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Dietary saponins from four popular herbal tea
exert prebiotic-like effects on gut microbiota in
C57BL/6 mice
Lei Chen a, William C.S. Tai a, W.L. Wendy Hsiao b,*
aCenter for Cancer & Inflammation Research, School of Chinese Medicine, Hong Kong Baptist University, 7
Baptist University Road, Kowloon Tong, Kowloon, Hong Kong, China
bState Key Laboratory of Quality Research in Chinese Medicine, Macau University of Science and Technology,
Avenida Wai Long, Taipa, Macau, China
ARTICLE INFO
Article history:
Received 2 April 2015
Received in revised form 19 June
2015
Accepted 24 June 2015
Available online 8 July 2015
ABSTRACT
Herbal saponins have raised considerable interest for their health-promoting effects, but
have not been examined for their role as prebiotics.This study aimed to investigate the impact
of saponins on gut microbiota in mice. Saponins from four herbal tea were chosen, i.e. sa-
ponins of ginseng (GS), red ginseng (RGS), notoginseng (NGS), and Gynostemma pentaphyllum
(GpS). PLS-DA plots of the faecal DNA fingerprints revealed that microbiota from the saponins-
treated and untreated mice clustered separately. Real time qPCR showed that some known
beneficial bacteria, Bacteroides, Lactobacillus and Bifidobacterium, were enhanced in the treat-
ment groups. GpS and NGS significantly increased the Bacteroidetes/Firmicutes ratio. Additionally,
Faecalibacterium prausnitzii, a bacterium associated with human intestinal health, was stimu-
lated by GpS treatment in a time-dependent manner. This study, for the first time,
demonstrated that the health-promoting effects of dietary saponins might be, in part, through
the manipulation of the gut microbiota to the benefit of the host.
© 2015 Elsevier Ltd. All rights reserved.
Keywords:
Gut microbiota
Herbal saponins
ERIC-PCR
Prebiotic-like effects
1. Introduction
Gut microbiota plays a vital role in health and disease. They
are essential for ensuring the proper functioning of meta-
bolic reactions, immune regulation, epithelial development, and
protection against pathogens (Bäckhed et al., 2012; Clemente,
Ursell, Parfrey, & Knight, 2012; Ohland & Jobin, 2015). Recent
findings have also reviewed the impact of diet on the gut
microbiota composition in humans and mice, thereby affect-
ing health outcomes (Graf et al., 2015; Scott, Gratz, Sheridan,
Flint, & Duncan, 2013; Tremaroli & Bäckhed, 2012). For example,
early studies have shown that obesity is associated with
changes in the proportion of Bacteroidetes to Firmicutes ob-
served both in humans and mice (Ley et al., 2005; Turnbaugh
et al., 2006). Interestingly, in a small scale human interven-
tion study, obese people taking low-calorie diet showed
increased abundance of Bacteroidetes concurrent with loss of
*Corresponding author. State Key Laboratory of Quality Research in Chinese Medicine, Macau University of Science and Technology, Avenida
Wai Long, Taipa, Macau, China. Tel.: +853 8897 2751; fax: +853 2882 5886.
E-mail address: wlhsiao@must.edu.mo (W.L.W. Hsiao).
Abbreviations: D0, day 0; D5, day 5; D10, day 10; D15, day 15; EDTA, ethylenediaminetetraacetic acid; ERIC, enterobacterial repetitive
intergenic consensus; GpS, Gynostemma pentaphyllum saponins; GS, ginseng saponins; MS, mass spectrometry; NGS, notoginseng sapon-
ins; PLS-DA, partial least squares discriminant analysis; qPCR, quantitative polymerase chain reaction; RGS, red ginseng saponins; UPLC,
ultra performance liquid chromatography
http://dx.doi.org/10.1016/j.jff.2015.06.050
1756-4646/© 2015 Elsevier Ltd. All rights reserved.
