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Procyanidin Oligomers Selectively and Intensively Promote Proliferation of Mouse Hair Epithelial Cells In Vitro and Activate Hair Follicle Growth In Vivo1

      We have previously reported that proanthocyanidins extracted from grape seeds possess growth-promoting activity toward murine hair epithelial cells in vitro and stimulate anagen induction in hair cycle progression in vivo. This report constitutes a comparison of the growth-promoting activity of procyanidin oligomers and the target cells of procyanidins in the skin. Results show that procyanidin dimer and trimer exhibit higher growth-promoting activity than the monomer. The maximum growth-promoting activity for hair epithelial cells with procyanidin B-2, an epicatechin dimer, reached about 300% (30 μM) relative to controls (=100%) in a 5 d culture. Optimum concentration of procyanidin C-1, an epicatechin trimer, was lower than that of procyanidin B-2; the maximum growth-promoting activity of procyanidin C-1 was about 220% (3 μM). No other flavonoid compounds examined exhibit higher proliferative activities than the procyanidins. In skin constituent cells, only epithelial cells such as hair keratinocytes or epidermal keratinocytes respond to procyanidin oligomers. Topical application of 1% procyanidin oligomers on shaven C3H mice in the telogen phase led to significant hair regeneration [procyanidin B-2, 69.6% ± 21.8% (mean ± SD); procyanidin B-3, 80.9% ± 13.0%; procyanidin C-1, 78.3% ± 7.6%] on the basis of the shaven area; application of vehicle only led to regeneration of 41.7% (SD = 16.3%). In this paper, we demonstrate the hair-growing activity of procyanidin oligomers both in vitro and in vivo, and their potential for use as agents to induce hair growth.

