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This study was conducted to evaluate the effect of Ecklonia cava, a marine alga native to Jeju Island in Korea, on the promotion of hair growth. When vibrissa follicles were cultured in the presence of E. cava enzymatic extract (which contains more than 35% of dieckol) for 21 days, E. cava enzymatic extract increased hair-fiber length. In addition, after topical application of the 0.5% E. cava enzymatic extract onto the back of C57BL/6 mice, anagen progression of the hair-shaft was induced. The treatment with E. cava enzymatic extract resulted in the proliferation of immortalized vibrissa dermal papilla cells (DPC). Especially, dieckol, among the isolated compounds from the E. cava enzymatic extract, showed activity that increased the proliferation of DPC. When NIH3T3 fibroblasts were treated with the E. cava enzymatic extract and the isolated compounds from the E. cava enzymatic extract, the E. cava enzymatic extract increased the proliferation of NIH3T3 fibroblasts, but the isolated compounds such as eckol, dieckol, phloroglucinol and triphlorethol-A did not affect the proliferation of NIH3T3 fibroblasts. On the other hand, the E. cava enzymatic extract and dieckol significantly inhibited 5α-reductase activity. These results suggest that dieckol from E. cava can stimulate hair growth by the proliferation of DPC and/or the inhibition of 5α-reductase activity.
Androgenetic alopecia (AGA), the most common type of alopecia, is a problem in men over the age of 40. Hair loss is a growing trend in the world, however, the prevention of this condition is not simple . Numerous possibilities have been discussed to treat hair loss . Nevertheless, only two FDA-approved drugs have been available for AGA patients, i.e., finasteride and minoxidil [3,4]. Finasteride is a type II 5α-reductase inhibitor. It can be used in prostatic hypertrophy  as well as in hair loss . Minoxidil was used as a drug to treat high blood pressure . However, it was also found to promote hair growth [8,9], although the mechanism of minoxidil-induced hair growth is not fully understood. In a previous study, minoxidil was shown to have proliferative and anti-apoptotic effects on dermal papilla cells . Moreover, it was also found to stimulate hair growth by the opening of ATP-sensitive K+-channel [11,12], the up-regulation of vascular endothelial growth factor (VEGF)  and the activation of the β-catenin pathway  in dermal papilla cells (DPC). The DPC consist of a cluster of specialized fibroblasts that play important roles in the regulation of the hair cycle through the secretion of diffusible proteins, such as insulin-like growth factor-1 (IGF-1) , hepatocyte growth factor (HGF) , VEGF  and transforming growth factor-β (TGF-β) [17,18].
Ecklonia cava, a brown alga, grows in the regions of Jeju Island in Korea. In previous reports, E. cava has been found to have effects such as anti-bacterial  and anti-inflammatory activity . E. cava has been reported to contain various phlorotannins—such as eckol, 8,8′-bieckol, 8,4″-dieckol, phlorofucofuroeckol A, triphlorethol-A, phloroglucinol, dioxinodehydroeckol, fucodiphlorethol G [21–24]. Eckol was reported to have radioprotective activities , dieckol to have anti-inflammatory effects , and triphlorethol-A to have antioxidant activity [22,23]. Recently, it has been found that phloroglucinol induced apoptosis . However, the effect and the underlying mechanisms of E. cava on hair growth have not been described. In this study, we examined the promotion effects and the mechanisms of E. cava extract and its components (eckol, dieckol, triphlorethol-A and phloroglucinol) on hair growth.
To determine whether E. cava enzymatic extract could induce hair growth, we examined the effect of E. cava enzymatic extract with the use of an organ culture of the rat vibrissa follicle. When the rat vibrissa follicles were treated with various concentrations of E. cava enzymatic extract for three weeks, in particular, the hair-fiber length with 1 μg/mL of E. cava enzymatic extract treatment showed a significant increase when compared to the control group (Figure 1). The result indicates that E. cava enzymatic extract contains components promoting hair growth.
To investigate whether anagen induction was promoted by E. cava enzymatic extract, we used C57BL/6 mice, since the dorsal hair is known to have a time-synchronized hair growth cycle . Shaved skin of telogen C57BL/6 mice is pink, which then darkens along with anagen initiation. As shown in Figure 2, the area of black skin was significantly larger (p < 0.05) in the 0.5% E. cava enzymatic extract treated group than in the control group at 26 days after depilation. Induction of the anagen phase in the 0.5% E. cava enzymatic extract treated group was observed to be faster than in the control group. The 5% Minoxidil (MINOXYLTM) treated group, a positive control group, showed gray skin from 13 days after depilation.
We examined the effects of E. cava enzymatic extract and its isolated compounds on the proliferation of DPC. When DPC were treated with E. cava enzymatic extract in the concentrations of 0.001, 0.01, 0.1, 1, 10 and 100 μg/mL, E. cava enzymatic extract significantly promoted the proliferation of DPC compared with the vehicle-treated control at all the concentrations, except the 100 μg/mL (Figure 3). We evaluated whether the isolated compounds from E. cava enzymatic extract such as eckol, dieckol, phloroglucinol and triphlorethol-A, could promote the proliferation of DPC. DPC were treated with eckol, dieckol, phloroglucinol and triphlorethol-A, individually, at 0.005, 0.01, 0.05, 0.1, 0.5, 1 and 10 μg/mL for 4 days. The dieckol increased the proliferation of DPC by 100.5%, 103.9%, 113.5%, 106.1%, 108.1%, 98.5% and 97.3%, respectively (Table 1). The eckol also promoted the proliferation of DPC by 100.8%, 106.1%, 120.3%, 108.5%, 107.8%, 105.4% and 104.1%, respectively (Table 1). However, phloroglucinol and triphlorethol-A did not affect the proliferation of DPC (Table 1).
Minoxidil, a hair-growth promoting agent, has a mitotic effect on NIH3T3 fibroblasts via KATP channel opening. Whether E. cava enzymatic extract, eckol, dieckol, phloroglucinol and triphlorethol-A could act as an opener of KATP channel, proliferation of NIH3T3 fibroblasts was examined. NIH3T3 fibroblasts were treated with E. cava enzymatic extract, eckol, dieckol, phloroglucinol and triphlorethol-A at 0.05, 0.1, 0.5, 1 and 10 μg/mL. The E. cava enzymatic extract significantly increased the proliferation of NIH3T3 fibroblasts by 119.6%, 118.8%, 116.3%, 113.7% and 77.4%, respectively (Figure 4A). To evaluate whether the E. cava-induced proliferation was mediated through KATP channel opening, NIH3T3 fibroblasts were pretreated with tolbutamide, a non-selective blocker of K+ channels. Tolbutamide inhibited the E. cava-induced proliferation of NIH3T3 fibroblasts (Figure 4B). Nevertheless, it is important to note that dieckol, a major component of the E. cava enzymatic extract, did not alter the proliferation of NIH3T3 fibroblasts (data not shown). Eckol, phloroglucinol and triphlorethol-A slightly increased the proliferation of NIH3T3 fibroblasts compared with the control group (data not shown).
5α-reductase activity is known to be important for preventing hair loss. We investigated the effects of E. cava enzymatic extract, eckol, dieckol, phloroglucinol and triphlorethol-A on the 5α-reductase activity using rat prostatic enzyme. As shown in Figure 5, the E. cava enzymatic extract, eckol and dieckol significantly inhibited 5α-reductase activities in a dose-dependent manner (Figure 5A–C). Especially, when the reaction mixture was incubated with 100 μg/mL of dieckol, its inhibition activity was similar to that of the finateride treated group, a positive control group (Figure 5C). However, phloroglucinol did not affect 5α-reductase activities (Figure 5D). 5α-Reductase activities in the triphlorethol-A treated group showed a slight inhibition (Figure 5E).
In this study, the hair growth promoting effect of E. cava enzymatic extract, which contains more than 35% of dieckol, was investigated in vitro and in vivo. To the best of our knowledge, this study is the first to demonstrate that E. cava enzymatic extract and dieckol, a component of E. cava, have the potential to promote hair growth via the proliferation of dermal papilla cells and/or the inhibition of 5α-reductase activity.
The hair growth cycles, in hair follicle organ cultures of the rat vibrissa follicles, have been reported to be synchronized according to their age  and the isolated rat vibrissa follicles could be maintained in vitro up to 23 days . Use of the organ culture methods to evaluate hair follicle growth is thought to be correlated with in vivo systems because the extent of hair growth can be observed as the sum of the function of each cell . We found that the E. cava enzymatic extract increased the hair-fiber length of follicles by 12.4% at 1 μg/mL, compared with the vehicle-treated control. To evaluate the in vivo effect of E. cava enzymatic extract on the induction of the anagen phase, the hair growth promoting effect on C57BL/6 mouse was examined. The hair growth stimulating in vitro effect of E. cava enzymatic extract was also observed in vivo using C57BL/6 mice.
The hair follicle consists of several distinct epithelial cells and DPC [31,32]. Regulation of hair growth depends on the balance between proliferation and apoptosis in the DPC . As shown in figure 3, E. cava enzymatic extract significantly increased the proliferation of DPC. We also examined whether the isolated compounds of E. cava extracts such as eckol, dieckol, phloroglucinol and triphlorethol-A, could increase the proliferation of DPC. We observed that eckol and dieckol increased the proliferation of DPC, whereas phloroglucinol and triphlorethol-A did not affect proliferation of DPC (Table 1).
