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J Plant Biotechnol 2022; 49(4): 316-324

Published online December 31, 2022

https://doi.org/10.5010/JPB.2022.49.4.316

© The Korean Society of Plant Biotechnology

A novel tetrapeptide for the treatment of hair loss identified in ginseng berry: in silico characterization and molecular docking with TGF-β2

Sung-Gyu Lee ・Sang Moon Kang ・Hyun Kang

Department of Medical Laboratory Science, College of Health Science, Dankook University, Cheonan-si, Chungnam, 31116, Republic of Korea
R&D Center, ANPEP Inc., Cheongju-si, Chungcheongbuk-do 28101, Korea

Correspondence to : e-mail: hkang@dankook.ac.kr

Received: 30 November 2022; Revised: 1 December 2022; Accepted: 1 December 2022

This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Hair loss causes psychological stress due to its effect on appearance. Therefore, the global market for hair loss treatment products is rapidly growing. The present study demonstrated that ginseng berry-derived and sequence-modified peptides promoted the proliferation rate of dermal papilla (DP) cells and keratinocytes, in addition to having antioxidant properties. Moreover, the potential role of these ginseng berry peptides as TGF-β2 antagonists was confirmed through in silico computer docking. In addition to promoting the growth of ,the ginseng berry-derived peptides also promoted the proliferation of keratinocytes experimental Particularly, an unmodified ginseng berry-derived peptide (GB-1) and two peptides with sequence modifications (GB-2 and GB-3) decreased ROS generation and exhibited a protective effect on damaged HaCaT keratinocytes. Computer-aided peptide discovery was conducted to identify the potential interactions of important proteins with transforming growth factor-beta 2 (TGF-β2), a key protein that plays a crucial role in the human hair growth cycle. Our results demonstrated that MAGH, an amino acid sequence present in herbal supplements and plant-based natural compounds, can inhibit TGF-β2.

Keywords ginseng berry, peptide, TGF-β2, hair loss, dermal papilla cell

Hair loss occurs in both men and women, and is a symptom of thinning and decreasing hair on the body or head, accompanied by features such as shrinking of hair follicles and reduction of hair follicles in the anagen phase (Ellis et al. 2002; Price 1999). Hair loss factors that have been identified so far include suppression of proliferation or functional decline of dermal papilla cells related to hair cycle control (Elliott et al. 1999), abnormal changes in hair cycle due to male hormones (Kaufman 2002) and reduced blood flow to the scalp (Kaufman 2002), anticancer peptides (Batchelor 2001; Botchkarev 2003), stress, physical stimulation, and environmental pollution (Aoki et al. 2003; Batchelor 2001). However, the exact mechanism of hair loss is not known exactly (Kaufman and Dawber 1999; Price 1999).

To date, only two peptides, finasteride (Propecia) and minoxidil (Rogain), have been approved by the Food and peptide Administration (FDA) for use. Finasteride and minoxidil were developed for the treatment of benign prostatic hyperplasia and hypertension, respectively, but both peptides are used as hair growth agents after their efficacy in promoting hair growth was found (Kaufman and Dawber 1999; Messenger and Rundegren 2004). Finasteride is known to improve androgenetic alopecia by inhibiting the activity of type II 5α-reductase and inhibiting the conversion of testosterone (T) to dihydrotestosterone (DHT) (Kaufman and Dawber 1999). Minoxidil is reported to promote cell proliferation by suppressing apoptosis of dermal papilla cells (Han et al. 2004), and to induce hair growth effects by opening ATP-sensitive potassium channels (Hamaoka et al. 1997) and activating Wnt/β-catenin pathway (Kwack et al. 2011).

