Comprehensive evaluation of antioxidant effects of Japanese Kampo medicines led to identification of Tsudosan formulation as a potent antioxidant agent

Article abstract of DOI:10.1007/s11418-018-1259-x

Oxidative stress due to the overproduction of reactive oxygen species plays an important role in the pathogenesis of various diseases. In the present study, we comprehensively evaluated the antioxidant activities of 147 oral formulations of Japanese traditional herbal medicines (Kampo medicines), representing the entire panel of oral Kampo medicines listed in the Japanese National Health Insurance Drug List, using in vitro radical scavenging assays, including the 2,2-diphenyl-1-picrylhydrazyl free radical scavenging activity assay, the superoxide anion scavenging activity assay, and the oxygen radical absorption capacity assay. Three of the formulations tested, namely, Tsudosan, Daisaikoto, and Masiningan, showed the most potent in vitro antioxidant activities and were selected for further investigation of their intracellular and in vivo antioxidant effects. The results of the 2′,7′-dichlorodihydrofluorescin diacetate assay demonstrated that all three Kampo medicines significantly inhibited hydrogen peroxide-induced oxidative stress in human hepatocellular liver carcinoma HepG2 cells. In addition, Tsudosan significantly increased the serum biological antioxidant potential values when orally administrated to mice, indicating that it also had in vivo antioxidant activity. The potent antioxidant activity of Tsudosan may be one of the mechanisms closely correlated to its clinical usage against blood stasis.

Full text of DOI:10.1007/s11418-018-1259-x

Journal of Natural Medicines
https://doi.org/10.1007/s11418-018-1259-x
ORIGINAL PAPER
Comprehensive evaluation of antioxidant efects of Japanese Kampo
medicines led to identifcation of Tsudosan formulation as a potent
antioxidant agent
Naoko Sato1,2 · Wei Li1 · Hiroaki Takemoto1 · Mio Takeuchi1 · Ai Nakamura1 · Emi Tokura1 · Chie Akahane1 ·
Kanako Ueno1 · Kana Komatsu1 · Noriko Kuriyama1 · Toshihisa Onoda1,2 · Koji Higai1 · Kazuo Koike1
Received: 13 August 2018 / Accepted: 15 October 2018
© The Japanese Society of Pharmacognosy and Springer Japan KK, part of Springer Nature 2018
Abstract
Oxidative stress due to the overproduction of reactive oxygen species plays an important role in the pathogenesis of various
diseases. In the present study, we comprehensively evaluated the antioxidant activities of 147 oral formulations of Japanese
traditional herbal medicines (Kampo medicines), representing the entire panel of oral Kampo medicines listed in the Japanese
National Health Insurance Drug List, using in vitro radical scavenging assays, including the 2,2-diphenyl-1-picrylhydrazyl
free radical scavenging activity assay, the superoxide anion scavenging activity assay, and the oxygen radical absorption
capacity assay. Three of the formulations tested, namely, Tsudosan, Daisaikoto, and Masiningan, showed the most potent
in vitro antioxidant activities and were selected for further investigation of their intracellular and in vivo antioxidant efects.
The results of the 2′,7′-dichlorodihydrofuorescin diacetate assay demonstrated that all three Kampo medicines signifcantly
inhibited hydrogen peroxide-induced oxidative stress in human hepatocellular liver carcinoma HepG2 cells. In addition,
Tsudosan signifcantly increased the serum biological antioxidant potential values when orally administrated to mice, indicat-
ing that it also had in vivo antioxidant activity. The potent antioxidant activity of Tsudosan may be one of the mechanisms
closely correlated to its clinical usage against blood stasis.
Keywords Kampo · Antioxidant · Daisaikoto · Tsudosan · Masiningan
Introduction
reduce oxidative stress by scavenging ROS has attracted the
interest of researchers. Although various antioxidants have
been adopted as drug candidates to date, the toxicity and
safety of synthetic antioxidants are still open to question,
as exemplifed by butylhydroxyanisole (BHA) and butylhy-
droxytoluene, both of which have been reported to be pos-
sible carcinogens [5]. Thus, natural medicines, with their use
recorded over long periods of medical practice, are consid-
ered to have the potential to be valuable sources of safe and
efective antioxidants.
Reactive oxygen species (ROS), including the superoxide
anion (O2−), hydrogen peroxide (H2O2), hydroxyl radi-
cal (·OH), and singlet oxygen (1O2), are generated during
the cellular metabolism of molecular oxygen [1] and play
important roles in cellular signaling and homeostasis [2, 3].
