Inhibitory activities of some traditional Chinese herbs against testosterone 5α-reductase and effects of Cacumen platycladi on hair re-growth in testosterone-treated mice

Journal of Ethnopharmacology 177 (2016) 1–9

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Inhibitory activities of some traditional Chinese herbs against testosterone 5α-reductase and effects of Cacumen platycladi on hair re-growth in testosterone-treated mice Bei Zhang a,1, Rong-weng Zhang b,1, Xi-quan Yin a, Zi-zhao Lao a, Zhe Zhang a, Qing-guang Wu a, Liang-wen Yu a, Xiao-ping Lai a,c, Yu-hua Wan b, Geng Li a,n a b c

Guangzhou University of Chinese Medicine, Guangzhou 510006, China Bawang (Guangzhou) Co. Ltd., Guangzhou 510440, China Dongguan Mathematical Engineering Academy of Chinese Medicine and Guangzhou University of Traditional Chinese Medicine, Dongguan 523808, China

art ic l e i nf o

a b s t r a c t

Article history: Received 2 March 2015 Received in revised form 10 August 2015 Accepted 5 November 2015 Available online 11 November 2015

Ethnopharmacological relevance: Many traditional Chinese medicines (TCM) have been used for hundreds of years for hair blackening and hair nourishing, and now many of them are commonly used in Chinese herbal shampoo to nourish the hair and promote hair growth. Aims of the study: The present study was performed to screen 5α-reductase (5αR) inhibitors from traditional Chinese medicines, evaluate its hair growth promoting activity in vivo, and further investigate its effects on androgen metabolism and the expression of 5αR II in hair follicles. Materials and methods: Nine TCM which were dried, ground and extracted by maceration with 75% ethanol or distilled water were used for screening 5αR inhibitors, and enzymes were extracted from the rat epididymis. The leaves of Platycladus orientalis (L.) Franco was used to evaluate the in vivo antiandrogenic activity. Skin color was observed daily and the hair re-growth was assessed by assigning the hair growth score. The longitudinal sections of hair follicles were used for observing follicle morphology, classifying of distinct stages of hair follicle morphogenesis and calculate the average score. The transverse sections were used for determination of hair follicle counts. Testosterone (T), Dihydrotestosterone (DHT) and Estradiol (E2) levels in serum and skin tissue were detected by ELISA kits. The immunofluorescence assay was used to detect the influence of CP-ext on 5αR expression in dorsal skin. Results: We found the extract of Ganoderma lucidum (GL-ext), Polygonum multiflori (PM-ext), Cacumen platycladi (CP-ext) and Cynomorium songaricum (CS-ext) showed stronger 5αR inhibitory activity. CP-ext (5 mg and 2 mg/mouse/day) could significantly shorten the time of the dorsal skin darkening and got longhaired (Po 0.01), and showed high hair re-growth promoting activity. Furthermore the histological data of hair follicles in each group showed that CP-ext could promote the growth of hair follicle and slowed down hair follicles enter the telogen. What's more CP-ext significantly reduced DHT levels and down-regulated the expression of 5αRⅡin skin (P o0.01). Conclusions: GL-ext, PM-ext, CP-ext and CS-ext showed strong 5αR inhibitory activity. CP-ext possesses high hair growth promoting activity in the in vivo androgen-sensitive mouse model via inhibiting the 5αR activity, decreasing the DHT levels and in turn suppressing the expression of 5αR. Our study may contribute to the development of a new generation of herbal supplements with clearer material basis of pharmacodynamic for treating androgenic alopecia (AGA). & 2015 Elsevier Ireland Ltd. All rights reserved.

Keywords: 5α-reductase Androgenic alopecia Traditional Chinese medicines Hair re-growth Dihydrotestosterone Immunofluorescence

1. Introduction 5αR is a key enzyme in the metabolism of androgens that converts testosterone (T) into the more potent androgen

n

Corresponding author. Fax: +86 2039358550. E-mail address: [email protected] (G. Li). 1 These authors contributed equally to this work, and are the co-first writers.

http://dx.doi.org/10.1016/j.jep.2015.11.012 0378-8741/& 2015 Elsevier Ireland Ltd. All rights reserved.

dihydrotestosterone (DHT) (Anderson and Liao, 1968; Bruchovsky and Wilson, 1968). It is a microsomal enzyme localized in the membrane of target cells. Three isozymes of 5αR are already known: the isozyme type I (5αRI), the isozyme type Ⅱ (5αRⅡ) and the isozyme type Ⅲ (5αRⅢ). 5αRI is mainly expressed in livers and non-genital skin, the optimal pH range it play a physiological role is 6–9, while 5αRⅡis the predominant isozyme detectable in androgen-dependent tissues such as the prostate, epididymis, seminal vesicles, and hair follicles and the optimum pH is 5.5

