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Effect of the herbal medicine Danggui Sini plus Wuzhuyu Shengji-
ang Tang on erythrocyte deformability in normal subjects: a
cross-over trial
JiEun Lee, Seung-Yeon Cho, Seong-Uk Park, Woo-Sang Jung, Sang-Kwan Moon, Jung-Mi Park, Chang-Nam Ko,
Ki-Ho Cho, Seungwon Kwon
aa
JiEun Lee, Seung-Yeon Cho, Seong-Uk Park, Woo-Sang
Jung, Sang-Kwan Moon, Jung-Mi Park, Chang-Nam Ko,
Ki-Ho Cho, Seungwon Kwon, Department of Cardiology
and Neurology, College of Korean Medicine, Kyung Hee
University, Seoul 02447, Korea
Correspondence to: Prof. Seungwon Kwon, Department
of Cardiology and Neurology, College of Korean Medicine,
Kyung Hee University, Seoul 02447, Korea.
kkokkottung@hanmail.net
Telephone: +82-02-958-9128
Accepted: December 25, 2017
Abstract
OBJECTIVE: To show whether Danggui Sini plus
Wuzhuyu Shengjiang Tang (DSWST) has any
transient effect on erythrocyte deformability in
normal subjects.
METHODS: A total of 25 subjects [mean age (27.8
± 1.8) years] was enrolled in this study. The study
was designed as a cross-over trial in which the
subjects took part for 2 d. On the first day, blood
samples were collected at baseline and 1-2 h after
administration of water, whereas, on the second
day, instead of water, the subjects were
administered DSWST after the baseline blood
sampling. The blood samples collected at baseline
and after the administration water or DSWST, were
examined for erythrocyte deformability.
RESULTS: The elongation index increased
significantly after 2 h (P = 0.009) compared to the
baseline after DSWST intake. However, after water
intake, there was no significant difference
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ISSN 0255-2922
© 2018 JTCM. This is an open access article under the CC BY-NC-ND license.
.
RESEARCH ARTICLE
TOPIC
observed. When comparing the percent change of
erythrocyte deformability between DSWST and
water, we found that after 2 h of administration,
DSWST improved erythrocyte deformability
significantly compared to water (P < 0.001).
CONCLUSION: DSWST has a transient effect on
erythrocyte deformability in normal subjects.
© 2018 JTCM. This is an open access article under the
CC BY-NC-ND license.
Keywords: Erythrocyte deformability; Peptide
elongation factors; Raynaud disease; Coldness of
hands and feet; Cross-over studies; Danggui Sini
plus Wuzhuyu Shengjiang Tang
INTRODUCTION
Korean herbal medicine is commonly used to treat
symptoms such as coldness of hands and feet.1
Coldness of hands and feet can be divided into two
major categories based on whether the symptoms
accompany Raynaud's phenomenon (RP), which is the
change of colors in fingers when exposed to cold. The
pathogenesis has not been clearly defined, but previous
studies suggest that through multiple mechanisms,
vascular abnormalities and decrease in microvascular
circulation contribute to these conditions.2 Danggui
Sini plus Wuzhuyu Shengjiang Tang (DSWST;
Dangguisayeokgaoshuyusaenggang-tang in Korean,
Tokishigyakukagoshuyushokyoto in Japanese), a
compound prepared from 9 plants, is one of the most
commonly used herbal medicine in these cases.1
DSWST was first mentioned in Shanghanlun, which
was written in the Later Han era (AD 25-220). In this
book, DSWST is based on the compound
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 JE Lee et al. / Research Article
Dangguisayuk-tang, which is prepared with 7 herbs:
Dazao (Fructus Jujubae), Guizhi (Ramulus
Cinnamomi), Baishao (Radix Paeoniae Alba), Danggui
(Radix Angelicae Sinensis), Mutong (Caulis Akebiae),
Gancao (Radix Glycyrrhizae) and Xixin (Herba Asari
Mandshurici). Dangguisayuk-tang is described to be
used on patients with complains of cold hands and
feet, and very weak pulse. DSWST, which is a
combination of Dangguisayuk-tang and, Evodiac
Fructus, and Zingiberis Rhizoma, has been mentioned
to be used when the Danguisayuk-tang symptoms are
more severe and the coldness of the body is chronic.
Traditional theories suggest that DSWST warms the
interior of the body1 by warming the meridian and
dispersing coldness. Other recent studies have shown
that DSWST prevents the cold syndrome3 and treats
dizziness with feelings of coldness.4 However, the
actual therapeutic mechanisms are not known yet.
In rats, peripheral circulation in the tail had improved
at 1 h after oral administration of DSWST.1 Moreover,
in another study conducted on young healthy subjects,
1 h following oral administration of DSWST, peripheral
blood flow in the fingers increasedsignificantly.5 Thus,
by improving peripheral circulation, DSWST may
have an effect on improving the symptoms related to
coldness of hands and feet.
