Journal of Functional Foods 30 (2017) 30–38

Contents lists available at ScienceDirect

Journal of Functional Foods

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

Agastache rugosa Kuntze extract, containing the active component

rosmarinic acid, prevents atherosclerosis through up-regulation of the
cyclin-dependent kinase inhibitors p21WAF1/CIP1 and p27KIP1
Jung-Jin Lee a, Ji-Hye Lee a, Min Jung Gu a, Joo-Hui Han b, Won-Kyung Cho a,⇑, Jin Yeul Ma a,⇑
a
Korean Medicine (KM) Application Center, Korea Institute of Oriental Medicine, Daegu 701-300, Republic of Korea
b
Department of Pharmacology, Chungnam National University College of Pharmacy, Daejeon 305-764, Republic of Korea

a r t i c l e i n f o a b s t r a c t

Article history:
Received 22 May 2016
Received in revised form 12 December 2016
Accepted 16 December 2016
Chemical compounds studied in this article:
Chlorogenic acid (Pubchem CID: 1794427)
Caffeic acid (Pubchem CID: 689043)
Calycosin-7-O-b-D-glucoside (Pubchem CID:
5318267)
Rosmarinic acid (Pubchem CID: 5281792)
Calycosin (Pubchem CID: 5280448)
Genistein (Pubchem CID: 5280961)
Agastache rugosa (Fisch. & C.A. Mey.) Kuntze (A. rugosa), commonly known as Korean mint, has tradition-
ally been used for both food and medicine, depending on which part of the plant is used. We investigated
the anti-proliferative activities of A. rugosa extract in vascular smooth muscle cells (VSMCs) and analysed
several molecular pathways to determine the active component of the extract. A. rugosa extract inhibited
PDGF-BB-stimulated VSMC proliferation and DNA synthesis. A. rugosa extract caused arrest of the cell
cycle at the G0/G1 transition by up-regulating the levels of p21WAF1/CIP1 and p27KIP1. Among the compo-
nents of the A. rugosa extract reported in a previous study, rosmarinic acid (RA) only inhibited DNA syn-
thesis and dose-dependently suppressed PCNA expression. These results indicate that A. rugosa extract,
which includes the active component RA, demonstrates anti-proliferative activity by arresting the cell
cycle at the G0/G1 phase through the upregulation of p21WAF1/CIP1 and p27Kip1 expression.
Ó 2016 Elsevier Ltd. All rights reserved.

Keywords:
Anti-proliferative activity
Agastache rugosa Kuntze
Cell cycle
Cyclin-dependent kinase inhibitor
Rosmarinic acid

1. Introduction response of atherosclerosis (Faxon et al., 2004; Ross, 1993). In nor-

The abnormal proliferation of aortic vascular smooth muscle
cells (VSMCs) is a major determinant of the plaque formation
Abbreviations: AKT, phosphatidylinositol 3-kinase-linked protein kinase; A.
rugose, Agastacherugosa Kuntze; BrdU, bromodeoxyuridine; CA, caffeic acid; CCK-8,
cell counting Kit-8; CDK, cyclin-dependent kinase; CKI, cyclin-dependent kinase
inhibitor; DAD, diode array UV/VIS detector; DMEM, Dulbecco’s modified Eagle’s
medium; ERK, extracellular signal regulated kinase 1/2; FBS, fetal bovine serum;
HPLC, high-performance liquid chromatographic; JNK, c-Jun N-terminal kinase;
PBS, phosphate-buffered saline; PCNA, proliferating cell nuclear antigen; PDGF,
platelet-derived growth factor; PI, propidium iodide; PLCc1, phospholipase C-c1;
RA, rosmarinic acid; Rb, retinoblastoma protein; SEM, standard error mean; UW,
ultrapure water; VSMC, vascular smooth muscle cell.
⇑ Corresponding authors.
E-mail addresses: mhjj3998@kiom.re.kr (J.-J. Lee), ideal0723@kiom.re.kr (J.-H.
Lee), guminjung@kiom.re.kr (M.J. Gu), han5621@cnu.ac.kr (J.-H. Han), wkcho@-
kiom.re.kr (W.-K. Cho), jyma@kiom.re.kr (J.Y. Ma).
mal vessels, VSMCs maintain a low mitotic level, but they can be
induced to proliferate and migrate by a variety of stimuli, such as
growth factors and cytokines, contributing to the formation of
atherosclerotic lesions (Deuel & Huang, 1984; Sachinidis, Locher,
Vetter, Tatje, & Hoppe, 1990). Among these growth factors,
platelet-derived growth factor (PDGF) causes atherosclerotic inti-
mal thickening, which can be observed after balloon angioplasty
(Sachinidis et al., 1990). VSMC proliferation induced by PDGF-BB
leads to the phosphorylation of tyrosine residues on the PDGF-BB
receptor, followed by the activation of early signalling molecules
such as phospholipase C (PLC) c1, extracellular signal-regulated
kinase (ERK) 1/2, and phosphatidylinositol 3-kinase (PI3K)-linked
protein kinase (AKT), resulting in cell cycle transitions (Bornfeldt
et al., 1995; Claesson-Welsh, 1994). Normally, VSMCs within artery
walls are arrested at the G0/G1 phase, which is a quiescent state

