Journal of Ethnopharmacology 155 (2014) 1291–1299

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Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jep

Research Paper

Comparative pharmacokinetics of rhein in normal

and loperamide-induced constipated rats and microarray analysis
of drug-metabolizing genes
Mei-Ling Hou a, Li-Wen Chang a, Chi-Hung Lin b, Lie-Chwen Lin c, Tung-Hu Tsai a,d,e,n
a
Institute of Traditional Medicine, National Yang-Ming University, Taipei, Taiwan
b
Institute of Microbiology and Immunology, National Yang-Ming University, Taipei, Taiwan
c
National Research Institute of Chinese Medicine, Taipei, Taiwan
d
Graduate Institute of Acupuncture Science, China Medical University, Taichung, Taiwan
e
Department of Education and Research, Taipei City Hospital, Taipei, Taiwan

art ic l e i nf o a b s t r a c t

Article history: Ethnopharmacological relevance: Rhein is a pharmacological active component found in Rheum palmatum
Received 6 May 2014 L. that is the major herb of the San-Huang-Xie-Xin-Tang (SHXXT), a medicinal herbal product used as a
Received in revised form remedy for constipation. Here we have investigated the comparative pharmacokinetics of rhein in
26 June 2014
normal and constipated rats. Microarray analysis was used to explore whether drug-metabolizing genes
Accepted 10 July 2014
Available online 19 July 2014
will be altered after SHXXT treatment.
Materials and methods: The comparative pharmacokinetics of rhein in normal and loperamide-induced
Keywords: constipated rats was studied by liquid chromatography with electrospray ionization tandem mass
Traditional Chinese medicine spectrometry (LC-MS/MS). Gene expression profiling in drug-metabolizing genes after SHXXT treatment
Rhein
was investigated by microarray analysis and real-time polymerase chain reaction (RT-PCR).
Loperamide-induced constipation
Results: A validated LC-MS/MS method was applied to investigate the comparative pharmacokinetics of
Microarray
Pharmacokinetics rhein in normal and loperamide-induced constipated rats. The pharmacokinetic results demonstrate that
the loperamide-induced constipation reduced the absorption of rhein. Cmax significantly reduced by 2.5-
fold, the AUC decreased by 27.8%; however, the elimination half-life (t1/2) was prolonged by 1.6-fold. Tmax
and mean residence time (MRT) were significantly prolonged by 2.8-fold, and 1.7-fold, respectively. The
volume of distribution (Vss) increased by 2.2-fold. The data of microarray analysis on gene expression
indicate that five drug-metabolizing genes, including Cyp7a1, Cyp2c6, Ces2e, Atp1b1, and Slc7a2 were
significantly altered by the SHXXT (0.5 g/kg) treatment.
Conclusion: The loperamide-induced constipation reduced the absorption of rhein. Since among the
25,338 genes analyzed, there were five genes significantly altered by SHXXT treatment. Thus, informa-
tion on minor drug-metabolizing genes altered by SHXXT treatment indicates that SHXXT is relatively
safe for clinical application.
& 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction physiological function of the bowel. Drugs commonly used include

Constipation is a frequent condition that is often treated pharma-
cologically (Muller-Lissner, 2013). Based on the Rome II diagnostic
criteria, constipation affects at least 8.5% of the individuals in Taiwan,
occurring more often in females and older persons (Chey et al., 2011;
Jong et al., 2010). The aim of treating constipation is to treat the
underlying causes, improve symptoms, and resume the normal
n
Corresponding author at: Institute of Traditional Medicine, School of Medicine,
National Yang-Ming University, 155, Li-Nong Street Section 2, Taipei 112, Taiwan.
Tel.: þ 886 2 2826 7115; fax: þ886 2 2822 5044.
E-mail address: thtsai@ym.edu.tw (T.-H. Tsai).
bulk laxatives, osmotic laxatives, non-absorbable sugar, stimulant
laxatives, cholinergic agents and other prokinetics agents. In East
Asian countries, traditional Chinese medicine (TCM) is another
option in addition to or instead of Western medicine for treating
constipation (Camilleri and Bharucha, 2010; Candy et al., 2011; Chey
et al., 2011). In many of these countries, especially Taiwan, TCMs are
frequently prescribed for the treatment of many chronic diseases
(Jong et al., 2010; Lai et al., 2012; Shih et al., 2012). According to a
survey from a cohort of one million randomly sampled cases from
the National Health Insurance database in Taiwan, the Chinese herbal
formula San-Huang-Xie-Xin-Tang (SHXXT) is widely used for the
treatment of constipation (Jong et al., 2010). This herbal formulation