One factor that can be used to evaluate micro-
circulation is erythrocyte deformability.6 Erythrocytes
have the ability to change shape under deforming
forces. Increase of erythrocyte deformability helps the
erythrocyte easily pass through a capillary smaller than
its size, which leads to improvement in blood flow.7
On the other hand, decrease in erythrocyte defor-
mability means increase in microcirculatory resistance,8
and can be seen not only in Raynaud's phenomenon
but also in diabetes, sepsis, sickle-cell disease,
peripheral vascular diseases and peripheral neuron
diseases etc.
It has been shown by some studies that, decrease of
erythrocyte deformability can be seen in RP patients.9
Park's study on stroke patients showed that, patients
with symptoms of coldness in their hands or feet (not
limited to Raynaud's phenomenon) showed decreased
erythrocyte deformability compared to the patients
without these symptoms.10 However, the effect of
DSWST, widely used for the treatment of RP patients,1,11
on erythrocyte deformability has not been shown by
any study. The aim of our study was to explore the
effects of DSWST on erythrocyte deformability.
MATERIALS AND METHODS
Subjects
Twenty-five healthy subjects (7 female, 18 male)
between the age groups of 25 to 35 [mean age (27.8 ±
1.8) years] were enrolled in this study. Subjects with a
history of cardiac disease, cerebrovascular disease,
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hypertension, diabetes mellitus, drug abuse, and
psychiatric problems; and subjects under any other
drug medication were excluded from the study.
Subjects who were unable to fast for 8 h before the
study were also excluded.
Preparation of Danggui Sini plus Wuzhuyu
Shengjiang Tang (DSWST)
The Department of Preliminary Pharmaceutical
Preparation of Kyung Hee University Korean Medical
Center (KHUKMC) prepared DSWST as dry extract
granules, 3 g per pouch. Although the original
DSWST in Shang Han Lun is composed of 9 herbs,
the DSWST used in this study is composed of 8 herbs:
Dazao (Fructus Jujubae) 5 g, Ganjiang (Rhizoma
Zingiberis) 4 g, Danggui (Radix Angelicae Sinensis) 3 g,
Guizhi (Ramulus Cinnamomi) 3 g, Baishao (Radix
Paeoniae Alba) 3 g, Wuzhuyu (Fructus Evodiae
Rutaecarpae) 2 g, Gancao (Radix Glycyrrhizae) 2 g and
Xixin (Herba Asari Mandshurici) 2 g. Akebiae Caulis
was excluded due to the minor toxicities of the plant.
These herbs were first cut into small pieces. The herbs
mixture was then extracted in a reflux condenser for
3 h with hot water. The solution was filtered through
filter paper and concentrated using a spray drying
process. The dry extracts were granulated using
3 binders, lactose, polyvinylpyrrolidone, dextrin and
ethanol. The ethanol was evaporated after binding.
Study design
This study was designed as a cross-over trial, where
subjects participated over a period of 2 d. On both
days, an 8-h fasting blood sample was taken from the
subjects at 8 o' clock in the morning. After first
sampling, subjects were given 150 mL of water on the
first day followed by second sampling after 1-2 h.
However, on the second day, instead of water, subjects
were administered DSWST orally, and the blood
sample was taken after same duration.
Measurement of erythrocyte deformability
The primary outcome of this study is the elongation
index (EI) of erythrocyte deformability. The EI is
defined as (L - W) / (L + W), where L is the major axis
and W is the minor axis of the erythrocyte ellipse at
the shear stress of 3 Pa. To measure the EI, a Rheoscan-
D microfluidic ektacytometer (Rheo Meditech, Seoul,
Republic of Korea) was used. One drop of blood was
obtained from each subject's fingertip (6.0 μL) and
was mixed with 0.5 mL of a highly viscous PVP solution
(31 mPa) in phosphate-buffered saline (0.14 mM). A
0.5 mL aliquot of this mixture was placed in the test
chamber of a disposable kit, which included a
microchannel (Rheo Meditech). Differential pressure
drove the mixture through the microchannel (0.2 mm
× 4 mm × 40 mm), and laser beam (wavelength, 635
nm) from a 1.5 mW laser diode passed through the
diluted mixture during the flow. The diffraction
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 JE Lee et al. / Research Article
pattern of the moving erythrocytes at multiple shear
stresses was projected onto a screen, and the images
were captured by a CCD-video camera every 0.5 s.
The images were analyzed using an ellipse-fitting
computer program. The average shear stress ranged
from 0 to 30 Pa, and the EI was calculated at 3 Pa. The
microchannel was discarded after each measurement.
Statistical analysis
The SPSS 12.0 (SPSS for Windows, Version 12.0.
Chicago, SPSS Inc. Chicago, IL, USA) was used for
statistical analysis. Data were summarized as mean ±
standard deviation or mean ± standard error of mean.
The changes in erythrocyte deformability from
baseline to post-treatment in each intervention were
compared by repeated measures analysis of variance
(ANOVA). To compare the percent change of
erythrocyte deformability between the DSWST intake
period and the water intake period, paired t-test was
performed. A value of P < 0.05 was considered
significant.
