Saw palmetto (serenoa repens); Genetik saç dökülmesine sebep olduğu bilimsel olarak kanıtlanmış tek etken DHT’dir. Saw palmettonun 5 alfa redüktazı engelleyerek DHT oluşumunu durdurduğu yönünde yapılmış çok sayıda bilimsel çalışma vardır. Okuyacağınız 3 yayın DHT’nin sebep olduğu prostat büyümesi ve saç dökülmesi üzerinedir.
5 alfa -reductase inhibition and hair growth promotion of some Thai plants traditionally used for hair treatment
Naphatsorn Kumara, Wandee Rungseevijitprapab, Nual-Anong Narkkhongc, Maitree Suttajit d, Q1 Chaiyavat Chaiyasut
Ethnopharmacological relevance: Many Thai traditional herbs have been used for hundreds of years for hair treatment and nourishment, including hair loss. However, scientific evidence about their mechanisms of action has not yet been elucidated. Aims of the study: The purpose of this research is to define the possible mechanisms involved in hair loss treatment of the selected plants by determining the 5 _-reductase enzyme inhibition and hair growth promoting activities, and the relationship between these two activities. Materials and methods: Seventeen Thai plants traditionally used for hair treatment were selected. The plants were dried, ground and extracted by maceration with ethyl alcohol. These extracts were further tested for 5 _-reductase inhibition using enzymes from rat livers. Hair growth promoting activity was tested in C57BL/6 mice. Results: Carthamus tinctorius L. was the most potent 5 _-reductase inhibitor, with a finasteride equiva-lent 5 _-reductase inhibitory activity (FEA) value of 24.30 ± 1.64 mg finasteride equivalent per 1 g crude extract. Phyllanthus emblica L. was the second most potent inhibitor, with FEA of 18.99 ± 0.40. Rhi-nacanthus nasutus (L.) Kurz. was the least potent 5 _-reductase inhibitor (FEA 10.69 ± 0.96). Carthamus tinctorius also was the most potent hair growth promoter in C57BL/6 mice. There were strong rela-tionships between 5 _-reductase inhibitory activity and hair growth promoting activity (r = 0.719), and between 5 _-reductase inhibitory activity and hair follicle count (r = 0.766). Conclusions: Ethanolic extract of Carthamus tinctorius was the most potent 5 _-reductase inhibitor and hair growth promoter. This discovery may lead to the development of new alternative medicines for hair loss prevention and treatment.
1. Introduction 24 Although hair loss may not be a life-threatening disorder, it has 25 a great impact on a person’s self-respect, mental health, and overall 26 quality of life. Within the disorder, androgenic alopecia (androge-27 netic alopecia or AGA) is the most common type of hair loss, which 28 affects large numbers of both men and women (Sinclair, 2004). AGA 29 can occur as early as the teen years, but usually begins in the later 30 years of life. It affects at least half of all men by the age of 50, and 31 up to 70% of 70-year-old men (Trüeb, 2002). AGA is an androgen- 32 ∗ Corresponding author. Tel.: +66 5394 4340; fax: +66 5389 4163. E-mail address: firstname.lastname@example.org (C. Chaiyasut). dependent and genetically acquired disorder, caused by excessive 33 activity of the 5 _-reductase enzyme in hair follicles (Sawaya, 1998). 34 It is usually observed that the hair follicles of AGA patients are 35 smaller than those in normal adults, which is a direct occurrence 36 of the hair miniaturization process caused by overactivity of dihy- 37 drotestosterone (Sinclair, 2004). 38 At present, there are some medicines that are used to treat 39 AGA. For example, 5 _-reductase inhibitors, finasteride and dutas- 40 teride, are used to treat androgen-related disorders (Robinson 41 et al., 2003). But these medicines have several undesirable side 42 effects: for example, impotence (erectile dysfunction), abnormal 43 ejaculation, decreased ejaculatory volume, abnormal sexual func- 44 tion, gynecomastia, testicular pain, impairment of muscle growth, 45 and severe myopathy (Lacy et al., 2008). Another medicine for
treating AGA is a topical minoxidil lotion. Minoxidil was first used as a vasodilator to treat cardiovascular disorders, but the 48 unexpected side effect of hirsutism led to its topical use as a 49 hair-growth stimulator. The mechanisms involved in AGA treat-50 ment are still unclear. It seems to open potassium channels and 51 increase the proliferation and differentiation of epithelial cells in 52 the hair shaft. However, local irritation, itching, dryness and ery-53 thema may occur when minoxidil is topically used, as well as 54 systemic side effects such as dizziness and tachycardia. Serious side 55 effects, such as an increase in left ventricular end-diastolic volume, 56 cardiac output, and left ventricular mass, have been reported with 57 the use of 2% minoxidil solution. Unfortunately, another poten-58 tial drawback of minoxidil therapy is the loss of newly grown 59 hair within one to three months after discontinuation of the 60 medicine (Abramowicz, 1998). 61 The 5 _-reductase enzyme (5 _R, EC 18.104.22.168; _4-3-oxo-steroid 62 5 _-oxidoreductase) is a microsomal enzyme that is responsi-63 ble for the reduction of 3-oxo-_4 steroidal compounds such as 64 testosterone, progesterone and corticosterone. In humans, 5 _R 65 plays a major role in the reduction of testosterone into a more 66 potent androgen, dihydrotestosterone (DHT), which is necessary 67 for normal male growth. However, high expression of DHT causes 68 androgen-related disorders such as acne, hirsutism, androgenic 69 alopecia, benign prostatic hyperplasia (BPH), and prostate cancer 70 (McGuire et al., 1960; Bruchovsky and Wilson, 1968). 