http://informahealthcare.com/phb ISSN 1388-0209 print/ISSN 1744-5116 online Editor-in-Chief: John M. Pezzuto Pharm Biol, Early Online: 1–6 ! 2015 Informa Healthcare USA, Inc. DOI: 10.3109/13880209.2014.959614
ORIGINAL ARTICLE
In vivo hair growth-stimulating effect of medicinal plant extract on BALB/c nude mice Shahnaz Begum, Li-Juan Gu, Mi-Ra Lee, Zheng Li, Jing-Jie Li, Md. Jamil Hossain, Yun-Bo Wang, and Chang Keun Sung
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Department of Food Science and Technology, College of Agriculture and Biotechnology, Chungnam National University, Daejeon, South Korea
Abstract
Keywords
Context: Chrysanthemum zawadskii var. latilobum (Asteraceae) (CZ) and Polygonum multiflorum Thunb. (Polygonaceae) (PM) have been used traditionally to treat different systemic diseases and acclaimed for various biological activities including hair growth. Objective: This study investigates the hair restoration efficacy of selected medicinal plant extracts on nude mice. Materials and methods: Nude mice genetically predisposed to pattern balding were used in this study. Topical methanol extracts of CZ and PM (10 mg/mouse/d) with standardized vehicle formulation, only vehicle (propylene glycol:ethanol:dimethyl sulfoxide, 67:30:3% v/v) and Minoxidil (2%) were applied daily for 40 consecutive days. Results: In our study, the maximum hair score (2.5 ± 0.29) was obtained in the CZ-treated group. Histological observation revealed a significant increase (p50.001) in the number of hair follicles (HF) in CZ-treated mice (58.66 ± 3.72) and Minoxidil-treated mice (40 ± 2.71). Subsequently, immunohistochemical analysis also confirmed the follicular keratinocyte proliferation by detection of BrdU-labeling, S-phase cells in Minoxidil and CZ-treated mouse follicular bulb and outer root sheaths. Conclusion: Our study revealed the underlying mechanism of stimulating hair growth in athymic nude mice by repair the nu/nu follicular keratin differentiation defect. Thus, the topical application of CZ may represent a novel strategy for the management and therapy of certain forms of alopecia.
BrdU, Chrysanthemum zawadskii, hair follicle, proliferation
Introduction Hair is one of the defining characteristics of mammals derived from the skin. It acts as a physical protector and thermal insulation of the skin also exerts as a dispersion organ of sweat and sebum (Schneider et al., 2009). While undesirable, rapid hair loss that occurs in both males and females, it has clinical implications, generally associated with diffuse thinning of scalp hair. The psychosocial impact of continuing hair loss may improve with any therapy that induces hair regrowth or retardation of further thinning. There is an everincreasing demand for drugs that manipulate scalp hair abundance and appearance. To date, minoxidil and finasteride are the only available synthetic drugs approved by US Food and Drug Administration. However, treatment of androgenic alopecia is still limited due to associated side effects and unpredicted efficacy (Libecco & Bergfeld, 2004).
Correspondence: Chang Keun Sung, Department of Food Science and Technology, College of Agriculture and Biotechnology, Chungnam National University, Daejeon 305-764, South Korea. Tel: +82 42 821 7145. Fax: +82 42 822 2287. E-mail: [email protected]
History Received 1 February 2014 Revised 22 July 2014 Accepted 26 August 2014 Published online 23 January 2015
Numerous pharmaceutical agents and hair transplant are promoted for the treatment of alopecia as well as improving scalp hair growth. A few of these include vitamins (vitamin D3), amino acids, but their efficacy remains dubious and transplant success is variable and expensive. But natural products have been quite prevalent in the hair care industry and nearly a thousand kinds of plant extracts have been examined with respect to hair growthpromoting activity, a few of them show tremendous potential as well (Takahashi et al., 1998). CZ has long been used as a traditional medicine for the treatment of common cold and other gastroenteric disorders and hypertension. Moreover, recent advanced research has reported the involvement of anti-inflammatory efficacy (Kim et al., 2012) and protective effects from liver damage (Seo et al., 2010). PM has also been reported as a potent hair growth promoter and is widely used for treating patients suffering from hair loss in East Asia (Park et al., 2011). Current research attempts to evaluate the response of these plant extracts associated with HF development and cycling. In this experiment, nude mice were used as a model for evaluating the efficacy of different plant extracts on hair growth. Usually, nude mice are restricted to cutaneous defects of the HF. Thus, this mouse develops HFs but these produce
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defective hair, due to a genetic defect of these mice. The fact is a genetically determined keratin disorder influences their development differentiation (Flanagan, 1966). No reports have been issued to date describing the potential role of natural extract (CZ and PM) on genetically distorted HF. Therefore, we reported the comparative efficacy of natural extracts to the restoration of the hair growth of nude mice.