Studies on many regulatory factors (fibroblast growth factor-7, Sonic hedgehog, and transforming growth factor-β) involved in the hair loss mechanism are being actively conducted. In particular, it has been continuously reported that the hair growth effect is regulated by factors related to the hair cycle of the growth phase, catagen phase, and telogen phase and signal transduction by their receptors (Cotsarelis and Millar 2001). In particular, it is known that the TGF-β signaling pathway plays an important role in the regulation of hair follicle formation and hair cycle (Paus et al 1997). Early in anagen, DHT stimulates the synthesis of TGF-β2 in dermal papilla cells. TGF-β2 inhibits epithelial cell proliferation and stimulates the synthesis of specific caspases. Then it triggers the intrinsic caspase network and consequently epithelial cells are eliminated through apoptosis (Tsuji et al. 2003).

Peptide is a molecule composed of two or more amino acids. It is composed of the same monomer as protein, but is involved in metabolism in the human body in a similar or different way to protein. Therefore, it is a compound that is used in various ways (Bergmann and Zervas 1932; Carpino and Han 1970). In particular, unlike proteins for which 3D structure is important, properties and physiological activities are influenced by the primary structure by sequence, so it has the advantage of being easy to fuse and mix with various compounds. In addition, peptides synthesized with the sequence of the active site of a protein show activity similar to that of the protein. In addition, peptides can be easily obtained compared to proteins and are advantageous in storage and activity maintenance, so they can be used in various fields (Kim et al. 2011; Moh et al. 2011).

Ginseng berry is a fruit of ginseng that can be seen in ginseng that is more than 3 years old and has been known to help improve skin. However, it has a disadvantage that it is difficult to store because it starts to bear fruit in mid-July of the year and falls off by itself after about 4 to 7 days and withers within a day. Ginseng berry is known to have the highest content of ginsenoside, the indicator component of ginseng, in the fruit of ginseng, 4-6 times more than in the root, and among them, ginsenoside Re is known to have the highest content. It is reported to be effective in anti-diabetes, sexual function improvement, skin improvement, and blood circulation improvement as it contains abundant natural ingredients (Choi et al. 2013; Kang and Park 2019; Kim et al. 2017). In the case of ginseng berry, various existing effects have been verified and it can show various pharmacological activities, so it seems to have a very high utilization value as a functional material.

Therefore, in this study, the cell proliferation rate and antioxidant effect were confirmed using dermal papilla cells and keratinocytes using ginseng berry-derived peptides and sequence-modified peptides confirmed in previous studies (Kang and Park 2019), and TGF-β2 antagonists it was intended to be used as basic data as a functional material for hair loss improvement in the future by verifying the possibility through 3D modeling.

Materials

The ginseng berry-derived peptides and sequence-modified peptides used in this study were the peptides provided by A&PEP Co., Ltd. (Ochang, Chungcheongbuk-do, Korea), and the peptide information is shown in Table 1. Dimethyl sulfoxide (DMSO), 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazol-ium Bromide (MTT), and 2’,7’-Dichlorodihydrofluorescein diacetate (DCFH-DA) were manufactured by Sigma Aldrich Chemical Co. (St. Louis, MO, USA). As reagents used for cell culture, fetal bovine serum (FBS), penicillin, and Dulbecco’s Modified Eagle Medium (DMEM; Gibco BRL, Gaithersberg, USA) were purchased by Gibco BRL Co. (Grand Island, NY, USA). The cell line used in the experiment, human dermal papilla (DP) cells, was used by Promocell GmbH (Heidelberg, Germany), and the HaCaT cell line, a human keratiocyte, was purchased from the Korea Cell Line Bank (Seoul, Korea).

Table 1 . Original and modified peptides of ginseng berry-derived peptides

Peptides nameModificationAmino acid sequenceChemical formulaMolecular weight (g/mol)
GB-1OriginalMAGHC16H26N6O5S414.48
GB-2Sequence modificationMAHG
GB-3Sequence modificationMHGA
GB-4Sequence modificationMHAG
GB-5Sequence modificationMGHA
GB-6Sequence modificationMGAH


Cell culture

DP cells and HaCaT cells were used as a culture medium by adding 10% FBS and 1% penicillin-streptomycin to DMEM medium, and subcultured once every 2 to 3 days in an incubator at 37°C and 5% CO2.