However, overproduction of ROS causes oxidative damage
to lipids, proteins, and nucleic acids, potentially leading to
the pathogenesis of various diseases, such as dyslipidemia,
cerebral infarction, cancer, among others [4]. Consequently,
the application of antioxidants as therapeutic agents to
In Japan, 148 formulations of Kampo medicines (Japanese
traditional medicines) are currently listed in the National
Health Insurance System Drug List and applied under clini-
cal conditions for the treatment of various diseases, includ-
ing many oxidative stress-related diseases, such as hyperlipi-
demia, arteriosclerosis, and diabetic complications. Despite
their extensive clinical usage, our knowledge of the antioxi-
dant activities of Kampo medicines is still quite limited. A
number of Kampo medicines, such as Saikokeishito [6] and
Hangeshashinto [7], have been individually investigated for
* Wei Li
liwei@phar.toho-u.ac.jp
1
2
Faculty of Pharmaceutical Sciences, Toho University,
Miyama 2-2-1, Funabashi, Chiba 274-8510, Japan
Toho University Sakura Medical Center, Shimoshidu 564-1,
Sakura, Chiba 285-8741, Japan
Vol.:(0123456789)
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Journal of Natural Medicines
their antioxidant activity. However, to our knowledge, the
antioxidant activity of these 147 formulations of Kampo
medicines has only been comprehensively investigated in
an in vitro oxygen radical absorption capacity (ORAC) assay
[8]; additional further cellular and animal experiments have
not been performed. In the study reported here, we evaluated
the antioxidant activities of Kampo medicines using three
diferent in vitro radical scavenging assays. The results indi-
cated that these formulations have intracellular antioxidant
activities against H2O2-induced oxidative stress, as well as
in vivo antioxidant efects in mice. The Kampo formulation
Tsudosan was the most potent antioxidant agent of the 147
formulations of oral Kampo medicines.
The sample concentrations were set to a concentration that
allowed data comparison among the diferent formulations.
DPPH free radical scavenging activity assay The DPPH
assay was performed as previously reported by Dudonné
et al. [9] with modifcations. The DPPH radical in MeOH
(0.15 mM, 90 µL) was mixed with 10 µL of aqueous solution
of each Kampo medicine. The samples were then incubated
for 30 min at room temperature, and the decrease in absorb-
ance at 515 nm was measured. BHA was used as the positive
control, and the half maximal (50%) efective concentration
(EC50) value was 26.7 1.3 μM. The concentration–activity
dependency relationship was examined at multiple concen-
trations ranging from 10 to 100 μU/mL. XY curves ftted
with the non-linear model and the EC50 values were calcu-
lated by the logistic curve ftting method using GraphPad
Prism® version 7 (GraphPad Software Inc., La Jolla, CA,
USA).
Materials and methods
Materials
Superoxide anion scavenging activity assay Superoxide
anion scavenging activity was determined by the WST
[2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulphophenyl)-
2H-tetrazolium, monosodium salt] reduction method, using
a superoxide dismutase (SOD) assay kit–WST (Dojindo
Molecular Technologies, Inc., Kumamoto, Japan) according
to the manufacturer’s protocol. BHA was used as the positive
control, and the EC50 value was 17.0 1.0 μM. The concen-
tration–activity dependency relationship was measured at
concentrations ranging from 0.42 to 83.3 μU/mL. XY curves
ftted with the non-linear model and the EC50 values were
calculated by the logistic curve ftting method (GraphPad
Prism® version 7; GraphPad Software Inc.).
In this investigation, we evaluated 147 formulations of
Kampo medicines, representing the entire panel of oral
Kampo medicines listed in the Japanese National Health
Insurance Drug List. These formulations are manufactured
by Tsumura & Co. (Tokyo, Japan), Kotaro Pharmaceuti-
cal Co., Ltd. (Osaka, Japan), Ohsugi Pharmaceutical Co.,
Ltd. (Osaka, Japan), Kracie Holdings, Ltd. (Tokyo, Japan),
Sanwa Shoyaku Co., Ltd. (Tochigi, Japan), Taikoseido Phar-
maceutical Co., Ltd. (Hyogo, Japan), or Toyo-Kampo Phar-
maceutical Co., Ltd. (Osaka, Japan), respectively.
Chemicals
Trolox (purity>98%) was purchased from Calbiochem (San
Diego, CA, USA); 2,2′-azobis(2-amidinopropane)dihydro-
chloride (AAPH) (purity> 97%), BHA (purity> 98%), 2,
and 2-diphenyl-1-picrylhydrazyl (DPPH) (purity > 95%)
were purchased from Wako Pure Chemical Industries, Ltd.
(Osaka Japan). Fluorescein sodium salt and all other rea-
gents were purchased from Sigma-Aldrich (St Louis, MO,
USA).
ORAC assay The ORAC assay is based on the scavenging
of peroxyl radicals generated by AAPH and was performed
as reported previously by Dudonné et al. [9] and Prior et al.
[10] with modifcations. Namely, 35 μL of the blank solu-
tion, trolox standard solutions (25.0, 12.5, and 6.25 µM), or
the sample solutions in 75 mM potassium phosphate bufer
(pH 7.0) was added to each well of a 96-well polypropylene
plate, followed by the addition of 115 µL of 94.4 nM fuores-
cein solution to each well. The plate was then covered with
a lid and incubated in the preheated (37 °C) fuorescence
reader (EnSpire Multimode Plate Reader; Perkin Elmer,
Yokohama, Japan) for 10 min, with shaking for 3 of the 10
min. The fuorescence (excitation 485 nm, emission 520 nm)
was measured every 2 min for up to 90 min after the addition
of the AAPH solution (31.7 mM, 75 μL) to each well of the
plate. The ORAC values were expressed as micromole trolox
equivalents per unit of Kampo medicine.
In vitro antioxidant activity
Sample preparation of Kampo medicines We defned the
amount of daily dose as 1 unit (U) for each Kampo medicine.
