Original Article

Yonago Acta medica 2016;59:67–80

Components of Boiogito Suppress the Progression of Hypercholesterolemia andFatty Liver Induced by High-Cholesterol Diet in Rats
Weibin Qian,* Junichi Hasegawa,* Xinrui Cai,*† Jie Yang,* Yoshitaka Ishihara,* Bingqiong Ping,*		
Satoshi Tsuno,* Yusuke Endo,* Akiko Matsuda*‡ and Norimasa Miura**Division of Pharmacotherapeutics, Department of Pathophysiological and Therapeutic Science, School of Medicine, Tottori University
Faculty of Medicine, Yonago 683-8503, Japan

ABSTRACT

were improved by treatment. Biomarkers of cholesterolsynthesis (lathosterol) and absorption (campesterol, betasitosterol) were lower in the treatment groups. Inflammatory-related factors (MCP1, CCR2 and TNF-alpha) andICAM-1 were ameliorated after treatment, especially by
ogi with ginger.
Conclusion Ogi, ogi with hesperidin or ginger have asimilar effect of BOT and ezetimibe on hypercholesterolemia and fatty liver. Ogi with ginger reveals a stronger
additive effect with no significant difference. However,as for the anti-inflammatory (MCP1, CCR2 and TNFalpha) and anti-arteriosclerotic (ICAM-1) effects, additive effects of ogi with ginger are more potent than thatof ogi alone or ezetimibe.

BackgroundOgi, one main component of boiogito(BOT), is reported to have an effect on hypercholesterolemia and NAFLD. In this experiment, we examined
effects of ogi on the progression of hypercholesterolemia
and fatty liver induced by high-cholesterol diet in rats
and compared with the effects of ogi combined withginger or hesperidin.
Methods Hypercholesterolemia and fatty liver were
induced by a high cholesterol diet in rats. Extract of ogi,
ogi with hesperidin, and ogi with ginger were added tothe high-cholesterol diet, respectively. Ezetimibe wasalso added to the high-cholesterol diet as a positive control. After 6 and 12 weeks, body, liver and adipose tissue
weights, blood chemistry, lipid-related and inflammatory-related factors were examined.
Result The high cholesterol diet increased body, liver
and adipose tissue weights, and serum cholesterol concentrations. Ogi, ogi with hesperidin or ginger and ezetimibe improved them. In the histological examinations,
we observed a significant improvement after treatment.
The lipid-related factors (RBP4, HFABP and CFABP)

Key words fatty liver; ginger; hesperidin; hypercholes-

terolemia; ogi
The increased prevalence of hypercholesterolemia andfatty liver diseases has provided an increasingly negative
connotation toward lipids and cardiovascular diseases(CVD), such as atherosclerosis and hypertension.1, 2
Non-alcoholic fatty liver disease (NAFLD) occurs more
frequently in patients with hypercholesterolemia.3 During the past few years, increasing evidence shows that
NAFLD has also become a multisystem disease that
affects many extra-hepatic organ systems, including
the heart and the vascular system.4 Much experimental,
clinical and epidemiological evidence supports a strong
association between NAFLD and CVD, especially atherosclerosis.5, 6
A lot of lipid metabolism-related molecules andinflammatory cytokines are associated with the progression of hypercholesterolemia and NAFLD. Retinol binding protein 4(RBP4) is a 21 kDa secreted protein that is
the transport protein for vitamin A (retinol) in blood.7 It
takes part in the control of metabolic and proliferativecell functions,8 including steatogenesis.9 The RBP4 genehas been linked to increasing fasting glucose levels10 and
related to hypercholesterolemia and NAFLD.11 Hearttype fatty acid binding protein (HFABP) and cutaneous
fatty acid-binding protein (CFABP) belong to the fatty
acid binding protein (FABP) super-family, which is as-†Present address: Shandong Academy Occupational Health andOccupational Medicine, Shandong Academy of Medical Sciences,
Jinan, Shandong 250002, China and ‡Present address: Division of
Fundamental Nursing, Faculty of Nursing, Nara Medical University,
Kashihara 634-8521, Japan
Corresponding author: Junichi Hasegawa, MD, PhD
hasegawa@med.tottori-u.ac.jp
Received 2015 December 15
Accepted 2016 January 4
Abbreviations: ALP, alkaline phosphatase; ALT, alanine aminotransferase; BOT, boiogito; BS, blood sugar C, control group;
CFABP, cutaneous fatty acid-binding protein; Cr, creatinine; CVD,
cardiovascular disease; FABP, fatty acid binding protein; H, highcholesterol diet group; HO, high-cholesterol diet with ogi group;
HOH, high-cholesterol diet with ogi and hesperidin group; HOG,
high-cholesterol diet with ogi and ginger group; HDL-C, high-density lipoprotein cholesterol; HE, high-cholesterol diet with ezetimibe
group; HFABP, heart fatty acid-binding protein; ICAM-1, intercellular adhesion molecule-1; LDH, lactate dehydrogenase; LDL-C, lowdensity lipoprotein cholesterol; MCP1, monocyte chemoattractant
protein-1; NAFLD, non-alcoholic fatty liver disease; RBP4, retinolbinding protein 4; RT-PCR, real-time polymerase chain reaction;
TBil, total bilirubin; TC, total cholesterol; TG, triglyceride; VEC,
vascular endothelial cells67