http://dx.doi.org/10.1016/j.jep.2014.07.022
0378-8741/& 2014 Elsevier Ireland Ltd. All rights reserved.
1292 M.-L. Hou et al. / Journal of Ethnopharmacology 155 (2014) 1291–1299
consists of rhizomes of Rheum palmatum L. (rhubarb), roots of
Scutellaria baicalensis Georgi and rhizomes of Coptis deltoidea C. Y.
Cheng & P. K. Hsiao, with a weight ratio of 2:1:1, respectively. Using
the ancient process of TCMs preparation, the herbs are mixed and
decocted with water before oral administration (Trivedi et al., 2007).
However, convenient pharmaceutical herbal products are also used
instead of the traditional herbal decocting process. Pharmaceutical
herbal products are mainly made by industrial manufacturing
methods of decoction, filtration, extraction, concentration, spray or
fluid bed granulation, coating and filling (Jong et al., 2010; Lai et al.,
2012; Shih et al., 2012).
Analytical methods have been reported to investigate the
pharmacokinetic screening of multiple constituents in Chinese
herbal formulae by HPLC-UV (Shia et al., 2011; Yin et al., 2009),
and HPLC-MS/MS (Shaw et al., 2012; Shi et al., 2011; Zan et al.,
2011). Furthermore, the assessment of rhein and/or its metabolites
from various Chinese herbal formulae in human and animals has
been studied (Jiang et al., 2012; Shia et al., 2011; Zan et al., 2011).
In our previous study, the pharmacokinetic data of rhein demon-
strate that the herbal formulae or the single herbal extract
provides significantly higher absorption rate than the pure com-
pound (Hou et al., 2014). This phenomenon suggests that the other
herbal ingredients of SHXXT and rhubarb extract significantly
enhance the absorption of rhein in rats. In addition, the herbal
formulae (SHXXT) are more efficient than the single herb (Rhu-
barb) or the pure compound (rhein) in rhein absorption (Hou
et al., 2014). According to our pharmacokinetic results for rhein,
Chinese herbal formulae containing various ingredients may have
an obvious impact on the contribution of a specific chemical to
absorption, producing additive, synergetic, or antagonistic effects.
In addition, studies on the comparative pharmacokinetic profiles
of drugs with the various body conditions, including acute blood
stasis, thrombotic focal cerebral ischemia, ulcerative colitis, and
acute pancreatitis have been reported (Dai et al., 2014; Feng et al.,
2013; Han et al., 2013; Zhu et al., 2014). Our hypothesis is that
whether may influence the pharmacokinetics of rhein after SHXXT
treatment. However, there is still limited information on the
comparative pharmacokinetic studies of Chinese herbal formulae
in normal and constipated rats and the impacts on pharmacoki-
netics of rhein in constipated animals.
Studies on gene expression profiling as an initial approach for
mechanistic studies of therapeutic prediction, drug development,
and the safety evaluation of herbal plants, herbal dietary supple-
ments and Chinese herbal formulae have been reported (Cheng
et al., 2010; Guo et al., 2010b). Analysis of the gene expression
profiles using microarrays, both in vitro and in vivo, has the
potential to be a practical approach for understanding the
mechanisms of toxicity and tumorigenicity (Wen et al., 2011). In
addition, investigation on gene expression changes in liver sam-
ples of rodents treated with Chinese herbal formulae can provide
information about its mechanism of action, such as the modula-
tion of drug metabolizing enzymes, which can potentially cause
herb–drug interactions and may be responsible for different types
of liver injury. We have proposed that analysis of alterations in
gene expression profiles can be an initial approach for determining
the mechanisms by which Chinese herbal formulae induced
hepatotoxicity. In the present study, gene expression changes
were analyzed with the focus on drug metabolizing genes in the
livers of rats treated orally with SHXXT.
Recently, using TCM for constipation treatment is an alternative
approach to Western medicine. However, the impacts of constipation
on the pharmacokinetic profiling of rhein after SHXXT treatment
remain unknown. In addition, concerns on safety evaluation of the
commercial pharmaceutical product, SHXXT, gene expression changes
focus on drug metabolizing genes in the livers of rats after SHXXT
treatment need to be further investigated. Hence, the aim of this study
is (i) to investigate the comparative pharmacokinetics of rhein from
SHXXT in normal and loperamide-induced constipated rats by LC-MS/
MS methods; and (ii) to explore gene expression profiling in drug-
metabolizing genes after SHXXT treatment.
2. Materials and methods
2.1. Chemicals and Reagents
The chemicals rhein and ibuprofen (internal standard) were
purchased from Sigma-Aldrich Chemicals (St. Louis, MO, USA). LC/
MS grade solvents were obtained from J.T. Baker, Inc. (Phillipsburg,
NJ, USA) and chromatographic reagents were obtained from Tedia
Co., Inc. (Fairfield, OH, USA). Triply deionized water (Millipore,
Bedford, MA, USA) was used for all preparations. The pharmaceu-
tical herbal product SHXXT manufactured in accordance with
Good Manufacturing Practice (GMP) for Chinese Crude Drugs
was obtained from pharmaceutical companies in Taiwan and has
been used medicinally for patients. The pharmaceutical herbal
product, SHXXT (product lot no. CP1403040), was purchased from
Sheng Chang Pharmaceutical Co., Ltd. (Taipei, Taiwan).
2.2. LC-MS/MS
The analytical method was carried out as in the previous study
(Hou et al., 2014). Briefly, the LC-MS/MS analysis was performed
using a Waters Acquity UPLC™ system (Waters, Manchester, UK)
consisting of a binary solvent manager, an automatic liquid
chromatographic sampler and a Waters Xevo™ tandem quadru-
pole mass spectrometer equipped with an electrospray ionization
(ESI) source. Separation was achieved using a Waters Acquity UPLC
type BEH C18 (100  2.1 mm, 1.7 mm) analytical column, main-
tained at 40 1C in a column oven. The mobile phase consisted of A
(0.1% formic acid in water) and B (0.1% formic acid in acetonitrile)
with a linear gradient elution of 15–85% (v/v) B at 0–8 min, and
15% B at 8–10 min. The flow rate was set at 0.25 mL/min, and the
injection volume was 5 mL. For operation in the MS/MS mode, the
electrospray ion source was operated with a negative ion mode.
The ESI parameters were set as follows: source temperature,
150 1C; desolvation temperature, 500 1C; desolvation gas flow,
800 L/h. The optimized cone voltages (CV) were 49 V and 15 V
for rhein and ibuprofen, respectively. The multiple reaction mon-
itoring (MRM) mode using specific precursor/product ion transi-
tions was employed for quantification. The molecular ions of rhein
and ibuprofen were fragmented at collision energies of 22 and
8 eV using argon as collision gas. Ion detection was performed by
monitoring the transitions: m/z 283.1-238.9 for rhein and m/z