http://dx.doi.org/10.1016/j.jff.2016.12.025
1756-4646/Ó 2016 Elsevier Ltd. All rights reserved.
 J.-J. Lee et al. / Journal of Functional Foods 30 (2017) 30–38
(Gordon, Reidy, Benditt, & Schwartz, 1990). Following VSMC prolif-
eration due to vascular damage, cell cycle progression is regulated
by cyclin-dependent kinases (CDKs) and cyclins (Collins, Jacks, &
Pavletich, 1997; Jacks & Weinberg, 1996). During VSMC prolifera-
tion, specific cyclin/CDK complexes modulate the orderly progres-
sion of the cell cycle. In particular, the transition from the G1 phase
to the S phase results in DNA replication and activation of the
retinoblastoma (Rb) and proliferating cell nuclear antigen (PCNA)
proteins through expression of the cyclin D/CDK4 and cyclin E/
CDK2 complexes (Dzau, Braun-Dullaeus, & Sedding, 2002). Impor-
tantly, the activity of these cyclin/CDK complexes is negatively reg-
ulated by CDK inhibitors (CKIs) such as the p21WAF1/CIP1 and p27KIP1
proteins (Denicourt & Dowdy, 2004; Zhan et al., 2003). In recent
reports, upregulation of p21WAF1/CIP1 and p27KIP1 expression was
shown to arrest cells at the G0/G1 transition, preventing the devel-
opment of atherosclerosis (Braun-Dullaeus et al., 2001; Fouty &
Rodman, 2003; Tanner et al., 2000; Yang et al., 1996).
Agastache rugose (Fisch. & C.A. Mey.) Kuntze (A. rugosa), a mem-
ber of the Labiatae mint family, is commonly known as Korean
mint and is distributed throughout Korea, Japan, China, and Siberia.
A. rugosa has traditionally been used as both food and medicine,
depending on which part of the plant is used (e.g., sprouts, shoots,
and aerial parts). In addition, A. rugosa has been reported to possess
a variety of pharmacological and physiological activities, including
anti-bacterial, anti-viral, anti-atherogenic, anti-fungal, anti-
tumour, and anti-inflammatory effects (Hong et al., 2001; Min,
Hattori, Lee, & Kim, 1999; Oh et al., 2005; Shin & Kang, 2003). A.
rugosa contains various polyphenol components, including caffeic
acid, kaempferol, rosmarinic acid, calycosin, apigenin, genistein,
and calycosin-7-O-b-D-glucoside (Desta et al., 2016). It was shown
that A. rugosa extract has anti-atherogenic effects on the inflamma-
tion response of endothelial cells (Hong et al., 2001), which is one
of the early stages of atherogenesis. In this study, we investigated
the effects of A. rugosa extract on abnormal proliferation in VSMCs,
which can lead to restenosis and atherosclerosis, we describe a
potential mechanism of action for these anti-proliferative effects,
and we identify the active component of A. rugosa.
2. Materials and methods
2.1. Materials and reagents
Agastache rugosa Kuntze was purchased from a Yeongcheon
Herb Market (Yeongcheon, Korea), and the identity of the plant
material was confirmed by Dr. Ki Hwan Bae of the College of Phar-
macy, Chungnam National University (Daejeon, Korea); a voucher
specimen has been stored in the herbal bank at the Korea Institute
of Oriental Medicine (Daejeon, Korea). Chlorogenic acid, rosmarinic
acid and caffeic acid were purchased from Sigma-Aldrich (St. Louis,
MO, USA). Calycosin and calycosin-7-O-b-D-glucoside were pur-
chased from Chemfaces (Hubei, China), and genistein was pur-
chased from TCI (Tokyo Chemical Industry Co., Ltd., Tokyo,
Japan). The purity of all the chemical reference substances was
greater than 95%. High-performance liquid chromatographic
(HPLC) grade acetonitrile and methanol were procured from J.T.
Baker Inc. (Philipsburg, NJ, USA), and formic acid was purchased
from Wako (P99.5%, Wako Pure Chemical Industries, Ltd., Osaka,
Japan). Ultrapure water (UW) was prepared using the Puris-Evo
UP Water system with Evo-UP Dio VFT and Evo-ROP Dico20 (Mirae
ST Co., Ltd., Anyang, Gyeonggi-do, Korea). UW was prepared to
have a resistivity of 18.2 MX cm 1 (Puris, Esse-UP Water system,
Mirae St Co., Anyang, Korea). Fetal bovine serum (FBS) and
phosphate-buffered saline (PBS) were purchased from HyClone
(Logan, UT, USA). Distilled water was filtered through a 0.45-lm
membrane filter from ADVANTEC (Tokyo, Japan) before analysis.
31
Dulbecco’s modified Eagle’s medium (DMEM) was purchased from
Lonza (Walkersville, MD, USA). Trypsin/EDTA and penicillin/strep-
tomycin were purchased from Gibco (Grand Island, NY, USA). anti-
phospho-extracellular signal regulated kinase 1/2 (ERK1/2), anti-
ERK1/2, anti-phospho-phospholipase C-c1 (PLCc1), anti-phospho-
p38, anti-p38, anti-phospho-phosphatidylinositol 3-kinase-linked
protein kinase B (Akt), anti-Akt, anti-phospho-c-Jun N-terminal
kinase (JNK), anti-JNK, anti-CDK2, anti-CDK4, anti-cyclin D1, anti-
cyclin E1, anti-phospho-retinoblastoma protein (Rb), anti-
proliferating cell nuclear antigen (PCNA), anti-p21, anti-p27, and
anti-b-actin antibodies were purchased from Cell Signalling Tech-
nology Inc. (Beverly, MA, USA). The Cell Counting Kit-8 (CCK-8)
was purchased from Dojindo Molecular Technologies (Rockville,
MD, USA). Platelet-derived growth factor (PDGF)-BB was obtained
from PEPROTECH Co. (Rocky Hill, NJ, USA). All other chemicals
were of analytical grade.
2.2. Agastache rugosa preparation
For the A. rugosa extraction, dried A. rugosa Kuntze (50.0 g) was
placed in 1 L of distilled water and extracted by heating for 3 h at
115 °C using a Gyeongseo Extractor (Cosmos-600, Inchon, Korea).
The extracted solution was filtered using a standard test sieve
(150 lm) (Retsch, Haan, Germany) and then freeze-dried and
maintained in a desiccator at 4 °C until use. The extraction yield
was 5.86%.
2.3. Cell culture
VSMCs, a primary cell type isolated from rat aorta via enzymatic
dispersion (Chamley, Campbell, McConnell, & Groschel-Stewart,
1977), were obtained from Biobud Co. (Seongnam-si, Gyeonggi-
do, Korea). VSMCs were cultured in DMEM (supplemented with
10% FBS, 100 IU/mL penicillin, 100 lg/mL streptomycin, 8 mM
HEPES, and 2 mM L-glutamine) at 37 °C in a humidified atmo-
sphere of 95% air and 5% CO2. The purity of the cultures was con-
firmed based on immunocytochemical localization of a-smooth
muscle actin. Our experiment used the VSMCs from passages 5–7.
2.4. Cell proliferation assay
VSMC proliferation was investigated as previously described
(Yu et al., 2008). In brief, VSMC proliferation was measured using
a colorimetric WST-1 assay with a cell counting kit (CCK-8)
(Dojindo Molecular Technologies, Rockville, MD, USA). VSMCs
were seeded into 96-well culture plates at 4  104 cells/mL and
then cultured in complete media (DMEM containing 10% FBS) at
37 °C for 24 h. After reaching approximately 70% confluence,
VSMCs were incubated with serum-free medium for 24 h, treated
with A. rugosa extract at various concentrations for another 24 h
(in newly made serum-free medium), and stimulated with PDGF-
BB (25 ng/mL). Then, after 22 h, WST-1 reagent (WST-1 premix)
was added to the medium, and the cells were incubated for an
additional 2 h. The absorbance was measured at 450 nm using a
microplate reader (Packard Instrument Co., Downers Glove, IL,
USA), according to the manufacturer’s instructions. The results
are expressed as percentages relative to the PDGF-BB-stimulated
control. In addition, VSMC proliferation was determined using
the crystal violet assay, which measures the levels of crystal violet
dye in cells (Gillies, Didier, & Denton, 1986). In brief, after 24 h of
