Journal of Functional Foods 45 (2018) 146–154

Contents lists available at ScienceDirect

Journal of Functional Foods

journal homepage: www.elsevier.com/locate/jff

Effect and interactions of Pueraria-Rehmannia and aerobic exercise on metabolic T

inflexibility and insulin resistance in ovariectomized rats fed with a high-fat diet
You Jin Kima,1, Hye Jin Kimb,1, Hyang Mok Oka, Hye Yun Jeonga, Won Jun Leeb, Connie Weaverc,
⁎
Oran Kwona,
a
Department of Nutritional Science and Food Management, Ewha Womans University, Seoul 03760, Republic of Korea
b
Department of Kinesiology and Sports Studies, Ewha Womans University, Seoul 03760, Republic of Korea
c
Department of Foods and Nutrition, Purdue University, West Lafayette, IN 47907, USA

A R T I C LE I N FO A B S T R A C T

Keywords: Due to limitations of estrogen replacement therapy, life style interventions have received a growing attention. This study
Aerobic exercise aimed to determine whether Pueraria-Rehmannia (PR) and/or aerobic exercise (Ex) could reduce cardiometabolic dys-
Foxglove function in an ovariectomized/high-fat diet rat model. PR ameliorated circulating levels of total cholesterol, LDL/HDL
Kudzu puerarin ratio, and leptin. It reduced fat mass, fat size, leptin gene expression, and macrophage infiltration in adipocytes. PR
Metabolic inflexibility
maintained leptin receptor gene expression in muscle tissues. It stimulated insulin-mediated GLUT4 translocation in
Insulin resistance
Ovariectomized rats
muscle and adipose tissues. PR plus Ex resulted in further improvement in glucose homeostasis by stimulating Akt-
mediated insulin receptor expression in the liver. Moreover, PR and/or Ex reduced vascular wall thickness and in-
tracellular cell adhesion molecule-1 production without causing uterotrophic effect. These results suggest that PR and Ex
may exert synergistic effects in modifying metabolic dysfunctions and insulin resistance, thus suppressing cardiovascular
risks in postmenopausal women through restoring insulin sensitivity and attenuating inflammation.

1. Introduction also known as an important source of puerarin, an isoflavone compound with

A decline in the level of circulating estrogen produces a wide array of
metabolic defects. It places postmenopausal women at greater risk of cardi-
ovascular disease (CVD), type 2 diabetes mellitus, and osteoporosis. Although
estrogen replacement therapy provides protective effects against these dis-
eases, the risk of oncogenicity and adverse outcomes associated with such
therapy limit its wide application in postmenopausal women (Lacey et al.,
2002). Alternatively, life style interventions such as diet and exercise with
little or no adverse effects have received a growing attention in recent years.
The most significant body of evidence about the effect of foods on post-
menopausal syndromes comes from genistein and daidzein (D'Anna et al.,
2007; Poluzzi et al., 2014), the two major phytoestrogens in soy. However,
convincing evidence is still lacking for other plant-derived dietary compo-
nents. Exercise also has emerged as an effective treatment for menopausal
women. However, interactions between diet and exercise on postmenopausal
syndromes are poorly understood.
The rhizome of Pueraria lobata is one of the most popular traditional foods
as well as folk medicine in Asian countries (Zhang, Lam, & Zuo, 2013). It is
weaker estrogenic activity compared to genistein or daidzein (Keung &
Vallee, 1998). The rhizome of Rehmannia glutinosa containing catalpol has
been used to treat diabetes in traditional Korean medicine (Kim, Jeong, &
Kim, 2016). Traditionally, kudzu has been mixed with foxglove to have
complementary effects. Our recent study has showed that a combination
supplement containing Pueraria-Rehmannia (PR) and/or aerobic exercise
(Ex) can mitigate the increase in bone turnover rate and induce favorable
architectural modification of trabecular bone through blockade of receptor
activator of nuclear factor-kappa B signaling in ovariectomized (OVX) rats
(Ok et al., 2015). Accumulating evidence has indicated that core components
of cardiometabolic syndrome are also risk factors for low bone mineral
density (BMD), suggesting the presence of shared common pathophysiolo-
gical mechanisms between CVD and BMD (McFarlane, 2006). Thus, we ex-
tended our previous experimental protocol on BMD in OVX rats to include
metabolic dysfunction and insulin resistance.
The objective of the present study was to determine whether PR
and/or Ex might have cardiometabolic health effects on OVX rats fed
with a high-fat diet (HFD). Comprehensive physiological profiling was

Abbreviations: Ex, aerobic exercise; GLUT4, glucose transporter 4; HFD, high-fat diet; HDL, high-density lipoprotein; HOMA-IR, homeostasis model assessment-insulin resistance; ICAM-
1, intracellular cell adhesion molecule-1; LDL, low-density lipoprotein; OVX, ovariectomized; PR, a combination supplement containing kudzu root (Pueraria lobata) plus foxglove
(Rehmannia glutinosa); SC, sham-control; TC, total cholesterol; TG, triglycerides
⁎
Corresponding authors at: Department of Nutritional Science and Food Management, Ewha Womans University, Seoul 03760, Republic of Korea.
E-mail address: orank@ewha.ac.kr (O. Kwon).
1
Both authors contributed equally.

https://doi.org/10.1016/j.jff.2018.04.006
Received 22 August 2017; Received in revised form 20 March 2018; Accepted 5 April 2018
Available online 10 April 2018
1756-4646/ © 2018 Elsevier Ltd. All rights reserved.
Y.J. Kim et al. Journal of Functional Foods 45 (2018) 146–154

Fig. 1. UPLC-Q-TOF/MS base peak intensity chromatograms of Pueraria lobata and Rehmannia glutinosa mixture extract in positive ion mode. Numbers for compounds
are the same as indicated in Supplementary Table 1. Metabolite classes shown here are: 1, Puerarin-O-glucoside; 2, 3′-Hydroxypuerarin; 3, 6″-O-Xylosylpuerarin; 4,
Puerarin; 5, 3′-Methoxypuerarin; 6, Daidzin; 7, Genistein 8-C-Apiosyl(1–6)-glucoside; 8, Puerarin pentose I; 9, Puerarin isomer; 10, Formononetin-8-C-Apiosyl(1–6)-
glucoside; 11, 4′-Methoxypuerarin; 12, 6″-O-Acetyl daidzin; 13, Puerarin pentose II; 14, Ononin; 15, Daidzein; 16, Biochanin A; 17, 5′-Hydroxyl oninin; 18, Genistein;
19, Formononetin; 20, Dihydroxy-prenylisoflavone; 21, Puerarone; 22, D-(+)-Raffinose; 23, Disaccharide derivatives; 24, Catalpol; 25, Kudzusaponin SA1; 26,
Saponin derivatives; 27, Soyasaponin I; 28, Saponin derivatives; 29, Pueroside A; 30, Pueroside B; 31, Sophoraside A or isomer; 32, Puerol B; 33–42, Unknown.

