Pulmonary, Gastrointestinal and Urogenital Pharmacology
Effects of ginseng saponins isolated from red ginseng on ultraviolet B-induced skin
aging in hairless mice
Young Gon Kima, Maho Sumiyoshia, Masahiro Sakanakaa, Yoshiyuki Kimurab,⁎
aDivision of Functional Histology, Department of Functional Biomedicine, Ehime University Graduate School of Medicine, Shitsukawa, Toon City, Ehime 791-0295, Japan
bDivision of Biochemical Pharmacology, Department of Basic Medical Research, Ehime University Graduate School of Medicine, Shitsukawa, Toon City, Ehime 791-0295, Japan
a b s t r a c ta r t i c l ei n f o
Received 5 June 2008
Received in revised form 26 September 2008
Accepted 10 November 2008
Available online 19 November 2008
It is well-known that chronic ultraviolet B (UVB) exposure at low-dose causes skin photoaging including
increases in skin thickness and wrinkle formation and reduction in skin elasticity. This study examined the
effects of total saponins and ginsenoside Rb1isolated from Red Ginseng roots on skin thickness, elasticity, and
wrinkle formation caused by long-term, low-dose UVB irradiation in hairless mice. The topical application of
inhibited increases in skin thickness and wrinkle formation and the reduction in skin elasticity induced by
long-term UVB irradiation. Furthermore, we examined the histological effects of total saponins and
ginsenoside Rb1in the skin of UVB-irradiated hairless mice. The increases in apoptotic, Ki-67-, and 8-
of total saponins and ginsenoside Rb1. Furthermore, total saponins and ginsenoside Rb1 prevented
the disruption of collagen fibers induced by the long-term UVB irradiation. Ginsenoside Rb1(100 fg, 10 pg,
and 1 ng/ml) increased the Bcl-2 expression level in UVB-treated human keratinocytes. The protectiveeffect of
suggest that the protectiveeffectof ginsenoside Rb1on skinphotoaging induced bychronic UVB exposuremay
be due to the increase in collagen synthesis and/or the inhibition of matrix metalloproteinase expression in
© 2008 Elsevier B.V. All rights reserved.
Red Ginseng root (Panax ginseng C.A. Meyer) is used clinically in
China, Korea, and Japan for various diseases including athero-
sclerosis, liver dysfunction, cerebrovascular diseases, hypertension,
and post-menopausal disorder (Yamamoto, 1988). Recently, we
reported that the promotion of burn wound healing by ginsenoside
Rb1might be due to the promotion of angiogenesis during skin
wound repair through stimulation of vascular endothelial growth
factor (VEGF) production and an increase in hypoxia inducible factor
(HIF)-1α expression in keratinocytes and the elevation of IL-1β
from macrophage accumulation in the burn wound area (Kimura
et al., 2006). Furthermore, the previous report showed that the
facilitating actions of ginsenoside Rb1 might be due to the pro-
motion of angiogenesis via the activation of basic fibroblast growth
factor (bFGF) through the increase in histamine released from mast
cells recruited by the stimulation of monocyte chemoattractant
protein-1 (MCP-1) as another mechanism (Kawahira et al., 2008). It
is well known that an increase in skin thickness and a reduction in
skin elasticity are caused by sun exposure. This phenomenon is
known as photoaging and is characterized by histological changes
including damage to collagen fibers and excessive deposition of
abnormal elastic fibers (Sams and Smith, 1961; Smith et al., 1962;
Uitto et al., 1989). We found recently that the protection by Red
Ginseng extract against acute UVB-irradiated skin aging, such as the
increase in skin thickness and pigmentation and the reduction in
skin elasticity, might be due to the inhibition of increases in skin
TGF-β1 content induced by UVB irradiation (Kim et al., 2008). This
study examined the effects of total saponins and ginsenoside Rb1
isolated from Red Ginseng roots on skin thickness, elasticity, and
wrinkles caused by the long-term, low-dose UVB irradiation in
2. Materials and methods
Total ginsengsaponins wereisolated by themethods described by
Shibata and co-workers (Nagai et al., 1971; Sanada et al., 1974a, b;
Shibata et al., 1985; Shibata, 2001). Briefly, ginsenoside Rb1(Fig. 1)
was isolated and purified from the total saponin fractions of roots of
European Journal of Pharmacology 602 (2009) 148–156
⁎ Corresponding author. Tel.: +81 89 960 5922; fax: +81 89 960 5239.
E-mail address: firstname.lastname@example.org (Y. Kimura).