Journal of Functional Foods 17 (2015) 892–902
Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/jff
ScienceDirec
t
body weight (Ley, Turnbaugh, Klein, & Gordon, 2006). Long-
term dietary habits have significant impact on human gut
microbiota. For example, children from a rural African village
where the daily dietary consumption is mainly plant polysac-
charides had a significant enrichment in Bacteroidetes and
depletion in Firmicutes in their faecal samples compared to
western European children. Interestingly, two unique genera,
Prevotella and Xylanibacter in Bacteroidetes, known to possess
genes for high-fibre degradation, were only found in African,
but not in European children in the cohort (De Filippo et al.,
2010). In a diet inventory study of 98 healthy human subjects
by Wu et al. (2011a), a positive correlation between Bacteroides-
enterotype and the intake of high-fat diet, while the Prevotella-
enterotype is associated with high-fibre diet based on the 16S
pyrosequencing data of the faecal samples from the volun-
teers. Similar findings on the dietary effect on gut microbiota
were also found in the reports by others (Kabeerdoss, Devi, Mary,
& Ramakrishna, 2012; Matijašic´ et al., 2014; Zimmer et al., 2012).
The gut microbiota has been long recognized for their role
in the transformation of dietary natural products into func-
tional metabolites to the host. For instance, the flavone baicalin,
isolated from the Radix Scutellariae, is hydrolysed by gut mi-
crobes to form aglycone, which is then absorbed and
subsequently conjugated back to baicalin (Akao et al., 2000).
Another study showed that chamomile tea, a functional food,
alters the metabolites and bacterial composition in the gut
(Wang et al., 2005). There were also a pool of literature dis-
cussing the metabolic activation of ginseng saponins
(ginsenosides) by intestinal bacteria (Bae, Han, Kim, & Kim,
2004; Hasegawa, Sung, Matsumiya, & Uchiyama, 1996; Lee
et al., 2009; Shin, Park, Sung, & Kim, 2003). The biotransfor-
mation by human intestinal bacteria of dietary phenolic
compounds such as the transformation of daidzein to equol,
and of flaxseeds to enterodiol, have also been reported (Matthies
et al., 2008; Wang et al., 2010; Woting, Clavel, Loh, & Blaut,
2010). Lately, with the advancement of genomic sequencing
and high-performance mass spectrometry, different in vitro
and in vivo studies have reinforced the role of microbiota in
the enhancement of the bioavailability and functionality of
dietary phtochemicals, including ginsenosides and polyphe-
nolic compounds (Chen & Sang, 2014; Chiou et al., 2014; Choi
et al., 2011; Kim et al., 2013; Song, Kim, & Kim, 2014; Wang,
Qi, Wang, & Li, 2011).
Prebiotics, including whole plant foods, polyphenols, and
fibre, are defined as non-digestible food ingredients that se-
lectively up-regulate the beneficial intestinal microbiota and
may contribute toward the health effects of these dietary
supplements (Bindels, Delzenne, Cani, & Walter, 2015; Roberfroid
et al., 2010; Tuohy, Conterno, Gasperotti, & Viola, 2012). Studies
have shown, for example, that faecal bifidobacterium is se-
lectively present in human subjects who consumed
oligofructose and inulin diets (Bouhnik et al., 2007;
Ramirez-Farias et al., 2009). Few cases of animal and human
studies have also revealed similar phenomena. For instance,
feeding of resveratrol, a polyphenolic compound found in
grapes, berries and various plants, has been found to in-
crease lactobacilli and bifidobacteria while at the same time
decreasing enterobacteria in colitis rats (Larrosa et al., 2009).
More recently, certain functional foods, such as Geranium
dielsianum tea,cassava bagasse flour and kiwifruit pectins, have
also been reported for their prebiotic effects (de Souza et al.,
2014; Ikeda et al., 2014; Parkar et al., 2010).