stimulation with PDGF-BB, the VSMCs was washed with ice-cold
PBS and then fixed in 4% paraformaldehyde (in PBS). The cells were
then stained with 0.5% crystal violet for 15 min, washed with
deionized water, and observed by microscopy (TH4-200; Olympus
Optical Co. Ltd., Tokyo, Japan).
32 J.-J. Lee et al. / Journal of Functional Foods 30 (2017) 30–38
2.5. Cell cytotoxicity
Cytotoxicity was assessed by treating VSMCs with A. rugosa
extract, and then using a colorimetric WST-1 assay to count cells.
The cells were exposed to A. rugosa extract at various concentra-
tion, or 10% Triton X-100 as a cytotoxic control, for various time
periods. WST-1 reagent was added to the medium, and the cells
were incubated for 1 h. Absorbance density was measured at
450 nm using a microplate reader. In addition, we also tested the
cytotoxicity of the A. rugosa extract using an Annexin V-FITC/PI
staining kit. The cell culture conditions were the same as described
for the cell proliferation assay. Following stimulation with PDGF-
BB (25 ng/mL) for 24 h, VSMCs were harvested by centrifugation
and then fixed with 70% ethanol for 48 h. Finally, the centrifuged
pellets were stained with Annexin V-FITC/PI solution at room tem-
perature for 15 min, according to manufacturer’s instructions, and
each cell nucleus was counted using flow cytometry (FACSCalibur,
Becton Dickinson and Co., Franklin Lakes, NJ, USA).
2.6. DNA synthesis
As previously described, the measurement of DNA synthesis
was monitored using a [3H]-thymidine incorporation assay (Yu
et al., 2008). The cell culture conditions for this assay were the
same as those described for the cell proliferation assay. [3H]-
thymidine (2 lCi/mL) was bound for 4 h before harvesting under
PDGF-BB-stimulated conditions (25 ng/mL) in serum-free medium.
The reaction was stopped by aspirating the medium and subjecting
the cultures to a series of washes with PBS containing ethanol/
ether (1:1, v/v) and 10% trichloroacetic acid on ice. Acid-insoluble
products containing [3H]-thymidine were extracted in 250 ll
0.5 M NaOH/well, and this solution was then combined with
3 mL scintillation cocktail (Ultimagold, Packard Bioscience, Meri-
den, CT, USA) and measured using a liquid scintillation counter
(LS3801, Beckman, Düsseldorf, Germany). In addition, to identify
the active components of A. rugosa extract, we investigated the
effects of two components, rosmarinic acid (RA) and caffeic acid
(CA), on DNA synthesis during VSMC proliferation using a BrdU
(bromodeoxyuridine) colorimetric kit (Roche, Basel, Switzerland).
After stimulation with PDGF-BB (25 ng/mL) for 24 h, VSMCs were
labelled with BrdU, according to manufacturer’s instructions, and
BrdU-labelled cells were measured using a microplate reader.
2.7. Cell cycle progression analysis
Cell cycle progression was examined as previously described
(Yu et al., 2008). The cell culture conditions were the same as
described for the cell proliferation assay. Following stimulation
with PDGF-BB (25 ng/mL) for 24 h, VSMCs were trypsinized and
centrifuged at 749g for 7 min. The centrifuged pellets were sus-
pended in 1 mL PBS (1), which was repeated twice, and then fixed
with 70% ethanol for 48 h. The fixed cells were briefly stirred and
centrifuged at 12400g for 5 min. The ethanol was removed, and
then the pellets were stained with 500 lL propidium iodide (PI)
solution (50 lg/mL PI in sample buffer containing 100 lg/mL
RNase A). Each sample was incubated at room temperature for
1 h. The PI-DNA complex in each cell nucleus was measured using
flow cytometry. The individual nuclear DNA content was reflected
by the incorporated PI fluorescence intensity. The rate of the cell
cycle based on the number of cells in the G0/G1, S, and G2/M phases
was determined using the Modfit LT software program.
2.8. Immunoblotting
Protein measurement by immunoblotting was performed as
previously described (Yu et al., 2008). VSMCs were stimulated with
PDGF-BB (25 ng/mL) for 5 min for ERK1/2 and PLCc1, 10 min for
JNK and p38, and 15 min for the Akt phosphorylation assays. To
measure CDK2, CDK4, cyclin D1, cyclin E1, p21, p27 and PCNA
expression as well as Rb phosphorylation, VSMCs were stimulated
with PDGF-BB (25 ng/mL) for 24 h. The levels of each protein were
normalized to the levels of b-actin or the respective total protein.
Band intensities were quantified using Scion Image for Windows
(Scion Corp., Frederick, MD, USA).
2.9. Immunofluorescence staining
VSMCs were cultured in 24-well plates on cover slips (8  104 -
cells/mL), stimulated with PDGF-BB (25 ng/mL) for 24 h, fixed with
PBS containing 2% paraformaldehyde for 10 min, and then blocked
with 5% bovine serum albumin in PBS for 1 h at room temperature.
The cells were incubated with primary antibodies (anti-p21 or
anti-p27) for 2 h, and then stained with Alexa Fluor 555-
conjugated secondary antibodies (Cell Signalling Technology Inc.,
Beverly, MA, USA). The cells were mounted in PI solution for
10 min and then observed by fluorescence microscopy (TH4-200;
Olympus Optical Co. Ltd., Tokyo, Japan).
2.10. Standard solution and Agastache rugosa extract preparation
Each standard was prepared as a stock solution at a concentra-
tion of 1 mg/mL using HPLC grade pure methanol, and all stock
solutions were stored at 4 °C before analysis. Dilutions of the stan-
dard solutions for chlorogenic acid, caffeic acid, calycosin-7-O-b-D-
glucoside, rosmarinic acid, calycosin and genistein at 1, 5, 50 and
100 lg/mL concentrations in pure methanol were used for calibra-
tion. The A. rugosa extract was prepared by dissolving 1 mg dry
powder in 1 mL 50% methanol/water (v/v), which was then
extracted via ultrasonication for 30 min and filtered through a
0.2-lm syringe membrane filter from Whatman Ltd (Maidstone,
UK) before injection for HPLC analysis
2.11. Chromatographic conditions
HPLC analysis was performed using a Dionex HPLC system (Dio-
nex Co., Sunnyvale, CA, USA), consisting of an Ultimate 3000 series
A binary pump, an auto-sampler, a column oven and a diode array
UV/VIS detector (DAD). The analytical data from the detector were
collected by using the Dionex Chromelon 7 software program. Tar-
get compounds were separated using a Luna C18 apparatus
(4.6 mm  250 mm id, 5 lm, Phenomenex Co., Torrance, CA,
USA), and the temperature of column was maintained at 40 °C.
The mobile phases were composed of formic acid/water (0.1%, v/
v); solvent A, and pure acetonitrile; solvent B (Desta et al., 2016).
The following gradient program was used: (B) = 10% (0–5 min),
10–13% (5–10 min), 13% (10–20 min), 13–20% (20–30 min), 20%
(30–50 min), 20–50% (50–55 min), 50% (55–60 min), 50–10% (60–
61 min) and 10% (61–75 min). Analysis was carried out at a flow
rate of 1.0 mL/min with DAD detection at 280 nm; the injection
volume was 10 lL. HPLC analysis was performed five times per
sample.
2.12. Statistical analysis
The data are expressed as the mean ± SEM values. A one-way
analysis of variance was used for multiple comparisons (GraphPad,
San Diego, CA, USA). Dunnett’s test was applied if a significant dif-
ference was observed among the treated groups. p < 0.05 was con-
sidered to be statistically significant.
 J.-J. Lee et al. / Journal of Functional Foods 30 (2017) 30–38 33