performed to identify the effect and interactions of PR and Ex on OVX
rats fed with HFD. It was also used to provide mechanistic insights into
the regulation of metabolic flexibility and insulin sensitivity in the liver,
muscle, periovular adipose tissues, and blood vessels.
2. Materials and methods
range of 150–1000 m/z. Tandem MS analysis was conducted with scan-type
turbo data-dependent scanning (DDS) under the same conditions used for MS
scanning. Metabolites were identified using standard compounds. When
standard compounds were unavailable, a tentative identification was made
bas on MS spectra using Combined Chemical Dictionary version 7.2
(Chapman and Hall/CRC), references, and an in-house library.

2.1. Test materials

PR containing aqueous extract from rhizomes of Pueraria lobata and
Rehmannia glutinosa was kindly provided by Neumed Co. (Seoul, Korea). The
preparation of PR has been described previously (Ok et al., 2015).
2.2. Chemical analysis of PR
Signature components of PR were extracted as described previously (Lee
et al., 2017). They were analyzed using an ultra-performance liquid chro-
matography-quadrupole-time of flight-mass spectrometry (UPLC/Q-TOF-MS)
system consisting of an ACQUITY UPLC SYSTEM (Waters, Milford, MA,
USA), a Q-TOF Premier MS (Waters Micromass Technologies, Manchester,
UK), a UV detector, and an ACQUITY UPLC BEH C18 column
(100 mm × 2.1 mm, 1.7 μm). Mobile phases consisted of the following: (A)
0.1% formic acid in water, and (B) acetonitrile containing 0.1% formic acid.
Elution condition was optimized as follows: 5% B (0–1 min), 5–100% B
(1–10 min), 100% B (10–11 min), 100–5% B (11–13 min), and 5% B
(13–14 min) at 0.3 mL/min. MS analysis was carried out in the range of
100–1000 m/z in both negative and positive ionization modes with capillary
voltage of 2.8 kV, cone voltage ≤40 V, desolvation gas flow rate of 700 L/h
at 200 °C, and ion source temperature of 100 °C.
PR was also analyzed by LC MS/MS using LTQ XL linear ion trap mass
spectrometer (Thermo Fischer Scientific, San Jose, CA, USA) coupled with a
diode array detector (200–600 nm) (Dionex Corporation, Sunnyvale, CA,
USA) and a Syncronis C18 UHPLC column (100 mm × 2.1 mm, 1.7 μm,
Thermo Fisher Scientific). The same mobile phases were used with the fol-
lowing elution condition: 10–100% B (0–15 min), 100% B (15–18 min), and
100–10% B (18–22 min) at 0.3 mL/min. Column temperature was main-
tained at 35 °C. An electrospray ionization (ESI) was used at capillary voltage
of 45 V, capillary temperature of 275 °C, source voltage of ± 5 kV, and mass
2.3. Experimental protocol
The experimental protocol was approved by the Institutional Animal Care
and Use Committee (IACUC) of Ewha Womans University. Female Sprague-
Dawley rats were OVX at age 8 weeks. After one-week of recovery, rats were
randomly divided into six groups and maintained on a HFD (D12451,
Research Diet; New Brunswick, NJ, USA) together with vehicle (negative
control), PR, or 17β-estradiol (positive control) in the absence of aerobic
exercise (HC, PR, and ES) or in the presence of aerobic exercise (Ex, PR-Ex,
and ES-Ex) for 8 weeks (n = 10 per group). One additional negative sham-
control (SC) group was fed with a normal diet (ND12450B, Research Diet)
without exercise. Rats in the PR and PR-Ex groups were orally gavaged once
daily with PR at 400 mg/kg body weight in PBS solution. Rats in the ES and
ES-Ex groups received oral administration of 17β-estradiol at 0.6 mg/kg body
weight daily. Rats in SC, HC, and Ex groups were gavaged with PBS only.
Exercise groups performed regular exercise using a treadmill (Panlab,
Barcelona, Spain) five times per week for the entire experimental period,
starting at 10o’clock in the morning. From the first to the 4th week, animals
ran at 15 m/min with 0° of inclination for 30 min per day. From the 5th to
the 8th week, running was progressed to 18 m/min with 0° of inclination for
40 min per day. All training rats were restrained from training 24 h before
sacrifice.
After rats were suffocated with CO2, blood samples were obtained and
centrifuged at 2,000 × g for 10 min at room temperature. The plasma was
then collected and stored in a freezer at −80 °C until use. Liver, muscle,
periovarian fat, aorta, and uterine were removed, weighed, and snap-frozen
in a freezer at −70 °C until use. Alternatively, they were fixed and preserved
in 4% formalin solution (Sigma, St. Louis, MO, USA) for histological analysis.

147
Y.J. Kim et al. Journal of Functional Foods 45 (2018) 146–154

(A) *
(B) *
250
* * 15 * *

Periovular fat mass (g)

Body weight gain (g)

200
10
150

100
5

50

0 0

x
C
x

Ex

PR
C
x
C

R
C

-E
E

-E

H
S

P
H

R
R

P
P
(C) SC HC Ex

PR PR-Ex 0.8
*
* *
0.6
Size of fat cell

0.4

0.2

0.0
C

Ex

x
SC

PR

-E
H

PR

Fig. 2. (A) Body weight gain, (B) Fat mass, and (C) Fat cell size in OVX/HFD rats at the end of a 8-week period of receiving PR and/or Ex. Values are means ± SEMs
(n = 10 per group). OVX, ovariectomized; HFD, high-fat diet; PR, a combination supplement containing Pueraria and Rehmannia; Ex, aerobic exercise; SC, sham-
operation with a low-fat diet; HC, OVX + HFD; PR, OVX + HFD + PR; Ex, OVX + HFD + Ex; PR-Ex, OVX + HFD+PR + Ex. *p < 0.05 versus HC, analysis of
variance with Dunnett’s post hoc test.