      Keywords

      Although many plant extracts have been tried for curing male pattern baldness, in most cases the efficacy was uncertain or the active materials were unknown. Minoxidil, which was originally synthesized as a potassium channel opener (
      • Buhl A.E.
      • Conrad S.J.
      • Waldon D.J.
      • Brunden M.N.
      Potassium channel conductance as a control mechanism in hair follicles.
      ) and initially prescribed for hypertension, is an FDA approved topical medication for curing male pattern baldness (
      • Kulick M.I.
      Topical minoxidil: its use in treatment of male pattern baldness.
      ;
      • Olsen E.A.
      Treatment of androgenetic alopecia with topical minoxidil.
      ). Recently, finasteride, which is a type II 5α-reductase inhibitor and was initially used for curing prostatic hypertrophy (
      • Dallob A.L.
      • Sadick N.S.
      • Unger W.
      • et al.
      The effect of finasteride, a 5α-reductase inhibitor, on scalp skin testosterone and dihydrotestosterone concentrations in patients with male pattern baldness.
      ;
      • Gormley G.J.
      Finasteride: a clinical review.
      ), has been approved by the FDA as an oral drug for curing male pattern baldness (
      • Kaufman K.D.
      • Olsen E.A.
      • Whiting D.
      • et al.
      Finasteride in the treatment of men with androgenetic alopecia.
      ). Several side-effects
      Wilson C, Walkden V, Powell S, Shaw S, Wilkinson J, Dawber R: Contact dermatitis in reaction to 2% topical minoxidil solution. J Am Acad Dermatol 24:661–662, 1991 (brief communications)
      2Wilson C, Walkden V, Powell S, Shaw S, Wilkinson J, Dawber R: Contact dermatitis in reaction to 2% topical minoxidil solution. J Am Acad Dermatol 24:661–662, 1991 (brief communications)
      and one accident have been reported with both agents (
      • McCormick M.A.
      • Forman M.H.
      • Manoguerra A.S.
      Severe toxicity from ingestion of a topical minoxidil preparation.
      ;
      • Boeck C.
      • Parker J.
      • Shank J.
      • Hordinsky M.
      Safety of long term therapy with 3% and 5% topical minoxidil in female androgenetic alopecia.
      ;
      • Nickel J.C.
      • Fradet Y.
      • Boake R.C.
      • et al.
      Efficacy and safety of finasteride therapy for benign prostatic hyperplasia: results of a 2-year randomized controlled trial (the PROSPECT study).
      ;
      • Wilton L.
      • Pearce G.
      • Edet E.
      • Freemantle S.
      • Stephens M.D.B.
      • Mann R.D.
      The safety of finasteride used in benign prostatic hypertrophy: a non-interventional observational cohort study in 14772 patients.
      ). It has been reported that cyclosporine A stimulates anagen induction (
      • Paus R.
      • Stenn K.S.
      • Link R.E.
      The induction of anagen hair growth in telogen mouse skin by cyclosporin A administration.
      ) and causes hirsutism (
      • Wysocki G.P.
      • Daley T.D.
      Hypertrichosis in patients receiving cyclosporine therapy.
      ). Capsaicin injected into telogen mice has been reported to cause anagen induction (
      • Paus R.
      • Heinzelmann T.
      • Schultz K.-D.
      • Furkert J.
      • Fechner K.
      • Czarnetzki B.M.
      Hair growth induction by substance P.
      ).
      More recently, we have found hair epithelial cell growth-promoting activity and ability to induce anagen phase in proanthocyanidins extracted from grape seeds (
      • Takahashi T.
      • Kamiya T.
      • Yokoo Y.
      Proanthocyanidins from grape seeds promote proliferation of mouse hair follicle cells in vitro and convert hair cycle.
      ) as an investigation of a wide range of plant extracts numbering more than a thousand. Proanthocyanidins are a species of phenolic compounds that take the form of polymers or oligomers built of flavan-3-ol units, such as catechin, epicatechin, gallocatechin, epigallocatechin, afzelechin, and epiafzelechin; whose molecules occasionally incorporate gallic acid (Porter 1994). Proanthocyanidins are very common in the plant kingdom. The significance of the existence of proanthocyanidins in plants is considered to be their anti-microbial action, their deterrence properties, and protection from ultraviolet rays (
      • Bernays E.A.
      • Driver G.C.
      • Bilgener M.
      Herbivores and plant tannins.
      ;
      • Scalbert A.
      Antimicrobial properties of tannins.
      ). Concerning the physiologic and pharmacologic effects of proanthocyanidins, the following activities have been reported: radical scavenging (
      • Vennat B.
      • Bos M.-A.
      • Pourrat A.
      • Bastide P.
      Procyanidins from tormentil: fractionation and study of the anti-radical activity towards superoxide anion.
      ), anti-oxidant (
      • Hong C.-Y.
      • Wang C.-P.
      • Huang S.-S.
      • Hsu F.-L.
      The inhibitory effect of tannins on lipid peroxidation of rat heart mitochondria.
      ), anti-mutagenic (
      • Liviero L.
      • Puglisi P.P.
      • Morazzoni P.
      • Bombardelli E.
      Antimutagenic activity of procyanidins from.
      ), anti-tumor-promoting (
      • Gali H.U.
      • Perchellet E.M.
      • Gao X.M.
      • Karchesy J.J.
      • Perchellet J.P.
      Comparison of the inhibitory effects of monomeric, dimeric, and trimeric procyanidins on the biochemical markers of skin tumor promotion in mouse epidermis.
      ), anti-fungal (
      • Eberhardt T.L.
      • Young R.A.
      Conifer seed cone proanthocyanidin polymers: characterization by 13C NMR spectroscopy and determination of antifungal activities.
      ), anti-viral (
      • Barnard D.L.
      • Smee D.F.
      • Huffman J.H.
      • Meyerson L.R.
      • Sidwell R.W.
      Antiherpesvirus activity and mode of action of SP-303, a novel plant flavonoid.
      ), anti-ulcer (
      • Vennat B.
      • Gross D.
      • Pourrat H.
      • Pourrat A.
      • Bastide P.
      • Bastide J.
      Anti-ulcer activity of procyanidins: preparation of water-soluble procyanidin-cimetidine complexes.
      ), anti-hypertensive activity (
      • Cheng J.-T.
      • Hsu F.-L.
      • Chen H.-F.
      Antihypertensive principles from the leaves of Melastoma candidum..
      ), and capillary protective action (
      • Facino R.M.
      • Carini M.
      • Aldini G.
      • Bombardelli E.
      • Morazzoni P.
      • Morelli R.
      Free radicals scavenging action and anti-enzyme activities of procyanidins from Vitis vinifera: a mechanism for their capillary protective action.
      ). There are no reports, however, on the hair-growing activity of proanthocyanidins, except for our previous report (
      • Takahashi T.
      • Kamiya T.
      • Yokoo Y.
      Proanthocyanidins from grape seeds promote proliferation of mouse hair follicle cells in vitro and convert hair cycle.
      ) on proanthocyanidins purified from grape seeds.
      We examined which type of proanthocyanidin molecules possess the highest hair-growing activity. We report here that procyanidin oligomers possess selective and intensive growth-promoting activity with respect to hair epithelial cells in vitro and stimulate anagen induction in vivo. As the mechanism of action of procyanidins, we expect there to be a direct effect on the follicular epithelium in the skin tissues.

      Materials and Methods

      Materials

      (–)-Epicatechin (Figure 1) and hesperetin were purchased from Sigma (St. Louis, MO). Chrysin, kaempferol (+)-taxifolin, and cyanidin chloride were purchased from Extrasynthese S.A. (Genay, France). Genistein and phloretin were purchased from Wako Pure Chemical Industries (Osaka, Japan). Quercetin was purchased from Nacalai Tesque (Kyoto, Japan).
      Figure thumbnail gr1
      Figure 1The structures of procyanidin monomer and oligomers. (a) (–)-Epicatechin; (b), procyanidin B-1; (c), procyanidin B-2; (d), procyanidin B-3; (e), procyanidin C-1.

