Polyphenols from the Heartwood of Cercidiphyllum japonicum and their Effects on Proliferation of Mouse Hair Epithelial Cells

• Abstract
• Introduction
• Materials and Methods
• Materials
• Cell culture
• Measurement of growth of hair epithelial cells by uptake of [³ H]-thymidine
• Colorimetric assay for cell proliferation by MTT
• Cell counting
• Extraction and isolation
• Results and Discussion
• Acknowledgements
• References

Abstract

The methanol extract of heartwood of Cercidiphyllum japonicum (Cercidiphyllaceae) stimulated proliferation of mouse hair epithelial cells, similar to minoxidil and procyanidin B-2. (+)-Taxifolin (2), quercetin (3), myricetin (4), (+)-dihydromyricetin (5) and gallic acid (6) were isolated from C. japonicum and showed significant proliferative activities on the hair epithelial cells.

Introduction

Recent progress in cell culture technique and genetic engineering has rapidly advanced basic research in hair growth and the hair follicle cycle. Minoxidil (Rogaine^®, Upjohn Co., Kalamazoo, MI, USA) an FDA-approved cure for male pattern baldness [1], stimulated the growth of mouse hair epithelial cells in vitro [2]. Procyanidin oligomers selectively and intensively promoted hair epithelial cell growth [3]. Furthermore, procyanidin B-2 significantly increased the number of hairs on human subjects versus a placebo control group [4].

Thus, in a search for accelerators of hair regrowth, proliferative effects on hair epithelial cells must be examined. A method for culturing cells from the murine hair apparatus has been established [5]. This culture system has been useful for estimating in vitro the pharmacological effects of possible agents [2], [6], [7].

In this report, we identify natural compounds in the methanol extract of heartwood of Cercidiphyllum japonicum (Cercidiphyllaceae, Japanese name: katsura) that promote the growth of mouse hair epithelial cells.

Materials and Methods

Materials

The wood of C. japonicum Siebold et Zucc. was collected from Kyushu university forests in Fukuoka, Japan and identified by Dr. S. Ohga. A voucher specimen (No 203) was deposited in the laboratory of Kyushu University. The chemicals used were fetal bovine serum (FBS) Invitrogen Corp. (Carlsbad, CA USA); bovine pituitary extract (BPE), Upstate Biotechnology (Lake Placid, NY USA); MCDB153, gallic acid and dimethyl sulfoxide (DMSO), Sigma Chemical Co. (St. Louis, MO USA); epidermal growth factor (EGF), hydrocortisone, insulin and minoxidil, Wako Pure Chemical Industries Co. (Osaka, Japan); Procyanidin B-2, Funakoshi Co. (Tokyo, Japan).

Cell culture

Mouse hair epithelial cells were isolated and cultured as described previously [5] with slight modifications. The skin specimens were obtained from the back area of 4-day-old Sea/ddy mice, cut into 5 mm widths, then soaked at Dullbecco’s modified Eagle’s medium (DMEM) containing 500 IU/ml dispase, 5 % fetal bovine serum (FBS) and 1 % v/v antibiotics at 4 °C for 17.5 h. The epidermis was peeled off, the remaining dermis layer was dispersed with DMEM containing 0.25 % collagenase at 37 °C for 1 h, and gently pipetted to obtain a dermal suspension. The dermal suspension was centrifuged at 1000 rpm for 5 min. The pellet was suspended in phosphate buffered saline without calcium or magnesium [PBS (-)]. The suspension was centrifuged at 400 rpm for 1 min to precipitate the hair follicle tissue. The hair follicle tissue was resuspended in PBS (-). This precipitation process was repeated four times. The hair follicle tissue was filtered through a 200 μm nylon mesh, and the hair follicle tissue caught on nylon mesh was recovered by back wash. The hair follicle tissue was treated with 0.05 % EDTA-0.25 % trypsin at 37 °C for 5 min and gently pipetted. The hair epithelial cells were suspended in DMEM containing 10 % FBS and 0.5 % antibiotics at the density of 5 × 10⁵ cells/ml. This hair follicle cell suspension was plated into type I collagen-coated 24 well plate (FALCON) with 1 ml/well and incubated at 37 °C in a humidified atmosphere of air containing 5 % CO₂. After 24 h incubation, the medium was discarded and the cells were washed with PBS (-). Then the cells were cultured with MCDB 153 (1 ml/well) containing 5 μg/ml insulin, 5 ng/ml EGF, 50 μg/ml bovine putative extract, 1.4 μg/ml hydrocortisone and 1 μl of EtOH solution with or without test compound.