At present, the prebiotic studies have been mainly focused
on the interaction of dietary polyphenols and intestinal
microbiota (Chen & Sang, 2014; Chiou et al., 2014; Duda-Chodak,
Tarko, Satora, & Sroka, 2015; Etxeberria et al., 2013). However,
the effects of saponins on the modulation of the intestinal
microbiota remain poorly understood. Like polyphenols, sa-
ponins are natural compounds commonly found in many
medicinal and edible plants. They are a group of amphiphilic
glycosides containing one or more sugar moieties bound to a
triterpene or steroid aglycone skeleton (Fig. 1). Herbal sapon-
ins exhibit diverse biological and pharmacological effects,
including antitumour, immunomodulatory, anti-inflammatory,
cardiovascular, hepatoprotective, cholesterol-lowering and an-
tiviral activities (Francis, Kerem, Makkar, & Becker, 2002;
Lacaille-Dubois & Wagner, 1996; Sparg, Light, & van Staden,
2004). For this study, we chose four herbal tea that are com-
monly used as dietary supplements and that are known to be
rich in saponins. They are ginseng (Panax ginseng) in two forms,
unprocessed and processed (known as red ginseng); notoginseng
(Panax notoginseng), and Gynostemma pentaphyllum (also named
jiaogulan). Although the chemical constituents of these four
herbs are similar in chemical structure, their health effects and
medicinal properties are different, as described below.
Ginseng (Panax ginseng C. A. Meyer, Araliaceae) is a general
tonic and adaptogen for the maintenance of host homeosta-
sis. It is one of the oldest and most studied medicinal herbs
in the world. Ginseng saponins, also referred to ginsenosides,
have been reported to exert various beneficial effects, includ-
ing anti-cancer, anti-diabetes, anti-aging and neuronal
protection effects (Kang & Min, 2012; Wee, Mee Park, & Chung,
2011). There are two kinds of ginseng products: ginseng (or also
called white ginseng) and red ginseng. Ginseng is the air-
dried raw ginseng, and red ginseng is produced by steaming
or heating processes. Recent reports have indicated that new
bioactive ginsenosides that not usually found in ginseng are
found in red ginseng (Kim et al., 2000).
Fig. 1 – Common skeletons of saponins. (A) Triterpenoid
saponins; (B) Steroid saponins.
893Journal of Functional Foods 17 (2015) 892–902
Notoginseng is the root of Panax notoginseng (Burk) F. H. Chen
(Araliaceae). It is known for its antihypertensive, antithrombotic,
anti-atherosclerotic and neuroprotective actions (Ng, 2006).The
saponins isolated from notoginseng include notoginsenosides,
ginsenosides and gypenosides (Dong et al., 2003).
Jiaogulan (Gynostemma pentaphyllum Makino, Cucurbitaceae)
is a perennial creeping plant. For centuries, the herbal tea made
from the aerial part of jiaogulan has been consumed in China
as a general tonic. Today, it is increasingly popular in Europe
and Northern America for lowering the serum lipid and cho-
lesterol levels (Wang, Chen, Hsieh, Cheng, & Hsu, 2002). Similar
to green tea, jiaogulan tea also possesses anti-oxidant and anti-
carcinogenic activities (Lin, Huang, & Lin, 2000; Wang et al.,
2002; Zhou, Wang, Zhou, & Zhang, 1998).
At present, remarkably few studies have examined the in-
fluence of herbal saponins on the composition of the gut
microbial community, indicating a serious gap in our under-
standing of the saponins–microbe interactions. In this study,
we aimed to investigate the impact of these four sources of
herbal saponins on the composition of the gut microbiota. The
saponins studied are the major active constituents of the four
commonly used dietary herbal tea, i.e. ginseng, red ginseng,
notoginseng and jiaogulan.
2. Methods and materials
2.1. Sources of herbal saponins
GS, RGS and NGS with 80% purity were purchased from Hongjiu
Biotech Company Ltd., Dalian, China. Standardization of the
saponins was performed by ultra high-performance liquid chro-
matography (UPLC)-mass spectrometry (MS) using 19 known
ginsenosides as standard markers. The detailed protocol and
the resulting chromatograms are presented in Supplementary
Fig. S1. GpS (85% purity) was purchased from the Hauduo
Natural Products (Guangzhou, China).The authentication and
chemical profiling of GpS were monitored according to Wu et al.
(2011b).