71 Many studies in Europe and the US have indicated that several 72 plants have the potential to inhibit 5 _R: for example, the Ameri-73 can dwarf palm (Serenoa repens or Sabal serrutala, saw palmetto) 74 which is rich in free fatty acids such as oleic, lauric, myristic and 75 linoleic acids, can inhibit 5 _R (Niederprûm et al., 1994). This plant 76 is extracted and developed into a variety of health supplements, 77 and is widely used in both Europe and the USA. The most popular 78 brand is Permixon®, a standardized saw palmetto extract whose 79 effect has been proven both in vitro, in vivo and in human clinical 80 trials (Di Silverio et al., 1998; Paubert-Braquet et al., 1998; Bayne 81 et al., 1999; Raynuad et al., 2002; Habib, 2009). Lingzhi mushroom 82 (Ganoderma lucidum) extract is also able to inhibit the 5 _R enzyme 83 (Fujita et al., 2005). Its triterpenoids may be responsible for this 84 action (Liu et al., 2006). 85 Some other plants with reported 5 _R inhibition activity are Piper 86 nigrum (Hirata et al., 2007), Alpinia officinarum (Kim et al., 2003), 87 Lygodium japonicum (Matsuda et al., 2002), Pleurotus ostreatus, and 88 Lentinula edodes (shiitake) (Fujita et al., 2005). 89 Additionally, many reports have indicated that plants or sub-90 stances with anti-5 _R activity can promote hair growth as well. 91 For example, Myrica rubra (red bayberry) bark (Matsuda et al., 92 2001), Thuja orientalis (white cedar) seed (Park et al., 2003), Piper 93 nigrum (black pepper) leaf (Hirata et al., 2007), Boehmeria niponon-94 ivea (Shimizu et al., 2000), and epigallocatechin-3-gallate (EGCG) 95 found in green tea (Kwon et al., 2007) are all able to promote hair 96 growth as well as inhibit the 5 _R enzyme. 97 In Thailand, several varieties of plants have been used for 98 preventing or treating hair loss, for hair nourishment, and for 99 improving the esthetic properties of hair (Boonyaprapas and 100 Chokchaijareonporn, 1996). These plants, the parts used, and their 101 method of uses are shown in Table 1. However, the possible mech-102 anisms involved in their treatment of hair loss have not yet been 103 elucidated. 104 This work aims to define the possible mechanisms of Thai tradi-105 tional plants which have been used as herbal remedies or cosmetics 106 to treat or prevent hair loss, to promote hair growth, to nourish 107 hair, or that have been used as ingredients in natural cosmetics. 108 This research will determine the 5 _R inhibitory and hair growth 109 promoting activity of these plants. The relationship between 5 _R 110 inhibition and the hair growth promoting activity of these plants 111 will also be investigated. 2. Materials and methods 112 2.1. Plant materials and extraction 113 Traditional plants, as shown in Table 1, were purchased from 114 local markets in Chiang Mai, Thailand. Since the plants were pur- 115 chased from local market as a fresh form, they were confirmed 116 by comparing with herbarium specimens at Faculty of Pharmacy, 117 Chiang Mai University, to ensure that the plants used in this exper- 118 iment were correct materials. Ethanol was used as an extraction 119 solvent due to the semipolar property of this solvent, which soluble 120 various phytochemical groups more than the use of polar solvents 121 like water or non-polar solvents. Moreover, using ethanolic extract 122 as an active ingredient in pharmaceutical and cosmetic products 123 provided more safety and compatibility than other organic sol- 124 vent. To extract the plants, they were separately dried at 45 ◦C in a 125 hot-air oven. Next, they were ground by using an electric grinder 126 and extracted by maceration with 95% ethyl alcohol. The extracts 127 were then evaporated to dryness under controlled pressure and 128 temperature using a rotary evaporator (Eyela, Tokyo, Japan). 129 2.2. Animals 130 Six-week-old male Sprague Dawley (SD) rats and seven-week- 131 old male C56BL/6Mlac mice were obtained from the National 132 Laboratory Animal Center, Bangkok, Thailand, and housed under 133 a 12 h light/dark cycle with free access to food and water. The pro- 134 tocol of this study was approved by the Animal Research Ethics 135 Committee of the Faculty of Pharmacy, Ubon Ratchathani Univer- 136 sity, Ubon Ratchathani, Thailand. 137 2.3. Chemicals 138 Dithiothreitol, sucrose, testosterone, finasteride and NADPH (all 139 of analytical grade) were purchased from Sigma Chemical (St. 140 Louis, MO). Methanol, dichloromethane and ethanol were pur- 141 chased from Fisher Scientific (Fair Lawn, NJ). Safflower yellow was 142 purchased from Tokyo Chemical Industry (Tokyo, Japan). Other 143 chemical compounds were purchased from Wako Pure Chemical 144 Industry (Osaka, Japan). 145 2.4. Method for determining the 5˛-reductase inhibitory activity 146 Rat microsomal suspension was prepared using the method 147 described in our previous paper (Kumar et al., 2011). Briefly, excised 148 SD rat livers were minced with scissors and homogenized in a solu- 149 tion composed of 0.32 M sucrose and 1 mM dithiothreitol in 0.02 M 150 phosphate buffer at pH 6.5. The liver homogenate was further cen- 151 trifuged twice at 4500 × g at 0 ◦C for 30 min each time. All of the 152 supernatants were collected and kept at −50 ◦C until used as an 153 enzyme source. 