K+ channel opening is involved in not only mitogenesis , but also hair growth . In previous studies, minoxidil was able to potentiate the mitogenic effects on NIH3T3 fibroblasts through the KATP channel opening . As shown in Figure 4, the E. cava enzymatic extract could promote the proliferation of NIH3T3 fibroblasts, which was inhibited by tobutamide, a non-selective blocker of K+ channels. The result indicates that the E. cava enzymatic extract can promote hair growth via the KATP channel opening.
Inhibition of 5α-reductase activity is important in preventing hair loss in AGA [36,37]. E. cava enzymatic extract significantly inhibited 5α-reductase activity in a dose dependent manner. Among the isolated compounds from E. cava enzymatic extract—eckol, dieckol, phloroglucinol and triphlorethol-A—dieckol was the most active. The results suggest that E. cava enzymatic extract and dieckol could have the potential for the treatment of AGA via the inhibition of 5α-reductase activities. Previous studies propose that AGA may be caused by DHT in different ways: The miniaturization of dermal papilla and hair follicles is induced by DHT, which leads to transition from anagen to catagen . DHT increases the levels of transforming growth factor-β1 (TGF-β1) and TGF-β2 in dermal papilla cells, which leads to decreased proliferation of epithelial cells [39,40]. Up-regulation of dickkopf related protein-1 (DKK-1) by DHT can cause repression of the growth of epithelial cells in hair follicles . In further studies, we need to examine whether that E. cava enzymatic extract and dieckol can regulate the levels of TGF-β1/β2 and DKK-1 in dermal papilla cells. The androgen action and gene expressions in dermal papilla cells (DPCs) from the human beard are known to be different from those in DPCs of the human scalp. In the future, therefore, although the structure of hair follicle in the human scalp is very similar to that of hair follicle in rat vibrissa, we need to examine whether E. cava extract and dieckol can promote hair growth of the human scalp.
The brown alga, E. cava, was collected along the coasts of Jeju Island in Korea, between February and May 2010 and taxonomically identified by Professor Ki Wan Lee. The samples were washed three times in tap water to remove any attached salt, epiphytes, and sand. Then, they were rinsed carefully with fresh distilled water, and maintained in a medical refrigerator at −20 °C. The frozen samples were then lyophilized and homogenized using a grinder prior to extraction.
We followed the methods reported in previous studies for the preparation of E. cava enzymatic extract . To briefly state the preparation procedure, fifty grams of E. cava were homogenized with water (2 L), and mixed with 500 μL of carbohydrate enzyme (celluclast 1.5L FG, Novozyme Nordisk, Bagsvaerd, Denmark). E. cava enzymatic extract was adjusted to be within the optimum pH and temperature range of the carbohydrate enzyme and enzymatic reactions were performed for 24 h. Following extraction, the extract was boiled for 10 min at 100 °C to inactivate the enzymes. Then, E. cava enzymatic extract was clarified by centrifugation (3000 rpm, for 20 min at 4 °C) to remove the residue. E. cava enzymatic extract was adjusted to pH 7.0.
Eckol, dieckol, phloroglucinol and triphlorethol-A were isolated from E. cava as previously described . In short, the dried E. cava was extracted three times with 80% aqueous EtOH, and was evaporated in a vacuum. The EtOH extract was then partitioned with EtOAc. The EtOAc fraction was subjected to silica and LH-20 column chromatography. The active compounds were finally purified by reversed-phase HPLC (ThermoFisher Scientific, San Jose, CA, USA), and the purified compounds were then confirmed by comparing their LC/MS, 1H NMR data to those in the existing literature .
Eckol: LC/MS data (M+, m/z: 372.0 calcd for C18H12O9). 1H NMR (400 MHz, DMSO-d6) δ 9.54 (1H, s, OH-9), 9.45 (1H, s, OH-4), 9.21 (2H, s, OH-2,7) 9.16 (2H, s, OH-3′,5′), 6.14 (1H, s, H-3), 5.96 (1H, d, J = 2.8 Hz, H-8), 5.80 (1H, d, J = 1.7 Hz, H-4′), 5.78 (1H, d, J = 2.8 Hz, H-6), 5.72 (2H, J = 1.7 Hz, H-2′,6′).
Dieckol: LC/MS data (M+, m/z: 742.0 calcd for C36H22O18). 1H NMR (400 MHz, DMSO-d6) δ 9.71(1H, s, OH-9), 9.61 (1H, s, OH-9″), 9.51 (1H, s, OH-4″), 9.46 (1H, s, OH-4), 9.36 (2H, s, OH-3″,5″), 9.28 (1H, s, OH-2″), 9.23 (1H, s, OH-2), 9.22 (1H, s, OH-7″), 9.15 (2H, s, OH-3′,5′) 6.17 (1H, s, H-3″), 6.14 (1H, s, H-3), 6.02 (1H, d, J = 2.7 Hz, H-8), 5.98 (1H, d, J = 2.7 Hz, H-8″), 5.95 (1H, s, H-2′, 6″′), 5.82 (1H, d, J = 2.7 Hz, H-6), 5.81 (1H, d, J = 2.7 Hz, H-6″), 5.80 (1H, t, J = 2.0 Hz, H-4′), 5.78 (2H, d, J = 2.0 Hz, H-2′,6′).
Phloroglucinol: LC/MS data (M+, m/z: 126 calcd for C6H6O3). 1H NMR (400 MHz, DMSO-d6) δ 8.97 (3H, s, OH-1,3,5), 5.66 (3H, s, H-2,4,5).
Triphlorethol-A: LC/MS data (M+, m/z: 374.0 calcd for C18H14O9). 1H NMR (400 MHz, DMSO-d6) δ 5.7 (1H, d, J = 2.7, H-3), 6.0 (1H, d, J = 2.9, H-5), 5.8 (1H, S, H-3′), 5.8 (1H, S, H-5′), 6.0 (1H, d, J = 2.2, H-2″), 5.9 (t, J = 2.2, H-4″), 6.0 (1H, d, J = 2.2, H-6″).
The purity of eckol, dieckol, phloroglucinol and triphlorethol-A was >95%, according to the peak area of all components absorbed at each specific wavelength in HPLC analysis. Their chemical structures are shown in Figure 6, and were freshly dissolved in dimethyl sulfoxide (DMSO) (Sigma, St. Louis, MO, USA) for subsequent treatment. Further, minoxidil sulfate and minoxidil were also dissolved in DMSO for subsequent treatment. The final concentration of DMSO was adjusted to 0.2% (v/v) in the following experiment. Tolbutamide was made up as a 410 mM stock solution in ethanol and added to the culture media in a final concentration of 0.25% ethanol.
Male Wistar rats (3 weeks of age) were supplied from Orient Bio (Seongnam, Gyeonggi, Korea). Six-week-old female C57BL/6 mice and 8-week-old male spargue-Dawley (SD) rats were purchased from Dae-Han Biolink (Eumsung, Chungbuk, Korea) and were provided with a standard laboratory diet and water ad libitum. All animals were cared for by using protocols (20100031) approved by the Institutional Animal Care and Use Committee (IACUC) of the Jeju National University.
Isolation of rat vibrissa follicles was performed as described previously . Briefly, rat vibrissa follicles were harvested from male Wistar rats that were 23 days old. To accomplish this, the rats were sacrificed under carbon dioxide (CO2). Next, both the left and right mystacial pads were removed from the rats and placed in a 1:1 (v/v) solution between Earle’s balanced salts solution (EBSS, Sigma, St. Louis, MO, USA) and PBS that contained 100 unit/mL of penicillin and 100 μg/mL of streptomycin. Anagen vibrissa follicles were then carefully dissected under a stereomicroscope (Olympus, Tokyo, Japan) from posterior parts of the mystacial pads, with considerable caution to remove the surrounding connective tissue without damaging the vibrissa follicle. Using this method, we were able to routinely isolate more than 40 follicles from each animal. The isolated follicles were then placed in separate wells in 24-well plates that contained 500 μL of Williams medium E (GIBCO Inc, Grand Island, NY, USA) supplemented with 2 mM L-glutamine (Gibco Inc, Grand Island, NY, USA), 10 μg/mL insulin (Sigma, St. Louis, MO, USA), 50 nM hydrocortisone (Sigma, St. Louis, MO, USA), 100 unit/mL penicillin and 100 μg/mL streptomycin at 37 °C. They were cultivated in an atmosphere comprised of 5% CO2 and 95% air. The isolated follicles were then treated with vehicle (DMSO diluted 1:1000 in Williams medium E) as a control and E. cava enzymatic extract (0.01, 0.1, 1 and 10 μg/mL). Minoxidil sulfate (Sigma, St. Louis, MO, USA) was used as a positive control in the culture systems (Buhl et al., 1990). The culture medium was changed every 3 days and photographs of the cultured vibrissa follicles were taken using a stereomicroscope, for 3 weeks. The length of the hair follicles was measured using a DP controller (Olympus, Tokyo, Japan).
Anagen was induced on the back skin of C57BL/6 mice that were in the telogen phase of the cycle by depilation, as described previously . Briefly, 6-week-old female C57BL/6 mice were allowed to adapt to their new environment for one week. The anagen was then induced in the back skin of the 7-week-old female C57BL/6 mice by shaving, which led to synchronized development of anagen hair follicles. From the following day (day 1), 0.2 mL of 0.5% E. cava enzymatic extract in 50% ethanol was topically applied every day for 33 days. 5% Minoxidil (MINOXYLTM; Hyundai Pharm. Co. Ltd., Cheonan, Chungnam, Korea) was used as a positive control. The back skin of the mice was then observed and photographed at 1, 7, 13, 20, 26 and 33 days after shaving. For the quantitative assessment, dotmatrix planimetry was performed .
Rat vibrissa immortalized dermal papilla cell line  was donated by the Skin Research Institute, Amore Pacific Corporation R & D Center, South Korea. The DPC were cultured in DMEM (Hyclone Inc., Logan, UT, USA), supplemented with 10% fetal bovine serum (Gibco BRL, Grand Island, NY, USA) and penicillin/streptomycin (100 unit/mL and 100 μg/mL, respectively), at 37 °C in a humidified atmosphere under 5% CO2.