2.4. Biochemical assays 2.5. Quantitative real-time PCR analysis

Plasma levels were biochemically determined using various com-
mercial kits. Glucose, total cholesterol (TC), triglycerides (TG), and
HDL assay kits were obtained from Asan Pharmaceutical Co. (Seoul,
Korea). Insulin and C-peptide kits were purchased from Mercodia
(Uppsala, Sweden). Leptin kit was obtained from Abcam (Cambridge,
UK). LDL level and homeostasis model assessment-insulin resistance
(HOMA-IR) were calculated using the following formulas:
LDL=TC−HDL−TG/5,
HOMO-IR=[fasting plasma glucose (mmol/L)
× fasting plasma insulin (μU/mL)]/22.5.
Total RNAs were extracted from the liver, muscle, and periovarian
fat using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA). RNA con-
centration and quality were measured at 260/280 nm using BioSpec-
nano (Shimadzu, Tokyo, Japan). A High Capacity RNA-to-cDNA kit
(Applied Biosystems, Foster City, CA, USA) was used for cDNA synth-
esis. Real-time quantitative PCR was performed in Step-One-Plus
system (Applied Biosystems). PCR was performed using TaqMan
(Applied Biosystems) to quantify expression levels of leptin [Lep;
Rn00565158_m1] and β-actin [Actb; Rn00667869_m1]. SYBR Green
master mix (Kapa Biosystems, Wilmington, MA, USA) was used for
leptin receptor (LepR) [(F) 5′-CCAGTACCCAGAGCCAAAGT-3′, (R)
5′-GGGCTTCACAACAAGCATGG-3′] and GAPDH [(F) 5′- TGCACCACC
AACTGCTTA-3′, (R) 5′-GGATGCAGGGATGATGTTC-3′].

148
Y.J. Kim et al. Journal of Functional Foods 45 (2018) 146–154

Table 1
Effect of PR and/or Ex on plasma parameters related to lipid and carbohydrate metabolism in OVX rats fed with a HFD.a
Parameters SC HC Ex PR PR-Ex p-Valueb

TG (mg/dL) 147.3 ± 5.6 141.8 ± 5.8 141.6 ± 5.3 155.3 ± 19.3 125.6 ± 4.6 0.335
TC (mg/dL) 125.1 ± 4.6 123.1 ± 2.8 110.9 ± 4.6 87.1 ± 3.5*** 81.9 ± 3.6*** < 0.0001
LDL (mg/dL) 30.6 ± 3.9 37.2 ± 4.9 27.8 ± 3.3 12.2 ± 5.1*** 12.0 ± 1.7*** < 0.0001
HDL (mg/dL) 64.3 ± 1.6 57.5 ± 4.8 54.2 ± 2.0 43.8 ± 2.4** 44.8 ± 2.8* < 0.0001
LDL/HDL ratio 0.5 ± 0.1 0.9 ± 0.3 0.5 ± 0.1 0.3 ± 0.1* 0.3 ± 0.0** 0.006
FBG (mg/dL) 8.8 ± 0.5** 12.8 ± 0.9 14.4 ± 0.7 11.5 ± 0.8 10.4 ± 0.8 < 0.0001
Insulin (μg/L) 1.8 ± 0.1 2.3 ± 0.2 2.1 ± 0.1 2.0 ± 0.3 1.5 ± 0.1* 0.034
C-peptide (nM/L) 1.0 ± 0.1* 1.6 ± 0.1 1.4 ± 0.1 1.2 ± 0.1 0.9 ± 0.0** < 0.0001
HOMA-IR 5.5 ± 0.3** 8.2 ± 0.7 7.9 ± 0.5 6.3 ± 0.9 5.1 ± 0.5** 0.001
Leptin (ng/mL) 4.8 ± 0.6** 14.8 ± 1.9 12.6 ± 2.8 6.6 ± 1.5** 3.6 ± 0.3** < 0.0001

a
Data are presented as means ± SEMs. PR, a combination supplement containing Pueraria and Rehmannia; Ex, aerobic exercise; OVX, ovariectomized; HFD,
high-fat diet; SC, sham-operation with a low-fat diet; HC, OVX + HFD; PR, OVX + HFD + PR; Ex, OVX + HC + Ex; PR-Ex, OVX + HFD + PR + Ex; TG, triglycerides;
TC, total cholesterol, FBG, fasting blood glucose; HOMA-IR, homeostasis model assessment-estimated insulin resistance. *p < 0.05, **p < 0.01, and ***p < 0.0001
versus HC, analysis of variance (ANOVA) with Dunnett’s post hoc test.
b
P-values refer to comparison among the five groups by ANOVA.