2.2. Animals and treatments
The animal welfare and experimental procedures were per-
formed strictly in accordance with the procedures approved
by the University Ethics Review Committee of Hong Kong Baptist
University for the care and use of laboratory animals. The
C57BL/6 mice were purchased from the Chinese University of
Hong Kong, and kept on a 12-h light/12-h dark cycle, 20–22 °C
temperature and 40–60% humidity with free access to food and
water. Mice were fed with a standard diet (PicoLab®Rodent Diet
20–5053, LabDiet, St. Louis, MO, USA). Saponins were dis-
solved in Milli-Q H2O at 50 mg/ml and then sterilized with
0.2 µm filter. Young male mice (8 week old) were randomly
divided into five experimental groups. Mice in all groups were
given daily single dose of herbal saponins (GS, RGS, NGS, and
GpS) at 500 mg/kg or Milli-Q H2O by gavage for 15 consecu-
tive days. Mice were not fasted before drug treatment. In order
to minimize the influence of food intake, the daily food intakes
of the mice were monitored using a comprehensive laboratory
animal monitoring system (CLAMS; Columbus Instruments, Co-
lumbus, OH, USA). The lowest level of food intake was
pinpointed in the afternoon hours, thus we set the timing of
daily drug feeding around 15:00. Two independent experi-
ments were performed with five mice per group for each
experiment (total 50 mice, 10 mice per group in total).Animal
faeces were collected from each individual mouse for two con-
secutive hours from 8:00 to 10:00 AM at day 0 (D0, before
treatment), day 5 (D5), day 10 (D10) and day 15 (D15) upon treat-
ment. The average amount of faeces collected from each mouse
was around 0.3 g. Well mixed faecal sample in 0.1 g from each
mouse was used for bacterial DNA extraction. The treatment
scheme is shown in Fig. 2A.
2.3. Bacterial genomic DNA extraction from faecal
samples
Total genomic DNA was isolated from faecal samples as de-
scribed by McCracken, Simpson, Mackie, and Gaskins (2001) and
Kong, Li, and Wu (2006) with slight modification. In brief, 0.1 g
faeces were vortexed in 4 ml sterile PBS (pH 7.4) for 5 min, then
centrifuged at 40 ×gfor 8 min to collect the upper phase con-
taining the bacteria. After repeating this procedure once, the
supernatants were combined and centrifuged at 2000 ×gfor
8 min. The supernatant was discarded.The bacterial pellets were
washed twice with PBS, then resuspended in 200 µl lysing buffer
I (150 mM NaCl; 100 mM EDTA (ethylenediaminetetraacetic acid);
pH 8.0) and 66.7 µl proteinase K (4 mg/ml) was then added. After
incubation at 55 °C for 2 h, 200 µl lysing buffer II (100 mM NaCl,
500 mM Tris-HCl, pH 8.0), plus 66.7 µl 10% SDS were then added
and incubated at room temperature for 5 min.The mixture was
extracted sequentially by phenol, phenol/chloroform/isoamyl
(25:24:1, v/v/v), chloroform/isoamyl (24:1, v/v), followed by two
volumes of cold ethanol and 1/10 volume of sodium acetate
(3 M, pH 5.2) for the precipitation of DNA.The solution was kept
overnight at −20 °C. Genomic DNA pellets were collected by cen-
trifuging at 15,000 ×gfor 15 min, and then washed twice with
cold 70% ethanol, dried, then dissolved in PCR H2O with 1.0 mg/
ml RNase A. The DNA concentration was determined by
NanoDrop 1000 spectrophotometry.
