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Chemical comparison of dried rehmannia root and prepared rehmannia root by UPLC-TOF MS and HPLC-ELSD with multivariate statistical analysis

  • Beijing Institute of radiation medicine

Abstract and Figures

To identify the chemical differences which lead to the different therapeutic effects of dried rehmannia root (DRR) and prepared rehmannia root (PRR), we compared the chemical composition of decoctions of randomly purchased DRR and PRR using ultra performance liquid chromatography (UPLC) coupled with time-of-flight mass spectrometry and high performance liquid chromatography (HPLC) coupled with evaporative light scattering detection (ELSD) with the aid of multivariate statistical analysis. Both approaches clearly revealed compositional and quantitative differences between DRR and PRR. UPLC-MS data indicated stachyose, rehmaionoside A (or rehmaionoside B), acteoside (or forsythiaside, or isoacteoside), 6-O-coumaroylajugol (or 6-O-E-feruloylajugol, or 6-O-Z-feruloylajugol) as important discriminators between DRR and PRR decoctions. HPLC-ELSD analysis showed that the content of fructose in the decoctions of PRR was about four times greater than that of DRR (P<10−5), while sucrose content in the decoctions of PRR was only about one seventh of that in DRR (P<0.01). Our results suggest that some compounds, such as fructose, stachyose and rehmaionoside, may be responsible for the differing therapeutic effects of DRR and PRR. Furthermore, improvements in quality control for PRR, which is currently lacking in the Chinese Pharmacopoeia, are recommended.
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Chemical comparison of dried rehmannia root and prepared
rehmannia root by UPLC-TOF MS and HPLC-ELSD with
multivariate statistical analysis
Qiande Liang
, Jing Ma
, Zengchun Ma
, Yuguang Wang
, Hongling Tan
Chengrong Xiao
, Ming Liu
, Beibei Lu
, Boli Zhang
, Yue Gao
Institute of Radiation Medicine, Academy of Military Medical Science, Beijing 100850, China
Tianjin University of Traditional Chinese Medicine, Tianjin 300193, China
Received 18 August 2012; revised 28 September 2012; accepted 22 October 2012
Rehmannia root;
Liquid chromatography;
Mass spectrometry;
Evaporated light
scattering detection;
Multivariate statistical
Abstract To identify the chemical differences which lead to the different therapeutic effects of
dried rehmannia root (DRR) and prepared rehmannia root (PRR), we compared the chemical
composition of decoctions of randomly purchased DRR and PRR using ultra performance liquid
chromatography (UPLC) coupled with time-of-flight mass spectrometry and high performance
liquid chromatography (HPLC) coupled with evaporative light scattering detection (ELSD) with
the aid of multivariate statistical analysis. Both approaches clearly revealed compositional and
quantitative differences between DRR and PRR. UPLC-MS data indicated stachyose, rehmaiono-
side A (or rehmaionoside B), acteoside (or forsythiaside, or isoacteoside), 6-O-coumaroylajugol
(or 6-O-E-feruloylajugol, or 6-O-Z-feruloylajugol) as important discriminators between DRR and
PRR decoctions. HPLC-ELSD analysis showed that the content of fructose in the decoctions of
PRR was about four times greater than that of DRR (Po10
), while sucrose content in the
Institute of Materia Medica, Chinese Academy of Medical Sciences
Chinese Pharmaceutical Association
Acta Pharmaceutica Sinica B
2211-3835 &2012 Institute of Materia Medica, Chinese Academy of Medical Sciences and Chinese Pharmaceutical Association. Production and
hosting by Elsevier B.V. All rights reserved.
Corresponding author.
E-mail address: (Yue Gao).
These authors contributed equally to this work.
Peer review under responsibility of Institute of Materia Medica, Chinese Academy of Medical Sciences and Chinese Pharmaceutical Association.
Acta Pharmaceutica Sinica B 2013;3(1):55–64
decoctions of PRR was only about one seventh of that in DRR (Po0.01). Our results suggest that
some compounds, such as fructose, stachyose and rehmaionoside, may be responsible for the
differing therapeutic effects of DRR and PRR. Furthermore, improvements in quality control for
PRR, which is currently lacking in the Chinese Pharmacopoeia, are recommended.
&2012 Institute of Materia Medica, Chinese Academy of Medical Sciences and Chinese Pharmaceutical
Association. Production and hosting by Elsevier B.V. All rights reserved.
1. Introduction
Dried rehmannia root (DRR) and prepared rehmannia root
(PRR) are herbal drugs that are frequently used in traditional
Chinese medicine (TCM). Although they are both prepared
from rehmannia root (Rehmannia glutinosa Libosch) and PRR
is prepared by further braising or steaming DRR
, they have
different therapeutic effects and are used for different ther-
apeutic purposes. According to the theory and practice of
TCM and the ancient Chinese philosophy, ‘‘Yin’’ is a concept
which is a contrary to ‘‘Yang’’ and ‘‘Yin’’ refers to the inner,
lower, weak, passive, tranquil or material aspects of things
(including the human body) while ‘‘Yang’’ refers to the outer,
upper, strong, positive, active or functional aspects of things.
Based on this concept, in addition to their common effect of
‘‘nourishing the body’s Yin’’, DRR possesses a ‘‘cold’’
character and can ‘‘reduce the body’s heat’’, while PRR
possesses a ‘‘warm’’ character as a simple tonic and it does
not ‘‘reduce the body’s heat’’ but can ‘‘replenish the blood and
reinforce the body’s essence and marrow’’
. These ‘‘pharma-
cological’’ differences have been documented in the TCM
literature for at least three hundred years
. In clinical practice,
DRR is used mainly for diseases showing ‘‘hot’’ symptoms,
such as feeling hot, deep-red tongue, fidgets, thirst and
exanthemata, while PRR is used mainly for diseases showing
‘‘blood-lacking’’ or ‘‘Yin-lacking’’ symptoms, such as feeling
faint, weak legs, yellow complexion, palpitations and tinnitus.
The reason for the difference in therapeutic effects between
DRR and PRR is currently not clear but surely lies in chemical
differences. A few studies concerning the chemical differences have
investigated the changes in
saccharide content and composition in rehmannia root prepara-
tions using high performance liquid chromatography (HPLC)
with differential refractive index detection during processing. They
found that peaks of glucose and fructose became outstanding
when DRR was steamed to PRR and deduced that defructosyla-
tion of stachyose, a major component of DRR, may take place
during steaming. The increase of fructose and decrease of
stachyose was also reported by Bian et al.
in 1995 using thin
layer chromatogram scan analysis. However, due to low resolution
or limited sensitivity of the above-mentioned methods, differences
in sugar content between DRR and PRR warrants verification by
more advanced methods. In 2010, Li et al.
explored potential
chemical markers using ultra high performance liquid chromato-
graphy coupled with time-of-flight mass spectrometry (TOF MS)
and multivariate statistical analysis (MVA) for the discrimination
of DRR from PRR. Leonuride or its isomer and 5-(a-D-
boxaldehyde were found as the most characteristic markers of
DRR and PRR. But their study did not report any differences in
sugar content or composition. Perhaps this was partly due to the
poor sensitivity of the electrospray ionization (ESI) mass spectro-
metric method for neutral carbohydrates
and the weak retention
of many neutral carbohydrates on the HSS T3
column used in
this study. Another reason may be that their sample extraction
method was ultrasonic extraction with methanol–water under
room temperature, rather than hot water extraction as carried out
by Wen et al.