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B. Zhang et al. / Journal of Ethnopharmacology 177 (2016) 1–9

(Asada et al., 2001; Zhu and Sun, 2005), which suggests that it is closely related to hair loss. And 5αRⅢ has been identified recently, which is overexpressed in the tissues of hormone-refractory prostate cancers (Uemura et al., 2008; Iwai et al., 2013). Studies have demonstrated that the generation of many diseases such as androgenic alopecia (McPhaul and Young, 2001), benign prostatic hyperplasia (Bartsch et al., 2000), acne (Förström, 1980), and female hirsutism (Faloia et al., 1998) is closely to the abnormal metabolism of androgens or the role of androgen, in which 5αR plays an important role. Seborrheic alopecia (SA), also known as androgenic alopecia (AGA) or male pattern alopecia (MPA) and female pattern alopecia (FPA) is characterized by greasy hair, dandruffy, itching, progressive alopecia in frontotemporal area and followed forming baldness. Although it may not be a life-threatening disorder, it has a great impact on a person's appearance, self-respect, mental health and overall quality of life. 5αR II, which converts T to DHT is the predominant form in hair follicles and plays a more critical role in the pathogenesis of SA. Although androgens mediate their activities by binding to the same androgen receptors (AR), the affinity of DHT is fivefold higher (Ellsworth et al., 1998). AR belongs to the nuclear receptor superfamily and functions as a transcription factor. Upon binding ligand, AR undergoes a conformational change, translocates to the nucleus, and binds to the specific DNA sequence of target genes. These result in positive (Promote hair growth) or negative (Inhibit hair growth) regulation of gene expression. Therefore, inhibition of 5αR II would thus be a good target for investigation of efficacy in SA. In fact, in recent years, many steroidal and non-steroidal 5αR inhibitors have been synthesized. Finasteride, a specific 5αRII inhibitor, has been used to treat androgen-related disorders and achieved outstanding results. However, these synthetic inhibitors often come with a lot of adverse reactions, for example: erectile dysfunction, abnormal sexual function, gynecomastia, impairment of muscle growth, and

so on (Lacy et al., 2008). Recently years, the researchers are focus on looking for new, effective and low toxicity 5αR inhibitors from natural plants for the treatment of SA, which is of great significance. And many plants were proved to have the potential to inhibit 5αR and promote hair growth, for example: Piper nigrum leaf (Hirata et al., 2007), Puerariae Flos (Kazuya et al., 2012), Carthamus tinctorius and Phyllanthus emblica (Kumar et al., 2012), and Rosmarinus officinalis Leaf (Kazuya et al., 2013). Traditional Chinese medical theory believes poliosis and lapsus pilorum are related to the weakness of liver, kidney and blood vigor. Therefore, the cure must rely on the reinforcement of the factors above. In China, many TCM have been used for nourishing the liver and kidney, promoting blood generation, cooling blood and blackening hair for hundreds of years (Huang et al., 2007; Zhang, 2010). Modern medical theory proves that many of them could promote hair follicle growth (Chen et al., 2010; Gu et al., 2007; Wu et al., 2006), which were used for the treatment of hair loss and applied in Chinese herbal shampoo.The Leaves of Platycladus orientalis (L.) Franco is a commonly used TCM in the treatment of hair loss, and previous experiments revealed that it mainly comprises volatile oils, flavonoids and tannins and could promote hair growth in mice (Chen et al., 2010; Shan et al., 2013). However, the possible mechanisms involved in their treatment of hair loss have not yet been elucidated. In this manuscript, we first report the results of the screening of the 5αR inhibitors from those crude drugs. In addition, an in vivo AGA model suppressed by testosterone was used to evaluate its hair growth promoting activity, and further investigate its effects on androgen metabolism and the expression of 5αR II in hair follicles. These crude drugs, their traditional efficacy and their applications are shown in Fig. 1 and Table 1.

Fig.1. Traditional Chinese medicines we used in the experiment.