MTT assay

DP cells were divided into 1 × 104 cells in 48 well-plate and cultured for 24 h. Cultured cells were treated with peptides at 50 and 100 μg/mL and cultured for 72 h. HaCaT cells were divided into 1 × 104 in 48 well-plate, cultured for 24 h, and then cultured cells were treated with peptides at 50 and 100 μg/mL and cultured for 24 h. After culturing for each cell line, 10 μL of 5 mg/mL MTT (Sigma) reagent was added and cultured for 4 h. After removing the supernatant, the formed formazan was dissolved in 100 μL of DMSO, and the absorbance was measured at 550 nm using an ELISA reader.

Measurement of reactive oxygen species (ROS) in cells

Intracellular ROS were measured using the principle that when intracellular ROS react with DCFH-DA (Sigma), they are oxidized to fluorescent DCF. As a control group, a control treated with 500 µM H2O2 without any treatment and a blank not treated with both the sample and 500 µM H2O2 were used. The cultured HaCaT cells were dispensed into a 96-well black plate so that the number of 1 × 104 cells per well was incubated for 24 h in a 37°C, 5% CO2 incubator, and then used for experiments. The cell line was treated with the peptide prepared at a concentration of 100 μg/mL, and pretreated for 3 h in a 37°C, 5% CO2 incubator. Thereafter, 500 μM H2O2 was treated for 3 h to induce ROS production. After 3 h, 25 μM DCFH-DA was treated for 30 min to stain. After that, fluorescence was measured with a fluorescence analyzer at excitation 485 nm and emission 528 nm. The results were expressed as the ROS production rate (%) by converting the fluorescence measurement value to 100% when H2O2 was not treated (Okimoto et al. 2000).

Virtual screening and ligand preparation

Peptides identified from Ginseng Berry is used for the virtual screening using default settings and 6 best hits has been obtained. These 10 compounds were subjected to site specific docking to finally select MAGH compounds with the best docking score for further analysis.

Molecular docking studies

The protein-ligand binding mechanism of the chosen protein ligand complexes was performed using Autodock 4.2 (Bikadi and Hazai 2009; Forli et al. 2016). The docking analyses were performed using semi-flexible docking approach. In this study proteins are kept rigid and ligands were kept flexible. The allowed degrees of freedom for ligand molecules are 10. The steps involving conversion of molecules into format, box type, grid box generation, etc. are specified by AutoDock. The docking poses with the least energy were analyzed using Discovery studio visualizer (Biovia 2017).

Stastical analysis

All experiments were repeated three times and expressed as mean ± SD. Statistical significance was verified by calculating the p-value using the student’s t-test of Sigma Plot (San Jose, CA, USA). Significance was indicated by *p < 0.05, **p < 0.01, and ***p < 0.001.

DP cells and HaCaT cells proliferation promoting effect

In order to confirm the effect of ginseng berry-derived peptides and sequence-modified peptides on the proliferation promoting effect of DP cells and HaCaT cells, peptides were treated with 50 and 100 μg/mL, and after 72 and 24 h, respectively, using MTT assay measured. As a result of the experiment, in DP cells, it was found that cell proliferation was promoted from a concentration of 50 μg/mL compared to the control group in all groups after 72 h (Fig. 1A). HaCaT cells also showed that cell proliferation was promoted by more than 100% in all groups. In particular, in the GB-6 group, treatment with 50 and 100 μg/mL showed the highest cell proliferation promoting effect at 125.61 ± 2.13 (p < 0.001) and 138.56 ± 0.90% (p < 0.001), respectively (Fig. 1B).