. However, we believe that hot water extraction is
more reasonable than room temperature methanol–water extrac-
tion, because water decoction is the routinely used procedure for
both DRR and PRR, and chemical differences in decoctions
prepared in this manner may correlate more closely with differ-
ences in therapeutic effects. Hence, in order to identify chemical
differences between DRR and PRR which may be responsible for
the differences in therapeutic effects, a more comprehensive
investigation is required between the decoctions of DRR and
PRR that includes both saccharide and non-saccharide
Liquid chromatography-mass spectrometry (LC-MS) with
MVA is a feasible strategy for chemical analysis of TCM drug
samples which are complex mixtures
. MVA is helpful in analysis
of the huge data matrix of multiple variables generated by LC-
MS. It finds out whether there is difference between groups of
samples utilizing principal component analysis (PCA) and which
signals or variables contribute the grouping by orthogonal partial
least-squares to latent structures discriminate analysis (OPLS-
DA). MS (especially high-resolution MS) provides further useful
qualitative information on the chemical substances producing the
signals present in this analysis.
In this study, with the aid of MVA, we employed both ultra
performance liquid chromatography (UPLC) coupled with
TOF-MS and HPLC coupled with evaporative light scattering
detection (ELSD), which is a suitable method for sugar
analysis, to compare the chemical compositions of decoctions
of DRR and PRR. Our goal was to acquire a relatively
comprehensive analysis of their chemical differences, which
may explain their differing therapeutic effects.
2. Materials and methods
2.1. Drugs and reagents
PRR and DRR samples (six each) were purchased from six
different drug stores in China (Table 1). D-Fructose was
purchased from Sinopharm Chemical Reagent Co., Ltd.
(Shanghai, China). D-Glucose and sucrose were purchased
from Beijing Chemical Reagent Company (Beijing, China).
Acetonitrile was of HPLC grade (Fisher Scientific, Fair Lawn,
New Jersey, USA). Deionized water was prepared with a
Millipore water purification system.
Qiande Liang et al.56
2.2. Analytical apparatus
A Waters Acquity UPLC system consisting of a binary solvent
manager, a sample manager and an ELSD detector, a Waters
SYNAPT HDMS quadrupole time-of-flight mass spectro-
meter and an analytical workstation with Waters MassLynx
4.1 software were used.
2.3. Sample preparation
20 g of each sample was weighed, soaked into 160 mL water
for 60 min at room temperature and boiled for 30 min.
The suspension was filtered and 100 mL water was added
for the second decoction with a duration of 20 min. The
filtered and mixed suspension from the two decoctions was
adjusted to a volume of 200 mL. 20 mL of the decoction was
precipitated by adding 80 mL alcohol and filtered. The filtrates
were evaporated to less than 1 mL at 50 1Cin vacuo. The
evaporated residue was dissolved with 50% acetonitrile into a
volumetric flask. The final volume of the sample solution was
set to 25 mL. Three standard compounds, D-fructose, D-
glucose and sucrose, were weighed and dissolved in 50%
acetonitrile to give serial concentrations within the range of
0.80–10.08 mg/mL, 0.80–20.08 mg/mL and 0.81–20.24 mg/
mL, respectively. The solutions of standards and samples
were filtered through a 0.45 mm (HPLC)/0.22 mm (UPLC)
membrane before injection into the UPLC or HPLC system.
2.4. UPLC-TOF MS analysis
Separations were carried out with a Waters Acquity HSS T3
column (100 mm 2.1 mm I.D., 1.8 mm). The mobile phase
consisted of (A) water containing 0.1% formic acid and (B)
acetonitrile containing 0.1% formic acid. The eluting conditions
were: isocratic 2% B (0–1 min), linear gradient from 2% to 5% B
(1–2 min), 5% to 12% B (2–5 min), 12% to 20% B (5–10 min),
20% to 30% B (10–12 min), 30% to 50% B
(12–13 min), 50% to 100% B (13–15 min), isocratic 100% B for
1 min, then back to 2% B in 1 min and isocratic 2% B for 3 min
before next run. The flow rate was 0.5 mL/min.
The column was maintained at 24 1C. The injection volume was
2mL. Mass spectrometry was performed with ESI source operat-
ing in negative ion mode. The lock mass compound was leucine
enkephalin (m/z554.2615). The lock spray reference scan
frequency was 20 s with reference cone voltage of 30 V. The
source temperature was set at 100 1C and the desolvation
temperature was set at 450 1C with desolvation gas flow of
900 L/h. The capillary voltage and cone voltage were set to
3 kV and 40 V, respectively. The collision energies were set as
6 V (trap)/4 V (transfer) with 2.00 mL/min trap gas flow. The LC-
MS data acquisition was controlled by MassLynx 4.1 software.
2.5. HPLC-ELSD analysis
Separations were carried out with a SHISEIDO carbohydrate
column (150 mm 2.0 mm I.D., 5 mm). The mobile phase was
an isocratic mixture of acetonitrile and water (80:20, v/v).
The column temperature was kept constant at 24 1C and the
flow rate was 0.6 mL/min. The drift tube temperature, gas
pressure and gain were 40 1C, 40 psi and 25, respectively. The
sample injection volume was 2 mL. The compounds were
identified by comparing their retention time values with those
of the standards (D-fructose, D-glucose and sucrose). The
HPLC-ELSD data acquisition was controlled by MassLynx
4.1 software.
2.6. Principal component analysis (PCA)
The PCA was carried out with the data of all samples. The
UPLC-MS data were processed by MarkerLynx XS and
EZinfo, sub modules of the MassLynx 4.1 software, to
accomplish signal deconvolution and MVA. The intensity
threshold was set to 100 counts. The HPLC-ELSD chromato-
grams were integrated by MassLynx software to give peak
area values of D-fructose, D-glucose and sucrose. PCA were
performed with UPLC-MS data (peak heights) and HPLC-
ELSD data (peak areas of D-fructose, D-glucose and sucrose)
separately to acquire an overview of trends or patterns.
The data scaling method was Pareto.