3. Results proliferative effects of the A. rugosa extract, we measured the phos-

3.1. Effects of A. rugosa extract on VSMC proliferation, cell viability and
early signal transduction pathway
To identify the effects of A. rugosa extract on VSMC prolifera-
tion, colorimetric WST-1 and crystal violet staining assays were
performed. The A. rugosa extract (10, 30 and 50 lg/mL) signifi-
cantly inhibited VSMC proliferation by at least 93% compared with
the PDGF-BB-stimulated controls (Fig. 1A(a)). In the crystal violet
staining assay, the A. rugosa extract inhibited VSMC proliferation,
consistent with the results of the WST-1 assay (Fig. 1A(b)). To
determine whether the anti-proliferative activity of A. rugosa
extract was due to cytotoxicity, we examined the cytotoxicity at
following treatment with 10, 30, and 50 lg/mL extract for various
times using a colorimetric WST-1 assay and Annexin V-FITC/PI
staining. The positive control (10% Triton-X) was found to be cyto-
toxic. By contrast, the A. rugosa extract did not appear to be cyto-
toxic, when measured by either apoptosis or necrosis (Fig. 1B). To
determine a potential molecular mechanism for the anti-
phorylation of PLCc1, ERK1/2, p38, JNK, and Akt in PDGF-BB-
stimulated VSMCs to investigate early signalling pathways in pro-
liferation. The A. rugosa extract significantly suppressed the phos-
phorylation of PLCc1, ERK, and Akt; however, the
phosphorylation of p38 and JNK was not affected by the extract
(Fig. 1C). These results suggest that A. rugosa extract exerts its
anti-proliferative activity via cell-cycle arrest, including the sup-
pression of cell cycle-related proteins and activating CKIs down-
stream of the early signalling pathways.
3.2. Effect of A. rugosa extract on DNA synthesis and cell cycle
progression
Next, we investigated the effects of A. rugosa extract on DNA
synthesis by measuring the incorporation of [3H]-thymidine in
cells. A. rugosa extract at concentrations of 10, 30, and 50 lg/mL
potently suppressed DNA synthesis by at least 86% compared with
the PDGF-BB-stimulated controls (Fig. 2A). Consistent with
observed suppression of DNA synthesis, the A. rugosa extract

Fig. 1. Effects of A. rugosa extract on VSMC proliferation and early signal transduction. Quiescent VSMCs cultured in serum-free medium were stimulated with 25 ng/mL
PDGF-BB for 24 h, and the effects of various concentrations of A. rugosa extract (10–30 lg/ml) were monitored. (A) Optimal densities were determined at 450 nm using the
WST-1 assay (n = 4) (a), and the inhibition of VSMC proliferation in the presence of A. rugosa extract was measured using crystal violet staining (n = 3) (b). (B) The cytotoxicity
of the A. rugosa extract in VSMCs was examined at three different concentrations (10, 30, and 50 lg/mL) for various times using a colorimetric WST-1 assay (a) and Annexin V-
FITC/PI staining (b) (n = 3). (C) The PDGF-induced phosphorylation of PLCc1, ERK1/2, p38, JNK, and Akt was measured using SDS-PAGE and immunoblotting (a). b-actin, ERK1/
2, p38, JNK, and Akt were used for normalization (n = 3) (b). The results were analysed by densitometry, and the values represent percentage relative to the PDGF-BB-
stimulated controls. The values represent the mean ± SEM. Significant differences from the PDGF-stimulated controls are shown indicated with asterisks: *p < 0.05 or
**
p < 0.01.
34 J.-J. Lee et al. / Journal of Functional Foods 30 (2017) 30–38