205.2-160.8 for ibuprofen. Ibuprofen was used as the internal
standard for negative ion mode determination. The software
program providing the data platform for spectral acquisition,
spectral presentation and peak quantification was the MassLynx
4.1 software package.
2.3. Method validation
The method validation assays for quantification of rhein in rat
plasma were carried out according to the currently accepted US
Food and Drug Administration (FDA) bioanalytical method valida-
tion guidance. The specificity was tested by screening six different
batches of drug-free rat plasma for the exclusion of any endogen-
ous co-eluting interference at the peak regions of rhein and
internal standard. The method validation assays were performed
as the previous study (Hou et al., 2014).
 M.-L. Hou et al. / Journal of Ethnopharmacology 155 (2014) 1291–1299
2.4. Pharmacokinetic study of rhein in normal rats
All animal experimental protocols were reviewed and approved
by the Institutional Animal Care and Use Committee (IACUC
number: 1011202) of National Yang-Ming University. Male specific
pathogen-free Sprague-Dawley rats were obtained from the
Laboratory Animal Center of the National Yang-Ming University,
Taipei, Taiwan. The animals had free access to food (laboratory
rodent diet 5P14, PMI Feeds, Richmond, IN) and water. For
pharmacokinetic study, Sprague-Dawley rats weighing 220 7
20 g were anesthetized with pentobarbital (50 mg/kg, i.p.) for
cannulation. A polyethylene tube (PE50) was implanted into the
left carotid artery for blood sampling. The cannula was exterior-
ized, fixed in the dorsal region of the neck. Patency of the tube was
maintained by flushing with heparinized saline (20 IU/mL). After
surgery, the rats were kept in cages individually and allowed to
recover for one day.
The SHXXT formulae suspended in water at doses of 5 g/kg was
individually administered to the rats by oral gavage. About 200 mL
of blood samples was withdrawn from the cannula implanted in
the carotid artery and placed into a heparin-rinsed vial at 0, 15, 30,
45, 60, 90, 120, 150, 180, 240, 300 and 360 min. The method of
plasma sample preparation was performed as the previous study
(Hou et al., 2014).
Pharmacokinetic calculations were performed on each indivi-
dual set of data using the pharmacokinetic software WinNonlin
Standard Edition, version 1.1 (Pharsight Corp., Mountain View, CA)
by noncompartmental methods.
2.5. Pharmacokinetics of rhein in constipated rats
Constipation was induced by loperamide administration (3 mg/
kg, s.c.) dissolved in normal saline administered to rats of the
experimental group twice per day at 09:00 and at 18:00 h on the
experimental days 1–5 (Wintola et al., 2010). The control rats were
administered with normal saline in the same manner as the
experimental rats. The passage of reduced, hard and dry pellets
indicated constipation in the rats. The water intake, food intake
and body weight gain of all the rats were recorded throughout the
experiment. The excreted fecal pellets of individual rats were
collected every day throughout the experiment, and total number,
weight and water content of the pellets were determined. The
fecal pellets were lyophilized thoroughly and their dry weights
were measured. The water content was calculated as the differ-
ence between wet and dry weights of the pellets.
While constipation was being induced, a polyethylene tube (PE
50) was implanted into the left carotid artery of the rat for blood
sampling. Pharmacokinetic study was performed as described
previously.
100 OH O OH
Relative Abundance
HO
O O
% m/z 283.1 → m/z 238.9
rhein
0 0
100 140 180 220
m/z, amu
Fig. 1. Chemical structures and mass spectra of (A) rhein, and (B) ibuprofen (internal standard; molecular weight 284, 206, respectively). Mass spectra of the analytes and
their product ions in UPLC-MS/MS with electrospray negative ion mode. The mass transitions of rhein, and ibuprofen were m/z 283.1-238.9, and 205.2-160.8, respectively.
1293
2.6. Gene expression profiling, drug administration and RNA
isolation
For adults, the powdered SHXXT formula of the pharmaceutical
herbal product for clinical applications is 1.5 g per time and 2–3
times daily. According to the dose translation (Reagan-Shaw et al.,
2008) from human to animal, the commercial SHXXT was sus-
pended in deionized water at dose of 0.5 g/kg for oral adminis-
tration. Thus, groups of two male Sprague-Dawley rats were
administered either vehicle (water, 10 mL/kg) or commercial
SHXXT powders (0.5 g/kg) in water by oral gavage once daily for
7 days. At two hours after drug administration of the 7th day,
about 1 g of tissue from the each liver lobe was collected from
each rat, placed in TRIzol (U.S. Patent no. 5,346,994) reagent and
stored at  80 1C until processed for RNA extraction and isolation.
The RNA concentration and purity were checked by OD260/OD280
(41.8) and OD260/OD230 (41.6), and the yield and quality were
accessed using an Agilent 2100 Bioanalyzer (Agilent Technologies,
Santa Clara, CA, USA).
2.7. Oligonucleotide DNA microarray, microarray analysis, real-time
polymerase chain reaction (RT-PCR) and data analysis
Gene expression profiling was performed using the Rat Whole
Genome OneArrayR v5 (Phalanx Biotech Group, Taiwan), contain-
ing 25,338 DNA oligonucleotide probes, with each probe a 60-mer
designed in the sense direction. Of the probes, 24,358 corre-
sponded to the annotated genes in RefSeq v42 and Ensembl v59
database. In addition, 980 control probes were also included.
Fluorescent aRNA targets were prepared from 2.5 μg total RNA
samples using OneArrayR Amino Allyl aRNA Amplification Kit
(Phalanx Biotech Group, Hsinchu, Taiwan) and Cy5 dyes (Amer-
sham Pharmacia, Piscataway, NJ, USA). Fluorescent targets were
hybridized to the Rat Whole Genome OneArrayR (ROA v1) with
Phalanx hybridization buffer using the Phalanx Hybridization
System, and the slides were dried by centrifugation and scanned
by an Axon 4000B scanner (Molecular Devices, Sunnyvale, CA,
USA). The Cy5 fluorescent intensities of each spot were analyzed
by GenePix 4.1 software (Molecular Devices). The technical repeat
data was tested by Pearson correlation coefficient calculation to
check the reproducibility (R value 40.975). The signal intensity of
each spot was loaded into a Rosetta Resolver SystemR (Rosetta
Biosoftware) to process data analysis. In brief, spots that passed
these criteria were normalized by the median scaling normal-
ization method. Normalized spot intensities were transformed to
gene expression log2 ratios between control and treatment groups
under Rosetta Resolver error model adjustment. Any spots with
log2 ratio Z1 or log2 ratior  1 and P-value o 0.05 were tested
for further analysis.
205.2
283.1 100
160.8
O
238.9 OH
%
m/z 205.2 → m/z 160.8
ibuprofen
260 150 170 190 210
m/z, amu
1294 M.-L. Hou et al. / Journal of Ethnopharmacology 155 (2014) 1291–1299