Materials and methods All animal care and use procedures were institutional protocols approved by the Institutional Animal Care and Use Committee (IACUC), Chungnam National University, Daejeon, South Korea (Code no. CNU-00244).
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Animals and treatment protocol Athymic BALB/c (nu/nu) male nude mice at 7 weeks of age were purchased from Dae-Han Biolink (Eumsung, Korea). As the nude mice are susceptible to infection by organisms, they were maintained successfully under specificpathogen-free (SPF) conditions in a laminar-flow rack and 12 h light: dark periods at 24 ± 2 C. These conditions also include sterile food and bedding, autoclaved or acidified water, frequent sterilization of water bottles, and cages. After adapting to new environmental conditions, five mice were allocated randomly into four groups. Topical application was performed when the synchronized telogen stage were observed in all the experimental groups. PM and CZ extracts (10 mg/mouse) were suspended in a standardize vehicle formulation and minoxidil (2%) were applied daily on the back skin of nude mice until completion of two hair growth generations.
Pharm Biol, Early Online: 1–6
Skin sample collection, histology, and immunostain for bromodeoxyuridine analysis Skin samples were collected on day 16 to observe the histological changes of the different treatment groups. The mice were given 50 mg/kg intra-peritoneal (IP) injections with 5-bromo-20 -deoxyuridine (BrdU; Sigma, St. Louis, MO) 2 h before being sacrificed. The specimens of dorsal skin were fixed in 10% buffered formalin at 4 C for 24 h, and then washed with PBS (pH 7.4). Fixed samples were dehydrated through an ascending series of graded ethanol, cleared in xylene, and embedded in paraffin blocks. Subsequently, samples were cut either longitudinally or transversely into 4-mm thick sections and stained with hematoxylin and eosin (H&E). The morphology and the number of HFs of nude mice were evaluated microscopically in 5 fields per section of the dorsal skin at a magnification of 100. BrdU incorporation was determined by immunohistochemical (IHC) staining of paraffin-embedded sections with mouse anti-BrdU antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Detection of follicular keratinocyte was confirmed by BrdU labeling of S-phase cells at a magnification of 400. Histological processing and digital photomicrographs were taken using the Leica application suite, Version 4.0.0, Leica Microsystems (Heerbrugg, Switzerland). Statistical analysis Differences between two groups were analyzed by Student’s t-test. ANOVA was followed after Duncan’s multiple comparison tests. All data are expressed as means ± SD. A p value of 50.05 was considered statistically significant.
Plant sample collection, extraction, and formulation Dried aerial parts of CZ and roots of PM were collected from the Jecheon Medicinal Herb Association, South Korea. The plant samples were authenticated by Professor Ki Hwan Bae (College of Pharmacy, Chungnam National University) where the voucher specimens were deposited. The dried samples were ground into powder (500 g) and extracted with 95% methanol at 60 C, after which the extraction liquid was filtered and evaporated under vacuum to dryness below 60 C. The resulting residue was weighed (yields of 13.32% for CZ and 11.23% for PM) and dissolved in a vehicle mixture (propylene glycol:ethanol:dimethyl sulfoxide, 67:30:3% v/v). Determination of hair length and coverage area After commencing daily treatment, images were taken in early hair existing phase from inter-scapular region of the skin on both hair-growth generations using KONG, Bom-Viewer Plus (Seoul, Korea). About six images at 80 were used with a Bomviewer Image Analyzer to measure hair length. The hair coverage condition of the nude mice was systematically evaluated three times per week. The data were recorded on a scale ranging from 0 to 4 with: ‘‘0’’ ¼ complete alopecia (no hair), ‘‘1’’ ¼525% hair coverage, ‘‘2’’ ¼ 25–50% hair coverage, ‘‘3’’ ¼450–75% hair coverage, and ‘‘4’’ ¼ normal hair density and 100% hair coverage.
Results Effects of CZ and PM on nude-mice hair growth We examined the in vivo hair-growth activity of the experimental nude mice, as they received vehicle formulated CZ and PM extracts, only vehicle control OR minoxidil. The vehicle-treated control group of nude mice exhibited particularly thin, transient, and irregular hair coats during the entire experimental period (Figure 1a). On day 12, the minoxidil and CZ-treated groups showed relatively dense hair on the head, extending to the posterior dorsum. In contrast, in the PM-treated mice, sparse hair-waves started on the neck and swept rapidly across the posterior dorsum (Figure 1a–c). In terms of the area of hair coverage, the CZtreated mice scored 2.5 ± 0.29 and 2 ± 0.41 in the first and second hair-growth generations, respectively (Figure 1b). Maximum hair coverage was observed in the minoxidil and CZ groups, in the first hair-growth generation (Figure 1a and b). Although the minoxidil-treated group showed a significant increase of hair coverage (score of 2.5 ± 0.28) in the first hair-growth phase, rapid hair loss reduced the score to 1.5 ± 0.29 in the next hair-growth phase (Figure 1b). Our results also showed that hair-length of the minoxidil and CZ-treated and CZ-treated mice increased significantly compared with the controls, in both hair-growth generations (Figure 1c).