Fig. 2. Effects of A. rugosa extract on cell cycle progression and cell cycle-related proteins. Quiescent VSMCs cultured in serum-starved medium were stimulated with 25 ng/
mL PDGF-BB, and the effects of A. rugosa extract (10–50 lg/ml) on DNA synthesis (n = 3) (A) and the cell cycle progression (B) (n = 5) were assessed. (C) The effects of A. rugosa
extract on cell cycle regulatory proteins stimulated by PDGF-BB, including cyclin D1/E1, CDK2/4, Rb, and PCNA as negative regulatory molecules, was measured using SDS-
PAGE followed by immunoblotting (n = 3) (a). b-actin was used for normalization (b). These results were analysed using densitometry, and the values represent the
percentage relative to PDGF-BB-stimulated controls. All values are expressed as the mean ± SEM. Significant differences from the PDGF-stimulated control are indicated with
asterisks: *p < 0.05 or **p < 0.01.

induced cell cycle arrest at the progression from G0/G1 to S phase
(Fig. 2B). Therefore, we next investigated the effect of A. rugosa
extract on the activation of positive regulatory proteins that are
involved in the G0/G1 arrest, such as CDK2, CDK4, cyclin D1, Cyclin
E1, PCNA and Rb. As shown in Fig. 2C, A. rugosa extract suppressed
the expression of the CDK 2-cyclin E1 and CDK 4-cyclin D1 com-
plexes in a concentration-dependent manner, and it also signifi-
cantly inhibited the phosphorylation of Rb protein, a major
modulator of the cell cycle transition network. Moreover, A. rugosa
extract potently suppressed the expression of PCNA, which is a
gene that synthesized following hyper-phosphorylation of the Rb
protein.
(Fig. 3A), indicating that the extract induces cell cycle arrest at
the G0/G1 phase is mediated by increasing the expression of p21
and p27. Additionally, to identify the direct activity on p21 and
p27 expression, we utilized an immunofluorescence assay. In this
experiment, A. rugosa extract at concentrations of 10, 30, and
50 lg/ml increased p27 expression by 268 ± 0.23, 255 ± 0.27, and
223 ± 0.07% compared with PDGF-BB–stimulated controls, respec-
tively (Fig. 3B(a and b)). A. rugosa extract also increased p21
expression by 230 ± 0.14, 169 ± 0.44, and 217 ± 0.08% compared
with the stimulated controls, respectively (Fig. 3B(c and d)). These
results indicate that the transition from the G0/G1 phase is sup-
pressed through increased p21 and p27 expression in the presence
of the A. rugosa extract.

3.3. Effect of A. rugosa extract on the expression of negative regulatory

proteins 3.4. HPLC analysis and determination of active components

Based on the effects of A. rugosa extract on cell cycle progres-
sion, we next investigated the effects of A. rugosa extract on the
expression of negative regulatory protein, such as p21 and p27
(Abukhdeir & Park, 2008). A. rugosa extract significantly increased
p21 and p27 expression in a concentration-dependent manner
The analysis methods and standards were as described previ-
ously (Desta et al., 2016). HPLC chromatogram characteristics of
the compounds (chlorogenic acid, caffeic acid, calycosin-7-O-
beta-D-glucoside, rosmarinic acid, calycosin, and genistein)
(Fig. 4B), including retention time and UV absorption, were deter-
 J.-J. Lee et al. / Journal of Functional Foods 30 (2017) 30–38 35

Fig. 3. Effects of A. rugosa extract on the degradation of p21WAF1/CIP1 and p27KIP1 mediated by PDGF-BB. Quiescent VSMCs cultured in serum-deprived medium were
stimulated with 25 ng/mL PDGF-BB. (A) The inhibitory effects of A. rugosa extract (10–50 lg/ml) on p21WAF1/CIP1 and p27Kip1 expression were examined using SDS-PAGE
followed by immunoblotting (a); b-actin was used for normalization (b), and results were analysed using densitometry. (C) p21WAF1/CIP1 and p27Kip1 expression was also
examined using an immunofluorescence assay. Total b-actin and the fluorescence levels of PI were used for normalization, and the levels of immunofluorescence were
analysed using densitometry; the values represent fold-changes relative to the stimulated control. The results are an average of three similar, independent experiments and
are expressed as the mean ± SEM. Significant differences relative to the PDGF-stimulated controls are indicated with asterisks: *p < 0.05 or **p < 0.01.

mined for each component in the sample. The resulting six peaks
had the following retention times: chlorogenic acid - 13.89 min,
caffeic acid – 17.53 min, calycosin-7-O-beta-D-glucoside –
32.22 min, rosmarinic acid – 47.07 min, calycosin – 57.29 min,
and genistein – 58.77 min. We show the chromatograms of the
standards and the samples at the retention times for caffeic acid
(17.56 min) and rosmarinic acid (47.10 min) (Fig. 4A). According
to the UV absorbance, the detection wavelength was set at
280 nm for the standard mixture and the samples. All calibration
curves for 6 compounds showed good linearity (r2 > 0.9992) using
the described HPLC method (Table 1). The results shown in Table 2
indicated that rosmarinic acid (RA) (100.8914 mg/g) and caffeic
acid (CA) (30.0459 mg/g) were present in the A. rugosa extract,
whereas the other components were not detected.
To determine the active component, we investigated the inhibi-
tory effects of RA and CA on DNA synthesis stimulated by PDGF-BB
using BrdU incorporation. The results showed that 40 lg/mL RA
significantly suppressed DNA synthesis compared with PDGF-BB-
stimulated controls (Fig. 4C). To identify the effects of RA and CA
on VSMC proliferation via cell cycle-related transduction path-
ways, we examined the activation of PCNA. We found that RA
inhibited PCNA expression in a dose-dependent manner, a gene
whose expression is based on the hyper-phosphorylation of Rb
during the early G0/G1 and the S phases, which is mediated by
PDGF-BB; by contrast, we observed no effect for CA on PCNA
expression (Fig. 4D). These results indicate that the aqueous
extract of A. rugosa included RA and CA as components, although
only RA showed specific activity on the suppression of DNA syn-
thesis and cell cycle progression during VSMC proliferation.
4. Discussion
During the pathogenesis of atherosclerosis and restenosis,
VSMC proliferation is promoted by various growth factors and
cytokines, including PDGF-BB (Dzau et al., 2002). Therefore, VSMC
proliferation is considered a key control point for the prevention
and treatment of atherosclerosis and restenosis (Uchida,
Sasahara, Morigami, Hazama, & Kinoshita, 1996). In this study,
we demonstrate that A. rugosa extract significantly inhibits VSMC
proliferation stimulated by PDGF-BB exposure. The present study
shows 3 major findings: (1) A. rugosa extract has an anti-
proliferative activity associated with PLCc1, ERK, and AKT on VSMC
proliferation. (2) This anti-proliferative activity of the A. rugosa
extract involves arrest at the transition from G0/G1 to S phase by
suppressing cell cycle-related proteins through the upregulation
of p21 and p27 expression. (3) Finally, we show that the aqueous
extract of A. rugosa contains RA and CA among its components,
as reported in a previous study (Desta et al., 2016), although only
RA appears to be an active component.
Endothelial cell dysfunction has been linked to several different
cytokines and growth factors through the inflammation response,
which ultimately causes the formation of atherosclerotic lesions
through VSMC proliferation (Ross, 1990; Ross, 1999). Therefore,
we investigated the inhibition of VSMC proliferation using the
aqueous extract of A. rugosa as a candidate substance for preven-
tion or treatment. A. rugosa extract showed anti-proliferative activ-
ity in PDGF-BB-stimulated VSMCs (Fig. 1A), and we show that this
effect was not due to cell cytotoxicity using annexin V-FITC/PI
staining (Fig. 1B). VSMC proliferation is primarily regulated by pro-
teins of the early signal transduction pathway, such as PLCc1,
ERK1/2, p38, JNK, and Akt (Sachinidis et al., 1990; Sears &
Nevins, 2002). In this study, A. rugosa extract significantly sup-
36 J.-J. Lee et al. / Journal of Functional Foods 30 (2017) 30–38

