Effects of Lycopersicon esculentum extract on hair growth and alopecia prevention

Jae-Suk Choi; Sung Kyu Jung; Min-Hee Jeon; Jin-Nam Moon; Woi-Sook Moon; Yi-Hwa Ji; In Soon Choi; Sang Wook Son

HAIR GROWTH–PROMOTING EFFECT OF L. ESCULENTUM EXTRACT 435 When the quantity of VEGF of mouse skin tissue treated with EAE, SCE, LTS, and min- oxidil was evaluated, the amount of RT-PCR products was found higher by 8.09%, 10.29%, 10.29%, and 5.14 % than that in the negative group. In KGF, the amount of RT-PCR products was higher by 9.29%, 11.43%, 12.14%, and 5.00% than that in the negative group. In IGF, the amount of RT-PCR products was higher by 12.68%, 15.49%, 13.38%, and 8.45% than that in the negative group. In TGF-β, the amount of RT-PCR products was higher by 8.46%, 10.80%, 10.00%, and 5.38% than that in the negative group. The mRNA expression of VEGF, KGF, and IGF-I in the 3% L. esculentum SCE group and 3% LTS group was found to be approximately two times more than those in the 3% minoxi- dil group. On the basis of these results, further tests were performed using LTS. Figure 1. Hair growth change of C57BL/6 mice after topical application of L. esculentum extracts at 4 weeks after treatment. Group 1: NC (ethanol) group 2: 3% L. esculentum EAE group 3: 3% L. esculentum SCE group 4: 3% LTS group 5: PC (3% minoxidil). Figure 2. Hair growth index of C57BL/6 mice after topical application of L. esculentum extracts for 4 weeks. Group 1: NC (ethanol) group 2: 3% L. esculentum EAE group 3: 3% L. esculentum SCE group 4: 3% LTS group 5: PC (3% minoxidil).

JOURNAL OF COSMETIC SCIENCE 436 HAIR GROWTH–PROMOTING EFFECT OF HAIR TONIC CONTAINING 3% LTS After daily treatment for 4 weeks the C57BL/6 female mice treated with the hair tonic containing 3% LTS show increased hair growth compared to the NC group. The PC group treated with hair tonic containing 3% minoxidil showed hair growth levels similar to the 3% LTS product (Figs 4 and 5). EFFECT OF TEST HAIR TONIC CONTAINING 3% LTS ON THE MRNA LEVEL OF GROWTH FACTORS According to Fig. 6, increases in IGF-1 and a decrease in VEGF and TGF-β expression were signifi cant for THT. No signifi cant increases were observed in KGF over the NC. Figure 3. Comparison of (A) VEGF, (B) KGF, (C) IGF-1 and (D) TGF-β expression in C57BL/6 mice after topical application of experimental materials, NC: negative control (10% ethanol as a vehicle), EAE: 3% L. esculentum ethyl acetate extract, SCE: 3% L. esculentum supercritical CO2 extract, LTS: 3% isolated lycopene Tween 80 solution, PC: positive control (3% minoxidil) for 4 weeks. *p 0.05, **p 0.01 compared with control. Figure 4. Hair growth change of C57BL/6 mice after topical application of hair tonic product with 3% LTS during 4 weeks. Group 1: NC (10% ethanol) group 2: THT (tonic product with 3% LTS) group 3: PC (3% minoxidil).