      Isolation of procyanidin B 1 [epicatechin-(4β→8)-catechin]

      ITwenty kiloliters of apple juice (Malus pumila Miller var. domestica Schneider, Fuji variety, commercial juice) was passed through a column (60 cmϕ × 88.5 cm) filled with Diaion HP-20 resin (Mitsubishi Kasei, Tokyo, Japan), equilibrated with demineralized water, then washed with 1000 liters of demineralized water and 500 liters of 15(vol/vol)% methanol. Five hundred liters of 45(vol/vol)% methanol was applied and the column was then eluted to produce 9450 g of a dry solid. Then, 1465 g of this was passed through a column (18 cmϕ × 39.3 cm) filled with Sephadex LH-20 (Pharmacia, Uppsala, Sweden) equilibrated with 25(vol/vol)% methanol, then washed with 20 liters of 25(vol/vol)% methanol and 20 liters of 50(vol/vol)% methanol. Twenty liters of 75(vol/vol)% methanol was passed through the column and the intended fraction was eluted to produce 233 g of a dry solid. Half of this was subjected to preparative high-performance liquid chromatography (150 mmϕ × 1000 mm, Soken, Japan; column packing was ODS silica gel, 50 μm particle size, KE-ODS-50 SRQ, YMC, Kyoto, Japan). The eluting conditions were as follows: flow-rate, 645 ml per min; room temperature; mobile phase A = 0.0001% acetic acid, mobile phase B = methanol; isocratic elution with A = 88%, B = 12%; monitored by a UV detector at a wavelength of 285 nm. A total of 107 g of procyanidin B-1 of purity greater than 94(wt/wt)% in dry weight was obtained from 20 kl of apple juice. The product was identified by mass spectrometry, 1H-NMR, and 13C-NMR (
      • Thompson R.S.
      • Jacques D.
      • Haslam E.
      • Tanner R.J.N.
      Plant proanthocyanidins. Part I. Introduction; the isolation, structure and distribution in nature of plant procyanidins.
      ;
      • Nonaka G.-I.
      • Hsu F.-L.
      • Nishioka I.
      Structures of dimeric, trimeric, and tetrameric procyanidins from.
      ).

      Isolation of procyanidin B 2 [epicatechin-(4β→8)-epicatechin]

      Procyanidin B-2 (Figure 1) was isolated using the same procedure as previously mentioned for procyanidin B-1 purification: namely, 213 g of procyanidin B-2 was obtained from 20 kl of apple juice. The product was identified by mass spectrometry, 1H-NMR, and 13C-NMR (
      • Thompson R.S.
      • Jacques D.
      • Haslam E.
      • Tanner R.J.N.
      Plant proanthocyanidins. Part I. Introduction; the isolation, structure and distribution in nature of plant procyanidins.
      ;
      • Morimoto S.
      • Nonaka G.-I.
      • Nishioka I.
      Tannins and related compounds. VIII. Isolation and characterization of flavan-3-ol glucosides and procyanidin oligomers from cassia bark (Cinnamomum cassia Blume).
      ).

      Isolation of procyanidin C 1 [epicatechin-(4β→8)-epicatechin-(4β→8)-epicatechin]

      Procyanidin C-1 (Figure 1) was isolated using the same procedure as previously described for procyanidin B-1 purification: namely, 21 g of procyanidin C-1 was obtained from 20 kl of apple juice. The product was identified by mass spectrometry, 1H-NMR, and 13C-NMR (
      • Nonaka G.-I.
      • Kawahara O.
      • Nishioka I.
      Tannins and related compounds. VIII. A new type of proanthocyanidin, cinchonains IIa and IIb from Cinchona succirubra.
      ;
      • Morimoto S.
      • Nonaka G.-I.
      • Nishioka I.
      Tannins and related compounds. VIII. Isolation and characterization of flavan-3-ol glucosides and procyanidin oligomers from cassia bark (Cinnamomum cassia Blume).
      ).

      Isolation of procyanidin B 3 [catechin-(4α→8)-catechin]

      Procyanidin B-3 (Figure 1) was isolated from barley grains as follows: 60 kg barley grains (Hordeum vulgare L. var. distichon Alefeld) was extracted with 90 kg of 70(wt/wt)% acetone at room temperature for 4 d. The extract was filtered through filter paper (No. 526, Advantec Toyo, Tokyo, Japan) to obtain an extract. The extract was then evaporated and passed through an HP-20 column (10 cmϕ × 50 cm size with a volume of 3925 ml) that had been equilibrated with demineralized water. After washing the column with 8 liters of 20(vol/vol)% methanol, 8 liters of 40(vol/vol)% methanol was applied to the column. The intended fraction was eluted to produce 23.4 g of a dry solid. The dry solid was dissolved in distilled water and then subjected to preparative high-performance liquid chromatography (ODS column, 15 μm particle size, 3 cmϕ × 30 cm, Nihon Waters, Tokyo, Japan) to obtain 1.4 g of procyanidin B-3. The product was identified by mass spectrometry, 1H-NMR, and 13C-NMR (
      • Thompson R.S.
      • Jacques D.
      • Haslam E.
      • Tanner R.J.N.
      Plant proanthocyanidins. Part I. Introduction; the isolation, structure and distribution in nature of plant procyanidins.
      ;
      • Brandon M.J.
      • Foo L.Y.
      • Porter L.J.
      • Meredith P.
      Proanthocyanidins of barley and sorghum; composition as a function of maturity of barley ears.
      ).

