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Evaluation of the Effect of Blackcurrant Products on Gut Microbiota and on Markers of Risk for Colon Cancer in Humans

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The purpose of this study was to determine in healthy humans whether First Leaf (FL; composed of blackcurrant extract powder, lactoferrin and lutein) and Cassis Anthomix 30 (CAM30; blackcurrant extract powder) can positively modify the colonic microbiota by enhancing the growth of the beneficial bacteria and inactivating the toxic bacterial enzymes which are known to be involved in colonic carcinogenesis. Thirty healthy adult male and female volunteers were recruited for this study. Fluorescent in situ hybridization was carried out to analyse the populations of fecal microbiota. Consumption of FL and CAM30 led to significant increases (P < 0.0001) in the population sizes of lactobacilli and bifidobacteria whereas the population sizes of Clostridium spp. and Bacteroides spp were decreased significantly (P < 0.0001). In addition, feeding of FL and CAM30 decreases the activity of β-glucuronidase (bacterial enzyme which is considered to be one of the enzymes that increases risk for colorectal cancer) and significantly decreased (P < 0.05) the fecal pH. In conclusion, the results of this study open up the possibility that consumption of FL and CAM30 can offer various benefits to human health through acting as novel prebiotic agents via increasing the numbers of beneficial bacteria (lactobacilli and bifidobacteria) in the gut. Copyright © 2013 John Wiley & Sons, Ltd.
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Evaluation of the Effect of Blackcurrant Products
on Gut Microbiota and on Markers of Risk for
Colon Cancer in Humans
Abdul-Lateef Molan,*
,#
Zhuojian Liu and Gabriel Plimmer
Institute of Food, Nutrition and Human Health, College of Health, Massey University, Palmerston North, New Zealand
The purpose of this study was to determine in healthy humans whether First Leaf (FL; composed of blackcurrant
extract powder, lactoferrin and lutein) and Cassis Anthomix 30 (CAM30; blackcurrant extract powder) can
positively modify the colonic microbiota by enhancing the growth of the beneficial bacteria and inactivating
the toxic bacterial enzymes which are known to be involved in colonic carcinogenesis. Thirty healthy adult male
and female volunteers were recruited for this study. Fluorescent in situ hybridization was carried out to analyse
the populations of fecal microbiota. Consumption of FL and CAM30 led to significant increases (P <0.0001) in
the population sizes of lactobacilli and bifidobacteria whereas the population sizes of Clostridium spp. and
Bacteroides spp were decreased significantly (P <0.0001). In addition, feeding of FL and CAM30 decreases
the activity of b-glucuronidase (bacterial enzyme which is considered to be one of the enzymes that increases risk
for colorectal cancer) and significantly decreased (P <0.05) the fecal pH. In conclusion, the results of this study
open up the possibility that consumption of FL and CAM30 can offer various benefits to human health through
acting as novel prebiotic agents via increasing the numbers of beneficial bacteria (lactobacilli and bifidobacteria)
in the gut. Copyright © 2013 John Wiley & Sons, Ltd.
Keywords: FL; CAM30; blackcurrant products; prebiotic activity; bacterial enzymes.
INTRODUCTION
The human colon is a rich microbial environment that
harbours a large and complex microbiota, and it is now
generally accepted that the composition of the human
intestinal microbiota has an important role in health and
disease (Guamer and Malagelada, 2003). The dietary
positive modulation of gut microbiota via increasing the
populations of the beneficial bacteria such as lactobacilli
and bidobacteria could lead to various health benets
through different mechanisms (Collins and Gibson,
1999). These mechanisms may include reducing gut pH
through stimulation the growth of the lactic acid-producing
microbiota (Langhendries et al., 1995), improvement of
immune function and stimulation of appropriate immuno-
modulatory cells (Isolauri et al., 1995) and direct antagonistic
effects on pathogens (Gibson and Wang, 1994).