The Kampo medicine under study was dissolved as 1/1000
of the daily dose in 1 mL of purifed water (concentration1
mU/mL) and extracted by sonication at room temperature
for 15 min. The mixture was then centrifuged at 12,000 rpm
for 15 min to remove insoluble matter afecting the measure-
ment, and the supernatant or supernatant diluted in purifed
water was used as the sample solution for in vitro assays.
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Journal of Natural Medicines
Evaluation of serum biological antioxidant potential values
in mice The biological antioxidant potential (BAP) value
of an ion represents the reducing ability of that ion against
active oxygen, which is used as an index of antioxidant
activity. In this study, we used the BAP test to examine
the concentration of antioxidants in the serum of mice that
reduced iron from the ferric (Fe3+) to ferrous (Fe2+) form
[12]. The experimental animals were randomly divided into
six experimental groups. The mice in the control group were
orally administered purifed water. Vitamin C (purity 99.6%,
Wako Pure Chemical Industries, Ltd.), as a positive control,
was administered to mice orally at a dose of 2.5 g/kg body
weight twice a day for 2 days. The Kampo medicines were
administered to mice orally at a dose of 2.5 g/kg body weight
twice a day for 2 days, i.e., the same dose as the positive
control. Thirty minutes after the fnal administration, the
whole blood of each mouse was collected from the jugu-
lar vein with the mice under dormicum (Nippon Zenyaku
Kogyo Co., Ltd., Fukushima, Japan)/butorphanol (Meiji
Seika Pharma Co., Ltd., Tokyo, Japan) mixed anesthesia
and ether anesthesia. The whole blood was centrifuged at
3000 rpm for 15 min at 4 °C to separate serum. The BAP
levels were measured using a free radical analyzer (FREE
Carrio Duo; Diacron Int. s.r.l., Grosseto, Italy) according to
the manufacturer’s instructions.
Intracellular antioxidant activities
Cells and cell culture Human hepatocellular carcinoma
HepG2 cell lines were purchased from the Japanese Collec-
tion of Research Bioresources Cell Bank/National Institutes
of Biomedical Innovation, Health and Nutrition (Tokyo,
Japan) and cultured in Dulbecco’s modifed Eagle’s medium
(DMEM) (Wako Pure Chemical Industries, Ltd.) supple-
mented with 10% fetal bovine serum (JRC Biosciences, Len-
exa, KS, USA), under a humidifed atmosphere containing
5% CO2 at 37 °C.
Analysis of intracellular ROS level Intracellular ROS level
was determined by 2′,7′-dichlorodihydrofuorescin diac-
etate (DCFH-DA) staining according to Higai et al. [11].
The sample concentrations were set to a concentration that
does not afect cell growth. HepG2 cells (2×105 cells/mL)
on 35-mm well plates (1 mL/well) were incubated with the
aqueous solution of the respective Kampo medicine (10 μU/
mL) for 24 h. After the culture medium was removed, the
cells were incubated with 100 μL of DCFH-DA solution
(50 μM in DMEM medium) for 15 min at 37 °C, following
which H2O2 was added to a fnal concentration of 1 mM. The
ROS level in the cells was estimated by scanning the fuo-
rescence of 2′,7′-dichlorofuorescein (DCF) using a model
LSMS 510 Meta confocal laser scan microscope (Carl Zeiss,
Oberkochen, Germany), and the time-dependent change of
fuorescence intensity was calculated at 0, 10, 20, 30, 40,
50 min using the image processing software ImageJ.
Statistical analysis
All results were expressed as the mean standard devia-
tion (SD). The data were analyzed using one-way analy-
sis of variance followed by Dunnett’s test with signifcance
set at p <0.05 or p <0.01 versus the vehicle or the New-
man–Keuls method with signifcance set at p < 0.05. All
statistical analyses were performed using GraphPad Prism®
version 7 (GraphPad Software Inc.).
WST‑1 cell proliferation assay HepG2 cells were seeded on
96-well culture plates (100 μL per well, 2×105 cell/mL) and
incubated for 6 h at 37 °C. The cells were then exposed to
diferent concentrations of sample solutions for 48 h. The
number of living cells was measured using a Premix WST-1
Cell Proliferation Assay System (TaKaRa, Shiga, Japan),
according to the manufacturer’s instructions.
Results and discussion
In vivo antioxidant efect
The frst step in our study was to comprehensively evalu-
ate the antioxidant activities of the 147 Kampo medicines
under study, which covered the entire panel of oral Kampo
medicines listed in Japanese National Health Insurance Drug
List, using three of the most common in vitro radical scav-
enging assays, namely, the DPPH, SOD, and ORAC assays
(Table 1). The DPPH assay is one of the simplest of antioxi-
dant assay methods, and its results represent the ability to
scavenge the stable free radical generated by DPPH [13]. The
SOD assay was carried out using a WST-1 based SOD inhi-
bition method to measure the scavenging ability of super-
oxide anion radicals, one species of ROS [14]. The ORAC
assay is a well-established method to measure the scaveng-
ing ability of peroxyl free radicals thermally generated from
Animals Four-week-old male ICR mice were purchased
from Clea Japan (Tokyo, Japan). Prior to experimentation,
the mice were acclimatized for 1 week to a temperature of
25 2 °C, humidity of 50 10%, and a 12/12-h light/dark
cycle. They were fed standard pellet chow and water ad libi-
tum. All behavioral observations were conducted between
1000 hours and 1500 hours. The measurement of biological
antioxidant potential was performed with nine mice in each
group. Experiments were performed in accordance with the
Toho University guidelines for animal care, handling, and
termination, which are in line with the international and
Japanese guidelines for animal care and welfare (Approval
no. 17-51-357).