Real-time polymerase chain reaction (RT-PCR) was used to verify intra- and inter-day precision (% RSD) and accuracy (% bias) values
the results for a selected group of genes whose expression changes of rhein in rat plasma were within 15%. These results show that the
were seen using microarray. Differentially expressed genes identified UPLC-MS/MS method is excellent for the quantitative analysis of
with microarray analysis were selectively confirmed by BIO-RAD rhein in rat plasma extracts.
MiniOption real-time PCR detection system. Five probes were used
in these assays, including ces2e (NM_001100477.1), cyp4a8 (NM_
3.2. Comparative pharmacokinetics of rhein in normal and
031605.2), cyp7a1 (NM_012942.1), cyp17a1 (NM_012753.1), and
constipated rats
cyp2c6 (XM_001066767.2). Each sample was tested in triplicate. Every
reaction well was performed by using 10 ng cDNA and 200 nM of
For constipation induction, loperamide reduced the number
forward and reverse primers. Quantitative PCR was performed at the
(467 4 vs. normal control, 6578), the weight (21.4 72.95 g vs.
conditions using 2X Fast SYBR Green PCR Master Mix (Applied
normal control, 24.3 7 1.88 g) and water content (5.84 72.20 g vs.
Biosystems, 4385610). Bio-rad CFX Manager Version 2.1 was used
normal control, 7.02 71.39 g) of fecal pellets. These results indi-
for experimental setup and data analysis for the MiniOpticon real-time
cated that the loperamide inhibits intestinal water secretion and
PCR detection system. qPCR data of target genes were relative
colonic peristalsis. This inhibition extends fecal evacuation time
quantifying normalized to reference gene, β-actin. Normalized Expres-
and delays intestinal luminal transit (Shimotoyodome et al., 2000;
sion (ΔΔCq) was used for relative gene expression analysis.
Wintola et al., 2010, 2011). In order to investigate whether