Fig. 1. Effect of ginseng berry-derived peptides and sequence-modified peptides on the proliferation of DP and HaCaT cells. (A) DP cells were treated with peptides (50 and 100 μg/mL) for 72 h. (B) HaCaT cells were treated with peptides (50 and 100 μg/mL) for 24 h. The proportion of living cells was measured via the MTT assay. All experiments were conducted in triplicate and the results are expressed as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001

Effects of ginseng berry-derived peptides and sequence-modified peptides on ROS generation induced by H2O2-induced oxidative stress

To investigate the effect of ginseng berry-derived peptides and sequence-modified peptides on the production of ROS, the peptides were pre-treated with 100 μg/mL for 3 h and then treated with 500 μM H2O2 for 3 h. Thereafter, the cells were stained with DCFH-DA to confirm the production of ROS. As a result, ROS generation was significantly (p < 0.001) increased (157.21 ± 4.70%) in the H2O2 (500 μM) treated group than in the H2O2 untreated group. However, in the groups treated with GB-1, GB-2, and GB-3, ROS generation was suppressed significantly (p < 0.01) with 127.55 ± 13.60, 131.81 ± 8.93, and 107.59 ± 16.03%, respectively (Fig. 2).

Fig. 2. Effects of ginseng berry-derived peptides and sequence-modified peptides on H2O2-induced ROS production in human HaCaT keratinocytes. The cells were pre-treated with or without peptides (100 μg/mL) for 3 h, after which they were induced with or without H2O2 (500 μM). After 3 h, the cells were harvested for ROS detection. ROS production was then measured as DCFH-DA-positive cell percentages. Similar results were obtained from three additional experiments. ###p < 0.001 compared to the cells without H2O2 treatment; **p < 0.01 and ***p < 0.001 compared to the cells treated with H2O2 alone

Protein modeling

The protein modeling for the TGF-β2 performed using Phyre2 webserver (Kelley et al. 2015) yielded a high confidence full length model. Fig. 3 shows the representation and of the TGF-β2 protein and MAGH ligand. Virtual screening and ligand selection. Initial virtual screening of peptides obtained from Ginsaeng berry yielded 6 best compounds. Top 6 hits obtained after virtual screening with respective binding energies in kcal/mol. These MAGH best hits were then subjected to site specific molecular docking Fig. 4 and 5. β-sheet formed TYR323, ILE324 and ASP325, α-helix fomed ALA305, ALA307, TYR 308 and CYS309. The compounds screened were mostly phytochemicals from natural sources which showed capability of biological activities such as anti-oxidants and anti-inflammatory.

Fig. 3. Three-dimensional structure of the TGF-β2 (A) and MAGH peptide (B)

Fig. 4. Docking pose and ligand interaction diagram of the TGF-β2 protein and the MAGH ligand (gray balls and sticks). The interacting residues are labeled and shown in pink

Fig. 5. Docking pose and ligand interaction diagram of the TGF-β2 protein and the MAGH ligand (A), close and extended TGF-β2 protein-MAGH ligand interaction (B), interaction group and interacting receptor group (C), and hydrophobic interaction (D)

Molecular docking studies

Molecular docking performed with AutoDock 4.2 (Bikadi and Hazai 2009) further strengthened our study in finding an effective peptide against this deadly disease. The peptide binding scores in the form of kcal/mol for all peptides.

Docking analyses and peptide interactions

The best compound obtained after docking which binds effectively with TGF-β2 is MAGH (the highest-ranked bound compound in all poses made as a cluster at the ligand binding sites of target proteins). The analyses were performed target by target to check the efficiency of the ligand and the state of interaction using Discovery studio visualizer (Biovia 2017) as mentioned here below: The docking TGF-β2 gave us the best hit with MAGH. Analysis of docking results revealed that the peptide binds in the active site of the protein, interacting with important residues with polar and non-polar interactions. Key interactions involved the presence of H-bond with Ser356, Ser357, Ser382, hydrophobic interactions with Leu385. Five different hydrophobic Interactions and two charge Interactions observed. The docking pose and ligand interaction diagram are displayed in Fig. 6. The minimum binding energy for this interaction was found to be 6.5 kcal/mol. The interaction inhibiting the TGF-β2 protein will help in the crucial and important step in the hair loss mechanism.