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Panax ginseng C.A. Meyer, Korean Red Ginseng, by repeated column
chromatography on silica gel with CHCl3–MeOH–H2O (65:35:10, v/v)
and octadecylsilyl silica with MeOH–H2O (1:1 to 7:3, v/v). The purity
of ginsenoside Rb1used in this study was more than 99.99%, as
determined by high-performance liquid chromatography. The total
ginsengsaponinwas dissolved in ethanol–propyleneglycol (7:3, v/v),
at the concentrations of 200 pg and 2 μg/ml, and ginsenoside Rb1was
also dissolved in the same solution, at the concentrations of 2 pg,
200 pg and 20 ng/ml. Sodium ascorbate (3 g) was dissolved in an
ethanol–propylene glycol mixture (100 ml) and used as a positive
control. Sample solutions (50 μl/mouse) were topically applied in the
dorsal region. Therefore, total ginseng saponin was applied at the
dose of 10 pg and 100 ng/mouse, ginsenoside Rb1was applied at the
dose of 100 fg, 10 pg and 1 ng/mouse, and vitamin C was applied at
1.5 mg/mouse. Rat monoclonal anti-mouse Ki-67 antibody, rabbit
polyclonal anti-rat biotin-labeled immunoglobulin antibody, and
peroxidase-labeled streptoavidin were purchased from DakoCyto-
mation (Kyoto, Japan). Anti-8-hydroxy-2′-deoxyguanosine (8-OHdG)
(clone N45.1) and rabbit anti-mouse collagen type I were purchased
from Japan Institute for the Control of Aging (Shizuoka, Japan) and
CEDARLANE Lab. Lt. (Ontario, Canada), respectively. Rabbit mono-
clonal anti-Bcl-2 (clone 50E3), rabbit polyclonal anti-Bax, and rabbit
polyclonal anti-Bak antibodies were purchased from Cell Signaling
Technology Inc. (Beverly, MA, USA). Mouse monoclonal anti-β-actin
antibody was purchased from Sigma. Other chemicals were of
Human keratinocytes, human keratinocyte basal medium (KB-2,
0.15 mM Ca2+), and KG-2 medium (KB-2 medium containing 10 μg/ml
of insulin, 0.1 ng/ml of human recombinant epidermal growth factor
(hEGF), 0.5 μg/ml of hydrocortisone, 50 μg/ml of gentamycin, 50 ng/ml
of amphotericin B, and 0.4% (v/v) bovine hypophysis extract) were
purchased from Kurabo Co. (Tokyo, Japan). To evaluate the effects of
natural compounds on UVB-induced biological changes, human
keratinocytes are used in a medium containing a low calcium
concentration (0.15 to 0.3 mM) and 80% subconfluent (Adhami et al.,
2003; Onoue et al., 2003; Wang and Kochevar 2005; Ishida and
Sakaguchi 2007). Therefore, we also examined the effects of ginseno-
side Rb1 on UVB-irradiated Bcl-2 and Bax expression in human
keratinocytes cultured under the conditions of a low calcium
concentration at 0.15 mM and 80% subconfluent.
Malealbino hairless HOS: HR-1 mice (5 weeks old) werepurchased
from Hoshino Laboratory Animals Co. Ltd. (Saitama, Japan), housed for
1 week in a temperature-controlled room at 25±1 °C and 60% relative
humidity, and given free access to standard laboratory diet and water
before the experiments. Mice were treated according to the Ethical
Guidelines of the Animal Center, Graduate School of Medicine, Ehime
University, and the experimental protocol was approved by the
Animal Studies Committee of Ehime University.
2.4. Measurement of the skin thickness, elasticity, and wrinkles induced
by UVB irradiation
To examine the effects of total ginseng saponins and ginsenoside
Rb1 on skin thickness, elasticity, and wrinkles induced by UVB
irradiation, a UVB lamp (15 W type, UV maximum wavelength
312 nm; UV intensity 100 μW/cm2; Ieda Boeki Co., Tokyo, Japan) was
used in this study. The time of UV irradiation was varied to control the
UVB energy applied to the dorsal region of each mouse. The value of
the minimal erythema dose (MED) per mouse was about 36 mJ/cm2.
Total ginseng saponins (10 pg and 100 ng/mouse), ginsenoside Rb1
(100 fg,10 pg, and 1 ng/mouse)and 3% vitamin C (1.5 mg/mouse) were
applied topically to the dorsal region of each mouse every day for
12 weeks. The initial dose of UVB was set at 36 mJ/cm2, which was
subsequently increased to 54 mJ/cm2at weeks 1–4, 72 mJ/cm2at
weeks 4–7, 108 mJ/cm2at weeks 7–10, and finally to 122 mJ/cm2at
weeks 10–12. The frequency of UVB irradiation was set at three times
per week before the topical application of vehicle (control), the
indicated amounts of total ginseng saponins, or ginsenoside Rb1. In
this protocol, wrinkles began to be observed macroscopically in the
dorsal region from about 6 weeks after the initiation of UVB
irradiation. The skin thickness and elasticity after UVB irradiation
were measured every week using a Quick Mini Caliper (Mitutoyo Co.,
Kanagawa, Japan) and a Digimatic Caliper (Mitutoyo Co., Kanagawa,
Japan), respectively. To evaluate the formation of wrinkles after the
UVB irradiation, each hairless mouse was anesthetized with an
intraperitoneal injection of pentobarbital (50 mg/kg body weight) at
6 and 9 weeks, and then the UVB-irradiated dorsal area (site of
wrinkle formation) was photographed. The degree of wrinkle
formationwasassessedfromthe photographof each animalaccording
to the grading scale described in Table 1, whereas the name of the
animal group was kept blind; this is a modification of the method
described by Bissett et al. (1987).
2.5. Measurement of TGF-β1 and glutathione in UVB-irradiated skin
At week 12, all mice were sacrificed by the overdose of pento-
barbital, and then all skin tissues were quickly removed. The removed
skin tissue (100 mg) was washed in phosphate buffered saline (PBS,
pH 7.0) and cut into small pieces, and then tissue protein extraction
reagent (T-PER) containing protease inhibitor (Pierce Co., Rockford, IL,
USA) (2 ml) was added to the skin tissue and the mixture was
homogenized. After the skin homogenate was centrifuged at 2000 ×g
Fig. 1. The structure of ginsenoside Rb1.
Grading of mouse skin wrinkles
No coarse wrinkles
A few shallow, coarse wrinkles across the back skin area are observed
occasionally (Bisset's Grade 1)
Shallow, coarse wrinkles across the back skin are observed on the whole
surface (Bisset's Grade 2)
Some deep, long wrinkles across the back skin are observed (Bisset's Grade 3)
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