154 5 _-reductase assay was performed according to our previous 155 paper (Kumar et al., 2011). Briefly, the reaction solution contained 156 0.2 ml of various plant extracts in 50% ethanol, 1.0 ml of 0.02 mM 157 phosphate buffer (pH 6.5), 0.3 ml of 500 ppm testosterone in 50% 158 ethanol, and 1.0 ml of rat microsomal suspension. Reactions were 159 then initiated by the addition of 0.5 ml of 0.77 mg/ml NADPH 160 in phosphate buffer, followed by incubation in a water bath at 161 37 ◦C for 30 min. The reactions were then stopped by adding 162 5.0 ml dichloromethane, and then adding 0.5 ml of 100 ppm propyl 163 p-hydroxybenzoate in 50% ethanol (as an internal standard for 164 HPLC). Four millilitres of the organic phase was decanted and 165 evaporated to dryness under controlled pressure. The residues 166 were collected and dissolved in 5.0 ml of methanol. An aliquot 167 of 10 _l was injected into the HPLC system (Agilent 1100 series, 168 using a Hypersil®-ODS column, 250 mm × 4.6 mm, 5 _M). The Table 1 Thai ethno-medicinal plants used in this experiment, their usage and method of preparation. Botanical name Family Part used, method of preparation, and ethno-medicinal uses Acacia concinna Wall. Leguminosae Dried pods are crushed and decocted with water; the juice is used as an anti-dandruff shampoo and for hair nourishment Alpinia galanga Willd. Zingiberaceae Fire-grilled rhizomes are crushed and filtered through cloth; the juice is applied on the scalp to kill fungi and to promote hair growth Andrographis paniculata Nees Acanthaceae Plants are boiled in water and filtered through cloth; the juice is applied on the hair as a hair rinse for hair growth promotion and hair loss prevention Averrhoa carambola L. Oxalidaceae Fruit juice is used as an anti-dandruff shampoo and for hair nourishment Carthamus tinctorius L. AsteraceaeFlowers are decocted in water and used as a hair rinse to enhance hair color Cassia siamea Lam. CeasalpiniaceaeLeaves are decocted in water and used as an anti-dandruff shampoo, and for oil control and hair nourishment. Citrus hystrix DC. Rutaceae Fresh fruits are squeezed and mixed with coconut (Cocos nucifera L.) juice, and used as a hair conditioner and to promote hair growth Clitoria ternatea L. Fabaceae Fresh flowers are crushed and filtered through cloth; the juice is applied on the scalp to promote hair growth. Cymbopogon citratus Stapf. Poaceae Whole plants are boiled in water and used as a hair rinse for oil control Ipomoea aquatica Forssk. Convovulaceae Leaves and stems are boiled in water and used as a hair rinse for hair loss treatment and hair conditioning Lawsonia inermis L. LythraceaeSun-dried leaves are soaked in water and applied to the hair as a hair-coloring agent Phyllanthus emblica L. Euphorbiaceae Dried fruits are fried in the sesame (Sesamum indicum L.) oil, and then applied to the hair to promote hair growth Rhinacanthus nasutus Kuntze Acanthaceae Whole plants are boiled and filtered through cloth; the juice is applied on the scalp to promote hair growth Sapindus rarak DC. Sapindaceae Dried fruits are crushed and soaked in water, the filtered through cloth and used as a shampoo Tinospora rumphii Boerl. Menispermaceae Vines are boiled and filtered through cloth; their juice is applied on the scalp to promote hair growth Trichosanthes cucumerina L. Curcubitaceae Fruits are peeled and chopped into small pieces, then applied throughout the scalp, left for a while, and rinse off with water to promote hair growth Zingiber officinale Roscoe ZingiberaceaeFire-grilled rhizomes are crushed and filtered through cloth; their juice is applied on the scalp to control scalp oil release and to promote hair growth mobile phase was a mixture of methanol and deionized water 170 (65:35) with a flow rate of 1.0 ml/min. A UV detector at 245 nm 171 was used to collect data. Finasteride was used as a standard 172 enzyme inhibitor. All of the results were expressed as finasteride 173 equivalent 5 _-reductase inhibitory activity (FEA) value (units of 174 mg finasteride equivalent per 1 g extract). 175 2.5. Method for determining hair growth promoting activity 176 Hair growth promoting activity of the extract was determined 177 by the method reported by Roh et al. (2002), with some modifica-178 tion. Briefly, 25 seven-week-old mice were randomly divided into 179 five groups for five treatments, as follows: vehicle control group, 180 positive control group, extract1 group, extract2 group, and extract3 181 group. Hair was removed from the 2 cm × 3 cm dorsal area of these 182 mice by using a depilatory cream. On the next day, 100 _l of the test 183 solution in a vehicle composed of propylene glycol: water: ethyl 184 alcohol in a ratio of 5:3:2 was applied. Minoxidil (2%) was used as 185 a positive control. A concentration of 1% w/w of each plant extract 186 was used. The hair growth promoting activity of the substances was 187 checked by the darkening of the dorsal skin, which indicated the 188 anagen phase of the hair follicles. Hair growth was measured at days 189 1, 7, 14, 21 and 28 by assigning a hair growth score, as follows: score 190 0 = no growth observed; 1 = up to 20% growth; 2 = 20–40% growth; 191 3 = 40–60% growth; 4 = 60–80% growth; and 5 = 80% to full growth 192 observed. 