W. Qian et al.

control group (C, n = 10), high-cholesterol diet group (H, n =10), high-cholesterol diet with ogi group (HO, n = 10), highcholesterol diet with ogi and hesperidin group (HOH, n =10), high-cholesterol diet with ogi and ginger group (HOG,n = 10), high-cholesterol diet with ezetimibe group (HE, n= 10). The rats in each group were numbered from 1 to 10.
Group C was fed a standard rat diet (moisture 8.83%, protein 25.13%, fat 4.92 %, fiber 4.42% and crude ash 6.86%,
CE-2; Japan Clea, Tokyo, Japan). A high-cholesterol diet11,20
was supplied for Group H; it was made by adding 2%cholesterol and 0.5% cholic acid to the standard diet. The
high-cholesterol diet with ogi was made by adding 1% ogi
extract to the high-cholesterol diet. The high-cholesterol dietwith ogi and hesperidin was made by adding 1% ogi extractand 0.08% hesperidin11 (Wako Pure Chemical Industries,
Osaka, Japan). The high-cholesterol diet with ogi and ginger
was made by adding 1% ogi extract and 1% ginger extract.
Ogi and ginger extracts were purchased from Tsumura &CO. (Tokyo). The high-cholesterol diet with ezetimibe wasmade by adding 0.0006% of ezetimibe (LKT laboratories,
St Paul, MN). The concentrations of the extracts administered were calculated based on body surface area. Adult patients take ogi, ginger extract, hesperidin and ezetimibe 15 g,
15 g, 1.2 g and 8 mg per day, respectively. The body surface
area conversion coefficient from humans to rats is 0.018.
The amount of feed for each rat was regulated to 25 g/day
and water was supplied ad libitum. Body weights, systolicand diastolic blood pressure and heart rate were measured
every week. Blood pressure and heart rate were measured
by a noninvasive computerized tail-cuff method (BP-98A;
Softron, Tokyo).

signed to transport fatty acids towards the mitochondria
for beta-oxidation and energy expenditure.12, 13 Recent
findings indicate that monocyte chemoattractant protein
1 (MCP1) and its receptor C-C chemokine receptor type
2 (CCR2) play a key role in hypercholesterolemia14 andCVD.15 Intercellular adhesion molecule-1 (ICAM-1) isover expressed in conditions with evidence of endothelial inflammation such as atherosclerosis.16 TNF-alphaplays a similar role with ICAM-1 in atherosclerosis.17
Therefore, in this study, we have monitored the expression of RBP4, HFABP, CFABP, MCP1, CCR2, ICAM-1
and TNF-alpha to detect the mechanisms of hypercholesterolemia and atherosclerosis.
Based on evidence from our previous study, we
proved that boiogito (BOT) has a protective effect on
the progression of hypercholesterolemia and fatty liver.
And the anti-inflammatory (MCP1, CCR2) and antiarteriosclerotic (ICAM-1) effects of BOT are morepotent than ezetimibe.18 Our results are consistent withanother previous study.19 Ogi (Astragalus membranaceus Bunge), one main component of BOT, has beenreported to have an effect on hypercholesterolemia and
NAFLD. Ogi showed statistically significant beneficial
effects on alanine aminotransferase (ALT) and glutamyltransferase activity,26 and significantly lowered plasma
total cholesterol by 45.8%, triglycerides by 30%, andlow-density lipoprotein-cholesterol (LDL-C) by 47.4%,
comparable to simvastatin.27 We have also proven thatginger (Zingiber officinale Roscoe) and hesperidin could
improve hypercholesterolemia and NAFLD by inhibiting both the synthesis and absorption of cholesterol andregulating the expression of mRNA for RBP4, HFABPand CFABP.11, 20 We have also proven that hesperidin
combined with ginger has a stronger effect than hesperidin alone on hypercholesterolemia.21 However, theinhibitory effect of the key components of BOT (ogi andginger) on hypercholesterolemia and NAFLD has not yet
been proven. Moreover, the synergistic action of ogi withhesperidin is unclear. In this experiment, we examinedthe effects of ogi on the progression of hypercholesterolemia and fatty liver induced by high-cholesterol diet inrats and compared with the effects of ogi combined withginger or hesperidin. Furthermore, we have studied themechanism of these kampo components on hypercholesterolemia and the protective effect on atherosclerosis.

Sample collection

On days 42 and 84, 5 rats of each group in the order of
how they were numbered were sacrificed by collecting
blood from the heart under pentobarbital anesthesia after fasting for 12 h. Liver tissue, adipose tissue aroundthe left kidney, and abdominal aorta were removed forspecimen, and then portions of the samples were stored
in a 10% formalin solution for hematoxylin and eosin(HE) and oil red O staining.22 The remaining samples
were immediately transferred into EP tubes containing
500 μL of RNA later (Ambion, Austin, TX), quickly
frozen in liquid nitrogen, and stored at –80 °C. Bloodchemistry was analyzed for rats using an auto analyzer
at an accredited clinical laboratory (SRL, Tokyo).

MATERIALS AND METHODS
Rats and feeding method

ICAM-1 and RBP4 enzyme-linked immunosorbent
assay (ELISA)

Sixty 8-week-age male Wistar rats (purchased from Charles
River Laboratories Japan) were acclimated in an air-conditioned room at 25 °C with 55% humidity and given standard
chow for 3 days. Then the rats were divided into six groups:

Serum samples were applied for an ELISA of ICAM-1(R&D, Minneapolis, MN) and RBP4 (Aviscera Bioscience),
according to the manufacturer’s instructions.
68Kampo components ameliorates dyslipidemia

RT-PCR
Total RNA from the liver and adipose tissue aroundthe left kidney was extracted using TRIzol reagent according to the manufacturer’s instructions (Promega,
Carlsbad, CA). A semiquantitative real-time polymerase
chain reaction (RT-PCR) was performed using LineGene (Toyobo, Tokyo) and SYBR Green I (Roche,
Basel, Switzerland). The detection was executed at theextension reaction stage in each cycle. The primer setsfor RBP4, HFABP, CFABP, MCP1, CCR2 and betaactin mRNA were all synthesized by Hokkaido System
Science (Sapporo, Japan). The sequence of each primer
is listed in Table1. Using the 2−ΔCT method, mRNA expression was semi-quantitatively measured as a relative
amount of each target RNA to a known housekeeping
gene (beta-actin) expression level.