2.6. Western blot analysis
Liver, muscle, and periovarian fat tissues were homogenized in
PRO-PREP protein extraction solution (iNtRON Biotechnology, Seoul,
Korea). Equal amounts of protein were resolved on SDS-PAGE gels and
then transferred to polyvinylidene difluoride (PVDF) membranes (Bio-
Rad Laboratories, Richmond, CA, USA). Membranes were blocked with
5% skim milk in 0.1% Tween-20 in TBS and probed with antibodies
against insulin receptor (IR) (Abcam), AKT, p-AKT(ser473), and β-actin
(Santa Cruz Biotechnology, Santa Cruz, CA, USA). These membranes
were then incubated with anti-rabbit or anti-mouse IgG horse-radish
peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology)
and then visualized with West One Western Blot Detection System
(iNtRON Biotechnology). Band intensities were determined with
ChemiDoc XRS System image reader followed by analysis using
Quantity One software (Bio-Rad Laboratories).
2.7. Histology and immunohistochemistry
Formalin-fixed and paraffin-embedded sections of liver, muscle,
periovarian fat tissues, aorta, and uterus were sliced at thickness of 5 μm.
Paraffin sections of periovarian fat tissues, aorta, and uterus were de-
paraffinized and stained with hematoxylin and eosin (H&E). For im-
munohistochemistry analysis, paraffin sections of muscle and perio-
varian fat were deparaffinized and probed with primary antibodies of
glucose transporter 4 (GLUT4) or F4/80 primary antibody (Abcam).
Sections were then washed thrice with 10 mM phosphate-buffered saline
containing 0.05% Tween 20 (PBST, pH 7.4) and incubated with a bio-
tinylated secondary antibody (Alexa 488-conjugated goat anti-rabbit IgG,
Invitrogen) targeting corresponding primary antibody. The complex was
then visualized using an Imaging System (Nikon, Tokyo, Japan).
Transverse segments of aorta were mounted onto cryostat chucks in
OCT embedding medium (Sakura Finetek, Torrance, CA, USA), snap frozen,
and cut with a cryostat microtome (Leica Microsystems, Wetzlar, Germany)
at −20 °C. Sections were incubated overnight with primary antibody di-
rected against intracellular cell adhesion molecule-1 (ICAM-1; Santa Cruz
Biotechnology) followed by incubation with fluorescein isothiocyanate-la-
beled secondary antibody. Sections were washed and mounted with fluor-
escent mounting medium (Dao Corporation, Carpinteria, CA, USA) for
analysis using a TX100 transmission fluorescence microscope (Nikon).
2.8. Statistical analysis
The normality of the data was assessed by Shapiro-Wilk test. Results
are presented as means ± standard error of means (SEMs). One-way
analysis variance (ANOVA) with Dunnett’s multiple comparison test
was performed to compare treatments (PR, Ex, and PR-Ex) with HC
group. Additionally, ANOVA with Tuckey multiple comparison test was
applied for individual between-group comparisons including positive
controls (Es and Es-Ex). All statistical analyses were performed using
Statistical Analysis Systems software, version 9.4 (SAS Institute, Cary,
NC, USA). Statistical significance was considered at p < 0.05.
3. Results
3.1. Signature components of PR
A total of 42 signature components were detected (Fig. 1 and Supporting
Information Table 1) using UPLC-Q-TOF-MS and UHPLC-LTQ-IT-MS/MS.
There were 21 isoflavones, 3 iridoid glycoside and saccharides, 4 triterpene
saponins, 4 lignans, and 10 non-identified components. Among these com-
ponents, puerarin and catalpol are the most well-known index materials for
quality control of rhizomes of Pueraria lobata (Miao et al., 2013; Peng et al.,
2011; Yan et al., 2013) and Rehmannia glutinosa (Tao et al., 2016), respec-
tively. Therefore, amounts of puerarin and catalpol were quantitatively de-
termined for standardization of PR. Results showed 49.1–73.7 mg/g of
puerarin and 1.6–9.8 mg/g of catalpol.
3.2. PR and/or Ex attenuates weight gain and reduces adipocyte size in
OVX/HFD rats
Significantly increased body weight gain (p < 0.0001) and periovular fat
mass (p = 0.042) were recorded in the HC group after 8-week of treatment
compared to those in the SC group. However, groups receiving PR or PR-Ex
had significantly lower body weight gain compared to the HC group (both
p < 0.0001), whereas Ex did not have a significant effect on weight gain by
itself (Fig. 2A). Consistent with this result, periovular fat mass was sig-
nificantly lower in PR or PR-Ex groups compared to that in the HC group
(both p < 0.0001). However, periovular fat mass was not significantly lower
in the Ex group compared to that in the HC group (Fig. 2B). Histological
analyses also revealed that adipocyte sizes were significantly increased in
OVX rats by HFD feeding (p = 0.002). However, they were reduced re-
markably in PR (p = 0.003) and PR-Ex (p = 0.001) group compared to those
in the HC group (Fig. 2C).
3.3. PR and/or Ex modulates plasma metabolic parameters in OVX/HFD rats
As shown in Table 1, OVX/HFD induced significantly higher levels of
leptin, fasting blood glucose (FBG), C-peptide (p < 0.0001 for all), and
HOMA-IR (p = 0.001) compared to SC, indicating that OVX/HFD could in-
duce metabolic dysfunction. Post-hoc analysis revealed that PR significantly
reduced TC (p < 0.0001), LDL (p < 0.0001), HDL (p = 0.007), LDL/HDL
(p = 0.020), and leptin (p = 0.004) levels compared to HC. PR-Ex showed
higher potency than PR. Levels of TC (p < 0.0001), LDL (p < 0.0001), HDL

149
Y.J. Kim et al. Journal of Functional Foods 45 (2018) 146–154

Fat tissue Liver Muscle

* *
3 * * * 8 * *
mRNA expression

mRNA expression
6

Leptin receptor
2
Leptin

1
2

0 0

x
C

R
C

x
HC

Ex
SC

PR

E
-E

-E
S

P
H

H
-E

R
PR

Fig. 3. Leptin/insulin sensitivity was determined in OVX/HFD rats at the end of a 8-week period of receiving PR and/or Ex.: (A) mRNA expression levels of leptin and
LepR in adipose tissue, liver, and skeletal muscle, (B) Immunohistochemical staining of GLUT4 in muscle and fat cells, and (C) Western blotting of insulin receptor
and AKT phosphorylation in liver and muscle. The second lane in the gel marked as “x” did not correspond to any group in this study. Values are means ± SEMs
(n = 10 per group). OVX, ovariectomized; HFD, high-fat diet; PR, a combination supplement containing Pueraria and Rehmannia; Ex, aerobic exercise; SC, sham-
operation with a low-fat diet; HC, OVX + HFD; PR, OVX + HFD + PR; Ex, OVX + HFD + Ex; PR-Ex, OVX + HFD + PR + Ex. *p < 0.05 versus HC, analysis of
variance with Dunnett’s post hoc test.