2.7. Orthogonal partial least-squares to latent structures
discriminate analysis (OPLS-DA)
Using the EZinfo module, OPLS-DA was carried out with
UPLC-MS data of 11 samples, DRR1–6 and PRR2–5, to
discover relatively important chemical markers. The PRR1
was excluded as an abnormal sample according to PCA
results. The data scaling method was Pareto.
Table 1 DRR and PRR samples analyzed in the present study.
Sample Location of purchase Time of purchase
DRR1 Kang Shou Tang drug store, Beijing 2009-10-8
DRR2 Xi Dan Jin Xiang drug store, Beijing 2009-10-8
DRR3 Gong Zhu Fen Tong Ren Tang drug store, Beijing 2009-10-8
DRR4 Kang Yu drug store, Beijing 2009-10-8
DRR5 Qian Men Tong Ren Tang drug store, Beijing 2009-10-8
DRR6 Bao Ding Tong Ren Tang drug store, Baoding City, Hebei Province 2009-10-2
PRR1 Kang Shou Tang drug store, Beijing 2009-10-8
PRR2 Xi Dan Jin Xiang drug store, Beijing 2009-10-8
PRR3 Gong Zhu Fen Tong Ren Tang drug store, Beijing 2009-10-8
PRR4 Kang Yu drug store, Beijing 2009-10-8
PRR5 Qian Men Tong Ren Tang drug store, Beijing 2009-10-8
PRR6 Bao Ding Tong Ren Tang drug store, Baoding City, Hebei Province 2009-10-2
Chemical comparison of dried rehmannia root and prepared rehmannia root by UPLC-TOF MS and HPLC-ELSD 57
2.8. Establishment of in-house library and preliminary
identification of chemical markers
By searching from Zhou et al.’s handbook
, PubMed and the
Chinese National Knowledge Infrastructure (CNKI) of Tsin-
ghua University, all compounds reported in the literature on
rehmannia root were summarized in a Microsoft Excel table to
establish an in-house library, which includes the name, mole-
cular formula and chemical structure of each published known
compound. The exact m/zvalues of putative deprotonated
quasi-molecular ions [M–H]
, the formic acid group adductive
ions [MþHCOO]
and the deprotonated dimeric ions [2M–
were calculated to compare with the observed exact m/z
values of those relatively important chemical makers discov-
ered by OPLS-DA. The possible identities of the markers were
deduced at mass accuracy of less than 3 ppm.
2.9. Determination of D-fructose, D-glucose and sucrose
Log/log calibration curves of D-fructose, D-glucose and
sucrose were obtained from the log values of the peak areas
over the log values of the concentrations of the standards.
Concentrations of D-fructose, D-glucose and sucrose in the
decoctions of the samples were calculated from this regression
analysis, and expressed as mean7standard deviation. The
significance of difference between two groups was analyzed
statistically by Student’s t-test.
3. Results and discussion
3.1. Data overview with PCA score plots
Figs. 1 and 2 show typical UPLC-MS and HPLC-ELSD
chromatograms. Figs. 3 and 4 show PCA score plots of
UPLC-MS data and HPLC-ELSD sugar data, respectively.
Distinct pattern of discrimination by the first principal
component scores (t[1]) between DRR and PRR samples can
be observed in both figures, i.e., the DRR and PRR samples
clustered towards opposite ends of the t[1] axis. The presence
of sample PRR1 markedly near the DRR cluster but far from
the PRR cluster in both figures suggests that PRR1 is likely to
be a substandard product or a product incorrectly handled in
some step during the course from factory to market. There-
fore, we have excluded sample PRR1 from further analysis.
3.2. Marker discovery by OPLS-DA and preliminary
assignment of identities
Fig. 5 shows the S-plots produced by the OPLS-DA of UPLC-
MS data with PRR1 excluded. Each green dot represents a
variable in the OPLS-DA model, i.e., a LC-MS peak with a
certain m/zvalue and a certain retention time (t
). The S-Plot
visualizes the variable influence in the model where the X-axis
represents contribution (covariance) and the Y-axis (spans
between 1 and 1) represents correlation (reliability)
Figure 1 Typical UPLC-MS BPI chromatograms of (a) DRR and (b) PRR. BPI chromatogram: base peak intensity chromatogram.
Qiande Liang et al.58
markers were indicated by those dots marked with red square,
which were automatically selected by the ‘‘select default’’ func-
tion of the software. These dots are the variables that differ-
entiate the most between the two groups and are therefore
relatively important. The t
and m/zvalue of these selected
variables and their identities tentatively assigned are listed in
Table 2. The identities of markers were assigned by searching
and matching the reported constituents of rehmannia root in the
in-house library at the mass accuracy of less than 3 ppm using
measured and theoretical exact m/zvalues. Among these selected
markers, stachyose (C
,MW¼666, peak 9)
noside A (or rehmaionoside B, C
,MW¼390, peak 11)
acteoside (or forsythiaside or isoacteoside, C
MW¼624, peak 12)
,6-O-coumaroylajugol (or 6-O-E-
feruloylajugol or 6-O-Z-feruloylajugol, C
peak 13)
were tentatively identified. The measured exact m/z
value of peak 2 and peak 6 approximate the theoretical exact m/z
value of [M–H]
of D-mannitol (C
double trisaccharide complex (C
with mass accuracy of 9.94 and 7.64 ppm, respectively. Fig. 6
shows the selected ion intensity trend plots of four tentatively
identified markers (peaks 9, 11, 12 and 13).
3.3. Contents of D-fructose, D-glucose and sucrose in
decoctions of DRR and PRR
Log/log calibration curves showed good linear relations
between the log values of the peak areas by HPLC-ELSD
and the log values of the concentrations of the standard
solutions. The regression equations were y¼0.6835x2.5027
¼0.9974) with linear range 0.80–10.08 mg/mL for D-fruc-
tose, y¼0.6228x2.0732 (r
¼0.9993) with linear range 0.80–
20.08 mg/mL for D-glucose, and y¼0.6443x2.2590
¼0.9993) with linear range 0.81–20.24 mg/mL for sucrose,
where xrepresents log value of peak area and yrepresents log
value of concentration. Concentrations of D-fructose, D-glu-
cose and sucrose in DRR and PRR with PRR1 excluded are
summarized in Fig. 7. The fructose content in the decoctions
of PRR (8.75 mg/mL) was about 4 times that in DRR
(2.29 mg/mL) (Po10
), while the sucrose content in the
decoctions of PRR (0.52 mg/mL) was only about one seventh
of that in DRR (3.55 mg/mL) (Po0.01). The glucose content
in the decoctions of PRR (9.08 mg/mL) increased approxi-
mately 57% as compared with DRR (5.80 mg/mL) (Po0.05).