Potential effects of hair growth on nude mice
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DOI: 10.3109/13880209.2014.959614
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Figure 1. (a) Comparative pattern of hair growth in different treatment groups. Mice were treated with different extracts with vehicle, Control (A), Minoxidil (B), P. multiflorum (C) and C. zawadaskii (D) topically applied for two hair growth generations. (b) Hair existing area. (c) Hair length of different treatment groups on first and second hair existing phases. Values are shown as mean ± SD. p 0.05, **p 0.001 compared with the corresponding control.
Effects on HF development and keratinocyte proliferation CZ and PM extracts stimulated HF development, as was evident from the photomicrographs obtained from the histological study. Follicular development was determined according to the guideline proposed by Paus et al. (1999). Using this guideline, we distinctly characterized the HF development in the control mice, which revealed histological signs of catagen VII stage, on day 16 (Figure 2a00 ). For the other treatment groups, the histology corresponded to the anagen stage VIII (for minoxidil-treated mice, Figure 2b), anagen stage VII (for ‘‘(PM-treated mice , Figure 2c’’), and anagen stage VIII (for CZ-treated-mice, Figure 2d00 ). These findings also validated the macroscopic observations (Figure 2a0 –d0 ) of the skin surfaces. We confirmed keratinocyte proliferation in the anagen HF by BrdU labeling of S-phase cells (Danilenko et al., 1995). BrdU-positive cells were abundant in both the hair matrix and the outer root sheaths of the minoxidil and CZ-treated mouse-skin specimens (Figure 2b000 and d000 ), but was not detected in the vehicle-only-treated mouse skin (Figure 2a000 ).
Effect on increase number of HF In terms of the number of HFs, the CZ-extract-treated group was found to be most effective among all the groups. A significant increase in the number of HFs was seen in the CZ-treated group (58.66 ± 3.72) and minoxidil-treated group (40 ± 2.71), compared with the controls (25 ± 2.82, Table 1). The representative photomicrograph of the CZ-treated mouseskin specimen also showed that an increased number of follicles existed (i.e., anagen induction occurred) in the deep subcutis (Figure 3d). In contrast, the catagen-phase micrograph of the control mouse-skin specimen (Figure 3a) showed that only a few follicles existed in the subcutis.
Discussion Learning about the normal development and synchronized cycling of HF between the developmental stages (anagen, catagen and telogen) is one of the key challenges for hair research. Most of our current knowledge of the substances which modulate hair growth in humans is derived from clinical observation, and studies with mice have also been
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Pharm Biol, Early Online: 1–6
Figure 2. Gross phenotype (a–d) and the highlighted area are enlarged in (a0 –d0 ) to show observation of dorsal skin. Skin specimen of dorsal skin of mice (a00 –d00 ) haematoxylin and eosin (H&E) stained histology. Arrows indicate distorted hair follicles (b00 , c00 ) and the straight hair shaft emerging through the epidermis (d00 ), Scale bar 500 mm. BrdU-labeled S-phase cells in the anagen hair bulb and outer root sheath (b000 , c000 and d000 , brown color), scale bar 50 mm.
Table 1. Effect of different treatments on the number of hair follicles. Number of hair follicle (HF) Group Control Minoxidil PM CZ
In subcutis
Total HF
12.33 ± 2.19 30.33 ± 2.84** 18.43 ± 2.09 39 ± 2.59**
25 ± 2.83 40.83 ± 3.8* 28 ± 2.72 58.67 ± 3.72**
The number of HFs in subcutis with respect to the total number of HFs (per field) (n ¼ 10) are expressed as the mean ± SD. *p 0.05, **p 0.001 compared with the corresponding control.
used to identify events associated with hair-follicle cycling. In genetically predisposed nude mice, sparsely distributed HFs are barely visible on the nude skin. Moreover, progressive shortening of successive anagen cycles also leads to excessive shedding of HFs. These changes in hair growth and development stages are crucial because active HFs are still present and cycling, even in the skin of bald scalps or mutant nude skin (Dawber, 1997; Olsen, 1994). In this study, we investigated the hair growth promoting effect of topical application of CZ and PM, on nude mice skin. Plants produce numerous bioactive compounds, and
Potential effects of hair growth on nude mice
DOI: 10.3109/13880209.2014.959614
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Figure 3. Transverse section of dorsal skin specimen of different treatment groups. Control (a), Minoxidil (b), P. multiflorum (c), C. zawadaskii (d) stained with haematoxylin and eosin (H&E). Dotted lines indicate the junction of dermis and subcutis. Scale bar 500 mm.