193 Digital images of total hair growth on day 28 were obtained 194 using a Coscam® USB-225 (Seoul, South Korea) with a 40× magni-195 fication lens. 196 2.6. Histological determination of hair follicles 197 After day 28, all of the mice were sacrificed. Their dorsal 198 skins were removed and then sectioned into two different pat-199 terns: transverse sections for determination of hair follicle count, 200 and longitudinal sections for overall histological assessment under 201 a light microscope (Olympus, Melville, NY). 202 2.7. Statistical analysis 203 All samples that tested for 5 _-reductase inhibition were ana- 204 lyzed in triplicate. All values were expressed as mean ± SD. To 205 compare several groups, analysis of variance was used. Signifi- 206 cant differences between means were determined by Duncan’s 207 multiple range test. Pearson’s correlation coefficient was used to 208 predict the relationship between 5 _-reductase inhibitory activity 209 and hair growth promoting activity. A probability value of p < 0.05 210 was adopted as the criteria for significant differences. 211 3. Results 212 3.1. 5˛-reductase inhibitory activity of the extract 213 Extraction yield of each plant was shown in Table 2. The micro- 214 somal suspension was prepared using the provided method, and 215 was assessed for soluble protein by the Lowry method (Lowry et al., 216 1951). Soluble protein was found to be 4.69 mg/ml. 217 The IC50 of finasteride, a well-known 5 _-reductase inhibitor, 218 was 0.39 _M. The inhibitory equation of finasteride was expressed 219 as: y = 166.78x − 15.285 (R2 = 0.999) with y representing % inhibi- 220 tion and x representing concentration of finasteride in _M. This 221 equation was used to calculate the results and was expressed as 222 finasteride equivalent inhibitory activity (FEA) value: the higher 223 the FEA value, the stronger the inhibitory activity of the extract. 224 The inhibitory activity of each plant extract is shown in Table 2. 225 FEA values of the extracts ranged from 10.69 to 24.30 mg FEA per 226 g extract (Table 2). The 5 _R inhibitory activity of each extract can 227 be arranged from higher to lower, as follows: Carthamus tinctorius 228 L., Phyllanthus emblica L., Cymbopogon citratus (DC.) Staphf., Alpinia 229 galanga Willd., Zingiber officinale Roscoe., Clitorea ternatea L. (CT), Extraction yield and 5 _-reductase inhibition activity of each plant extract reported as FEA (finasteride equivalent 5 _-reductase inhibition activity) value. Plants Extraction yield (%) Finasteride equivalent 5 _-reductase inhibition ability: FEA value (mg finasteride/1 g crude extract)a Carthamus tinctorius L. 19.25 24.30 ± 1.64a Phyllanthus emblica L. 21.63 18.99 ± 0.40b Cymbopogon citratus (DC.) Staphf. 4.00 18.55 ± 0.78b Alpinia galanga Swartz. 5.88 18.54 ± 0.85b Zingiber officinale Roscoe. 8.44 18.32 ± 0.82b Clitorea ternatea L. (CT) 18.61 15.39 ± 0.67c Citrus hystrix DC. 8.24 13.72 ± 0.79d Trichosanthes cucumerina L. 2.75 13.37 ± 0.84d Tinospora rumphii Boerl. 3.88 13.33 ± 0.30d Ipomoea aquatica Forssk. 1.90 13.16 ± 0.43d Averrhoa carambola L. 4.65 13.12 ± 0.87d Andrographis paniculata Nees 2.94 13.01 ± 0.81d Cassia siamea Lam. 2.18 12.87 ± 1.12d Acacia concinna Wall. 15.23 12.78 ± 0.87d Sapindus rarak DC. 2.67 12.81 ± 0.84d Lawsonia inermis Linn. 16.60 12.58 ± 0.45d Rhinacanthus nasutus (L.) Kurz. 4.60 10.69 ± 0.96e a Values in table expressed as mean ± SD of triplicate experiments. Means in column with different letters are significantly different (p < 0.05). Citrus hystrix DC., Trichosanthes cucumerina L., Tinospora rumphii 231 Boerl., Ipomoea aquatica Forssk., Averrhoa carambola L., Andro-232 graphis paniculata Nees, Cassia siamea Lam., Acacia concinna Wall., 233 Sapindus rarak DC., Lawsonia inermis Linn., and Rhinacanthus nasu-234 tus (L.) Kurz., respectively. In this experiment, Carthamus tinctorius 235 was the strongest 5 _R inhibitor, and Rhinacanthus nasutus was the 236 weakest 5 _R inhibitor. There were no significant differences in 237 5 _R inhibitory activity in Phyllanthus emblica, Cymbopogon citratus, 238 Alpinia galanga, and Zingiber officinale, and between Citrus hys-239 trix, Trichosanthes cucumerina, Tinospora rumphii, Ipomoea aquatica, 240 Averrhoa carambola, Andrographis paniculata, Cassia siamea, Acacia 241 concinna, Sapindus rarak and Lawsonia inermis. 242 For confirmation of the enzyme inhibitory activity of Carthamus 243 tinctorius, safflower yellow, a major compound found in the florets, 244 was further tested for enzyme inhibitory activity by IC50 determi-245 nation; the IC50 of safflower yellow was 119.9 ppm (FEA value of 246 12.74). 247 3.2. Hair growth promoting activity of the extracts 248 The three plants with the highest 5 _R inhibitory activity, 249 Carthamus tinctorius, Phyllanthus emblica and Clitorea ternatea, were 250 further tested for hair growth promoting activity (Fig. 1). At day 28, 251 it was found that Carthamus tinctorius demonstrated the highest 252 hair growth promoting activity, followed by Clitorea ternatea and 253 Phyllanthus emblica. As shown in Fig. 1, the normal hair growth rate 254 of the mice was seen in the vehicle curve. In minoxidil-treated mice, 255 it was found that minoxidil constantly promote hair growth of the 256 mice. Plant extracts can promote the hair growth during the first 257 14 days of the experiment, while during the last 14 days the hair 258 growth rates were constant. Among these extracts, Carthamus tinc-259 torius had the highest hair growth promoting activity. Additionally, 260 Phyllanthus emblica and Clitorea ternatea did not show any differ-261 ence in hair growth rate increment in the first 14 days, but over the 262 last 14 days Clitorea ternatea tended to increase the hair growth 263 rate more than Phyllanthus emblica. There was a strong correlation 264 between FEA value and hair growth promoting activity (r = 0.719) 265 at day 14 of the treatment. 