보문(Article)
Fermentation Microbiology (FM) | Fermentation Technology
Microbiol. Biotechnol. Lett. 2022; 50(4): 533-547
https://doi.org/10.48022/mbl.2206.06011
Received: June 29, 2022; Revised: November 26, 2022; Accepted: November 29, 2022
Hair Growth Effect of TS-SCLF from Schisandra chinensis Extract Fermented with Lactobacillus plantarum
Young Min Woo1†, Jae Yong Seo2,3†, Soo-ya Kim1†, Ji Hyun Cha1, Hyun Dae Cho2, Young Kwon Cha2, Ju Tae Jeong2, Sung Min Park4, Hwa Sun Ryu4, Jae Mun Kim4, Moon Hoy Kim4, Hee-Taek Kim5, Yong-Min Kim6, Kwang Sik Joo7, Sun Mi Lee7, JungNo Lee4*, and Andre Kim1,8*
1Department of Pharmaceutical Engineering, College of Medical and Life Science, Silla University, Busan 46958, Republic of Korea
2Creative Innovation Research Center, Cosmecca Korea, Co., Ltd., Gyeonggi-do 13488, Republic of Korea
3Department of Cosmetics Engineering, College of Engineering, Konkuk University, Seoul 05029, Republic of Korea
4Bio Convergence R&D Center, CoseedBioPharm, Co., Ltd., Chungbuk 28161, Republic of Korea
5Department of Korean Medical Ophthalmology & Otolaryngology & Dermatology, College of Korean Medicine, Semyung University, Chungbuk 27136, Republic of Korea
6Department of Cosmetic Sciences and Beauty Biotechnology, Semyung University, Chungbuk 27136, Republic of Korea
7R&D Center, TS Trillion, Co., Ltd., Seoul 07206, Republic of Korea
8Hankook Liposome Co., Ltd., Busan 46702, Republic of Korea
Correspondence to :
JungNo Lee, jnlee2000@hanmail.net
Andre Kim, adrk@silla.ac.kr
†These authors contributed equally to this work.
Abstract
This study investigated the hair growth effect of Schisandra chinensis extract (TS-SC) and TS-SC fermented by Lactobacillus plantarum (TS-SCLF) on human dermal papilla cells (hDPCs). The production of vascular endothelial growth factor (VEGF), insulin-like growth factor 1 (IGF-1), keratinocyte growth factor/fibroblast growth factor 7 (KGF/FGF-7) and hepatocyte growth factor (HGF), transforming growth factor beta 1 (TGF-β1) were examined. The secretion rates of VEGF and KGF/FGF-7 were high in TS-SC, and the secretion rates of IGF-1 and HGF were high in TS-SCLF. TGF-β1 was inhibited in a concentration-dependent manner in all samples. Gene expression of VEGF, IGF-1, KGF, HGF and alkaline phosphatase, relevant to hair growth, were examined. The data revealed that TS-SC and TS-SCLF successfully promoted hair growth in hDPCs. The IGF-1 gene was expressed in a dose-dependent manner in TS-SCLF. These results indicate that TS-SC and TS-SCLF fermented extract effectively promoted hair growth and gene expression relevant to hair growth in hDPCs. Used in clinical trials the test substance ‘CMK-LPF01’ showed a statistically significant increase in the number of hairs at 8 weeks, 16 weeks, and 24 weeks compared to before product use, and a change in hair growth, a secondary efficacy evaluation variable. Through additional research in the future, it is expected that “CMK-LPF01” can be developed as a functional material that can help alleviate symptoms of hair loss.
Keywords: Schisandra chinensis, Lactobacillus plantarum, VEGF, IGF-1
Graphical Abstract
Introduction
Hair not only protects the human skull but also acts as an important factor in terms of perceived beauty. Due to the improvement in our living standards, awareness of the appearance of hair is increasing, and the desire for cleanliness and beauty is changing [1−3]. Damage to hair health can result from artificial factors, such as chemical procedures and incorrect management; environmental factors, such as UV rays, humidity, seasonal changes, and air pollution; and biological factors, such as personal health conditions, stress, and aging [4]. The hair cycle can be divided into the anagen stage (hair growth), the catagen stage (a temporary halt in hair growth), the telogen stage (removal of old hair), and the new anagen stage (generation of new hair) [5−7]. As hair loss progresses, the hair becomes thinner, the anagen stage becomes shorter in the hair cycle, and the telogen stage becomes longer [8−10].
Treatments for thinning hair/hair loss to date include drug administration or application, phototherapy, immunotherapy, hair transplantation, and the like. Since the approval of minoxidil liniment for the treatment of androgenetic alopecia, it has been used worldwide as the main treatment for androgenetic alopecia, together with oral finasteride [11−14]. Although the exact mechanism of hair growth has not been elucidated, it is thought that increased nutrient supply through vasodilation and the effect of opening potassium channels together induce hair growth [15]. However, interest in natural products is increasing because drug therapy requires long-term use and is accompanied by several side effects, such as cardiovascular disorders, skin irritation, decreased sexual function, teratogenicity, itching, and dry dermatitis [16, 17]. Many studies have been conducted on the hair growth effect using black soybean extract [18], hydrolysate of lactic acid bacteria [19, 20], and
Materials and Methods
Preparation of TS-SC and TS-SCLF extracts
TS-SC and TS-SCLF were provided by CoseedBioPharm Co. Ltd. (Korea). According to the information provided,
TS-SC structure identification
The TS-SC extract was subjected to silica column chromatography and Sephadex LH-20 column chromatography to separate the compound, and the structure of the compound was confirmed using 1H and 13C NMR (Fig. 1). Compound 1 was obtained as a white-yellow amorphous powder. Through 1H-, 13C-NMR, it was a lignan-based compound having four methoxy groups, two methyl groups, one deoxymethylene group [δ H 5.94 (2H,s), δ C 101], and two single aromatic benzene protons. As expected, it was confirmed as gomisin N through the literature.
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Figure 1.Structures of compounds isolated from S. chinensis fruit.