Rockville, MD) was used to analyze the integrating optical density of target band.
Immunohistochemical studies

During the immunohistochemical analyses, 4% paraformaldehyde-fixed aorta tissue specimens were processed.
Anti-ICAM-1 (Abcam, Cambridge, United Kingdom) wasused. As a negative control, tissues were stained without
the primary antibody. The optical densities were measured
by Image-Pro Plus v 6.0 software (Media Cybernetics,
Rockville, MD).
The condition of LC/MS/MS analysis

Lathosterol, campesterol and beta-sitosterol were analyzed
using a HPLC Prominence (Shimadzu, Kyoto, Japan) coupled with a mass spectrometer (AB SCIEX QTRAP 5500
LC/MS/MS System, SCIEX, Tokyo). In brief, separationsof lathosterol, campesterol and beta-sitosterol were carried out using a Poroshell 120 EC-C18 column 2.1 × 150
mm, 2.7 μm particle size (Agilent Technologies, CA). The
elution (150 µL/min) was completed at 40 °C in 25 minwith linear gradient from 20% eluent B (ethanol) to 80%
of eluent B (acetonitrile).The injection volume was 10µL. The LC–MS instrument was operated in the positive
atmospheric pressure chemical ionization (APCI) modewith corona current of 3 μA, and capillary temperatureof 500 °C. Declustering and entrance potential were set
at 60 and 10 V, respectively. MS/MS spectra at m/z 315
were obtained, and parameters for the specific MRM
transition were optimized. The collision energy was 30
V. MRM transition of 315.0 → 176.0 was found to be theoptimal transitions.

Table 1. The sequence of each PCR primer
Gene

Primer

RBP4

Forward 5´-gacaaggctcgtttctctgg-3´Reverse 5´-gactcgtcccttggctgtag-3´Forward 5´-ctagcatgagggaagcaagg-3´Reverse 5´-tgcttcatccagacaagtgg-3´
Forward 5´-gggctggctcttaggaagat-3´Reverse 5´-aaaacacggtcgtcttcacc-3´
Forward 5´-ctgtagcatccacgtgctgt-3´Reverse 5´-tgctgctggtgattctcttg-3´Forward 5´-gatcctgcccctacttgtca-3´Reverse 5´-agatgagcctcacagcccta-3´Forward 5´-gtagccatccaggctgtgtt-3´Reverse 5´-ccctcagatgggcacagt-3´

HFABP
CFABP
MCP1
CCR2
Beta-actin

Statistical analyses

CCR2, C-C chemokine receptor type 2; CFABP, cutaneous fatty
acid-binding protein; HFABP, heart fatty acid-binding protein;
MCP1, monocyte chemoattractant protein-1; PCR, polymerase
chain reaction; RBP4, retinol-binding protein 4.

The data are expressed as the means ± standard deviation. Statistical comparisons were made using the two independent samples t-test by SPSS 11.0J (IBM, Armonk,
NY). P < 0.05 was considered statistically significant.

Western blotting

RESULTS

Proteins from the liver were extracted using CelLytic
MT according to the manufacturer’s instructions (Sigma,
St. Louis, MO). We performed western blotting with
20 mg/mL of proteins and the i-Blot gel transfer system(Invitrogen, Tokyo). Anti- TNF-alpha (1:500 dilutions,
Sigma) and beta-actin (MBL, Nagoya, Japan) antibodies
were used in the study according to the manufacturer’s
instructions. Chemiluminescent signals were detected
within 1 min with LAS-4000 (Fujifilm, Tokyo). The gelswere run under the same experimental conditions. The
expression of TNF-alpha was normalized by beta-actin.
The Gel-Pro Analyzer 4.0 software (Media Cybernetics,

Histological examination of liver tissues

From hematoxylin-eosin staining we can found thatthe fatty degeneration (steatosis) of liver was observedin high-cholesterol diet-fed Groups, but not in Group
C. The tiny and large vacuoles as well as pleomorphic
nuclei were more conspicuous in treatment groups thanin Group H (Fig. 1A). Oil red O staining revealed thesame changes that the livers in all high-cholesterol dietsupplemented groups were filled with microvesicular or
macrovesicular fat deposits; they were depicted as reddish deposits (Fig. 1B). Groups HOG and HE seems toshow better effect (× 400).
69

Fig. 1.
W. Qian et al.

A
A

6W

12W

C

H

HO

HOH

HOG

HE

B
B

6W

12W

6W

12W

6W

12W

H

C

HOH

HO

HE

HOG

Fig. 1. Histopathological examination of liver.
A: Fatty degeneration (steatosis) of the liver is observed in the high-cholesterol diet–fed groups (H, HO, HOH, HOG and HE), but not inGroup C in hematoxylin-eosin stained tissues. Fatty changes increased with time to a greater extent in Group H than Groups HO, HOH,
HOG and HE. B: Oil red O staining revealed more lipid droplets (stained red) to be accumulated in vacuoles in Group H than othergroups. 6W: 6 weeks, 12W: 12 weeks. C: control, standard diet for 6 and 12 weeks (n = 5); H: high-cholesterol diet for 6 and 12 weeks (n =5); HO: high-cholesterol diet with ogi for 6 and 12 weeks (n = 5); HOH: high-cholesterol diet with ogi and hesperidin for 6 and 12 weeks(n = 5); HOG: high-cholesterol diet with ogi and ginger for 6 and 12 weeks (n = 5); HE: high-cholesterol diet with ezetimibe for 6 and 12
weeks (n = 5). Bars express 25 μm.

70Kampo components ameliorates dyslipidemia

6W

12W

6W

C

H

HO

HOH

HOG

HE

12W

Fig. 2. Histopathological examination of adipose tissue.
The larger fat cells were observed in high-cholesterol diet-fed groups (H, HO, HOH, HOG and HE), but not in Group C (55.16 ± 7.21 μm
and 55.78 ± 6.58 μm after 6 and 12 weeks respectively) in hematoxylin-eosin stained tissues. Fat cells enlarged with time to a greater extent in Group H (90.91 ± 12.73 and 94.03 ± 8.54 μm in diameter after 6 and 12 weeks respectively) than Groups HO, HOH, HOG and HE(54.94 ± 9.22 μm and 59.16 ± 8.93 μm, 53.28 ± 8.40 μm and 56.88 ± 7.99 μm, 46.09 ± 7.66 μm and 54.54 ± 8.91 μm, 58.91 ± 8.25 μm and59.06 ± 7.00 μm in diameter after 6 and 12 weeks respectively). 6W: 6 weeks, 12W: 12 weeks. C: control, standard diet for 6 and 12 weeks(n = 5); H: high-cholesterol diet for 6 and 12 weeks (n = 5); HO: high-cholesterol diet with ogi for 6 and 12 weeks (n = 5); HOH: highcholesterol diet with ogi and hesperidin for 6 and 12 weeks (n = 5); HOG: high-cholesterol diet with ogi and ginger for 6 and 12 weeks (n= 5); HE: high-cholesterol diet with ezetimibe for 6 and 12 weeks (n = 5). Bars express 50 μm.