150
Y.J. Kim et al.
Fig. 3. (continued)
(p = 0.013), LDL/HDL (p = 0.003), leptin (p < 0.0001), insulin
(p = 0.012), C-peptide (p < 0.0001), and HOMA-IR (p = 0.002) in PR-Ex
were significantly lower than those in HC group. However, Ex did not affect
plasma parameters.
3.4. PR and/or Ex reduces leptin and insulin resistance in muscle, adipose
tissue, and liver of OVX/HFD rats
To further investigate the process involved in fat mass reduction and
metabolic restoration, leptin mRNA expression in adipose tissue and
LepR mRNA expression in muscle and liver were determined. For leptin
mRNA expression, OVX/HFD-induced increase (p = 0.007) was de-
creased by Ex (p = 0.008), PR (p = 0.001), and PR-Ex (p = 0.005) to
almost normal level. For LepR mRNA expression, neither an increase in
the HC group nor a decrease in the PR and PR-Ex group was statistically
significant in the liver. Only Ex showed a significant decrease compared
to HC group (p = 0.039). In contrast, OVX/HFD-induced increase was
statistically significant in muscle (p = 0.003) while PR-Ex significantly
reduced its level (p = 0.014) (Fig. 3A).
The effect of PR and/or Ex on insulin resistance was then investigated
using visualized and quantitative data. GLUT4 is usually stored in in-
tracellular vesicles in the cytoplasm. However, it is translocated to the cell
membrane upon insulin stimulation (Chiang et al., 2001). Our data showed
that OVX/HFD-induced suppression of GLUT4 trafficking to the plasma
membrane in fat cells and muscle was significantly increased by PR
(both p < 0.0001), Ex (p < 0.0001 and p = 0.0015), and PR-Ex (both
p < 0.0001) compared to that in the HC group (Fig. 3B). Furthermore, IR
protein expression in the liver was significantly increased by PR (p = 0.038),
Ex (p = 0.015), and PR-Ex (p < 0.0001) compared to that in the HC group.
OVX/HFD-induced suppression (p = 0.006) of Akt phosphorylation was sig-
nificantly (p < 0.0001) increased by PR-Ex compared to that in the HC
group (Fig. 3C). On the basis of additional statistical analysis of cardiome-
tabolic biomarkers, estrogen-mimicking effect of PR and additive effect be-
tween PR and Ex were suggested (Supporting Information Table 2).
3.5. PR and/or Ex inhibits macrophage infiltration into fat tissues and
modulates aortic inflammation in OVX/HFD rats
Next, we determined whether decrease in plasma metabolic parameters
was associated with decreased inflammation. Infiltrated macrophages in
periovular fat cells were visualized after immunohistochemical staining with
F4/80. This analysis revealed that the expression of F4/80 protein was in-
creased sharply in the HC group. However, it almost completely disappeared
after PR and/or Ex treatment (Fig. 4A). Next, immunofluorescent signal of
adhesion molecule ICAM-1 in aorta was measured. A strong signal was seen
on endothelial cells in the HC group. However, it was considerably reduced in
PR and PR-Ex groups compared with that in the HC group (Fig. 4B).
151
Journal of Functional Foods 45 (2018) 146–154
3.6. PR and/or Ex shows a reduction in vascular wall thickness, but not in
uterotrophic activity, in OVX/HFD rats
Finally, changes in phenotype for vascular and uterine thickness were
determined using visualized data. Although not statistically significant, the
vascular wall was thicker in the HC group than that in all other treatment
groups investigated, including positive groups (Fig. 5A). In contrast, the
thickness of the uterine wall was significantly reduced in the HC group. PS
(p = 0.0206), PR-Ex (p = 0.0002), ES (p < 0.0001), and ES-Ex
(p < 0.0001) groups showed statistically significant increase in the thickness
of uterine wall compared to the HC group (Fig. 5B). However, the level of
increase was the most noticeable in the ES group.
4. Discussion
In the present study, we demonstrated that OVX combined with
HFD exacerbated estrogen deficiency-related metabolic inflexibility
based on biochemical, molecular, and histological data of blood, liver,
muscle, adipose tissue, blood vessel, and uterus. However, overall data
from this study supported our hypothesis that PR and/or Ex could exert
favorable effect on metabolic dysfunctions and insulin resistance, thus
suppressing the cardiovascular risks in OVX/HFD rats.
The first finding of this study was that OVX/HFD-induced increases in
body weight, periovular fat mass, fat cell size, and circulating levels of TC and
LDL/HDL ratio were differently modulated by PR, Ex, and PR-Ex. Althogh PR
was effective in attenuating these measures, Ex failed to alter plasma meta-
bolic parameters by itself. This is consistent with results of previous studies
showing that moderate-intensity aerobic exercise alone does not significantly
alter blood lipid profiles in overweight/obese postmenopausal women in a
one-year exercise trial (Mohanka et al., 2006). However, a combination of PR
with EX showed better results by additive effects on insulin secretion, acti-
vation, and resistance. OVX/HFD-induced increase in circulating insulin, C-
peptide, and HOMA-IR were restored to almost normal level by PR-Ex. These
results are in line with a previous study showing that OVX-induced dyslipi-
demia and insulin resistance can be attenuated by estrogen replacement plus
endurance exercise (Pighon, Gutkowska, Jankowski, Rabasa-Lhoret, &
Lavoie, 2011; Weigt et al., 2013).
Impaired leptin sensitivity has been reported to be a contributing factor to
excess fat accumulation caused by estrogen deficiency (Ainslie et al., 2001).
In this study, we found that there was a consistent decrease of periovular fat
mass/size of fat cell and circulating leptin in the PR-Ex and PR groups. Based
on gene expressions in various tissues, we demonstrated that the most sen-
sitive response to all treatments was observed in leptin mRNA in fat cells
while the least response was observed in LepR mRNA in muscle. Our results
also showed that OVX/HFD-induced suppression of insulin-dependent GLUT4
translocation to the plasma membrane was effectively modified by PR, Ex,
and PR-Ex in fat cells and skeletal muscle. Although the most efficient
Y.J. Kim et al. Journal of Functional Foods 45 (2018) 146–154

(A)
SC HC EX PR

PR-Ex ES ES-Ex

(B)

SC HC EX PR

PR-ExES ES-Ex

Fig. 4. Immunohistochemical analysis for (A) Macrophage infiltration of periovular fat tissue and (B) Aortic ICAM-1 in OVX/HFD rats at the end of 8-week period of receiving PR
and/or Ex. Arrows highlight the presence of macrophage infiltrated in fat tissues in panel (A) and ICAM-1 protein in panel (B). ICAM-1, intracellular cell adhesion molecule-1;
OVX, ovariectomized; HFD, high-fat diet; PR, a combination supplement containing Pueraria and Rehmannia; Ex, aerobic exercise; SC, sham-operation with a low-fat diet; HC,
OVX + HFD; PR, OVX + HFD + PR; Ex, OVX + HFD + Ex; PR-Ex, OVX + HFD + PR + Ex. *p < 0.05 versus HC, analysis of variance with Dunnett’s post hoc test.