Compound 1 (gomisin N) [29]: 1H-NMR (700 MHz, CDCl3) δ :
0.74 (3H, d,
13C-NMR (175 MHz, CDCl3) δ : 151.5 (C-1), 140.3 (C-2), 152.0 (C-3), 111.0 (C-4), 134.8 (C-5), 39.3 (C-6), 33.3 (C-7), 40.6 (C-8), 35.9 (C-9), 136.9 (C-10), 103.1 (C-11), 148.4(C-12), 134.2 (C-13), 140.9 (C-14), 121.1 (C-15), 123.8 (C-16), 21.6 (CH3-17), 12.2 (CH3-18), 101.0 (OCH2O), 61.0, 60.4, 59.8, 56.0 (OCH3 × 4)
Changes in gomisin N content before and after fermentation
Gomisin N content of TS-SC extract and TS-SCLF after fermentation was quantitatively analyzed using HPLC (2695 Separations Module, Waters, USA). Optima pak 5 μm 4.5 × 250 mm (Kanto Chemical, Japan) was used for the column, and Waters 2998 PDA Detector was used as the detector. The detection wavelength was detected at 254 nm, 10 μl of the sample was injected, and the column temperature was analyzed at 35℃. Acetonitrile was used for mobile phase A and water was used for mobile phase B. Mobile phase A was 70% and mobile phase B 30%, maintained at a rate of 1.0 ml/min for 25 min, and analysis was carried out.
Cell culture
Human dermal papilla cells (hDPCs) were purchased from CEFO Co., Ltd. (Korea). The cultured cells were sub-cultured in our laboratory. Supplement (CB-HDPGM) and antibacterial-antifungal solutions were added to the basal medium (CB-HDP-GM). All reagents were purchased from CEFO Co., Ltd. (Korea). During the culture, 5% CO2 was continuously supplied while maintaining the temperature at 37℃.
Cell cytotoxicity
An MTT assay was used for cell proliferation effect. The hDPCs were cultured in 96-well plates (6 × 103 cells/ well, in 100 μl medium) for 24 h and then treated with TS-SC and TS-SCLF (0−500 μg/ml) for 24 h. MTT reagent (1 μg/ml) was added to the cell preparations, and plates were incubated for an additional 4 h. Dimethyl sulfoxide (200 μl/well) was added to dissolve formazan crystals. Absorbance was measured at 540 nm with a microplate reader (Thermo Fisher Scientific, USA). Each treatment group underwent the experimental treatment three times, and the cell proliferation effect on the hydrolysates was represented as a percentage.
Measurement of growth factor
The hDPCs were plated at 1 × 106 cells/ml in a 60-mm dish and incubated overnight. The cells were treated with minoxidil (30 μM), TS-SC and TS-SCLF (0.1, 1, and 10 μg/ml) and incubated for an additional 48 h. Secretion of growth factors into extracellular medium was determined by an ELISA Kit (R&D Systems, USA). The VEGF, insulin-like growth factor 1 (IGF-1), transforming growth factor beta 1 (TGF-β1), keratinocyte growth factor/fibroblast growth factor 7 (KGF/FGF-7), and hepatocyte growth factor (HGF) concentrations were measured. Next, 50 μl of assay diluent were added to 200 μl of standard and culture samples, reacted at room temperature (RT) for 2 h, and then washed 3 times. After that, 200 μl of conjugate was added and reacted at RT for 2 h again. After washing 3 times, 200 μl of substrate solution was added and reacted at RT for 20 min. Then, 50 μl of stop solution was added to stop the reaction. The absorbance was measured three times per sample.
RT-PCR
The hDPCs were plated at 1 × 106 cells/ml in a 60-mm dish and incubated overnight. The cells were treated with minoxidil (30 μM), TS-SC and TS-SCLF (0.1, 1, and 10 μg/ml) and incubated for an additional 48 h. Total RNA from the hDPCs was extracted using TRizol™ reagent (Invitrogen, USA), in accordance with the manufacturer’s instructions. The RT-PCR reaction was performed with 1 μg of total RNA, 3 μl of forward primer, 3 μl of reverse primer, and 20 μl of reaction mixture, which was provided by AccuPower RT/PCR PreMix (Bioneer, Korea). Then, PCR was performed in a total mixture volume of 50 μl for 35 cycles at 94℃ for 30 sec, 56℃ for 30 sec, and 72℃ for 1 min. In addition, amplified cDNA products were separated on 2% agarose gel by electrophoresis. The primer sequences of the amplified genes are presented in Table 1. Each sample was analyzed in triplicate, and target genes were normalized to the reference housekeeping gene—glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Thereafter, fold differences were calculated for each treatment group using normalized CT values for the control. To quantitatively determine the RT-PCR results, the densitometry data for band intensities in different sets of experiments were generated by analyzing the gel images on image processor software (NEOimage, ver 2.4). The density values of the target genes were normalized to an endogenous reference, GAPDH.
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Table 1 . Primers and expected size for RT-PCR analysis.
Gene Forward primer (from 5’ to 3’) Reverse primer (from 3’ to 5’) PCR product size (bp) VEGF CCT GGT GGA CAT CTT CCA GGA GTA CC GAA GCT CAT CTC TCC TAT GTG CTG GC 196 IGF-1 TCA ACA AGC CCA CAG GGT AT ACT CGT GCA GAG CAA AGG AT 307 KGF GAC ATG GAT CCT GCC AAC TT AAT TCC AAC TGC CAC TGT CC 304 HGF CGA GGC CAT GGT GCT ATA CT ACA CCA GGG TGA TTC AGA CC 296 ALP CAA ACC GAG ATA CAA GCA CTC CC CGA AGA GAC CCA ATA GGT AGT CCA C 196 GAPDH ACC ACA GAC CAT GCC ATC AC TCC ACC ACC CTG TTG CTG TA 450
Clinical efficacy evaluation
Institutional review board (IRB) review. This clinical trial study was conducted after obtaining approval from the independent bioethics committee of the testing institution for human application (Protocol No. CMK-LPF01).