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Journal of Natural Medicines
Table 1 In vitro antioxidant activity of the 147 formulations of oral Kampo medicines tested in this study
Number
Name of oral Kampo formulation
DPPH (%)
SOD (%)
ORAC (µM TE/U)
100 μU/mL
50 μU/mL
82.1 0.5
83.3 μU/mL
41.6 μUnit/mL
1
2
3
4
5
6
7
8
Anchusanc
30.4 1.7
35.7 1.8
92.8 0.3
52.7 1.3
34.3 0.5
70.1 2.2
52.6 0.6
23.8 11.0
33.5 0.7
91.2 0.3
7.4 1.7
70.7 8.3
37.2 10.3
98.8 0.7
84.0 2.1
57.3 7.0
83.7 2.8
52.2 3.3
67.3 10.6
45.7 7.0
98.3 1.8
56.8 5.3
76.2 2.3
79.4 5.8
87.1 1.4
77.8 4.5
96.3 1.4
45.2 1.6
91.8 4.3
94.9 1.7
96.0 4.3
100.0 1.4
91.3 0.5
84.1 0.2
66.9 6.9
93.5 0.8
82.7 2.0
80.5 1.7
82.6 1.7
88.9 3.1
81.4 2.2
25.6 0.4
81.5 3.1
80.0 3.0
69.2 1.5
72.1 2.2
89.5 1.4
87.6 3.6
78.6 1.8
97.9 1.6
70.2 1.5
74.5 2.1
70.2 2.4
95.2 1.1
76.7 18.7
94.0 2.0
79.8 4.4
89.1 0.3
93.1 7.0
1384 13
11 72
Bakumondotoc
Bofutsushosana
Boiogitoa
88.7 1.3
8306 29
1419 47
2545 75
2759 135
1931 252
1310 63
3444 178
4314 98
Bukuryoinc
Bukuryoingohangekobokutof
Bushirichutod
Byakkokaninjintoc
Chikujountantof
Choijokitof
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
83.0 0.1
71.2 0.4
89.1 0.3
79.3 1.3
Choreitoc
0 0.0
Choreitogoshimotsutof
Chotosanf
44.4 0.8
47.2 0.7
62.1 0.3
58.8 1.4
91.0 0.2
35.2 1.8
90.8 0.8
92.6 0.2
90.3 2.1
94.1 0.9
79.9 1.4
45.0 0.9
21.6 1.7
92.8 0.3
63.2 1.4
68.5 2.4
87.3 1.5
85.4 2.2
62.6 1.9
37.1 2.3
91.6 0.9
66.6 0.6
65.3 2.1
52.2 2.5
71.5 1.0
90.3 0.7
63.4 1.6
92.9 0.3
49.5 2.6
76.4 3.0
41.8 1.0
86.2 1.4
60.0 0.2
94.2 0.7
82.1 2.4
93.2 1.6
91.4 0.1
774 69
2505 83
278 46
3759 213
9498 158
741 35
Choyotoc
Daibofutof
Daijokitoa
Daikenchutoc
Daiobotampitoc
Daiokanzotoa
Daisaikotoc
63.8 0.5
69.6 0.5
92.4 0.5
82.9 0.5
78.4 0.4
84.7 1.5
96.9 1.9
91.1 0.9
76.4 2.4
3563 291
5426 105
11567 61
5769 121
2579 120
1942 271
1006 27
5943 265
1929 177
3497 26
2079 123
1450 124
3314 93
2022 186
3770 22
3000 25
2472 197
3059 60
3512 125
4277 277
2864 131
6853 38
1662 167
2962 117
2124 73
5680 413
2314 19
4674 229
2813 122
5713 242
6596 288
Daisaikotokyodaioc
Eppikajutsutoc
Gokotob
Goreisanc
Gorinsanf
53.4 0.9
78.5 1.8
Goshajinkiganf
Goshakusanc
Goshuyutoc
Hachimijiogana
Hainosankyutoa
Hangebyakujutsutemmatoc
Hangekobokutoc
Hangeshashintoc
Heiisana
53.0 0.8
Hochuekkitoc
Inchingoreisanf
Inchinkotoa
59.1 1.0
89.7 0.8
Ireitof
Jidabokuippof
Jiinkokatof
92.7 1.7
Jiinshihotof
Jinsoinf
Jizusoippof
77.1 1.2
87.9 2.6
Jumihaidokutoa
Junchotof
81.0 1.4
Juzentaihotoc
Kakkonkajutsubutod
Kakkontoc
61.4 0.3
61.1 0.8
87.3 0.3
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Journal of Natural Medicines
Table 1 (continued)
Number
Name of oral Kampo formulation
DPPH (%)
SOD (%)
ORAC (µM TE/U)
100 μU/mL
50 μU/mL
56.3 0.3
83.3 μU/mL
41.6 μUnit/mL
92.9 0.9
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
Kakkontokasenkyushin’ia
Kambakutaisotoc
Kamikihitoa
92.2 0.5
31.6 1.7
48.7 1.2
91.4 0.1
15.7 2.0
91.3 0.3
35.7 2.9
93.5 0.4
93.6 2.7
92.0 0.9
90.6 1.1
90.6 0.8
77.0 0.9
92.5 0.7
77.0 2.7
91.4 0.5
76.6 1.2
93.0 0.9
91.1 0.0
33.3 0.7
91.7 0.6
22.4 0.7
4.1 1.4
96.3 3.5
66.0 4.9
50.0 4.7
78.0 1.3
25.5 5.8
88.6 2.2
11.4 3.5
99.0 3.6
96.1 3.4
94.2 2.2
92.8 2.7
87.0 7.7
100.0 7.0
99.7 8.6