2.8. Statistical analysis

SHXXT (5 g/kg, p.o.)
Data are expressed as the mean 7 SD or mean 7SEM. Compar- SHXXT (5 g/kg, p.o.)
ison between two groups was performed using the unpaired + Loperamide (3 mg/kg, s.c., bid x 5)
Student's t-test, and differences were considered statistically 100
significant when p o0.05.

Rhein concentration (μg/mL) 10

3. Results

3.1. LC-MS/MS and method validation

1
In order to investigate the comparative pharmacokinetics of
rhein in normal and constipated rats, a sensitive and reliable
analytical method was developed and validated. The validated
LC-MS/MS methods for rhein analysis have been reported in our 0.1
previous study (Hou et al., 2014) and applied for further pharma-
cokinetic studies. Briefly, the MRM mode provided high selectivity
and sensitivity for the quantification assay (Fig. 1). The represnta-
tive chromatograms of rhein and ibuprofen (IS) under the opti- 0.01
mized conditions are shown in Fig. 2 and the results show that no 0 60 120 180 240 300 360
interferences existed under the present analytical conditions. The Time (min)
linear regression analysis results showed that the coefficient
Fig. 3. Mean plasma concentration-time profile of rhein after the Chinese herbal
estimation of the standard curve was 4 0.995. The data showed formula San-Huang-Xie-Xin-Tang (SHXXT; 5 g/kg, p.o.) was given alone (●) or
excellent reproducibility. The limit of detection (LOD) and quanti- loperamide-induced (3 mg/kg, s.c., twice per day for 5 days) constipated rats (○).
fication (LOQ) of rhein were 50 and 100 ng/mL, respectively. The Each point represents mean7 SEM (n ¼6).