2.4. ERIC (Enterobacterial Repetitive Intergenic
Consensus)-PCR
ERIC sequences are non-coding, highly conserved intergenic
repeated sequences that reside in the genome of various bac-
terial species in addition to enterobacteria (Wilson & Sharp,
2006). ERIC-PCR was used to detect the gut microbiome
using faecal genomic DNA as the template and a pair of
ERIC specific primer sequences: ERIC 1R (5′-ATGTAA
GCTCCTGGGGATTCAC-3′) and ERIC 2 (5′-AAGTAAGTG
ACTGGGGTGAGCG-3′)(
Versalovic, Koeuth, & Lupski, 1991). The
25 µl reaction mixture contained 5 µl5×PCR reaction buffer,
200 µM dNTP, 2.5 mM Mg2+, 0.4 µM primers, 1 unit HotstartTaq
polymerase, and 50 ng faecal genomic DNA. PCR was per-
formed using the following protocol: 94 °C for 5 min, followed
by 35 cycles of 95 °C for 50 s, 49 °C for 30 s, 46 °C for 30 s
and 72 °C for 3 min, and then a final extension at 72 °C for 9 min
(Peng et al., 2009). 10 µl of each PCR product was loaded
onto a 2% (w/v) agarose gel containing 0.5 µg/ml ethidium
894 Journal of Functional Foods 17 (2015) 892–902
bromide and run for 40 min at 100 V in 1 ×TAE buffer. A DNA
ladder (0.1–10.0 kb) was used as the DNA marker (NEB, N3200).
The agarose gels were photographed using a Gel DocTM
XR +System (Bio-Rad, Hercules, CA, USA).
2.5. Application of PLS-DA for ERIC-PCR data analysis
Partial least squares discriminant analysis (PLS-DA) is one of
the most widely used methods in multivariate classification.
In this study, we applied PLS-DA to evaluate the similarity of
microbial composition between the control and treatment
groups based on the ERIC-PCR data. The banding patterns of
the ERIC-PCR products (see Fig. 2) were photographed and digi-
tized using the Image Lab 3.0 system (Bio-Rad) to generate the
data based on the sum of the distance and the intensity of each
DNA band within each sample lane. The scores were sub-
jected to PLS-DA plot using the SIMCA-P 12.0 tool (Umetrics,
Umea, Sweden).
2.6. Quantitative real time PCR (qPCR)
The abundance of specific bacteria was measured by qPCR using
Applied Biosystems ViiA™ 7 PCR system (Carlsbad, CA, USA)
with taxon-specific 16S rRNA gene primers (Invitrogen, Carls-
bad, CA, USA). A universal primer set was used to detect the
16S rRNA gene of total bacteria, and used to calculate the rela-
tive abundance of specific bacteria group.The sequences of the
primers used were listed in Supplementary Table S1. Briefly,
the qPCR was carried out using Power SYBR® Green PCR Maser
Mix (Applied Biosystems Inc.) with 5 ng faecal genomic DNA
and 200 nM of each primer.The amplification conditions were
as follows: 95 °C for 10 min, followed by 40 cycles of 95 °C for
15 s and 60 °C for 1 min. Using the same batch of genomic DNA
from each faecal sample, qPCR was performed to determine
the amount of the following bacteria: Firmicutes,Bacteroidetes,
Bacteroides,Lactobacillus,Bifidobacterium,Clostridium Cluster IV
and Faecalibacterium prausnitzii.Ten samples were used for each
experimental group.The comparative Ct method (2-ΔΔCt method)
was applied to determine the relative change of specific bac-
teria in the faeces of individual mouse before (D0) and after
treatment. ΔΔCt =(Cttreatment_specific bacteria -Ct
treatment_total bacteria)-
(CtD0_specific bacteria -Ct
D0_total bacteria).
2.7. Statistical analysis
The data obtained from two independent experiments, a total
of ten mice per group were analysed and presented as
mean ±SEM. Statistical comparisons were performed using re-
peated measures ANOVA followed by Dunnett’s post test with
the GraphPad Prism version 5.00 (GraphPad Software, San Diego,
CA, USA) at P values of <0.001 (***), <0.01 (**) or <0.05 (*).
3. Results
3.1. Herbal saponins altered the profiles of faecal
microbiota
To investigate the impact of herbal saponins on the gut
microbiota, faecal samples were collected from the control and
four experimental groups at D0, D5, D10 and D15 (Fig. 2A). The
food intake, body weight and faeces production of mice in all
five groups were monitored throughout the experimental period.
No significant changes in either food intake or body weight were
observed in any of the groups (Fig. 2B,C). As for faecal samples,
the faecal microbial fingerprints of ERIC-PCR showed an average
of 19 bands per sample, ranging from approximately 100 to
3000 bp with various intensities (Fig. 2D–H).The resulting PCR
gel images were converted to digitized profiles and analysed
using PLS-DA tool. Each data point represented faecal microbiota
from an individual mouse, 10 mice for each group. The data
showed that all treatment groups formed distinct clusters com-
pared to the non-treatment control group (Fig. 3).