The proliferation of DPC was evaluated by measuring the metabolic activity using a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) . Briefly, DPC at 1.0×104 cells/mL were seeded into 96-well plate, then cultured for 24 h in a serum-free DMEM, and then treated with vehicle (DMSO diluted 1:1000 in serum-free DMEM) as a control, E. cava extract (0.001~100 μg/mL), eckol (0.005~10 μg/mL), dieckol (0.005~10 μg/mL), phloroglucinol (0.005~10 μg/mL), triphlorethol-A (0.005~10 μg/mL) and minoxidil sulfate (1 μM), for 4 days. After incubation, 0.1 mg (50 μL of a 2 mg/mL solution) of MTT (Sigma, St. Louis, MO, USA) was added to each well, and the cells were then incubated at 37 °C for 4 h. Next, the plates were centrifuged at 1000 rpm for 5 min at room temperature and the media was then carefully aspirated. 200 μL of DMSO was then added to each well to dissolve the formazan crystals and the absorbance of the plates, at 540 nm, was then read immediately on a microplate reader (BioTek Instrument, Inc., Winooski, VT, USA). All experiments were performed three times and the mean absorbance values were calculated. The results are expressed as a percentage of absorbance caused by treatment with the extract or the active component compared to those of the vehicle treated controls.
The mouse embryonic NIH3T3 fibroblasts were purchased from ATCC (Rockville, MD, USA) and cultured in ATCC-formulated Dulbecco’s Modified Eagle’s Medium (DMEM), supplemented with 10% (v/v) heat-activated bovine calf serum (BCS), 100 unit/mL penicillin and 100 μg/mL streptomycin at 37 °C atmosphere and 5% CO2.
The proliferation of NIH3T3 fibroblasts was also evaluated by measuring the metabolic activity using MTT assay . NIH3T3 fibroblasts, at 1.0 × 104 cells/mL, were seeded into a 96-well plate. Cells were incubated for 24 h with DMEM supplemented with 10% BCS, then washed with phosphate buffered saline (PBS, Sigma, St. Louis, MO, USA). The cells were maintained with DMEM supplemented with 10% BCS or 1.5% BCS and treated with vehicle (DMSO) as a control, E. cava extract (0.05~10 μg/mL), eckol (0.05~10 μg/mL), dieckol (0.05~10 μg/mL), phloroglucinol (0.05~10 μg/mL), triphlorethol-A (0.05~10 μg/mL) and minoxidil (75 μM), for 4 days. To clarify whether proliferation of NIH3T3 fibroblasts is regulated by KATP channel opening, NIH3T3 fibroblasts were pretreated with tolbutamide (2 mM), a non-selective blocker of K+ channel, for 30 min prior to incubation with E. cava enzymatic extract for 4 days. All experiments were performed three times and the mean absorbance values were calculated. The results are expressed as the percentage in the absorbance caused by treatment with the extract or the active component compared to those of the vehicle untreated controls.
Male SD rats (8 weeks) were sacrificed with carbon dioxide (CO2). The prostates of rats were dissected, freed of their capsules, then washed with saline, and stored at −80 °C. Frozen tissues were thawed on ice and procedures were carried out at 4 °C. The tissues were homogenized with Polytron homogenizer (Brinkman Instruments, Wesrbury, NY, USA) in 5–6 tissue volumes of medium A (0.32 M sucrose, 1 mM dithiothreitol (DTT), 0.2 mM phenylmethylsulfonylfluoride (PMSF), and 20 mM potassium phosphate buffer pH 6.6). The homogenates were centrifuged at 100,000 g for 60 min. The pellets were recovered, washed with three tissue volumes of medium A and centrifuged two additional times at 400 g at 0 °C for 10 min. The washed pellets were suspended in medium A and stored at −80 °C until use. The suspension (2.5 mg protein/mL for Rat prostates, determined by the Bradford method) was used as source of 5α-reductase.
5α-reductase activities were assayed as previously described . The reaction mixture contained a final volume of 500 μL: one millimole DTT, 40 mM potassium phosphate buffers, 2 mM NADPH, Testosterone including 120 n Ci [1,2,6,7-3H]. The reaction in triplicate was started when it was added to the rat prostatic enzyme fraction (250 μg protein), 0.2% DMSO as a control, E. cava extract (10, 30, 50, 70 and 100 μg/mL), eckol (10, 70 and 100 μg/mL), dieckol (10, 70 and 100 μg/mL), phloroglucinol (10, 70 and 100 μg/mL) and triphlorethol-A (10, 70 and 100 μg/mL). Finasteride 2 nM (MERCK SHARP & DOHME, South Granville, Australia) was used as a positive control. The mixture was incubated at 37 °C for 60 min, and then stopped by adding 1 mL of ethyl acetate and mixing for 1 min. After centrifugation at 1000 g for 5 min, the organic phase was removed which then was dried under a heating plate, dissolved in 50 μL of ethyl acetate containing 500 μg/mL testosterone and 500 μg/mL dihydrotestosterone (DHT) and applied to a silica gel 60 F254 TLC plate (Merck, Darmstadt, Germany). The plate was developed in a solvent system consisting of an ethyl acetate:cyclohexane (1:1) solution, the plate then was air dried. Testosterone was visibly seen under the UV light (254 nm) and DHT was detected using 10% H2SO4 solution via posteriorly heating the plate. Under these conditions, DHT will be shown as a dark yellow color. Areas containing androgen were removed and the strips were soaked in the 5 mL of ULTIMA GOLDTM Cocktails (PerkinElmer, Inc., Waltham, MA, USA) and the radioactivity level was then measured via a liquid scintillation counter (Packard Bioscience, Meriden, CT, USA). The activity of 5α-reductase was expressed as the ratio [DHT/(T + DHT)] × 100.
Each experiment was performed at least in triplicate. Results are expressed as mean ± SD or mean ± SE from three separate experiments. The Student’s t test and one-way ANOVA test were used to determine the statistical significance.
In conclusion, this study demonstrated that dieckol, a principal component of E. cava, could stimulate hair growth through the proliferation of dermal papilla cells and the inhibition of 5α-reductase activity. These finding indicate that dieckol from E. cava enzymatic extract is a possible therapeutic compound for treatment of hair loss.
This research was a part of the project titled “Development of product and material promoting hair-growth from Jeju marine algae” funded by the Ministry of Land, Transport and Maritime Affairs, Korea.
Figure 1. Hair growth effect of Ecklonia cava enzymatic extract on rat vibrissa follicles. (A,B) Individual vibrissa follicles from Wistar rats were micro dissected and then cultured in William’s E medium at 37 °C under 5% CO2. Vibrissa follicles were treated with various concentrations of E. cava enzymatic extract and minoxidil sulfate, as indicated. All experiments were performed in triplicate. The difference in the length of vibrissa follicles of the control group on day 21 was taken to be 100%. Data are presented as the percentage of the length of the treated follicles based on the mean length of the control follicles ± SE. * p < 0.05, ** p < 0.01 vs. control.
Figure 2. The effect of Ecklonia cava enzymatic extract on the anagen induction in C57BL/6 mice. After shaving, the back skins were treated with 0.5% E. cava enzymatic extract, vehicle and 5% minoxidil every day for 33 days. (A) The back skins were photographed at 1, 7, 13, 20, 26 and 33 days after depilation; (B) On day 26, the quantitative assessment of anagen induction analyzed via dotmatrix planimetry was performed. The transparency was put on a photo of a mouse to mark the areas that were in different stages (pink = telogen, anagen = black). Afterward a dotmatrix (sheet with a uniform defined dot pattern) was placed under the marked foil to calculate the percentages of the regions of interest by counting the dots. The percentage of anagen induction was calculated by the equation [(black skin/total skin) × 100]. Data are presented as the mean ± SE (n = 6). * p < 0.05, ** p < 0.01, *** p < 0.001 vs. vehicle treated control.
Figure 3. The effect of Ecklonia cava enzymatic extract on the proliferation of dermal papilla cells. Rat vibrissa immortalized DPC (1.0 × 104 cells/mL) were plated in 96 well plates. DPC were treated with various concentrations of E. cava enzymatic extract and minoxidil sulfate, as indicated. Cell proliferation was measured using a MTT assay for 4 days. All experiments were performed in triplicate. Data are presented as the mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control.
Figure 4. The effect of Ecklonia cava enzymatic extract on the proliferation of NIH3T3 fibroblasts. (A,B) Mouse embryonic NIH3T3 fibroblasts (1.0 × 104 cells/mL) were plated in 96 well plates. NIH3T3 fibroblasts were treated with various concentration of E. cava enzymatic extract, as indicated. Stimulation with minoxidil served as a positive control. (B) NIH3T3 fibroblasts were pretreated with tolbutamide for 30 min prior to incubation with E. cava enzymatic extract. Cell proliferation was measured using a MTT assay for four days. All experiments were performed in triplicate. Data are presented as the mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control; ### p < 0.001 vs. E. cava-treated group.
Figure 5. Inhibition effects of Ecklonia cava enzymatic extract and its isolated compounds on 5α-reductase activities. (A-E) Assay of 5α-reductase inhibition was performed using a crude extract of rat prostate. The reaction mixture contained [1,2,6,7-3H] testosterone, prostatic enzyme and E. cava enzymatic extract or its isolated compounds (eckol, dieckol, phloroglucinol and triphlorethol-A). The conversion rate of testosterone (T) to dihydrotestosterone (DHT) was calculated by the equation [DHT/(T + DHT)]. Inhibition activity (%) was expressed as a percentage of reduced conversion rate compared to the control. The inhibition activity of control group was regarded as 0% (not shown). Finasteride was used as a positive control. Data are presented as the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. finasteride; # p < 0.05, ## p < 0.01, ### p < 0.001 vs. control.
Figure 6. Structures of eckol, dieckol, phloroglucinol and triphlorethol-A.
Table 1. The effects of isolated compounds from Ecklonia cava enzymatic extract on the proliferation of dermal papilla cells.
The proliferation of the control group was regarded as 100% (not shown). All experiments were performed in triplicate. Data are presented as the mean ± SD.
***p < 0.001 vs. control.
© 2012 by the authors; licensee Molecular Diversity Preservation International, Basel, Switzerland. This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).