Blackcurrant (Ribes nigrum) berries or its products
are potent antioxidants. Miller and Rice-Evans (1997)
reported that a blackcurrant drink (Ribena
W
) has higher
antioxidans activity than orange and apple. Recently, it
has been reported that long-term consumption of both
the boysenberry and blackcurrant drinks raised the
plasma total antioxidant capacity of the study participants
suggesting that boysenberry and blackcurrant may help
protect against oxidative stress-related health conditions
(McGhie et al., 2007). More recently, Lyall et al. (2009)
reported that consumption of blackcurrant anthocyanins
alleviate exercise-induced oxidative stress, and may, if
given at the appropriate amount and time, complement
the ability of exercise to enhance immune responsiveness
to potential pathogens.
Our previous study (Molan et al., 2010) provided the
rst evidence that consumption of FL and CAM30 can
beneficially affect fecal parameters related to colon cancer
risk in rats via decreasing the activity of the bacterial
b-glucuronidase enzyme (an enzyme important in carcin-
ogen activation) and fecal pH. In addition, our results
demonstrated the ability of FL and CAM30 to act as
prebiotics, as evidenced by their capacity to increase
significantly the numbers of beneficial bacteria such as
lactobacilli and bidobacteria.
b-glucuronidase interferes with the detoxication process
by uncoupling glucuronides and consequently deconjugating
potential toxins, increasing the formation of carcinogens
in the bowel and promoting the enterohepatic recirculation
of toxins, hormones and various drugs in the body
(Kuhn, 1998; LoGuidice et al., 2012). Furthermore, research
correlates elevated levels of b-glucuronidase with increased
colon cancer risk and that excessive b-glucuronidase activity
may be a primary factor in the aetiology of colon cancer
(Kim and Jin, 2001; Gill and Rowland, 2002; De Moreno
de LeBlanc and Perdigon, 2005).
The purpose of the present study was to measure the effect
of FL and CAM30 on the numbers of fecal lactobacilli and
bidobacteria using molecular method (fluorescent in situ
hybridization; FISH) in healthy subjects, numbers of fecal
Bacteroides spp. and Clostridium spp. using FISH method.
The effect of these products on some markers for the risk
of colon cancer such as the bacterial b-glucuronidase
enzyme and the fecal pH was also investigated.
* Correspondence to: Abdul-Lateef Molan, Institute of Food, Nutrition
and Human Health, College of Health, Massey University, Palmerston
North, New Zealand.
E-mail: A.L.Molan@massey.ac.nz
#
Present address: Department of Biology, College of Sciences, Diyala
University, Diyala, Iraq
PHYTOTHERAPY RESEARCH
Phytother. Res. 28: 416422 (2014)
Published online 15 May 2013 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/ptr.5009
Copyright © 2013 John Wiley & Sons, Ltd.
Received 14 January 2013
Revised 04 April 2013
Accepted 08 April 2013
SUBJECTS AND METHODS
Nutritional supplementation and study design. The
commercial products First Leaf (FL; composed of
blackcurrant extract powder, lactoferrin and lutein is
developed by Four Leaf Japan Co., Ltd. Japan) and
Cassis Anthomix 30 (CAM30; blackcurrant extract powder
developed by Just the Berries Ltd. New Zealand) were
encapsulated in gelatine. Study participants were given
a total of four capsules per day.
Volunteers were recruited from the general population
through the local newspaper, and Massey University
Campus advertising. Thirty healthy volunteers aged
2060 years were recruited for the study, and all the
subjects (16 women, 14 men) completed the study. They
were randomly divided into FL (8 women, 7 men) and
CAM30 groups (8 women, 7 men). In group one, the
subjects were given the recommended dosage of FL
(1500 mg/day; 375 mg 4 capsules) while the subjects
in group two were given the recommended dosage of
CAM30 (672 mg/day; 168 mg 4 capsules) according
to the instruction from the funding company. Both
products contain the same amount of blackcurrant
powder (672 mg).
The study was divided into three steps: a 2-week base-
line step [Day (D) -14 to D0; period 1], a 2-week
supplementation step (D0-D14; period 2) and a 2-week
wash-out step (D14-D28; period 3). Stool samples were
collected at baseline (d0; period 1), and after 2 weeks
(d14; period 2) of products ingestion, followed by
samples at week 6 (d28; period 3). Every volunteer
was provided with a cooler lled with ice for storage of
the sample until delivery to the laboratory. All samples
were delivered on ice within 12 h of defecation and then
stored at 80
o
C until processing with FISH method as
described below.