their fruitful application may promote hair regrowth by upregulating hair development. In this context, natural products are being studied by researchers because there is a thriving clinical therapy that has already been proved, even in the form of only a crude preparation (Zhang et al., 2013). The mouse mutant ‘‘nude’’ is hairless, as described by Flanagan (1966) and athymic (Pantelouris, 1968). The hair defect of the nu/nu mutant is reported to be caused by imperfect keratinization in the hair shaft (Flanagan, 1966; Rigdon & Packchanian, 1974), which causes the hair to break off at the skin level. The macroscopic appearance of hair growth of the control nude mice was documented as a few short, crippled, bent hair shafts emerging from the HFs. Very few follicles, of locally variable density, occur in the dorsal skin (Ko¨pf-Maier et al., 1990; Militzer, 2001; Rigdon & Packchanian, 1974); and only during a short anagen growth phase (Iversen & Iversen, 1967). Here, we were able to observe an incomparable hair growth pattern in CZ-treated mice with other treated groups (Figure 1). This consequence has raised the possibility that CZ might induce some signals that regulate the follicle to continue the growth phase of the anagen stage (Hozumi et al., 1994). Skin specimens of the CZ-treated mice were also found to have abundant BrdU-positive keratinocytes in their hair bulbs and outer root sheaths. One hypothesis is that temporary keratinization in the HF gives strength to hair shafts that rise above the skin surface (Watanabe et al., 1991). Moreover, in this study we have closely observed two generations of hair growth, as well as macroscopic analysis of hair growth parameters that included changing patterns of nude mice hair growth, area of hair coverage, and length of hair shaft. The hair shaft of CZtreated mice achieved a substantial increase in length and became both thicker and smoother, whereas control hair shafts were transient, aberrant, short and irregular in shape, all suggesting a deeply impaired keratinization process (Hozumi et al., 1994). Histological study of the different treatment groups showed that the CZ-treated group had an increased number of HFs in the deep subcutis, and had completely developed HFs that corresponded to the anagen phase of the hair-growth cycle (Ogawa & Hattori, 1983; Zhang et al., 2013). Further detailed clinical trials and screening by chemical analysis will be necessary to identify the bioactive components in the extract. This knowledge will be used to prove the biological activity of the CZ extract, and to show whether the whole extract, rather than individual components, acts against alopecia.
Conclusion The methanol extract of CZ and PM were assayed to explore their bioactivities on nude mouse HF. The results indicate that CZ extract has potential for HFs restoration by inducing the follicular keratinocyte proliferation and increasing the number of HFs. This study may provide useful information and indicate directions for further exploring potential nutraceutical and pharmaceutical applications in hair-growth research.
Acknowledgements We would like to express gratitude to Dr. Kang Ju Choi, a generous person for the support and willingness to spend valuable time is gratefully acknowledged.
Declaration of interest The authors report no conflicts of interest.
Reference Danilenko DM, Ring BD, Yanagihara D, et al. (1995). Keratinocyte growth factor is an important endogenous mediator of hair follicle growth, development, and differentiation. Am J Pathol 147:145–54. Dawber R. (1997). Disease of the Hair and the Scalp. UK: Blackwell Publishing. Flanagan SP. (1966). ‘Nude’, a new hair less gene with pleiotropic effects in the mouse. Genet Res 8:295–309. Hozumi Y, Imaizumi T, Kondo S. (1994). Effect of cyclosporin on hair-existing area of nude mice. J Dermatol Sci 7:33–8. Iversen U, Iversen OH. (1967). Cycles of hair growth in hairless mice. Acta Path Microbial Scand 69:50–62. Kim Y, Han J, Sung J, et al. (2012). Anti-inflammatory activity of Chrysanthemum zawadskii var. latilobum leaf extract through haem oxygenase-1 induction. J Funct Foods 4:474–9. Ko¨pf-Maier P, Mboneko VF, Merker HJ. (1990). Nude mice are not hairless. A morphological study. Acta Anat (Basel) 139:178–90. Libecco JF, Bergfeld WF. (2004). Finasteride in the treatment of alopecia. Expert Opin Pharmacother 5:933–40. Militzer K. (2001). Hair growth pattern in nude mice. Cells Tissues Org 168:285–94. Ogawa H, Hattori M. (1983). Regulation mechanisms of hair growth. Curr Probl Dermatol 11:159–70. Olsen EA. (1994). Disorders of Hair Growth: Diagnosis and Treatment. New York: McGraw-Hill. Pantelouris EM. (1968). Absence of thymus in a mouse mutant. Nature 217:370–1. Park HJ, Zhang N, Park DK. (2011). Topical application of Polygonum multiflorum extract induces hair growth of resting hair follicles through upregulating Shh and b-catenin expression in C57BL/6 mice. J Ethnopharmacol 135:369–75. Paus R, Mu¨ller-Ro¨ver S, Van der Veen C, et al. (1999). A comprehensive guide for the recognition and classification of distinct stages of hair follicle morphogenesis. J Invest Dermatol 113:523–32.