266 Total hair growth of the mice is shown in Fig. 2. Fig. 2A shows 267 normal hair growth of mice receiving the vehicle, while Fig. 2B 268 shows the increased hair growth from minoxidil. All three plants 269 extracts were able to promote hair growth better than minoxidil 270 (Fig. 2C–E). 271 3.3. Histological determination of hair follicles 272 The mean hair follicle count, obtained from a transverse sec- 273 tion of the dorsal skin area, is shown in Table 3. In the vehicle 274 control mice, the mean active hair follicle count was 24.2 ± 2.8 275 hair follicles per selected area under 100× magnification by light 276 microscope. The mice receiving minoxidil, Carthamus tinctorius, 277 Phyllanthus emblica and Clitorea ternatea had 36.3 ± 4.1, 69.5 ± 7.6, 278 46.4 ± 3.0, and 52.5 ± 6.1 hair follicles per area, respectively. It was 279 found that mice that received Carthamus tinctorius had the highest 280 number of active hair follicles in their skin. 281 The morphological structure of the skin, obtained from a longi- 282 tudinal section of the dorsal skin, is shown in Fig. 3. Mice receiving 283 Carthamus tinctorius (Fig. 3C) had more hair follicles than mice 284 receiving Clitorea ternatea (Fig. 3E), Phyllanthus emblica (Fig. 3D), 285 minoxidil (Fig. 3B) and the vehicle (Fig. 3A). 286 There was a strong relationship between 5 _-reductase 287 inhibitory activity (as FEA value) and hair follicle number (r = 0.766). 288 4. Discussion 289 For determination of 5 _-reductase inhibitory activity, radioim- 290 munoassay (RIA) is the most widely accepted method. However 291 RIA, which uses a radioactive compound, requires many complexes 292 instruments and other equipment. Although immunoassay is a 293 fast and easy method, there is a cross-reactivity of many andro- 294 gens (Lootens et al., 2008). Matsuda et al. (2001) developed a 295 simple isocratic HPLC method. In our previous paper, we mod- 296 ified the detection wavelength from 254 nm to 245 nm which 297 encounters less interference from the reaction system (Kumar 298 et al., 2011). In this experiment, finasteride has IC50 at 0.39 _M, 299 Table 3 Effects of vehicle, minoxidil, Carthamus tinctorius L., Phyllanthus emblica L., and Cli-torea ternatea L. (CT) on hair follicle count in C57BL/6 mice. Test substances Hair follicles counta Vehicle (propylene glycol:water:ethanol) 24.2 ± 2.8a Minoxidil 36.3 ± 4.1b Carthamus tinctorius L. 69.5 ± 7.6c Phyllanthus emblica L. 46.4 ± 3.0d Clitorea ternatea L. (CT) 52.5 ± 6.1e a Values in table expressed as mean ± SD of five mice. Means in column with different letters are significantly different (p < 0.05) which is comparable to a previous report of 0.34 _M (Park et al., 300 2003). The most potent 5 _R inhibitor in this experiment was 301 Carthamus tinctorius (safflower), which contains safflower yellow 302 as a major compound in the florets (Duke, 1992; Fan et al., 2009). 303 This suggested that the synergistic interaction of some other phyto-304 chemicals in the ethanolic extract of Carthamus tinctorius, including 305 flavonoids – for example, carthamin, carthamidin, isocarthamidin, 306 6-hydroxykaempferol compounds, etc. – may result in the high-307 est inhibitory activity. The other active plants contained different 308 classes and amounts of phytochemicals, which may result in the 309 same inhibitory potency. Besides that, the crude extract of each 310 plant contains many kinds of phytochemicals, some of which may 311 be active against the enzyme inhibition, or even promote the activ-312 ity of the enzyme. The balance of those two chemicals resulted in 313 the FEA values seen in this experiment. For confirmation, partial 314 or full purification of the extract needs to be performed in order 315 to define which classes of phytochemicals have the great potency. 316 This may lead to the development of new alternative medicines to 317 treat androgen-related disorders, especially androgenic alopecia. 318 For decades, black C57BL/6 mice have been widely used to evalu-319 ate the hair growth promoting activity of many compounds (Datta 320 et al., 2009). Since C57BL/6 mice contain no melanocytes on the 321 skin, the melanogenesis of these mice occurs only in the hair folli-322 cles. Melanogenesis in these pigmented mice is strongly related to 323 the hair growth cycle. Melanins are produced only in the anagen 324 phase, and production stops at the beginning of the catagen phase 325 (Slominski et al., 1994). For this reason, C57BL/6 mice are the most 326 useful in vivo model for testing of hair growth promoting activity. 