Pre-enrolment screening. The trial was preceded by a
screening procedure that included completion of health
and dietary/lifestyle questionnaire. This study has been
reviewed and approved by the Massey University
Human Ethics Committee: Southern A, application 10/10.
All the participants gave their written informed consent.
Participants were instructed to keep their usual die-
tary habits for the duration of the study, except for
avoiding intake of vitamin supplements, foods rich in
anthocyanins (red-coloured fruits and vegetables, all
kinds of berries, grapes, red wine and berry juices) and
fermented dairy products containing viable bidobacteria
or lactobacilli because they have been shown to lead to
increase in fecal bidobacteria and lactobacilli within a
few days of consumption. Other exclusion criteria were: a
history of gastrointestinal diseases other than appendicitis,
alcohol (more than 2 units/day), smoking, endocrine
disease, diagnosis of any form of cancer, vascular disease
or diabetes mellitus, antibiotics or laxatives taken during
the month before the study, other medications taken
during the study period and recent major surgery.
Measurements
FISH analysis of microbiota in feces. The probes used in
the study were Bif164, Bac303, His150 and Lab158, spe-
cicforbidobacteria, bacteroides, clostridia (perngens/
histolyticum subgroup) and lactobacilli/enteroccocus,
respectively. These were commercially synthesised and
labelled with the fluorescent dye Cy3 (GeneWorks,
Australia). The samples were prepared for FISH analysis
as described by Dinoto et al. (2006) with some modica-
tions. In short, fecal samples were prepared by mixing 1
g with 9 ml of sterile ice-cold phosphate buffer (PBS;
pH 7.2). The mixture was homogenized by vortexing
with a dozen glass beads for 3 min, the fecal debris was
removed by centrifugation at low speed (700 g) and the
bacteria containing supernatant was xed in 4% (w/v)
paraformaldheyde in PBS (pH 7.2) overnight at 4
o
C.
Fixed samples were washed in PBS and stored in a
known volume of 50% (v/v) ethanol-PBS at 20C until
time of hybridization. Aliquots (5 ml) of xed bacterial
cells were applied to Teon-coated microscopic slides
(BIOLAB, New Zealand; 10 wells) and air dried. The
bacterial cells were then dehydrated with a series of
solutions containing 50%, 80% and 99.5% ethanol
(3 min for each concentration). The bacterial cells xed
on the glass slides were hybridized by addition of 8 ml
of hybridization buffer (0.9 M NaCl, 0.01% sodium
dodecyl sulfate, 20 mM Tris-HCl, 20% deionized
formamide; pH 7.2) with 0.5 ml of Cy3-labeled oligonu-
cleotide specic probes (50 ng/ml). The slides were
hybridized at 46C for 2 h in a plastic box containing
wet sponges (soaked in hybridization buffer). After
hybridization, the slides were rinsed with warm hybridi-
zation buffer at 48C and washed in pre-warmed washing
buffer (225 mM NaCl, 0.01% sodium dodecyl sulfate,
20 mM Tris-HCl; pH 7.2) for 20 min at 48C. After
washing step, the slides were rinsed with ice-cold distilled
water and thoroughly dried before being observed using
auorescence scanning microscope.
The dried slides were examined with an Olympus
BX51 microscope, under 400X magnication. The
images were captured using an Optronics MagnaFIRE
SS99802 digital camera with MagnaFIRE frame-grabbing
software on a Pentium IV computer (Manawatu Micros-
copy and Imaging Centre, Massey University, Palmerston,
New Zealand). Fluorescent cells were counted automat-
ically in 10 randomly selected elds/slide using ImageJ
(Abramoff et al., 2004).