Selection of testers. A total of 58 male and female patients aged between 19 and 54 were recruited for this clinical trial after gaining approval from the Clinical Trial Review Committee. The subjects satisfied selection criteria for basic and specific androgenetic and female pattern alopecia through the recruitment notice for this clinical trial. The male subjects were diagnosed as 2, 2A or higher by the Norwood–Hamilton classification, or 1 or higher by the Ludwig classification. Diagnosed female subjects were selected. Subjects were targeted who would not use special hair products or hair care and manipulation during the study period and who would maintain the same hair shape and color as at the time of the first visit. These voluntarily consented in writing for the human application study. Patients with hair loss diseases such as alopecia areata, telogen alopecia, and scar alopecia were excluded, to ensure accurate comparative analysis. Other criteria for exclusion included severe acute kidney, heart, or other chronic diseases in the last 6 months that could affect test results, impaired blood sugar levels, uncontrolled hypertension, psychiatric disorders, and infectious skin diseases. Pregnant or lactating women and those who were planning to become pregnant within a month were also excluded, as were patients with severe dermatitis, scalp psoriasis, and scalp infection, those who had undergone surgical treatment for hair loss, such as hair transplantation or scalp reduction, within the last 6 months, and subjects who had taken or were using topical hair growth agents, topical steroids, and wool or hair growth agents in the past 1 month. Those taking oral dutasteride or finasteride, spironolactone, cimetidine, diazoxide, cyclosporine, ketoconazole, etc. were also excluded. Based on the opinion of the person in charge of the human application study, those who lacked nutrition, who were judged to be unable to comply or deemed unsuitable by other doctors, and among the clinical trial volunteers, the relevant subjects were excluded from the study.
Prescription of the test substance. A pale yellow liquid wash off-type shampoo formulation containing TS-SCLF as an active ingredient was prepared and used in the test. Subjects participating in the clinical trial were instructed to use the shampoo once a day for a total of 24 weeks, according to the researchers’ guidance, after they provided written informed consent.
Evaluation by phototricogram. Subjects participating in the clinical trial underwent a phototrichogram using a folliscope at each visit from the second visit onwards so that the number of their hairs could be evaluated. Two dots with a diameter of 1 mm left and right were tattooed at intervals of 1 cm on the hair loss area of the parietal region, and the same area was marked for each measurement. For objective measurement, one investigator measured the same site during the second visit and at 8 weeks, 16 weeks, and 24 weeks.
Subject evaluation. For hair growth change and adverse reaction evaluation, a questionnaire on scalp safety was prepared for the subjects. After the start of the human application test, a questionnaire was compiled to evaluate the overall condition of the hair, including the degree of hair loss, the appearance of the hair (thickness, quality, quantity), and the degree of scalp irritation, and improvement and satisfaction were evaluated step by step.
Researcher evaluation. A questionnaire on hair growth change and adverse reaction evaluation was prepared for the researchers by photographing scalp safety. After the start of the human application test, a questionnaire was compiled to evaluate the overall condition of the hair, including the degree of hair loss and the appearance of the hair (thickness, quality, quantity), and the degree of improvement was evaluated at visits 3, 4, and 5 by stage. Two researchers conducted an individual evaluation, and for objective measurement, photos of the hair loss site taken at visits 3, 4, and 5 were evaluated collectively at the end of the study.
Efficacy evaluation. The Safety group refers to subjects who participated in the human application test, used the product at least once in the test, and confirmed safetyrelated data at least once. The ITT group refers to subjects who were randomly assigned to this human application study and used the product for human application testing at least once, and for whom primary efficacy evaluation data were collected at least once. The PP group consisted of subjects who successfully completed the study according to the human application test protocol among the subjects included in the ITT group.
The ITT and PP groups were targeted for efficacy data analysis, with the ITT group the main analysis group. Safety data were evaluated for the Safety group, and demographic and other underlying characteristics were analyzed for the ITT group. Only in the efficacy data of the ITT group, if a missing value occurred or a subject dropped out before the human application study was completed, last observation carried forward analysis was applied, and the most recent data obtained before the occurrence of a missing value or subject dropout was replaced. Interim analyses were not performed.
Statistics. All results are indicated in terms of means and standard deviations. The data obtained from this study were first analyzed through a one-way analysis of variance using SPSS 25 (SPSS Inc., USA) and then tested using Duncan’s multiple test at
Results
Changes in gomisin N content before and after fermentation
Fig. 2 shows the results of quantitative analysis of gomisin N content in TS-SC extract and TS-SCLF after fermentation using HPLC. Both show a retention time of 28.7 min. Gomisin N at 2008.927 ppm in TS-SC extract and 2552.395 ppm in TS-SCLF were analyzed, indicating that the content of gomisin N also increased during fermentation with
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Figure 2.Response surface plots and contour plots for the effect of extraction variables on gomisin N.
Cell cytotoxicity of the extracts
Cell cytotoxicity was measured by MTT assay (Fig. 3). The TS-SC and TS-SCLF were treated with hDPCs at 0−500 μg/ml and incubated for 24 h at 37℃. The analysis showed more than 94% viability, up to a concentration of 31.3 μg/ml. Therefore, an experiment was conducted using the TS-SCLF extract at a concentration of 25 ppm, which is non-toxic to the shampoo used in clinical trials.
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Figure 3.Cell cytotoxicity was measured by MTT assay.
Human dermal papilla cells (hDPCs) were treated with different dosages of TS-SC (A) and TS-SCLF (B). The values are the means ± SD of three independent experiments, and the cell proliferation effect on the hydrolysates was represented as a percentage (*p < 0.05). Values are the means ± SD of three independent experiments.
Effects of TS-SC and TS-SCLF on growth factors
The results for the production of growth factors in hDPCs are shown in Fig. 4. When hDPCs were treated with 30 μM of minoxidil, a VEGF concentration of 203.63 pg/ml was measured, which was 15.60% higher than that of the control group (Fig. 4A). Treatment with TSSC 0.1 μg/ml exhibited a concentration of 278.06 pg/ml, which was 57.85% higher than that of the control group, which was 36.55% higher than that of the minoxidil group. The TS-SCLF 0.1 μg/ml exhibited a concentration of 243.96 pg/ml, which was 38.49% higher than that of the control group, which was 19.8% higher than that of the minoxidil group. VEGF is known to induce hair growth by participating in vasodilation, and the extract is thought to affect hair growth at certain concentrations [30].
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Figure 4.Stimulation of growth factor secretion by TS-SC and TS-SCLF.