91.3 1.5
98.3 2.1
84.0 2.2
86.0 0.5
94.6 1.3
45.8 12.3
94.0 0.8
46.5 5.5
5594 82
1792 41
2165 149
1429 24
654 119
5622 99
481 144
2154 139
3691 111
887 102
926 238
2981 489
2676 99
1803 12
1285 51
Kamishoyosanc
Kanzotob
54.3 0.5
59.8 1.2
Keigairengyotoa
Keihitof
Keimakakuhantog
Keishakuchimotod
Keishibukuryoganc
Keishibukuryogankayokuininf
Keishikajutsubutoc
Keishikakakkontog
Keishikakobokukyonintog
Keishikaogitog
Keishikaryojutsubutoa
Keishikaryukotsuboreitoc
Keishikashakuyakudaiotof
Keishikashakuyakutoc
Keishininjintof
Keishitoc
53.8 1.2
61.6 1.9
76.4 1.5
47.7 4.4
52.6 0.6
88.6 1.4
85.1 1.6
92.3 1.2
70.2 2.8
61.4 4.6
77.7 0.8
66.6 0.7
84.8 1.0
51.8 0.8
50.5 1.2
3285
0
9
0
91.7 0.1
51.4 0.0
3573 167
4842 588
912 126
3080 253
837 34
79.6 1.3
71.9 3.6
52.4 1.2
Kihitof
Kikyosekkoc
0.0
0.0
0
0
0 0
Kikyotof
6.5 1.5
745 200
3179 207
7560 153
Kososanc
53.7 0.8
92.1 0.5
66.7 1.0
98.1 0.4
45.3 1.5
32.4 0.2
40.6 1.3
69.2 0.7
92.6 0.3
51.9 1.5
14.1 0.7
85.4 1.7
22.0 1.4
81.7 1.9
92.9 1.3
91.1 0.6
94.3 0.6
46.4 1.6
39.8 1.1
91.6 1.4
65.6 1.0
26.3 0.4
64.5 0.6
32.9 0.7
71.0 2.8
100.0 1.7
71.5 3.4
94.9 0.8
87.9 2.9
55.3 5.4
94.8 4.3
81.0 1.0
48.8 2.6
85.7 2.9
43.9 7.3
89.1 1.3
3.2 1.4
84.3 2.9
98.1 2.9
87.3 1.6
94.8 2.5
73.1 3.7
71.2 2.7
95.7 2.0
78.7 1.8
69.4 6.4
80.3 2.1
69.8 4.6
Kumibinrotoc
90.1 0.6
74.7 1.5
93.5 3.2
90.4 1.7
Kyukichoketsuine
Kyukikyogaitoc
Makyokansekitoc
Makyoyokukantoc
Maobushisaishintoa
Maotoc
2578
4622
4
2
4150 333
399
1
44.7 4.9
797 1202
478 79
13409 244
1758 306
1237 112
3485 104
222 12
2895 77
3339 16
2965 20
2353 97
4034 130
1069 75
6255 79
Masiningana
92.5 0.6
Mokuboitoc
Nichintof
Nijutsutof
Ninjintof
Ninjin’yoeitoc
Nyoshinsanf
62.1 0.4
54.4 0.7
66.9 0.9
84.8 0.6
72.0 1.8
Ogikenchutof
Ogontod
Orengedokutoa
Orentoa
Otsujitoc
73.3 0.8
90.2 0.8
Rikkosanf
3090 2
2258 117
309 77
Rikkunshitoc
Rokumiganf
Ryokankyomishingenintoa
1899
1
1 3

Journal of Natural Medicines
Table 1 (continued)
Number
Name of oral Kampo formulation
DPPH (%)
SOD (%)
ORAC (µM TE/U)
100 μU/mL
50 μU/mL
83.3 μU/mL
41.6 μUnit/mL
97
98
99
Ryokeijutsukantoa
Ryokyojutsukantoa
Ryutanshakantoc
Saibokutof
45.1 0.2
18.9 1.4
90.8 1.6
95.0 1.1
78.2 3.8
62.6 0.4
91.4 0.4
91.7 0.2
91.1 1.8
85.0 1.5
86.4 0.0
43.1 1.4
36.8 0.7
64.5 0.7
92.4 0.4
93.0 0.7
25.5 1.4
90.7 0.5
59.7 1.2
91.5 0.1
88.0 1.0
75.0 0.1
37.1 0.9
13.5 0.9
44.7 0.6
54.1 0.5
88.5 1.8
94.3 1.3
6.7 1.0
79.7 3.7
15.8 11.1
92.0 0.4
92.0 1.0
97.0 1.6
86.3 0.8
86.8 3.2
84.4 2.9
93.8 1.0
91.7 1.7
91.1 1.8
83.1 3.3
10.9 1.6
74.9 2.0
96.0 1.6
85.7 3.0
46.6 1.2
95.9 1.7
78.6 0.2
94.0 3.5
94.1 1.5
93.0 0.4
52.5 14.5
58.3 4.9
63.0 1.0
87.9 0.3
92.4 0.2
88.9 2.3
10.1 10.2
48.5 8.4
91.3 5.2
57.5 0.75
87.7 3.0
86.2 0.3
80.4 1.0
86.5 1.8
86.2 2.2
67.4 12.7
99.0 1.8
76.2 1.4
86.8 1.1
84.7 6.5
89.3 0.8
92.6 1.0
84.1 0.2
100.0 3.8
92.1 1.0
97.5 3.8
465 58
1776 116
6466 33
4246 90
5577 183
3113 108
5335 484
2926 184
8117 80
3629 40
2403 133
2286 302
905 37
3741 124
4995 168
4483 259
1396 195
1972 241
2262 33
1966 188
1044 84
3896 32
1109 70
1040 42
64.3 0.6
59.7 1.4
89.8 0.3
80.8 3.0
72.8 3.7
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
133
134
135
136
137
138
139
140
141
142
143
144
Saikantoc
Saikokaryukotsuboreitof