Fig. 6. In silico molecular 3D docking pattern of the TGF-β2 protein and molecular surface shape feature of the MAGH ligand

Ligand interaction site were A:GLN383:CG - A:DG3: O1P, A:GLN383:CD - A:DG3:P, A:GLN383:CD - A:DG3: O1P, A:GLN383:OE1 - A:DG3:P, A:GLN383:OE1 - A:DG3: O1P, A:LYS412:CB - A:DG3:O2P, A:LYS412:CG - A:DG3: O2P

A:LYS412:CD - A:DA2:C2’, A:LYS412:CD - A:DA2:H2’2, A:LYS412:CD - A:DG3:O2P

A:LYS412:CD - A:DG3:H8, A:LYS412:CE - A:DA2:C2’, A:LYS412:CE - A:DA2:C1’

A:LYS412:CE - A:DA2:H2’1, A:LYS412:CE - A:DA2:H2’2, A:LYS412:NZ - A:DA2:C2’

A:LYS412:NZ - A:DA2:C1’, A:LYS412:NZ - A:DA2:N9, A:LYS412:NZ - A:DA2:C8

A:LYS412:NZ - A:DA2:H2’1, A:LYS412:NZ - A:DA2:H8, A:LYS412:HZ2 - A:DA2:C8

A:DA2:H5’1 - A:GLN383:O, A:LYS412:NZ - A:DA2:O2P, A:LYS412:NZ - A:DG3:O2P

A:SER356:OG - A:ACT4:O2P, A:SER356:OG - A:ACT4:O5’, A:LYS412:NZ - A:DA2:O5’

A:ACX1:H3’ - A:SER382:OG, A:DA2:H4’ - A:GLN383:O, A:DA2:H8 - A:DA2:05’