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HAIR GROWTH–PROMOTING EFFECT OF L. ESCULENTUM EXTRACT 435 When the quantity of VEGF of mouse skin tissue treated with EAE, SCE, LTS, and min- oxidil was evaluated, the amount of RT-PCR products was found higher by 8.09%, 10.29%, 10.29%, and 5.14 % than that in the negative group. In KGF, the amount of RT-PCR products was higher by 9.29%, 11.43%, 12.14%, and 5.00% than that in the negative group. In IGF, the amount of RT-PCR products was higher by 12.68%, 15.49%, 13.38%, and 8.45% than that in the negative group. In TGF-β, the amount of RT-PCR products was higher by 8.46%, 10.80%, 10.00%, and 5.38% than that in the negative group. The mRNA expression of VEGF, KGF, and IGF-I in the 3% L. esculentum SCE group and 3% LTS group was found to be approximately two times more than those in the 3% minoxi- dil group. On the basis of these results, further tests were performed using LTS. Figure 1. Hair growth change of C57BL/6 mice after topical application of L. esculentum extracts at 4 weeks after treatment. Group 1: NC (ethanol) group 2: 3% L. esculentum EAE group 3: 3% L. esculentum SCE group 4: 3% LTS group 5: PC (3% minoxidil). Figure 2. Hair growth index of C57BL/6 mice after topical application of L. esculentum extracts for 4 weeks. Group 1: NC (ethanol) group 2: 3% L. esculentum EAE group 3: 3% L. esculentum SCE group 4: 3% LTS group 5: PC (3% minoxidil).

JOURNAL OF COSMETIC SCIENCE 436 HAIR GROWTH–PROMOTING EFFECT OF HAIR TONIC CONTAINING 3% LTS After daily treatment for 4 weeks the C57BL/6 female mice treated with the hair tonic containing 3% LTS show increased hair growth compared to the NC group. The PC group treated with hair tonic containing 3% minoxidil showed hair growth levels similar to the 3% LTS product (Figs 4 and 5). EFFECT OF TEST HAIR TONIC CONTAINING 3% LTS ON THE MRNA LEVEL OF GROWTH FACTORS According to Fig. 6, increases in IGF-1 and a decrease in VEGF and TGF-β expression were signifi cant for THT. No signifi cant increases were observed in KGF over the NC. Figure 3. Comparison of (A) VEGF, (B) KGF, (C) IGF-1 and (D) TGF-β expression in C57BL/6 mice after topical application of experimental materials, NC: negative control (10% ethanol as a vehicle), EAE: 3% L. esculentum ethyl acetate extract, SCE: 3% L. esculentum supercritical CO2 extract, LTS: 3% isolated lycopene Tween 80 solution, PC: positive control (3% minoxidil) for 4 weeks. *p 0.05, **p 0.01 compared with control. Figure 4. Hair growth change of C57BL/6 mice after topical application of hair tonic product with 3% LTS during 4 weeks. Group 1: NC (10% ethanol) group 2: THT (tonic product with 3% LTS) group 3: PC (3% minoxidil).