Bacterial enzyme activities. The b-glucuronidase and
b-glucosidase activities were determined aerobically
according to the method of De Preter et al. (2008) with
some modications. Briey, fecal samples (100 mg/ml)
were suspended in cold 0.1M potassium phosphate
buffer (pH 7.0). The mixture was homogenized by
vortexing, and the fecal suspension was mixed with equal
volume of 0.1 M phosphate buffer (pH 7) containing 0.2%
Triton-X-100 to disrupt the bacterial cells. The mixture
was centrifuged at 1200 g for 15 min at 4
o
C. The super-
natant was used for the assessment of b-glucuronidase
and b-glucosidase activities.
b-Glucuronidase. Fecal supernatant (10 ml) was added
to 40 ml reaction mixture (0.1M potassium phosphate
buffer, 0.1mM ethylenediaminetetraacetic acid and
0.05mM phenolphthalein b-glucuronide) in a 96-well
microplate. The enzyme reaction was run for 60 min at
37
o
C and was stopped by addition of 250 ml of 0.2M
glycine buffer (pH 10.4) containing 0.2M NaCl. For each
sample, a control (fecal supernatant with reaction
mixture without phenolphthalein b-glucuronide substrate)
417BLACKCURRANT PRODUCTS AND GUT MICROBIOTA
Copyright © 2013 John Wiley & Sons, Ltd. Phytother. Res. 28: 416422 (2014)
was determined which was incubated for the same time
period. The amount of phenolphthalein released from
the enzymatic reaction was quantied by measuring the
ultraviolet absorption at 550 nm using a plate reader
(ELX 808, automated microplate reader, BIO-TEK
Instruments, INC, Vermont, USA). Concentrations were
calculated, after correction for controls, from a standard
curve of phenolphthalein and were expressed as milligrams
of phenolphthalein per gram feces per hour.
This assay is based on the assumption that the b-
glucuronidase enzyme present in the fecal sample
deconjugates phenolphthalein from the phenolphthalein-
containing substrate. Thus, the amount of phenolphthalein
released is representative of the enzyme activity (De Preter
et al., 2008).
b-Glucosidase. Fecal supernatant (10 ml) was added to
40 ml reaction mixture (0.1M phosphate-buffered saline,
1mM p-nitrophenyl b-pyranoside) in a 96-well microplate.
The reaction was run for 60 min at 37
o
C and was stopped
by addition of 250 ml of 0.1M NaOH. For each sample, a
control (fecal supernatant with reaction mixture without
p-nitrophenyl b-pyranoside substrate) was determined
which was incubated for the same time period. Ultraviolet
absorption was measured at 450 nm.
Concentrations were calculated, after correction for
controls, from a standard curve of p-nitrophenol and
were expressed as milligrams of p-nitrophenol per gram
feces per hour.
Stool pH. An association between high colonic (or
stool) pH and increased risk of colon cancer has been
reported (Isikawa et al., 1995). Accordingly, the pH of
each stool sample was measured in order to test if there
is any link between the concentrations of b-
glucuronidase, b-glucosidase, the numbers of lactobacilli
and bidobacteria and the stool pH. The pH was mea-
sured using a digital pH-meter at room temperature
(2022
o
C).
Statistical analysis. Before analyses, bacterial numbers
were log transformed to improve normality. Fecal
concentrations of Bidobacterium spp., Lactobacillus
spp., Clostridium spp. and Bacteroides spp. were expressed
as log bacterial cells/g wet weight. Data are presented as
means SEM. Repeated measures analysis of variance
(ANOVA) was used to compare: (i) the bacterial popu-
lation levels, bacterial enzymes and pH at d0 (period 1)
in each group with treatment as factor and (ii) the same
parameters between d0 and d14 (period 2) and between
d0 and d28 (period 3) in each group with time and
treatment as factors. Following a significant F test
(P<0.05), one-way ANOVA and Tukeys test were
performed to test for significant differences among
means. SAS version 9.2 for Windows was used for this
analysis, and the differences were considered statistically
significant at P <0.05.
RESULTS
Prebiotic and antimicrobial activities
The results of this study showed that consumption of FL
(composed of blackcurrant extract powder, lactoferrin
and lutein) and Cassis Anthomix 30 (CAM30; blackcurrant
extract powder) does not decrease the population sizes
of the beneficial bacteria such as lactobacilli and
bidobacteria. Although the numerical differences
between the treatment period (period 2) and the baseline
period (period 1) are small, consumption of these products
has led to significant increases in the population sizes of
Lactobacillus spp. and Bidobacterium spp. (P <0.0001).