enhancement was achieved by Ex in fat cells, GLUT4 translocation to plasma
membrane was more pronounced by PR and PR-Ex than that by Ex in muscle
fibers. We noted that, unlike in fat cells, sugar was used to produce energy in
muscle fibers. This result is supported by our previous findings showing that
PR and Ex can improve fat oxidation through inducing plasma-membrane-
associated fatty acid binding protein (FABPpm) (Kim, Yoon, Kwon, & Lee,
2016). We used soleus muscle fibers due to their higher contents of type I
muscle fibers. GLUT4 has been reported to be more highly expressed in type I
muscle fiber (Miura, Kai, Ono, & Ezaki, 2003). Furthermore, we demon-
strated that PR, Ex, and PR plus Ex were all effective in enhancing insulin
receptor protein expression in the liver. However, Aktser473 phosphorylation,
a key downstream mediator of insulin receptor signaling cascade (Dominici,
Hauck, Argentino, Bartke, & Turyn, 2002), was significantly increased only
by PR-Ex, suggesting that the additive effect between PR and Ex on further
improvement of insulin resistance might be attributed to the activation of
insulin signaling pathway through Akt phosphorylation in the liver.
Previous studies have reported that menopause-induced adipocyte
hypertrophy and hypertriglyceridemia are linked to inflammation in en-
dothelial cells and adipocytes, thus attenuating the risk of CVD (Gao et al.,
2006). Here, our results showed that OVX/HFD-induced macrophage (F4/80-
positive cells) infiltration in periovular adipose tissue almost completely
disappeared after treatment with PR, Ex, or both. This result implicates im-
provement of whole-body insulin sensitivity (Kawanishi, Yano, Yokogawa, &
Suzuki, 2010). Our results also showed that PR and PR-Ex could significantly
attenuate OVX/HFD-induced inflammation in vascular endothelium, as de-
monstrated by the reduction in ICAM-1. When results of the present study are
viewed collectively, PR might have a beneficial effect in terms of improving
inflammatory responses and metabolic abnormalities.
Our additional statistical analysis allowed between-group comparisons
including positive controls. Result confirmed several potential benefits of
using PR and/or Ex. First, we found that PR and/or Ex resembled estrogen in
their ability to lower OVX/HFD-induced insulin resistance and metabolic
abnormalities. However, unlike estrogen, PR, Ex, or PR-Ex did not exert such
a dramatic increase in uterine wall thickness, an adverse effect of estrogen

152
Y.J. Kim et al. Journal of Functional Foods 45 (2018) 146–154

(A)
SC HC EX PR

Bar; 100 m

PR-Ex ES ES-Ex
120

Aorta thickness (μm)

100

80

60

R
x
C

x
S
E

-E
-E
S

P
H

ES
P
(B)
SC HC EX PR

Thickness

Bar; 500 m

PR-Ex ES ES-Ex
1.2 *
*
*
Uterine thickness (mm)

*
*
0.8

0.4

0
x
C

x
C

S
R

E
E

-E
S

P
H

-
R

S
P

Fig. 5. Hematoxylin eosin staining for thickness measurement of (A) Vascular wall and (B) Uterus in OVX/HFD rats at the end of 8-week period of receiving PR and/
or Ex. Scale bar represents 100 μm in panel (A) and 500 μm in panel (B). OVX, ovariectomized; HFD, high-fat diet; PR, a combination supplement containing Pueraria
and Rehmannia; Ex, aerobic exercise; SC, sham-operation with a low-fat diet; HC, OVX + HFD; PR, OVX + HFD + PR; Ex, OVX + HFD + Ex; PR-Ex,
OVX + HFD + PR + Ex. *p < 0.05 versus HC, analysis of variance with Dunnett’s post hoc test.

often observed in previous studies (Han et al., 2012; Way, Keating, Baker,
Chuter, & Johnson, 2016). Second, we found an additive effect of PR plus Ex
on insulin resistance and metabolic abnormalities by direct comparison be-
tween PR group and PR-Ex group.
Using UPLC/Q-TOF-MS, we demonstrated that PR contained multiple
components including isoflavones, iridoid glycoside, triterpene saponins, and
lignans. Bioactivities of these individual components have been reported in
several independent experiments: improvement of insulin sensitivity by
puerarin (Wu et al., 2013) and genistein (Arunkumar, Karthik, & Anuradha,
2013); enhancement of GLUT4 translocation by puerarin (Zhao & Zhou,
2012), genistein (Ha et al., 2012; Wang et al., 2013; Weigt et al., 2015),
diadzein (Cheong et al., 2014), and catalpol (Shieh et al., 2011); and re-
ductions in ICAM-1 by genistein (Zhang, Zheng, Zhao, Guo, & Chen, 2013).
However, effects of PR might be attributed to synergistic effects of multiple
constituents in the preparation (Zhou et al., 2016). Therefore, it is a limitation
of the present study not exploring the mechanisms of synergistic actions from
multiple components of PR.
5. Conclusions
Our results revealed that PR could mimic estrogen based on a direct
comparison with 17β-estradiol. Although almost all tissues might be linked to
estrogen action, we focused on the cardiometabolic effect of PR in this study.
PR reduced fat mass and adipocyte size, increased leptin and insulin re-
sistance, and attenuated inflammation in OVX/HFD rats. Furthermore, ad-
ditive effect on insulin resistance was observed when PR was combined with
regular aerobic exercise, thereby regulating whole body metabolic home-
ostasis (Stern, Rutkowski, & Scherer, 2016). Results of the present study
provide a crucial basis for understanding the underlying mechanism related
to the effect and interactions of PR and regular exercise. This can be used to

