any of the groups
(Fig. 2B and D).
Fig. 2. Effects of one-week and two-week dietary supplementation with DFO on fecal pellet weight and fecal water content in mice. Mice were treated with (A and B)
0.2 mL DW, DFO (100, 500, and 1000 mg/kg, p.o.), FOS (1000 mg/kg, p.o.), and bifidobacteria (10
9
CFU, p.o.) for a week, then (C and D)0.2 mL DW, DFO (500 mg/
kg, p.o.) and FOS (1000 mg/kg, p.o.) for two weeks. Each bar of the data represents means ± SEM (n = 6 –10). *P< 0.05 and **P<0.01 compared to vehicle
control group (DW).
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3.2. Effects of DFO on evacuation time and gut transit
Marker meals were used to demonstrate the action of DFO on upper
gut and total gut transit time. The total gut transit time of the vehicle
control was approximately 230 min. Among the groups supplemented
with DFO at 1000 mg/kg for a week and 500mg/kg for two weeks, this
time was reduced by approximately 30%. These reduced times were
similar to the total gut transit times observed in the groups treated with
the reference prebiotic and the reference probiotic (Fig. 3A and C).
Upper gut transit, defined as the proportion of the small intestinal
length transited by a charcoal meal, was 56.03 ± 2.80% among the
mice supplemented with DW (vehicle control) for a week and
61.01 ± 4.40% among the mice supplemented with DW for two weeks.
These values significantly increased in every experimental group except
the group treated with DFO at 100 mg/kg for a week (Fig. 3B and D).
However, compared to the control group, evacuation time slightly in-
creased among mice treated for one week with DFO at 100 and 500 mg/
kg but decreased among other groups supplemented for one week.
Evacuation time was not significantly different compared to the control
group among mice treated with 500 mg/kg of DFO for two weeks
(Fig. 1B and D).
3.3. Effects of DFO on colonic motility
Since gut transit time decreased after supplementation with DFO, it
is possible that DFO could increase gut motility. In the spatiotemporal
maps, two patterns of motility were observed. The initial pattern was
the propagation or peristaltic contractions, motility induced by food
pellets. In the group treated with 1000 mg/kg DFO for a week, the
pellets were pushed at 0.6 mm/s (Fig. 4D) by aboral propagation. This
fecal pellet velocity was a significant increase compared to the control
group. Meanwhile, administration of 500 mg/kg DFO and 1000 mg/kg
FOS for two weeks also increased fecal pellet velocity, but not as sig-
nificantly (Fig. 5D). The results suggested that the reference prebiotics
and probiotics had similar or even greater effects than DFO, with the
exception of 1000 mg/kg FOS treatment for a week (Fig. 4D). The
second pattern of motility consisted of shallow circular muscle con-
tractions separated by short phases of relaxation. Because the con-
tractions pushed the pellets forward and backward (non-propagation
contraction or segmentation), the contraction velocity could not be
calculated. By comparison with the vehicle control, a significant in-
crease in the total number of contractions (Fig. 4A) was observed after
treatment with 1000 mg/kg DFO for a week. The contribution to this
total increase was especially noticeable in the non-propagation pattern
(Fig. 4B). This response was similar to the results of the week long
treatments with the reference prebiotic and probiotic (Fig. 4A and B).
However, the number of propagation contractions was not significantly
different (Fig. 4C). Similarly, two weeks of treatment with 500 mg/kg
DFO and 1000 mg/kg FOS yielded a significant increase in the total
number of contractions and non-propagation contractions but not
propagation pattern contractions (Fig. 5A–C). Spatiotemporal maps of
these colonic motility patterns can be found in Supplemental Figs. S4
and S5.
3.4. Effects of DFO supplementation for one week on proximal and distal
colonic circular and longitudinal SM contractions
The mean amplitude of contraction of the circular SM from proximal
colon segments from the control group was 30.27 ± 4.99%. Among all
the other groups, the mean amplitude increased. These increases ranged
Fig. 3. Effects of one-week and two-week
dietary supplementation with DFO on Evan-
blue total gut transit time and charcoal meal
upper gut transit in mice. Mice were treated
with (A and B)0.2 mL DW, DFO (100, 500 and
1000 mg/kg, p.o.), FOS (1000 mg/kg, p.o.),
and bifidobacteria (10
9
CFU, p.o.) for a week,
then (C and D)0.2 mL DW, DFO (500 mg/kg,
p.o.) and FOS (1000 mg/kg, p.o.) for two
weeks. (A and C) Each bar of the total gut
transit time represents the mean of the total gut
transit time (min) ± SEM (n = 4–6). (B and D)
Each bar of the upper gut transit represents the
mean of the percentage of the small intestine
length traveled by the charcoal plug ± SEM
(n = 7–11). *P< 0.05, **P< 0.01 and
***P< 0.001 compared to vehicle control
group (DW).