      Isolation and culturing of hair epithelial cells

      Mouse hair epithelial cells were isolated and cultured according to the method reported by
      • Tanigaki N.
      • Ando H.
      • Ito M.
      • Hashimoto A.
      • Kitano Y.
      Electron microscopic study of cultured cells from the murine hair tissues: cell growth and differentiation.
      with suitable modifications. The dorsal skin was peeled from 4 d old C3H/HeNCrj mice (Charles River Japan, Kanagawa, Japan), cut into about 5 mm widths, then dipped into Eagle’s minimum essential medium containing 500 IU dispase per ml (from Bacillus polymyxa, Godo Shusei, Tokyo, Japan), 60 μg kanamycin per ml, and 5% fetal calf serum at 4°C for 16 h. The epidermis was peeled off, and the remaining dermis layer was dispersed with Dulbecco’s modified Eagle medium (DMEM) containing 0.25% collagenase (from Streptomyces parvulus, Nitta Gelatin, Osaka, Japan), 50 U penicillin per ml, 50 μg streptomycin per ml, and 10% fetal calf serum at 37°C for 1 h. This dermis suspension was filtered through a 212 μm nylon mesh, and the filtrate was centrifuged at 1000 r.p.m. for 5 min. The pellet was suspended in phosphate-buffered saline without calcium and magnesium. The suspension was left to stand for 15 min, whereby the hair follicle tissue was precipitated. The hair follicle tissue was resuspended in phosphate-buffered saline without calcium and magnesium and then precipitated. This precipitation process was repeated three times. The hair follicle tissue was finally incubated in 0.05% EDTA-0.25% trypsin at 37°C for 5 min. The hair follicle cells were suspended in DMEM supplemented with 50 U penicillin per ml, 50 μg streptomycin per ml, and 10% fetal calf serum at the density of 3 × 105 cells per ml. This hair follicle cell suspension was pipetted into a 24 well Type I collagen-coated plate (2 cm2 per well, Iwaki Glass, Chiba, Japan) with 1 ml per well and incubated in a humidified atmosphere containing 5% CO2 at 37°C for 24 h. After 24 h incubation, the medium was exchanged with MCDB153 (Kyokuto Pharmaceutical, Tokyo, Japan) (
      • Boyce S.T.
      • Ham R.G.
      Calcium-regulated differentiation of normal human epidermal keratinocytes in chemically defined clonal culture and serum-free serial culture.
      ) containing 5 mg bovine insulin per liter, 5 μg mouse EGF per liter, 40 mg bovine pituitary extract per liter, 10 mg human transferrin per liter, 0.4 mg hydrocortisone per liter, 0.63 μg progesterone per liter, 14 mg O-phosphorylethanolamine per liter, 6.1 mg ethanolamine per liter, 50 U penicillin per ml, and 50 μg streptomycin per ml. It was then further incubated in a humidified atmosphere containing 5% CO2 at 37°C for 5 d. During incubation, the medium was removed and replaced with fresh medium every other day.

      Isolation and culturing of dermal papilla cells from rat vibrissae

      Dermal papillae were isolated from PVG/Sea rat (10 wk old, male, purchased from Japan SLC, Shizuoka, Japan; breeding by Seiwa Experimental Animal Research Laboratory, Fukuoka, Japan) vibrissa follicles according to the method of
      • Jahoda C.
      • Oliver R.F.
      The growth of vibrissa dermal papilla cells.
      . The rats were anesthetized and the whisker follicles were dissected. The lower halves of the follicle were sectioned and placed into DMEM containing 50 U penicillin per ml, 50 μg streptomycin per ml, and 20% fetal calf serum. For the following procedure, we used a needle (24G × 1″, 0.55 × 25 mm, Terumo, Tokyo, Japan) and scalpel (Mini no. 15 disposable scalpel, Feather, Osaka, Japan) for the isolation of dermal papillae from vibrissa follicles. Under a stereoscopic microscope, holding the vibrissa follicle by the needle, we cut horizontally just above the hair bulb with the scalpel, and pulled out the hair shaft using a pair of tweezers. Then, holding the hair bulb with the needle, we peeled and turned over the outer root sheath around the dermal papilla using another needle from the upper side. The exposed dermal papillae were sectioned by needle and transferred on a needle tip to the 6 well culture plate (9.6 cm2 per well, Falcon no. 3046, Nippon Becton Dickinson, Tokyo, Japan). After rubbing the dermal papilla onto the culture plate and leaving it to stand for cultivation in DMEM containing 50 U penicillin per ml, 50 μg streptomycin per ml, and 20% fetal calf serum, dermal papilla cells grew out from the original sections. After 2 wk of culture, the culture plate became subconfluent, and 0.02% EDTA-0.1% trypsin treatment was carried out. By adding DMEM containing 50 U penicillin per ml, 50 μg streptomycin per ml, and 20% fetal calf serum, a cell suspension containing 3 × 104 cells per ml was prepared. The dermal papilla cell suspension was pipetted into a 24 well culture plate (2 cm2 per well, Falcon no. 3047, Nippon Becton Dickinson) with 1 ml per well and incubated in a humidified atmosphere containing 5% CO2 at 37°C for 7 d. During incubation, the medium was removed and replaced with fresh medium every other day.

      Isolation and culturing of mouse epidermal keratinocytes

      Keratinocytes were obtained from the EDTA-trypsin treated epidermis of C3H/HeNCrj mouse and cultured in a 24 well collagen Type I coated plate (at the initial cell density of 5 × 104 cells per cm2, Iwaki Glass, Japan) in the same manner described at hair epithelial cells using an DMEM for the first 24 h and MCDB153 (containing the same additives as that of the hair epithelial cells) for the next 6 d.