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HAIR GROWTH–PROMOTING EFFECT OF L. ESCULENTUM EXTRACT 433 of distilled water were distributed into a glass beaker. A 0.3 g nicotine amide and 0.5 g salicylic acid were added and dissolved, and then the fi nal volume was brought up to 100 ml immediately. The formulations studied were prepared in a PRIMIX RM homomixer (PRIMIX Co., Ltd., Osaka, Japan) at 500 rpm within 5 min and supplemented with 3% (w/w) of LTS. A placebo formulation was prepared without LTS. HISTOLOGICAL ANALYSIS OF HAIR FOLLICLES The substances and test materials were applied topically on the back skin of the mice, once a day for 4 weeks. After week 4, all of the mice were sacrifi ced. Their dorsal skins were removed and fi xed in 4% formaldehyde solution and embedded in paraffi n. The fragments were sectioned into two different patterns: transverse sections for determina- tion of hair follicle count and longitudinal sections for the overall histological assessment. The 3-μm sections were stained with hematoxylin–eosin and toluidine blue and exam- ined under a light microscope (Magnifi cation: ×200) (Olympus, Melville, NY). DRAIZE SKIN IRRITATION TEST The irritation potentials of the test hair tonic solution with 3% LTS were evaluated accord- ing to the method of Draize (26) with slight modifi cation (27). Briefl y, two male New Zealand White (NZW) rabbits weighing 2.5–3.0 kg were acclimatized for 5 days before starting the study. The back of each rabbit was clipped free of hair and then divided into four sectors as shown in Fig. 8: after abrasion and application of hair tonic (upper left), after abrasion and no application of hair tonic (upper right), no abrasion and application of hair tonic (lower left), and no abrasion and no application of hair tonic as NC (lower right). The area of each sector was 6.25 cm2 (2.5 cm ×2.5 cm). For abrasion, several layers of skin were removed with adhesive tape from one half (upper side) of the shaved backs. Adhesive tape stripping was done about fi ve times. A 0.5 ml of the test hair tonic solution with 3% LTS was applied once, uniformly, on the left side only of the hair-free skin of each rabbit. Table I Nucleotide Sequence of the Primers Used For PCR Amplifi cations in This Study Growth Factor Primer Sequence VEGF Forward ACS CGG TGG TGG AAG AAG AG Reverse CAA GTC TCC TGG GGA CAG AA KGF Forward ACG AGG CAA AGT GAA AGG GA Reverse TGC CAC AAT TCC AAC TGC CA IGF-1 Forward TCA TGT CGT CTT CAC ACC TCT TCT Reverse CCA CAC ACG AAC TGA AGA GCA T TGF-β Forward GCG GCA GCT GTA CAT TGA CT Reverse ACT GTG TGT CCA GGC TCC AA GAPDH Forward CAA TGA ATA CGG CTA CAG CAA C Reverse AGG GAG ATG CTC AGT GTT GG

JOURNAL OF COSMETIC SCIENCE 434 Skin was observed for erythema or edema as required by the Draize test (0: no erythema or no edema 1: barely perceptible erythema or edema 2: well-defi ned erythema or slight edema 3: moderate to severe erythema or moderate edema 4: severe erythema or edema) at 24 and 72 h after application (28,29). The Draize test was done and evaluated accord- ing to the Korea Food and Drug Administration guideline. ANALYSIS BY HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY FOR THE QUANTIFICATION OF LYCOPENE The quantifi cation analysis of the lycopene of the LTS was performed using a Waters 600E HPLC system (Waters Co., Milford, MA) equipped with Waters 486 UV detector, as de- scribed by Rho et al. (23). The chromatographic analysis was conducted using a reverse- phase ZORBAX Eclipse plus C18 column (4.6 mm × 250 mm Agilent, Santa Clara, CA) with 5 μm particles. The characterization of the lycopene extracts was performed in isocratic mode and the mobile phase used was methanol/THF (90:10 v/v). Lycopene samples were dissolved in 20 μl of a methanol/hexane (1:2 v/v) solution. Chromatographic separation of extracts was performed at a constant fl ow rate of 1 ml/min. Lycopene was detected at 472 nm. For quantitative analysis, standard lycopene (Sigma L9879 Sigma-Aldrich, Ltd., St. Louis, MO) was also analyzed using the high-performance liquid chromatography (HPLC) system under the same conditions. STATISTICAL ANALYSIS Analysis of variance as a statistical analysis was performed using SPSS (version 12.00 SPSS Inc. Chicago, IL). A value of p 0.05 was considered statistically signifi cant. RESULTS YIELD OF L. ESCULENTUM EXTRACT The yields of EAE, SCE, and LTS of L. esculentum (100 g) were 3.8 g, 0.75 g, and 0.014 g, respectively. HAIR GROWTH–PROMOTING EFFECT OF L. ESCULENTUM EXTRACT After 4 weeks of daily topical treatment on C57BL/6 female mice, 3% (w/w) formulations of EAE, SCE, and LTS all showed hair growth–promoting activity greater than the NC. The hair growth–promoting activity of LTS was also similar to that of the 3% Minoxidil control (PC) (Figs 1 and 2). EFFECT OF L. ESCULENTUM EXTRACT ON THE MRNA LEVEL OF GROWTH FACTORS According to Fig. 3, increases in VEGF and IGF-1 were signifi cant only for SCE and LTS. Increases in KGF were signifi cant only for LTS, and there were no signifi cant increases in TGF-β over the NC.