Histological examination of adipose tissue

in Groups HO, HOH and HOG after 6 weeks (P < 0.05).

The larger fat cells were observed in high-cholesterol
diet-fed groups (H, HO, HOH, HOG and HE), but not inGroup C. It was more notable in Group H than Groups
HO, HOH, HOG and HE (Fig. 2, × 400).

Blood chemistry and cholesterol concentrations

As Table 3 shows, the high-cholesterol diet increase theconcentrations of LDL-C, TC, ALT, ALP, LDH andreduce the concentrations of HDL-C in Group H compared with Group C after 6 and 12 weeks in serum (P< 0.05). Compared with Group H, the concentrations of
LDL-C and TC decreased in Groups HOH, HOG, HE
after 6 weeks and Groups HO, HOH, HOG, HE after 12
weeks (P < 0.05), while the concentration of HDL-C has
a opposite trends (P < 0.05). After 12 weeks, the activities of ALT, ALP, LDH reduced in different degree inGroups HO, HOH, HOG, HE compared with Group H(P < 0.05). The BS, TBil, TG and Cr have no significant
differences during the experiment.

Body, liver, and adipose tissue weights

During the whole experiment, the amount of feed foreach rat was regulated to 25g/day and there was no
significant difference in each group. As Table 2 shows,there were no significant differences in the baseline ofbody weights at the beginning of the experiment. Highcholesterol diet increased body, liver and adipose tissue
weights in Group H compared with Group C both after
6 and 12 weeks (P < 0.05). Body, liver and adipose tissue weights were significantly lower in Groups HO,
HOH, HOG, HE than Group H after 6 and 12 weeks (P< 0.05). Liver weights in Group HE were lower than that71

W. Qian et al.

Lathosterol, Campesterol and beta-Sitosterol concentrations determined by LC/MS/MS

which were lower in the treatment groups (HO, HOH,
HOG and HE) than Group H (P < 0.05).

Concentrations of serum lathosterol decreased in theGroup HO, HOH, HOG and HE compared with GroupH (Table 4, P < 0.05). The same trend was found in theconcentrations of serum campesterol and beta-sitosterol,

ICAM-1 and RBP4 in serum

ICAM-1 concentrations were increased in Group H compared with Groups C, HO, HOH, HOG and HE group

Table 2. Body, liver and Lipid weightsCBody
weights (g)Liverweights (g)Adipose tissue
weights (g)0W 308.68 ± 6.72
6W 453.03 ± 16.69
12W 507.97 ± 24.12
6W 11.66 ± 1.09
12W 13.26 ± 1.48
6W
2.59 ± 0.67
12W
3.53 ± 0.55

H

HO

HOH

HOG

HE

315.86 ± 8.56
310.47 ± 9.23
310.67 ± 8.77
311.73 ± 7.96
311.57 ± 7.59
503.12 ± 17.27* 456.91 ± 17.68† 456.20 ± 22.70† 453.88 ± 20.55† 451.48 ± 18.23†
576.09 ± 20.54* 497.23 ± 20.29† 503.52 ± 29.72† 506.20 ± 15.24† 510.63 ± 8.27†
19.62 ± 1.90*
16.39 ± 1.03†
17.21 ± 1.09†
16.79 ± 2.15†
13.24 ± 1.76†‡§||
28.99 ± 4.42*
19.90 ± 5.03†
20.44 ± 3.06†
20.22 ± 3.32†
16.79 ± 3.40†
3.74 ± 0.40*
2.35 ± 0.70†
2.78 ± 0.89†
2.09 ± 0.69†
2.58 ± 0.48†
5.88 ± 0.50*
2.09 ± 0.50†
2.99 ± 0.74†
3.35 ± 0.30†
2.20 ± 0.90†||

C: control, standard diet for 6 and 12 weeks (n = 5); H: high-cholesterol diet for 6 and 12 weeks (n = 5); HO: high-cholesterol diet with ogi
for 6 and 12 weeks (n = 5); HOH: high-cholesterol diet with ogi and hesperidin for 6 and 12 weeks (n = 5); HOG: high-cholesterol dietwith ogi and ginger for 6 and 12 weeks (n = 5); HE: high-cholesterol diet with ezetimibe for 6 and 12 weeks (n = 5). *P < 0.05 vs. Group C,†P < 0.05 vs. Group H, ‡P < 0.05 vs. Group HO, §P < 0.05 vs. Group HOH, ||P < 0.05 vs. Group HOG. Data are expressed as the means ±standard deviation. 0W: 0 week, 6W: 6 weeks, 12W: 12 weeks.

Table 3. Blood chemistry and cholesterol concentrations

LDL-C (mg/dL)
HDL-C (mg/dL)
TC (mg/dL)
TG (mg/dL)
ALT (U/L)
ALP (U/L)
TBil (mg/dL)
Cr (mg/dL)
LDH (U/L)
BS (mg/dL)

6W
12W
6W
12W
6W
12W
6W
12W
6W
12W
6W
12W
6W
12W
6W
12W
6W
12W
6W
12W

C

H

HO

HOH

HOG

HE

9.20 ± 3.19
9.20 ± 1.30
17.20 ± 4.02
19.00 ± 2.35
56.80 ± 15.01
55.60 ± 8.20
20.75 ± 5.50
26.80 ± 8.81
30.00 ± 3.67
30.40 ± 4.16
192.00 ± 25.50
202.00 ± 25.29
0.05 ± 0.01
0.06 ± 0.01
0.34 ± 0.06
0.37 ± 0.05
156.00 ± 18.46
177.50 ± 64.57
103.40 ± 7.54
95.80 ± 10.66

53.00 ± 17.90*
65.67 ± 15.95*
6.40 ± 1.67*
13.00 ± 1.63*
150.25 ± 34.06*
160.75 ± 48.75*
18.60 ± 8.11
31.80 ± 2.59
50.80 ± 11.30*
168.00 ± 53.84*
372.75 ± 32.47*
515.67 ± 36.09*
0.03 ± 0.01
0.04 ± 0.01
0.31 ± 0.03
0.41 ± 0.09
516.67 ± 32.75*
599.00 ± 19.80*
92.80 ± 6.14
101.80 ± 6.14