These results show that the increase in fructose and decrease in
sucrose when DRR is converted to PRR is significant and
remarkable. The change in the content of these sugars does
not contradict the findings of Wen et al.
and Bian et al.
is much clearer now from our results. The preparation process
increases the amount of fructose in the PRR decoction while
eliminating most of the sucrose in the DRR decoction,
indicating that the saccharide changes caused by preparation
are substantial and distinctive.
3.4. Physiological and pharmaceutical indications
It is interesting that our findings on chemical markers in this
study differ largely from study of Li et al.
reported in 2010
which also used negative ion mode UPLC-TOF MS with MVA.
In their study, six markers were selected from the OPLS-DA S-
Plot, including a(t
2.34 min, m/z449.1283, matching 5-(a-D-
boxaldehyde), b(t
4.11 min, m/z236.0553, unidentified), c(t
2.41 min, m/z465.1236, unidentified), d(t
3.66 min, m/z
393.1360, matching leonuride or its isomer), e(t
3.64 min, m/
z393.1361, matching leonuride or its isomer) and f(t
6.51 min,
m/z345.1549, matching rehmapicroside). The major difference
between our research methods is that they used ultrasonic
extraction with methanol–water under room temperature, while
we used hot water extraction which we deemed to be relatively
closer to clinical practice than their method.
Our study shows distinct changes in saccharide content in the
decoctions after DRR is converted to PRR, i.e., the contents of
monosaccharide, especially fructose, increased while the contents
of some oligosaccharides decreased. The content of fructose and
glucose increased by 282% and 57%, respectively, while the
content of sucrose decreased by 85%. Peak 9 (C
Figure 2 Typical HPLC-ELSD chromatograms of (a) standard
mixture of D-fructose, D-glucose and sucrose, (b) DRR and
(c) PRR.
Chemical comparison of dried rehmannia root and prepared rehmannia root by UPLC-TOF MS and HPLC-ELSD 59
MW¼666) matches stachyose, whose signal intensity dropped
from tens (DRR) to zero (PRR). Stachyose is a tetrasaccharide
reported as one of the most abundant components in DRR with
the content of 30–60%
. The peak matches verbascose (peak
14, C
,MW¼828, mass accuracy ¼1.69 ppm, t
min), a pentasaccharide in DRR
, and also shows a descending
trend (Fig. 8). Although it was not automatically selected, it has a
correlation of 0.9056 and is very near those selected peaks in the
S-Plot (Fig. 5, dot marked with black square). The increase in
fructose and glucose as well as the decrease in stachyose after
DRR conversion to PRR has been reported in literature
confirmed by our results. However the decrease of sucrose and
verbascose has not been reported.
Our previous study on Si-Wu-Tang (SWT) showed that
fructose is a hematopoiesis-promoting compound
a major constituent in SWT, a traditional Chinese formula
consisting of PRR, angelica root, chuanxiong rhizoma and
paeonia root, which possesses hematopoiesis-promoting activity
and is used mainly for ‘‘blood-enrichment’’. Similarly to SWT
administration, the oral administration of fructose increased the
number of peripheral leukocytes and four types of progenitor
cells in bone marrow of irradiated mice (anemia model),
including colony-forming unit-granulocyte-macrophages
(CFU-GM), colony-forming unit-mature erythroid cells (CFU-
E), colony-forming unit-immature erythroid cells (BFU-E), and
colony-forming unit-multipotential cells (CFU-mix)
. Our study
further showed that the free fructose and glucose in SWT
decoction is almost completely provided by PRR
. According
to the conventional theory of TCM which uses ‘‘Jun’’
(‘‘emperor’’, the most important ingredient or the ingredient
plays a central role in a formula), ‘‘Chen’’ (‘‘minister’’), ‘‘Zuo’’
(‘‘assistor’’), and ‘‘Shi’’ (‘‘emissary’’) to identify the importance
and roles of different ingredient drugs in a formula, PRR is the
‘‘Jun’’ in the SWT formula
. Therefore, an interesting coin-
cidence appeared that the ‘‘Jun’’ drug (PRR) in a hematopoiesis-
promoting formula (SWT) provides most of the hematopoiesis-
promoting constituent (fructose). The present study showed a
tremendous increase of fructose content in the decoction after
DRR is converted to PRR. According to the principles and
practice of TCM, PRR is basically used for ‘‘blood-enrichment’’
Figure 3 PCA score plot of UPLC-MS data.
Figure 4 PCA score plot of HPLC-ELSD sugar data.
Figure 5 OPLS-DA S-plot of UPLC-MS data excluded PRR1. Peak 9 matches stachyose. Peak 11 matches rehmaionoside A or B. Peak
12 matches acteoside or forsythiaside or isoacteoside. Peak 13 matches 6-O-Coumaroylajugol or 6-O-E-Feruloylajugol or 6-O-Z-
Feruloylajugol. Peak 14 matches verbascose. Identities of other peaks were not assigned.
Qiande Liang et al.60
not ‘‘heat-reduction’’, while DRR is used for ‘‘heat-reduction’’
not ‘‘blood-enrichment’’. Therefore, another interesting coinci-
dence appeared that a hematopoiesis-promoting constituent
(fructose) increases tremendously when a ‘‘non-blood-enrich-
ment’’ drug (DRR) is prepared to become a ‘‘blood-enrichment’’
drug (PRR). These two coincidences support the speculation that
fructose is an important hematopoiesis-promoting constituent in
SWT as well as in PRR, and at the same time, provide a possible
explanation for why PRR is a ‘‘blood-enriching’’ drug while
DRR is not. The possible mechanism for the hematopoiesis-
Figure 6 Selected ion intensity trend plots of tentatively identified markers automatically selected from OPLS-DA S-plots. (a) Peak 9, m/z
665.2135; (b) Peak 11, m/z435.2227; (c) Peak 12, m/z623.1983; (d) Peak 13, m/z523.1813.
Table 2 Automatically selected variables indicating potential makers in OPLS-DA S-plots and their preliminarily assigned
Assigned identity Molecular
Theoretical exact m/zMass
1 0.50 195.0495 NA
2 0.51 181.0694 NA
3 0.57 105.0179 NA
4 0.57 447.1347 NA
5 0.60 579.1773 NA
6 0.60 1007.3380 NA
7 0.66 799.2383 NA
8 0.81 755.2392 NA
9 0.84 665.2135 Stachyose C
711.2195 1331.4359 0.75 8,15
10 7.66 183.0995 NA
11 8.81 435.2227 Rehmaionoside A or B C
389.2175 435.2230
779.4429 0.69 8,12
12 10.84 623.1983 Acteoside or
or isoacteoside
669.2031 1247.4030 1.12 8,13,14
13 11.84 523.1813 6-O-coumaroylajugol or
6-O-E-feruloylajugol or
569.1870 1047.3709 0.57 8
The underlined theoretical exact m/zvalue is the one matching the measured value. NA, not assigned.