Measurement of growth of hair epithelial cells by uptake of [³ H]-thymidine

After incubation for 4 days on a 24 well plate described above, [³ H]-thymidine (8 kBq/well) (statistic ability, 1.81TBq/mmol) (Amersham International, Bucks, U.K.) was added in the plate, and then incubated for 24 h at 37 °C in 5 % CO₂. Then the medium was removed and washed with PBS (-) two times. The DNA in each well was precipitated by the addition of a 10 % solution of trichloroacetic acid for 30 min at 4 °C before digestion in 500 μl of 1 N sodium hydroxide for 24 h at 37 °C. The incorporation rate of [³ H]-thymidine was counted by a liquid scintillation counter. Minoxidil and procyanidin B-2 were used as positive controls.

Colorimetric assay for cell proliferation by MTT

The degree of cell growth was determined by means of the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. MTT reagent (Sigma) was dissolved in PBS at a concentration of 5 mg/ml, filtered through a 0.22 μm membrane filter (cellulose acetate, DISMIC-13 cp; Advantec, Tokyo, Japan), and added 5 % (v/v) to the culture medium. The culture plate was further incubated in a humidified atmosphere containing 5 % CO₂ at 37 °C for 4 h. After removing the medium, 1.0 ml of 0.04 N HCl per 2 cm² well were added and the absorbance was measured at 570 nm relative to 640 nm.

Cell counting

After culturing in MCDB 153, the cells were harvested from the 24-well plate at day 6 and counted in a haemocytometer chamber.

The data were analyzed by Student’s t-test, differences of P < 0.05 being regarded as significant.

Extraction and isolation

The heartwood portion of C. japonicum was chipped and milled to pass a # 40 screen. The milled heartwood (60 g) was extracted with methanol (300 ml) at room temperature (about 22 °C) for 24 h and then filtered. The extractions were performed for 3 times and methanol extracts were pooled together, and then concentrated in vacuo at 40 °C to give 2.6 g (dry weight, 4.3 % of milled heartwood). The dry methanol extract (2.5 g) was suspended in water and partitioned with n-hexane, diethyl ether and ethyl acetate, successively to give n-hexane-soluble (0.01 g, 4.0 % of the methanol extract), diethyl ether-soluble (1.11 g, 45 %), ethyl acetate-soluble (0.54 g, 22 %) and aqueous portion (0.72 g, 28 %), respectively. The diethyl ether-soluble portion (0.4 g) was fractionated into 8 fractions (fr. A-H) by column chromatography on silica gel (Wakogel C-200, Wako, Osaka, Japan) (50 g) eluted with ethyl acetate, n-hexane and methanol as follows. [ethyl acetate : n-hexane = 1 : 1, 200 ml (fr. A: 38 mg, TLC, n-hexane : ethyl acetate (1 : 2), R_f value 0.53∼0.60)→50 ml (fr. B: 41 mg, R_f 0.50∼0.55)→50 ml (fr. C: 24 mg, R_f 0.40∼0.53)→50 ml (fr. D: 4 mg, R_f 0.30∼0.45)→100 ml and ethyl acetate 150 ml (fr. E: 171 mg, R_f 0.10∼0.35)→50 ml (fr. F: 31 mg, R_f < 0.15)→200 ml (fr. G:14 mg, R_f < 0.10)→400 ml (fr. H: 61 mg, R_f < 0.05)]. (2R, 3R)-(+)-Aromadendrin (1) [8] (34.5 mg); [α]²⁵ _D: + 25° (MeOH; c 0.5), (2R,3R)-(+)-taxifolin (2) [9] (15 mg), [α]²⁵ _D: + 6.3° (MeOH; c 0.7), quercetin (3) [10] (7.5 mg), myricetin (4) [11] (13 mg), (2R, 3R)-(+)-dihydromyricetin (5) [12] (37 mg), [α]²⁵ _D: + 10° (MeOH; c 0.9), gallic acid (6) (8 mg) and kaempferol (7) [13] (5 mg) were isolated by preparative HPLC (Inertsil PREP-ODS: 20 mm i. d. × 250 mm) using 0.1 %TFA-CH₃OH gradient (1 from fr. A, B and C, 2 from fr. C and D, 3 from fr. C, 4 from fr. D and E, 5 from fr. E, 6 from fr. E and F) and identified by comparison with their authentic samples or published data [8], [9], [10], [11], [12], [13].