Saikokeishikankyotoc
Saikokeishitoc
Saikoseikantoc
Saireitof
52.2 0.4
44.4 0.4
61.2 0.9
87.7 1.8
75.1 2.9
79.2 1.8
Sammotsuogontof)
San’oshashintoc
Sansonintoa
Seihaitof
Seijobofutoa
53.6 0.3
46.4 1.0
77.0 1.4
85.5 1.9
Seishinrenshiinf
Seishoekkitof
Senkyuchachosanf
Shakanzotoc
63.3 0.8
49.2 1.0
Shakuyakukanzobushitod
Shakuyakukanzotoc
Shichimotsukokatoa
Shigyakusanf
86.4 1.6
79.0 1.1
71.1 6.0
Shikunshitof
Shimbutoc
0 0
1329 101
5237 77
Shimotsutoc
Shimpitoc
82.1 2.5
67.4 1.0
Shin’iseihaitoa
Shireitoa
74.2 0.2
49.7 2.8
5119 1
498 103
1091 52
2214 48
829 19
Shishihakuhitoc
Shofusanc
34.1 0.5
71.2 1.4
4.6 1.8
Shohangekabukuryotoc
Shokenchutoc
91.5 1.2
80.6 3.4
87.0 1.3
62.6 2.0
87.9 1.9
43.4 2.1
91.8 1.0
54.5 1.2
65.0 1.9
90.8 0.4
94.4 1.1
91.0 0.9
80.2 1.7
90.2 2.2
90.3 2.6
92.6 1.6
1607 20
5295 95
4646 55
2699 207
3229 100
2602 57
6678 261
2589 174
2367 46
211 167
1890 20
2882 34
2354 54
28660 355
4247 71
6991 119
Shomakakkontof
Shosaikotoc
Shosaikotokakikyosekkof
Shoseiryutoc
Sokeikakketsutof
Tokakujokitoc
Tokiinshif
89.1 0.2
95.4 2.4
79.4 0.8
Tokikenchutof
Tokishakuyakusanc
Tokishakuyakusankabushia
Tokishigyakukagoshuyushokyotof
Tokitof
39.2 0.6
55.7 1.0
48.9 0.2
Tsudosanc
92.0 1.2
39.6 3.1
76.8 0.8
92.6 0.7
81.1 3.3
83.9 8.0
Unkeitoc
Unseiinc
1 3

Journal of Natural Medicines
Table 1 (continued)
Number
Name of oral Kampo formulation
DPPH (%)
SOD (%)
ORAC (µM TE/U)
100 μU/mL
50 μU/mL
50.6 0.7
83.3 μU/mL
41.6 μUnit/mL
145
146
147
Yokuinintof
90.6 0.6
75.8 4.6
93.0 3.4
89.3 3.3
79.2 7.8
86.6 0.3
5398 165
3381 326
3559 103
Yokukansana
Yokukansankachimpihangec
46.7 0.9
Data are presented as the mean of triplicate measurements for each group standard deviation (SD)
DPPH 2,2-Diphenyl-1-picrylhydrazyl free radical scavenging activity rate, ORAC oxygen radical absorbance capacity, SOD (sodium dioxide
dimutase) superoxide anion scavenging activity rate, TE trolox equivalents, μU microunit
aOhsugi Pharmaceutical Co., Ltd. (Osaka, Japan)
bKracie Holdings, Ltd. (Tokyo, Japan)
cKotaro Pharmaceutical Co., Ltd. (Osaka, Japan)
dSanwa Shoyaku Co., Ltd. (Tochigi, Japan)
eTaikoseido Pharmaceutical Co., Ltd. (Hyogo, Japan)
fTsumura & Co. (Tokyo, Japan)
gToyo-Kampo Pharmaceutical Co., Ltd. (Osaka, Japan)
AAPH [15, 16]. We found that at a fnal concentration of
0.5 mU/mL, fve formulations of the 147 Kampo medicines
tested, namely, Masiningan, Tsudosan, Daisaikoto, Keishi-
kashakuyakudaioto, and Kumibinroto, completely scav-
enged the DPPH free radical (inhibition ratio>90%). At a
fnal concentrations of 41.6 μU/mL, ten Kampo medicines,
namely, Daisaikoto, Jidabokuippo, Tsudosan, Kumibinroto,
Tokakujokito, Kyukikyogaito, Daisaikotokyodaio, Otsujito,
Keishibukuryogan, and Kakkontokasenkyusini, had com-
pletely scavenged this superoxide anion radical (inhibition
ratio>90%). The concentration dependence of the top fve
formulations of Kampo medicines was then evaluated in the
DPPH assay, at concentrations ranging from 10 to 100 μU/
mL (Fig. 1a), and in the SOD assay, at concentrations rang-
ing from 0.42 to 83.3 μU/mL (Fig. 1b). The EC50 and ORAC
values of the fve formulations of Kampo medicines showing
the most potent activities in each assay are shown in Table 2.