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from 37.00 ± 7.67%, for the group treated with 100 mg/kg DFO for a
week, to 92.63 ± 5.16 after 7-day treatment with 10
9
CFU bifido-
bacteria. After supplementation for a week with 500 and 1000 mg/kg
DFO, the contraction amplitude was significantly higher than that of
the control group. This result was similar to the result for prebiotic FOS
(Fig. 6A). The representative traces of the contractions of proximal
colonic circular SM of mice treated with DW, DFO, FOS, and bifido-
bacteria for 7 days were shown in Supplemental Fig. S6. In the long-
itudinal SM proximal colon segments, the mean amplitude of contrac-
tion of the control group was 36.27 ± 7.79% but, after every 7-day
treatment the mean amplitudes of contractions increased. These in-
creases were especially significant in the groups supplemented with
1000 mg/kg of DFO (70.55 ± 8.31%) and 10
9
CFU bifidobacteria
(79.21 ± 8.98). Treatment with 1000 mg/kg FOS did not significantly
enhance the amplitude of contraction in longitudinal SM (Fig. 6A). The
representative traces of the contractions of proximal colonic long-
itudinal SM of mice treated for 7 days were shown in Supplemental Fig.
S7. The mean frequency of the spontaneous contractions in both cir-
cular and longitudinal SM proximal colon segments in any experimental
group was not significantly different from the control group (Fig. 6C).
In contrast, the durations of contraction in longitudinal SM were sig-
nificantly longer in the groups treated with 1000 mg/kg DFO and bi-
fidobacteria than they were in the control group. Meanwhile, supple-
mentation with 1000 mg/kg FOS significantly increased the duration of
contraction only in the circular SM (Fig. 6E). We also investigated the
effect of DFO on the contraction of the distal colonic SM (Supplemental
Fig. S8 and S9). In both circular and longitudinal SM segments
(Fig. 6B), the mean amplitude of contractions significantly increased,
compared to the control group, only in the group treated with bifido-
bacteria. Similarly, the mean frequency of spontaneous contractions in
both circular and longitudinal SM showed no significant change in re-
sponse to DFO and FOS when compared to the results from the control
groups (Fig. 6D). The duration of contractions of circular muscle sig-
nificantly increased in the groups treated with 1000 mg/kg of DFO and
bifidobacteria, whereas the contraction duration of longitudinal muscle
increased in the groups treated with 1000 mg/kg FOS and bifido-
bacteria (Fig. 6F).
3.5. Effects of DFO supplementation for two weeks on proximal and distal
colonic circular and longitudinal SM contractions
After treatment with 500 mg/kg of DFO and 1000 mg/kg of FOS for
two weeks, the amplitude of proximal colonic contractions was sig-
nificantly higher than the amplitude of the control group in both cir-
cular and longitudinal SM (Fig. 7A). DFO and FOS increased the fre-
quency of circular SM contractions when compared to the control group
(Fig. 7C) and also increased the duration of contractions in longitudinal
SM (Fig. 7E). The representative traces of the contractions of proximal
Fig. 4. Effects of a week of dietary supplementation with DFO on the number of contractile responses for 30 min in the entire colon. Mice were treated with 0.2 mL
DW, DFO (100, 500 and 1000 mg/kg, p.o.), FOS (1000 mg/kg, p.o.), and bifidobacteria (10
9
CFU, p.o.) for a week: (A) Number of total contractions, (B) Number of
non-propagation contractions, (C) Number of propagation contractions, and (D) Velocity of fecal pellet propulsion through whole colon. Data are means ± SEM
(n = 5). *P< 0.05, **P< 0.01 and ***P< 0.001 compared to control group (DW).
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colonic circular and longitudinal SM of mice treated with DW, DFO, and
FOS for 14 days were shown in Supplemental Fig. S10 and S11. In the
distal colon, in the groups treated with 500 mg/kg DFO and 1000 mg/
kg FOS, the amplitude of contractions in circular SM was significantly
higher than the amplitude of the control group. The amplitude of
contractions in longitudinal SM showed no change (Fig. 7B). In contrast
to the amplitude, no significant difference was measured in the fre-
quency of contractions after administration of DFO and FOS (Fig. 7D).