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HAIR GROWTH–PROMOTING EFFECT OF L. ESCULENTUM EXTRACT 437 When the quantity of VEGF of mouse skin tissue treated with the test hair tonic product that contained 3% LTS or 3% minoxidil was evaluated, the amount of RT-PCR product- treated hair tonic with LTS was lower by 42.31% and 3.85% than that in the NC and PC, respectively. In KGF, the amount of RT-PCR products was higher by 41.67% and 133.33% than that in the NC and PC, respectively. In IGF-1, the amount of RT-PCR products was higher by 17.86% than that in the NC, and higher by 257.14% than that in the PC, respectively. In TGF-β, the amount of RT-PCR products was lower by 45.84% than that in the NC and lower by 15.28% than that in the PC, respectively (Fig. 6). ANAGEN INDUCTION AND HAIR RESTORATION OF FEMALE C57BL/6 MICE BY TEST HAIR TONIC PRODUCT WITH 3% LTS The morphological structure of the skin, obtained from a longitudinal section of the dorsal skin is shown in Fig. 7. In NC, early anagen was observed. Further, dyeing by toluidine blue was slightly observed in the epidermal cells of hair follicles undergoing anagen induction and Figure 5. Hair growth index of C57BL/6 mice after topical application of hair tonic product with 3% LTS for 4 weeks. Group 1: NC (10% ethanol) group 2: THT (tonic product with 3% LTS) group 3: PC (3% minoxidil). Figure 6. Comparison of (A) VEGF, (B) KGF, (C) IGF-1, and (D) TGF-β expression in C57BL/6 mice after topical application of experimental materials, NC: negative control (10% ethanol as a vehicle), THT (hair tonic with 3% LTS), PC: positive control (3% minoxidil) for 4 weeks. *p 0.05, **p 0.01 compared with control.

JOURNAL OF COSMETIC SCIENCE 438 in growing hair follicles. Otherwise, 3% minoxidil (PC) showed most hair follicles were fully induced and growing into the subcutaneous region. In addition, the staining by toluidine blue was distinctly observed in the growing inner and outer root sheaths of the hair bulb. Findings with the 3% LTS-treated samples were very similar to the minoxidil control. However, in the test hair tonic experiment, the anagen induction rate for the 3% LTS product was slower than that of the minoxidil control, but greater than that of the NC (Fig. 7). DRAIZE SKIN IRRITATION STUDY The test hair tonic with 3% LTS showed no signs of irritation on abraded or intact back skin of two male NZW rabbits at 24 or 72 h after treatment (Fig. 8). LYCOPENE QUANTIFICATION OF L. ESCULENTUM EXTRACT We used HPLC to confi rm the lycopene quantifi cation of the 3% LTS. Lycopene was identifi ed by comparing the retention times of the pigment in the extraction mixture with those of respective standard compounds (Sigma products). The HPLC chromato- gram is shown in Fig. 9. When the area of the lycopene peak was calculated and compared with standard compounds, the all-trans-lycopene and cis-lycopene content of 3% LTS was 3.63 and 1.65 μg/ml, respectively. DISCUSSION There are various forms of alopecia, the most common being “AGA,” which affects mil- lions of men and women. For both men and women, AGA may begin as early as the teen Figure 7. Hematoxylin–eosin (A–C) and toluidine blue (D–F) staining of the skin sections. The NC: nega- tive control (10% ethanol as a vehicle A, D), test hair tonic product contained 3% LTS (B, E) or 3% min- oxidil (C, F) were applied topically on the back skin of the mice, once a day for 4 weeks. Magnifi cation: ×200.

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