      Isolation and culturing of mouse dermal fibroblasts

      Dermal fibroblasts were obtained from the EDTA-trypsin treated dermis of C3H/HeNCrj mouse and cultured in a 24 well culture plate (at the initial cell density of 1.5 × 104 cells per cm2, Falcon no. 3047) using a DMEM (supplemented with 50 U penicillin per ml, 50 μg of streptomycin per ml, and 10% fetal calf serum) in a humidified atmosphere containing 5% CO2 at 37°C for 5 d.

      Colorimetric assay for cell proliferation by MTT

      The degree of cell growth was determined from an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay (
      • Carmichael J.
      • DeGraff W.G.
      • Gazdar A.F.
      • Minna J.D.
      • Mitchell J.B.
      Evaluation of a tetrazolium-based semiautomated colorimetric assay: assessment of chemosensitivity testing.
      ). To summarize, MTT reagent (5 mg per ml, dissolved in water and filtered through a 0.2 μm membrane filter) was added by a volume of 1/100 to the culture medium and further incubated in a humidified atmosphere containing 5% CO2 at 37°C for 4 h. After removing the medium, the formed dye was extracted with acidic isopropanol containing 0.1 N HCl (adding 0.4 ml per 2 cm2 of well), and the absorbance was measured at 570 nm relative to 640 nm.

      Preparation methods of topically applied agents for in vivo evaluation

      Fourteen grams of ethyl alcohol, 0.2 g of procyanidin oligomers, 2 g of 1,3-butylene glycol, 0.1 g of N-acetylglutamine-isostearyl (Kyowa Hakko Kogyo, Tokyo, Japan), 0.05 g of polyoxyethylene (25) glyceryl monopyroglutamate monoisostearate (Nihon Emulsion, Tokyo, Japan), and 3.65 g of pure water were mixed together, and the solids were dissolved to prepare a sample solution for the in vivo mice test.
      A vehicle without procyanidins was used as the control. Minoxidil (Upjohn Company, Kalamazoo, MI; purchased from Sigma) agent was prepared in the same way as the procyanidin-containing agent.

      Test for hair-growing activity to induce anagen phase in mice

      With reference to the method introduced by
      • Hattori M.
      • Ogawa H.
      Biochemical analysis of hair growth from the aspects of aging and enzyme activities.
      , the hair-growing activity to induce anagen phase in mice was measured. In this test, 8 wk old male C3H/HeS1c (Japan SLC, Shizuoka, Japan) mice whose hair cycle was in the telogen stage were used. The mice were grouped into multiple groups; each one comprised of four or five mice. The hair on the back of each mouse was carefully shaven with an electric shaver so as not to injure or stimulate the skin. (Otherwise stimulation would raise background activity for control.) Two hundred microliters of test sample was applied onto the shaven area once a day. On the nineteenth day of the test, the mouse back skins were observed and photographed; then the skin was peeled from the back of each mouse and photographed.