A:DA2:H2 - A:ASP357:OD2, A:DG3:H5’1 - A:GLN383:OE1, A:DG3:H1 - A:SER356:OG

A:DG3:H8 - A:DG3:O5’, A:ACX1:N9 - A:DA2, A:SER356:O - A:DG3, A:DA2 - A:DG3

A:DA2 - A:DG3, A:DA2 - A:DG3, A:DA2 - A:DG2, A:DA2 - A:LYS412, A:ACX1:HTER

A:ACX1:O1P, A:ACX1:OP2, A:ACX1:O5’, A:ACX1:O4’, A:ACX1:O3, A:DA2:O4’, A:DA2:O3’, A:DA2:N7, A:DA2: N1, A:DA2:N3, A:DA2:H61, A:DA2:H62, A:DG3:O1P, A:DG3:O4’, A:DG3:O3’, A:DG3:N7, A:DG3:O6, A:DG3:N3, A:DG3:H1, A:DG3:H21, A:DG3:H22, A:ACT4:O1P, A:ACT4: O4’, A:ACT4:N9, A:ACT4:HACP, A:SER382:C - A:DA2: O1P, A:SER382:O - A:DA2:P, A:SER382:O - A:DA2:O1P, A:SER382:O - A:DA2:O2P, A:SER382:O - A:DA2:O5’, A:SER382:CB - A:DA2:O1P, A:SER382:CB - A:DA2:O1P, A:GLN383:N - A:DA2:O1P, A:GLN383:CA - A:DA2:C3’, A:GLN383:CA - A:DA2:H3’, A:GLN383:CB - A:DA2:H3’, A:GLN383:CG - A:DA2:C3’, A:GLN383:CG - A:DA2:O3’, A:GLN383:CG - A:DG3:P, A:GLN383:CG - A:DG3:O1P, A:GLN383:CD - A:DG3:P, A:GLN383:CD - A:DG3:O1P, A:GLN383:OE1 - A:DG3:P, A:GLN383:OE1 - A:DG3:O1P, A:LYS412:CB - A:DG3:O2P, A:LYS412:CG - A:DG3:O2P, A:LYS412:CD - A:DA2:C2’, A:LYS412:CD - A:DA2:H2’2, A:LYS412:CD - A:DG3:O2P, A:LYS412:CD - A:DA2:H8, A:LYS412:CE - A:DA2:C2’, A:LYS412:CE - A:DA2:C1’, A:LYS412:CE - A:DA2:H2’1, A:LYS412:CE - A:DA2:H2’2, A:LYS412:NZ - A:DA2:C2’, A:LYS412:NZ - A:DA2:C1’, A:LYS412:NZ - A:DA2:N9, A:LYS412:NZ - A:DA2:C8, A:LYS412:NZ - A:DA2:H2’1, A:LYS412:NZ - A:DA2:H8, A:LYS412:HZ2 - A:DA2:C8, A:DA2:H5’1 - A:GLN383:O, A:LYN412:NA - A:DA2:O2P, A:LYN412:NA - A:DG3:O2P, A:SER356:OG - A:ACT4:O2P, A:SER356:OG - A:ACT4:O5’, A:LYN412:NZ - A:DA2:O5’, A:ACX1:H3’ - A:SER382:OG, A:DA2:H4’ - A:GLN383:O, A:DA2:H8 - A:DA2:O5’, A:DA2:H2 - A:ASP357:OD2, A:SER356:CA - A:ACT4:N9, A:SER356:C - A:ACT4:N9, A:SER356:CB - A:ACT4:C2’, A:SER356:CB - A:ACT4:C1’, A:SER356:CB - A:ACT4:N9, A:SER356:CB - A:ACT4:H2’1, A:ASP357:N - A:ACT4:O4’, A:ASP357:N - A:ACT4:C1’, A:ASP357:N - A:ACT4:N9, A:ASP357:CA - A:DG3:C2, A:ASP357:CA - A:DG3:N2, A:ASP357:CA - A:DG3:H21, A:ASP357:C - A:DG3:C2, A:ASP357:C - A:DG3:N2, A:ASP357:C - A:DG3:H21, A:ASP357:C - A:DG3:H22, A:ASP357:C - A:DG3:C2, A:ASP357:O - A:DG3:N2, A:ASP357:O - A:DG3:H21, A:ASP357:O - A:DG3:H22, A:ASP357:CB - A:DG3:C2, A:ASP357:CB - A:DG3:N2, A:ASP357:CB - A:DG3:N3, A:ASP357:CB - A:DG3:H21, A:ASP357:HN - A:ACT4:O4’, A:THR358:N - A:DG3:N2, A:THR358:N - A:DG3:H22, A:SER382:CA - A:DA2:O1P, A:SER382:C - A:DA2:P, A:SER382:C - A:DA2:O1P, A:SER382:O - A:DA2:P, A:SER382:O - A:DA2:O1P, A:SER382:O - A:DA2:O2P. From this, TGF-β2 : MGAH key interacting residues are GLN383, LYS412, SER356 SER382, ASP357 and THR358.

In recent years, the number of people suffering from hair loss has increased, and the number of women suffering from hair loss has also increased. It is known that hair growth and hair cycle control occur by various mechanisms, such as the action of various growth factors and their receptors, and the action of hormones. In particular, hair matrix cells of hair follicles composed of epithelial cells and dermal papilla cells composed of mesenchymal cells act as pivotal elements in the formation and growth of hair (Ellis et al. 2002). In the anagen phase of the hair follicle, proliferation of constituent cells of the hair follicle, including keratinocytes of the matrix surrounding the dermal papilla cells, occurs (Elliott et al. 1999).

In this study, to find new peptides that can promote hair growth, the cell proliferation effects of ginseng berry-derived peptides and sequence-modified peptides on DP cells and HaCaT cells were investigated. As a result, DP cells and HaCaT cells showed cell proliferation promoting effects in all groups (Fig. 1).