100 100 100

2 2

1
Relative Abundance

% % %
1

0 0 0
2 4 6 8 10 2 4 6 8 10 2 4 6 8 10
Fig. 2. Representative UPLC-MS/MS chromatograms of: (A) blank rat plasma sample; (B) blank rat plasma sample spiked with rhein (2500 ng/mL); (C) rat plasma sample
containing rhein (869 ng/mL) collected at 90 min after the Chinese herbal formula San-Huang-Xie-Xin-Tang administration (SHXXT; 5 g/kg, p.o.). 1: rhein (retention time:
5.57 min); 2: ibuprofen (internal standard; 2500 ng/mL, retention time: 6.95 min).
 M.-L. Hou et al. / Journal of Ethnopharmacology 155 (2014) 1291–1299
pharmacokinetic profiling of rhein is be altered under constipa-
tion, the pharmacokinetics of rhein in normal and constipated rats
was investigated. The given dose of SHXXT for comparison was
followed as in our previous study (Hou et al., 2014). The mean
plasma concentration-time profiles of rhein after oral dosage of
SHXXT (5 g/kg) to normal or constipated rats (n ¼6) are illustrated
in Fig. 3, and their pharmacokinetic parameters were calculated, as
presented in Table 1. As Fig. 3 shows, the phenomenon of the
concentration vs. time curve of rhein is different in normal and
constipated rats. With the combination of loperamide and SHXXT,
Cmax of rhein was markedly reduced. Meanwhile, changes in the
pharmacokinetic parameters of rhein under constipation com-
pared with the normal group were as follows: Cmax was signifi-
cantly reduced by 2.5-fold (p o0.05); otherwise, Tmax was
significantly prolonged by 2.8-fold (p o0.05). The AUC of rhein
was decreased by 27.8%. The elimination half-life (t1/2) was
prolonged by 1.6-fold and the volume of distribution (Vss) was
increased by 2.2-fold. In addition, the mean residence time (MRT)
was significantly prolonged by 1.7-fold (p o0.05). After comparing
the pharmacokinetic results of the normal and constipated rats
with t-test, it is found that the important pharmacokinetic para-
meters Cmax, Tmax, and MRT were markedly different in the
constipated rats.
3.3. Gene expression profiling
High-density oligonucleotide microarray analysis was used for
the determination of gene expression in rat liver after treatment
with SHXXT. Total RNA was isolated from liver samples of control
rats and rats treated with SHXXT (0.5 g/kg, p.o., daily for 7 days).
The overall quality of data from the microarrays was first assessed
by hierarchical cluster analysis (HCA). The log2 intensity of the
entire gene set including 25,338 probes was scaled by Z-score
transformation, and then these values were hierarchically clus-
tered using a distance metric of 1  R, where R is the Pearson
correlation coefficient between two samples, and the average
linkage (Fig. 4). As expected, the two technical replicates for the
same RNA samples were tightly grouped, indicating close similar-
ity between them. For advanced data analysis, all biological
replicates were pooled and calculated to identify differentially
expressed genes based on the threshold of fold change and
p-value. A subset of differential genes was selected for clustering
analysis. For this microarray project, the number of genes clus-
tered was 190.
The intensities of gene expression data were also analyzed by
principal component analysis (PCA). PCA uses analysis of the
Table 1
Comparative pharmacokinetic parameters of rhein after oral administration of
SHXXT (5 g/kg, p.o.) in normal and loperamide-induced constipated rats.
PK parameters SHXXT (5 g/kg, p.o.) SHXXT (5 g/kg, p.o.)
þ loperamide
Cmax (mg/mL) 41.8 710.7 16.9 7 4.16n
Tmax (min) 15 70 42.5 7 11.9n
t1/2 (min) 50.2 72.73 79.2 7 16.1
AUC (min mg/mL) 1712 7575 1236 7 235
CL (mL/min/kg) 9.22 71.44 11.17 2.18
MRT (min) 50.2 74.14 837 10.3n
Vss (L/kg) 0.69 70.12 1.50 7 0.52
Data expressed as mean 7 S.E.M. (n¼ 6). The constipation was induced by loper-
amide (3 mg/kg, s.c.) twice per day for 5 days and then SHXXT (5 g/kg, p.o.) was
administered for pharmacokinetic study. Cmax, the peak plasma concentration of a
drug after administration; Tmax, the time point of maximum plasma concentration;
t1/2, elimination half-life; AUC, area under the concentration vs. time curve; CL,
total body clearance; MRT, mean residence time; Vss, volume of distribution.
n
Significantly different from SHXXT alone at p o 0.05.
1295
principal sources of variance in data and displays this information
graphically in a two-dimensional or three-dimensional form, and
it is commonly used to classify gene expression profiles (Guo et al.,
2010a). Fig. 5 displays a PCA 3D view using the first three principal
components for gene expression profiles from the samples. The
PCA results show that the variable of the first three principal
components (PC1, PC2, and PC3) for this study were 38.2, 28.0, and
17.9%, respectively. These results of PCA are consistent with the
results from HCA. Less separation was observed between control
and SHXXT-treated groups based on PCA.
The normalized data were used for selection of differentially
expressed genes (DEGs). A 2-fold change in gene expression was
used to compare the controls and a P-value less than 0.05 for the
difference as minimum requirements for the selection of DEGs.
Based on these two criteria, a total of 110, 126, 136, and 82 genes,
respectively, in SHXXT-treated groups were up-regulated and
identified as DEGs. However, a total of 88, 140, 142, and 105 genes,
respectively, in SHXXT-treated groups were down-regulated and
identified as DEGs.
Five drug-metabolizing genes were significantly altered by the
SHXXT (0.5 g/kg) treatment. Of the five altered genes, four were
up-regulated and one was down-regulated. As tabulated in
Table 2, among the five drug-metabolizing enzyme associated
genes, two genes (Cyp7a1 and Cyp2c6) are Phase I metabolizing
enzymes; one gene (Ces2e) is Phase II metabolizing enzymes, and
two genes (Atp1b1 and Slc7a2) are transporters (Phase III).
RT-PCR assay was used to verify the results for a selected group
of genes whose expression changes were seen observed using
microarrays. Three drug-metabolizing genes were selected for the
RT-PCR validation and the results are shown in Table 2. Based on
triplicate measurements for each RNA sample, the genes Cyp7a1
and Cyp2c6 were over-expressed, whereas Ces2e was down-
expressed. As expected, the RT-PCR data were in good agreement
with the microarray data (Table 2).
4. Discussion
In the present study, the validated LC-MS/MS methods were
applied for study on pharmacokinetics of rhein in normal and
constipated rats, and furthermore, the MRM data demonstrated
that the quantitative mass transition of rhein is consistent with the
previous reports (Jiang et al., 2012; Zan et al., 2011). Although the
pharmacokinetics of SHXXT prepared by decoction and macera-
tion have been investigated (Yin et al., 2009; Zan et al., 2011;
Zhang et al., 2013), the pharmacokinetic results demonstrate that
different preparation methods affected the pharmacokinetic char-
acteristics of active constituents in SHXXT. Thus, we investigated
the pharmacokinetics of SHXXT preparations from pharmaceutical
companies instead of preparing SHXXT decoctions ourselves.
According to our previous pharmacokinetic study results, the
herbal formulae (SHXXT) are more efficient than the single herb
(rhubarb) or the pure compound (rhein) in rhein absorption (Hou
et al., 2014). In addition, our results demonstrate that the herbal
formulae (SHXXT) contains multiple herbs that have synergistic
effects according to one of the basic concepts of oriental medicine,
the sovereign, minister, assiatant and courier principles. However,
there is still limited information on the comparative pharmacoki-
netic studies of Chinese herbal formulae in normal and consti-
pated rats and the impacts on pharmacokinetics of rhein in
constipated animals.
It is well documented that loperamide abolishes experimental
osmotic diarrhea by acting on intestinal motility, and consequently
reducing the flow entering the colon (Schiller et al., 1984). Reduced
fecal excretion in loperamide-administered rats compared to
normal rats has confirmed that loperamide induced constipation
1296 M.-L. Hou et al. / Journal of Ethnopharmacology 155 (2014) 1291–1299