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Rigdon RH, Packchanian A. (1974). Histologic study of the skin of congenitally athymic ‘nude’, mice. Tex Rep Biol Med 32:711–23. Schneider MR, Schmidt-Ullrich R, Paus R. (2009). The hair follicle as a dynamic miniorgan. Curr Biol 19:132–42. Seo JY, Lim SS, Park J, et al. (2010). Protection by Chrysanthemum zawadskii extract from liver damage of mice caused by carbon tetrachloride is maybe mediated by modulation of QR activity. Nutr Res Pract 4:93–8.
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Takahashi T, Kamiya T, Yokoo Y. (1998). Proanthocyanidins from grape seeds promote proliferation of mouse hair follicle cells in vitro and convert hair cycle in vivo. Acta Derm Venereol 78:428–32. Watanabe S, Mochizuki A, Wagatsuma K, et al. (1991). Hair growth on nude mice due to cyclosporine. Australas J Dermatol 18:714–19. Zhang NN, Park DK, Park HJ. (2013). Hair growth-promoting activity of hot water extract of Thuja orientalis. BMC Complement Altern Med 13:9.
ORIGINAL ARTICLE
In vivo hair growth-stimulating effect of medicinal plant extract on BALB/c nude mice Shahnaz Begum, Li-Juan Gu, Mi-Ra Lee, Zheng Li, Jing-Jie Li, Md. Jamil Hossain, Yun-Bo Wang, and Chang Keun Sung
Pharmaceutical Biology Downloaded from informahealthcare.com by Selcuk Universitesi on 01/24/15 For personal use only.
Department of Food Science and Technology, College of Agriculture and Biotechnology, Chungnam National University, Daejeon, South Korea
Abstract
Keywords
Context: Chrysanthemum zawadskii var. latilobum (Asteraceae) (CZ) and Polygonum multiflorum Thunb. (Polygonaceae) (PM) have been used traditionally to treat different systemic diseases and acclaimed for various biological activities including hair growth. Objective: This study investigates the hair restoration efficacy of selected medicinal plant extracts on nude mice. Materials and methods: Nude mice genetically predisposed to pattern balding were used in this study. Topical methanol extracts of CZ and PM (10 mg/mouse/d) with standardized vehicle formulation, only vehicle (propylene glycol:ethanol:dimethyl sulfoxide, 67:30:3% v/v) and Minoxidil (2%) were applied daily for 40 consecutive days. Results: In our study, the maximum hair score (2.5 ± 0.29) was obtained in the CZ-treated group. Histological observation revealed a significant increase (p50.001) in the number of hair follicles (HF) in CZ-treated mice (58.66 ± 3.72) and Minoxidil-treated mice (40 ± 2.71). Subsequently, immunohistochemical analysis also confirmed the follicular keratinocyte proliferation by detection of BrdU-labeling, S-phase cells in Minoxidil and CZ-treated mouse follicular bulb and outer root sheaths. Conclusion: Our study revealed the underlying mechanism of stimulating hair growth in athymic nude mice by repair the nu/nu follicular keratin differentiation defect. Thus, the topical application of CZ may represent a novel strategy for the management and therapy of certain forms of alopecia.
BrdU, Chrysanthemum zawadskii, hair follicle, proliferation
Introduction Hair is one of the defining characteristics of mammals derived from the skin. It acts as a physical protector and thermal insulation of the skin also exerts as a dispersion organ of sweat and sebum (Schneider et al., 2009). While undesirable, rapid hair loss that occurs in both males and females, it has clinical implications, generally associated with diffuse thinning of scalp hair. The psychosocial impact of continuing hair loss may improve with any therapy that induces hair regrowth or retardation of further thinning. There is an everincreasing demand for drugs that manipulate scalp hair abundance and appearance. To date, minoxidil and finasteride are the only available synthetic drugs approved by US Food and Drug Administration. However, treatment of androgenic alopecia is still limited due to associated side effects and unpredicted efficacy (Libecco & Bergfeld, 2004).