      Results

      We compared the growth-promoting activity on hair epithelial cells by procyanidins of different degrees of polymerization, and of different flavan-3-ol units: catechin, epicatechin, or a combination of both. For the degree of polymerization, we compared the growth-promoting activity of the monomer, dimer, and trimer: we report on the example of (–)-epicatechin, procyanidin B-2 [epicatechin-(4β→8)-epicatechin], and procyanidin C-1 [epicatechin-(4β→8)-epicatechin-(4β→8)-epicatechin] (Figure 2). For the combination of flavan-3-ol units, we report on procyanidin B-1 [epicatechin-(4β→8)-catechin], procyanidin B-2 [epicatechin-(4β→8)-epicatechin], and procyanidin B-3 [catechin-(4α→8)-catechin] (Figure 3).
      Figure thumbnail gr2
      Figure 2Procyanidin dimer and trimer more intensively promote growth of hair epithelial cells than the procyanidin monomer. Growth-promoting activities for hair epithelial cells relative to controls (=100%) are shown. Procyanidins were added to the culture during the last 5 d. For the control, a medium without procyanidins was used. ●, (–)Epicatechin;▴, procyanidin B-2;▪, procyanidin C-1;×, minoxidil. Results are represented as the mean ± SD (n = 6) carried out with primary cultures prepared from 50 neonatal mice.
      Figure thumbnail gr3
      Figure 3Procyanidin dimeric isomers with different flavan-3-ol units also promote the growth of hair epithelial cells. Growth-promoting activities for hair epithelial cells relative to controls (=100%) are shown. Procyanidins were added to the culture during the last 5 d. For the control, a medium without procyanidins was used. ●, Procyanidin B-1;▴, procyanidin B-2;▪, procyanidin B-3. Results are represented as the mean ± SD (n = 6) carried out with primary cultures prepared from 50 neonatal mice.
      Of (–)-epicatechin, procyanidin B-2, and procyanidin C-1, procyanidin B-2 shows the highest growth-promoting activity of about 300% relative to controls (= 100%) in a 5 d culture of hair epithelial cells. The optimum concentration of procyanidin C-1 was lower than that of procyanidin B-2, but the maximum growth-promoting activity of procyanidin C-1 relative to controls (=100%) was about 220%, lower than that of procyanidin B-2; however, at concentrations from 0.3–3 μM, the growth-promoting activity of procyanidin C-1 was higher than that of procyanidin B-2. The activity of (–)-epicatechin was lower than those of procyanidin B-2 and procyanidin C-1 (Figure 2). No significant difference in growth-promoting activity was observed among procyanidin B-1, procyanidin B-2, and procyanidin B-3 (Figure 3); their maximum growth-promoting activities were higher than those of the monomer or trimer of procyanidins. On the other hand, minoxidil was less effective in this cell culture system, demonstrating about 160% proliferative activity at a concentration of 400 μM (Figure 2). The micrograph of hair epithelial cells from mice cultured in procyanidin B-2 containing medium is shown in Figure 4.
      Figure thumbnail gr4
      Figure 4Micrographs of hair epithelial cells from mice cultured in MCDB-153 medium for 5 d. (a) Control, (b) 30 μM procyanidin B-2. Scale bar: 100 μm.
      We compared the growth-promoting activity of procyanidins on hair epithelial cells with the activities of other flavonoid compounds. Among flavonoids, we selected typical compounds from each group: flavones, isoflavones, flavonols, flavanones, dihydroflavonols, dihydrochalcones, and anthocyanidins. From flavones we chose chrisin; from isoflavones, genistein; from flavonols, kaempferol and quercetin; from flavanones, hesperetin; from dihydroflavonols, taxifolin; from dihydrochalcones, phloretin; and from anthocyanidins, cyanidin chloride. Figure 5 shows the growth-promoting activity on hair epithelial cells by these flavonoid compounds. Among the examined flavonoid compounds, procyanidin was the only compound that showed a high level of growth-promoting activity on hair epithelial cells: greater than 300% (procyanidin B-2).
      Figure thumbnail gr5
      Figure 5Procyanidins selectively promote the growth of hair epithelial cells. Other flavonoid compounds examined, however, only slightly stimulate the growth of hair epithelial cells. Maximum growth-promoting activities relative to controls (=100%) are shown. Flavonoid compounds were added to the culture during the last 5 d. For the control, we used a medium to which an amount of 1/100 volume of dimethyl sulfoxide (DMSO) (for chrisin, genistein, kaempferol, quercetin, hesperetin, taxifolin, phloretin, and cyanidin chloride) or water (for procyanidin B-2) was added in place of flavonoid containing solution. We observed no significant difference between DMSO control and water control. Brackets show optimum concentration. Results are represented as the mean ± SD (n = 6) carried out with primary cultures prepared from 50 neonatal mice.
      On the other hand, procyanidins did not affect the proliferation of dermal papilla cells (Figure 6).
      Figure thumbnail gr6
      Figure 6Dermal papilla cells did not respond in cell growth by procyanidins with different degrees of polymerization. Growth-promoting activities for dermal papilla cells relative to controls (=100%) are shown. Procyanidins were added to the culture during the last 6 d. For the control, a medium without procyanidins was used. ●, (–)-Epicatechin;▴, procyanidin B-2;▪, procyanidin C-1. Results are represented as the mean ± SD (n = 6) carried out with secondary cultures prepared from 20 follicles of two rats.

      Procyanidin dimer and trimer selectively and intensively promote growth of mouse keratinocytes

      We examined the effect of procyanidins on both types of skin cells: epithelial cells and mesenchymal cells. Specifically, we examined the effect of procyanidins on the proliferation of keratinocytes and fibroblasts.
      Figure 7 shows the effect of procyanidins on keratinocyte cell growth. Among (–)-epicatechin, procyanidin B-2, and procyanidin C-1, procyanidin B-2 showed the highest growth-promoting activity of about 250% relative to controls (=100%) at concentrations of 0.3–100 μM. The growth-promoting activity of procyanidin C-1 was about 210% relative to controls (=100%), lower than that of procyanidin B-2, at concentrations of 0.1–10 μM. The optimum concentration of procyanidin C-1 was lower than that of procyanidin B-2. The activity of (–)-epicatechin was relatively lower than the activities of procyanidin B-2 and procyanidin C-1. This proliferative pattern, related to the degree of polymerization of procyanidins, resembles that of hair epithelial cells.
      Figure thumbnail gr7
      Figure 7Procyanidin dimer and trimer intensively promote the growth of mouse keratinocytes to a greater extent than the procyanidin monomer. Growth-promoting activities for keratinocytes relative to controls (=100%) are shown. Procyanidins were added to the culture during the last 6 d. For the control, a medium without procyanidins was used. ●, (–)-Epicatechin;▴, procyanidin B-2;▪, procyanidin C-1. Results are represented as the mean ± SD (n = 6) carried out with primary cultures prepared from 50 neonatal mice.
      On the other hand, dermal fibroblast growth was not affected by the procyanidins examined (Figure 8).
      Figure thumbnail gr8
      Figure 8Dermal fibroblasts did not respond when exposed to procyanidins with different degrees of polymerization. Growth-promoting activities for dermal fibroblasts relative to controls (=100%) are shown. Procyanidins were added to the culture during the last 4 d. For the control, a medium without procyanidins was used. ●, (–)-Epicatechin;▴, procyanidin B-2;▪, procyanidin C-1. Results are represented as the mean ± SD (n = 6) carried out with primary cultures prepared from 50 neonatal mice.