Reactive oxygen species (ROS) is an unstable free radical, which is generated by oxidation and stress in cells. Due to various physical, chemical, and environmental factors, ROS are formed by superoxide radical, hydroxyl radical, hydrogen peroxide, or hydroxyl peroxide. When it is changed into very ROS such as hydroxyl peroxide and singlet oxygen, it acts on lipids, proteins, sugars, and DNA that make up the cells of our body. It is known to cause various diseases as well as aging. Excessive oxidative stress during the process of melanogenesis in growing hair causes hair follicle cell death, resulting in hair loss (Peters 2006), and mutations in nuclear and mitochondrial DNA, resulting in canities and alopecia. (Nixhimura et al. 2005; Tobin et al. 2001).

DCFH-DA was used to measure ROS level changes in HaCaT cells. HaCaT was first tested by dividing into control group, H2O2 treatment group, and ginseng berry-derived peptides and sequence-modified peptides + H2O2 treatment group. And as a result of measuring the ROS level by staining with DCFH-DA, ROS generation was significantly (p < 0.001) increased (157.21 ± 4.70%) in the H2O2 (500 μM) treated group than in the H2O2 untreated group. However, in the groups treated with GB-1, GB-2, and GB-3, ROS generation was suppressed significantly (p < 0.01) with 127.55 ± 13.60, 131.81 ± 8.93, and 107.59 ± 16.03%, respectively (Fig. 2). Here, increased ROS induce DNA damage (Kang et al. 2012). In addition, it was confirmed that ROS production was reduced by treatment with GB-1, GB-2, and GB-3, which showed the protective effect of damaged HaCaT.

In hair follicle cells, TGF-β2 inhibits epithelial cell proliferation and stimulates the synthesis of specific caspases. It then triggers the endogenous caspase network and consequently epithelial cells are eliminated through apoptosis (Tsuji et al. 2003). Therefore, we performed a computer-aided peptide discovery process for important proteins involved in the mechanism of action of TGF-β2 as a biomarker of hair loss.

TGF-β2 : MAGH complex form have strong H-bond D(H)..A(max dist) 3,4, salt bridge D(H)...A(max dist) 4, week H-bond D(H)..A(max dist) 3.8, acceptor-acceptor (max dist) 3, hydrogen-bong D-H 90, strong H-bond D(H)..A(max dist) 3,4, salt bridge D(H)...A(max dist) 4, week H-bond D(H)..A(max dist) 3.8, acceptor-acceptor (max dist) 3, hydrogen-bong D-H-A(min deg) 90, hydrogen-bong D-H-A(max deg) 180, hydrogen-bong D-H-Y(min deg) 90, hydrogen-bong D-H-Y(max deg) 180, hydrogen-bong X-D-A(min deg) 90, hydrogen-bong X-D-A(max deg) 90, hydrogen-bong D-A-Y(min deg) 90, hydrogen-bong D-A-Y(max deg) 180, acceptor-acceptor A-Lp-Lp-A torsion(max deg) 15, acceptor-acceptor A-Lp-A(min deg) 90, acceptor-acceptor A-Lp-A(max deg) 180, hydrogen bond with Sp donor X-D-A(min deg) 135, donor-donor H-H-D(min deg) 120, fluorine non-bond(max dist) 3.7, halogen (Cl,Br,I) VDW fraction(max) 1, halogen (Cl,Br,I) bond C-X-B(min deg) 120, halogen (Cl,Br,I) bond C-X-B(max deg) 180, halogen (Cl,Br,I) bond X-B-Y(min deg) 75, halogen (Cl,Br,I) bond X-B-Y(max deg) 180

Charge-charge(max dist) 5.6, Pi-Cation(max dist) 5, Pi-Cation(max deg) 40

Pi-Donor(max dist) 4.2 Pi-Donor(max deg) 45, Pi-Donor deviation(max deg) 40

Pi-Lone Pair(max dist) 3 Pi-Lone Pair(max deg) 45, Pi-Lone Pair devlation(max deg)40, Pi-Sigma(max dist) 4, Pi-Sigma(max deg) 45, Pi-Sulfur edge-on(min deg) 70