Fig. 4. HPLC analysis chromatogram and determination of A. rugosa extract components. (A) HPLC chromatogram at 280 nm of a standard mixture (1 – chlorogenic acid; 2 –
caffeic acid (CA); 3 – calycosin-7-O-beta-D-glucoside; 4 – rosmarinic acid (RA); 5 – calycosin; and 6 – genistein) as well as an extract of A. rugosa extract prepared from a dry
powder. (B) Chemical structure of reference compounds in Agastache rugosa Kuntze. (C) The effect of RA and CA on DNA synthesis was examined using BrdU incorporation. (D)
PCNA was measured using SDS-PAGE followed by immunoblotting. b-actin was used for normalization, and the results were analysed using densitometry; the values
represent the percentage relative to the PDGF-stimulated controls. The results are an average of three similar, independent experiments and are expressed as the mean ± SEM.
Significant differences relative to the controls are indicated with asterisks: *p < 0.05 or **p < 0.01.

Table 1 tion of anti-proliferative genes and up-regulation of p21 (Lee &

Calibration curve (n = 5) parameters used to analyse the dry powder Agastache rugosa Moon, 2005; Meloche & Pouyssegur, 2007). Akt is phosphorylated
Kuntze water extract sample.
through the PI3K pathway and also acts as an important regulator
Compound Linear range (lg/ Regression r2b of cell cycle progression, cell survival and migration (Higaki &
ml) equationa Shimokado, 1999; Jung, Haendeler, Goebel, Zeiher, & Dimmeler,
Chlorogenic acid 1–100 y = 0.1967 x 0.9992 2000; Zhang, Zhou, Li, & Xu, 2011). In this study, PLCc1, ERK1/2
+ 1.4612 and Akt phosphorylation were stimulated by PDGF-BB, and these
Caffeic acid 1–100 y = 0.4622 x 0.9998
effects were significantly inhibited by A. rugosa extract (Fig. 1C).
+ 3.6760
Calycosin-7-O-b-D- 1–100 y = 0.2870 x 0.9997 These results indicate that the anti-proliferative effects of A. rugosa
glucoside + 2.3524 extract on VSMCs act, at least in part, by suppressing the phospho-
Rosmarinic acid 1–100 y = 0.5231 x 1.0000 rylation of PLCc1, ERK1/2 and Akt. Therefore, the transitional arrest
+ 3.4463
of cell cycle progression in the presence of A. rugosa extract is con-
Calycosin 1–100 y = 0.4327 x 0.9997
+ 3.4463 sistent with its effects on cell cycle-related proteins and negative
Genistein 1–100 y = 0.4965 x 0.9997 regulatory proteins.
+ 4.0113 In VSMCs, proliferation is modulated by both positive and neg-
a
In the regression equation y = ax + b, y: Peak area, x: concentration (lg/ml).
ative regulatory proteins, such as cyclins, CDKs, Rb, PCNA, and CKIs,
b
r2: the square value of the correlation coefficient of the equation. and thus, these regulatory proteins are important targets for the
inhibition of proliferation (Braun-Dullaeus et al., 2004; Dzau
et al., 2002; Sherr, 1995; Sherr & Roberts, 1999). The cell cycle tran-
pressed the phosphorylation of PLCc1, ERK1/2 and Akt. Generally, sition from G0/G1 to S phase is mediated by the CDK2-cyclin E1 and
PLCc1 phosphorylation modulates a variety of downstream growth CDK4-cyclin D1 complexes, and these complexes are suppressed
factor-induced signal transduction pathways (Heldin, Ostman, & by p21 and p27 expression, which act as a negative regulators,
Ronnstrand, 1998). ERK1/2 is an essential modulator of the transi- arresting the cell cycle at G0/G1 phase (Abukhdeir & Park, 2008;
tion from G0/G1 to S phase, which is related to the activation of Sherr & Roberts, 1999). A. rugosa extract potently inhibited DNA
negative regulators of cell cycle progression, including the induc- synthesis, arrested the cell cycle transition at the G0/G1 phase
 J.-J. Lee et al. / Journal of Functional Foods 30 (2017) 30–38 37

Table 2
Quantification results from dry powder Agastache rugosa Kuntze water extract sample.

Compound Retention time (min) Area (mAU) Amount (mg/g) Amount average (mg/g) S.D. R.S.D. (%)
Chlorogenic acid N.D. N.D. N.D. N.D. N.D. N.D.
Caffeic acid 17.55 17.5570 30.03 30.0459 0.09 0.29
17.57 17.5730 30.07
17.57 17.5700 30.06
17.61 17.6130 30.15
17.50 17.5030 29.92
Calycosin-7-O-b-D-glucoside N.D. N.D. N.D. N.D. N.D. N.D.
Rosmarinic acid 47.00 47.1230 100.94 100.8914 0.21 0.20
47.13 47.0930 100.87
47.03 47.1100 100.91
47.11 47.2170 101.16
47.21 46.9670 100.58
Calycosin N.D. N.D. N.D. N.D. N.D. N.D.
Genistein N.D. N.D. N.D. N.D. N.D. N.D.