153
Y.J. Kim et al.
improve cardiometabolic health of women during menopausal transition and
postmenopausal period. Future intervention studies are warranted to confirm
these findings for postmenopausal subjects. In addition, a comprehensive
systematic analysis is needed to clarify the component or ingredient and
mechanisms involved in the influence of PR on cardiometabolic health.
Conflicts of interest
The authors have no conflicts of interest related to this study to disclose.
Acknowledgments
This study was supported by the Bio-synergy Research Project
(NRF2012M3A9C4048761) through the National Research Foundation
funded by the Ministry of Science ICT and Future Planning, Republic of
Korea. It was also supported by a grant (2017R1A6A3A11034115 to YJ Kim)
of the Basic Science Research Program funded by the Ministry of Education,
Republic of Korea.
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in the
online version, at http://dx.doi.org/10.1016/j.jff.2018.04.006.
References
Ainslie, D. A., Morris, M. J., Wittert, G., Turnbull, H., Proietto, J., & Thorburn, A. W.
(2001). Estrogen deficiency causes central leptin insensitivity and increased hy-
pothalamic neuropeptide Y. International Journal of Obesity and Related Metabolic
Disorders, 25(11), 1680–1688. http://dx.doi.org/10.1038/sj.ijo.0801806.
Arunkumar, E., Karthik, D., & Anuradha, C. V. (2013). Genistein sensitizes hepatic insulin
signaling and modulates lipid regulatory genes through p70 ribosomal S6 kinase-1
inhibition in high-fat-high-fructose diet-fed mice. Pharmaceutical Biology, 51(7),
815–824. http://dx.doi.org/10.3109/13880209.2013.766896.
Cheong, S. H., Furuhashi, K., Ito, K., Nagaoka, M., Yonezawa, T., Miura, Y., & Yagasaki, K.
(2014). Daidzein promotes glucose uptake through glucose transporter 4 transloca-
tion to plasma membrane in L6 myocytes and improves glucose homeostasis in Type
2 diabetic model mice. Journal of Nutritional Biochemistry, 25(2), 136–143. http://dx.
doi.org/10.1016/j.jnutbio.2013.09.012.
Chiang, S. H., Baumann, C. A., Kanzaki, M., Thurmond, D. C., Watson, R. T., Neudauer, C. L., ...
Saltiel, A. R. (2001). Insulin-stimulated GLUT4 translocation requires the CAP-dependent
activation of TC10. Nature, 410(6831), 944–948. http://dx.doi.org/10.1038/35073608.
D'Anna, R., Cannata, M. L., Atteritano, M., Cancellieri, F., Corrado, F., Baviera, G., ...
Squadrito, F. (2007). Effects of the phytoestrogen genistein on hot flushes, en-
dometrium, and vaginal epithelium in postmenopausal women: A 1-year randomized,
double-blind, placebo-controlled study. Menopause, 14(4), 648–655. http://dx.doi.
org/10.1097/01.gme.0000248708.60698.98.
Dominici, F. P., Hauck, S., Argentino, D. P., Bartke, A., & Turyn, D. (2002). Increased insulin
sensitivity and upregulation of insulin receptor, insulin receptor substrate (IRS)-1 and IRS-2
in liver of Ames dwarf mice. Journal of Endocrinology, 173(1), 81–94.
Gao, H., Liang, M., Bergdahl, A., Hamrén, A., Lindholm, M. W., Dahlman-Wright, K., &
Nilsson, B. O. (2006). Estrogen attenuates vascular expression of inflammation as-
sociated genes and adhesion of monocytes to endothelial cells. Inflammation Research,
55(8), 349–353. http://dx.doi.org/10.1007/s00011-006-5194-z.
Ha, B. G., Nagaoka, M., Yonezawa, T., Tanabe, R., Woo, J. T., Kato, H., ... Yagasaki, K.
(2012). Regulatory mechanism for the stimulatory action of genistein on glucose
uptake in vitro and in vivo. Journal of Nutritional Biochemistry, 23(5), 501–509.
http://dx.doi.org/10.1016/j.jnutbio.2011.02.007.
Han, Y., Li, X., Zhou, S., Meng, G., Xiao, Y., Zhang, W., ... Ji, Y. (2012). 17ss-estradiol
antagonizes the down-regulation of ERalpha/NOS-3 signaling in vascular endothelial
dysfunction of female diabetic rats. PLoS One, 7(11), e50402. http://dx.doi.org/10.
1371/journal.pone.0050402.
Kawanishi, N., Yano, H., Yokogawa, Y., & Suzuki, K. (2010). Exercise training inhibits
inflammation in adipose tissue via both suppression of macrophage infiltration and
acceleration of phenotypic switching from M1 to M2 macrophages in high-fat-diet-
induced obese mice. Exerc Immunol Rev, 16, 105–118.
Keung, W. M., & Vallee, B. L. (1998). Kudzu root: An ancient Chinese source of modern
antidipsotropic agents. Phytochemistry, 47(4), 499–506.
Kim, H. J., Yoon, H. M., Kwon, O., & Lee, W. J. (2016). The effect of pueraria lobata/
rehmannia glutinosa and exercise on fatty acid transporters expression in ovar-
iectomized rats skeletal muscles. Journal of Exercise Nutrition & Biochemistry, 20(3),
32–38. http://dx.doi.org/10.20463/jenb.2016.09.20.3.5.
Kim, H. Y., Jeong, H. J., & Kim, H. M. (2016). Antidepressant-like effect of Ikwitang
involves modulation of monoaminergic systems. Molecular Medicine Reports, 13(3),
2815–2820. http://dx.doi.org/10.3892/mmr.2016.4809.
Lacey, J. V., Mink, P. J., Lubin, J. H., Sherman, M. E., Troisi, R., Hartge, P., ... Schairer, C.
(2002). Menopausal hormone replacement therapy and risk of ovarian cancer. JAMA,
288(3), 334–341.
154
Journal of Functional Foods 45 (2018) 146–154
Lee, Y. J., Ahn, Y., Kwon, O., Lee, M. Y., Lee, C. H., Lee, S., ... Kim, J. Y. (2017). Dietary
wolfberry extract modifies oxidative stress by controlling the expression of in-
flammatory mRNAs in overweight and hypercholesterolemic subjects: a randomized,
double-blind placebo-controlled trial. Journal of Agriculture and Food Chemistry,
65(2), 309–316. http://dx.doi.org/10.1021/acs.jafc.6b04701.
McFarlane, S. I. (2006). Bone metabolism and the cardiometabolic syndrome:
Pathophysiologic insights. Journal of the Cardiometabolic Syndrome, 1(1), 53–57.
Miao, W. J., Wang, Q., Bo, T., Ye, M., Qiao, X., Yang, W. Z., ... Guo, D. A. (2013). Rapid
characterization of chemical constituents and rats metabolites of the traditional Chinese
patent medicine Gegen-Qinlian-Wan by UHPLC/DAD/qTOF-MS. Journal of Pharmaceutical