FL-ext 55.0

HE-ext 44.0

RC-ext 18.6

RA-ext 32.0

CT-ext 18.4

CS-ext 21.0

CP-ext 14.0

GL-ext 2.7 PM-ext 18.0

2.1. Reagents Easy-Lowyer protein assay kit was obtained from Beijing ComWin Biotech Co., Ltd. (Beijing, China). ELISA kits of T, DHT and E2 were purchased from BlueGene Biotech. (Shanghai, China). SRD5A2 antibody was purchased from Biorbyt (San Franciso, USA). Goat anti-rabbit IgG-FITC was purchased from Santa Cruz Biotechnology, Inc (California, USA). Dithiothreitol (DTT), Glycerol, Testosterone, NADPH, Phenylmethanesulfonyl fluoride (PMSF), Tris–HCl buffer (pH 5.5), Methanol and ethanol were purchased from Nanjing Senbeijia Biotechnology Co., Ltd. (Nanjing, China). 2.2. Animals Male Sprague Dawley rats (9 weeks of age) and male C57BL/ 6NCrSlc (C57) mice (7 weeks of age) were obtained from central animal house facility of Guangzhou University of Chinese Medicine. They were acclimatized in an air conditioned room at 227 2 °C, relative humidity of 50710%, 12/12-h light/dark cycles. All the animals were free to standard laboratory feed and tap water before experiment. The studies were approved by the Animal Ethics Committee of Guangzhou University of Chinese Medicine. 2.3. Plant and drugs The TCMs used in this experiment, as shown in Fig. 1 and Table 1, were locally purchased from Guangdong Province Traditional Chinese Medical Hospital (Guangzhou, China), identified and authenticated by Prof. Lai Xiao-ping in the School of Chinese Materia Medica, Guangzhou University of Chinese Medicine. The voucher specimens (No. 2013120301, 2013120302, 2013120303, 2013120304, 2013120305, 2013120306, 2013120307, 2013120308, 2013120309) were deposited in the herbarium of Guangzhou University of Chinese Medicine for further study. Finasteride was purchased from Merck Pharmaceutical Co., Ltd., Hangzhou, China (Standard: 5 mg/tablet, batch number: J013788). 2.4. Preparation of the plant extract All crude drugs were pulverized and extracted by using 75% Ethanol for 2.5 h with the heating reflux method (CT-ext was extracted with distilled water for 0.5 h). The extract solution was filtered and concentrated by using a rotary evaporator. Then the corresponding extracts were dried in a vacuum oven, and the extract powder was collected and weighed by an analytical balance to calculate the extraction yield (Table 1) according to formulaⅠ. The total flavonoids in CP-ext were detected at 254 nm by the ultraviolet spectrophotometric, using quercetin as reference solution. The content of total flavonoids was 0.251%. Ⅰ Extraction yield (%)¼ Extract powder weight (g)/crude drugs weight (g)*100% Finally, the extract powder was dissolved in 75% ethanol before the experiment. The reaction concentrations in vitro were 2.5 mg/mL, 0.5 mg/mL, 0.1 mg/mL and 0.02 mg/mL, respectively. 2.5. Method for determining the 5α-reductase inhibitory activity

Lycium barbarum L.

Eclipta prostrata (L.) L.

Ligusticum chuanxiong hort.

Angelica sinensis (Oliv.) Diels

Carthamus tinctorius L.

Polyporaceae Polygonaceae

Ganoderma lucidum Polygonum multiflorum Thunb. Platycladus orientalis (L.) Franco Cynomorium songaricum Rupr.

Fruiting body Ling Zhi Lucid Ganoderma Replenishing qi and calming the nerves (Liu and Tang, 2009) Radix He Show Wu Radix Polygoni Multiflori Nourishing the liver and kidney, darkening hair. Commonly used in Chinese herbal shampoo to nourish the hair and promote hair growth (Gu et al., 2008) Cupressaceae leaf Ce Bai Ye Cacumen Platycladi Darkening hair and promoting hair growth, Commonly used in Chinese herbal shampoo (Huang et al., 2007) Cynomoriaceae Stems Suo Yang Herba cynomorii Nourishing the kidney, Commonly used in Chinese herbal shampoo to nourish the hair (Sun and Chang, 2005) Asteraceae Flowers Hong Hua Flos Carthami Promoting blood circulation, promote hair follicle growth, commonly used in Chinese herbal shampoo (Wu et al., 2006) Umbelliferae Radix Dang Gui Radix Angelicae Ainensis Nourishing and activating blood.Used as a hair conditioner to nourish the scalp and supply nutrition hair needs (Wu et al., 2006) Umbelliferae Rhizoma Chuan Xiong Szechwan Lovage Promoting blood circulation, promote hair follicle growth (Gu et al., 2007) Rhizome Asteraceae Herba Mo Han Lian Yerbadetajo Herb Nourishing the liver and kidney, darkening hair (Li and Wang, 2010). Commonly used in Chinese herbal shampoo to nourish the hair and promote hair growth Solanaceae Fructus Gou Qi Zi Fructus Lycii Nourishing the liver and kidney, darkening hair (Zhu, 2005). Commonly used in Chinese herbal shampoo to nourish the hair and promote hair growth

English name Local name Family

Part used

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2. Materials and methods

Botanical name

Table 1 Traditional Chinese medicine used in this experiment, their usage and extraction yield.