3.2. qPCR analysis of the effect of saponins on the
relative abundance of Bacteroidetes and Firmicutes in
faecal microbiota
Firmicutes and Bacteroidetes are the two bacterial phyla that domi-
nated in the gut microbiota of the healthy mice.To learn more
about the compositions of the faecal microbiota upon saponin
treatment, we carried out qPCR with 16S rRNA gene specific
primers to identify the presence of Firmicutes and Bacteroidetes
in the faecal samples. In contrast to the control group, the rela-
tive abundance of Firmicutes was significantly decreased in the
GpS treatment group (Fig. 4A), whereas the relative abun-
dance of Bacteroidetes was significantly enriched in the GpS and
NGS treatment groups (Fig. 4B). In addition, GpS- and NGS-
treated mice showed a time-dependent shift in the faecal
Bacteroidetes/Firmicutes ratio in favour of Bacteroidetes in the
course of 15-day treatment (Fig. 4C).
3.3. qPCR analysis of the effect of saponins on the levels
of common commensal bacteria
Bacteroides, consisting well-known beneficial bacteria, is a pre-
dominant genus within Bacteroidetes phylum in the intestinal
tract. Both GpS- and NGS-fed mice showed an increased level
of Bacteroides (Fig. 5A), which was in line with the enrich-
ment of Bacteroidetes in the GpS and NGS groups shown in Fig. 4.
Two common beneficial genera, Bifidobacterium and Lactobacil-
lus were also assessed.The qPCR results showed that GpS, NGS
and GS effectively increased Lactobacillus (Fig. 5B), whereas NGS
and RGS significantly enhanced the level of Bifidobacterium
(Fig. 5C). In addition, the level of Clostridium Cluster IV, which
is one of the major clusters of butyrate-producing bacteria, was
markedly higher in RGS-treated mice than any other treat-
ment groups (Fig. 5D). Furthermore, within the Clostridium
Cluster IV, an anti-inflammatory commensal bacterial species,
Faecalibacterium prausnitzii, was significantly enhanced in the
GpS-group (Fig. 5E). The above results indicated that the in-
gested herbal saponins can indeed modulate beneficial bacteria
in the gut of the host.
4. Discussion
Triterpene saponins have been recognized as the main con-
stituents contributing to the health benefits of many dietary
895Journal of Functional Foods 17 (2015) 892–902
Fig.2–Atime course study of the faecal microbiota of mice from different herbal saponins treatment groups. (A) Treatment
scheme; (B) Diet consumption; (C) Body weight; (D–H) ERIC-PCR fingerprints of the faecal microbiota of individual C57BL/6
mice fed with water control (D), GpS (E), NGS (F), RGS (G), and GS (H). ERIC-PCR data of faecal samples from three
representative mice obtained at D0, D5, D10 and D15 are shown in the charts. A total of ten mice were used for each group.
A1–A3, B1–B3, C1–C3, D1–D3 and E1–E3 are the representative ERIC-PCR gel images of three mouse faecal samples.
896 Journal of Functional Foods 17 (2015) 892–902
and medicinal plants. Ginseng, both raw and processed,
notoginseng and jiaogulan are among the most common
saponin-rich herbal tea used in China and Southeastern Asian.
These four herbs share some common saponins; however, they
have their own unique profile of saponins.This can somehow
explain the overlapping biological activities of the four herbs,
as well as the specific health benefits and pharmacological func-
tions of each herb.
Prebiotics are considered as nondigestible food ingredi-
ents that can stimulate the growth of beneficial intestinal
bacteria, including bifidobacteria and lactic acid bacteria to the
benefit of the host health (Bindels et al., 2015; Roberfroid et al.,
2010; Tuohy et al., 2012). Synergism between prebiotics and
probiotics has been also revealed regarding its impact on health
promotion and immunomodulation (Patel, 2015). In the past
ten years, prebiotic research has mainly focused on fibre and
polyphenolic compounds. The potential role of triterpenoid sa-
ponins has been overlooked.