54.75 ± 18.91
29.60 ± 10.11†
6.60 ± 1.14
17.25 ± 0.96†
124.00 ± 33.09
83.80 ± 13.05†
19.75 ± 4.57
23.00 ± 6.96
70.00 ± 2.00
35.67 ± 3.79†
363.67 ± 8.14
258.80 ± 29.18†
0.041 ± 0.02
0.05 ± 0.02
0.42 ± 0.10
0.40 ± 0.10
262.00 ± 32.08†
192.00 ± 48.08†
89.60 ± 10.41
78.00 ± 15.07

35.75 ± 6.24†‡
41.00 ± 9.49†‡
12.00 ± 2.74†‡
18.00 ± 1.00†
106.00 ± 20.83†
115.60 ± 23.54†
21.20 ± 4.92
28.50 ± 8.76
52.00 ± 3.39‡
70.60 ± 19.19†
327.00 ± 29.46†
289.33 ± 27.75†
0.04 ± 0.01
0.05 ± 0.01
0.32 ± 0.05
0.33 ± 0.03
277.33 ± 25.48†
214.00 ± 16.46†
108.33 ± 4.51
82.20 ± 13.74

28.33 ± 3.06†‡
9.00 ± 1.22†‡§||
31.75 ± 9.84†
28.20 ± 2.17†§
13.80 ± 4.15†‡ 16.80 ± 2.28†‡§
16.00 ± 1.00†
17.75 ± 2.06†
90.00 ± 13.53†‡ 54.60 ± 7.16†‡§||
120.00 ± 40.28† 94.00 ± 9.43†
15.25 ± 3.40
13.00 ± 5.57
39.00 ± 10.10
17.40 ± 11.59
46.33 ± 9.29‡
91.33 ± 20.53†‡§||
55.00 ± 14.14†
36.50 ± 10.47†
314.33 ± 28.94†‡ 390.33 ± 20.74§||264.33 ± 38.89† 265.25 ± 39.66†
0.03 ± 0.02
0.06 ± 0.00
0.06 ± 0.02
0.05 ± 0.01
0.41 ± 0.02
0.36 ± 0.04
0.32 ± 0.07
0.35 ± 0.07
356.33 ± 33.56†‡§342.67 ± 35.84†‡§318.00 ± 87.68† 282.33 ± 82.71†
104.80 ± 7.56
93.67 ± 6.11
75.00 ± 13.17
103.20 ± 18.90

C: control, standard diet for 6 and 12 weeks (n = 5); H: high-cholesterol diet for 6 and 12 weeks (n = 5); HO: high-cholesterol diet with ogi
for 6 and 12 weeks (n = 5); HOH: high-cholesterol diet with ogi and hesperidin for 6 and 12 weeks (n = 5); HOG: high-cholesterol dietwith ogi and ginger for 6 and 12 weeks (n = 5); HE: high-cholesterol diet with ezetimibe for 6 and 12 weeks (n = 5). *P < 0.05 vs. Group C,†P < 0.05 vs. Group H, ‡P < 0.05 vs. Group HO, §P < 0.05 vs. Group HOH, ||P < 0.05 vs. Group HOG. Data are expressed as the means ±standard deviation. 6W: 6 weeks, 12W: 12 weeks.
ALP, alkaline phosphatase; ALT, alanine aminotransferase; BS, blood sugar; Cr, creatinine; HDL-C, high-density lipoprotein cholesterol;
LDH, lactate dehydrogenase; LDL-C, low-density lipoprotein cholesterol; TBil, total bilirubin; TC, total cholesterol; TG, triglyceride.

72

Kampo components ameliorates dyslipidemia

Fig. 3.

Table 4. Lathosterol, campesterol, and beta-sitosterol concentrations in serum determined by LC/MS/MS-2 μg/mL)

Lathosterol (10Campesterol (μg/mL)
beta-Sitosterol (μg/mL)

C

H

HO

HOH

HOG

HE

1.81 ± 0.29
1.19 ± 0.46
0.77 ± 0.27

3.84 ± 0.43*
2.26 ± 0.18*
2.51 ± 0.35*

2.53 ± 0.88†
1.36 ± 0.36†
1.46 ± 0.39†

2.55 ± 0.37†
1.50 ± 0.33†
1.47 ± 0.11†

2.40 ± 0.41†
1.40 ± 0.84†
0.92 ± 0.24†‡§

2.09 ± 0.79†
1.29 ± 0.04†
0.95 ± 0.51†‡§

C: control, standard diet for 6 and 12 weeks (n = 5); H: high-cholesterol diet for 12 weeks (n = 5); HO: high-cholesterol diet with ogi for 12
weeks (n = 5); HOH: high-cholesterol diet with ogi and hesperidin for 12 weeks (n = 5); HOG: high-cholesterol diet with ogi and ginger
for 12 weeks (n = 5); HE: high-cholesterol diet with ezetimibe for 12 weeks (n = 5). *P < 0.05 vs. Group C, †P < 0.05 vs. Group H, ‡P < 0.05
vs. Group HO, §P < 0.05 vs. Group HOH, ||P < 0.05 vs. Group HOG. Data are expressed as the means ± standard deviation.

*

9

ICAM-1
†

20
ng/mL

†

RBP4

*

†

†

*

†

†

6

†‡

ng/mL

30

10

†
† †‡

† †

3

0

0
C

H

HO

HOH HOG

C

HE

H

HO

HOH HOGHE

Fig. 3. ICAM-1 and RBP4 concentrations in serum.
ICAM-1 and RBP4 concentrations in standard diet group (C, n = 5), high-cholesterol diet group (H, n = 5), high-cholesterol diet with ogigroup (HO, n = 5), high-cholesterol diet with ogi and hesperidin group (HOH, n = 5), high-cholesterol diet with ogi and ginger group (HOG,n = 5) and high-cholesterol diet with ezetimibe group (HE, n = 5). Dotted and shaded columns represent 6 and 12 weeks, respectively. *P< 0.05 vs. Group C, †P < 0.05 vs. Group H, ‡P < 0.05 vs. Group HO. 6W: 6 weeks, 12W: 12 weeks.
ICAM-1, intercellular adhesion molecule-1; RBP4, retinol-binding protein 4.

after 12 weeks (Fig. 3, P < 0.05). Compared with othertreatment groups, Group HOG seemed to have the best
effect. RBP4 concentrations were increased in Group
H compared with the other groups, including Groups
HOG and HE after 6 weeks (P < 0.05) and Groups HO,
HOH, HOG and HE after 12 weeks (P < 0.05). It hasless concentration in Group HOG than Group HO after
12 weeks (P < 0.05).