Two weeks after ceasing the supplementation (period 3)
with FL and CAM30, there was no significant increase in
the fecal counts of Lactobacillus spp. and Bidobacterium
spp. (Table 1) when compared with the baseline period
(period 1).
Consumption of FL and CAM30 led to a significant
reduction in the population sizes of fecal Clostridium
spp. and Bacteroides spp. (P <0.0001) relative to the
numbers at the baseline period (Table 1). No signicant
differences in the numbers of Bacteroides spp. or
Bacteroides spp. were observed between period 1 and
period 3.
Activity of microbial enzymes (b-glucuronidase and
b-glucosidase)
b-glucuronidase. The results indicate that consumption
of the FL and CAM30 by healthy subjects can benecially
affect fecal parameters related to colon cancer risk.
Consumption of FL and CAM30 clearly lowered the
activity of b-glucuronidase in the feces of the subjects
(Fig. 1). The decrease in activity was statistically signi-
cant (P <0.05) in comparison with the baseline period.
The levels of b-glucuronidase at the end of
supplementation with FL and CAM30, expressed as a
Table 1. Enumeration of Lactobacillus,Bidobacterium,Clostridium and Bacteroides species (Log
10
cells/g of wet feces) from the fecal
samples hybridized with genus-specic oligoneucleotide probes in FISH analysis. The volunteer groups were given First Leaf (FL; four
capsules of 375 mg each/day) or Cassis Anthomix 30 (CAM30; four capsules of 168 mg each/day) daily for 14 consecutive days
Microbial group
FL CAM30
Period 1* Period 2 Period 3 Period 1 Period 2 Period 3
Lactobacillus spp. 7.54 0.02
a
7.96 0.02
b
7.60 0.03
a
7.54 0.04
a
7.89 0.04
b
7.64 0.04
a
Bifidobacterium spp. 7.93 0.04
a
8.5 0.03
b
8.05 0.06
a
8.07 0.06
a
8.3 0.05
b
8.15 0.07
ab
Clostridium spp. 8.60 0.02
a
8.03 0.03
b
8.51 0.04
a
8.5 0.02
a
8.1 0.03
b
8.47 0.03
a
Bacteroides spp. 8.40 0.03
a
7.94 0.05
b
8.27 0.06
a
8.36 0.02
a
7.79 0.04
b
8.32 0.03
a
*The data are expressed as means standard errors of the means (n =15 subjects/group and 6 replicates per subject). For each product,
means with different superscript letters within a row are significantly different at P 0.05.
418 A-L. MOLAN ET AL.
Copyright © 2013 John Wiley & Sons, Ltd. Phytother. Res. 28: 416422 (2014)
percentage of baseline concentrations, decreased by 25.5%
and 24.4%, respectively (data not shown). Moreover,
b-glucuronidase levels decreased by 20.9% and
10.6% of baseline levels after the washout period in
subjects consuming FL and CAM30, respectively (data
not shown).
b-glucosidase. Consumption of FL and CAM30 led to
significant increases (P <0.01) in the activity of b-
glucosidase in comparison with the baseline (period 1)
values (Fig. 2). Two weeks after termination of the
supplementation (washout, period 3) with CAM30, the
activity of this enzyme was still significantly higher
(P <0.05) than the baseline values.
b-glucosidase levels increased by 32.8 and 30.3% after
consumption of FL and CAM30 (period 2), respectively,
when compared with the baseline levels (data not shown).
Two weeks after termination of the supplementation with
FL and CAM30, the levels of b-glucosidase in the feces
were still higher than the baseline period by 21.4% and
21.0%, respectively.
Fecal pH. It can be seen from Fig. 3 that consumption
of FL and CAM30 resulted in a significant decrease
(P <0.05) in the fecal pH when compared with the
baseline values.