The duration of contractions of the longitudinal SM of DFO- and FOS-
treated mice was significantly longer than the control group (Fig. 7F).
The representative traces of the contractions of distal colonic circular
and longitudinal SM of mice treated for 14 days were shown in Sup-
plemental Fig. S12 and S13.
3.6. Effects of DFO on colonic smooth muscle histology
To determine whether DFO increased the thickness of the colonic
SM, the histological characteristics of the colonic wall were determined.
There was no evidence that consumption of 500 mg/kg DFO over the
two week period had irritated the colonic mucosa. The morphology of
the simple squamous epithelium was normal. The number of goblet
cells present in the mucosal layer was comparable to the numbers
present in FOS- and DW-treated groups (Fig. 8A, arrows). Regarding the
muscular layer, there was no significant difference in the thickness of
the SM layer in the DFO-treated group compared to both FOS- and DW-
treated groups (Fig. 8B).
4. Discussion
The present study of mice found that dietary supplementation with
DFO accelerated upper gut transit, which reduced travel time of the
content to the colon and also reduced total gut transit time. This result
is consistent with the results from the reference FOS- and bifido-
bacteria-treated groups and supplementation with short-chain FOS and
products containing Lactobacillus or Bifidobacterium species also re-
duced intestinal transit time in human adults [33,34]. Reduced in-
testinal transit time was reported in malabsorptive states and diarrhea
symptoms [35]. However, in this study, the BW of DFO-treated groups
did not change compared to the control group and no diarrheal feces
were observed. DFO is a non-digestible, fermentable, and soluble short-
chain carbohydrate. When consumed, around 50% of DFO is estimated
to reach the colon. The rest is hydrolyzed by salivary and pancreatic α-
amylases (16%), gastric juice (2.5%), and small intestinal brush-border
enzymes (30%). In the intestine, this soluble fiber has an appreciable
water holding capacity that increases fecal mass. However, the water
holding capacity of DFO is known to be less than that of other fibers,
such as wheat fiber [36]. Fecal pellet wet weight significantly increased
in DFO- and FOS-treated groups in this study, compared to the control
group. However, the percentages of fecal water content were not sig-
nificantly different. These results are consistent with the finding of a
previous study that fecal water content in dogs was not influenced by
FOS treatment [37].
Increased colonic content stimulated peristaltic or propagation
Fig. 5. Effects of DFO dietary supplementation for two weeks on the number of contractile responses for 30 min in the entire colon. Mice were treated with 0.2 mL
DW, DFO (500 mg/kg, p.o.) and FOS (1000 mg/kg, p.o.) for two weeks: (A) Number of total contractions, (B) Number of non-propagation contractions, (C) Number
of propagation contractions, and (D) Velocity of fecal pellet propulsion through whole colon. Data are means ± SEM (n = 5–6). *P< 0.05 compared to control
group (DW).
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contractions and also accelerated intestinal transit [38]. Peristaltic
contraction reduced the colonic diameter without occluding the lumen.
Thus, these movements did not empty the proximal colon in a single
sweep but rather slowly pushed small amounts of content into the distal
colon. Even though the propagation pattern of the DFO-treated groups
was not significantly different when compared to the control group, a
trend of increase was observed and fecal pellet velocity significantly
increased after one week of treatment with 1000 mg/kg DFO. The effect
of DFO on propagation pattern was comparable to the result obtained
from bifidobacteria but not the result of FOS, which enhanced only the
non-propagation pattern. Distention by natural fecal pellets was a major
trigger for neurally mediated colonic propulsion [39,40]. Thus, it could
reasonably be suggested that peristaltic contractions in the colon were
the result of a distension-triggered motor pattern generator mediated by
the enteric nervous system [41].