The results presented here are to address the interaction
between the gut microbiota and dietary herbal saponins, and
determine whether herbal triterpenoid saponins can act as
prebiotics by influencing the host intestinal microbiota. In this
study, we reported the changes of the faecal microbiota ob-
tained from mice receiving daily oral administration of 500 mg/
kg of herbal saponins over a period of 15 days.The dosage used
in the study seems to be at the high side; however, it caused
no adverse effect to the animals (Fig. 2). One of the known traits
of saponins is poor bioavailability and they are hard to be ab-
sorbed through the intestinal wall (Yu, Chen, & Li, 2012). This
might be one of the reasons that the mice can tolerate oral
dosage of saponins up to 1000 mg/kg for long duration of treat-
ment without adverse effect (Attawish et al., 2004; Chiranthanut
Fig. 3 – PLS-DA score plots of the ERIC-PCR data. The gel images were digitized using the Image Lab 3.0 system (Bio-Rad).
Based on the distance and the intensity of each DNA bands, the SIMCA-P 12.0 tool was applied to obtain the PLS-DA score
plots. (A) Control vs. GpS; (B) Control vs. NSG; (C) Control vs. RGS; (D) Control vs. GS. n =10/group.
897Journal of Functional Foods 17 (2015) 892–902
et al., 2013). It is known that some dietary phytochemicals are
poorly absorbed and the bioavailability of ingested bioactive
food compounds is a complex and challenging process (Rein
et al., 2013). Interestingly, some studies showed that the
bioavailability of a single bioactive compound is much poorer
than it is consumed with the whole plant extract (Cheng, Lin,
Lin, & Tsai, 2014; Keung, Lazo, Kunze, & Vallee, 1996; Vissers,
Bozonet, Pearson, & Braithwaite, 2011), suggesting that the con-
sumption of whole herbal tea may show a greater bioavailability
than that of saponins alone. The potential synergistic effects
of saponins and other phytonutrients on gut microbiota would
be of interest for further investigation.
In order to evaluate the changes of microbial profile
responding to saponin administration, PLS-DA was applied
based on the ERIC-PCR fingerprints of the faecal DNA
samples between the treatment and the control groups. The
data showed that all treatment groups formed distinct
clusters compared to the non-treatment control group.
Fig. 4 – Relative abundance of Firmicutes (A) and Bacteroidetes (B) in the faeces of control and herbal saponins-treated mice.
(C)Bacteroidetes/Firmicutes ratio. Bacterial genomic DNA was extracted from the faecal samples of mice at D0, D5, D10 and
D15, and the abundance of Firmicutes and Bacteroidetes was determined by qPCR with each phylum-specific 16S rRNA gene
primers. The relative abundance of the specific bacteria was normalized to that of the total faecal bacteria, and expressed as
fold change over the D0 sample of each mouse. All of the data are presented as the mean ±SEM (* P <0.05, ** P <0.01 versus
D0 samples); n =10/group.
Fig. 5 – Effect of herbal saponins on common commensal bacteria in the faecal samples of mice. (A)Bacteroides;(B)
Lactobacillus;(C)Bifidobacterium;(D)Clostridium Cluster IV; (E)Faecalibacterium prausnitzii. qPCR was used to determine the
number of bacteria with each taxon-specific 16S rRNA gene primers and normalized to that of the total faecal bacteria, and
expressed as fold change over the D0 sample of each mouse. All of the data are presented as the mean ±SEM (* P <0.05, **
P<0.01 versus D0 samples); n =10/group.
898 Journal of Functional Foods 17 (2015) 892–902
Furthermore, the gut microbiota responded differently to each
herbal saponins (Fig. 3). Interestingly, RGS and GS, which share
similar chemical profiles, displayed similar profiles in the mi-
crobial composition in relationship to the control group
(Fig. 3C,D).