lowercased:
this study was conducted to evaluate the effect of ecklonia cava, a marine alga native to jeju island in korea, on the promotion of hair growth. when vibrissa follicles were cultured in the presence of e. cava enzymatic extract (which contains more than 35% of dieckol) for 21 days, e. cava enzymatic extract increased hair-fiber length. in addition, after topical application of the 0.5% e. cava enzymatic extract onto the back of c57bl/6 mice, anagen progression of the hair-shaft was induced. the treatment with e. cava enzymatic extract resulted in the proliferation of immortalized vibrissa dermal papilla cells (dpc). especially, dieckol, among the isolated compounds from the e. cava enzymatic extract, showed activity that increased the proliferation of dpc. when nih3t3 fibroblasts were treated with the e. cava enzymatic extract and the isolated compounds from the e. cava enzymatic extract, the e. cava enzymatic extract increased the proliferation of nih3t3 fibroblasts, but the isolated compounds such as eckol, dieckol, phloroglucinol and triphlorethol-a did not affect the proliferation of nih3t3 fibroblasts. on the other hand, the e. cava enzymatic extract and dieckol significantly inhibited 5α-reductase activity. these results suggest that dieckol from e. cava can stimulate hair growth by the proliferation of dpc and/or the inhibition of 5α-reductase activity.
androgenetic alopecia (aga), the most common type of alopecia, is a problem in men over the age of 40. hair loss is a growing trend in the world, however, the prevention of this condition is not simple . numerous possibilities have been discussed to treat hair loss . nevertheless, only two fda-approved drugs have been available for aga patients, i.e., finasteride and minoxidil [3,4]. finasteride is a type ii 5α-reductase inhibitor. it can be used in prostatic hypertrophy  as well as in hair loss . minoxidil was used as a drug to treat high blood pressure . however, it was also found to promote hair growth [8,9], although the mechanism of minoxidil-induced hair growth is not fully understood. in a previous study, minoxidil was shown to have proliferative and anti-apoptotic effects on dermal papilla cells . moreover, it was also found to stimulate hair growth by the opening of atp-sensitive k+-channel [11,12], the up-regulation of vascular endothelial growth factor (vegf)  and the activation of the β-catenin pathway  in dermal papilla cells (dpc). the dpc consist of a cluster of specialized fibroblasts that play important roles in the regulation of the hair cycle through the secretion of diffusible proteins, such as insulin-like growth factor-1 (igf-1) , hepatocyte growth factor (hgf) , vegf  and transforming growth factor-β (tgf-β) [17,18].
ecklonia cava, a brown alga, grows in the regions of jeju island in korea. in previous reports, e. cava has been found to have effects such as anti-bacterial  and anti-inflammatory activity . e. cava has been reported to contain various phlorotannins—such as eckol, 8,8′-bieckol, 8,4″-dieckol, phlorofucofuroeckol a, triphlorethol-a, phloroglucinol, dioxinodehydroeckol, fucodiphlorethol g [21–24]. eckol was reported to have radioprotective activities , dieckol to have anti-inflammatory effects , and triphlorethol-a to have antioxidant activity [22,23]. recently, it has been found that phloroglucinol induced apoptosis . however, the effect and the underlying mechanisms of e. cava on hair growth have not been described. in this study, we examined the promotion effects and the mechanisms of e. cava extract and its components (eckol, dieckol, triphlorethol-a and phloroglucinol) on hair growth.
to determine whether e. cava enzymatic extract could induce hair growth, we examined the effect of e. cava enzymatic extract with the use of an organ culture of the rat vibrissa follicle. when the rat vibrissa follicles were treated with various concentrations of e. cava enzymatic extract for three weeks, in particular, the hair-fiber length with 1 μg/ml of e. cava enzymatic extract treatment showed a significant increase when compared to the control group (figure 1). the result indicates that e. cava enzymatic extract contains components promoting hair growth.
to investigate whether anagen induction was promoted by e. cava enzymatic extract, we used c57bl/6 mice, since the dorsal hair is known to have a time-synchronized hair growth cycle . shaved skin of telogen c57bl/6 mice is pink, which then darkens along with anagen initiation. as shown in figure 2, the area of black skin was significantly larger (p < 0.05) in the 0.5% e. cava enzymatic extract treated group than in the control group at 26 days after depilation. induction of the anagen phase in the 0.5% e. cava enzymatic extract treated group was observed to be faster than in the control group. the 5% minoxidil (minoxyltm) treated group, a positive control group, showed gray skin from 13 days after depilation.
we examined the effects of e. cava enzymatic extract and its isolated compounds on the proliferation of dpc. when dpc were treated with e. cava enzymatic extract in the concentrations of 0.001, 0.01, 0.1, 1, 10 and 100 μg/ml, e. cava enzymatic extract significantly promoted the proliferation of dpc compared with the vehicle-treated control at all the concentrations, except the 100 μg/ml (figure 3). we evaluated whether the isolated compounds from e. cava enzymatic extract such as eckol, dieckol, phloroglucinol and triphlorethol-a, could promote the proliferation of dpc. dpc were treated with eckol, dieckol, phloroglucinol and triphlorethol-a, individually, at 0.005, 0.01, 0.05, 0.1, 0.5, 1 and 10 μg/ml for 4 days. the dieckol increased the proliferation of dpc by 100.5%, 103.9%, 113.5%, 106.1%, 108.1%, 98.5% and 97.3%, respectively (table 1). the eckol also promoted the proliferation of dpc by 100.8%, 106.1%, 120.3%, 108.5%, 107.8%, 105.4% and 104.1%, respectively (table 1). however, phloroglucinol and triphlorethol-a did not affect the proliferation of dpc (table 1).
minoxidil, a hair-growth promoting agent, has a mitotic effect on nih3t3 fibroblasts via katp channel opening. whether e. cava enzymatic extract, eckol, dieckol, phloroglucinol and triphlorethol-a could act as an opener of katp channel, proliferation of nih3t3 fibroblasts was examined. nih3t3 fibroblasts were treated with e. cava enzymatic extract, eckol, dieckol, phloroglucinol and triphlorethol-a at 0.05, 0.1, 0.5, 1 and 10 μg/ml. the e. cava enzymatic extract significantly increased the proliferation of nih3t3 fibroblasts by 119.6%, 118.8%, 116.3%, 113.7% and 77.4%, respectively (figure 4a). to evaluate whether the e. cava-induced proliferation was mediated through katp channel opening, nih3t3 fibroblasts were pretreated with tolbutamide, a non-selective blocker of k+ channels. tolbutamide inhibited the e. cava-induced proliferation of nih3t3 fibroblasts (figure 4b). nevertheless, it is important to note that dieckol, a major component of the e. cava enzymatic extract, did not alter the proliferation of nih3t3 fibroblasts (data not shown). eckol, phloroglucinol and triphlorethol-a slightly increased the proliferation of nih3t3 fibroblasts compared with the control group (data not shown).
5α-reductase activity is known to be important for preventing hair loss. we investigated the effects of e. cava enzymatic extract, eckol, dieckol, phloroglucinol and triphlorethol-a on the 5α-reductase activity using rat prostatic enzyme. as shown in figure 5, the e. cava enzymatic extract, eckol and dieckol significantly inhibited 5α-reductase activities in a dose-dependent manner (figure 5a–c). especially, when the reaction mixture was incubated with 100 μg/ml of dieckol, its inhibition activity was similar to that of the finateride treated group, a positive control group (figure 5c). however, phloroglucinol did not affect 5α-reductase activities (figure 5d). 5α-reductase activities in the triphlorethol-a treated group showed a slight inhibition (figure 5e).
in this study, the hair growth promoting effect of e. cava enzymatic extract, which contains more than 35% of dieckol, was investigated in vitro and in vivo. to the best of our knowledge, this study is the first to demonstrate that e. cava enzymatic extract and dieckol, a component of e. cava, have the potential to promote hair growth via the proliferation of dermal papilla cells and/or the inhibition of 5α-reductase activity.
the hair growth cycles, in hair follicle organ cultures of the rat vibrissa follicles, have been reported to be synchronized according to their age  and the isolated rat vibrissa follicles could be maintained in vitro up to 23 days . use of the organ culture methods to evaluate hair follicle growth is thought to be correlated with in vivo systems because the extent of hair growth can be observed as the sum of the function of each cell . we found that the e. cava enzymatic extract increased the hair-fiber length of follicles by 12.4% at 1 μg/ml, compared with the vehicle-treated control. to evaluate the in vivo effect of e. cava enzymatic extract on the induction of the anagen phase, the hair growth promoting effect on c57bl/6 mouse was examined. the hair growth stimulating in vitro effect of e. cava enzymatic extract was also observed in vivo using c57bl/6 mice.
the hair follicle consists of several distinct epithelial cells and dpc [31,32]. regulation of hair growth depends on the balance between proliferation and apoptosis in the dpc . as shown in figure 3, e. cava enzymatic extract significantly increased the proliferation of dpc. we also examined whether the isolated compounds of e. cava extracts such as eckol, dieckol, phloroglucinol and triphlorethol-a, could increase the proliferation of dpc. we observed that eckol and dieckol increased the proliferation of dpc, whereas phloroglucinol and triphlorethol-a did not affect proliferation of dpc (table 1).
k+ channel opening is involved in not only mitogenesis , but also hair growth . in previous studies, minoxidil was able to potentiate the mitogenic effects on nih3t3 fibroblasts through the katp channel opening . as shown in figure 4, the e. cava enzymatic extract could promote the proliferation of nih3t3 fibroblasts, which was inhibited by tobutamide, a non-selective blocker of k+ channels. the result indicates that the e. cava enzymatic extract can promote hair growth via the katp channel opening.
inhibition of 5α-reductase activity is important in preventing hair loss in aga [36,37]. e. cava enzymatic extract significantly inhibited 5α-reductase activity in a dose dependent manner. among the isolated compounds from e. cava enzymatic extract—eckol, dieckol, phloroglucinol and triphlorethol-a—dieckol was the most active. the results suggest that e. cava enzymatic extract and dieckol could have the potential for the treatment of aga via the inhibition of 5α-reductase activities. previous studies propose that aga may be caused by dht in different ways: the miniaturization of dermal papilla and hair follicles is induced by dht, which leads to transition from anagen to catagen . dht increases the levels of transforming growth factor-β1 (tgf-β1) and tgf-β2 in dermal papilla cells, which leads to decreased proliferation of epithelial cells [39,40]. up-regulation of dickkopf related protein-1 (dkk-1) by dht can cause repression of the growth of epithelial cells in hair follicles . in further studies, we need to examine whether that e. cava enzymatic extract and dieckol can regulate the levels of tgf-β1/β2 and dkk-1 in dermal papilla cells. the androgen action and gene expressions in dermal papilla cells (dpcs) from the human beard are known to be different from those in dpcs of the human scalp. in the future, therefore, although the structure of hair follicle in the human scalp is very similar to that of hair follicle in rat vibrissa, we need to examine whether e. cava extract and dieckol can promote hair growth of the human scalp.
the brown alga, e. cava, was collected along the coasts of jeju island in korea, between february and may 2010 and taxonomically identified by professor ki wan lee. the samples were washed three times in tap water to remove any attached salt, epiphytes, and sand. then, they were rinsed carefully with fresh distilled water, and maintained in a medical refrigerator at −20 °c. the frozen samples were then lyophilized and homogenized using a grinder prior to extraction.
we followed the methods reported in previous studies for the preparation of e. cava enzymatic extract . to briefly state the preparation procedure, fifty grams of e. cava were homogenized with water (2 l), and mixed with 500 μl of carbohydrate enzyme (celluclast 1.5l fg, novozyme nordisk, bagsvaerd, denmark). e. cava enzymatic extract was adjusted to be within the optimum ph and temperature range of the carbohydrate enzyme and enzymatic reactions were performed for 24 h. following extraction, the extract was boiled for 10 min at 100 °c to inactivate the enzymes. then, e. cava enzymatic extract was clarified by centrifugation (3000 rpm, for 20 min at 4 °c) to remove the residue. e. cava enzymatic extract was adjusted to ph 7.0.
eckol, dieckol, phloroglucinol and triphlorethol-a were isolated from e. cava as previously described . in short, the dried e. cava was extracted three times with 80% aqueous etoh, and was evaporated in a vacuum. the etoh extract was then partitioned with etoac. the etoac fraction was subjected to silica and lh-20 column chromatography. the active compounds were finally purified by reversed-phase hplc (thermofisher scientific, san jose, ca, usa), and the purified compounds were then confirmed by comparing their lc/ms, 1h nmr data to those in the existing literature .
eckol: lc/ms data (m+, m/z: 372.0 calcd for c18h12o9). 1h nmr (400 mhz, dmso-d6) δ 9.54 (1h, s, oh-9), 9.45 (1h, s, oh-4), 9.21 (2h, s, oh-2,7) 9.16 (2h, s, oh-3′,5′), 6.14 (1h, s, h-3), 5.96 (1h, d, j = 2.8 hz, h-8), 5.80 (1h, d, j = 1.7 hz, h-4′), 5.78 (1h, d, j = 2.8 hz, h-6), 5.72 (2h, j = 1.7 hz, h-2′,6′).
dieckol: lc/ms data (m+, m/z: 742.0 calcd for c36h22o18). 1h nmr (400 mhz, dmso-d6) δ 9.71(1h, s, oh-9), 9.61 (1h, s, oh-9″), 9.51 (1h, s, oh-4″), 9.46 (1h, s, oh-4), 9.36 (2h, s, oh-3″,5″), 9.28 (1h, s, oh-2″), 9.23 (1h, s, oh-2), 9.22 (1h, s, oh-7″), 9.15 (2h, s, oh-3′,5′) 6.17 (1h, s, h-3″), 6.14 (1h, s, h-3), 6.02 (1h, d, j = 2.7 hz, h-8), 5.98 (1h, d, j = 2.7 hz, h-8″), 5.95 (1h, s, h-2′, 6″′), 5.82 (1h, d, j = 2.7 hz, h-6), 5.81 (1h, d, j = 2.7 hz, h-6″), 5.80 (1h, t, j = 2.0 hz, h-4′), 5.78 (2h, d, j = 2.0 hz, h-2′,6′).
phloroglucinol: lc/ms data (m+, m/z: 126 calcd for c6h6o3). 1h nmr (400 mhz, dmso-d6) δ 8.97 (3h, s, oh-1,3,5), 5.66 (3h, s, h-2,4,5).
triphlorethol-a: lc/ms data (m+, m/z: 374.0 calcd for c18h14o9). 1h nmr (400 mhz, dmso-d6) δ 5.7 (1h, d, j = 2.7, h-3), 6.0 (1h, d, j = 2.9, h-5), 5.8 (1h, s, h-3′), 5.8 (1h, s, h-5′), 6.0 (1h, d, j = 2.2, h-2″), 5.9 (t, j = 2.2, h-4″), 6.0 (1h, d, j = 2.2, h-6″).