Results and Discussion

We employed the assay of Tanigaki et al. [2], [5], a strong tool for developing new and effective hair growth agents. In fact, this assay led to the discovery of procyanidin B-2 as a hair growth agent [3]; also with this assay, minoxidil showed proliferative activity on mouse hair epithelial cells [2]. In several reports, the MTT assay [3] or cell counting method in a haemocytometer chamber [2], [6] has been used to evaluate proliferation activity of minoxidil on hair epithelial cells. In our preliminary experiment, the MTT assay method showed low sensitivity against the proliferation activity of minoxidil, while the cell counting method was too troublesome. We therefore investigated the applicability of the [³ H]-thymidine incorporation method to the evaluation of proliferative activity on mouse hair epithelial cells (Fig. [1]). In a time course experiment, minoxidil (70 μg/ml, 334 μM) stimulated proliferation of cultured cells after day 3 and seemed to extend the cell proliferation period, possibly by delaying cell differentiation. These results agree with the previous studies using either MTT [3] or cell counting [2]. Also, procyanidin B-2, which stimulated proliferation of mouse hair epithelial cells in vitro and hair growth in vivo [3], showed growth phenomena similar to those of minoxidil (Fig. [1]). On the assumption that these effects of both agents are important to hair growth activities in vivo, we adopted the [³ H]-thymidine incorporation method on cultivation day 4 to search for natural ingredients for a hair growth agent.

As a result, we found that the methanol extract of heartwood of C. japonicum (0.1, 1.0, 10 μg/ml) promoted the growth of mouse hair epithelial cells on day 4. The proliferative activity of the methanol extract was 148 (0.1 μg/ml), 140 (1.0 μg/ml) and 150 % (10 μg/ml) compared to control (100 %). That of minoxidil (70 μg/ml) was 140 %. In our time course investigation, this extract extended cell proliferation to a period similar to that of minoxidil (Fig. [1]). C. japonicum may contain compounds that behave similarly to minoxidil. The extract was portioned by n-hexane, diethyl ether and ethyl acetate. The respective proliferative activities of n-hexane-soluble, diethyl ether-soluble, ethyl acetate-soluble and the aqueous portion were, at 0.1 μg/ml, 127, 147, 121 and 117 % that of the control. The diethyl ether-soluble portion, having the highest proliferative activity, was further separated by silica gel CC, yielding 8 fractions (fr. A-H), all of which showed proliferation activity at 0.5 or 5.0 μg/ml (data not shown). Further fractionation by preparative HPLC isolated the main components in each fraction, giving (2R,3R)-(+)-aromadendrin (1), (2R,3R)-(+)-taxifolin (2), quercetin (3), myricetin (4), (2R,3R)-(+)-dihydromyricetin (5) and gallic acid (6) (Fig. [2]). We then determined that the components stimulating proliferation of mouse hair epithelial cells were: (+)-taxifolin (2) (10 μM), quercetin (3) (1.0 μM), myricetin (4) (1.0 and 10 μM), (+)-dihydromyricetin (5) (10 μM) and gallic acid (6) (1.0, 10 μM) (Fig. [3]). These activities were nearly equal to or more effective than minoxidil (70 μg/ml, 0.33 mM).