A common ingredient of Kampo medicines showing high
in vitro antioxidant intensity is Rhei Rhizoma. Rhei rhizome,
one of the widely used crude drugs found in Kampo medi-
cines, has been reported to have various biological efects,
such as purgative, antipyretic, anti-infammatory, antiangio-
genic, and antineoplasmic activities [17]. Zhong et al. dem-
onstrated that anthraquinones have a protective efect against
oxidative stress-related endothelial cell injury [18]. Bupleuri
Radix and Scutellariae Radix, which are components of
the Kampo formulation Daisaikoto, have been reported to
have a potential to protect hepatic cells from free radicals
in endotoxemia [19]. In other studies, the Kampo formula-
tion Shosaikoto was found to scavenge superoxide anions,
hydroxyl radicals, DPPH radicals in a dose-dependent man-
ner [20], and the Daisaikoto formulation limited the devel-
opment of arteriosclerosis in the aorta of KHC rabbits and
showed an antioxidant capacity for low-density lipoprotein
A
100
Masiningan
50
Tsudosan
Daisaikoto
Keishikashakuyakudaioto
Kumibinroto
0
10
100
Concentration (µUnit/mL)
B
Daisaikoto
Jidabokuippo
Tsudosan
100
50
0
Kumibinroto
Tokakujokito
1
10
100
Concentration (µUnit/mL)
Fig. 1 Dose–response curve of the fve formulations of Kampo medi-
cines showing the most potent activities in the DPPH (2,2-diphenyl-
1-picrylhydrazyl) free radical scavenging activity assay (a) and in the
superoxide anion scavaging activity (SOD sodium oxide dimutase)
assay (b)
1 3

Journal of Natural Medicines
Table 2 In vitro antioxidant activity of the fve formulations of Kampo medicines with the highest activity
DPPH
SOD
ORAC
Kampo formulation
EC50 (μU/mL)
Kampo formulation
EC50 (μU/mL)
Kampo formulation
ORAC (µM TE/U)
Masiningan
Tsudosan
Daisaikoto
Keishikashakuyakudaioto
Kumibinroto
23.2 0.9
23.2 0.6
23.9 1.1
25.2 1.1
26.9 0.9
Daisaikoto
Tsudosan
Tokakujokito
Jidabokuippo
Kumibinroto
6.2 1.1
6.3 1.9
6.3 1.1
6.8 1.1
14.3 4.5
Tsudosan
28660 355
13409 244
11567 61
9498 158
8306 29
Masiningan
Daisaikoto
Daijokito
Bofutsushosan
Data represent the mean of triplicate measurements of each group SD
EC50 Half maximal (50%) efective concentration
[21]. Magnoliae Cortex is present in Kampo formulations
Tsudosan and Masiningan. Son et al. reported that two major
compounds of Magnoliae Cortex, namely, magnolol and
honokiol, inhibited the production of inducible nitric oxide
synthase and tumor necrosis factor alpha in their experiment
system using RAW264.7 cells [22], and Chin et al. reported
that these two compounds also eliminated hydroxyl radi-
cals and inhibited lipid hyperoxidation in rats, resulting in
prevention of liver damage by the peroxide radical [23].
Our assay results show that the Tsudosan, Daisaikoto, and
Fig. 2 Intracellular antioxidant
efect of the Kampo formula-
tions Tsudosan, Daisaikoto, and
Masiningan on human hepato-
cellular carcinoma HepG2 cells.
a The level of reactive oxygen
species (ROS) in H2O2-treated
HepG2 cells over time, b
A
determination of ROS in the
HepG2 cells 50 min following
the addition of H2O2, by fuores-
cent microscopic study. Data
are presented as the mean
standard deviation (SD) of three
measurements (n=3). Statistical
signifcance was determined
with Dunnett’s test. Double
asterisks indicate signifcance
vs. the control group at p<0.01
B
0 min
50 min
Control
Daisaikoto
Tsudosan
Masiningan
+H2O2
1 3

Journal of Natural Medicines
Masiningan formulations were the most potent in vitro anti-
oxidants of the Kampo formulations tested; we therefore
selected these for further investigation of their intracellular
and in vivo antioxidant activities.