The increased ratio of Firmicutes to Bacteroidetes has been
reported to positively correlate with several diseases and symp-
toms, such as obesity (Ley et al., 2005, 2006) and irritable
syndrome (Rajilic-Stojanovic et al., 2011). In our study, the
Bacteroidetes/Firmicutes ratio was elevated after the adminis-
tration of GpS and NGS, indicating that GpS and NGS may
play a role to revert the aberrant shift of Bacteroidetes to
Firmicutes. Certain gut Bacteroides has been linked to various
health benefits. For example, Bacteroides alleviates obesity-
associated metabolic syndromes (Ridaura et al., 2013). Certain
species of Bacteroides such as B. acidifaciens can promote IgA
production (Yanagibashi et al., 2012). In our study, the level of
Bacteroides was significantly enhanced in mice feeding with
GpS or NGS. Bifidobacterium and Lactobacillus are commonly
consumed as probiotics (Yang, 2008), and were stimulated
after administration of the tested saponins. The stimulation
of Lactobacillus was more prominent with GpS and GS, while
the enhancement of Bifidobacterium was stronger with NGS
and RGS treatments. One of the beneficial effects of Bacteroi-
des and Bifidobacterium, as revealed in recent reports, is the
ability to metabolize ginsenoside Rb1 to compound K, which
exhibits potent pharmacological effects in antitumour, anti-
inflammatory, and anti-allergic activities (Bae, Park, & Kim,
2000; Kim et al., 2013). In addition, our results appear to echo
the results of studies on the effects of polyphenol-rich green
and black tea extracts on enhancing the Bifidobacterium species
and the associated bifidogenic effects (Jin, Touyama, Hisada,
& Benno, 2012; van Duynhoven et al., 2013). This similar
effect suggests that the dietary herbal saponins may possess
prebiotic potential, at least in this aspect.
Another interesting finding was the enhancement effect
of GpS on a butyrate producing bacterium, Faecalibacterium
prausnitzii. Butyrate is a short chain fatty acid (SCFA) derived
from the microbial metabolites of dietary fibre in the gut.
Butyrate exhibits a wide range of health effects from anti-
inflammatory properties to enhancement of intestinal barrier
function (Frank et al., 2007; Hamer et al., 2008). Due to its
multiple epigenetic effects, butyrate has been well docu-
mented for various diseases prevention and treatment (Berni
Canani, Di Costanzo, & Leone, 2012). Faecalibacterium prausnitzii
is the most important butyrate-producing bacterium. It belongs
to the Clostridium cluster IV, one of the main sources of butyrate-
producing microbes (Van den Abbeele et al., 2013), which
was also found significantly elevated in the RGS-treated mice.
F. prausnitzii has been reported to ameliorate dysbiosis and
mediate protective effects in patients with Crohn’s disease
(Sokol et al., 2008). The presence of F. prausnitzii is directly
associated with the reduction of low-grade inflammation in
obesity and diabetes independently of calorie intake (Furet
et al., 2010). Thus, modulation of F. prausnitzii by exogenous
substances may have preventive or therapeutic applications
to human health. Collectively, the current findings strongly
suggest that the saponins tested in this study may contrib-
ute to their health-promoting effects through the modulation
of the beneficial gut commensal microbiota.
5. Conclusion
This study examined the impact of administration of the four
herbal saponins on common commensal bacteria in a mam-
malian gut. The tested dietary saponins exerted prebiotic-
like effects, enhancing bacteria known to be beneficial to the
host. These findings have important implications. First, it sug-
gests that the dietary saponins can be considered as a new
group of prebiotics. Second, the study provides a new insight
to the long-recognized beneficial effects of dietary herbal sa-
ponins through the understanding of the role of gut microbiota.
Third, the study also suggests that herbal saponins along with
the prebiotics may have important clinical implications in
human gut health.
Conflict of interest
The authors have no conflicts of interest to declare.
Acknowledgments
This study was supported by the Macau Science and Technol-
ogy Development Fund 015/2014/A1 and the Research Grants
Council of Hong Kong under GRF260413 to WL Wendy Hsiao.
We also thank Dr. Martha Dahlen for her editing work.
Appendix: Supplementary material
Supplementary data to this article can be found online at
doi:10.1016/j.jff.2015.06.050.
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