Chemical comparison of dried rehmannia root and prepared rehmannia root by UPLC-TOF MS and HPLC-ELSD 61
promoting effect of fructose was discussed in our previous
The molecular structures of stachyose, verbascose, raffinose
(also a constituent of rehmannia root
) and sucrose all contain a
terminal fructose group. Our results indicate decreases in
stachyose, verbascose and sucrose after DRR is converted to
PRR. It does not contradict Wen et al.’s viewpoint
defructosylation may take place during the preparation process.
Stachyose, verbascose and raffinose belong to the galactosido-
sucrose series of sugars which consist of a1,6-linked chains of
D-galactose attached to the 6-glucosyl position of sucrose. These
are non-digestible by human digestive juices and may promotes
flatulence, but can be preferentially consumed by potentially
remedial intestinal bacteria in the colon
. A US patent of 2007
reported that orally administered stachyose has an antiallergenic
action, particularly in suppressing atopic dermatitis
. Hypogly-
cemic activity was also reported for stachyose and Rehmannia
glutinosa oligosaccharides, the majority of which is sta-
. Whether the special ‘‘cold’’ character and ther-
apeutic effect of ‘‘reducing the body’s heat’’ of DRR is involved
with antiallergenic or hypoglycemic activities requires further
Peak 11 matching rehmaionoside A or B decreased after
preparation of PRR from DRR. The pharmacological effect
of rehmaionoside B was reported by Nakase et al.
in 1991.
They investigated the effects of rehmaionoside B and some
other constituents of rehmannia root on bladder and urethral
smooth muscle strips of mice, and rehmaionoside B most
effectively inhibited the contraction induced by noradrenaline
in urethral smooth muscle. Rehmaionoside B not only
inhibited the spontaneous contraction but also slightly relaxed
the normal tone of the bladder smooth muscle. They con-
cluded that the effect of rehmaionoside B on the bladder was
an indomethacin-like action. As is known, indomethacin is
outstanding for potent anti-inflammatory, analgesic and anti-
pyretic effects
. Interestingly, it has been reported that the
aqueous extract of DRR showed markedly hypotensive,
sedative and anti-inflammatory effects
but the extract of
PRR was ineffective in preventing the development of edema
in arthritic rats and in acute or chronic inflammation
Although Nakase et al.’s report
did not mention whether
rehmaionoside B has hypotensive, sedative or anti-
inflammatory effects, the close relationship between smooth
muscle and blood pressure, and the outstanding anti-
inflammatory effect of indomethacin prompted us to surmise
that rehmaionoside B may contribute to DRR’s hypotensive
or anti-inflammatory activity. It is necessary and worthwhile
to test this hypothesis in future studies. If this hypothesis is
true, we can further surmise that the special ‘‘cold’’ character
and therapeutic effect of ‘‘reducing the body’s heat’’ is, to
some extent, the results of the activity exerted by rehmaiono-
side as an indomethacin-like hypotensive or anti-inflammatory
drug. Further studies are required to verify these hypotheses.
Peak 12 showed an increasing trend after DDR is converted to
PRR, but it is more difficult to surmise its pharmacological
indication because it corresponds to three isomeric compounds in
rehmannia root, i.e., acteoside, forsythiaside and isoacteoside.
Acteoside was reported to exhibit anti-inflammatory, antinephri-
tic, anti-hepatotoxic, antiproliferative, cytotoxic against various
tumor cells, antimetastastic, immunomodulatory and antioxidant
. Isoacteoside also showed an antiproliferative
Figure 8 Selected ion intensity trend plots of a peak (t
¼0.59 min, m/z¼827.2683) identified tentatively as verbascose.
Figure 7 Concentrations of D-fructose, D-glucose and sucrose in
the decoctions of DRR and PRR excluding PRR1. Data
expressed as mean7S.D., n¼6 for DRR, n¼5 for PRR.
Qiande Liang et al.62
. It is reported that forsythiaside has strong antioxidant,
antibacterial and antiviral activities, and also exhibits neuropro-
tective properties and a slow relaxation effect against
norepinephrine-induced contraction of rat aorta
also corresponds to three isomeric compounds reported in
rehmannia root, i.e., 6-O-coumaroylajugol, 6-O-E-feruloylajugol
and 6-O-Z-feruloylajugol, which showed a decreasing trend after
DDR is converted to PRR. Unfortunately, we have not found
any pharmacological reports concerning these compounds.
Another notable point is that the quality standard of PRR in
the Chinese Pharmacopoeia which controls the quality of the
preparation process as well as the quality of the final product
currently is lacking a quality control compound for this prepara-
. The quality of PRR is routinely assessed based on subjective
indices such as color, smell and glutinosity. The findings of this
study suggest that fructose, stachyose and rehmaionoside A or B
could be considered as potential quality control compounds due to
their characteristic contents in DRR and PRR.
4. Conclusions
In the present study, a chemical comparison between decoctions
of DRR and PRR by UPLC-TOF MS and HPLC-ELSD with
MVA was explored. Our results show characteristic changes in
the content of major monosaccharides and oligosaccharides as
DRR is converted to PRR, indicate a possible special role for
fructose, stachyose and rehmaionoside in the differing therapeu-
tic effects of DRR and PRR, and suggest potential quality
control markers for assessing PRR and DRR.
The authors are grateful for financial support from the
National Nature Science Foundation of China (Grant Nos.
81073161, 81130067 and 30730112), the National Basic
Research Program of China (Grant No. 2011CB505304) and
the Natural Science Foundation of Beijing (Grant No.
7112110), and for technical support from Mr. Yong Wang
and other technologists of Waters China Ltd.
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... The difference in their efficacy is related to the change in composition caused by processing. Liang et al. (2013) confirmed significant differences in the composition of fructose, rehmaionoside, and stachyose in DRR and PRR, which may cause their different efficacy. Catalpol, an iridoid glycoside component, is also decomposed through steaming and is not suitable as a characteristic component of PRR. ...
