HAIR GROWTH–PROMOTING EFFECT OF L. ESCULENTUM EXTRACT 431 On the basis of this reasoning, much research has been performed for natural crude drugs having 5α-reductase inhibitory activities (15–17). However, there remains a demand for a highly effective 5α-reductase inhibitor or hair growth promoter with an excellent safety and effi cacy profi le. L. esculentum is one of the most popular vegetables and lycopene is the principle carot- enoid causing the characteristic red hue of L. esculentum (18). Several reports show that diet rich in lycopene has benefi cial effects on human health (19,20). A possible role has been suggested for L. esculentum and its products in preventing cardiovascular disease (19,21) and protecting against some types of cancer (20,22). Especially, epidemiological studies have consistently showed an association between high intakes of lycopene and reduced prostate cancer risk. The lycopene contributed to the reduction of prostate cancer by interfering with logical testosterone activation by down- regulating 5α-reductase, and consequently reduced steroid target genes expression (22). On the basis of previous reports, our attention was drawn to the possibility of hair growth–promoting activity of L. esculentum extract, which contains high level of lyco- pene, regulating 5α-reductase activity. Therefore, in this study we evaluated the hair growth–promoting effect and the effect on several growth factors involved in hair growth of L. esculentum extract and isolated lycopene in C57BL/6 mice. MATERIALS AND METHODS SAMPLE PREPARATION AND MATERIALS L. esculentum was provided by Chal-Tomato Co., Ltd. (Daejeo, Busan, Korea). Fresh L. escu- lentum was thoroughly washed with tap water. After washing, the seeds were removed and the L. esculentum was chopped into cubes. Then, the cubes were freeze-dried for 3 days until a moisture content of ca. 0.8% was reached. The freeze-dried L. esculentum were then ground in a mill to pass through a 500-mesh sieve and then stored at −20 °C until use (23). PREPARATION OF THE L. ESCULENTUM EXTRACT The ethyl acetate extract (EAE) of L. esculentum and the isolated (=semipurifi ed) lycopene from L. esculentum was prepared according to the method of Roh et al. (23). In brief, EAE was ex- tracted from 100 g of the powdered freeze-dried L. esculentum with 1 L of ethyl acetate on the orbital shaking machine (30 rpm) for 30 min. Then, the resulting L. esculentum EAE was evaporated at the reduced pressure of up to 10% of the initial volume. Isolated lycopene (0.14 g 77.0%) from of L. esculentum EAE, using an antisolvent (methanol) salting-out method was prepared, and then dissolved in 100 ml Tween 80. The resulting lycopene Tween 80 solution was designated as LTS. Supercritical CO2 extraction of L. esculentum powder (100 g) was performed at 45 °C in 300 bars for 4 h, according to the method of Cadoni et al. (24). ANIMALS Twenty-fi ve female C57BL/6 mice (5 weeks old upon receipt SLC, Shizuoka, Japan) were treated after acclimatizing to the laboratory conditions for 7 days. Animals were allocated

JOURNAL OF COSMETIC SCIENCE 432 fi ve per polycarbonate cage in a temperature (20°C) and humidity (40–45%)-controlled room. The light/dark cycle was 12:12 h and food (Samyang, Wonju, Korea) and water were supplied ad libitum. DETERMINATION OF HAIR GROWTH–PROMOTING ACTIVITY Hair growth–promoting activity of the L. esculentum extract was determined by the method reported by Roh et al. (25), with some modifi cations. Briefl y, 6-week-old C57BL/6 mice were randomly divided into fi ve groups for fi ve treatments, as follows: the negative control (NC) group (10% ethanol as a vehicle), positive control (PC) group (3% minoxidil), L. esculentum extract 1 group [3% (w/w) of EAE], extract 2 group [3% (w/w) of supercritical CO2 extract (SCE)], and extract 3 group [3% (w/w) of LTS]. Hair was removed from the 2 cm × 3 cm dorsal area of these mice by shaving carefully with an electric clipper. The substances and test materials were applied topically on the back skin of the mice, once a day for 4 weeks. The hair growth–promoting activity of the substances was checked by the darkening of the dorsal skin, which indicated the anagen phase of the hair follicles. The hair growth scoring was performed by two independent dermatologists, who were unaware of treatment regimen and we used the average of the two scores. Hair growth was measured at every 1 week during 4 weeks by assigning a hair growth index, as follows: score 0 = no growth observed 1 = up to 20% area of skin covered with hair 2 = 20–40% area of skin covered with hair 3 = 40–60% area of skin covered with hair 4 = 60–80% area of skin covered with hair and 5 = 80% to full area of skin covered with hair observed. Digital images of total hair growth on day 28 were obtained using Nikon Coolpix P100 (Nikon Co., Tokyo, Japan). RNA EXTRACTION AND REAL-TIME RT-PCR Total RNA extraction was performed with Trizol reagent (Life Technologies, Gaithers- burg, MD), and the cDNA was synthesized by a reverse transcription reaction using the RNA PCR kit (Applied Biosystems Roche Inc., Foster City, CA) in a 20 μl mixture containing 1 μg RNA, 50 mM KCl, 10 mM Tris/HCl, 5 mM MgCl2, 1 mM of each dNTPs, oligo-(dT) primers, 20 units of RNAse inhibitor, and 50 units of MuLV reverse transcriptase. Nucleotide sequence of the primers used in this study is shown in Table I. The reaction mixture was incubated for 60 min at 42 °C, then heated at 90°C for 7 min in a thermocycler (GeneAmp PCR system 9600 PerkinElmer, Roche Molecular System, Branchburg, NJ). Real-time PCR was performed using a lightcycler instrument using FastStart DNA Master SYBR Green PCR kit (Roche, Mannheim, Germany). Quantifi - cation of the VEGF, keratinocyte growth factor (KGF), IGF-I, and TGF-β mRNA ex- pression was corrected by glyceraldehyde 3-phosphate dehydrogenase (GAPDH). TEST HAIR TONIC WITH L. ESCULENTUM EXTRACT For possible future application in hair growth–promoting agents and pharmaceutical products for hair growth and alopecia prevention, the test hair tonic solution with LTS was prepared and evaluated for hair growth–promoting activity. At fi rst, 70 ml aliquots