N.D.: Not detected.

(Fig. 2A and B), and strongly inhibited CDK2, CDK4, cyclin E1 and
cyclin D1 expression in a dose-dependent manner. Treatment also
resulted in lower phosphorylation levels for Rb and decreased
expression of PCNA (Fig. 2C), which is a product of phosphorylated
Rb-mediated gene expression during the cell cycle transition from
the G0/G1 to S phase (Tomita et al., 2005). These results suggest
that arrest at the G0/G1 phase might function through CKIs such
as the Cip/Kip and INK4 family (Sherr and Roberts, 1999). A. rugosa
extract not only increased the expression of p21WAF1/CIP1 and
p27KIP1 in our immunoblotting assays (Fig. 3A) in a
concentration-dependent manner, it also increased signal intensity
in our immunofluorescence assays, which we used to directly visu-
alize p21WAF1/CIP1 and p27KIP1 expression (Fig. 3B). These results
suggest that the anti-proliferative activity of A. rugosa extract is
associated with the increase of p21WAF1/CIP1 and p27KIP1 expression,
consistent with the suppression of cell cycle-related proteins dur-
ing the G0/G1 to S transition.
Additionally, to identify the active component responsible for
the anti-proliferative activity of A. rugosa extract, we tested several
components of A. rugosa extract that were reported in an earlier
study, including chlorogenic acid, caffeic acid (CA), calycosin-7-
O-beta-D-glucoside, rosmarinic acid (RA), calycosin, and genistein
(Fig. 4B) (Desta et al., 2016). Of these components, only CA and
RA were detected in the aqueous extract of A. rugosa via HPLC anal-
ysis (Fig. 4A). Importantly, 40 lM RA significantly inhibited BrdU
incorporation, consistent with decreased DNA synthesis, whereas
CA showed no effect (Fig. 4C). Moreover, only RA was able to sup-
press the expression of PCNA in a concentration-dependent man-
ner (Fig. 4D). These results indicate that RA is the primary active
component in the aqueous portion of the A. rugosa extract
(Table 2).
5. Conclusions
This study indicates that A. rugosa extract, and RA in particular,
inhibit VSMC proliferation by arresting the cell cycle transition
from the G0/G1 phase to the S phase via the inhibiting the proteins
was related the G0/G1 phase, finally, these effects are due to the up-
regulation of p21WAF1/CIP1 and p27KIP1 expression. Therefore, we
cautiously suggest that A. rugosa extract may be useful as a preven-
tive agent or food supplement for the management of atherosclero-
sis and restenosis.
Conflicts of interest
The authors declare no conflicts of interest.
Acknowledgements
This work was supported by a grant (K16281) awarded to the
KIOM from the Ministry of Science, ICT and Future Planning (MISP),
Republic of Korea.
References
Abukhdeir, A. M., & Park, B. H. (2008). P21 and p27: Roles in carcinogenesis and
drug resistance. Expert Reviews in Molecular Medicine, 10, e19.
Bornfeldt, K. E., Raines, E. W., Graves, L. M., Skinner, M. P., Krebs, E. G., & Ross, R.
(1995). Platelet-derived growth factor. Distinct signal transduction pathways
associated with migration versus proliferation. Annals of the New York Academy
of Sciences, 766, 416–430.
Braun-Dullaeus, R. C., Mann, M. J., Seay, U., Zhang, L., von Der Leyen, H. E., Morris, R.
E., & Dzau, V. J. (2001). Cell cycle protein expression in vascular smooth muscle
cells in vitro and in vivo is regulated through phosphatidylinositol 3-kinase and
mammalian target of rapamycin. Arteriosclerosis, Thrombosis, and Vascular
Biology, 21, 1152–1158.
Braun-Dullaeus, R. C., Mann, M. J., Sedding, D. G., Sherwood, S. W., von der Leyen, H.
E., & Dzau, V. J. (2004). Cell cycle-dependent regulation of smooth muscle cell
activation. Arteriosclerosis, Thrombosis, and Vascular Biology, 24, 845–850.
Chamley, J. H., Campbell, G. R., McConnell, J. D., & Groschel-Stewart, U. (1977).
Comparison of vascular smooth muscle cells from adult human, monkey and
rabbit in primary culture and in subculture. Cell and Tissue Research, 177,
503–522.
Claesson-Welsh, L. (1994). Platelet-derived growth factor receptor signals. Journal of
Biological Chemistry, 269, 32023–32026.
Collins, K., Jacks, T., & Pavletich, N. P. (1997). The cell cycle and cancer. Proceedings of
the National Academy of Sciences of the United States of America, 94, 2776–2778.
Denicourt, C., & Dowdy, S. F. (2004). Cip/Kip proteins: More than just CDKs
inhibitors. Genes & Development, 18, 851–855.
Desta, K. T., Kim, G. S., Kim, Y. H., Lee, W. S., Lee, S. J., Jin, J. S., ... Shin, S. C. (2016). The
polyphenolic profiles and antioxidant effects of Agastache rugosa Kuntze
(Banga) flower, leaf, stem and root. Biomedical Chromatography, 30, 225–231.
Deuel, T. F., & Huang, J. S. (1984). Platelet-derived growth factor. Structure, function,
and roles in normal and transformed cells. Journal of Clinical Investigation, 74,
669–676.
Dzau, V. J., Braun-Dullaeus, R. C., & Sedding, D. G. (2002). Vascular proliferation and
atherosclerosis: New perspectives and therapeutic strategies. Nature Medicine,
8, 1249–1256.
Faxon, D. P., Fuster, V., Libby, P., Beckman, J. A., Hiatt, W. R., Thompson, R. W., ...
Loscalzo, J. (2004). Atherosclerotic vascular disease conference: Writing group
III: Pathophysiology. Circulation, 109, 2617–2625.
Fouty, B. W., & Rodman, D. M. (2003). Mevastatin can cause G1 arrest and induce
apoptosis in pulmonary artery smooth muscle cells through a p27Kip1-