In the current experiment (Fig. [1]), the proliferation effects of procyanidin B-2 were below those of Ref. [3] (about 300 % to control). This difference seems attributable partly to the methodological difference between the MTT assay (Ref. [3]) and the [³ H]-thymidine incorporation assay. However, the precise reason cannot be explained, because the mode of action of procyanidin B-2 on hair epithelial cells remains obscure. Also, it should be noted that minoxidil required extremely higher concentrations for action than did the tested polyphenols (see Figs. [1] and [3]), suggesting that the polyphenols and minoxidil act on the hair epithelial cells partly in different ways; this difference is also obscure.

The active compounds isolated (2 - 6) showed proliferation activity by the assay for thymidine incorporation, but they still may interfere with thymidine metabolism (shunt of the salvage pathway or reduction in thymidine catabolism [14] and shifting growth to dependency on de novo synthesis of thymidine [15]), thus increasing the [³ H]-thymidine incorporation without stimulating proliferative growth. This led us to set up additional comparative studies using different methods (MTT and cell counting) based on different principles in order to rule out any technical artifact or bias in measurements. We measured the effect of gallic acid (6), representative of compounds (2 - 6), on hair epithelial cell growth, and found significant increases in both cell numbers and MTT activities (Fig. [4]). We concluded that gallic acid (6) directly affects the growth of mouse hair epithelial cells. Other isolated compounds (2 - 5) were expected easily to behave as (6) did because of their structural similarities as polyphenols. Also, the morphology of hair epithelial cells cultured in medium containing C. japonicum extract and the isolated active compounds (2 - 6) were rounded, characteristic of undifferentiated juvenile cells (data not shown), like those treated by minoxidil [2] and procyanidin B-2 [3]. This suggests that the extract and isolates of C. japonicum may prevent cell differentiation and extend the growth phase.

Polyphenols isolated from C. japonicum not only promote proliferation on mouse hair epithelial cells but perform several other biological activities, such as antioxidation [16], [17]. However, the mechanism underlying their proliferation abilities remains unclear. We are now investigating which moiety in polyphenols causes proliferative activity and its mode of action. Fuller molecular knowledge of the regulation of hair epithelial cell proliferation may provide a more rational and scientific approach to designing safe and effective hair growth control agents.

Acknowledgements

The authors are grateful to Dr. K. Nakaoji and Dr. O. Mori for technical advice, and to Dr. S. Ohga for providing and identifying the plant material. This work was supported in part by the Japan Society for the Cosmetology Research Foundation (Tokyo, Japan).