Human dermal papilla cells (hDPCs) were treated with compounds for 48 h. Secreted levels of vascular endothelial growth factor (VEGF) (A), insulin-like growth factor 1 (IGF-1) (B), keratinocyte growth factor/fibroblast growth factor-7(KGF/FGF-7) (C) and hepatocyte growth factor (HGF) (D) were determined in supernatants by ELISA. The control group was an untreated sample and the positive control group (#) was a treated minoxidil 30 μM (*p < 0.05, **p < 0.01, ***p < 0.001). All values are expressed as means ± SD (n = 3).
In the results of IGF-1 (Fig. 4B), the TS-SCLF 0.1 and 1 μg/ml showed concentrations of 1118.69 and 1132.13 pg/ml, which were 10.83% and 12.16% higher than the minoxidil 30 μM treatment group and 30.23% and 31.80% higher than the control group, respectively. IGF-1 plays a role in activating cells in the hair root region, and it is thought that the TS-SCLF has a significant role in the secretion of IGF-1.
KGF and HGF have been reported as peripherally secreted growth factors that promote hair growth [31, 32]. KGF/FGF-7 was 68.70 pg/ml in the control group and 97.52 pg/ml in the minoxidil 30 μM treatment group, which was 41.94% higher in the minoxidil-treated group (Fig. 4C). In the 0.1 μg/ml of the TS-SC treatment group, the concentration was 112.95 pg/ml, which was 64.41% higher than the control group. In the 0.1 μg/ml of the TS-SCLF treatment group, the concentration was 107.77 pg/ml, which was 56.86% higher than the control group. As a result of HGF measurement, when minoxidil was treated with 30 μM, the concentration of 1613.31 pg/ ml was 7.65% higher than that of the control group (Fig. 4D). Treatment with 0.1 μg/ml of TS-SC showed a concentration of 1773.03 pg/ml, and 18.31% higher HGF was secreted compared to the control group. In the 10 μg/ml of the TS-SCLF treatment group, the concentration was 1859.94 pg/ml, which was 24.11% higher than the control group.
Growth factors, such as fibroblast growth factor-1 (FGF-1), -2, -7, and -10 and epidermal growth factor (EGF), also promote cell cycle proliferation, improve hair loss, and promote hair cell regeneration, both in and outside the body [31, 32]. TGF-β1 is known for inducing hair loss by interfering with hair follicle growth and causing entrance into the catagen stage faster than normal [33−35]. The 10 μg/ml of TS-SCLF treatment group showed a concentration of 271.32 pg/ml, which was 43.95% lower than the control group and 27.63% lower than the minoxidil 30 μM group (Fig. 5).
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Figure 5.Inhibition of growth factor secretion by TS-SC and TS-SCLF.
Human dermal papilla cells (hDPCs) were treated with compounds for 48 h. Secreted levels of transforming growth factor-beta 1 (TGF-β1) on Gomisin N of hDPCs. The control group was an untreated sample and the positive control group (#) was a treated minoxidil 30 μM (*p < 0.05, **p < 0.01, ***p < 0.001). All values are expressed as means ± SD (n = 3).
Effects of TS-SC and TS-SCLF on gene expression
The expression rate of secreted growth factor after TSSC and TS-SCLF treatment in hDPCs was measured (Fig. 6). VEGF was 39.22% when treated with 0.1 μg/ml of TS-SC, which showed a 10.52% higher expression rate than the control group (28.70%) and a 5.66% higher expression rate than the minoxidil group (34.36%). IGF-1 was also found to be 30.95% in the TS-SC 10 μg/ml group, which was 8.83% higher than the control group (22.53%). The highest expression was shown in the TSSCLF 10 μg/ml group at 36.98%, which was 14.45% higher than the control group. KGF was 16.79% in the minoxidil 30 μM group, which was 4.81% higher than the control group (11.98%). Finally, the highest expression was shown in the TS-SC 0.1 μg/ml group at 20.27%. For the HGF measurement, all groups showed higher expression rates than the control group. The control group was determined at 61.51%, and the 30 μM minoxidil group was 69.59%. TS-SCLF 10 μg/ml was 74.22%. TSSC 0.1 μg/ml was 79.10%, which was the highest expression. Alkaline phosphatase (ALP) is considered a useful marker when estimating the hair growth-inducing activity of hair follicle mesenchymal cells. The ALP secretion rate was 10.91% in the control group and 20.88% at 30 μM of minoxidil. At 0.1 μg/ml, TS-SC was 21.51%, indicating a 10.60% higher secretion rate than the control. It is known that the activity of ALP is strong in the anagen stage and weak in the catagen stage [36].
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Figure 6.Effects of TS-SC and TS-SCLF on the mRNA level of growth factors in hDPCs by RT-PCR analysis.
Quantification of RTPCR products for growth factors using a NEOimage program. The amount of RT-PCR product for each growth factor was corrected according to the quantity of GAPDH and expressed as a percentage of the control (*p < 0.05). Values are the means ± SD of three independent experiments.
Clinical efficacy evaluation
Characteristics of clinical trial subjects. A total of 56 patients were enrolled in the clinical trial for efficacy evaluation, and among the subjects, 3 each dropped out of the test group and control group, so a total of 56 people, 28 in the final test group and 28 in the control group, participated in the study (Table 2). The average age of the patients participating in the study was 42.32 ± 5.68 (years) in the test group and 43.29 ± 4.74 (years) in the control group, and there was no statistically significant difference between the two groups (age:
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Table 2 . Age and gender distribution of patients.
Test group Control group Total p -valueN=28 N=28 N=56 N (%) N (%) N (%) Age (year) N 28 28 56 0.4933a Mean±SD 42.32 5.68 43.29 4.74 42.80 5.21 Median 42.50 44.00 43.00 Min, Max 27.00 51.00 33.00 49.00 27.00 51.00 Gender N 28 28 56 0.3424b Male 5 (17.86) 8 (28.57) 13 (23.21) Female 23 (82.14) 20 (71.43) 43 (76.79) a: Independent two-sample t-test.
b: Chi-square test.