DISCUSSION
This study has identied FL (composed of blackcurrant
extract powder, lactoferrin and lutein) and Cassis
Anthomix 30 (CAM30; blackcurrant extract powder)
as good prebiotics that can significantly promote the
growth of beneficial bacteria such as bidobacteria and
lactobacilli and lower the numbers of other bacteria
such as bacteroides and clostridia. Such ndings suggest
that these products might have the potential to promote
the survival, colonization and activity of beneficial
bacteria in the gastrointestinal tract, thus potentially
improving their beneficial effects. Our previous results
(Molan et al., 2010) showed that gavaging rats with FL
and CAM30 resulted in a significant increase in the
numbers of bidobacteria and lactobacilli and a signicant
decrease in the numbers of bacteroides and clostridia.
Many health benets have been associated with
bidobacteria and lactobacilli in the gut microbiota such
as inhibition of gut pathogens (Gibson and Wang, 1994),
prevention of colon cancer (Reddy, 1998; Burns and
Rowland, 2000; Geier et al., 2006), synthesis of vitamins
and enhancing the immune system (Gill, 1998).
FL is a supplement containing lutein, lactoferrin and
blackcurrant extract, and consequently, the health
benets shown in the present study may be related to
Figure 1. b-glucuronidase enzyme activity (mg/g wet feces) in
feces of two groups of healthy volunteers supplemented with First
Leaf (panel A) or CAM30 (panel B) before (period 1), during (period
2) and after (period 3) treatment. Values are meansstandard
errors of the means (n = 15 subjects/group; six replicates per
subject). * P 0.05 versus the baseline (period 1).
Figure 2. b-glucosidase enzyme activity (mg/g feces) in feces of
two groups of healthy volunteers supplemented with First Leaf
(panel A) or CAM30 (panel B) before (period 1), during (period 2)
and after (period 3) treatment. Values are means standard errors
of the means (n = 15 subjects/group; six replicates per subject).
*P<0.05 versus the baseline (period 1).
419BLACKCURRANT PRODUCTS AND GUT MICROBIOTA
Copyright © 2013 John Wiley & Sons, Ltd. Phytother. Res. 28: 416422 (2014)
one or a combination of these ingredients. Lactoferrin is
a multifunctional protein (Valenti and Antonini, 2005),
and it is known to have antibacterial activity towards a
wide range of bacterial pathogens (Kai et al., 2002), broad
spectrum antiviral activity (Florisa et al., 2003; Seganti
et al., 2004), and antifungal effects (Samaranayake et al.,
2001). The antibacterial and antifungal effects are mainly
linked to iron sequestration and destabilization of the
bacterial and fungal membranes while the antiviral effect
is mainly related to inhibition of viral host cell interaction
through blocking of host cell heparan sulfate or interaction
with viral surface proteins (Jenssen and Hancoc, 2009).
Lutein is a potent antioxidant (Zhang et al., 1991)
and some studies have shown that its high dietary
intake enhances immune function and also has been
associated with risk reduction of many chronic dis-
eases, including age-related macular degeneration,
cancer and cardiovascular diseases (Hadden et al.,
1999). Moreover, some studies have shown that a
supplement containing lutein, zeaxanthin and
blackcurrant extract has beneficial effects on visual
functioning (Richer et al., 2004).
Our previous study (Molan et al., 2010) provided the
rst evidence that consumption of FL and CAM30 can
beneficially affect fecal parameters related to colon
cancer risk in rats via decreasing the activity of the
bacterial b-glucuronidase enzyme. Some studies correlate
elevated levels of b-glucuronidase with increased colon
cancer risk (Kim and Jin, 2001; De Moreno de LeBlanc
and Perdigon, 2005).
The present study conrmed the results of the previous
study and showed that FL and CAM30 can decrease the
activity of b-glucuronidase and might therefore reduce
the carcinogenicity of feces. This could be perceived as
potentially beneficial for the subjects.
Kim and Jin (2001) measured the fecal b-glucuronidase
activity of patients with colon cancer and healthy con-
trols in order to determine the relationship between
the uctuation of intestinal bacterial b-glucuronidase
and colon cancer. The authors stated that the fecal
b-glucuronidase activity of patients with colon cancer
was 1.7 times higher than that of the healthy controls,
and when the fecal specimens were sonicated, the
enzyme activity of patients with colon cancer was
12.1 times higher than that of the healthy controls. It
was concluded that potent b-glucuronidase activity is
a prime factor in the etiology of colon cancer.