Traditional efficacy and their applications

Extraction yield (%)

B. Zhang et al. / Journal of Ethnopharmacology 177 (2016) 1–9

As reported in the previous study (Normington and Russell, 1992; Iwai et al., 2013) that type II 5αR isozyme was abundant in the rat epididymis. The epididymis of 20 rats (10 weeks of age after 1 week of acclimation) were dissected and homogenized with a blender in precooled 1 M Tris–HCl buffer (pH 5.5) containing 1 mM PMSF fluoride, 1 mM DTT, and 10% glycerol cocktail.

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The epididymis homogenate was centrifuged by Heraeus Laboratory Centrifuge (Model Biofuge Pico, Heraeus, Germany) at 4 °C 3000g for 10 min, the supernatant was centrifuged at 4 °C 10000g for 50 min to obtain a crude enzyme suspension. The protein concentration of the enzyme solution was determined by the method of Lowry (Iwai et al., 2013). The enzyme solution was kept at 80 °C until used as an enzyme source. Inhibition assay of testosterone 5αR enzyme reaction was performed according to the previous method reported (Matsuda et al., 2001; Mitamura et al., 2005; Kazuya et al., 2012; Kumar et al., 2012) with minor modifications. Briefly, the reaction solution contained 50 μL extracts, 300 μL 1 M Tris–HCl buffer (pH 5.5), 50 μL 1 mM testosterone in 75% ethanol and 500 μL enzyme solution (Diluted to a certain concentration with the Tris buffer pH 5.5). Reactions were then initiated by the addition of 100 μL 1 mM NADPH. The mixture was incubated in a incubators at 37 °C for 30 min. The reactions were then stopped by adding 1 mL Methanol, mixed, and centrifuged at 5000 rpm for 15 min. Separate the supernatant and filter with 0.22 μm microporous membrane. And an aliquot of 20 μL was injected into the HPLC system under the following conditions: Diamonsil™ C18 column (250 mm  4.6 mm, 5 μm); column temperature, room temperature; mobile phase, MeOH/water (7:3, v/v); flow rate, 1.0 mL/min; detection, UV at 242 nm. Finasteride was used as a standard enzyme inhibitor. Inhibition rate was calculated according to the following formula. Ⅰ conversion rate (%) of testosterone to DHT¼ (Concentration of T at 0 min- Concentration of T at 30 min)/Concentration of T at 0 min*100% Ⅱ Inhibition (%) ¼{(Concentration variation of T for control groups in 30 min- Concentration variation of T for sample groups in 30 min)/Concentration variation of T for control groups in 30 min}*100% 2.6. Effects of CP-ext on hair growth promoting activity of testosterone-treated C57 mice Hair growth promoting activity of these extracts were determined as reported previously (Kazuya et al., 2012; Kumar et al., 2012). The C57 mice (seven-week-old) were randomly divided into 5 groups (8 mice per group): Normal group, Model group, Positive group, CP high dose group (CP-H), CP low dose group (CP-L). The 2 cm  2.5 cm dorsal hair of these mice was depilated by wax/resin mixture (1:1). Beginning the next day, 100 μL 0.05% testosterone solution (dissolved in 75% Ethanol) was applied topically to the shaved dorsum once a day for 28 days with a pipette except the Normal group. After 30 min of testosterone treatment, 100 μL of the corresponding drug solutions were applied topically as follows: Positive group given 2% Finasteride (dissolved in 75% ethanol solution), CP groups given CP solution (Shan et al., 2013) (dissolved in 75% ethanol, Concentration: 50 mg/mL, 20 mg/mL; Dosage: 5 mg/mouse/day, 2 mg/mouse/day), Normal group and Model group were given 75% ethanol solution as control. Skin color was observed daily, and the times when the skin got dark and the hair grew were also recorded. The hair re-growth was assessed in each mouse at 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 and 28 days by assigning a hair growth score, as follows: score0 ¼ no growth observed; 1 ¼up to 20% growth; 2 ¼20–40% growth; 3 ¼40–60% growth; 4 ¼60–80% growth; and 5¼ 80% to full growth observed. 2.7. HE staining determination effects of CP-ext on hair follicles At the termination of the experiment, all of the mice were sacrificed. The dorsal skin of each animal was dissected and then sectioned into two different patterns (both fixed in 4% paraformaldehyde). After 24 h, the skin samples were proceeded for H

