Strain and medium used
Lactobacillus plantarum was purchased from KCCM (KCCM 11322, Korea Federation of Culture Collection, Seoul, Korea). MRS broth (Difco, Detroit, MI, USA) was used as the lactic acid bacteria culture medium and was anaerobically cultured at 37 °C for 48 h.
Production of Lactobacillus plantarum protein enzyme hydrolysates
The protease activity of L. plantarum, and Alcalase (Novozyme Co., Bagsvaerd, Denmark), bromelain (Novozyme), and Protamex (Novozyme), which are different types of protease, were reacted with the cultured L. plantarum by adjusting the active optimal temperature and pH (Alcalase, 50℃, pH 7; bromelain, 45℃, pH 7; Protamax, 60℃, pH 7). They were added to make a 1% protein concentration and hydrolyzed for 2 h (LP-A: L. plantarum hydrolysates by Alcalase; LP-B: L. plantarum hydrolysates by bromelain; LP-P: L. plantarum hydrolysates by Protamex). The protein concentration was determined by drawing a standard curve using the Bradford method with bovine serum albumin (BSA) at R2 = 0.9951 as a standard sample. These L. plantarum protein enzyme hydrolysates were freeze-dried and used as samples.
Cell culture
In this study, MG-63 osteoblast-like cells were used as controls for hDPC. The human osteoblast-like MG-63 cells used in this study were purchased from Korea Cell Line Bank (KCLB, Seoul, Korea), and the human dermal papilla cells (hDPC) were purchased from Cell Engineering for Origin (CEFO Co., Seoul, Korea). The cultured cells were subcultured in our laboratory. Ten percent FBS (Gibco, Grand Island, NY, USA) and 1% antibacterial-antifungal solution were added to the DMEM (Gibco, Grand Island, NY, USA). During the culture, 5% CO2 was continuously supplied while maintaining the temperature at 37 °C.
Cell toxicity and cell growth rate after treatment of Lactobacillus plantarum protein enzyme hydrolysates
A CCK assay was used to verify cytotoxicity. MG-63 cells and hDPC were dispensed at a concentration of 6 × 103 cells/well on a 96-well plate. After 24 h, the L. plantarum protein enzyme hydrolysates were added at concentrations of 0, 31.25, 62.5, 125, 250, and 500 μg/mL and cultured for another 24 h. After the culture, a CCK reagent (Dongin LS, Seoul, Korea) was added, and the hydrolysates were cultured for 2 h at 37 °C in a 5% CO2 incubator and measured at 450 nm using an ELISA reader. Each treatment group underwent the experimental treatment three times, and the cell proliferation effect on the hydrolysates was represented as a percentage.
To determine the cell growth rate, MG-63 cells were plated on a 100-mm dish at a cell number of 1 × 106 cells/mL, and hDPC was plated on a 100-mm dish at a cell number of 1 × 105 cells/mL. The L. plantarum protein hydrolysates were reacted with tryphan blue solution at 1:1, and the unstained live cells were counted every other day for 10 days and observed at × 20 magnification using an inverted microscope.
Measurement of growth factor
The vascular endothelial growth factor (VEGF) and insulin-like growth factor 1(IGF-1) concentrations were measured using the ELISA Kit (R & D Systems, Minneapolis, MN, USA). Fifty microliters of assay diluent was added to 200 μL of standard and culture samples, reacted at 37 °C for 2 h, and then washed three times. After that, 200 μL of conjugate was added and reacted at 37 °C for 2 h again. After washing three times, 200 μL of substrate solution was added and reacted at 37 °C for 20 min. Then, 50 μL of stop solution was added to stop the reaction. The absorbance was measured three times, and the absorbance value was calculated by subtracting the.
Experimental animals
Five-week-old C57BL/6 male mice were purchased from Samtako Bio Korea (O-San, Korea). The animals were allowed to eat food and drink water freely at a temperature of 24 °C ± 0.5 °C, a humidity of 55–65%, and 12 h of light cycle. They were used for the experiment after a 7-day period of adaptation.
Application of the sample
To apply the resting period during the circulation period of the hair, the back hair was first removed using an electric razor, and then the remaining hair was removed using a depilatory (Niclean, Ildong Pharmaceutical). There was no damage to the skin during hair removal, and continuous observation showed no inflammation. For each group, 100 μL of each extract and 5% minoxidil (Hyundai Pharm. Co., Korea) were applied to the backs whose hair had been removed twice a day (at 10:00 a.m. and 6:00 p.m.) for 14 days. Distilled water was used as a control.
Visual observation of hair growth
To verify the hair growth with the naked eye at 0, 4, 7, 10, and 14 days after the start of the experiment, Zoletil (Virbac, Paris, France) and Rumpun (Bayel Korea Co. Seoul, Korea) were mixed at 9:1, and the mice were anesthetized (10/10 g) by intraperitoneal injection and then photographed. The hair growth of each group was rated by visual observation as 0–9% (0 points), 10–19% (1 point), 20–29% (2 points), 30–39 (3 points), 40–49% (4 points), 50–59% (5 points), 60–69% (6 points), 70–79% (7 points), 80–89% (8 points), and 90–100% (9 points). On the 14th day of the experiment, the mice were killed by cervical dislocation, the dorsal tissues were removed, and the inside of the extracted skin was visually observed.
Statistical processing
All experimental results are expressed as means and standard deviations. A one-way ANOVA was conducted for the resulting data using SPSS 20 (SPSS Inc., Chicago, IL, USA), and the data were tested with Duncan’s multiple test at p < 0.05.
