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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: Panax ginseng; 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- REFERENCES
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