& E staining. A light microscope was used for overall histological assessment. The longitudinal sections were used for observing follicle morphology. The hair follicle staging was scored based on MullerRover' morphological stage (Paus et al., 1999; Müller-Röver et al., 2001), 60 follicles was randomly selected from each mouse to classify of distinct stages of hair follicle morphogenesis and calculate the average score: Ⅵ growth period is 100, early degenerative is 200, mid regression is 300, late degenerative is 400. The transverse sections were used for determination of hair follicle count (Dhanotia et al., 2011). 2.8. Effects of CP on T, DHT and E2 levels in serum and skin tissue Blood samples of overnight fasted mice were directly obtained from the post-ocular venous plex, and allowed to clot at room temperature for 1 h before they were centrifuged by Heraeus Laboratory Centrifuge (Model Biofuge Pico, Heraeus, Germany) at 3500 rpm at the temperature of 4 °C for 15 min to separate the serum. Their dorsal skins were removed, weighed and homogenized with a blender in cooled PBS (1:10, w/v). The homogenate was centrifuged under the same conditions. Then the T, DHT and E2 levels in the serum and skin homogenates were detected by standard diagnostic test ELISA kits according to the manufacturer's instructions. 2.9. Immunofluorescence detecting effects of CP on expression of 5αreductase in skin tissue The dorsal skin of each animal was dissected and made of frozen tissue sections by CM-1850 freezing microtome (Leica, Germany) at  25 °C, the frozen sections were 4% paraformaldehyde fixed and then incubated in 5% BSA, 0.2%Triton X-100 and 1  PBS to permeabilize the tissue sections. Followed, the frozen sections were incubated with the SRD5A2 antibody at 1/200 dilution overnight at 4 °C, then incubated with the secondary antibody goat anti-rabbit IgG-FITC at room temperature for half an hour (Avoid light).The LSM 700 fluorescence microscopy (Zeiss, Germany) was used to observe and take pictures, the Image J software system was used to measure fluorescence intensity. 2.10. Statistical analysis All data were expressed as means 7S.E.M. Analysis was performed by one-way ANOVA for multiple comparisons, and significant difference between control and experimental groups, using SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA). Mann– Whitney U-test was performed in case of variance heterogeneity. P-values less than 0.05 were considered to be statistically significant.

3. Results 3.1. Inhibitory activities of plant extracts on 5α-reductase Finasteride, a specific 5αR II inhibitor, has been used to treat androgen-related disorders. Table 2 showed that it could inhibit the 5αR activity in a dose-dependent manner, and the IC50 value measured by the model was 212 nM, which was comparable to the previously reported 237 nM (Mitamura et al., 2005), indicating that the in vitro screening model we established was convenient, stable and reliable and could be widely used to screen inhibitors. The results in our study suggested that among the crude drugs tested, GL-ext, PM-ext, CP-ext and CS-ext showed stronger inhibitory activity compared to the other plants as 85.86%, 90.25%,

B. Zhang et al. / Journal of Ethnopharmacology 177 (2016) 1–9

Table 2 Inhibitory activities of Finasteride in vitro against 5αR. Samples

Concentration (μmol/L)

Control – Finasteride 0.02 0.1 0.5 1

Conversion rate of T Inhibition (%) IC50 (n to DHT (%) mol/L) 55.26 7 0.95 41.777 1.76nn 31.977 3.03nn 12.137 1.74nn 9.02 7 1.72nn

– 24.42 42.15 78.04 83.67

– 212

Data were presented as mean7 S.E.M. (n¼ 3). nn

Po 0.01 compared with the Normal group.