and Biomedical Analysis, 72, 99–108. http://dx.doi.org/10.1016/j.jpba.2012.09.015.
Miura, S., Kai, Y., Ono, M., & Ezaki, O. (2003). Overexpression of peroxisome proliferator-
activated receptor gamma coactivator-1alpha down-regulates GLUT4 mRNA in ske-
letal muscles. Journal of Biological Chemistry, 278(33), 31385–31390. http://dx.doi.
org/10.1074/jbc.M304312200.
Mohanka, M., Irwin, M., Heckbert, S. R., Yasui, Y., Sorensen, B., Chubak, J., ... McTiernan,
A. (2006). Serum lipoproteins in overweight/obese postmenopausal women: A one-
year exercise trial. Medicine and Science in Sports and Exercise, 38(2), 231–239. http://
dx.doi.org/10.1249/01.mss.0000184584.95000.e4.
Ok, H. M., Gebreamanuel, M. R., Oh, S. A., Jeon, H., Lee, W. J., & Kwon, O. (2015). A
root-based combination supplement containing pueraria lobata and rehmannia glu-
tinosa and exercise preserve bone mass in ovariectomized rats fed a high-fat diet.
Calcified Tissue International, 97(6), 624–633. http://dx.doi.org/10.1007/s00223-
015-0057-7.
Peng, J. B., Jia, H. M., Liu, Y. T., Zhang, H. W., Dong, S., & Zou, Z. M. (2011). Qualitative
and quantitative characterization of chemical constituents in Xin-Ke-Shu prepara-
tions by liquid chromatography coupled with a LTQ Orbitrap mass spectrometer.
Journal of Pharmaceutical and Biomedical Analysis, 55(5), 984–995. http://dx.doi.org/
10.1016/j.jpba.2011.03.045.
Pighon, A., Gutkowska, J., Jankowski, M., Rabasa-Lhoret, R., & Lavoie, J. M. (2011).
Exercise training in ovariectomized rats stimulates estrogenic-like effects on expres-
sion of genes involved in lipid accumulation and subclinical inflammation in liver.
Metabolism, 60(5), 629–639. http://dx.doi.org/10.1016/j.metabol.2010.06.012.
Poluzzi, E., Piccinni, C., Raschi, E., Rampa, A., Recanatini, M., & De Ponti, F. (2014).
Phytoestrogens in postmenopause: The state of the art from a chemical, pharmaco-
logical and regulatory perspective. Current Medicinal Chemistry, 21(4), 417–436.
Shieh, J. P., Cheng, K. C., Chung, H. H., Kerh, Y. F., Yeh, C. H., & Cheng, J. T. (2011).
Plasma glucose lowering mechanisms of catalpol, an active principle from roots of
Rehmannia glutinosa, in streptozotocin-induced diabetic rats. Journal of Agriculture
and Food Chemistry, 59(8), 3747–3753. http://dx.doi.org/10.1021/jf200069t.
Stern, J. H., Rutkowski, J. M., & Scherer, P. E. (2016). Adiponectin, leptin, and fatty acids
in the maintenance of metabolic homeostasis through adipose tissue crosstalk. Cell
Metabolism, 23(5), 770–784. http://dx.doi.org/10.1016/j.cmet.2016.04.011.
Tao, J. H., Zhao, M., Wang, D. G., Yang, C., Du, L. Y., Qiu, W. Q., & Jiang, S. (2016).
Biotransformation and metabolic profile of catalpol with human intestinal microflora by
ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass
spectrometry. Journal of Chromatography B, Analytical Technologies in Biomedical Life Science,
1009–1010, 163–169. http://dx.doi.org/10.1016/j.jchromb.2015.12.007.
Wang, M., Gao, X. J., Zhao, W. W., Zhao, W. J., Jiang, C. H., Huang, F., ... Liu, K. (2013).
Opposite effects of genistein on the regulation of insulin-mediated glucose home-
ostasis in adipose tissue. British Journal of Pharmacology, 170(2), 328–340. http://dx.
doi.org/10.1111/bph.12276.
Way, K. L., Keating, S. E., Baker, M. K., Chuter, V. H., & Johnson, N. A. (2016). The Effect
of Exercise on Vascular Function and Stiffness in Type 2 Diabetes: A Systematic
Review and Meta-analysis. Current Diabetes Review, 12(4), 369–383.
Weigt, C., Hertrampf, T., Flenker, U., Hulsemann, F., Kurnaz, P., Fritzemeier, K. H., &
Diel, P. (2015). Effects of estradiol, estrogen receptor subtype-selective agonists and
genistein on glucose metabolism in leptin resistant female Zucker diabetic fatty (ZDF)
rats. Journal of Steroid Biochemistry and Molecular Biology, 154, 12–22. http://dx.doi.
org/10.1016/j.jsbmb.2015.06.002.
Weigt, C., Hertrampf, T., Kluxen, F. M., Flenker, U., Hulsemann, F., Fritzemeier, K. H., &
Diel, P. (2013). Molecular effects of ER alpha- and beta-selective agonists on reg-
ulation of energy homeostasis in obese female Wistar rats. Molecular and Cellular
Endocrinology, 377(1–2), 147–158. http://dx.doi.org/10.1016/j.mce.2013.07.007.
Wu, K., Liang, T., Duan, X., Xu, L., Zhang, K., & Li, R. (2013). Anti-diabetic effects of
puerarin, isolated from Pueraria lobata (Willd.), on streptozotocin-diabetogenic mice
through promoting insulin expression and ameliorating metabolic function. Food and
Chemistry Toxicology, 60, 341–347. http://dx.doi.org/10.1016/j.fct.2013.07.077.
Yan, Y., Chai, C. Z., Wang, D. W., Yue, X. Y., Zhu, D. N., & Yu, B. Y. (2013). HPLC-DAD-Q-
TOF-MS/MS analysis and HPLC quantitation of chemical constituents in traditional
Chinese medicinal formula Ge-Gen Decoction. Journal of Pharmaceutical and
Biomedical Analysis, 80, 192–202. http://dx.doi.org/10.1016/j.jpba.2013.03.008.
Zhang, H. P., Zheng, F. L., Zhao, J. H., Guo, D. X., & Chen, X. L. (2013). Genistein inhibits
ox-LDL-induced VCAM-1, ICAM-1 and MCP-1 expression of HUVECs through heme
oxygenase-1. Archives of Medical Research, 44(1), 13–20. http://dx.doi.org/10.1016/
j.arcmed.2012.12.001.
Zhang, Z., Lam, T. N., & Zuo, Z. (2013). Radix Puerariae: An overview of its chemistry,
pharmacology, pharmacokinetics, and clinical use. The Journal of Clinical
Pharmacology, 53(8), 787–811.
Zhao, Y., & Zhou, Y. (2012). Puerarin improve insulin resistance of adipocyte through
activating Cb1 binding protein path. Chinese Journal of Integrative Medicine, 18(4),
293–298. http://dx.doi.org/10.1007/s11655-011-012-1058-2.
Zhou, W., Wang, J., Wu, Z., Huang, C., Lu, A., & Wang, Y. (2016). Systems pharmacology
exploration of botanic drug pairs reveals the mechanism for treating different dis-
eases. Scientific Reports, 6, 36985. http://dx.doi.org/10.1038/srep36985.