Functional evaluation
Primary efficacy evaluation. Change in the total number of hairs was the primary efficacy evaluation variable of this human application test. This was the confirmed through phototrichogram after 24 weeks of use compared to before the product was used for the human application test (Baseline), and whether the test product is superior to the control product was to prove it (Fig. 7).
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Figure 7.Change in total hair count confirmed through phototrichogram after 24 weeks compared to baseline.
All values are expressed as means ± SD (n = 28).
Looking at the results of the analysis of the use of products for human application testing in the ITT group, summarized in Table 3, the number of hairs in the test group did not increase significantly compared to the number of hairs in the control group before product use (
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Table 3 . Changes in total hair count confirmed through phototrichogram after 24 weeks compared to baseline.
Visit ITT PP Test group Control group Test group Control group N=28 N=28 N=28 N=28 Hair density Baseline 101.36±23.57 105.75±27.73 101.46±24.44 105.96±27.34 8 weeks 103.11±21.10 104.39±29.55 103.23±21.76 104.50±26.35 16 weeks 104.25±20.89 103.86±27.10 104.46±21.57 103.96±26.81 24 weeks 106.89±20.02 101.43±25.91 107.27±20.64 101.58±25.50 Improvement rate 8 weeks 1.73 -1.29 1.74 -1.38 16 weeks 2.85 -1.79 2.96 -1.89 24 weeks 5.97 -4.09 5.73 -4.13 Within-group comparative significance 8 weeks <0.0001d 0.0074d <0.0001d 0.0044c 16 weeks <0.0001d 0.0012d <0.0001d 0.0014c 24 weeks <0.0001d <0.0001d <0.0001d <0.0001d Significance between groups Full view 0.5257a 0.5344a 8 weeks 0.8418a 0.8505a 16 weeks 0.9518a 0.9412a 24 weeks 0.3815a 0.3805a Improvement rate (%) = [(average value after n week − average value of baseline)/average value of baseline] × 100.
a: Repeated measures ANOVA, post hoc Bonferroni correction.
c: Paired t-test.
d: Wilcoxon’s signed rank test.
Looking at the results of the analysis of the use of the product for the human application test in the PP group, the number of hairs in the test group did not increase significantly compared to the number of hairs in the control group before the product was used (
Secondary efficacy assessment. The secondary efficacy evaluation variable of this human application test was the evaluation by the subjects of changes in hair growth. After 8 weeks, 16 weeks, and 24 weeks of use, a subject evaluation questionnaire was conducted by the test and control groups. Table 4 summarizes the comparisons within each group of the subjects’ evaluation of changes in hair growth, and Table 5 summarizes the comparisons between groups.
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Table 4 . Changes in total hair count confirmed through phototrichogram after 24 weeks.
Visit ITT PP Test group Control group Test group Control group N=28 N=28 N=28 N=28 Mean±SD Mean±SD Mean±SD Mean±SD Do you think your hair loss symptoms have improved? 8 weeks 0.25±0.44 0.29±0.53 0.23±0.43 0.27±0.53 16 weeks 0.82±0.61 0.43±0.50 0.81±0.63 0.42±0.50 24 weeks 0.96±0.58 0.71±0.60 0.92±0.56 0.73±0.60 Within-group comparative significance 8 weeks 0.0156 d 0.0215 d 0.0313 d 0.0391 d 16 weeks <0.0001 d 0.0005 d <0.0001 d 0.0010 d 24 weeks <0.0001 d <0.0001 d <0.0001 d <0.0001 d Do you think the hair on the top of the head has become plentiful? 8 weeks 0.11±0.42 0.18±0.48 0.08±0.39 0.19±0.49 16 weeks 0.57±0.57 0.32±0.48 0.58±0.58 0.35±0.49 24 weeks 0.61±0.63 0.43±0.50 0.58±0.64 0.42±0.50 Within-group comparative significance 8 weeks 0.3750 d 0.1250 d 0.6250 d 0.1250 d 16 weeks <0.0001 d 0.0039 d 0.0001 d 0.0039 d 24 weeks <0.0001 d 0.0005 d 0.0002 d 0.0010 d Do you think the hair at the front of the headline has become plentiful? 8 weeks 0.25±0.59 0.29±0.46 0.23±0.59 0.27±0.45 16 weeks 0.82±0.67 0.32±0.48 0.81±0.69 0.35±0.49 24 weeks 1.00±0.72 0.32±0.55 0.96±0.72 0.35±0.56 Within-group comparative significance 8 weeks 0.0625 d 0.0078 d 0.1094 d 0.0156 d 16 weeks <0.0001 d 0.0039 d <0.0001 d 0.0039 d 24 weeks <0.0001 d 0.0078 d <0.0001 d 0.0078 d Do you think the number of hairs falling out per day has decreased? 8 weeks 0.14±0.71 0.32±0.61 0.12±0.71 0.31±0.62 16 weeks 0.68±0.61 0.43±0.50 0.65±0.63 0.42±0.50 24 weeks 0.93±0.60 0.61±0.63 0.92±0.63 0.62±0.64 Do you think your hair has become thicker? 8 weeks 0.4316 d 0.0225 d 0.5898 d 0.0386 d 16 weeks <0.0001 d 0.0005 d <0.0001 d 0.0010 d 24 weeks <0.0001 d <0.0001 d <0.0001 d 0.0001 d Is your hair thicker? 8 weeks 0.32±0.48 0.11±0.31 0.31±0.47 0.12±0.33 16 weeks 0.50±0.58 0.25±0.52 0.50±0.58 0.27±0.53 24 weeks 0.43±0.57 0.29±0.60 0.42±0.58 0.31±0.62 Within-group comparative significance 8 weeks 0.0039 d 0.2500 d 0.0078 d 0.2500 d 16 weeks 0.0002 d 0.0313 d 0.0005 d 0.0313 d 24 weeks 0.0010 d 0.0313 d 0.0020 d 0.0313 d
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Table 5 . Subject comparison results for changes in hair growth.