Although our knowledge of the change in serum BAP
values after the administration of Kampo medicines in ani-
mal experiments is still quite limited, it has been reported
that the administration of the Kampo formulations
Tokakujokito and Keisibukuryogan increased rat serum
BAP values and improved blood stasis [25]. The Kampo
Tsudosan formulation consists of ten crude drugs: glycyr-
rhiza (Glycyrrhizae Radix), immature orange (Aurantii
Fructus Immaturus), safower (Carthami Flos), magno-
lia bark (Magnoliae Cortex), sappan wood (Sappan Lig-
num), rhubarb (Rhei Rhizoma), citrus unshiu peel (Citri
Unshiu Pericarpium), Japanese angelica root (Angelicae
Acutilobae Radix), anhydrous sodium sulfate (Sal Mira-
bilis Anhydricus), and akebia stem (Akebia Caulis). This
formulation is considered to be one of strongest Kampo
medicines against blood stasis and is used clinically to
treat chronic infammatory diseases. The increase in mice
serum BAP values following treatment with the Kampo
Tsudosan formulation in our study may be closely related
to its blood stasis efect.
We evaluated the intracellular antioxidant activities of
these three Kampo medicines against H2O2-induced oxida-
tive stress in human hepatocellular liver carcinoma HepG2
cells using the DCFH-DA assay. DCFH-DA is the most
widely used cell-permeable probe for detecting intracellu-
lar H2O2 and oxidative stress. In this assay, DCFH-DA is
hydrolyzed intracellularly to the DCFH carboxylate anion
and subsequently oxidized to fuorescent DCF. Each of the
three Kampo medicines was pre-incubated with the HepG2
cells, and then removed from the extracellular component.
We then measured the time-course of the reductive efect of
each intercellular Kampo medicine on H2O2-induced DCF
fuorescence intensity. As shown in Fig. 2a, the addition of
H2O2 to the HepG2 cells resulted in a notable induction of
DCF fuorescence intensity after 30 min, but this intensity
was signifcantly reduced in those cells pre-incubated with
the selected Kampo formulations at the fnal concentration
of 10 μU/mL. At the time-point of 50 min, there were fewer
cells stained with DCF among the HepG2 cells treated with
the Tsudosan formulation than among the (untreated) con-
trols (Fig. 2a, b). It should be noted that cytotoxicity was
not observed when the cells were pre-incubated with the
selected Kampo formulations. These results reveal that the
Tsudosan, Daisaikoto, and Masiningan formulations efec-
tively inhibited H2O2-induced oxidative stress intracellularly.
Since all three Kampo formulations exhibited intracel-
lular antioxidant activities, their antioxidant efects were
subsequently evaluated in vivo in mice using the BAP test,
which is based on the ability of a plasma blood sample to
reduce ferric ions to ferrous ions as a FRAP assay (ferric
reducing ability of plasma). BAP values indicate systemic
antioxidative properties and refect the reduction potential
of the serum. In simple terms, non-enzymatic antioxidants,
such as vitamin C, can be described as reductants, and inac-
tivation of oxidants by reductants can be described as redox
reactions. The oxidative stress index is an arbitrary param-
eter derived from BAP values to represent an individual
compound’s antioxidant potential against the condition of
oxidative stress [24]. The Tsudosan, Daisaikoto, and Masin-
ingan formulations and vitamin C were orally administrated
to mice at a dose of 2.5 g/kg body weight, twice a day for
2 days. The serum BAP values were measured. In compari-
son to the control group, the serum BAP values were sig-
nifcantly increased in those groups of mice orally adminis-
trated the Tsudosan formulation and vitamin C, respectively,
whereas the groups of mice administrated the Daisaikoto and
Masiningan formulations tended to show an increased serum
BAP value (Fig. 3).
In summary, comprehensive evaluation of the in vitro
antioxidant activities of 147 formulations of oral Kampo
medicines resulted in the identifcation of formulations
Tsudosan, Daisaikoto and Masiningan having the most
potent antioxidant activity. Further investigation of the
intercellular and in vivo antioxidant activities of these
three Kampo medicines led to the identifcation of the
Tsudosan formulation as a potent antioxidant agent. The
results of our study suggest that the clinical usage of the
Fig. 3 Values of the biological antioxidant potential (BAP) following
the administration of selected Kampo formulations (Tsudosan, Dai-
saikoto, Masiningan) and the non-enzymatic antioxidant vitamin C.
Data are presented as the mean SD of nine measurements (n=9).
Statistical signifcance was determined with Dunnett’s test. Single
and double asterisks indicate signifcance vs. the control group at *p
<0.05 and **p <0.01, respectively
1 3

Journal of Natural Medicines
11. Higai K, Satake M, Nishioka H, Azuma Y, Matsumoto K (2008)
Glycated human serum albumin enhances macrophage infamma-
tory protein-1β mRNA expression through protein kinase C-δ and
NADPH oxidase in macrophage-like diferentiated U937 cells.
Biochim Biophys Acta 1780(2):307–314
Tsudosan formulation may be closely correlated to its
potent antioxidant efects.
Acknowledgements This investigation was supported by JSPS KAK-
ENHI 15K08003, and the Joint Research grants of Toho University
Faculty of Pharmaceutical Sciences.
12. Pasquini A, Luchetti E, Marchetti V, Cardini G, Iorio EL (2008)
Analytical performances of d-ROMs test and BAP test in canine
plasma. Defnition of the normal range in healthy labrador dogs.
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Compliance with ethical standards
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Conflict of interest The authors declare no confict of interest.
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