327 The conversion of hair follicles into the anagen phase can be easily 328 seen by the blackening of their skin. Moreover, it was found that 329 most hair follicles of C57BL/6 mice at seven weeks of age are in the 330 telogen phase of the hair cycle. Since it is known that melanogen-331 esis in C57BL/6 mice occurs only during the anagen phase of the 332 hair growth cycle, the blackening of the dorsal area indicated that 333 the plant extracts are able to stimulate the anagen phase of the hair 334 growth cycle in these mice. In this experiment, Carthamus tinctorius 335 showed the best ability to stimulate hair follicle growth, and hence 336 was the most potent hair growth promoter. Since hair growth is an 337 active process, the vehicle treated group served as a control group 338 for determination of the normal mouse hair growth rate by using 339 the slope of the hair growth curve. Minoxidil, a well-known topical 340 medicine for treating AGA, increased the hair growth rate in a con- 341 stant manner throughout the experiment. Surprisingly, the three 342 extracts (Carthamus tinctorius, Phyllanthus emblica and Clitorea ter- 343 natea) seemed to increase the rate of hair growth for only the first 344 14 days of the experiment; during the last 14 days the growth rate 345 appeared to be constant. This is a different result from that obtained 346 in a previous study by Hirata et al. (2007), using methanolic extract 347 of Piper nigrum leaf as a test compound, where incremental hair 348 growth was seen throughout the treatment period. 349 The histological data of hair follicles in each group showed that 350 the mechanism of Carthamus tinctorius and other plants in hair 351 growth promoting activity may be due to an increase in an active 352 hair follicle, and as anagen promoter. Since the activity of 5 _R in 353 hair follicles causes hair follicle miniaturization (Sinclair, 2004), 354 this implies that with lower 5 _R activity in hair follicles, larger hair 355 follicles and consequently larger hair shafts are obtained. This may 356 explain the relationship between FEA value and hair growth pro- 357 moting activity. The Pearson’s correlation coefficient between FEA 358 and hair follicle count suggested that the higher the FEA, the higher 359 the number of hair follicles, leading to increased hair growth. 360 In this experiment, none of the plant extracts applied to mice 361 caused erythema, redness, drying or scaling as was the case with 362 the minoxidil group. This indicated that plant extracts in a suitable 363 vehicle may be useful as an alternative topical medicine to minox- 364 idil therapy. Moreover, some plants with high anti-5 _R activity in 365 this experiment may have the potential for development as herbal 366 supplements for treating androgenic alopecia. 367 5. Conclusions 368 In conclusion, ethanolic extract of Carthamus tinctorius is the 369 most active 5 _-reductase inhibitor and hair growth promoter, com- 370 pared to finasteride and minoxidil, respectively. The plant extracts 371 showed strong relationships between 5 _-reductase inhibitory and 372 hair growth promoting activity, and between 5 _-reductase inhi- 373 bition and the number of hair follicles. This indicates that plant 374 extracts may be beneficial as an alternative medicine. Our group 375 focused on using plant extracts as cosmeceuticals for prevention 376 and treatment of hair loss. To achieve a practical alternative topi- 377 cal treatment for hair loss, Carthamus tinctorius is currently being developed as a suitable hair formulation, using nanoparticles to deliver active substances directly to the hair follicles. 380 Acknowledgements 381 Kumar N. would like to thank the Office of Higher Education 382 Commission, Thailand for supporting by grant fund under the pro-383 gram Strategic Scholarships for Frontier Research Network for the 384 Join Ph.D. Program Thai Doctoral degree for this research. This 385 research was also supported by Office of the National Research 386 Council of Thailand, Faculty of Pharmacy, Ubon Ratchathani Univer-387 sity and the Graduate School, Chiang Mai University. The authors 388 would like to thank Thongchai Boonsorn for helping in the prepa-389 ration of mice dorsal area skin section. 390 References 391 Abramowicz, M., 1998. Propecia and rogain extra strength for alopecia. The Medical 392 Letter 40, 25–27. 393 Bayne, C.W., Donnelly, F., Ross, M., Habib, F.K., 1999. Serenoa repens (Permixon): a 394 5alpha-reductase types I and II inhibitor-new evidence in a coculture model of 395 BPH. Prostate 40, 232–241. 396 Boonyaprapas, N., Chokchaijareonporn, O. (Eds.), 1996. SamoonPrai Maipeunban 397 (Herbal plants, in Thai). Faculty of Pharmacy, Mahidol University, Thailand. 398 Bruchovsky, N., Wilson, J.D., 1968. The conversion of testosterone to 5-alpha-399 androstan-17-beta-ol-3-one by rat prostate in vivo and in vitro. Journal of 400 Biological Chemistry 243, 2012–2021. 401 Datta, K., Singh, A.T., Mukherjee, A., Bhat, B., Ramesh, B., Burman, A.C., 2009. 402 Eclipta alba extract with potential for hair growth promoting activity. Journal of 403 Ethnopharmacology 124, 450–456. 404 Di Silverio, F., Monti, S., Sciara, A., 1998. Effects of long-term treatment with Serenoa 405 rapens (Permixon) on the concentrations and regional distribution of androgens 406 and epidermal growth factor in benign prostatic hyperplasia. Prostate 37, 77–83. 407 Duke, J.A., 1992. Handbook of Phytochemical Constituents of GRAS Herbs and Other 408 Economic Plants. CRC Press, Boca Raton, Florida. 409 Fan, L., Zhao, H.-Y., Xu, M., Zhou, L., Guo, H., Han, J., Wang, B.