the purity of eckol, dieckol, phloroglucinol and triphlorethol-a was >95%, according to the peak area of all components absorbed at each specific wavelength in hplc analysis. their chemical structures are shown in figure 6, and were freshly dissolved in dimethyl sulfoxide (dmso) (sigma, st. louis, mo, usa) for subsequent treatment. further, minoxidil sulfate and minoxidil were also dissolved in dmso for subsequent treatment. the final concentration of dmso was adjusted to 0.2% (v/v) in the following experiment. tolbutamide was made up as a 410 mm stock solution in ethanol and added to the culture media in a final concentration of 0.25% ethanol.
male wistar rats (3 weeks of age) were supplied from orient bio (seongnam, gyeonggi, korea). six-week-old female c57bl/6 mice and 8-week-old male spargue-dawley (sd) rats were purchased from dae-han biolink (eumsung, chungbuk, korea) and were provided with a standard laboratory diet and water ad libitum. all animals were cared for by using protocols (20100031) approved by the institutional animal care and use committee (iacuc) of the jeju national university.
isolation of rat vibrissa follicles was performed as described previously . briefly, rat vibrissa follicles were harvested from male wistar rats that were 23 days old. to accomplish this, the rats were sacrificed under carbon dioxide (co2). next, both the left and right mystacial pads were removed from the rats and placed in a 1:1 (v/v) solution between earle’s balanced salts solution (ebss, sigma, st. louis, mo, usa) and pbs that contained 100 unit/ml of penicillin and 100 μg/ml of streptomycin. anagen vibrissa follicles were then carefully dissected under a stereomicroscope (olympus, tokyo, japan) from posterior parts of the mystacial pads, with considerable caution to remove the surrounding connective tissue without damaging the vibrissa follicle. using this method, we were able to routinely isolate more than 40 follicles from each animal. the isolated follicles were then placed in separate wells in 24-well plates that contained 500 μl of williams medium e (gibco inc, grand island, ny, usa) supplemented with 2 mm l-glutamine (gibco inc, grand island, ny, usa), 10 μg/ml insulin (sigma, st. louis, mo, usa), 50 nm hydrocortisone (sigma, st. louis, mo, usa), 100 unit/ml penicillin and 100 μg/ml streptomycin at 37 °c. they were cultivated in an atmosphere comprised of 5% co2 and 95% air. the isolated follicles were then treated with vehicle (dmso diluted 1:1000 in williams medium e) as a control and e. cava enzymatic extract (0.01, 0.1, 1 and 10 μg/ml). minoxidil sulfate (sigma, st. louis, mo, usa) was used as a positive control in the culture systems (buhl et al., 1990). the culture medium was changed every 3 days and photographs of the cultured vibrissa follicles were taken using a stereomicroscope, for 3 weeks. the length of the hair follicles was measured using a dp controller (olympus, tokyo, japan).
anagen was induced on the back skin of c57bl/6 mice that were in the telogen phase of the cycle by depilation, as described previously . briefly, 6-week-old female c57bl/6 mice were allowed to adapt to their new environment for one week. the anagen was then induced in the back skin of the 7-week-old female c57bl/6 mice by shaving, which led to synchronized development of anagen hair follicles. from the following day (day 1), 0.2 ml of 0.5% e. cava enzymatic extract in 50% ethanol was topically applied every day for 33 days. 5% minoxidil (minoxyltm; hyundai pharm. co. ltd., cheonan, chungnam, korea) was used as a positive control. the back skin of the mice was then observed and photographed at 1, 7, 13, 20, 26 and 33 days after shaving. for the quantitative assessment, dotmatrix planimetry was performed .
rat vibrissa immortalized dermal papilla cell line  was donated by the skin research institute, amore pacific corporation r & d center, south korea. the dpc were cultured in dmem (hyclone inc., logan, ut, usa), supplemented with 10% fetal bovine serum (gibco brl, grand island, ny, usa) and penicillin/streptomycin (100 unit/ml and 100 μg/ml, respectively), at 37 °c in a humidified atmosphere under 5% co2.
the proliferation of dpc was evaluated by measuring the metabolic activity using a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (mtt) . briefly, dpc at 1.0×104 cells/ml were seeded into 96-well plate, then cultured for 24 h in a serum-free dmem, and then treated with vehicle (dmso diluted 1:1000 in serum-free dmem) as a control, e. cava extract (0.001~100 μg/ml), eckol (0.005~10 μg/ml), dieckol (0.005~10 μg/ml), phloroglucinol (0.005~10 μg/ml), triphlorethol-a (0.005~10 μg/ml) and minoxidil sulfate (1 μm), for 4 days. after incubation, 0.1 mg (50 μl of a 2 mg/ml solution) of mtt (sigma, st. louis, mo, usa) was added to each well, and the cells were then incubated at 37 °c for 4 h. next, the plates were centrifuged at 1000 rpm for 5 min at room temperature and the media was then carefully aspirated. 200 μl of dmso was then added to each well to dissolve the formazan crystals and the absorbance of the plates, at 540 nm, was then read immediately on a microplate reader (biotek instrument, inc., winooski, vt, usa). all experiments were performed three times and the mean absorbance values were calculated. the results are expressed as a percentage of absorbance caused by treatment with the extract or the active component compared to those of the vehicle treated controls.
the mouse embryonic nih3t3 fibroblasts were purchased from atcc (rockville, md, usa) and cultured in atcc-formulated dulbecco’s modified eagle’s medium (dmem), supplemented with 10% (v/v) heat-activated bovine calf serum (bcs), 100 unit/ml penicillin and 100 μg/ml streptomycin at 37 °c atmosphere and 5% co2.
the proliferation of nih3t3 fibroblasts was also evaluated by measuring the metabolic activity using mtt assay . nih3t3 fibroblasts, at 1.0 × 104 cells/ml, were seeded into a 96-well plate. cells were incubated for 24 h with dmem supplemented with 10% bcs, then washed with phosphate buffered saline (pbs, sigma, st. louis, mo, usa). the cells were maintained with dmem supplemented with 10% bcs or 1.5% bcs and treated with vehicle (dmso) as a control, e. cava extract (0.05~10 μg/ml), eckol (0.05~10 μg/ml), dieckol (0.05~10 μg/ml), phloroglucinol (0.05~10 μg/ml), triphlorethol-a (0.05~10 μg/ml) and minoxidil (75 μm), for 4 days. to clarify whether proliferation of nih3t3 fibroblasts is regulated by katp channel opening, nih3t3 fibroblasts were pretreated with tolbutamide (2 mm), a non-selective blocker of k+ channel, for 30 min prior to incubation with e. cava enzymatic extract for 4 days. all experiments were performed three times and the mean absorbance values were calculated. the results are expressed as the percentage in the absorbance caused by treatment with the extract or the active component compared to those of the vehicle untreated controls.
male sd rats (8 weeks) were sacrificed with carbon dioxide (co2). the prostates of rats were dissected, freed of their capsules, then washed with saline, and stored at −80 °c. frozen tissues were thawed on ice and procedures were carried out at 4 °c. the tissues were homogenized with polytron homogenizer (brinkman instruments, wesrbury, ny, usa) in 5–6 tissue volumes of medium a (0.32 m sucrose, 1 mm dithiothreitol (dtt), 0.2 mm phenylmethylsulfonylfluoride (pmsf), and 20 mm potassium phosphate buffer ph 6.6). the homogenates were centrifuged at 100,000 g for 60 min. the pellets were recovered, washed with three tissue volumes of medium a and centrifuged two additional times at 400 g at 0 °c for 10 min. the washed pellets were suspended in medium a and stored at −80 °c until use. the suspension (2.5 mg protein/ml for rat prostates, determined by the bradford method) was used as source of 5α-reductase.
5α-reductase activities were assayed as previously described . the reaction mixture contained a final volume of 500 μl: one millimole dtt, 40 mm potassium phosphate buffers, 2 mm nadph, testosterone including 120 n ci [1,2,6,7-3h]. the reaction in triplicate was started when it was added to the rat prostatic enzyme fraction (250 μg protein), 0.2% dmso as a control, e. cava extract (10, 30, 50, 70 and 100 μg/ml), eckol (10, 70 and 100 μg/ml), dieckol (10, 70 and 100 μg/ml), phloroglucinol (10, 70 and 100 μg/ml) and triphlorethol-a (10, 70 and 100 μg/ml). finasteride 2 nm (merck sharp & dohme, south granville, australia) was used as a positive control. the mixture was incubated at 37 °c for 60 min, and then stopped by adding 1 ml of ethyl acetate and mixing for 1 min. after centrifugation at 1000 g for 5 min, the organic phase was removed which then was dried under a heating plate, dissolved in 50 μl of ethyl acetate containing 500 μg/ml testosterone and 500 μg/ml dihydrotestosterone (dht) and applied to a silica gel 60 f254 tlc plate (merck, darmstadt, germany). the plate was developed in a solvent system consisting of an ethyl acetate:cyclohexane (1:1) solution, the plate then was air dried. testosterone was visibly seen under the uv light (254 nm) and dht was detected using 10% h2so4 solution via posteriorly heating the plate. under these conditions, dht will be shown as a dark yellow color. areas containing androgen were removed and the strips were soaked in the 5 ml of ultima goldtm cocktails (perkinelmer, inc., waltham, ma, usa) and the radioactivity level was then measured via a liquid scintillation counter (packard bioscience, meriden, ct, usa). the activity of 5α-reductase was expressed as the ratio [dht/(t + dht)] × 100.
each experiment was performed at least in triplicate. results are expressed as mean ± sd or mean ± se from three separate experiments. the student’s t test and one-way anova test were used to determine the statistical significance.
in conclusion, this study demonstrated that dieckol, a principal component of e. cava, could stimulate hair growth through the proliferation of dermal papilla cells and the inhibition of 5α-reductase activity. these finding indicate that dieckol from e. cava enzymatic extract is a possible therapeutic compound for treatment of hair loss.
this research was a part of the project titled “development of product and material promoting hair-growth from jeju marine algae” funded by the ministry of land, transport and maritime affairs, korea.
figure 1. hair growth effect of ecklonia cava enzymatic extract on rat vibrissa follicles. (a,b) individual vibrissa follicles from wistar rats were micro dissected and then cultured in william’s e medium at 37 °c under 5% co2. vibrissa follicles were treated with various concentrations of e. cava enzymatic extract and minoxidil sulfate, as indicated. all experiments were performed in triplicate. the difference in the length of vibrissa follicles of the control group on day 21 was taken to be 100%. data are presented as the percentage of the length of the treated follicles based on the mean length of the control follicles ± se. * p < 0.05, ** p < 0.01 vs. control.
figure 2. the effect of ecklonia cava enzymatic extract on the anagen induction in c57bl/6 mice. after shaving, the back skins were treated with 0.5% e. cava enzymatic extract, vehicle and 5% minoxidil every day for 33 days. (a) the back skins were photographed at 1, 7, 13, 20, 26 and 33 days after depilation; (b) on day 26, the quantitative assessment of anagen induction analyzed via dotmatrix planimetry was performed. the transparency was put on a photo of a mouse to mark the areas that were in different stages (pink = telogen, anagen = black). afterward a dotmatrix (sheet with a uniform defined dot pattern) was placed under the marked foil to calculate the percentages of the regions of interest by counting the dots. the percentage of anagen induction was calculated by the equation [(black skin/total skin) × 100]. data are presented as the mean ± se (n = 6). * p < 0.05, ** p < 0.01, *** p < 0.001 vs. vehicle treated control.
figure 3. the effect of ecklonia cava enzymatic extract on the proliferation of dermal papilla cells. rat vibrissa immortalized dpc (1.0 × 104 cells/ml) were plated in 96 well plates. dpc were treated with various concentrations of e. cava enzymatic extract and minoxidil sulfate, as indicated. cell proliferation was measured using a mtt assay for 4 days. all experiments were performed in triplicate. data are presented as the mean ± sd. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control.
figure 4. the effect of ecklonia cava enzymatic extract on the proliferation of nih3t3 fibroblasts. (a,b) mouse embryonic nih3t3 fibroblasts (1.0 × 104 cells/ml) were plated in 96 well plates. nih3t3 fibroblasts were treated with various concentration of e. cava enzymatic extract, as indicated. stimulation with minoxidil served as a positive control. (b) nih3t3 fibroblasts were pretreated with tolbutamide for 30 min prior to incubation with e. cava enzymatic extract. cell proliferation was measured using a mtt assay for four days. all experiments were performed in triplicate. data are presented as the mean ± sd. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control; ### p < 0.001 vs. e. cava-treated group.
figure 5. inhibition effects of ecklonia cava enzymatic extract and its isolated compounds on 5α-reductase activities. (a-e) assay of 5α-reductase inhibition was performed using a crude extract of rat prostate. the reaction mixture contained [1,2,6,7-3h] testosterone, prostatic enzyme and e. cava enzymatic extract or its isolated compounds (eckol, dieckol, phloroglucinol and triphlorethol-a). the conversion rate of testosterone (t) to dihydrotestosterone (dht) was calculated by the equation [dht/(t + dht)]. inhibition activity (%) was expressed as a percentage of reduced conversion rate compared to the control. the inhibition activity of control group was regarded as 0% (not shown). finasteride was used as a positive control. data are presented as the mean ± sd of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. finasteride; # p < 0.05, ## p < 0.01, ### p < 0.001 vs. control.
figure 6. structures of eckol, dieckol, phloroglucinol and triphlorethol-a.
table 1. the effects of isolated compounds from ecklonia cava enzymatic extract on the proliferation of dermal papilla cells.
the proliferation of the control group was regarded as 100% (not shown). all experiments were performed in triplicate. data are presented as the mean ± sd.
***p < 0.001 vs. control.
© 2012 by the authors; licensee molecular diversity preservation international, basel, switzerland. this article is an open-access article distributed under the terms and conditions of the creative commons attribution license (http://creativecommons.org/licenses/by/3.0/).