ITT PP Test group - Control group Test group - Control group 8weeks 16 weeks 24 weeks 8 weeks 16 weeks 24 weeks Do you think your hair loss symptoms have improved? 0.7275b 0.0164b 0.1150b 0.7188b 0.0274b 0.2285b Do you think the hair on the top of the head has become plentiful? 0.5429b 0.0946b 0.3185b 0.3405b 0.1451b 0.4460b Do you think the hair at the front of the headline has become plentiful? 0.6849b 0.0038b 0.0002b 0.6614b 0.0129b 0.0010b Do you think the number of hairs falling out per day has decreased? 0.2438b 0.1295b 0.0518b 0.2262b 0.1944b 0.0804b Is your hair thicker? 0.0542b 0.0625b 0.2011b 0.0952b 0.1011b 0.3134b
Looking at the results of the intragroup comparative analysis of the five questionnaire items for each visit in the ITT group summarized in Table 6, the subject evaluation for the item “Do you think the symptoms of hair loss have improved” was shown to be a statistically significant improvement when comparing Baseline-16 weeks with Baseline-24 weeks. The subject evaluation for the item “Do you think the hair in the parietal region has become abundant?” did not show a statistically significant improvement when comparing the test group with the control group after Baseline-8 weeks, but after Baseline-16 weeks and Baseline-24 weeks, it was evaluated as a statistically significant improvement in comparison after week. The subject evaluation for the items “Do you think the hair in the front hairline has become abundant?” and “Do you think the amount of hair falling out per day has decreased?” showed a statistically significantly improvement in the comparison group after Baseline-8 weeks. However, it was evaluated that there was a statistically significant improvement in comparisons after Baseline-16 weeks and Baseline-24 weeks. In the case of the control group, a statistically significant improvement was evaluated after Baseline-8 weeks, Baseline-16 weeks, and Baseline-24 weeks. The subjects’ evaluation of the item “Do you think the hair has become thicker?” was evaluated as a statistically significant improvement in comparison with the test group after Baseline-8, Baseline-16, and Baseline-24 weeks. In the case of the control group, there was no statistically significant improvement in comparison after Baseline-8 weeks, but it was evaluated as a statistically significant improvement in comparison after Baseline-16 weeks and Baseline-24 weeks. Looking at the results of intragroup comparative analysis of the five questionnaire items for each visiting week in the PP group, there were no statistically significant differences compared to the ITT group.
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Table 6 . Comparison result of researcher evaluation by photography.
ITT PP Test group Control group Test group Control group N=28 N=28 N=28 N=28 Mean±SD Mean±SD Mean±SD Mean±SD Have your hair loss symptoms improved? 8 weeks 0.54±0.050 0.02±0.62 0.54±0.50 -0.02±0.61 16 weeks 0.79±0.46 -0.23±0.54 0.81±0.44 -0.25±0.56 24 weeks 0.88±0.51 -0.02±0.80 0.87±0.53 -0.06±0.80 within-group comparative significance 8 weeks <0.0001 d 0.8333 d <0.0001 d 1.0000 d 16 weeks <0.0001 d 0.0044 d <0.0001 d 0.0044 d 24 weeks <0.0001 d 0.8605 d <0.0001 d 0.6034 d Is the hair in the parietal area fuller? 8 weeks 0.21±0.41 -0.04±0.38 0.19±0.40 -0.04±0.39 16 weeks 0.30±0.46 -0.14±0.44 0.29±0.46 -0.15±0.46 24 weeks 0.48±0.69 -0.02±0.59 0.48±0.70 -0.06±0.57 within-group comparative significance 8 weeks 0.0005 d 0.7266 d 0.0020 d 0.7266 d 16 weeks <0.0001 d 0.0386 d <0.0001 d 0.0386 d 24 weeks <0.0001 d 1.0000 d <0.0001 d 0.6291 d Is your hair at the front of your head fuller? 8 weeks 0.05±0.23 0.00±0.19 0.06±0.24 0.00±0.20 16 weeks 0.29±0.46 0.04±0.19 0.31±0.47 0.04±0.19 24 weeks 0.41±0.50 0.05±0.35 0.40±0.50 0.06±0.37 within-group comparative significance 8 weeks 0.2500 d 1.0000 d 0.2500 d 1.0000 d 16 weeks <0.0001 d 0.5000 d <0.0001 d 0.5000 d 24 weeks <0.0001 d 0.4531 d <0.0001 d 0.4531 d Is your hair thicker? 8 weeks 0.30±0.54 0.05±0.44 0.33±0.55 0.02±0.42 16 weeks 0.34±0.61 -0.16±0.42 0.37±0.63 -0.17±0.43 24 weeks 0.43±0.50 -0.18±0.54 0.46±0.50 -0.19±0.56 within-group comparative significance 8 weeks <0.0001 d 0.5488 d <0.0001 d 1.0000 d 16 weeks <0.0001 d 0.0117 d <0.0001 d 0.0117 d 24 weeks <0.0001 d 0.0309 d <0.0001 d 0.0309 d C: paired t-test.
D: Wilcoxon’s signed rank test.
Looking at the comparison results between the subject evaluation groups for the change in hair growth in the ITT group summarized in Table 5, the item “Do you think the symptoms of hair loss have improved?” was found to be statistically significant. For the item “Do you think the hair at the frontal line has become abundant?” the difference between the test group and the control group was statistically significant at 16 weeks and 24 weeks after using the product. Looking at the comparison results between the subject evaluation groups on the change in hair growth in the PP group, there were no statistically significant differences compared to the ITT group.
The secondary efficacy evaluation variable of this human application test was evaluation by the researchers using photography. After 8 weeks, 16 weeks, and 24 weeks of using the product in the test group and control group, each subject was evaluated by two researchers. Prior to the significance test of the investigators’ evaluation, the agreement was verified through the agreement test between the two researchers, and then the significance was tested using the average value between them. Table 6 shows the comparison between the test group and the control group, and Table 7 shows the comparison between the groups.
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Table 7 . Comparison result of researcher evaluation by photography.
Evaluation Test group – Control group 8 weeks 16 weeks 24 weeks ITT Have your hair loss symptoms improved? <0.0001a <0.0001a <0.0001a Is the hair in the parietal area fuller? 0.0016a <0.0001a 0.0002a Is your hair at the front of your head fuller? 0.1823a 0.0003a <0.0001a Is your hair thicker? 0.0135a <0.0001a <0.0001a PP Have your hair loss symptoms improved? <0.0001a <0.0001a <0.0001a Is the hair in the parietal area fuller? 0.0046 <0.0001a <0.0001a Is your hair at the front of your head fuller? 0.1825 0.0003a 0.0002a Is your hair thicker? 0.0027 <0.0001a <0.0001a a: Independent two-sample t-test.
b: Mann-Whitney U test.