References

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Prof. Ryuichiro Kondo

Laboratory of Systematic Forest and Forest Products Science

Department of Forestry and Forest Products Sciences

Faculty of Agriculture

Kyushu University

6-10-1 Hakozaki

Higashi-ku

Fukuoka, 812-8581

Japan

Phone: /Fax: +81-92-642-2811

Email: kondo@brs.kyushu-u.ac.jp

References

• 1 Katz H I. Topical minoxidil: review of efficacy and safety.  Cutis. 1989;  43 94-8
  PubMedSearch in Google Scholar
• 2 Tanigaki-Obana N, Ito M. Effects of cepharanthine and minoxidil on proliferation, differentiation and keratinization of cultured cells from the murine hair apparatus.  Archives of Dermatological Research. 1992;  284 290-6
  CrossrefPubMedSearch in Google Scholar
• 3 Takahashi T, Kamiya T, Hasegawa A, Yokoo Y. Procyanidin oligomers selectively and intensively promote proliferation of mouse hair epithelial cells in vitro and activate hair follicle growth in vivo .  The Journal of Investigative Dermatology. 1999;  112 310-6
  PubMedSearch in Google Scholar
• 4 Takahashi T, Kamimura A, Yokoo Y, Honda S, Watanabe Y. The first clinical trial of topical application of procyanidin B-2 to investigate its potential as a hair growing agent.  Phytotherapy Research. 2001;  15 331-6
  CrossrefPubMedSearch in Google Scholar
• 5 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.  Archives of Dermatological Research. 1990;  282 402-7
  CrossrefPubMedSearch in Google Scholar
• 6 Nakaoji K, Nayeshiro H, Takahashi T. Norreticuline and reticuline as possible new agents for hair growth acceleration.  Biological & Pharmaceutical Bulletin. 1997;  20 586-8
  PubMedSearch in Google Scholar
• 7 Shirai A, Ikeda J, Kawashima S, Tamaoki T, Kamiya T. KF19418, a new compound for hair growth promotion in vitro and in vivo mouse models.  Journal of Dermatological Science. 2001;  25 213-8
  CrossrefPubMedSearch in Google Scholar
• 8 Janes N F, Morgan J WW. The occurrence of dihydrokampferol in Nothofagus species.  Journal of the Chemical Society. 1960;  2560-5
  PubMedSearch in Google Scholar
• 9 Kondo T. On the chemical constituents of the heartwood of Distylium racemosum S. et Z.  Journal of the Faculty of Agriculture, Kyushu University. 1951;  10 79-99
  PubMedSearch in Google Scholar
• 10 Imamura H, Kurosu H, Takahashi T. The chemistry of wood extractives. XVIII. The heartwood constituents of Rhus javanica L.  Mokuzai Gakkaishi. 1967;  13 295-9
  PubMedSearch in Google Scholar
• 11 Mabry T J, Markham K R, Thomas M B. In The systematic identification of flavonoids. Springer-Verlag New York Inc 1970: 309
  Search in Google Scholar
• 12 Miller J M, Bohm B A. Flavonoids of Laptarrhena pyrolifolia. .  Phytochemistry. 1979;  18 1412-3
  CrossrefPubMedSearch in Google Scholar
• 13 Schultz T P, Harms W B, Fisher T H, Mcmurtrey K D, Minn J, Nicholas D D. Durability of angiosperm heartwood-The importance of extractives.  Holzforschung. 1995;  49 29-34
  PubMedSearch in Google Scholar
• 14 Schwartz P M, Kugelman L C, Coifman Y, Hough L M, Milstone L M. Human keratinocytes catabolize thymidine.  The Journal of Investigative Dermatology. 1988;  90 8-12
  PubMedSearch in Google Scholar
• 15 Schwartz P M, Milstone L M. Dipyridamole potentiates the growth-inhibitory action of methotrexate and 5-fluorouracil in human keratinocytes in vitro .  The Journal of Investigative Dermatology. 1989;  93 523-7
  PubMedSearch in Google Scholar
• 16 vanAcker S ABE, vandenBerg D J, Tromp M NJL, Griffioen D H, vanBennekom W P, vanderVijgh W JF, Bast A. Structural aspects of antioxidant activity of flavonoids.  Free Radical Biology and Medicine. 1996;  20 331-42
  CrossrefPubMedSearch in Google Scholar
• 17 Okuda T. Novel aspects of tannins-renewed concepts and structure-activity relationships.  Current Organic Chemistry. 1999;  3 609-22
  PubMedSearch in Google Scholar

Prof. Ryuichiro Kondo

Laboratory of Systematic Forest and Forest Products Science

Department of Forestry and Forest Products Sciences

Faculty of Agriculture

Kyushu University

6-10-1 Hakozaki

Higashi-ku

Fukuoka, 812-8581

Japan

Phone: /Fax: +81-92-642-2811

Email: kondo@brs.kyushu-u.ac.jp