In addition to distending the colonic wall by volume, DFO might
alter bowel motility by changing the colonic environment. In vitro
studies showed that bacterial fermentation of oligosaccharides in-
creased production of SCFAs, lowering colonic pH [42–44]. The lower
pH stimulated the growth of lactobacilli and bifidobacteria and sup-
pressed the growth of harmful bacteria [43]. Increasing fermentation
by-products such as gas and SCFAs could increase stool bulk and also
stimulate gut motility [45]. There are many conflicting studies about
the effects of prebiotics and probiotics on GI motility. Some of these
studies suggested that these supplements increased intestinal motility,
while others concluded the opposite [46–48]. Ingestion of Lactobacillus
reuteri reduced the amplitude of colonic contractions at both constant
and increased luminal pressure, which is required to induce phasic
contractions in rats [46]. On the other hand, the administration of
fermented milk prepared with Lactobacillus casei enhanced colonic
propulsive contraction and defecation rate in pigs [47]. Another study
proposed that healthy newborns fed with breast milk had softer stools
Fig. 6. Effects of a week of DFO supplementation on spontaneous (A, C and E) proximal and (B, D and F) distal colonic circular and longitudinal SM contractions in
mice. Mice were treated with 0.2 mL DW, DFO (100, 500 and 1000 mg/kg, p.o.), FOS (1000 mg/kg, p.o.), and bifidobacteria (10
9
CFU, p.o.) for a week. Values are
means ± SEM (n = 10) and are expressed as (A and B) a percentage of the maximum amplitude of contraction, (C and D) contractions/min, and (E and F) the
duration of contractions in seconds. *P< 0.05, **P< 0.01 and ***P< 0.001 compared to vehicle control group (DW).
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and higher stool frequency than newborns fed with bovine milk. GOS
and FOS supplementation reduced stool consistency but increased stool
frequency [48]. In agreement with previous studies, we found that DFO
accelerated gut transit by increasing intestinal motility, fecal pellet
velocity, and also the number of colonic contractions, especially non-
propagation pattern contractions.
Non-propagation or segmenting contractions cause mixing and local
circulation of contents. This pattern may slow gut transit to counteract
the strong effect of DFO on peristaltic contraction. Therefore, in this
study, no adverse effects from DFO supplementation, such as diarrhea
or malabsorption, were observed. Normally the anal canal is closed by
internal anal sphincter contraction. When the rectum is distended, the
internal sphincter relaxes by reflex. Rectal distention elicits a sensation
that signals the urge for defecation, which is prevented by the external
anal sphincter. The contraction of the external sphincter is maintained
by reflex activation through dorsal roots in the sacral spinal cords. Since
there was no significant difference in evacuation time in this study, we
concluded that DFO and the other treatments in this study did not affect
neural control over defecation.
Delayed motility or transit of colonic contents may lead to con-
stipation. There is a direct correlation between increased dietary fibers
or prebiotics, increased colonic intraluminal bulk, and enhanced co-
lonic transit or motility. Many studies reported that prebiotics improve
health in a similar way to probiotics but are cheaper, safer, and easier to
incorporate into the diet. However, excessive intake of short-chain
carbohydrates can cause undesirable side effects, such as flatulence,
bloating, rumbling, cramps, and liquid stools, which are all caused by
gas formation and the osmotic effects of certain fermentation products.
Fortunately, 1000 mg/kg/day or less of DFO was usually well-tolerated
by the mice in our study.
Colonic SM contractions are organized to allow optimal absorption
of water and electrolytes, net aboral movement of contents, and the
Fig. 7. Effects of two-week dietary supplementation with DFO on spontaneous (A, C and E) proximal and (B, D and F) distal colonic circular and longitudinal SM
contractions in mice. Mice were treated with 0.2 mL DW, DFO (500 mg/kg, p.o.) and FOS (1000 mg/kg, p.o.) for two weeks. Values are means ± SEM (n = 8) and
are expressed as (A and B) a percentage of the amplitude of maximum of contraction, (C and D) contractions/min and (E and F) the duration of contraction in
seconds. *P< 0.05 and **P< 0.01 compared to vehicle control group (DW).
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Fig. 8. Histological cross-section images (Conventional H&E and PAS staining) of (A) mouse colon paraffin sections and (B) colonic SM thickness of DFO-treated
mice. Mice were orally administered with 0.2 mL DW, DFO (500 mg/kg)or FOS (1000 mg/kg)for two weeks. Arrows in an upper row and a middle row in (A)
represent epithelium and number of goblet cells, respectively; a lower row shows thickness of muscular layer; scale bar =20 μm.
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storage and orderly evacuation of feces. The muscularis layers display
two distinct motility patterns: propagated peristaltic contractions,
which involve the coordinated contractions of the longitudinal and
circular SM; and non-propagated segmentation contractions, which
mainly involve the circular muscle layer. This study showed that DFO
treatment for a week increased the force and duration of contractions in
both circular and longitudinal SM in the proximal colon but only in-
creased the duration of circular SM contractions in the distal colon.
After two weeks of treatment with DFO, the contraction frequency of
circular SM had increased in the proximal but not the distal colon.