-R., Guo, D.-A., 410 2009. Qualitative evaluation and quantitative determination of 10 major active 411 components in Carthamus tinctorius L. by high-performance liquid chromatog-412 raphy coupled with dioade array detector. Journal of Chromatography A 1216, 413 2063–2070. 414 Fujita, R., Liu, J., Shimizu, K., Konishi, F., Noda, K., Kumamoto, S., Ueda, C., Tajiri, H., 415 Kaneko, S., Suimi, Y., Kondo, R., 2005. Anti-androgenic activities of Ganoderma 416 lucidum. Journal of Ethnopharmacology 102, 107–112. 417 Habib, F.K., 2009. Serenoa repens: the scientific basis for the treatment of benign 418 prostatic hyperplasia. European Urology Supplement 8, 887–893. 419 Hirata, N., Tokunaga, M., Naruto, S., Iinuma, M., Matsuda, H., 2007. Testosterone 420 5 _-reductase inhibitory active constituents of Piper nigrum leaf. Biological and 421 Pharmaceutical Bulletin 30, 2402–2405. 422 Kim, Y.-U., Son, H.K., Son, H.K., Ahn, M.-J., Lee, S.S., Lee, S.K., 2003. Inhibition of 423 5alpha-reductase Activity by Diarylheptanoids from Alpinia officinarum [Online]. 424 Available: http://www.ncbi.nlm.nih.gov/pubmed/12567285 (6 June, 2010). 425 Kumar, T., Chaiyasut, C., Rungseevijitprapa, W., Suttajit, M., 2011. Screening of 426 steroid 5 _-reductase inhibitory activity and total phenolic content of Thai 427 plants. Journal of Medicinal Plants Research 5, 1265–1271. Kwon, O.S., Han, J.H., Yoo, H.J., Chung, K.H., Cho, K.H., Eun, H.C., Kim, K.H., 2007. 428 Human hair growth enhancement in vitro by green tea epigallocatechin-3- 429 gallate. Phytomedicine 14, 551–555. 430 Lacy, C.F., Armstrong, L.L., Goldman, M.P., Lance, L.L., 2008. Drug Information Hand- 431 book with International Trade Names Index, 17th ed. LexiComp Inc., United 432 States, pp. 652–653. 433 Liu, J., Kurashiki, K., Shimizu, K., Kondo, R., 2006. Structure-activity relationship for 434 inhibition of 5(-reductase by triterpenoids isolated from Ganoderma lucidum. 435 Bioorganic and Medicinal Chemistry 14, 8654–8660. 436 Lootens, L., Eenoo, P.V., Meuleman, P., Leroux-roels, G., Thuyne, W.V., Delbeke, F.T., 437 2008. Development and validation of quantitative gas chromatography–mass 438 spectrometry method for the detection of endogenous androgens in mouse 439 urine. Journal of Chromatography A 1178, 223–230. 440 Lowry, O.H., Rosbrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measure- 441 ment with the Folin phenol reagent. Journal of Biological Chemistry 193, 442 265–275. 443 Matsuda, H., Yamazaki, M., Matsuo, K., Asanuma, Y., Kubo, M., 2001. Anti-androgenic 444 activity of Myricae cortex-isolation of active constituents from bark of Myrica 445 rubra. Biological and Pharmaceutical Bulletin 24, 259–263. 446 Matsuda, H., Yamazaki, M., Naruto, S., Asanuma, Y., Kubo, M., 2002. Anti-androgenic 447 and hair growth promoting activities of Lygodii spora (spore of Lygodium japon- 448 icum) I. Active constituents inhibiting testosterone 5 _-reductase. Biological and 449 Pharmaceutical Bulletin 25, 622–626. 450 McGuire, J.S., Hollis, V.W., Tomkin, G.M., 1960. Some characteristics of the micro- 451 somal steroid reductases of a rat liver. Journal of Biological Chemistry 235, 452 112–117. 453 Niederprûm, H.J., Schweikert, H.U., Zânker, K.S., 1994. Testosterone 5-reductase 454 inhibition by free fatty acids from Sabal serrulata fruits. Phytomedicine 1, 455 127–133. 456 Park, W.-S., Lee, C.-H., Lee, B.-G., Chang, I.-S., 2003. The extract of Thujae occiden- 457 talis semen inhibited 5-reductase and adrochronogenetic alopecia of B6CBAF1/j 458 hybrid mouse. Journal of Dermatological Science 31, 91–98. 459 Paubert-Braquet, M., Cousse, H., Raynaud, J-P., Mencia-Huerta, J.M., Braquet, P., 1998. 460 Effect of lipidosterolic extract of Serenoa repens (Permixon®) and its major com- 461 ponents on basic fibroblast growth factor-induced proliferation of cultures of 462 human prostate biopsies. European Urology 33, 340–347. 463 Raynuad, J.-P., Cousse, H., Martin, P.-M., 2002. Inhibition of type 1 and type 2 5 _- 464 reductase by free fatty acids, active ingredients of Permixon®. Journal of Steroid 465 Biochemistry and Molecular Biology 82, 233–239. 466 Robinson, A.J., DeLucca, I., Drummond, S., Boswell, G.A., 2003. Steroidal nitrone 467 inhibitor of 5 _-reductase. Tetrahedron Letter 44, 4801–4804. 468 Roh, S.-S., Kim, C.D., Lee, M.-H., Hwang, S.-L., Rang, M.-J., Yoon, Y.-K., 2002. The 469 hair growth promoting effect of Sophora flavescens extract and its molecular 470 regulation. Journal of Dermatological Science 30, 43–49. 471 Sawaya, M.E., 1998. Novel agents for the treatment of alopecia. Seminars in Cuta- 472 neous Medicine and Surgery 17, 276–283. 473 Shimizu, K., Kondo, R., Sakai, K., Shoyama, Y., Sato, H., Ueno, T., 2000. Steroid 474 5(-reductase inhibitory activity and hair regrowth effects of an extract 475 from Boehmeria nipononivea. Bioscience, Biotechnology, and Biochemistry 64, 476 875–877. 477 Sinclair, R.D., 2004. Male androgenetic alopecia. Journal of Men’s Health and Gender 478 1, 319–327. 479 Slominski, A., Paus, R., Plonka, P., Chakraborty, A., Maurer, M., Pruski, D., Lukiewicz, 480 S., 1994. Melanogenesis during the anagen–catagen–telogen transformation 481 of the murine hair cycle. The Journal of Investigative Dermatology 102, 482 862–869. 483 Trüeb, R.M., 2002. Molecular mechanisms of androgenetic alopecia. Experimental 484 Gerontology 37, 981–990.