Thus, lower activity of this enzyme can be considered
beneficial in terms of the risk of colon cancer (Gill and
Rowland, 2002). Moreover, De Moreno de LeBlanc
and Perdigon (2005) reported that a number of bacterial
enzymes including b-glucuronidase and nitroreductase
play an important role in cancer development as
they hydrolyse carcinogenic compounds and that
consumption of yoghurt starter bacteria is able to re-
duce the activity of these enzymes, indicating a possi-
ble mechanism by which probiotics can prevent
colorectal cancer.
The results of the present study have provided the
rst evidence that consumption of FL and CAM30 by
healthy subjects can beneficially affect fecal parameters
related to colon cancer risk such as b-glucuronidase
and fecal pH. The low activity of b-glucuronidase
detected in this study may indicate that the FL
and CAM30 could have anticarcinogeneic capacity,
but this needs to be conrmed in animal and human
trials. These results may be explained by the signicant
reduction in the populations of Bacteroides spp.
and Clostridium spp. This enzyme is generated by a
wide range of gut bacteria including Escherichia coli,
some species of Bacteroides and Clostridium (Gloux
et al., 2011).
The proposed ability of FL and CAM30 to minimize
the risk factors of colon cancer relies mainly on the
proven prebiotic activity, via increasing the populations
of the beneficial bacteria such as lactobacilli and
bidobacteria in rats (Molan et al., 2010) and humans
(present study), on the capacity to reduce the numbers of
pathogenic bacteria in rats (Molan et al., 2010) and humans,
especially those generating the harmful b-glucuronidase
enzyme, and on the ability to reduce the fecal pH in both
rats and humans. Reddy (1999) reported that increased
numbers of bidobacteria/lactobacilli within the gut
microbiota may reduce either the numbers and/or
activities of putrefactive bacteria in the colon, leading to
a reduction in enzyme activities involved in carcinogenic
pathways as well as toxic metabolites such as amines,
indoles, p-cresol and skatol.
Although the precise mechanism by which lactic acid
bacteria may inhibit colon cancer are unknown, Geier
et al. (2006) reported in their review that the strategy of
using probiotics, prebiotics and synbiotics in the treatment
of colorectal cancer has the potential to inhibit the
development and progression of neoplasia via mechanisms
including; decreased intestinal inammation, enhanced
immune function and anti-tumorigenic activity, binding
Figure 3. Fecal pH values of healthy volunteers given First Leaf
(panel A) or CAM30 (panel B) before (period 1), during (period 2)
and after (period 3) treatment. Values are means standard errors
of the means (n = 15 subjects/group). * P 0.05 versus the baseline
(period 1).
420 A-L. MOLAN ET AL.
Copyright © 2013 John Wiley & Sons, Ltd. Phytother. Res. 28: 416422 (2014)
to potential food carcinogens including toxins found in
meat products and a reduction in bacterial enzymes
which hydrolyse precarcinogenic compounds, such as b-
glucuronidase.
The results of the present study showed that FL and
CAM30 supplementation significantly decreased the
fecal pH value. This nding supports the results of our
previous study (Molan et al., 2010). It has been found
that fecal pH tended to be higher in subjects with
colorectal cancer than in those with adenomas or with
no colorectal disease (Thornton, 1981; Isikawa et al.,
1995). It has been reported that a high colonic pH
promotes co-carcinogen formation from bacterially
degraded bile acids or cholesterol and that acidication
of the colon either by dietary bre or milk may prevent
this process (Thornton, 1981).
The increase in the activity of b-glucosidase during
the consumption of FL and CAM30 is probably due to
the stimulation of the growth of lactic acid bacteria
(prebiotic activity), which have high levels of b-glucosidase
activity in comparison with the other members of the gut
microbiota (Saito et al., 1992). Previous studies have shown
that oligosaccharides have the ability to increase the activity
of this enzyme, and this has been attributed to the ability of
oligosaccharides to stimulate the growth of lactic acid bacte-
ria (Saito et al., 1992; Rowland and Tanaka, 1993). Marteau
et al. (1990) found an increase in fecal b-glucosidase activity
in subjects consuming milk fermented with Lactobacillus
acidophilus and Bidobacterium bidum. Hughes and
Rowland (2001) reported that the increase in rat colonic
b-glucosidase activity during the oligosaccharide diets
(chicory fructans-oligofructose and inulin) may represent
a prebiotic effect.