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Free goat clipart - Clipart Collection | Goat clip art black and, goat clip art, boer goat clipart clipart panda.
HddFhm proudly present free goat clipart free vector. Stop your search here! 1-24 of 2965362 in cdr, svg, jpg, png design format. Commercial use of free goat clipart is prohibited. Download now free goat clipart.

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free goat clipart - clipart collection | goat clip art black and, goat clip art, boer goat clipart clipart panda.
hddfhm proudly present free goat clipart free vector. stop your search here! 1-24 of 2965362 in cdr, svg, jpg, png design format. commercial use of free goat clipart is prohibited. download now free goat clipart.


text: Building construction work. The project is for the refurbishment and extension of Telford Ice Rink to provide new build soft play and associated catering facilities, shell for future energy centre and shell and core restaurant. The contract will also include for a refurbishment of the existing ice rink to form shell and core restaurant provision and replacement ice rink plant including replacement ice pad. It is proposed to undertake the works as one contract on a phased basis with an ice rink closure of 4 months or less. The contract will be JCT Design and Build including amendments.

lowercased: building construction work. the project is for the refurbishment and extension of telford ice rink to provide new build soft play and associated catering facilities, shell for future energy centre and shell and core restaurant. the contract will also include for a refurbishment of the existing ice rink to form shell and core restaurant provision and replacement ice rink plant including replacement ice pad. it is proposed to undertake the works as one contract on a phased basis with an ice rink closure of 4 months or less. the contract will be jct design and build including amendments.


text:
around three of the four walls, allowing cats to access the top of the Maker armoire.
As well as lots of details to make it aesthetically pleasing. Including this operable wheel!
The first step to greater cat mobility throughout the room is here!

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around three of the four walls, allowing cats to access the top of the maker armoire.
as well as lots of details to make it aesthetically pleasing. including this operable wheel!
the first step to greater cat mobility throughout the room is here!


text: Copyright © 2005...2019 KOMPASS Ukraine. All rights reserved. Sitemap | MySQL: 9/0.163s.

lowercased: copyright © 2005...2019 kompass ukraine. all rights reserved. sitemap | mysql: 9/0.163s.


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Heel measures approximately 2 inches" ⛳CONFORTABLE: Padded Memor..
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Heel measures approximately 2 inches" ⛳CONFORTABLE: Padded Memory Foam Ins..
La Melodie Girls' Hold & Stretch ballet dancing Tight, 86% Nylon + 14% Spandex, high elastic seamles..