Correspondence: Chang Keun Sung, Department of Food Science and Technology, College of Agriculture and Biotechnology, Chungnam National University, Daejeon 305-764, South Korea. Tel: +82 42 821 7145. Fax: +82 42 822 2287. E-mail: [email protected]
History Received 1 February 2014 Revised 22 July 2014 Accepted 26 August 2014 Published online 23 January 2015
Numerous pharmaceutical agents and hair transplant are promoted for the treatment of alopecia as well as improving scalp hair growth. A few of these include vitamins (vitamin D3), amino acids, but their efficacy remains dubious and transplant success is variable and expensive. But natural products have been quite prevalent in the hair care industry and nearly a thousand kinds of plant extracts have been examined with respect to hair growthpromoting activity, a few of them show tremendous potential as well (Takahashi et al., 1998). CZ has long been used as a traditional medicine for the treatment of common cold and other gastroenteric disorders and hypertension. Moreover, recent advanced research has reported the involvement of anti-inflammatory efficacy (Kim et al., 2012) and protective effects from liver damage (Seo et al., 2010). PM has also been reported as a potent hair growth promoter and is widely used for treating patients suffering from hair loss in East Asia (Park et al., 2011). Current research attempts to evaluate the response of these plant extracts associated with HF development and cycling. In this experiment, nude mice were used as a model for evaluating the efficacy of different plant extracts on hair growth. Usually, nude mice are restricted to cutaneous defects of the HF. Thus, this mouse develops HFs but these produce
2
S. Begum et al.
defective hair, due to a genetic defect of these mice. The fact is a genetically determined keratin disorder influences their development differentiation (Flanagan, 1966). No reports have been issued to date describing the potential role of natural extract (CZ and PM) on genetically distorted HF. Therefore, we reported the comparative efficacy of natural extracts to the restoration of the hair growth of nude mice.
Materials and methods All animal care and use procedures were institutional protocols approved by the Institutional Animal Care and Use Committee (IACUC), Chungnam National University, Daejeon, South Korea (Code no. CNU-00244).
Pharmaceutical Biology Downloaded from informahealthcare.com by Selcuk Universitesi on 01/24/15 For personal use only.
Animals and treatment protocol Athymic BALB/c (nu/nu) male nude mice at 7 weeks of age were purchased from Dae-Han Biolink (Eumsung, Korea). As the nude mice are susceptible to infection by organisms, they were maintained successfully under specificpathogen-free (SPF) conditions in a laminar-flow rack and 12 h light: dark periods at 24 ± 2 C. These conditions also include sterile food and bedding, autoclaved or acidified water, frequent sterilization of water bottles, and cages. After adapting to new environmental conditions, five mice were allocated randomly into four groups. Topical application was performed when the synchronized telogen stage were observed in all the experimental groups. PM and CZ extracts (10 mg/mouse) were suspended in a standardize vehicle formulation and minoxidil (2%) were applied daily on the back skin of nude mice until completion of two hair growth generations.
Pharm Biol, Early Online: 1–6
Skin sample collection, histology, and immunostain for bromodeoxyuridine analysis Skin samples were collected on day 16 to observe the histological changes of the different treatment groups. The mice were given 50 mg/kg intra-peritoneal (IP) injections with 5-bromo-20 -deoxyuridine (BrdU; Sigma, St. Louis, MO) 2 h before being sacrificed. The specimens of dorsal skin were fixed in 10% buffered formalin at 4 C for 24 h, and then washed with PBS (pH 7.4). Fixed samples were dehydrated through an ascending series of graded ethanol, cleared in xylene, and embedded in paraffin blocks. Subsequently, samples were cut either longitudinally or transversely into 4-mm thick sections and stained with hematoxylin and eosin (H&E). The morphology and the number of HFs of nude mice were evaluated microscopically in 5 fields per section of the dorsal skin at a magnification of 100. BrdU incorporation was determined by immunohistochemical (IHC) staining of paraffin-embedded sections with mouse anti-BrdU antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Detection of follicular keratinocyte was confirmed by BrdU labeling of S-phase cells at a magnification of 400. Histological processing and digital photomicrographs were taken using the Leica application suite, Version 4.0.0, Leica Microsystems (Heerbrugg, Switzerland). Statistical analysis Differences between two groups were analyzed by Student’s t-test. ANOVA was followed after Duncan’s multiple comparison tests. All data are expressed as means ± SD. A p value of 50.05 was considered statistically significant.