CONCLUSIONS
Consumption of FL and CAM30 led to significant
increases in the population sizes of both lactobacilli and
bidobacteria when compared to the baseline period,
and this prebiotic activity may explain the high activity
of b-glucosidase, which is generated mainly by these
beneficial bacteria. In addition, consumption of FL and
CAM30 led to a significant reduction in the activity of
b-glucuronidase, a proposed marker for colon cancer. The
results also showed that FL and CAM30 supplementation
significantly decreased the fecal pH value. The results of
this study open up the possibility that consumption of FL
and CAM30 can offer various benets to human health
through acting as novel prebiotic agents (via increasing
the numbers of lactobacilli and bidobacteria in the gut)
and may have anticancer activity via lowering the
fecal pH and decreasing the activity of the bacterial
b-glucuronidase enzyme. It is important to mention that
these results need to be conrmed in another randomized,
double-blind, placebo-controlled, and crossover human
study involving healthy volunteers.
Acknowledgements
This study was supported by Four Leaf Japan Co., Ltd.and Just the
Berries Ltd..
Conf lict of Interest
The authors declare that there are no conicts of interest about the
present study.
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Abramoff, M.D., Magelhaes, P.J., Ram, S.J. "Image Processing with ImageJ". Biophotonics International, volume 11, issue 7, pp. 36-42, 2004.
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The first function attributed to lactoferrin (Lf), an iron binding protein belonging to the non-immune natural defences, was antimicrobial activity that depended on its capacity to sequester iron. Iron-independent microbicidal activities, requiring direct interaction between this cationic protein and microbial surface components, were later demonstrated. Many other anti-microbial and anti-viral functions have since been ascribed to Lf. In mucosal secretions, iron and Lf modulate the motility and aggregation of pathogenic bacteria. Lf inhibits bacterial adhesion on abiotic surfaces through ionic binding to biomaterials, or specific binding to bacterial structures or both. Lf inhibition of bacterial adhesion to host cells requires Lf binding to bacteria and/or host cells. Lf hinders microbial internalization by binding to both glycosaminoglycans and bacterial proteins which can be degraded by Lf-mediated proteolysis. Moreover, Lf internalisation and localisation to the host cell nuclei could modulate bacterial entry into cells through gene regulation. Finally, the capability of Lf to exert antiviral activity, through its binding to host cells and/or viral particles, strengthens the idea that it is an important brick in the mucosal wall, effective against both microbial and viral attacks.
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An optimally functioning immune system is essential for protection against infectious diseases and cancers. Deficiency in any component of the immune system can predispose an individual to a greater risk of infection or may enhance the severity of disease. Several studies have shown that dietary supplementation with lactic acid bacteria (LAB) can be used to promote health and well-being; LAB are normal components of the human intestinal flora and are commonly used as starter cultures in dairy products. Consumption of LAB has been associated with a variety of health benefits including enhanced immune performance, increased resistance to infectious diseases, alleviation of food allergies and the suppression of cancer development. The precise mechanisms by which LAB act on the immune system are not fully understood. However, there is sufficient evidence to suggest that LAB exert their immunity enhancing effects by augmenting both non-specific (e.g. phagocyte function, NK-cell activity) and specific (e.g. antibody production, cytokine production, lymphocyte proliferation, delayed-type hypersensitivity) host immune responses. It is important to note that most of the evidence supporting these immunoenhancing effects is derived from in vitro or animal studies and there is a scarcity of carefully designed and properly controlled clinical studies demonstrating immune health benefits for humans, especially healthy human subjects. This article discusses the impact of LAB on different immune functions, the role of LAB-induced immunoenhancement in disease resistance, and highlights gaps in our knowledge that need further research.