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heel measures approximately 2 inches" ⛳confortable: padded memor..
la melodie girl footed ballet tights ultra soft dance tight for girls/toddlers/kids/boys..
☘opaque tights with no-roll waistband; 81% nylon,19% spandex. ☘ultra..
la melodie girls' pro footed ballet tights, dance tight for toddlers/kids/boys (1 pair)..
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nylon/spandex covered elastic torso band and features a memorystretch front l..
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material: polyester, spandex, lace sleeveless, delicate lace back with wide c..
happy cherry kids latin dress,girls sleeveless rumba samba costume dance dress..
capezio jr. footlight character shoe..
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heel measures approximately 2 inches" ⛳confortable: padded memory foam ins..
la melodie girls' hold & stretch ballet dancing tight, 86% nylon + 14% spandex, high elastic seamles..


text:
Thanks to a 2008 post at Garvey Cey Russell Lopes for pairing these double-play shots. Field and sight lines match so closely, it's safe to say they're from the same White Sox-Royals game.
Chris Stufflestreet (RIP) tried to sort out which game we're seeing in two 1973 Topps Photography posts: Double Play Starter? and Mystery Solved! The artificial turf and mix of uniform numbers led to general agreement on a spring training game at Terry Field, Kansas City's home from 1969-87. But when's the game from, 1973, 1972, or earlier?
There's a business reason so much 1973 Topps photography looks dodgy. MLB owners locked the players union out of team facilities for most of February, closing off a large window for spring training photo shoots. 1973's bargaining agreement (signed Feb 25) established tenets still in place today--salary arbitration, "5 and 10," contract tender deadlines--but the month-long delay left teams less than two weeks to prep players for games. The slim pickings for new photos likely pushed Topps into lower-quality archive shots and lots of player airbrushing.
Thanks to an eBay listing for Chicago's 1973 Spring Training schedule and Google news search, we can check these game situations for 1973, as KC hosted Chicago twice that spring in Ft. Myers.
March 11: Royals win 4-3 in 10 innings (news story and box score), but Mike Andrews didn't play.
March 20: KC prevails 4-3 in late-innings again, this time with Andrews in the lineup...but not at 2B (news story and box score).
How about 1972 spring training, when White Sox #5 was Tony Muser and KC #33 was Bob Oliver? I couldn't find a spring schedule on eBay this time, but located two Ft. Myers match-ups in the news archive.
March 12: Chicago beats KC 5-1 (news story and box score). Andrews plays 2B and homers, Patek plays SS, first basemen Muser and Oliver both reach base, and each team turns a double play.
March 16: White Sox win 9-5 (news story, back one page from link), but Andrews doesn't play.
Given available info, I conclude 1973 Topps #42 and #334 show double plays from Chicago's 5-1 win over KC on March 12, 1972, as Freddie Patek and Mike Andrews force runners Tony Muser and Bob Oliver at second. Batters were likely White Sox catcher Ed Herrmann and Royals 2B Cookie Rojas.
UPDATE: Also read Bruce Markusen's dive into Mike Andrews' roller-coaster baseball story. Great stuff.
oh man - spring training sleuthing. that is awesome.
Thanks! Going through Google News felt old school, like working with rolls of library microfiche.

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thanks to a 2008 post at garvey cey russell lopes for pairing these double-play shots. field and sight lines match so closely, it's safe to say they're from the same white sox-royals game.
chris stufflestreet (rip) tried to sort out which game we're seeing in two 1973 topps photography posts: double play starter? and mystery solved! the artificial turf and mix of uniform numbers led to general agreement on a spring training game at terry field, kansas city's home from 1969-87. but when's the game from, 1973, 1972, or earlier?
there's a business reason so much 1973 topps photography looks dodgy. mlb owners locked the players union out of team facilities for most of february, closing off a large window for spring training photo shoots. 1973's bargaining agreement (signed feb 25) established tenets still in place today--salary arbitration, "5 and 10," contract tender deadlines--but the month-long delay left teams less than two weeks to prep players for games. the slim pickings for new photos likely pushed topps into lower-quality archive shots and lots of player airbrushing.
thanks to an ebay listing for chicago's 1973 spring training schedule and google news search, we can check these game situations for 1973, as kc hosted chicago twice that spring in ft. myers.
march 11: royals win 4-3 in 10 innings (news story and box score), but mike andrews didn't play.
march 20: kc prevails 4-3 in late-innings again, this time with andrews in the lineup...but not at 2b (news story and box score).
how about 1972 spring training, when white sox #5 was tony muser and kc #33 was bob oliver? i couldn't find a spring schedule on ebay this time, but located two ft. myers match-ups in the news archive.
march 12: chicago beats kc 5-1 (news story and box score). andrews plays 2b and homers, patek plays ss, first basemen muser and oliver both reach base, and each team turns a double play.
march 16: white sox win 9-5 (news story, back one page from link), but andrews doesn't play.
given available info, i conclude 1973 topps #42 and #334 show double plays from chicago's 5-1 win over kc on march 12, 1972, as freddie patek and mike andrews force runners tony muser and bob oliver at second. batters were likely white sox catcher ed herrmann and royals 2b cookie rojas.
update: also read bruce markusen's dive into mike andrews' roller-coaster baseball story. great stuff.
oh man - spring training sleuthing. that is awesome.
thanks! going through google news felt old school, like working with rolls of library microfiche.


text:
Valley Grain CO Trucking is a Trucking business in Beardsley, MN.
Valley Grain CO Trucking, a stable growing company under Trucking, is being run by Robert Haukos Full Name Report. Since , their sales have been favorable and consistent at $100.000 to $499.999. Company location coordinates are: 45.55644179999999,-96.7.
It can be reached by dialing (320) 265-0190 Full Phone Report, and you could get in touch with their sales team in 606 Garfield St S, Beardsley, Minnesota MN 56211. Visit their website at: to know more about the company’s history, products and services, as well as frequently asked questions and privacy policies.
You can learn more by checking their classification codes: SIC – and NAIC – . Call the hotline stated above for faster transaction and queries. Valley Grain CO Trucking would like to hear from you.

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valley grain co trucking is a trucking business in beardsley, mn.
valley grain co trucking, a stable growing company under trucking, is being run by robert haukos full name report. since , their sales have been favorable and consistent at $100.000 to $499.999. company location coordinates are: 45.55644179999999,-96.7.
it can be reached by dialing (320) 265-0190 full phone report, and you could get in touch with their sales team in 606 garfield st s, beardsley, minnesota mn 56211. visit their website at: to know more about the company’s history, products and services, as well as frequently asked questions and privacy policies.
you can learn more by checking their classification codes: sic – and naic – . call the hotline stated above for faster transaction and queries. valley grain co trucking would like to hear from you.


text:
Are you having difficulty using the Fashion Effect Store promo code?
First of all, visit Fashion Effect Store. Select your desired product/s and buy them. Add the product to the shopping cart.
Check out promos & deals at Fashion Effect Store today! At Fashion Effect Store, it is currently at its best price ever.
Use Fashion Effect Store coupons before it expires, so act as soon as possible Best sellers will be the first to go.
Great bargains at Fashion Effect Store, come check it out! groundbreaking bargain for only a limited period. Best sellers will be the first to go.
Spend less on select items when you use Fashion Effect Store coupons. it is a great time to buy. Guaranteed to make your heart beat with these deals.
Treat yourself to huge savings with Fashion Effect Store coupons! Guaranteed to make your heart beat with these deals.
Submit a new coupon for Fashion Effect Store and help others save!
Have you got any working Fashion Effect Store code? Please submit the code using the form and help others like you.

lowercased:
are you having difficulty using the fashion effect store promo code?
first of all, visit fashion effect store. select your desired product/s and buy them. add the product to the shopping cart.
check out promos & deals at fashion effect store today! at fashion effect store, it is currently at its best price ever.
use fashion effect store coupons before it expires, so act as soon as possible best sellers will be the first to go.
great bargains at fashion effect store, come check it out! groundbreaking bargain for only a limited period. best sellers will be the first to go.
spend less on select items when you use fashion effect store coupons. it is a great time to buy. guaranteed to make your heart beat with these deals.
treat yourself to huge savings with fashion effect store coupons! guaranteed to make your heart beat with these deals.
submit a new coupon for fashion effect store and help others save!
have you got any working fashion effect store code? please submit the code using the form and help others like you.


text:
How To Stop Receiving Annoying Text Message On Etisalat. guess you have been receiving annoying text message from etisalat network. most of them are either from lotto, random subscription message, quote, bible passage etc. at times you may even wonder how come you have been subscribed to any of the packages because you keep getting series of deduction message from your account balance. even when you tried to call customer care service, they will tell you that you are not subscribed to any of their packages or probably tell you they will deactivate it as soon as you hung up the call. well, if you are in this shoe and you wish to stop receiving annoying text message from etisalat, then this post will guide you through on how to get that done.
Text STOP to 224 .
You will receive a message saying your request will be Answered in the next 7 days.
Just relax and wait within 7 days. You will be deactivated from receiving annoying text message from your etisalat network.
No Responses to "How To Stop Receiving Annoying Text Message On Etisalat"

lowercased:
how to stop receiving annoying text message on etisalat. guess you have been receiving annoying text message from etisalat network. most of them are either from lotto, random subscription message, quote, bible passage etc. at times you may even wonder how come you have been subscribed to any of the packages because you keep getting series of deduction message from your account balance. even when you tried to call customer care service, they will tell you that you are not subscribed to any of their packages or probably tell you they will deactivate it as soon as you hung up the call. well, if you are in this shoe and you wish to stop receiving annoying text message from etisalat, then this post will guide you through on how to get that done.
text stop to 224 .
you will receive a message saying your request will be answered in the next 7 days.
just relax and wait within 7 days. you will be deactivated from receiving annoying text message from your etisalat network.
no responses to "how to stop receiving annoying text message on etisalat"


text:
In the replies, you can see an old comment by Urre reading, "But it's sadly discontinued at the moment due to unforeseen bugs. I announced it too early."
His thread is about the trials and tribulations he and Lord Havoc of [url=http://icculus.org/twilight/darkplaces/]DarkPlaces[/url] went through to get to the place their at now. The physics are mind blowing but Urre's delivery is smooth.
It's a long read but really inspiring.
pavlvs[/url] made [url=http://pavlvs.nexuizninjaz.com/video/Octobre.avi]an amazing video for October (Octobre)[/url].
Speaking of ninja videos, toolbox put together the montage below. He composed the music as well!
I've been doing some work on the Nexuiz Ninjaz SVN servers, you should see them improving more and more, less and less bugs as I get comfortable admining all of them. If you have any questions, comments or concerns, feel free to post them in the [url=http://www.nexuizninjaz.com/forum/showthread.php?tid=534]Nexuiz Ninjaz SVN servers thread[/url].
[url=http://www.nexuizninjaz.com/forum/member.php?action=profile&uid=104]mikeeusa[/url] did a revision of his [url=http://maps.nexuizninjaz.com/slimepipectf6.pk3]giant slimepipectf6 map[/url], [url=http://maps.nexuizninjaz.com/slimepipesmallctf6.pk3]slimepipesmallctf6[/url] which is a bit more manageable. Out of all his maps, this is surely the most "Nexuiz" of them all. Screenshots below.