Looking at the results of the researcher evaluation within the ITT group summarized in Table 6, in the test group, after 8 weeks of using the product, the evaluation score decreased over time in all items except for “Is the hair in the front hairline?” increased, and the results for each time point were evaluated as having significantly improved. Looking at the results of the researcher evaluation within the PP group, there were no statistically significant differences compared to the ITT group.
Looking at the comparison between the researcherevaluated groups in the ITT group summarized in Table 7, in the case of “Are the hair loss symptoms improved?” “Is the hair in the parietal region rich?” and “Is the hair thicker?” there were statistically significant differences at all visit time points. In the case of “Is the frontal hairline abundant?” there was no significant difference at the time point after 8 weeks of using the product, but there was a statistically significant difference at the time points after 16 weeks and 24 weeks. Looking at the comparison between the investigator evaluation groups in the PP group, there were no statistically significant differences compared to the ITT group.
Discussion
In this study, the effect of hair growth in hDPCs was analyzed using the TS-SCLF fermented extract obtained by fermenting with
After hDPC was treated with minoxidil, TS-SC, and TS-SCLF fermented extracts, growth factors were measured by ELISA using the culture supernatant. VEGF has been reported as a vasodilator, which improves blood circulation by dilating blood vessels to promote hair growth and differentiation of hair follicle cells. IGF-1 is produced only in mesenchymal cells, and its receptors exist in epithelial cells. In particular, in the case of hair follicles, it has been reported that IGF-1 secreted from DPCs not only promotes proliferation of epithelial cells in culture but also significantly increases the length of hair follicle tissue. IGF-1 is an important growth factor for regulating hair growth. TGF-β has many effects on cell growth, death, differentiation, and morphology. TGF-β1 inhibits the growth of hair follicles and induces hair loss.
After hDPC was treated with minoxidil, TS-SC, and TS-SCLF fermented extract, mRNA expression was measured. As a result, VEGF, IGF-1, KGF, HGF, and ALP all showed higher expression rates than the control and minoxidil groups. ALP is used as a marker for estimating hair growth-inducing activity, and when 0.1 μg/ ml of TS-SC was treated, the mRNA expression rate was 10.60% higher than in the control group.
Efficacy and safety related to hair loss prevention were evaluated when using “CMK-LPF01,” a material for alleviating symptoms in hair loss patients. The data obtained from the subjects of the human application test were largely analyzed in three types: Safety, Intention To Treat (ITT), and Per Protocol (PP). There was no difference between the groups in terms of age, sex, past and current medical history of the registered subjects in their basic information, and their history of taking the preceding and concomitant drugs.
Summarizing the above results, “CMK-LPF01,” a material for alleviating hair loss symptoms, did not show a statistically significant level (
Acknowledgments
This study was supported by the Technology Innovation Program (10077377, Development of hair and anti-aging cosmetic materials using lactic acid bacteria enzyme hydrolysates) funded By the Ministry of Trade, Industry & Energy (MOTIE, Korea) and supported by the Brain Busan 21+ project (BB21+).
Conflict of Interest
The authors have no financial conflicts of interest to declare.
References
- Lee KM, Lee JS, Oh JY, Kim YC. 2010. The hair growth effect of
Polygoni mulitiflori radix water extracts in C57BL/6 mice.J. Cosmetol. Sci. 6 : 419-426.
- Do EJ, Hwang MY, Kim SY, Lee JS, Yang DS, Yang CH,
et al . 2011. The effect ofGyungokgo-gamibang extract on hair growth and protein expression in mice.Korean J. Herbol. 26 : 9-14. - Ryu EM, Shin HJ. 2011. Hair growth effect of ethyl acetate and water fractions of
Sparassis crispa extracts on hair-removed C57BL/6 mice.Asian J. Beauty Cosmetol. 9 : 1-10. - Park HY, Kim SN, Kang BH, Lee JH. 2010. Experimental studies of
Glycine max Merr. (black bean),Triticum aestivum L. (wheat) andOryza sativa L. (rice bran) extracts on the effects of hair growth activity and physical properties.Korean J. Oriental Med. 16 : 167-173. - Ryu EM, Shin HJ, Na MS. 2010. Effect of
Sparassis crispa extracts on hair growth inhibition of hair-removed C57BL/6 mice.Asian J. Beauty Cosmetol. 8 : 1-11. - Park KS, Park DH. 2016. Comparison of
Saccharina japonica -Undaria pinnatifida mixture and minoxidil on hair growth promoting effect in mice.Arch. Plast. Surg. 43 : 498-505.
- Kim JY, Shin JY, Choi YH, Nam YJ, Lee SY, Jeon JH,
et al . 2019. Hair growth promoting effect ofHottuynia cordata extract in cultured human hair follicle dermal papilla cells.Biol. Pharm. Bull. 42 : 1665-1673.
- Ryu EM, Seo GW, Kee KH, Shin HJ. 2013. Effect of ethanolic extract from wheat sprout on hair growth of C57BL/6 mouse.
Korean J. Aesthet. Cosmetol. 11 : 1051-1057. - Kim JH, Lee SO, Beik GY, Kim KJ. 2021. Effects of
Aucklandia lappa Decne. extract on hair growth in depilated CL57BL/6 mice.Korean J. Clin. Lab. Sci. 53 : 88-95.
- Chen Y, Huang J, Chen R, Yang L, Wang J, Liu B,
et al . 2020. Sustained release of dermal papilla-derived extracellular vesicles from injectable microgel promotes hair growth.Theranostics 10 : 1454-1478.
- Han JH, Kwon OS, Chung JH, Cho KH, Eun HC, Kim KH. 2004. Effect of minoxidil on proliferation and apoptosis in dermal papilla cells of human hair follicle.
J. Dermatol. Sci. 34 : 91-98.
- Oh HJ, Park HK, Ly SY. 2012. An assessment of hair loss ameliorating effect of medicinal plant extracts, amino acid and vitamin complex in human subject.