< 0.05), and then continued to increase after 12 weeks
compared to all the other 5 groups (P < 0.05). Comparedwith the other groups, the mRNA expression of CFABP
in liver in Group H were higher than that in the othergroups after 12 weeks (P < 0.05), whereas they were notobvious after 6 weeks. Compared with the other groups,
the mRNA expression of MCP1 in liver in Group H
remained high during the entire experiment, especiallyafter 12 weeks. And Group HOG revealed a better effectthan Groups HO and HE at 12 weeks (P < 0.05). The expressions of CCR2 in Groups HO, HOH and HOG werelower than that in Group H (P < 0.05) after 6 and 12
weeks, but in Group HE it was not significantly reduced
than that in Group H after 6 and 12 weeks.
The same trend was found in adipose tissue aroundmRNA expression in liver and adipose tissue

As Fig. 4 shows, RBP4 mRNA expression in liver inGroup H was significantly increased compared to thatin other groups (P < 0.05). HFABP mRNA expression
in Group H was significantly increased compared tothat in Groups C, HOH, HOG and HE after 6 weeks (P73

W. Qian et al.

LiverFig. 4.

1.8
*

1.5

†

0.9

*

RBP4

*

†

*

1.2

Adipose tissue

1.5

RBP4

†

†

†

†

†

†

†

0.6

†

1

†

†
†
†‡

†

0.5

0.3
0
1.8

C

H
*

HO
HOH
HFABP

HE

0
1.5

C

1.2

†

*

1.2

HOG

†‡

†‡

†‡

†

†‡

†

H
*

HO
HOH
HFABP

HE

†

†

†

*

HOG

†

0.9

†‡§

0.6

0.6

0.3
0.0

C

H

1.6

HO

HOG

HE

†

*

0

†

†

†

0.8

0.4

0.4

1.6

C

H

HO

*

HOH

HOG

HE

MCP1

*

†

†

1.2

†

†‡¶

†

0

1.6

0.8

0.4

0.4

C

H

1.6

HO

*

1.2

HOH

HOG

HE

†

†

0.0

†
†

†

0.8

0.8

0.4

0.4

0.0

C

H

HO

HOH

HOG

HE

0

HOG

HE

†

†

HOG

HE

*

C

H

HO
†

*

†

†

C

H

HO

*

†

H

†
†¶

HOH

†‡¶

HOG

HE

CCR2

*

C

HOH

MCP1

*

1.2

†¶

HOH

†

†

1.6

CCR2

*

HO

CFABP

1.2

0.8

0.0

H

1.2

0.8

0.0

C

1.6

CFABP

*

1.2

HOH

†

HO

†

†¶

†¶

†‡¶

HOH

HOG

HE

Fig. 4. Changes of mRNA expression with time in liver and adipose tissue.
Levels of RBP4, HFABP, CFABP, MCP1 and CCR2 against beta-actin mRNA expression are shown as above histogram. The left-side
graphs show the expression in liver. The right-side graphs show the expression in adipose tissue around the left kidney. C: control, standard diet for 6 and 12 weeks (n = 5); H: high-cholesterol diet for 6 and 12 weeks (n = 5); HO: high-cholesterol diet with ogi for 6 and 12
weeks (n = 5); HOH: high-cholesterol diet with ogi and hesperidin for 6 and 12 weeks (n = 5); HOG: high-cholesterol diet with ogi andginger for 6 and 12 weeks (n = 5); HE: high-cholesterol diet with ezetimibe for 6 and 12 weeks (n = 5). Dotted and shaded columns represent 6 and 12 weeks, respectively. *P < 0.05 vs. Group C, †P < 0.05 vs. Group H, ‡P < 0.05 vs. Group HO, §P < 0.05 vs. Group HOH, ¶P< 0.05 vs. Group HE. 6W: 6 weeks, 12W: 12 weeks.
CCR2, C-C chemokine receptor type 2; CFABP, cutaneous fatty acid-binding protein; HFABP, heart fatty acid-binding protein; MCP1,
monocyte chemoattractant protein-1; RBP4, retinol-binding protein 4.

74

Kampo components ameliorates dyslipidemia

the left kidney. The up-regulation of RBP4, HFABPand CFABP mRNA expression were found in Group
H. After treatment, they were decreased. MCP1 andCCR2 mRNA expression was not observed differences
between Group H and Group HE. However Group HOG
showed significant differences compared with othertreatment groups (P < 0.05).

staining) was found on the whole layers of thoracic aortas in Group C. Significantly positive ICAM-1 immunostaining was observed in Group H and mainly localizedon the endothelial layers. Positive ICAM-1 immunostaining was also observed in Groups HO, HOH, HOG
and HE, but less than Group H (× 400). Mean opticaldensity values of ICAM-1 are showed in Fig. 5B. The
photographs generated were quantitatively analyzed theoptical density of ICAM-1 by Image - Pro Plus. After
treatment, the expressions in Groups HO, HOH, HOG
and HE were decreased than Group H. And Group HOG

Fig. 5.
ICAM-1immunostaining
expression in aorta
As shown in Fig. 5A, after 12 weeks, seldom ICAM1immunostaining (Red-brown deposits indicate positive

AA

C

H

HO

HOH

HOG

HE

BB

0.028

*
†

†

0.021

†

†‡¶

0.014

0.007

0.000

C

H

HO

HOH

HOG

HE

Fig. 5. ICAM-1 expression in aorta after 12 weeks.
A: Immunohistochemistry analyses of ICAM-1 in aorta (The arrows indicate positive expressions). B: Mean optical density values of
ICAM-1. The optical density of ICAM-1 were quantitatively analyzed with Image - Pro Plus after 12 weeks. C: control, standard diet for
12 weeks (n = 5); H: high-cholesterol diet for 12 weeks (n = 5); HO: high-cholesterol diet with ogi for 12 weeks (n = 5); HOH: high-cholesterol diet with ogi and hesperidin for 12 weeks (n = 5); HOG: high-cholesterol diet with ogi and ginger for 12 weeks (n = 5); HE: highcholesterol diet with ezetimibe for 12 weeks (n = 5). *P < 0.05 vs. Group C, †P < 0.05 vs. Group H, ‡P < 0.05 vs. Group HO, ¶P < 0.05 vs.
Group HE. Bars express 25 μm.