      Procyanidin oligomers stimulate anagen induction in hair cycle progression in murine model

      C3H mouse dorsal hair is known to have a time-synchronized hair cycle. From about 18–21 d of age and 47–95 d of age, the dorsal hairs are in the telogen phase (
      • Hattori M.
      • Ogawa H.
      Biochemical analysis of hair growth from the aspects of aging and enzyme activities.
      ). The test compound was topically applied from the eighth to the tenth week (19 d application) during the second telogen phase, and the hair-covered area at the tenth week was evaluated.
      In this assay system, minoxidil gave a positive response. After a 19 d application of 1% minoxidil-containing agent, about 80% (81.2% ± 10.5%, average ± SD) of the shaven area was covered with hair. The control group to which vehicle was applied, on the other hand, showed little hair growth: only about 40% (41.7% ± 16.3%) of the shaven area was covered with hair on day 19. The groups to which 1% procyanidin oligomers had been applied showed an extensive growth area [procyanidin B-2, 69.6% ± 21.8% (average ± SD); procyanidin B-3, 80.9% ± 13.0%; procyanidin C-1, 78.3% ± 7.6%] on day 19 (Figure 9).
      Figure thumbnail gr9
      Figure 9Procyanidin oligomers are able to induce anagen phase efficiently in hair cycle progression in the murine model to the same degree as minoxidil. Photographs were taken after the topical application of test agents for 19 d. Test agents were applied to 8 wk old C3H telogen mice (♂), 200 μl per d per mouse. (a) Vehicle; (b) 1% minoxidil; (c) 1% procyanidin B-2; (d) 1% procyanidin B-3; (e) 1% procyanidin C-1.
      These results demonstrate that procyanidin oligomers, such as procyanidin B-2, procyanidin B-3, or procyanidin C-1, possess marked hair-growing activity to induce anagen phase in vivo. On the other hand, neither (+)-catechin nor (–)-epicatechin, a flavan-3-ol unit of procyanidins, stimulated anagen induction in vivo (data not shown).DC

      Discussion

      Proanthocyanidins have been used as medications aimed at protecting the capillary vessels (
      • Dartenuc J.-Y.
      • Marache P.
      • Choussat H.
      Résistance capillaire en gériatrie etude d’un microangioprotecteur = Endotélon.
      ), as cosmetics to protect the skin (
      • Wayne Z.
      • Elementals L.L.C.
      • Scottsdale A.Z.
      Pycnogenol and skincare.
      ), and as antioxidants in foods and beverages; however, there has been very little information gathered on the correlation between their degree of polymerization and properties.
      • Barnard D.L.
      • Smee D.F.
      • Huffman J.H.
      • Meyerson L.R.
      • Sidwell R.W.
      Antiherpesvirus activity and mode of action of SP-303, a novel plant flavonoid.
      report that proanthocyanidin polymers possess anti-viral activities against the herpes virus.
      • Ariga T.
      • Koshiyama I.
      • Fukushima D.
      Antioxidative properties of procyanidins B-1 and B-3 from azuki beans in aqueous systems.
      report that its anti-oxidant properties increase in proportion to the degree of polymerization. The profile of the proanthocyanidins from grape seeds in which we first discovered their hair-growing activity is as follows: the constitutive monomers were catechin and epicatechin, the degree of polymerization was 3.5, and the galloylation rate was 25% at the molar ratio per constitutive flavan-3-ol unit (
      • Takahashi T.
      • Kamiya T.
      • Yokoo Y.
      Proanthocyanidins from grape seeds promote proliferation of mouse hair follicle cells in vitro and convert hair cycle.
      ). Here, we investigated which proanthocyanidin molecules possess the highest hair-growing activity. It was revealed that among the monomer, dimer, and trimer of procyanidins, procyanidin dimer possesses the highest growth-promoting activity. The order of optimum concentration showing the maximum proliferative activity was trimer < dimer < monomer. In in vivo hair-growing activity to induce anagen phase, the intensity of dimers and trimers was almost equal; however, neither (–)-epicatechin nor (+)-catechin, monomers of proanthocyanidins, stimulated anagen induction. In in vitro and in vivo assays, we could find no difference between dimeric isomers composed of different monomers: procyanidin B-1, procyanidin B-2, and procyanidin B-3. The excision of gallate using tannase from proanthocyanidins purified from grape seeds raises the activity in vitro and in vivo (data not shown). Our in vitro studies led to the discovery of no other active flavonoid compounds. The procyanidin compounds we examined were built only of flavan-3-ol, so hair-growing activity may be associated with this structure.