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Rehmanniae Radix (RR, the dried tuberous roots of Rehmannia glutinosa (Gaertn.) DC.) is an important traditional Chinese medicine distributed in Henan, Hebei, Inner Mongolia, and Northeast in China. RR is frequently used to treat diabetes mellitus, cardiovascular disease, osteoporosis and aging-related diseases in a class of prescriptions. The oligosaccharides and catalpol in RR have been confirmed to have neuroprotective effects. However, there are few studies on the anti-Alzheimer’s disease (AD) effect of oligosaccharides in Rehmanniae Radix (ORR). The chemical components and pharmacological effects of dried Rehmannia Radix (DRR) and prepared Rehmannia Radix (PRR) are different because of the different processing methods. ORR has neuroprotective potential, such as improving learning and memory in rats. Therefore, this study aimed to prove the importance of oligosaccharides in DRR (ODRR) and PRR (OPRR) for AD based on the Caenorhabditis elegans (C. elegans) model and the different roles of ODRR and OPRR in the treatment of AD. In this study, we used paralysis assays, lifespan and stress resistance assays, bacterial growth curve, developmental and behavioral parameters, and ability of learning and memory to explore the effects of ODRR and OPRR on anti-AD and anti-aging. Furthermore, the accumulation of reactive oxygen species (ROS); deposition of Aβ; and expression of amy-1, sir-2.1, daf-16, sod-3, skn-1, and hsp-16.2 were analyzed to confirm the efficacy of ODRR and OPRR. OPRR was more effective than ODRR in delaying the paralysis, improving learning ability, and prolonging the lifespan of C. elegans. Further mechanism studies showed that the accumulation of ROS, aggregation, and toxicity of Aβ were reduced, suggesting that ORR alleviated Aβ-induced toxicity, in part, through antioxidant activity and Aβ aggregation inhibiting. The expression of amy-1 was downregulated, and sir-2.1, daf-16, sod-3, and hsp-16.2 were upregulated. Thus, ORR could have a possible therapeutic effect on AD by modulating the expression of amy-1, sir-2.1, daf-16, sod-3, and hsp-16.2. Furthermore, ORR promoted the nuclear localization of daf-16 and further increased the expression of sod-3 and hsp-16.2, which significantly contributed to inhibiting the Aβ toxicity and enhancing oxidative stress resistance. In summary, the study provided a new idea for the development of ORR.
... RRP is good at nourishing yin and supplementing blood, boosting essence, and supplementing bone marrow [16]. Up to now, over 140 compounds have been isolated and identified, such as polysaccharides, oligosaccharides, glycosides, iridoid glycosides, flavonoid, phenol glycoside ionone, furfurals and microelements [17][18][19]. ...
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In traditional Chinese medicine (TCM), type 2 diabetes mellitus (T2DM) is regarded as Xiao Ke disease. Liuwei Dihuang pills (LWP), a classical TCM formula, with the function of nourishing kidney yin, has been used for treating Xiao Ke disease in clinic. In this review, we systematically highlighted recent evidence on LWP and T2DM data from clinical and animal studies, summarized the clinical application, pharmacological mechanism and the active compounds of LWP for the treatment of T2DM. This systematic review will provide an insightful understanding of TCM formulas, pharmacological mechanisms, medicinal-disease interactions, and will lay a foundation for the development of new drug therapy for T2DM.
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Based on the theory of activating spleen and generating blood, this study explored the effect of Rehmanniae Radix Prae-parata on the spleen metabolome of the rat model with blood deficiency syndrome.The rat model of blood deficiency syndrome was established by combining with cyclophosphamide(CTX) and N-acetyl-phenylhydrazine(APH), and the metabolomes of the spleen samples were analyzed with ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF-MS).Principal component analysis(PCA) and orthogonal partial least squares-discriminant analysis(OPLS-DA) were carried out for the metabolite profiles of spleen samples.The MEV heatmap and metabolic network were established based on the potential biomarkers.Finally, the blood routine indexes were combined with the metabolomic profile to reveal the mechanism of Rehmanniae Radix Praeparata in activating spleen and generating blood.The treatment with CTX and APH decreased the blood routine indexes such as white blood cell count(WBC), red blood cell count(RBC), platelet(PLT), and hematocrit(HCT), indicating that the rat model of blood deficiency syndrome was successfully established.The administration of Rehmanniae Radix Praeparata significantly improved the blood routine indexes, which suggested that Rehmanniae Radix Praeparata played a role in replenishing blood.In addition, the metabolomics analysis identified 41 potential biomarkers.The PCA and MEV heatmap also showed significant improvement effect of Rehmanniae Radix Praeparata on the spleen metabolic profile.These potential biomarkers were mainly involved in tricarboxylic acid cycle, niacin and nicotinamide metabolism, phenylalanine metabolism, tyrosine metabolism, taurine and hypotaurine metabolism, and sphingolipid metabolism.Therefore, we hypothesize that Rehmanniae Radix Praeparata may regulate energy metabolism, peripheral blood production, and oxidative injury of hemocytes to tonify blood.
Xiaoer Huanglong Granule is the only Chinese Patent Medicine widely used for treating attention deficit hyperactivity disorder. However, not much is known on the bioactive components and pharmacokinetics of Xiaoer Huanglong Granule even after it was successfully introduced into clinical use. This study analyzed the components in the medication and rat plasma after oral administration with the help of UNIFI platform and Masslynx. A total of 119 and 37 components were detected in the medication and plasma, respectively, using ultra performance liquid chromatography ‐ tandem mass spectrometer. We established a rapid and sensitive simultaneous determination of 1 triterpene saponin, 3 monoterpene glycosides, and 3 lignans in rat plasma by solid‐phase extraction. The determination was accomplished within 7.50 min via gradient elution. The values of lower limit of quantification were validated at 0.08 ng/mL for tenuifolin, 0.8 ng/mL for lactiflorin, 1.828 ng/mL for albiflorin, 2 ng/mL for paeoniflorin, gomisin B, and gomisin D, 10 ng/mL for schisandrin. The results from validations of other methods were all acceptable (RSD ≤ 14.94%). This is the first report on the identification and pharmacokinetics studies of components in Xiaoer Huanglong Granule. Moreover, the pharmacokinetic behavior of lactiflorin was studied for the first time. This article is protected by copyright. All rights reserved
Wangbi Capsule is a compound preparation composed of 17 traditional Chinese medicines, which is widely used in the treatment of rheumatoid arthritis. In our study, a rapid and straightforward analytical method was successfully established by using Ultra performance liquid chromatography coupled with Fourier transform ion cyclotron resonance mass spectrometry technology to quickly separate and identify the chemical constituents of Wangbi Capsule. The chromatography was performed on a Universal XB-C18 column (2.1 mm×150 mm, 3 μm) by the mobile phase containing acetonitrile (A)-0.1% formic acid and water (B) at the flow rate of 0.2 mL/min. The positive and negative ions of the samples were analyzed by extraction ion chromatography. Based on accurate mass spectrometry informations such as excimer ions and fragment ions, combined with the reference materials and the reference literature, 140 compounds in the Wangbi Capsule were identified or preliminarily characterized. The results of the study show that Wangbi Capsules mainly contain 3 types of nucleosides, 31 types of alkaloids, 12 types of organic acids, 29 types of flavonoids, 12 types of monoterpene glycosides, 30 types of coumarins, 10 types of chromones, 3 types of saponins, 6 kinds of phenylethanoid glycosides and other chemical components. The method established in this study can quickly and efficiently identify the chemical constituents in Wangbi Capsules. It is hoped that it will have a certain significance for the characterization of other Chinese medicine components and lay the foundation for pharmacological research and subsequent in vivo studies of Wangbi Capsules.