Inhibition of Inflammatory Gene Expression in Keratinocytes Using a Composition Containing Carnitine, Thioctic Acid and Saw Palmetto Extract
Sridar Chittur,1 Brian Parr,1 and Geno Marcovici2
1State University of New York (SUNY), Albany, NY, USA
Advanced Restoration Technologies, Inc., 9035 North 15th Place, Phoenix, AZ 85020, USA
Received 29 December 2008; Accepted 8 July 2009
Copyright © 2011 Sridar Chittur et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Chronic inflammation of the hair follicle (HF) is considered a contributing factor in the pathogenesis of androgenetic alopecia (AGA). Previously, we clinically tested liposterolic extract of Serenoa repens(LSESr) and its glycoside, β-sitosterol, in subjects with AGA and showed a highly positive response to treatment. In this study, we sought to determine whether blockade of inflammation using a composition containing LSESr as well as two anti-inflammatory agents (carnitine and thioctic acid) could alter the expression of molecular markers of inflammation in a well-established in vitro system. Using a well-validated assay representative of HF keratinocytes, specifically, stimulation of cultured human keratinocyte cells in vitro, we measured changes in gene expression of a spectrum of well-known inflammatory markers. Lipopolysaccharide (LPS) provided an inflammatory stimulus. In particular, we found that the composition effectively suppressed LPS-activated gene expression of chemokines, including CCL17, CXCL6 and LTB(4) associated with pathways involved in inflammation and apoptosis. Our data support the hypothesis that the test compound exhibits anti-inflammatory characteristics in a well-established in vitro assay representing HF keratinocyte gene expression. These findings suggest that 5-alpha reductase inhibitors combined with blockade of inflammatory processes could represent a novel two-pronged approach in the treatment of AGA with improved efficacy over current modalities.
The pathogeneses of benign prostatic hyperplasia (BPH) and androgenetic alopecia (AGA) are mediated in part by the transcriptional pathways downstream of the steroid hormone androgen receptor (AR). The predominant ligand in these tissues is dihydrotestosterone (DHT), which is formed by the conversion of the inactive form of testosterone (T) and is catalyzed by the enzyme 5-alpha reductase (5-AR).
Anti-androgens and inhibitors of 5-AR have proven effective in the treatment of BPH as well as AGA, attesting to their common disease mechanisms. Both the pharmaceutical compound, finasteride (Proscar or Propecia) and the liposterolic extract of Serenoa repens (LSESr) have shown efficacy in the treatment of BPH and AGA. Notably, in a direct comparison of LSESr against finasteride, it has been reported that LSESr exhibited a 3-fold greater inhibition of 5-AR in in vitro assays .
Finasteride (at a dose of 5 mg, as in Proscar) is used as the treatment of choice for BPH, particularly in the USA. A number of well-controlled studies point to its efficacy in ameliorating the signs and symptoms of BPH . In large, double-blind, placebo-controlled clinical studies recruiting over 1600 patients, it was shown that the administration of finasteride reduced the size of the prostate by a mean of 22%, following 6 months of therapy . Likewise, multiple well-controlled clinical trials reinforce the utility of LSESr in the setting of BPH, predominantly in Europe . Investigators have found that LSESr is well tolerated and has greater efficacy than placebo and similar efficacy to finasteride in improving symptoms in men with BPH .
During the course of a clinical trial of Proscar for BPH, it was noted serendipitously that there was a cessation of hair loss in study subjects receiving drug . Therefore, finasteride (at a dose of 1 mg; as in Propecia) was subsequently investigated in clinical trials for the treatment of men with AGA. In affected individuals, long-term treatment with finasteride 1 mg/day over 5 years was well-tolerated, led to visible improvements in scalp hair growth and slowed the further progression of hair loss that occurred without treatment . LSESr is well known for its role in BPH as a 5-AR inhibitor, leading us to postulate a similar effect in AGA.
Previously, we tested LSESr and its glycoside, β-sitosterol, in subjects with AGA and showed a highly positive response to treatment. The blinded investigative staff assessment reported that 60% of study subjects dosed with the active study formulation were rated as improved at the conclusion of the trial and established the effectiveness of naturally occurring 5-AR inhibitors against AGA for the first time .
Notwithstanding, the common mechanism of androgens in their pathogenesis, several lines of emerging evidence suggest that both BPH and AGA are also associated with significant dysregulation in the expression of inflammatory cytokines . For example, gene expression profiling of prostate tissue from BPH patients revealed molecular signatures containing genes associated with inflammation . Likewise, chronic inflammation of the hair follicle (HF) is also considered a contributing factor in AGA . Another recent study reported a relationship between moderate to extensive alopecia and chronic low-grade inflammation . Histologically, it has been shown that in scalp biopsies from AGA patients, sustained follicular inflammation with connective tissue remodeling eventually results in permanent hair loss and, thus, is described as a possible cofactor in the complex etiology of the disorder .
On the basis of such findings, the utility of treating BPH-affected patients with anti-inflammatory agents in combination with 5-AR inhibitors is currently under investigation. In one study, combination therapy with alpha(1)-adrenergic receptor antagonists [alpha(1)-ARAs] and the 5-AR inhibitor finasteride was significantly more effective than either component alone in reducing BPH-related symptoms (