75

ig. 6.

W. Qian et al.

was significantly lower compared with Groups HO andHE (P < 0.05).

ated the serum lipid and lipoprotein levels as well as
the steatotic process by biochemical and histologicalmethods, respectively.23 The results showed that fatty
liver was well developed with long-term high cholesterol diets, and hepatocytes were filled with many lipid
droplets. This process was more evident in the portal
zones, where fat hepatocytes were more numerous. It
has reported that the abnormal serum levels of HDLC and LDL-C are associated with NAFLD and CVD.24
Statins, hydroxyl-methylglutaryl-coenzyme A (HMGCoA) reductase inhibitors, are one kind of lipid-loweringdrugs, which can inhibit cholesterol synthesis. They canwell decrease LDL-C and increase HDL-C. However,they sometimes caused rhabdomyolysis and hepatitis.
Kampo medicines are considered as a safe way to harmonize the abnormal serum levels of HDL-C and LDLC and to minimize the side effects. We have alreadyproved that BOT can improve blood chemistry and cholesterol concentrations.18 In this experiment, we foundthat ogi, ogi with hesperidin, ogi with ginger and ezetimibe could decrease the concentrations of LDL-C and
TC and increase HDL-C. ALT and ALP activities (after
6 weeks) in Group HE were significantly elevated than
kampo groups. The reason may be kampo medicines
have fewer side effects than ezetimibe. The marked
elevation of ALT and ALP activities in Group H wereconsistent with our previous study.18 However, the ALT
and ALP activities in Group HE were decreased after 12
weeks, which was probably due to the individual differences in the rats.
Lipid-soluble molecules share several aspects ofphysiology due to common adaptations that allow themto function in a hydrophilic environment. RBP4, thetransport protein for vitamin A (retinol) in blood, plays
the role during hypercholesterolemia through binding to
specific nuclear receptors like the peroxisome proliferator-activated receptor (PPAR) and the retinoid X receptor(RXR).25 PPAR could directly regulate RBP4 expression, at least in certain tissues.26 On the other hand, theincrease of RBP4 levels may potentially enhance retinoid
availability for RXR-PPAR action, by promoting retinoltransport to the periphery of the body.27 RBP4 has also
been identified as an adipokine, which is involved in
the modulation of glucose metabolism.28 Graham et al29
reported that increased levels of RBP4 were associatedwith obesity and hypercholesterolemia. Thus, the measurement of serum or plasma RBP4 levels is a helpful
way for revealing metabolic disorders. In our experiment, we also found the increased trend of RBP4 inserum as well as mRNA expression of liver and adipose
with high cholesterol diet rats. Ogi, ogi with hesperidin,
ogi with ginger and ezetimibe could decrease the expres-

TNF-alpha western blotting expression in liver

After 12weeks, the TNF-alpha protein levels in liverof Groups HO, HOH, HOG and HE were significantly
inhibited compared with that of Group H (Fig. 6A, P< 0.05). It has less expression of TNF-alpha protein inGroup HOG than Group HO and HE (Fig. 6B, P < 0.05).
Blood pressure and heart rate

No significant changes in systolic or diastolic bloodpressure and heart rates were observed during the experiment (data not shown).

AA
TNF-alphaBeta-actin
C

BB

2.0

H

HO

HOH

HOG

HE

*

1.5

†

†

1.0

†

†‡¶

HOH

HOG

0.5
0.0

C

H

HO

HE

Fig. 6. TNF-alpha expression in liver.
A: Western blot analysis of TNF-alpha expression in liver. B: The
TNF-alpha expression levels were quantitatively analyzed with
Gel-Pro Analyzer 4. C: control, standard diet for 12 weeks (n = 5);
H: high-cholesterol diet for 12 weeks (n = 5); HO: high-cholesterol
diet with ogi for 12 weeks (n = 5); HOH: high-cholesterol diet withogi and hesperidin for 12 weeks (n = 5); HOG: high-cholesterol
diet with ogi and ginger for 12 weeks (n = 5); HE: high-cholesterol
diet with ezetimibe for 12 weeks (n = 5). *P < 0.05 vs. Group C,†P < 0.05 vs. Group H, ‡P < 0.05 vs. Group HO, ¶P < 0.05 vs.
Group HE.

DISCUSSION
A high cholesterol diet can lead to high lipid levels andestablish fatty liver model in rats. Del Moral ML et aladministered a high cholesterol diet in rats, and evalu76