Plant sample collection, extraction, and formulation Dried aerial parts of CZ and roots of PM were collected from the Jecheon Medicinal Herb Association, South Korea. The plant samples were authenticated by Professor Ki Hwan Bae (College of Pharmacy, Chungnam National University) where the voucher specimens were deposited. The dried samples were ground into powder (500 g) and extracted with 95% methanol at 60 C, after which the extraction liquid was filtered and evaporated under vacuum to dryness below 60 C. The resulting residue was weighed (yields of 13.32% for CZ and 11.23% for PM) and dissolved in a vehicle mixture (propylene glycol:ethanol:dimethyl sulfoxide, 67:30:3% v/v). Determination of hair length and coverage area After commencing daily treatment, images were taken in early hair existing phase from inter-scapular region of the skin on both hair-growth generations using KONG, Bom-Viewer Plus (Seoul, Korea). About six images at 80 were used with a Bomviewer Image Analyzer to measure hair length. The hair coverage condition of the nude mice was systematically evaluated three times per week. The data were recorded on a scale ranging from 0 to 4 with: ‘‘0’’ ¼ complete alopecia (no hair), ‘‘1’’ ¼525% hair coverage, ‘‘2’’ ¼ 25–50% hair coverage, ‘‘3’’ ¼450–75% hair coverage, and ‘‘4’’ ¼ normal hair density and 100% hair coverage.
Results Effects of CZ and PM on nude-mice hair growth We examined the in vivo hair-growth activity of the experimental nude mice, as they received vehicle formulated CZ and PM extracts, only vehicle control OR minoxidil. The vehicle-treated control group of nude mice exhibited particularly thin, transient, and irregular hair coats during the entire experimental period (Figure 1a). On day 12, the minoxidil and CZ-treated groups showed relatively dense hair on the head, extending to the posterior dorsum. In contrast, in the PM-treated mice, sparse hair-waves started on the neck and swept rapidly across the posterior dorsum (Figure 1a–c). In terms of the area of hair coverage, the CZtreated mice scored 2.5 ± 0.29 and 2 ± 0.41 in the first and second hair-growth generations, respectively (Figure 1b). Maximum hair coverage was observed in the minoxidil and CZ groups, in the first hair-growth generation (Figure 1a and b). Although the minoxidil-treated group showed a significant increase of hair coverage (score of 2.5 ± 0.28) in the first hair-growth phase, rapid hair loss reduced the score to 1.5 ± 0.29 in the next hair-growth phase (Figure 1b). Our results also showed that hair-length of the minoxidil and CZ-treated and CZ-treated mice increased significantly compared with the controls, in both hair-growth generations (Figure 1c).
Potential effects of hair growth on nude mice
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DOI: 10.3109/13880209.2014.959614
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Figure 1. (a) Comparative pattern of hair growth in different treatment groups. Mice were treated with different extracts with vehicle, Control (A), Minoxidil (B), P. multiflorum (C) and C. zawadaskii (D) topically applied for two hair growth generations. (b) Hair existing area. (c) Hair length of different treatment groups on first and second hair existing phases. Values are shown as mean ± SD. p 0.05, **p 0.001 compared with the corresponding control.
Effects on HF development and keratinocyte proliferation CZ and PM extracts stimulated HF development, as was evident from the photomicrographs obtained from the histological study. Follicular development was determined according to the guideline proposed by Paus et al. (1999). Using this guideline, we distinctly characterized the HF development in the control mice, which revealed histological signs of catagen VII stage, on day 16 (Figure 2a00 ). For the other treatment groups, the histology corresponded to the anagen stage VIII (for minoxidil-treated mice, Figure 2b), anagen stage VII (for ‘‘(PM-treated mice , Figure 2c’’), and anagen stage VIII (for CZ-treated-mice, Figure 2d00 ). These findings also validated the macroscopic observations (Figure 2a0 –d0 ) of the skin surfaces. We confirmed keratinocyte proliferation in the anagen HF by BrdU labeling of S-phase cells (Danilenko et al., 1995). BrdU-positive cells were abundant in both the hair matrix and the outer root sheaths of the minoxidil and CZ-treated mouse-skin specimens (Figure 2b000 and d000 ), but was not detected in the vehicle-only-treated mouse skin (Figure 2a000 ).
Effect on increase number of HF In terms of the number of HFs, the CZ-extract-treated group was found to be most effective among all the groups. A significant increase in the number of HFs was seen in the CZ-treated group (58.66 ± 3.72) and minoxidil-treated group (40 ± 2.71), compared with the controls (25 ± 2.82, Table 1). The representative photomicrograph of the CZ-treated mouseskin specimen also showed that an increased number of follicles existed (i.e., anagen induction occurred) in the deep subcutis (Figure 3d). In contrast, the catagen-phase micrograph of the control mouse-skin specimen (Figure 3a) showed that only a few follicles existed in the subcutis.