Fig. 4. Hierarchical cluster analysis (HCA) of expression profiles for the control and SHXXT-treated groups, which as treated by the Chinese herbal formula San-Huang-Xie-
Xin-Tang (SHXXT; 0.5 g/kg, p.o., daily for 7 days). HCA was applied across the SHXXT-treated and control groups using a combined list genes identified to be altered
statistically significantly in at least one of the samples studied relative to control. Each column represents the results from an individual hybridization. Each row represents
the log2 intensity values of the 4 samples for one particular gene. Samples are labeled according to the convention of control groups (N1 and N2) and SHXXT-treated groups
(SH1 and SH2)_Technical replicate number. Green indicates genes that are down-regulated; red indicates genes that are up-regulated in treated animals relative to control
animals. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
 M.-L. Hou et al. / Journal of Ethnopharmacology 155 (2014) 1291–1299 1297

(Shimotoyodome et al., 2000; Wintola et al., 2010, 2011). Accord-
ing to our results, for inducing constipation, subcutaneous admin-
istration with loperamide (3 mg/kg) reduced the number (467 4
vs. normal control, 6578), the weight (21.4 72.95 g vs. normal
control, 24.3 71.88 g) and water content (5.84 72.20 g vs. normal
control, 7.02 71.39 g) of fecal pellets compared with the control
rats. Many studies on the laxative effects of aqueous plant extracts
or traditional Chinese formula have been reported (Kakino et al.,
2010; Kakino et al., 2012; Kim et al., 2013; Meite et al., 2010;
Wintola et al., 2010, 2011; Wu et al., 2010; Wu et al., 2011), though
there is limited information on the pharmacokinetics of aqueous
plant extracts or traditional Chinese formula under constipated
conditions. Additionally, investigations on the comparative phar-
macokinetic profiles of drugs with the various body conditions
have been reported (Dai et al., 2014; Feng et al., 2013; Han et al.,
2013; Zhu et al., 2014). In the previous study, it was found that the
absorption times of rhein and chrysophanol were accelerated and
the absorption amount of these two compounds was increased in
rabbits with acute blood stasis, suggesting that rhein and chryso-
phanol would possibly be the two effective compounds in Quyu
Qingre granules (Dai et al., 2014). Otherwise, there are no studies
concerning about the comparative pharmacokinetics of rhein in
PC2
PC1
PC3
Fig. 5. Principal component analysis (PCA) of gene expression profiles for the
control (N) and SH groups, which as treated by the Chinese herbal formula San-
Huang-Xie-Xin-Tang (SHXXT; 0.5 g/kg, p.o., daily for 7 days). The intensity of the
entire gene set was used; no specific cutoff was applied for the analysis. The
autoscaled method was used for PCA. The purple and green dots indicate control
group. The red and blue dots indicate the SHXXT-treated group. (For interpretation
of the references to color in this figure legend, the reader is referred to the web
version of this article.)
normal and constipated animals. As shown in Fig. 3, the
concentration-time curve of rhein is different from that of normal
rats, indicating that constipation affects the pharmacokinetics of
rhein. Although the pharmacokinetic parameters of rhein under
constipation have been changed, only Cmax, Tmax, and MRT of rhein
have a significant difference from the normal control group. This
suggests that constipation reduced the rhein absorption and
retained the rhein excretion in the gastrointestinal system. It is
known that loperamide-induced delays in colonic transit is a
spastic constipation due to its inhibition on stool frequency in
humans (Camilleri, 2011) and the increase of colonic contractions
in animals (Kim et al., 2013); thus, it is speculated that the MRT of
rhein was prolonged significantly by 1.7-fold (p o0.05) due to the
slow transit of intestinal motility induced by loperamide admin-
istration. Meanwhile, according to our pharmacokinetic results,
Cmax of rhein was significantly reduced by 2.5-fold (p o0.05),
indicating that constipation influenced the absorption of rhein
from the gastrointestinal system. The reduced absorption of rhein
may illustrate that the AUC of rhein in plasma was not higher in
constipated rats with prolonged intestine retention time. As
expected, the elimination half-life (t1/2) was prolonged by 1.6-fold,
while Tmax and volume of distribution (Vss) was increased by 2.8-
fold (p o0.05) and 2.2-fold, respectively.
Recent studies on the molecular mechanisms of traditional
Chinese medicinal formulae (Cheng et al., 2010; Cheng et al., 2008;
Wen et al., 2011) or herbal extracts (Guo et al., 2010a; Guo et al.,
2010b; Guo et al., 2010c) using gene expression microarray have
been reported. The results of Guo et al. (Guo et al., 2010a)
indicated that drug-metabolizing genes were significantly altered
in response to Ginkgo Biloba extracts treatments. In addition,
pathway and network analyses were applied to investigate the
gene relationships, functional clustering, and mechanisms
involved in Ginkgo Biloba extracts. In addition, these results of
analyses demonstrate that Ginkgo Biloba-related drug metaboliz-
ing enzymes may cause herb–drug interactions and contribute to
hepatotoxicity. Furthermore, the outcomes of pathway and net-
work analysis may be used to elucidate the toxic mechanisms of
Ginkgo Biloba (Guo et al., 2010a). Possible mechanisms for SHXXT
and the relationship between SHXXT and its herbal components in
HepG2 cells by microarray technique have been investigated
(Cheng et al., 2008). The results indicated that SHXXT and its
components had a unique anti-proliferation pattern through the
p53 signaling pathway and the DNA damage signaling pathway in
HepG2 cells (Cheng et al., 2008). Because metabolic activation of
chemicals is very important for liver toxicity and herb–drug
interactions, and because alteration of drug metabolizing genes
is suspected to contribute to the toxicity of SHXXT, we first
investigated the gene expression changes of drug-metabolizing
enzymes and found that the expression of a relatively small
number of drug-metabolizing genes was altered. Generally,
the expression of differentially regulated genes obtained from