Table 3 Inhibitory activities of plant extracts in vitro against 5αR. Samples Concentration (mg/mL) Control GL-ext

PM-ext

HE-ext

FL-ext

CP-ext

CS-ext

CT-ext

RA-ext

RC-ext

– 0.02 0.1 0.5 2.5 0.02 0.1 0.5 2.5 0.02 0.1 0.5 2.5 0.02 0.1 0.5 2.5 0.02 0.1 0.5 2.5 0.02 0.1 0.5 2.5 0.02 0.1 0.5 2.5 0.02 0.1 0.5 2.5 0.02 0.1 0.5 2.5

Conversion rate of T to DHT (%)

Inhibition (%)

52.52 7 0.57 17.63 7 2.60nn 15.277 1.69nn 14.08 7 1.51nn 7.42 7 1.08nn 29.38 7 1.68nn 16.89 7 2.78nn 6.90 7 2.71nn 5.127 1.78nn 51.20 7 1.08 45.95 7 0.32nn 39.56 7 2.77nn 37.30 7 2.93nn 47.52 7 1.16nn 42.977 0.41nn 40.99 7 1.53nn 38.29 7 0.68nn 17.84 7 1.82nn 11.05 7 4.65nn 8.767 4.71nn 8.69 7 7.20nn 22.54 7 3.68nn 21.45 7 6.07nn 9.81 7 1.21nn 5.377 3.83nn 44.017 3.08 38.337 3.29nn 18.34 7 3.67nn 18.58 7 1.27nn 45.167 2.64 44.077 0.98 43.377 2.15n 39.60 7 3.24nn 45.707 2.96 45.23 7 3.94 44.84 7 1.81 42.88 7 0.77n

– 66.44 70.94 73.20 85.86 44.06 67.84 86.87 90.25 1.74 11.82 24.07 28.42 8.80 17.54 20.62 26.51 62.69 76.90 81.69 81.84 52.88 55.15 79.50 88.78 7.97 19.84 61.65 61.14 5.56 7.83 9.30 17.19 4.44 5.42 6.23 10.34

Data were presented as mean7 S.E.M. (n¼ 3). nn n

Po 0.01 compared with the Normal group. Po 0.05 compared with the Normal group.

81.84%, 88.87% of inhibition rate respectively (Table 3). Our study first discovered CP-ext was a significant 5αR inhibitor, and traditional Chinese medical theory believes Cacumen platycladi can cool blood, blacken hair and used for hair loss and premature graying. Therefore, elucidating the possible mechanisms involved in its treatment of hair loss is of great significance. So we intend to focus on Cacumen platycladi in further studies. 3.2. Hair growth promoting activity of CP-ext in testosterone-treated C57 mice Since CP-ext showed high 5αR inhibitory activity and its traditional use in hair lose, the in vivo anti-androgenic activity were

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examined by using a hair re-growth assay in testosterone-sensitive male C57 strain mice. Dorsal hairs of 7-week-old C57 strain mice enter the telogen phase (Skin color was pink, Fig. 2A) and were moved into the anagen phase (Skin color was black, Fig. 2B) after depilating by wax/resin mixture (1:1). As shown in Fig. 2D and E, compared with the Normal group, testosterone treatment markedly shortened the anagen phase and suppressed hair re-growth (P o0.01). However, the application of CP-ext (5 mg and 2 mg/ mouse/day) could significantly shorten the time of the dorsal skin darkening and got longhaired (P o0.01). Further hair growth promoting activity were tested by assigning a hair growth score every other day from the 8th day, it was found that CP-L and CP-H showed high hair growth promoting activity (Fig. 3),which revealed that CP extract showed a significant anti-androgenic activity in the in vivo mouse model. 3.3. Effects of CP-ext on histopathology of skin sections The morphology of hair follicle, obtained from a longitudinal section of the dorsal skin, is shown in Fig. 4A–E. Mice in Normal group had more hair follicles, the formation of melanin was obvious and dermal papilla grew downward into the dermis (Fig. 4A). While in the Model group, hair follicles were sparse and degradation with less melanin and located at the junction of the epidermis and dermis (Fig. 4B). Mice receiving CP had more hair follicles than mice in the Model group (Fig. 4D and E). Then we randomly selected 60 follicles from each mouse to classify of distinct stages of hair follicle morphogenesis and calculate the average score based on Müller-Röver' morphological stage. The mean score of mice in Normal group was 122, indicated hair follicle was in Ⅵ growth period and early degenerative. While the score of mice in Model group was 312.33, significantly increased (P o0.01), demonstrated hair follicle was in mid regression and late degenerative. The mean score decreased notably (Po 0.01) when mice receiving CP treatment (Fig. 4F). The transverse sections were used for determination of hair follicle count (Fig. 5A–E). In the Normal group mice, the mean hair follicle count was 72.4 per selected area under 50  magnification by light microscope. The mean hair follicle count of the Model group was 20.8, significantly decreased compared with the Normal group (Po 0.01). The mice receiving finasteride, CP-H, CP-L had 64.2, 68.2 and 59.0 hair follicles per area, respectively, significantly increased compared with the Model group (Po 0.01). 3.4. Effects of CP-ext on androgen levels In order to investigate the effect of 5αR inhibitory on hormone metabolism, the levels of T, DHT and E2 in serum and skin homogenates were detected by standard diagnostic test ELISA kits. Fig. 6A–C Showed that the levels of T, DHT and E2 in serum had no significant change compared with the Normal group (P 40.05). T, DHT and E2 levels in skin homogenates increased significantly compared with the Normal group (P o0.05), while DHT levels in CP-H and CP-L groups decreased significantly compared with the Model group (P o0.01), and E2 levels in CP-H and CP-L groups increased significantly compared with the Model group (Po 0.01) (Fig. 6D–F). The result indicated that mice receiving CP treatment did not affect serum androgen levels, but could significantly reduce DHT levels in skin. 3.5. Effects of CP-ext on the expression of 5α-reductase In order to investigate whether the inhibitor CP affect the expression of 5αR, the immunofluorescence assay was used to detect 5αR expression in dorsal skin. Fig. 7 showed the expression intensity of 5αR in each group, and the blue fluorescence image of