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in the replies, you can see an old comment by urre reading, "but it's sadly discontinued at the moment due to unforeseen bugs. i announced it too early."
his thread is about the trials and tribulations he and lord havoc of [url=http://icculus.org/twilight/darkplaces/]darkplaces[/url] went through to get to the place their at now. the physics are mind blowing but urre's delivery is smooth.
it's a long read but really inspiring.
pavlvs[/url] made [url=http://pavlvs.nexuizninjaz.com/video/octobre.avi]an amazing video for october (octobre)[/url].
speaking of ninja videos, toolbox put together the montage below. he composed the music as well!
i've been doing some work on the nexuiz ninjaz svn servers, you should see them improving more and more, less and less bugs as i get comfortable admining all of them. if you have any questions, comments or concerns, feel free to post them in the [url=http://www.nexuizninjaz.com/forum/showthread.php?tid=534]nexuiz ninjaz svn servers thread[/url].
[url=http://www.nexuizninjaz.com/forum/member.php?action=profile&uid=104]mikeeusa[/url] did a revision of his [url=http://maps.nexuizninjaz.com/slimepipectf6.pk3]giant slimepipectf6 map[/url], [url=http://maps.nexuizninjaz.com/slimepipesmallctf6.pk3]slimepipesmallctf6[/url] which is a bit more manageable. out of all his maps, this is surely the most "nexuiz" of them all. screenshots below.


text:
What is the faculty's mission?
How do I find out more about PWC?
Who succeeds in this major?
What skills will I learn as a PWC major?
What courses will I take as a PWC major?
What work will I do in these courses?
What minors correlate with the PWC major?
What writing opportunities exist outside the classroom?
What careers do PWC majors pursue?
What job titles do PWC graduates obtain?
Where do I look for job opportunities?
Do PWC majors have internships?
Dr. Amanda Bemer is an Associate Professor of English and the Director of the Professional Writing and Communication major at SMSU.The Faculty Directory provides more information on her background.
Students in our department will meet and work with professors from varying departments such as English, Fine Arts, and Communications. Also, students will meet writers and professionals in the field and have the opportunity to hear visiting writers and scholars from the Upper Midwest and around the country.
For further information, call the SMSU English Department at 507-537-7155, or Dr. Amanda Bemer at 507-537-7159. She may also be reached by e-mail at Amanda.Bemer@smsu.edu.
Students with strengths in writing and communicating in a variety of texts do particularly well in this major.
Beside writing coursework, assignments and projects students complete involve a range of technology and mediums to complete work. For example, Adobe Illustrator to create a graphic logo, WordPress to launch a blog, and Microsoft Word redlining feature to copyedit a document. In the area of communication, students will develop, demonstrate, and evaluate their presentations as well as their peers.
To see work completed by PWC majors, visit the Examples of Professional Writing page.
PWC majors take a variety of writing, communication, and visual art courses to fulfill the major's requirements. The SMSU English Department Course Catalog and the Required Course Descriptions page specifies the required courses and their descriptions.
Many courses include individual and group writing assignments, projects, and presentations. The Examples of Professional Writing page gives specific student examples from required PWC courses.
Students can add any minor, and PWC graduates have minors from a variety of disciplines and departments. Some of the common minors for PWC students include Literature, Marketing, Public Relations, Communication Studies, Graphic Design, and Advertising Design Communication.
Students have the opportunity to be involved in several student publications including The Spur, Perceptions Student Literary Journal, and Yellow Medicine Review. Students may also work as tutors in the SMSU Writing Center.
PWC graduates pursue career opportunities in a variety of fields including: journalism, periodical and book publishing, software publishing, advertising and marketing, computer systems design, instructional design, social media writing, corporate communications, corporate training, government agencies, and other not-for-profit agencies. To learn more about the pay scale for these types of jobs, visit the College Salary Report webpage.
A PWC graduate may become a/an: Journalist, Technical Writer, Technical Editor, Freelance Writer, Copy Editor, Communications Manager, Social Media Writer, Writer/Analyst, Translation Coordinator, Medical Writer, Publisher, Information Designer/Developer, Public Relations Specialist, Reviewer, Document Designer, Corporate Trainer, Magazine Editor, Newspaper Editor, Grant Writer, or Non-Profit Organization Writer.
Many SMSU students use Mustang Jobs to find job opportunities. Look at the On-Campus, Internship, and Career Opportunities page for more job opportunity options.
Yes, many PWC majors complete internships during the summer or the academic year. Students can enroll to receive school credit for their internships. More information is given in the Internship Guidelines. The On-Campus, Internship, and Career Opportunities page has resources to assist in finding an internship.
A PWC graduate can be employed by a variety of different companies, including a local newspaper, a software company, the manufacturing industry, the medical and healthcare industry, a non-profit organization, a local, state or federal government agency, an advertising company, a college/university, a law firm, a publishing company, a magazine, a scholarly journal, or a radio station.

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what is the faculty's mission?
how do i find out more about pwc?
who succeeds in this major?
what skills will i learn as a pwc major?
what courses will i take as a pwc major?
what work will i do in these courses?
what minors correlate with the pwc major?
what writing opportunities exist outside the classroom?
what careers do pwc majors pursue?
what job titles do pwc graduates obtain?
where do i look for job opportunities?
do pwc majors have internships?
dr. amanda bemer is an associate professor of english and the director of the professional writing and communication major at smsu.the faculty directory provides more information on her background.
students in our department will meet and work with professors from varying departments such as english, fine arts, and communications. also, students will meet writers and professionals in the field and have the opportunity to hear visiting writers and scholars from the upper midwest and around the country.
for further information, call the smsu english department at 507-537-7155, or dr. amanda bemer at 507-537-7159. she may also be reached by e-mail at amanda.bemer@smsu.edu.
students with strengths in writing and communicating in a variety of texts do particularly well in this major.
beside writing coursework, assignments and projects students complete involve a range of technology and mediums to complete work. for example, adobe illustrator to create a graphic logo, wordpress to launch a blog, and microsoft word redlining feature to copyedit a document. in the area of communication, students will develop, demonstrate, and evaluate their presentations as well as their peers.
to see work completed by pwc majors, visit the examples of professional writing page.
pwc majors take a variety of writing, communication, and visual art courses to fulfill the major's requirements. the smsu english department course catalog and the required course descriptions page specifies the required courses and their descriptions.
many courses include individual and group writing assignments, projects, and presentations. the examples of professional writing page gives specific student examples from required pwc courses.
students can add any minor, and pwc graduates have minors from a variety of disciplines and departments. some of the common minors for pwc students include literature, marketing, public relations, communication studies, graphic design, and advertising design communication.
students have the opportunity to be involved in several student publications including the spur, perceptions student literary journal, and yellow medicine review. students may also work as tutors in the smsu writing center.
pwc graduates pursue career opportunities in a variety of fields including: journalism, periodical and book publishing, software publishing, advertising and marketing, computer systems design, instructional design, social media writing, corporate communications, corporate training, government agencies, and other not-for-profit agencies. to learn more about the pay scale for these types of jobs, visit the college salary report webpage.
a pwc graduate may become a/an: journalist, technical writer, technical editor, freelance writer, copy editor, communications manager, social media writer, writer/analyst, translation coordinator, medical writer, publisher, information designer/developer, public relations specialist, reviewer, document designer, corporate trainer, magazine editor, newspaper editor, grant writer, or non-profit organization writer.
many smsu students use mustang jobs to find job opportunities. look at the on-campus, internship, and career opportunities page for more job opportunity options.
yes, many pwc majors complete internships during the summer or the academic year. students can enroll to receive school credit for their internships. more information is given in the internship guidelines. the on-campus, internship, and career opportunities page has resources to assist in finding an internship.
a pwc graduate can be employed by a variety of different companies, including a local newspaper, a software company, the manufacturing industry, the medical and healthcare industry, a non-profit organization, a local, state or federal government agency, an advertising company, a college/university, a law firm, a publishing company, a magazine, a scholarly journal, or a radio station.


text:
Three rented mega-sized cold-storage cells have earned their permanent place right next to the 6,000 m2 production halls of a Coolworld customer. Each cell has a cubic capacity of 100 cubic metres. The company has decided to rent for a longer period, which is quite uncommon, but this enables this customer to wait for the right moment to make new investments in permanent cooling areas.
This is a meat-processing company with 80 employees. Through continuous innovation, this company manages to introduce new, ready to eat convenience products into the market time and again. The company was established in 1973 and now supplies to bulk users, caterers and gastronomic sectors throughout Europe. And the company now also successfully serves the retail world, enabling this Coolworld customer to grow substantially.
The three mega-sized cold-storage cells enable the meat-processing company to perfectly handle any variations in the stock requirement for different end products. Of course, these installations comply with the most stringent HACCP standards, giving them the same quality level as permanent cold stores.

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three rented mega-sized cold-storage cells have earned their permanent place right next to the 6,000 m2 production halls of a coolworld customer. each cell has a cubic capacity of 100 cubic metres. the company has decided to rent for a longer period, which is quite uncommon, but this enables this customer to wait for the right moment to make new investments in permanent cooling areas.
this is a meat-processing company with 80 employees. through continuous innovation, this company manages to introduce new, ready to eat convenience products into the market time and again. the company was established in 1973 and now supplies to bulk users, caterers and gastronomic sectors throughout europe. and the company now also successfully serves the retail world, enabling this coolworld customer to grow substantially.
the three mega-sized cold-storage cells enable the meat-processing company to perfectly handle any variations in the stock requirement for different end products. of course, these installations comply with the most stringent haccp standards, giving them the same quality level as permanent cold stores.


text: In order to facilitate the process of compliance with Article 51 of the Council of Europe Convention on the Prevention and Combating Violence against Women and Domestic Violence (Istanbul Convention) on risk assessment and risk management, MIGS carried out a study on “Risk Assessments of Domestic Violence”, with funding by the National Machinery of Women’s Rights of the Ministry of Justice and Public Order. The study examines and analyses existing risk assessments mechanisms on domestic abuse both in Europe and internationally that have shown a high rate of success, as well as the legislative and other measures required to ensure effective implementation of such mechanisms. The report also provides specific recommendations on the implementation of a risk assessment and risk management mechanism in Cyprus, taking into account the legislative framework and the specific country context.

lowercased: in order to facilitate the process of compliance with article 51 of the council of europe convention on the prevention and combating violence against women and domestic violence (istanbul convention) on risk assessment and risk management, migs carried out a study on “risk assessments of domestic violence”, with funding by the national machinery of women’s rights of the ministry of justice and public order. the study examines and analyses existing risk assessments mechanisms on domestic abuse both in europe and internationally that have shown a high rate of success, as well as the legislative and other measures required to ensure effective implementation of such mechanisms. the report also provides specific recommendations on the implementation of a risk assessment and risk management mechanism in cyprus, taking into account the legislative framework and the specific country context.