      Target cells

      Skin is composed of keratinocytes, which are epithelial cells, and fibroblasts, which are mesenchymal cells. Hair follicles consist of the outer root sheath, the inner root sheath, and the bulb matrix, which are epithelial cells, and the dermal papillae, which are mesenchymal cells. It is thought that the interaction between epithelial cells and mesenchymal cells is important in the progress of the hair cycle. It is reported that androgen receptors exist in the dermal papilla (
      • Hibberts N.A.
      • Howell A.E.
      • Randall V.A.
      Balding hair follicle dermal papilla cells contain higher levels of androgen receptors than those from non-balding scalp.
      ); it is speculated that secretion of several growth factors and cytokines from the hair follicle, such as the dermal papilla, the outer root sheath, the inner root sheath, or the bulb matrix, affect epidermal cell growth at the bulb region, and promote the hair cycle (
      • Messenger A.G.
      The control of hair growth: an overview.
      ;
      • Danilenko D.M.
      • Ring B.D.
      • Pierce G.F.
      Growth factors and cytokines in hair follicle development and cycling: recent insights from animal models and the potentials for clinical therapy.
      ). In this paper, we have demonstrated that procyanidin oligomers directly promote the proliferation of the hair follicular epithelium and do not affect dermal papilla cells at least in proliferation level. On the other hand, we also demonstrated that procyanidin oligomers promote the proliferation of keratinocytes in skin. From this, we conclude that procyanidins directly affect the growth of the epithelial cells in skin. Interestingly, the pattern of proliferative activity of keratinocytes in relation to the polymerization degree of procyanidins was similar to that for hair epithelial cells (Figure 2, Figure 7).

      Assumed mechanism of action of procyanidins

      It is known that minoxidil causes the proliferation of hair epithelial cells in vitro (
      • Tanigaki-Obana N.
      • Ito M.
      Effects of cepharanthine and minoxidil on proliferation, differentiation and keratinization of cultured cells from the murine hair apparatus.
      ;
      • Buhl A.E.
      • Waldon D.J.
      • Kawabe T.T.
      • Holland J.M.
      Minoxidil stimulates mouse vibrissae follicles in organ culture.
      ). We have illustrated that procyanidin oligomers possess high growth-promoting activity on hair keratinocytes and epidermal keratinocytes, which are classified as epithelial cells. It is noteworthy that the morphology of hair epithelial cells cultured in a procyanidin-containing medium takes on the rounded appearance characteristic of undifferentiated juvenile cells (Figure 4).
      Stem cell existence was assumed at the bulge area of the infundibular region of the outer root sheath by
      • Cotsarelis G.
      • Sun T.-T.
      • Lavker R.M.
      Label-retaining cells reside in the bulge area of pilosebaceous unit: implications for follicular stem cells, hair cycle, and skin carcinogenesis.
      . So, the outer root sheath appears to be important in the progress of the hair cycle. It is assumed that the growth-promoting effects of procyanidins on the outer root sheath cells switch the bulb region to the growing phase by some mechanism, causing the follicular hair cycle to convert from the telogen phase to the anagen phase. We are now investigating the relationship between protein kinase C-inhibiting activity by procyanidins (
      • Kashiwada Y.
      • Nonaka G.-I.
      • Nishioka I.
      • Ballas L.M.
      • Jiang J.B.
      • Janzen W.P.
      • Lee K.-H.
      Tannins as selective inhibitors of protein kinase C.
      ;
      • Cui C.B.
      • Tezuka Y.
      • Yamashita H.
      • Kikuchi T.
      • Nakano H.
      • Tamaoki T.
      • Park J.-H.
      Constituents of a fern, Davallia mariesii Moore. V. Isolation and structures of davallin, a new tetrameric proanthocyanidin, and two new phenolic glycosides.
      ;
      • Polya G.M.
      • Foo L.Y.
      Inhibition of eukaryote signal-regulated protein kinases by plant-derived catechin-related compounds.
      ) and hair growing activity.

      Potential for the use of procyanidins as agents inducing hair growth

      We have demonstrated that procyanidin oligomers possess both hair epithelial cell growth-promoting activity and hair-growing activity to induce anagen phase in vivo. The mechanism of androgenetic alopecia is now speculative. Androgenetic alopecia is defined as “vellus transformation” (
      • van Scott E.J.
      • Ekel T.M.
      Geometric relationships between the matrix of the hair bulb and its dermal papilla in normal and alopecic scalp.
      ). For the mechanism of the action of minoxidil, no hormonal effect, but the direct effect on the follicular epithelium has been confirmed (
      • Kulick M.I.
      Topical minoxidil: its use in treatment of male pattern baldness.
      ;
      • Olsen E.A.
      Treatment of androgenetic alopecia with topical minoxidil.
      ). It is reported that minoxidil induces a significant shortening of the telogen phase (
      • Uno H.
      • Cappas A.
      • Schlagel C.
      Cyclic dynamics of hair follicles and the effect of minoxidil on the bald scalps of stumptailed macaques.
      ;
      • Mori O.
      • Uno H.
      The effect of topical minoxidil on hair follicular cycles of rats.
      ). It is thus possible to assume that procyanidin oligomers, which directly affect hair epithelial cell growth and stimulate anagen induction in vivo, will be effective for curing androgenetic alopecia.
      We have already performed toxicologic studies (
      • Takahashi T.
      • Yokoo Y.
      • Inoue T.
      • Ishii A.
      Toxicological studies on procyanidin B-9 for external application as a hair growing agent.
      ) and a human pilot clinical study using procyanidin B-2, and confirmed that it is safe enough for topical use of procyanidin B-2 and effectiveness in curing androgenetic alopecia.
      Takahashi T, Yokoo Y, Watanabe Y: A pilot clinical study of topical procyanidin B-2 with the aim of considering its application as a hair growing agent. (manuscript in preparation).
      We are now improving the industrial process for manufacturing procyanidin oligomers, and are planning a large-scale human clinical test.

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