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B. Zhang et al. / Journal of Ethnopharmacology 177 (2016) 1–9

Fig. 2. Effect of CP-ext on the hair cycle of C57 mice model. (A) Dorsal skin color of 7-week-old C57 strain mice is pink, (B) Dorsal skin of C57 mice become dark in the anagen phase, (C) Dorsal hair grew. (D and E) showed the times when the skin got dark and the hair grew in each group of mice. Results were presented as mean 7 S.E.M. (n¼8). **P o 0.01 compared with the Normal group; ##Po 0.01 compared with the Model group. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 3. Hair growth promoting effect of CP-ext in C57 mice, hair growth score was test every other day from the 8th day.

skin tissue showed the immunoreactivity of 5αR. Fig. 8 was the mean fluorescence intensity measured by the image J software systems.Compared with the Normal group, the expression of 5αR up-regulated significantly in the Model group (P o0.01).while in the CP-H and CP-L groups the expression of 5αR down-regulated significantly compared with the Model group (P o0.01).

4. Discussion In the present study, the simple accurate HPLC method was used for determination of 5αR inhibitory activity, the conditions of the reaction system was explored and a stable in vitro screening model was established for screening inhibitors from some TCM. Our research found that GL-ext, PM-ext, CP-ext and CS-ext were potential 5αR inhibitors. In China, these drugs have been

commonly used for blackening and nourishing hair, promoting hair growth, and now many of them are applied in Chinese herbal shampoo. Our study first discovered CP-ext was a significant 5αR inhibitor, and elucidating the possible mechanisms involved in its treatment of hair loss is of great significance.So further experiments were done over CP-ext. The dorsal skin of the 7-week-old C57 mouse enters the telogen phase, and the skin color was pink (Müller-Röver et al., 2001). When depilated by wax/resin mixture, the hair follicles converse into the anagen phase and can be easily seen by the blackening of their skin. In this experiment, CP-H and CP-L significantly shortened the time the skin darkening and grew hair, indicated that CP-ext was able to stimulate the anagen phase of the hair growth cycle in these mice, there was no statistical significance between the high and low dose. Further hair growth promoting activity were tested by assigning a hair growth score and found that testosterone treatment markedly suppressed hair re-growth, while finasteride, CP-H and CP-L showed high hair growth promoting activity, which revealed that CP-ext showed a significant anti-androgenic activity in the in vivo mouse model. 5αR converts T to DHT, which combine with the same AR and formation of conjugates thereby cause hair follicle miniaturization and enter the telogen (Anderson and Liao, 1968; Rathnayake, Sinclair, 2010). Therefore, lower 5αR activity in hair follicles may be due to an increase in active hair follicle. The histological data of hair follicles in each group showed that hair follicle in Model group was sparse and mainly in mid regression and late degenerative, CP-H and CP-L could promote the growth of hair follicle and slowed down hair follicles enter the telogen. Our study implied that the mechanism of CP-ext in hair growth promoting activity may be due to an increase in active hair follicle, and as an anagen promoter. DHT plays a critical role in the pathogenesis of AGA. on the one