Table 2
Drug metabolizing genes altered by 0.5 g/kg SHXXT treatment in rat liver and the gene expression fold changes of RT-PCR in comparison with the microarray.

Gene Gene bank ID Microarray fold P-value Gene description RT-PCR fold change
symbol change

Phase I metabolism
Cyp7a1 PH_rn_0010923 2.38 0.0007 Cytochrome P450, family 7, subfamily a, polypeptide 1 1.62 7 0.55
Cyp2c6 PH_rn_0018070 2.06 0.0114 Cytochrome P450, family 2, subfamily c, polypeptide 6 0.81 7 0.61
Phase II metabolism
Ces2e PH_rn_0013262  19.1 o 0.0001 Carboxylesterase 2E  1.80 7 0.31
Phase III metabolism
Atp1b1 PH_rn_0002686 2.32 o 0.0001 ATPase, Naþ /K þ transporting, beta 1 polypeptide
Slc7a2 PH_rn_0018451 2.18 0.0001 Solute carrier family 7 (cationic amino acid transporter, y þ system), member 2

The symbol of minus (  ) indicates down-regulation.

1298 M.-L. Hou et al. / Journal of Ethnopharmacology 155 (2014) 1291–1299
a microarray should be validated by real-time polymerase chain
reaction or Northern blot. As expected, the RT-PCR data were
consistent with the microarray data. Overall, five drug-
metabolizing genes including Cyp7a1, Cyp2c6, Ces2e, Atp1b1,
and Slc7a2 were significantly altered by the SHXXT (0.5 g/kg)
treatment. This is the first report to reveal gene expression
profiling in drug metabolizing genes after SHXXT treatment.
5. Conclusions
An UPLC-MS/MS method for the quantification of rhein and its
application to determine the comparative pharmacokinetics of
rhein in normal and constipated rats have been developed and
validated. In addition, the method was applied for a pharmacoki-
netic study in freely moving rats. The comparative pharmacoki-
netic parameters of rhein in normal and constipated rats after oral
dosage with the Chinese herbal formulae (SHXXT) are here
reported for the first time. These pharmacokinetic parameters
may be used as reference for the clinical prescription compatibility
of traditional Chinese medicine prescriptions. Constipation
reduced the absorption of rhein and the MRT of rhein was
significantly prolonged. Gene expression profiling illustrated that
five drug metabolizing enzymes that may affect the contribution
to therapeutic effects, hepatotoxicity and safety evaluation, were
altered by SHXXT treatment.
Acknowledgment
Funding for this study was provided in part by research grants
from the National Science Council (NSC102-2113-M-010-001-MY3)
Taiwan; and TCH10301-62-021; TCH103-02 from Taipei City Hos-
pital, Taipei, Taiwan.
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