Discussion Learning about the normal development and synchronized cycling of HF between the developmental stages (anagen, catagen and telogen) is one of the key challenges for hair research. Most of our current knowledge of the substances which modulate hair growth in humans is derived from clinical observation, and studies with mice have also been
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Figure 2. Gross phenotype (a–d) and the highlighted area are enlarged in (a0 –d0 ) to show observation of dorsal skin. Skin specimen of dorsal skin of mice (a00 –d00 ) haematoxylin and eosin (H&E) stained histology. Arrows indicate distorted hair follicles (b00 , c00 ) and the straight hair shaft emerging through the epidermis (d00 ), Scale bar 500 mm. BrdU-labeled S-phase cells in the anagen hair bulb and outer root sheath (b000 , c000 and d000 , brown color), scale bar 50 mm.
Table 1. Effect of different treatments on the number of hair follicles. Number of hair follicle (HF) Group Control Minoxidil PM CZ
In subcutis
Total HF
12.33 ± 2.19 30.33 ± 2.84** 18.43 ± 2.09 39 ± 2.59**
25 ± 2.83 40.83 ± 3.8* 28 ± 2.72 58.67 ± 3.72**
The number of HFs in subcutis with respect to the total number of HFs (per field) (n ¼ 10) are expressed as the mean ± SD. *p 0.05, **p 0.001 compared with the corresponding control.
used to identify events associated with hair-follicle cycling. In genetically predisposed nude mice, sparsely distributed HFs are barely visible on the nude skin. Moreover, progressive shortening of successive anagen cycles also leads to excessive shedding of HFs. These changes in hair growth and development stages are crucial because active HFs are still present and cycling, even in the skin of bald scalps or mutant nude skin (Dawber, 1997; Olsen, 1994). In this study, we investigated the hair growth promoting effect of topical application of CZ and PM, on nude mice skin. Plants produce numerous bioactive compounds, and
Potential effects of hair growth on nude mice
DOI: 10.3109/13880209.2014.959614
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Figure 3. Transverse section of dorsal skin specimen of different treatment groups. Control (a), Minoxidil (b), P. multiflorum (c), C. zawadaskii (d) stained with haematoxylin and eosin (H&E). Dotted lines indicate the junction of dermis and subcutis. Scale bar 500 mm.
their fruitful application may promote hair regrowth by upregulating hair development. In this context, natural products are being studied by researchers because there is a thriving clinical therapy that has already been proved, even in the form of only a crude preparation (Zhang et al., 2013). The mouse mutant ‘‘nude’’ is hairless, as described by Flanagan (1966) and athymic (Pantelouris, 1968). The hair defect of the nu/nu mutant is reported to be caused by imperfect keratinization in the hair shaft (Flanagan, 1966; Rigdon & Packchanian, 1974), which causes the hair to break off at the skin level. The macroscopic appearance of hair growth of the control nude mice was documented as a few short, crippled, bent hair shafts emerging from the HFs. Very few follicles, of locally variable density, occur in the dorsal skin (Ko¨pf-Maier et al., 1990; Militzer, 2001; Rigdon & Packchanian, 1974); and only during a short anagen growth phase (Iversen & Iversen, 1967). Here, we were able to observe an incomparable hair growth pattern in CZ-treated mice with other treated groups (Figure 1). This consequence has raised the possibility that CZ might induce some signals that regulate the follicle to continue the growth phase of the anagen stage (Hozumi et al., 1994). Skin specimens of the CZ-treated mice were also found to have abundant BrdU-positive keratinocytes in their hair bulbs and outer root sheaths. One hypothesis is that temporary keratinization in the HF gives strength to hair shafts that rise above the skin surface (Watanabe et al., 1991). Moreover, in this study we have closely observed two generations of hair growth, as well as macroscopic analysis of hair growth parameters that included changing patterns of nude mice hair growth, area of hair coverage, and length of hair shaft. The hair shaft of CZtreated mice achieved a substantial increase in length and became both thicker and smoother, whereas control hair shafts were transient, aberrant, short and irregular in shape, all suggesting a deeply impaired keratinization process (Hozumi et al., 1994). Histological study of the different treatment groups showed that the CZ-treated group had an increased number of HFs in the deep subcutis, and had completely developed HFs that corresponded to the anagen phase of the hair-growth cycle (Ogawa & Hattori, 1983; Zhang et al., 2013). Further detailed clinical trials and screening by chemical analysis will be necessary to identify the bioactive components in the extract. This knowledge will be used to prove the biological activity of the CZ extract, and to show whether the whole extract, rather than individual components, acts against alopecia.
Conclusion The methanol extract of CZ and PM were assayed to explore their bioactivities on nude mouse HF. The results indicate that CZ extract has potential for HFs restoration by inducing the follicular keratinocyte proliferation and increasing the number of HFs. This study may provide useful information and indicate directions for further exploring potential nutraceutical and pharmaceutical applications in hair-growth research.
Acknowledgements We would like to express gratitude to Dr. Kang Ju Choi, a generous person for the support and willingness to spend valuable time is gratefully acknowledged.
Declaration of interest The authors report no conflicts of interest.
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