RESULTS
After DSWST intake, the elongation index (EI) of
erythrocyte deformability increased after 1 and 2 h,
whereas it showed a tendency to decrease after water
intake (Table 1). Compared to baseline, the difference
was significant after 2 h after DSWST intake (P =
0.009) ([Figure 1A). However, EI showed no sig-
nificance compared to baseline after water intake.
When comparing the percent change of EI between
DSWST and water intake, we found that after 2 h,
DSWST improved erythrocyte deformability sig-
nificantly compared to water (P < 0.001, Figure 1B).
Table 1 Elongation Index (EI) of erythrocyte deformability
Time DSWST Control
Baseline 0.327±0.014 0.333±0.016
After 1 h 0.330±0.014 0.330±0.016
After 2 h 0.334±0.014 0.327±0.016
Notes: DSWST: administration of Danggui Sini plus Wuzhuyu
Shengjiang Tang extraction 3 g with 150 mL of water. Control:
administration of 150 mL of water. The data is presented as
mean ± standard deviation.
DISCUSSION
In Korean medicine, herbal medicine is widely used to
treat symptoms such as sensitivity to coldness. There
are many medicines to choose from, that differ for
each patient, but DSWST, which is a compound
prepared from 9 plants, is one of the most used herbal
medicine in these cases1. Despite the fact that DSWST
has been used for treating RP for a long time1, the
mechanisms underlying the effects have not been
clearly reported. In one in vitro study, it was found that
DSWST inhibits cold-induced responses in endothelial
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0.340 DSGOST
a A
0.335 Water
0.330
EI
0.325
0.320
0.315
Baseline 1h 2h
4 DSGOST
b B
3 Water
2
1
0
%
Baseline 1h 2h
－1
－2
－3
－4
Figure 1 Elongation index and change of erythrocyte
deformability
A: elongation index; B: change of erythrocyte deformability.
DSWST: administration of Danggui Sini plus Wuzhuyu
Shengjiang Tang extraction 3 g with 150 mL of water.
Control: administration of 150 mL of Water. EI: elongation
index of erythrocyte deformability. % : percent change of
erythrocyte deformability compared to baseline. DSWST:
Danggui Sini plus Wuzhuyu Shengjiang Tang. Vertical bars
represent standard error of the mean. aP = 0.009 by
repeated measure analysis of variance (compared to
baseline). bP < 0.001 by paired t-test (compared to the water
intake).
cells and vascular smooth muscle cells.10
In this study, it was shown that DSWST has a
transient effect on erythrocyte deformability in young
healthy subjects. After 2 h following oral
administration of DSWST, the elongation index of
erythrocyte deformability improved significantly
compared to baseline (P = 0.009), and the percent
change was also significant compared to the water
intake (P < 0.001).
The results of this study suggest that DSWST could be
used for treatment of diseases associated with reduced
erythrocyte deformability, such as RP. In this study,
DSWST improved microcirculation by affecting the
erythrocyte itself, since erythrocyte deformability is a
property determined by geometric and material
properties of the erythrocyte cell membrane.7 However,
a previous in vitro study,12 explored the effects of
DSWST on endothelial cells and vascular smooth
muscle cells. It showed that the compound inhibits
cold-induced activation of RhoA in both vascular
smooth muscle cells and endothelial cells, and blocks
endothelin-1-mediated paracrine path for cold
response on vessels. RhoA is a GTPase protein that
activates Rho kinase, which eventually leads to
reorganization of the actin cytoskeleton of cells.
Activation of Rho/Rho-kinase signaling pathway has
771 October 15, 2018 | Volume 38 | Issue 5 |
 JE Lee et al. / Research Article
been reported to decrease erythrocyte deformability,
hence inhibiting the activation of RhoA may help
increase the erythrocyte deformability.13 As mentioned
before, the mechanisms behind the Raynaud's
phenomenon is complex and the pathophysiology is
contributed by imbalance between vasoconstriction
and vasodilation, and reduced blood flow.11 Thus,
DSWST may have effects on various mechanisms
including Rho/Rho-kinase signaling pathway.However,
this conclusion must be drawn with caution and
further studies are needed to fully understand the
therapeutic mechanisms of DSWST.
It is also hard to say which bioactive components of
DSWST are responsible for these effects. A previous
study has shown that angelica extract prevents the
decrease of erythrocyte deformability in vitro, so
Angelica Radix may play a key role in effecting
erythrocyte deformability.14 Further studies will be
needed to find out thebioactive components
responsible for these effects. This study was performed
on healthy young patients, whose erythrocyte
deformability was in the normal range. To prove the
effects of DSWST on erythrocyte deformability,
patient-based trials will be needed. Further, this study
only showed the acute effects of DSWST. Hence trials
will be needed to show the long-term effects of the
compound.
In conclusion, this study showed that DSWST has a
transient effect on erythrocyte deformability in young
healthy subjects. Thus, DSWST improves micro-
circulation by improving erythrocyte deformability and
may be effective for patients with diseases associated
with decreased erythrocyte deformability.
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