Promotion of hair growth by ginseng radix on cultured mouse vibrissal hair follicles
PHYTOTHERAPY RESEARCH Phytother. Res.17, 797–800 (2003)
PROMOTION OF HAIR GROWTH BY GINSENG RADIX
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/ptr.1241 Promotion of Hair Growth by Ginseng Radix on Cultured Mouse Vibrissal Hair Follicles Hideaki Matsuda1*, Miho Yamazaki1, Yusuke Asanuma2 and Michinori Kubo1 1Faculty of Pharmaceutical Sciences, Kinki University, 3-4-1, Kowakae, Higashiosaka, Osaka, 577-8502, Japan 2Daiichi Pharmaceutical Co., Ltd., 3-14-10, Nihonbashi, Chuo-ku, Tokyo, 103-8234, Japan A 70% methanol extract from red ginseng (steamed and dried roots of Panax ginseng C. A. Meyer, a kind of Ginseng Radix) had superior activity to that of white ginseng (peeled and dried root of P. ginseng, another kind of Ginseng Radix) in a hair growth promoting assay using mouse vibrissal follicles in organ culture. Of the major constituents of P. ginseng, ginsenoside-Rb (G-Rb ) exhibited activity, but ginsenoside-Rg (G-Rg ) and -Ro (G-Ro) were ineffective. Additionally, 20(S)-ginsenoside-Rg (20(S)-G-Rg ) formed by the processing of red ginseng from the crude root of P. ginseng also showed hair growth promoting activity. These results indicate that Ginseng Radix possesses hair growth promoting activity, and its bioactive components are partially attributable to the ginseng saponin components mentioned above. Copyright 2003 John Wiley & Sons, Ltd. Keywords:Panaxginseng; Ginseng Radix; ginsenoside; hair growth activity; organ culture.
activity (Kubo et al., 1988). In order to confirm the hair
INTRODUCTION
growth promoting activity of Ginseng Radix and toidentify the active constituents, a newer in vitro and/or
Ginseng Radix is an important crude drug that has been
in vivo hair growth evaluation system was sought.
used from ancient times to improve constitutional ten-
In recent years, the study of hair growth promoting
dencies to poor body condition, to promote appetite,
agents has greatly advanced. Several animal models,
to increase vitality and to reduce over-sensitivity to cold.
beginning with the C3H mouse (Hattori and Ogawa,
Pharmacological evidence shows that ginseng improves
1983) have been used experimentally to evaluate the
blood circulation and accelerates both metabolism and
extent of hair growth. Newer techniques for evaluation
digestion. In the Japanese Pharmacopoeia Fourteenth
based on cell cultures, such as dermal papillae cells,
Edition (2001), two kinds of Ginseng Radix, namely,
hair matrix cells and outer root sheath cells (ORSCs)
red ginseng (steamed and dried root of Panax ginseng)
have been developed to analyse quantitatively the
and white ginseng (peeled and dried root of P. ginseng,
extent of hair growth in vitro (Messenger, 1984; Arase
without steaming) are described. Many pharmacological
et al., 1991; Fujie et al., 1993). Among the cells that
and chemical studies have been carried out on these
constitute hair tissue it is possible to perform a succes-
two differently processed roots. Conclusive evidence
sive culture of dermal papillae cells and ORSCs,
indicates that red ginseng is superior to white ginseng
however, this has not yet been confirmed in the case
as the former contains numerous effective components
of matrix cells. Because of the complicated structure
(Matsuura et al., 1984; Kitagawa et al., 1983; Samukawa
of hair tissue, it is presumed that even if epithelial or
et al., 1995) and possesses stronger physiological
mesenchymal cells could be cultivated separately clear
activity compared with the latter (Matsuda et al., 1985,
results about hair growth would not be forthcoming.
1986, 1987a, 1987b; Matsuda and Kubo, 1983, 1984,
More recently, organ culture methods of hair folli-
1985). Ginsenoside-Rg , an artifact formed in the pre-
cles have been developed to evaluate quantitatively the
paration of red ginseng from crude root of P. ginseng
extent of hair growth in vitro (Jindo and Tsuboi, 1997;
(Kitagawa et al., 1983, 1989; Kasai et al., 1983), has been
Imai et al., 1993; Jindo et al., 1993; Philpott et al., 1990).
reported to show inhibitory effects on blood aggrega-
The organ culture method separately cultivates each
tion (Yamamoto et al., 1988) and on the metastasis of
individual hair follicle. This evaluation system is thought
tumour cells (Sato et al., 1994). Furthermore, it increases
to be correlated with in vivo systems because the ex-
blood circulation (Matsuda et al., 2000). In our prelimi-
tent of hair growth can be observed as the sum of the
nary hair growth promoting assay of several Chinese
functions of each cell (Jindo and Tsuboi, 1997). Thus,
crude drugs using the ddY strain mouse model, it has
the activity of two kinds of Ginseng Radix and major
been found that a 50% ethanol extract from Gin-
ginseng saponins were examined using the organ
seng Radix (roots of P. ginseng) exhibited significant
culture of vibrissal hair follicles. This report deals withthe hair growth promoting effect of Ginseng Radix onvibrissal hair follicles (excised from the upper lip region
* Correspondence to: Dr H. Matsuda, Faculty of Pharmaceutical Sciences,Kinki University, 3-4-1 Kowakae, Higashiosaka, Osaka, 577-8502, Japan.
of mice) in order to explore natural resources involved
in the acceleration and promotion of hair growth.