Kampo components ameliorates dyslipidemia

sions of the up-regulated RBP4, suggesting their specificeffects in alleviating hypercholesterolemia to be partly
induced through a lowering of the RBP4 expressions.
HFABP is a 15 kDa small protein consisting of 132
amino acids.33 It is rich in the myocardium34 and produced by skeletal muscle,35 cardiomyocytes, kidney distal tubular cells,36 and specific parts of the brain.37 And
CFABP is abundant in the psoriatic skin.38 HFABP and
CFABP are thought to play key roles in fatty acid metabolism, like fatty acid storage and transport.38 FABPs
are also expressed in mononuclear cells (predominantly
monocytes and macrophages).30 FABPs mRNA expressions have been reported to increase in abdominal
white adipose tissue in rats fed a high cholesterol dietfor 30 days.39 They were reported to be up-regulated inliver and adipose tissue of rats fed high-cholesterol dietfor 12 weeks. Our previous study proved that HFABPand CFABP maintained high expression levels for 12
weeks.11, 18 Here, this phenomenon was also demonstrated. HFABP and CFABP mRNA expressions in the liver
and adipose tissue of high-cholesterol diet group wereincreased compared to Group C while decreased after
treatment.
Intestinal cholesterol absorption is a kind of active
and selective process, which mediated by several transporter proteins located at the intestinal brush border
membrane. Cholesterol and plant sterols are taken up
by the enterocyte through the Niemann-Pick C1 Like 1
transporter.40 Moreover, the ATP-binding cassette (ABC)transporters G5 and G8 actively induced plant sterols efflux and, to lesser extent cholesterol, back into the intestinal lumen. In addition, ABCG5 and ABCG8 are located at the canalicular membranes of hepatocytes, where
they facilitate efflux of cholesterol and plant sterols into
bile.41 Animal cell membranes normally contain only asingle major class of sterol, cholesterol.42 However, plant
cell membranes typically contain a complex mixtureof sterols, such as lathosterol, campesterol and betasitosterol,42, 43 which have similar structure with cholesterol. The serum lathosterol concentration is an indicatorof whole-body cholesterol synthesis and can reveal theexpression of cholesterol synthesis.44 However, campesterol and beta-sitosterol are known to reduce blood cholesterol levels, and indicated cholesterol absorption.45–47
It is reported that high cholesterol absorption and lowcholesterol synthesis are associated with increased severity of coronary artery disease.48 However, in an experiment, which takes plasma non-cholesterol sterols as
a diagnostic tool, Noto et al also found increases in bothcholesterol synthesis and absorption in pediatric hypercholesterolemia as we found in the high-cholesterol dietgroup.49 In this experiment, concentrations of these threebiomarkers were increased in Group H, while decreased
after treatment, consistent with our previous research, inwhich concentrations of lathosterol, campesterol and beta-sitosterol were simultaneously increased in high cholesterol diet rats.11 Brahma Naidu P et al also proved thatsynthesis of cholesterol was increased in high fat dietrats, which was probably due to the increase HMG CoA
reductase in liver.50 So, we have demonstrated that ogi,
ogi with hesperidin, and ogi with ginger have a similareffect with BOT and ezetimibe on hypercholesterolemia
and fatty liver. These kampo components can efficiently
reduce cholesterol concentrations by inhibiting both absorption and synthesis by adjusting mRNA or enzymes
associated with cholesterol absorption or synthesis.
A lot of evidences showed that inflammatory factors could take part in the occurrence and developmentof hypercholesterolemia, NAFLD and atherosclerosis.6
MCP1 and its receptor CCR2 are two valuable inflammation related cytokines associated with NAFLD andatherosclerosis.51 They can be engendered by vascular endothelial cells, monocytes and vascular smoothmuscle cells.52 It was reported that they would increaseby hypercholesterolemia and obesity.53, 54 TNF-alpha,participated in many inflammatory diseases, was also
up-regulated on atherosclerosis.55 In our experiment,
kampo treatment groups could down-regulate the MCP1
and CCR2 mRNA expression in both liver and adipose
tissue. Ezetimibe showed a reduced trend however
not significantly different. Ogi showed a stronger antiinflammatory effect when combined with ginger thanother treatment groups. A similar effect was revealedon the protein expression of TNF-alpha in liver. GroupHOG had advantage on anti-inflammatory effect during
the whole experiment.
ICAM-1, one of the markers of endothelial cell
activation, is expressed in endothelial cells, which has
participated in neutrophil as well as monocyte migration, migration and adhesion of endothelial cells.56 Somestudies proved that the concentration of serum ICAM-1
correlated with the expression of ICAM-1 in the media
and the vasa vasorum.57 Therefore, it is a reliable measure using the concentration of serum ICAM-1 to detect
the expression of the molecule in the tissue of the aortic
wall. It was reported that ICAM-1 was up-regulated
after vascular injury in neointimal and medial smoothmuscle cells.58 And experiment evidences also showedthat the expressions of both serum ICAM-1 and tissue
ICAM-1 were increased in the early stages of atherosclerosis rats.57 In our experiment, both the concentrationof serum ICAM-1 and expression of ICAM-1 in aorta
were increased in Group H. Kampo components andezetimibe could down-regulate the high expression with77

W. Qian et al.

high-cholesterol diet. Ogi combined with ginger still
showed advantage both in serum and aorta. We speculate that down-regulated ICAM-1 may be one of themechanisms to prevent atherosclerosis of ogi, ogi withhesperidin and ezetimibe, especially ogi combined withginger.
Our previous study proved that BOT has a protective
effect on the progression of hypercholesterolemia andfatty liver induced by high-cholesterol diet in rats. BOT
is composed of ogi (Astragalus membranaceus Bunge),boi (Sinomenium acutum Rehder et Wilson), sojyutsu(Atractylodes lancea De Candolle), ginger (Zingiber officinale Roscoe), taiso (Zizyphus jujuba Miller var. inermis Rehder) and kanzo (Glycyrrhiza uralensis Fischer).
Ogi and ginger are the key components of BOT. Wang
D et al. reported that ogi could significantly decrease
concentrations of TC and LDL-C and improved the atherosclerosis profile.59 Matsuda A et al. also found thatginger tended to reduce RBP4 mRNA expression levelsin the liver and visceral fat in hyperlipidemia.20 Wang
X et al. revealed that hesperidin could improve hypercholesterolemia and fatty liver by inhibiting both thesynthesis and absorption of cholesterol.11 However few
studies have assessed the mechanism of ogi on hypercholesterolemia atherosclerosis as well as the synergistic
action between ogi and hesperidin or ginger.
In the present findings, three kampo component
groups showed a similar effect with BOT and ezetimibe
on hypercholesterolemia and fatty liver. These kampo
components could efficiently reduce cholesterol concentrations by inhibiting both absorption and synthesis. The
synergistic action of ogi with ginger showed stronger effect but had no significant difference. On the other hand,ogi combined with ginger revealed the most powerfulanti-arteriosclerotic effect than other kampo components
groups as well as ezetimibe group. According to ogi andginger are the key components of BOT and the results
above, we speculate that ogi has the similar effects with
BOT both on the progression of hypercholesterolemia
and arteriosclerosis, while ginger maybe helpful for other components of BOT especially on anti-inflammation
and anti-arteriosclerosis.

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Acknowledgments: We would like to express our sincere appreciation for the fellowship and research grant from the Japan
Research Foundation of Clinical Pharmacology (JRFCP). And
we thank for Dr. K. Nagata’s assistance for LC/MS/MS studies.
The authors declare no conflict of interest.

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