Korean J. Hum. Ecol. 21 : 587-596.
- McClellan KJ, Markham A. 1999. Finasteride.
Adis Drug Evaluation 57 : 111-126.
- Hsu CL, Liu JS, Lin AC, Yang CH, Chung WH, Wu WG. 2014. Minoxidil may suppress androgen receptor-related functions.
Oncotarget 5 : 2187-2197.
- Shapiro J, Price VH. 1998. Hair regrowth: Therapeutic agents.
Dermatol. Clin. 16 : 341-356.
- An GS, Hwang IC. 2009. Hair loss prevention and hair growth promotion by herb extracts which contain the cultured Korean wild ginseng.
J. Korea Soc. Beauty Art 10 : 221-226. - Choi HM, Hwang SJ, Lee JS, Do EJ, Kim MY, Kim MR. 2011. Effects of hair tonic and food including Korean medicinal herbs on hair growth in an alopecia model of C57BL/6 mice.
Korean J. Herbol. 26 : 119-124. - Jeon HY, Kim SH, Kim CW, Shin HJ, Seo DB, Lee SJ. 2011. Hair growth promoting effect of black soybean extract
in vitro andin vivo .Korean J. Food Sci. Technol. 43 : 747-753.
- Woo YM, Kim OJ, Jo ES, Jo MY, Ahn MY, Lee YH,
et al . 2019. The effect ofLactobacillus plantarum hydrolysates promoting VEGF production on vascular growth and hair growth of C57BL/6 mice.J. Anal. Sci. Technol. 10 : 18-27.
- Joo SS. 2011.
In vitro andin vivo hair growth promotion effects ofLactobacillus plantarum -fermented plant extracts (MBN).Korean J. Food Sci. Technol. 43 : 381-386.
- Kim JY, Shin JY, Choi UH, Jang M, Nam YJ, Lee SY,
et al . 2019. Hair growth promoting effect ofHoyyuynia cordata extract in cultured human hair follicle dermal papilla cells.Biol. Pharm. Bull. 42 : 1665-1673.
- Nagappan A, Jung DY, Kim JH, Lee HY, Jung MH. 2018. Gomisin N alleviates ethanol-induced liver injury through ameliorating lipid metabolism and oxidative stress.
Int. J. Mol. Sci. 19 : 2601.
- Lee JN, Ryu HS, Kim JM, Jung TH, Park SM, Lee YM. 2017. Antimelanogenic effect of gomisin N from
Schisandra chinensis (Turcz.) Baillon (Schisandraceae) in melanoma cells.Arch. Pharm. Res. 40 : 807-817.
- Oh SY, Kim YH, Bae DS, Um BH, Pan CH, Kim CY,
et al . 2010. Antiinflammatory effects of gomisin N, gomisin J, and schisandrin C isolated from the fruit ofSchisandra chinensis .Biosci. Biotechnol. Biochem. 74 : 90597-1.
- Jang MK, Yun YR, Kim SH, Kim JH, Jung MH. 2016. Protective effect of gomisin N against endoplasmic reticulum stressinduced hepatic steatosis.
Biol. Pharm. Bull. 39 : 832-838.
- Heo JH, Park JG, Cheon HJ, Kim YS, Kang SS, Hung TM,
et al . 2006. Hepatoprotective activities of gomisin A and gomisin N.Korean J. Pharmacogn. 37 : 294-301. - Cho HD, Jeong JY, Ryu HS, Lee JN, Park SM. 2020. Dermal papilla cells proliferation constituent of
Schisandra chinensis fruits and optimization using response surface methodology.J. Soc. Cosmet. Sci. Korea 46 : 415-424. - Woo YM, Kim OK, Jo ES, Jo MY, Ahn MY, Lee YH,
et al . 2019. The effect ofLactobacillus plantarum hydrolysates promoting VEGF production on vascular growth and hair growth of C57BL/6 mice.J. Anal. Sci. Technol. 10 : 1-9.
- Gnabre J, Unlu I, Chang TC, Lisseck P, Bourne B, Scolnik R,
et al . 2010. Isolation of lignans fromSchisandra chinensis with antiproliferative activity in human colorectal carcinoma: Structureactivity relationships.J. Chromatogr. B 878 : 2693-2700.
- Madaan A, Joshi V, Kishore A, Verma R, Singh AT, Jaggi M,
et al . 2017.In vitro hair growth promoting effects of naringenin and hesperetin on human dermal papilla cells and keratinocytes.Am. J. Dermatol. Venereol. 6 : 51-57. - Wakame K, Nakata A, Sato K, Mihara Y, Takanari J, Sato A,
et al . 2017. Comparison of effects on gene expression activity of lowmolecular-weight lychee fruit polyphenol (Oligonol®), adenosine, and minoxidil in human dermal papilla cells.J. Biomed. Transl. Res. 3 : 5-12.
- Tsuboi R, Sato C, Kurita Y, Ron D, Rubin JS, Ogawa H. 1993. Keratinocyte growth factor (FGF-7) stimulates migration and plasminogen activator activity of normal human keratinocytes.
J. Invest. Dermatol. 101 : 49-53.
- Hue JJ, Jo BK, Kang BS, Kim JH, Nam SY, Yun YW,
et al . 2010. Effect of sodium silicate on hair growth in C57BL/6 mice.Lab. Anim. Res. 26 : 55-62.
- Kim YE, Choi HC, Nam G, Choi BY. 2019. Costunolide promotes the proliferation of human hair follicle dermal papilla cells and induces hair growth in C57BL/6 mice.
J. Cosmet. Dermatol. 18 : 414-421.
- Paus R, Foitzik K, Welker P, Bulfone-Paus S, Eichmuller S. 1997. Transforming growth factor-β receptor type I and type II expression during murine hair follicle development and cycling.
J. Invest. Dermatol. 109 : 518-526.
- Yamauchi K, Kurosaka A. 2009. Inhibition of glycogen synthase kinase-3 enhances the expression of alkaline phosphatase and insulin-like growth factor-1 in human primary dermal papilla cell culture and maintains mouse hair bulbs in organ culture.
Arch. Dermatol. Res. 301 : 357-365.