independent pathway. Circulation Research, 92, 501–509.
Gillies, R. J., Didier, N., & Denton, M. (1986). Determination of cell number in
monolayer cultures. Analytical Biochemistry, 159, 109–113.
Gordon, D., Reidy, M. A., Benditt, E. P., & Schwartz, S. M. (1990). Cell proliferation in
human coronary arteries. Proceedings of the National Academy of Sciences of the
United States of America, 87, 4600–4604.
Heldin, C. H., Ostman, A., & Ronnstrand, L. (1998). Signal transduction via platelet-
derived growth factor receptors. Biochimica et Biophysica Acta, 1378, F79–F113.
Higaki, M., & Shimokado, K. (1999). Phosphatidylinositol 3-kinase is required for
growth factor-induced amino acid uptake by vascular smooth muscle cells.
Arteriosclerosis, Thrombosis, and Vascular Biology, 19, 2127–2132.
38 J.-J. Lee et al. / Journal of Functional Foods 30 (2017) 30–38
Hong, J. J., Choi, J. H., Oh, S. R., Lee, H. K., Park, J. H., Lee, K. Y., ... Oh, G. T. (2001).
Inhibition of cytokine-induced vascular cell adhesion molecule-1 expression;
possible mechanism for anti-atherogenic effect of Agastache rugosa. FEBS
Letters, 495, 142–147.
Jacks, T., & Weinberg, R. A. (1996). Cell-cycle control and its watchman. Nature, 381,
643–644.
Jung, F., Haendeler, J., Goebel, C., Zeiher, A. M., & Dimmeler, S. (2000). Growth factor-
induced phosphoinositide 3-OH kinase/Akt phosphorylation in smooth muscle
cells: Induction of cell proliferation and inhibition of cell death. Cardiovascular
Research, 48, 148–157.
Lee, B., & Moon, S. K. (2005). Ras/ERK signaling pathway mediates activation of the
p21WAF1 gene promoter in vascular smooth muscle cells by platelet-derived
growth factor. Archives of Biochemistry and Biophysics, 443, 113–119.
Meloche, S., & Pouyssegur, J. (2007). The ERK1/2 mitogen-activated protein kinase
pathway as a master regulator of the G1- to S-phase transition. Oncogene, 26,
3227–3239.
Min, B. S., Hattori, M., Lee, H. K., & Kim, Y. H. (1999). Inhibitory constituents against
HIV-1 protease from Agastache rugosa. Archives of Pharmacal Research, 22,
75–77.
Oh, H. M., Kang, Y. J., Kim, S. H., Lee, Y. S., Park, M. K., Heo, J. M., ... Chang, K. C. (2005).
Agastache rugosa leaf extract inhibits the iNOS expression in ROS 17/2.8 cells
activated with TNF-alpha and IL-1beta. Archives of Pharmacal Research, 28,
305–310.
Ross, R. (1990). Mechanisms of atherosclerosis – a review. Advances in Nephrology
from the Necker Hospital, 19, 79–86.
Ross, R. (1993). The pathogenesis of atherosclerosis: A perspective for the 1990s.
Nature, 362, 801–809.
Ross, R. (1999). Atherosclerosis – an inflammatory disease. The New England Journal
of Medicine, 340, 115–126.
Sachinidis, A., Locher, R., Vetter, W., Tatje, D., & Hoppe, J. (1990). Different effects of
platelet-derived growth factor isoforms on rat vascular smooth muscle cells.
Journal of Biological Chemistry, 265, 10238–10243.
Sears, R. C., & Nevins, J. R. (2002). Signaling networks that link cell proliferation and
cell fate. Journal of Biological Chemistry, 277, 11617–11620.
Sherr, C. J. (1995). Mammalian G1 cyclins and cell cycle progression. Proceedings of
the Association of American Physicians, 107, 181–186.
Sherr, C. J., & Roberts, J. M. (1999). CDK inhibitors: Positive and negative regulators
of G1-phase progression. Genes & Development, 13, 1501–1512.
Shin, S., & Kang, C. A. (2003). Antifungal activity of the essential oil of Agastache
rugosa Kuntze and its synergism with ketoconazole. Letters in Applied
Microbiology, 36, 111–115.
Tanner, F. C., Boehm, M., Akyurek, L. M., San, H., Yang, Z. Y., Tashiro, J., ... Nabel, E. G.
(2000). Differential effects of the cyclin-dependent kinase inhibitors p27(Kip1),
p21(Cip1), and p16(Ink4) on vascular smooth muscle cell proliferation.
Circulation, 101, 2022–2025.
Tomita, H., Osanai, T., Toki, T., Maeda, N., Murakami, R., Chen, Z., ... Okumura, K.
(2005). Roxithromycin is an inhibitor of human coronary artery smooth muscle
cells proliferation: A potential ability to prevent coronary heart disease.
Atherosclerosis, 182, 87–95.
Uchida, K., Sasahara, M., Morigami, N., Hazama, F., & Kinoshita, M. (1996).
Expression of platelet-derived growth factor B-chain in neointimal smooth
muscle cells of balloon injured rabbit femoral arteries. Atherosclerosis, 124,
9–23.
Yang, Z. Y., Simari, R. D., Perkins, N. D., San, H., Gordon, D., Nabel, G. J., & Nabel, E. G.
(1996). Role of the p21 cyclin-dependent kinase inhibitor in limiting intimal cell
proliferation in response to arterial injury. Proceedings of the National Academy
of Sciences of the United States of America, 93, 7905–7910.
Yu, J. Y., Lee, J. J., Lim, Y., Kim, T. J., Jin, Y. R., Sheen, Y. Y., & Yun, Y. P. (2008). Genistein
inhibits rat aortic smooth muscle cell proliferation through the induction of
p27kip1. Journal of Pharmacological Sciences, 107, 90–98.
Zhan, Y., Kim, S., Izumi, Y., Izumiya, Y., Nakao, T., Miyazaki, H., & Iwao, H. (2003).
Role of JNK, p38, and ERK in platelet-derived growth factor-induced vascular
proliferation, migration, and gene expression. Arteriosclerosis, Thrombosis, and
Vascular Biology, 23, 795–801.
Zhang, B. C., Zhou, Z. W., Li, X. K., & Xu, Y. W. (2011). PI-3K/AKT signal pathway
modulates vascular smooth muscle cells migration under cyclic mechanical
strain. VASA, 40, 109–116.