Short-chain carbohydrates, including DFO, are very rapidly fermented
in the terminal ileum and proximal colon to produce SCFAs. Therefore,
SM in the proximal colon should be affected much more than SM in the
distal colon [36]. However, the underlying mechanisms that control the
effects of DFO on colonic SM contraction are still unknown.
Recent research found that specific SCFAs, such as butyrate, in-
creased cholinergic-mediated colonic circular SM contractions in rats
[49]. Hurst and co-workers also reported that butyrate, acetate, and
propionate in the colonic lumen have different effects on proximal and
distal colonic contractions and that these effects were dependent on
chain length. The net effect of SCFAs on contraction depended on the
balance of SCFAs produced when gut microbiota ferment non-digestible
carbohydrates [14]. The occurrence of contractions is always influ-
enced by slow wave activity in the colonic SM cell membrane. The
contractions, however, are initiated by a spike in potential activity,
which occurs when slow waves reach the electrical threshold. Thus,
slow wave frequency sets the maximum frequency of contractions. The
force and duration of muscle contractions are directly related to the
amplitude and frequency of spike potential. The spike potential oc-
currence depended heavily on neuronal and hormonal activities as well
as local chemical agents, but slow waves were extremely regular [50].
Aligned with our results, the effects of DFO might regulate the amount
of spiking but less so the threshold of the slow wave. Therefore, DFO
affects mostly the strength and duration of contractions rather than the
frequency.
Coordinated intestinal circular and longitudinal SM contractions
produce caudal propulsion of luminal contents via peristalsis. Excitatory
factors such as SCFAs from the bifidogenic effect and distention from
the osmotic effect may act on free fatty acid and stretch receptors at the
intestinal epithelia. They stimulate 5-hydroxytryptamine, which acti-
vates CGRP-containing neurons, a series of interneurons, and motor
neurons to release acetylcholine, tachykinin, or substance P orad to the
luminal stimulus. These neurotransmitters cause circular SM contrac-
tions and longitudinal SM relaxation. Vasoactive intestinal peptide and
nitric oxide are released to the luminal stimulus and stimulate circular
SM relaxation and longitudinal SM contractions [50].
Some studies confirmed that prebiotic supplements or SCFAs could
change the colonic structure. Butyrate, for example, exerted a trophic
effect in colonocytes. However, in the present study, DFO did not show
trophic effects on the gut wall nor change SM thickness. Previous stu-
dies also reported that a high fiber diet increased both secretory activity
and numbers of mucin-secreting goblet cells in the colon of rats [51].
Conversely, our study showed no DFO effect on either epithelium or
goblet cell numbers. Since intestinal mucin in colonic mucosa plays a
cytoprotective role against a variety of luminal hazards, and since
goblet cell numbers were altered during intestinal infections, based on
general criteria in histomorphological scores for intestinal inflamma-
tion, changes in epithelial or mucosal architecture did not result from
consumption of DFO [52,53]. These findings may confirm the safety of
this product as a supplement.
Taken together, these data suggest that in addition to desirable
prebiotic properties [25], DFO also acts as a bulk-forming laxative
which absorbs water from the intestinal lumen to increase fecal mass
(osmotic effect) as well as a stimulant laxative that increases intestinal
motility in mice. We also showed an association between DFO ingestion
and alteration to colonic SM contractility. These findings confirm that
DFO may be suitable to supplement prebiotic/probiotic/symbiotic
products and laxative products. DFO may also be a promising nutri-
tional therapy for GI motility disorders such as constipation and IBS.
Nevertheless, further investigation is required to identify the under-
lying mechanisms responsible for changes in GI motility induced by diet
or gut bacteria.
Disclosure
None of the authors had conflicts of interest throughout the study
process.
Authorship contributions
The authors’responsibilities:
P.K., S.P., C.T., S.W., and N.C. - conceptualized and designed re-
search;
P.K., S.K., K.B., F.H., and C.T. - conducted research;
S.W. - provided essential reagents;
P.K., S.K., K.B., F.H., and C.T. - analyzed data;
P.K., C.T. and N.C. - wrote the paper;
P.K. - reviewed final content;
All authors: reviewed and approved final version of the manuscript.
Acknowledgements
This work was supported by a grant from Thailand Research Fund
(P.K., grant number MRG5980042), (N.C. is a TRF Senior Research
Scholar, grant number TRF; RTA6080007). The authors are grateful for
Publication Clinic of Prince of Songkla University and Mr. Thomas
Coyne for providing assistance in proofreading and providing feedback
on the manuscript.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.biopha.2019.108821.
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