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Background Rehmanniae Radix (RR), an herb with numerous pharmacological effects, is widely used in traditional Chinese medicine for the treatment of blood deficiency syndrome, either alone or in combination with other herbs. However, the mechanism by which processed Rehmanniae Radix (PRR) improves blood enrichment efficacy has not been clearly defined. Methods Ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass (UPLC-Q-TOF/MS) and biochemical methods were combined to explore the hematopoietic functional mechanisms of PRR on blood deficiency in a rat model, as well as the potential active ingredient for blood enrichment efficacy. The pharmacological effects of PRR were evaluated on a rat blood deficiency model induced by cyclophosphamide in combination with 1-acetyl-2-phenylhydrazine. The blood routine index, including white blood cell (WBC), red blood cell (RBC), and platelet (PLT) counts, as well as hemoglobin (HGB) level, and the changing metabolite profile based on urine and serum were assessed. Nontargeted metabolomic studies, combined with biochemical analyses, were employed to clarify pharmacological mechanisms. Results PRR significantly increased the blood routine index levels and reversed the levels of SOD, GSH, and ATP. The PRR group was similar to the control group, as determined from the metabolic profile. All of the 60 biomarkers, representing the typical metabolic characteristics of the blood-deficient rat model, mainly involved energy metabolism dysfunction, the peripheral circulation system, and oxidative damage in the body. This improvement may be attributed to changes in polysaccharide and sixteen non-polysaccharide compounds in PRR, which were caused by processing RR with rice wine. Conclusions The strategies of integrated metabolomic and biochemical analyses were combined, revealing the biological function and effective mechanism of PRR.
Ethnopharmacological relevance Radix Rehmanniae (RR) is the tuber root of Rehmannia glutionsa Libosch, which was firstly recorded in Shennong's Classic of Materia Medica (《神农本草经》). RR is a non-toxic and wide used traditional Chinese medicine. RR has the effect of clearing heat, generating essence, cooling blood, stopping bleeding, nourishing yin and blood, and filling marrow. It is used in clinic in the form of processed decoction pieces, including Dry Radix Rehmnniae (DRR) and Rehmanniae Radix Praeparata (RRP). The application of RR in traditional Chinese medicine (TCM) prescriptions can treat various diseases, such as anemia, irregular menstruation, deficiency of liver yin, renal failure and so on. Aim of review This paper aims to provide a comprehensive and productive review of RR, which mainly contains botanical characteristics, processing methods, traditional application, chemical composition, quality control and pharmacological action. Materials and methods Literature search was conducted through the Web of Science, Baidu Scholar, ScienceDirect, PubMed, CNKI, and WanFang DATA using the keywords “Radix Rehmnniae”, “Rehmanniae Radix Praeparata”, “processing”, “clinical application”, “chemical composition”, “quality control”, and “pharmacological action”. In addition, information was collected from relevant textbooks, reviews, and documents. Results RR is a traditional Chinese herbal medicine with clinical value and rich resources. More than 100 components have been isolated and identified from RR. It has multiple pharmacological actions, such as hemostasis, antioxidation, anti-osteoporosis, lowering blood sugar, improving renal function, anti-inflammation, protecting neuronal function, antidepression and anti-anxiety. DRR and RRP are two different processed products of RR. After processing, there are great changes in property, taste, efficacy, clinical application, chemical composition and pharmacological action. At present, identifying chemical constituents of RR and its medicinal value has been deeply studied. However, there is a lack of research on the reasons for the differences in pharmacological effects between DRR and RRP. The reasons for these differences need to be further verified. Catalpol, the active component of RR, has been studied extensively in the literature, but the pharmacological effects of catalpol cannot represent the pharmacological effects of the whole RR. In the future, effective components such as rehmannioside D, polysaccharide, total glycosides, and effective parts in RR need to be further studied and developed. The pharmacodynamic material basis and mechanism of RR need to be further discussed. The scientific connotation and processing methods of RRP need to be studied and standardized.
Ethnopharmacological relevance: In traditional Chinese medicine (TCM) and modern pharmacodynamics, dried Rehmannia Radix (DRR) possesses prominent anti-thrombotic activity that decreases after processing by nine steaming and drying cycles to develop processed Rehmannia Radix (PRR). Due to the complexity of the DRR components, the chemical mechanism leading to efficacy changes of DRR caused by processing is still unclear. Aim of study: This study aimed to trace the anti-thrombotic active compounds of DRR and different degrees of processed RR (PRR) and to evaluate the synergistic effects among different active components. Materials and methods: The anti-thrombotic active chemical fraction of DRR extracts was evaluated. Targeted fractions of the processed products of RR were prepared at different processing stages. The changes in monosaccharides, oligosaccharides and secondary metabolites during processing were characterized by multidimensional high-performance liquid chromatography (HPLC). The anti-thrombotic effects of targeted fractions of different RR samples were evaluated by analyzing the length of tail thrombus (LT) and serum biochemical indicators in carrageenan-induced tail-thrombus mice. The spectrum-effect relationships were investigated by partial least squares regression (PLSR) analysis and gray correlation analysis (GRA). Finally, the active compounds were screened by spectrum-effect relationship analysis and validated in vivo, and their synergistic effects were determined by Webb's fraction multiplication method. Results: Six ingredients highly associated with anti-thrombotic activities were screened out by the spectrum-effect relationship analysis, of which oligosaccharides (stachyose, sucrose and raffinose) and iridoid glycosides (catalpol, leonuride and melitoside) possessed a synergistic effect on tumor necrosis factors (TNF-α), interleukin 1β (IL-1β) and plasminogen activator inhibitor 1 (PAI-1)/tissue-type plasminogen activator (t-PA) ratio in vivo with synergistic coefficient (SC)>1. Conclusion: The main material basis of the anti-thrombotic activities of DRR is oligosaccharide components of stachyose, raffinose and sucrose, iridoid glycosides components of catalpol, leonuride and melittoside. The two kinds of components exert synergistic anti-thrombotic effects by inhibiting the expression of inflammatory factors and regulating the balance of the fibrinolysis system.