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Fig. 4. Effect of CP-ext on morphology of hair follicle. (A) Normal group, (B) Model group, (C) Finasteride group, (D) CP-ext high dose group, (E) CP-ext low dose group. (F) Staging scores of follicle morphology. Digital images were obtained under the light microscope with 50  magnification. Results were presented as mean7 S.E.M. (n¼ 5). **P o 0.01 compared with the Normal group; ##Po 0.01 compared with the Model group.

hand 5αRⅡ has a higher affinity for steroid and the Km is approximately nM range, while Km of 5αRⅠis approximately μM range. On the other hand 5αR II is the predominant form in hair follicles, which converts T to DHT, although androgens mediate their activities by binding to the same AR, the affinity of DHT is fivefold higher. How the 5αR inhibitor CP-ext affects androgen metabolism in the blood and skin tissue? The present study showed that testosterone treatment markedly increased the DHT levels in the dorsal skin, CP-H and CP-L could significantly decrease the DHT levels by inhibiting the activity of 5αR without dose-dependent manner. However, CP-ext had almost no effect on androgen levels in the blood. Our study suggested that the increased androgen levels in the partial may be the main cause of AGA. Previous studies (Berman et al., 1995; Silver et al., 1994) demonstrated that both T and its metabolite DHT could induce the expression of SRD5A2 gene, and confirmed that 5αR inhibitors could lead to the down-regulation of SRD5A2 expression, but not

completely disappeared. In our study, the immunofluorescence assay was used to detect the influence of CP-ext on 5αRⅡ expression in dorsal skin. We found that testosterone treatment markedly increased the 5αRⅡ expression, which was closely related to the high levels of DHT. When treated with CP-ext, the expression of 5αRⅡ down-regulated markedly. In this study, we discovered several potential 5αR inhibitors, which may lead to the development of new natural medicines to treat androgen-related disorders, especially AGA. However, active substances in the crude extract were not clear, it is necessary to identify the standardized phyto-constituents, analysis structureactivity relationship and determine the binding targets in future.

5. Conclusions In conclusion, GL-ext、PM-ext、CP-ext and CS-ext showed strong 5αR inhibitory activity. CP-ext possesses high hair growth

Fig. 5. Effect of CP-ext on hair follicle count. (A) Normal group, (B) Model group, (C) Finasteride group, (D) CP-ext high dose group, (E) CP-ext low dose group, (F) The number of hair follicles. Digital images were obtained under the light microscope with 50  magnification. Results were presented as mean 7S.E.M. (n¼5). **Po 0.01 compared with the Normal group; ##Po 0.01 compared with the Model group.

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B. Zhang et al. / Journal of Ethnopharmacology 177 (2016) 1–9

Fig. 6. Effect of CP-ext on androgen levels in serum and skin. (A) T levels in serum, (B) DHT levels in serum, (C) E2 levels in serum, (D) T levels in skin, (E) DHT levels in skin, (F) E2 levels in skin. Results were presented as mean 7 S.E.M. (n¼ 8). *P o0.05 compared with the Normal group, **P o0.01 compared with the Normal group; ##Po 0.01 compared with the Model group.

promoting activity in the in vivo androgen-sensitive mouse model via inhibiting the 5αR activity, decreasing the DHT levels and in turn suppressing the expression of 5αR. This indicates that plant

extracts may be beneficial as an alternative medicine. Moreover, our group is focused on using plant extracts as Yukon hair fluid for prevention and treatment of hair loss, some plants with high anti-

Fig. 7. Immunofluorescence assay detecting the expression of 5α-reductase. (A1–E1) The blue fluorescence image of skin tissue showed the immunoreactivity of 5αreductase, (A2–E2) The green fluorescence image of skin tissue showed the DAPI staining for nucleus. (A3–E3) The overlapping of the fluorescence image of skin tissue. Digital images were obtained under the fluorescence microscope with 280  magnification.

B. Zhang et al. / Journal of Ethnopharmacology 177 (2016) 1–9

Fig. 8. Image J software systems measuring the mean fluorescence intensity. Results were presented as mean7 S.E.M. (n¼ 3). **P o0.01 compared with the Normal group; ##Po 0.01 compared with the Model group.

5αR activity in this experiment may contribute to the development of a new generation of herbal supplements with clearer material basis of pharmacodynamic for treating AGA.

Conflict of interest None.

Acknowledgments This work was supported by Grants from Science & Technology Cooperation Program of Hong Kong, Macao and Taiwan of The Chinese Ministry of Science (No. 2014DFH30010), Science & Technology Planning Project of Guangdong Province of China (No. 2012A030100003) and Guangdong Province Universities and Colleges Pearl River Scholar Funded Scheme (2011).

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