Copyright 2003 John Wiley & Sons, Ltd. Phytother. Res. 17, 797–800 (2003)
Copyright 2003 John Wiley & Sons, Ltd.
steel mesh and lens paper were placed. Three vibrissal
MATERIALS AND METHODS
hair follicles were placed on the lens paper in each dishand immersed into the medium so as to be covered
Plant materials. Red ginseng and white ginseng orig-
with a thin layer of medium. Each group consisted of
inating from the roots of P.ginseng, cultivated in
four dishes. After pre-incubation for 12 h, the med-
Korea, were provided by Japan Korea Red Ginseng
ium was exchanged for one containing each sample
solution. The samples dissolved with 50% ethanolwere added to the medium (final concentration: sam-
Preparation of 70% methanol extract from Red Ginseng
ples; 1–50 µg/mL, ethanol; 0.2% (v/v)). The medium was
or White Ginseng and ginsenosides. The crushed roots
changed during the culture period (72 h) every 24 h.
of red ginseng or white ginseng were extracted twice
Human hepatocyte growth factor/scatter factor (HGF/
with 70% methanol, under reflux, for 2 h. These ex-
SF, R&D Systems Inc., MN, USA) was used as a posi-
tracts were evaporated under reduced pressure and
tive control (Jindo and Tsuboi, 1997).
then lyophilized to give two 70% methanol extracts(RG-ext, yield: 32.0%, WG-ext, yield: 19.9%). G-Rb ,
Measurement of length of vibrissal hair follicle. The
G-Rg , G-Ro and 20(S)-G-Rg were isolated from red
individual hair follicles were photographed immediately
ginseng by the method previously described (Kitagawa
prior to and 48 h and 72 h after the start of incubation
et al., 1983, 1989). The ginsenoside content in these
(magnification × 20). Changes in hair length were cal-
extracts was determined using the high performance
culated from the photographs and expressed as mean ±
liquid chromatography (HPLC) method as described
SE of 11–12 vibrissal hair follicles.
by Samukawa et al. (1995). The HPLC conditions wereas follows: apparatus, Shimadzu LC-6A system equipped
Statistical analysis. The experimental data were tested
with a Shimadzu SPD-6A detector, Shimadzu SLC-6A
for statistical significance using Bonferroni/Dunn’s
system controller, Shimadzu CTO-6A column oven and
Shimadzu chromatopac C-R3A; column, SuperspherRP-18(e) (4.0 i.d. × 250 mm, Merck); eluent, (A)CH CN–H O–0.1% H PO (21:72:8 v/v), (B) CH CN,
flow rate 0.8 mL/min [linear gradient flow programme:
RESULTS AND DISCUSSION
(A) 0–19 min; 100%, 19–20 min; 100%–90%, 20–73 min90%, 73–103 min; 90%–70%, 103–130 min; 70%];
As shown in Table 1, RG-ext promoted hair growth
column temperature: programme (0–30 min; 35 °C, 30–
in a dose-dependent manner after both 48 h and 72 h
60 min; 55 °C, 60–130 min; 35 °C; detection, UV 202 nm;
of culture. It exhibited its most significant effect at a
retention time (min), G-Rb ; 68, G-Rg 27, G-Ro; 72 and
dosage of 50 µg/mL. The effect of WG-ext was lower
20(S)-G-Rg ; 123. The results are shown in Table 3.
compared with that of RG-ext. These results suggest
that ginsenosides play a major role in Ginseng Radix
Animals. B6C3F mice were used for all experimental
mediated hair growth promotion since they are found
procedures. Three to eight-day-old pups with their
more abundantly in RG-ext compared with WG-ext.
mothers were purchased from Japan SLC (Shizuoka,
To date, 26 saponins have been isolated and ident-
Japan). The mice were maintained in an air-conditioned
ified in Ginseng Radix. These saponins are divided
room with light from 7 a.m. to 7 p.m. The room tem-
broadly into three groups termed panaxadiols, pana-
perature (about 23 °C) and humidity (about 60%) were
xatriols and oleananes based on the basic structure
controlled automatically. Laboratory pellet chows (Labo
of aglycones produced by hydrolysis. Among these
MR Stock, Nihon Nosan Kogyo) and water were given
saponins, G-Rb in the panaxadiol saponins, G-Rg in
freely. Nine-day-old mice were used for experiments.
the panaxatriol saponins and G-Ro (a peculiar com-ponent in Ginseng Radix) in the oleanane saponins are
Organ culture of mouse vibrissal hair follicles. Hair
relatively abundant in Ginseng Radix. In order to clarify
growth activity using an organ culture of mouse vibrissal
the active ingredient, the hair growth promoting effects
hair follicles was performed according to the method
of these three saponins were investigated.
described by Jindo and Tsuboi (1997) with minor modi-
As shown in Table 2, G-Rb promoted hair growth
fications. Under the stereomicroscope, normal anagen
in a dose-dependent manner, with the most significant
vibrissal hair follicles were obtained from these mice
effect seen at 10 µg/mL after 48 h culture. G-Rg and
under ether anaesthesia using a scalpel and tweezers.