Silver nanoparticles (AgNPs) are widely sought after for a variety of biomedical and environmental applications due to their antimicrobial and catalytic properties. We present here a green and simple synthesis of AgNPs utilizing traditional Chinese medicinal herbs. The screening of 20 aqueous herb extracts shows that Sheng Di Huang (Rehmannia glutinosa) had the most promising potential in producing AgNPs of 30 ± 6 nm, with narrow size distribution and high crystallinity. The antimicrobial activities of these AgNPs conducted on E. coli cells were found to be superior in comparison to poly(vinylpyrrolidone)‐capped AgNPs synthesized using common chemical method. Additionally, the AgNPs obtained possess excellent catalytic performance in the reduction of 4‐nitrophenol to 4‐aminophenol. We compared the phytochemical and FTIR spectral analyses of the herb extract before and after synthesis, in order to elucidate the phytochemicals responsible for the reduction of Ag+ ions and the capping of the AgNPs produced.
This work explores the effects of dietary Radix Rehmanniae Preparata polysaccharide (RRPP) supplementation on the growth performance, nonspecific immune responses, immune- and growth-related gene expression and disease resistance to Aeromonas hydrophila in Luciobarbus capito. Diets containing five concentrations of 0%, 0.05%, 0.1%, 0.2% and 0.4% RRPP were fed to fish for 60 d. The results indicated that the growth performance significantly increased in the 0.1%, 0.2% and 0.4% RRPP groups compared with that in the control (P < 0.05). The activities of serum lysozyme (LAZ), acid phosphatase (ACP), superoxide dismutase (SOD), alkaline phosphatase (AKP) and total protein (TP) were significantly increased in the appropriate RRPP supplemented groups (P < 0.05). With respect to immune- and growth-related genes, such as interleukin (IL)-1β, IL-8, tumor-necrosis factor (TNF)-α, interferon (IFN)-γ, growth hormone (GH), insulin-like growth factor (IGF)-I and IGF-II, up-regulation were observed in the three organs (kidney, spleen, gut) of the fish fed with RRPP, compared with the control. In contrast, the mRNA expression of IL-10 and transforming-growth factor (TGF)-β were downregulated. After challenge with A. hydrophila, the final survival rate was significantly higher in fish fed the RRPP supplement than that in the control group (P < 0.05). In conclusion, RRPP enhanced the growth performance, immune response and disease resistance of Luciobarbus capito, with the greatest effects at 0.2% RRPP.
The water extract of the roots of Rehmannia glutinosa LIBOS. forma hueichingensis HSIAO was fractionated by the chromatographies on two types of ion-exchange resin. Fifteen amino acids and D-glucosamine were found and determined in the basic fraction. Phosphoric acid was detected and estimated as a component of the acidic fraction. Neutral fraction, the main fraction of the water extract, was mostly constituted with carbohydrates. D-Glucose, D-galactose, D-fructose, sucrose, raffinose, manninotriose, stachyose, verbascose and D-mannitol were identified and determined. It was found that stachyose is the main component of the water extract and the yield of the substance was 48.3% from the dried material.
We have investigated the effects of rehmaionoside B and other constituents of Rehmaniae radix on bladder and urethral smooth muscle strips of mice. Of the five constituents investigated, rehmaionoside B (1 mg/mL) most effectively inhibited the contraction induced by noradrenaline (10 μg/mL) in urethral smooth muscle. These constituents, especially rehmaionoside B, not only inhibited the spontaneous contraction but slightly relaxed the normal tone of the bladder smooth muscle. The relaxation induced by rehmaionoside B (0.1 mg/mL) corresponded to 26.4% of the isoproterenol (1 μg/mL) response. Rehmaionoside B also inhibited the PGF2α-induced spontaneous contraction of the bladder. The treatment with rehmaionoside B significantly inhibited arachidonic acid-induced contraction of the bladder. This response was similar to that induced by indomethacin. Rehmaionoside B inhibited both urethral and bladder smooth muscles, which may affect micturition. The effect of rehmaionoside B on the bladder was an indomethacin-like action.
Revenue enhancement and value creation are core issues of mergers and acquisitions (M&A). Revenue enhancing synergy associated with cross-industry M&A is supported by Asian emerging markets. Both within-industry M&A and cross-industry M&A deals realise significant positive abnormal returns. The difference between the two categories of M&A is statistically significant in a three-day window, but not statistically significant in a two-day window. Information leakages may be driving the larger valuation effects because a three-day window includes one day before the announcement date. Since large firms tend to diversify their business, the result that cross-industry M&A deals realise lower abnormal returns than within-industry may be driven by the firm size effect.
Our previous study showed that free fructose is an important active constituent responsible for Si-Wu-Tang's (SWT) effect promoting hematopoiesis and immunity. However, the contribution from SWT's four ingredient drugs to the free fructose content in the SWT decoction was not clear. To answer this question, in this study, the fructose, glucose and sucrose content in the SWT decoction, in the decoctions of each single ingredient drug, and in the decoctions of the four formulae lacking each single ingredient drug were determined by HPLC-ELSD. The results showed that the fructose and glucose content in the decoction of single Rehmanniae Radix were almost the same as those in the SWT decoction. In the single Rehmanniae Radix decoction concentrations were: 4.25 ± 0.53 mg/mL for fructose, and 3.43 ± 0.60 mg/mL for glucose; in the SWT decoction concentrations were: 4.10 ± 0.43 mg/mL for fructose, and 3.42 ± 0.32 mg/mL for glucose, while the content of fructose and glucose in the decoctions of single Angelica Radix, single Paeoniae Radix, single Chuanxiong Rhizoma and the formula lacking Rehmanniae Radix were either very small or undetectable. On the other hand, the fructose and glucose content in the decoctions of the formulae lacking Angelica Radix, lacking Paeoniae Radix and lacking Chuanxiong Rhizoma also were approximately the same as those in the SWT decoction. This indicated that Rehmanniae Radix provides most of the free fructose and glucose in the SWT decoction, and therefore plays an important role in SWT's effect promoting hematopoiesis and immunity. As for sucrose in the SWT decoction, Angelica Radix was shown to be a major donor.
Our previous study showed that fructose is an important active constituent that is responsible for Si-Wu-Tang's (SWT) effects promoting hematopoiesis and immunity. In order to provide primary data for analysis of the mechanism of fructose's bioactivity, the concentration of serum fructose in rats after a single oral administration dose of Si-Wu-Tang was determined. The concentration of serum fructose in fasting rats was 0.34 ± 0.24 mg/dL. After oral administration of 7.2 mL per kg body weight of SWT extract (1 mL extract corresponds to 1 g SWT dried herbs), serum fructose levels reached a peak concentration of 1.03 ± 0.25 mg/dL within 60 min, and then declined to the baseline level within 180 min, a pattern which is similar to the one reported for oral administration of pure fructose. The peak concentration was only 2-3 times higher than the baseline serum fructose concentration. These results showed that the increase of blood fructose concentration after oral administration of SWT is small and transient, which is very probably due to the quick metabolism of fructose by the liver. We suggest, for future research, it is necessary to consider the probability that fructose's bioactivity on hematopoiesis and immunity is not exerted by fructose in its original form, but after it is metabolized by the liver.