G-Ro, however, did not exhibit any similar effects. It
The vibrissal hair follicles were removed from the
has been reported that G-Rb can promote nerve fibre
upper lip region. A total of nine intact follicles from
growth (Saito et al., 1977), protein synthesis (Shibata
the two ventral and dorsal rows nearest the eye were
et al., 1976) and RNA polymerase activity (Iijima et al.,
harvested from each pad. Follicles from each litter of
1976). In the organ culture system of mouse vibrissal
pups were pooled. Isolated vibrissal hair follicles of mice
hair follicles, it can be assumed that G-Rb acts as an
were placed in RPMI 1640 medium (Gibco™, Invitro-
activator for promoting hair growth at a cellular level.
gen Corp., CA USA) containing 50 unit/mL penicillin
From the results in Table 3, it seems quite probable
and 50 µg/mL streptomycin (penicillin–streptomycin,
that Rb is one of active ingredients because it is present
Gibco™, Invitrogen Corp.). After washing with RPMI
in relatively large quantities in RG-ext.
1640 medium, the vibrissal hair follicles were cultured
Some of the panaxadiol saponins (including
in RPMI 1640 medium at 31 °C in 95% O –5% CO .
ginsenoside-Rb , -Rb , -Rc and -Rd) are known to be
Each organ culture dish (Falcon 3037, Becton-Dickinson
converted to 20(S)- and 20(R)-G-Rg during the pro-
Labware, Franklin Lakes, NJ, USA) contained 0.75 mL
cessing of the crude root of P. ginseng to red gin-
of the medium in the centre well, over which a stainless
seng (Kitagawa et al., 1983, 1989; Kasai et al., 1983).
Copyright 2003 John Wiley & Sons, Ltd. Phytother. Res. 17, 797–800 (2003)
PROMOTION OF HAIR GROWTH BY GINSENG RADIX
Table 1. Hair growth effect of 70% methanol extract from red ginseng (RG-ext) or white ginseng (WG-ext) on organ culture of mouse vibrissal hair follicles
Individual vibrissal hair follicles from B6C3F mouse were micro-dissected and cultured in
RPMI 1640 medium at 31 °C in 95% O –5% CO . After pre-incubation for 12 h, medium was
exchanged for medium containing sample solutions. The individual hair follicles were photo-graphed immediately prior to 48 h and 72 h after the start of incubation. Changes in hair lengthwere calculated from the photographs and reported as mean ± SE of 11–12 vibrissal hairfollicles. Significantly different from the control group. a p < 0.05. Table 2. Hair growth effects of ginsenoside-Rb (G-Rb ), -Rg (G-Rg ) and -Ro (G-Ro) on organ culture of mouse vibrissal hair follicles
According to the method in Table 1. Each value represents the mean ± SE of 11–12 vibrissalhair follicles. Significantly different from the control group. a p < 0.05. Table 3. Ginsenoside contents in red ginseng (RG-ext) or white ginseng (WG-ext)
Values are average calculated from three measurements of each ginseng (n = 3). Table 4. Hair growth effect of 20(S)-ginsenoside-Rg (20(S)-G-Rg ) and human hepatocyte growth factor/scatter factor (HGF/SF) on organ culture of mouse vibrissal hair follicles
According to the method in Table 1. Each value represents the mean ± SE of 11–12 vibrissalhair follicles. Significantly different from the control group. a p < 0.01.
20(S)-G-Rg significantly promoted hair growth at
a dosage of 10 µg/mL after 48 h of culture (Table 4). CONCLUSIONS
As shown in Table 4, a positive control, HGF/SF,showed 8.4% and 4.2% activation after 48 and 72 h,
Commercially available hair care agents include anti-
inflammatory, antiandrogenic and antibacterial agents
Copyright 2003 John Wiley & Sons, Ltd. Phytother. Res. 17, 797–800 (2003)
along with vitamins and moisturizers. Ginseng Radix
tional energy and thereby stimulate hair growth. The
extracts are also found in such hair care agents.
results presented here indicated that Ginseng Radix
However, no detailed explanations are given about
possesses hair growth promoting activity and that G-
the mechanism of hair growth or the major active
Rb may be one of the active constituents of Ginseng
constituents. Increased blood circulation around the
Radix in the mouse vibrissal hair follicle organ culture
head dermis may provide hair roots with addi-
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In Proceeding of the Sixth International Tinnitus Seminar, Cambridge 1999 Intracranial pressure as a generator of aural noises: Improved differential diagnosis will facilitate effective treatments Marchbanks R.J. Consultant Scientist Director, Non-invasive Intracranial Assessment Unit (NIPA), Medical Physics Department, Southampton University Hospital, Southampton SO16 6YD Keywords: intr