Pi-Sulfur face-on(max dist) 4.5, Pi-Sulfur face-on(max deg) 25

Pi-Pi centroid (max dist) 6, Pi-Pi closest atom(max dist) 4.5, Stacked Pi-Pi theta(max deg) 50, Stacked Pi-Pi gamma(max deg) 35, T-shaped Pi-Pi theta(max deg) 30, T-shaped Pi-Pi gamma(min deg) 55, Stacked Pi-Amide theta(max deg) 40

Stacked Pi-Amide gamma(max deg) 20, Alkyl centroid (max dist) 5.5, Sulface area scale factor 1 and X -7.20457, Y 14.0845, Z 8371292.

Structural biology approach which has been working with all diseases in the past has been found to be the fastest, cheapest and reliable to discover peptideagainst deadly diseases. We performed computer-aided peptide discovery process against the important proteins involved in the mechanism of action for TGF-β2. Our results show that MAGH, a herbal supplement and a plant based natural compound has the capability to inhibit TGF-β2 target proteins.

This research was supported by the Ministry of Trade, Industry & Energy (MOTIE), Korea Evaluation Institute of Industrial Technology (KEIT) through the Encouragement Proꠓgram for Bio industry core technology development project (Project NO: 20009105).

The authors declare that they have no conflict of interest.

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Article

Research Article

J Plant Biotechnol 2022; 49(4): 316-324

Published online December 31, 2022 https://doi.org/10.5010/JPB.2022.49.4.316

Copyright © The Korean Society of Plant Biotechnology.

A novel tetrapeptide for the treatment of hair loss identified in ginseng berry: in silico characterization and molecular docking with TGF-β2

Sung-Gyu Lee ・Sang Moon Kang ・Hyun Kang

Department of Medical Laboratory Science, College of Health Science, Dankook University, Cheonan-si, Chungnam, 31116, Republic of Korea
R&D Center, ANPEP Inc., Cheongju-si, Chungcheongbuk-do 28101, Korea

Correspondence to:e-mail: hkang@dankook.ac.kr

Received: 30 November 2022; Revised: 1 December 2022; Accepted: 1 December 2022

This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Hair loss causes psychological stress due to its effect on appearance. Therefore, the global market for hair loss treatment products is rapidly growing. The present study demonstrated that ginseng berry-derived and sequence-modified peptides promoted the proliferation rate of dermal papilla (DP) cells and keratinocytes, in addition to having antioxidant properties. Moreover, the potential role of these ginseng berry peptides as TGF-β2 antagonists was confirmed through in silico computer docking. In addition to promoting the growth of ,the ginseng berry-derived peptides also promoted the proliferation of keratinocytes experimental Particularly, an unmodified ginseng berry-derived peptide (GB-1) and two peptides with sequence modifications (GB-2 and GB-3) decreased ROS generation and exhibited a protective effect on damaged HaCaT keratinocytes. Computer-aided peptide discovery was conducted to identify the potential interactions of important proteins with transforming growth factor-beta 2 (TGF-β2), a key protein that plays a crucial role in the human hair growth cycle. Our results demonstrated that MAGH, an amino acid sequence present in herbal supplements and plant-based natural compounds, can inhibit TGF-β2.

Keywords: ginseng berry, peptide, TGF-&beta,2, hair loss, dermal papilla cell

Introduction

Hair loss occurs in both men and women, and is a symptom of thinning and decreasing hair on the body or head, accompanied by features such as shrinking of hair follicles and reduction of hair follicles in the anagen phase (Ellis et al. 2002; Price 1999). Hair loss factors that have been identified so far include suppression of proliferation or functional decline of dermal papilla cells related to hair cycle control (Elliott et al. 1999), abnormal changes in hair cycle due to male hormones (Kaufman 2002) and reduced blood flow to the scalp (Kaufman 2002), anticancer peptides (Batchelor 2001; Botchkarev 2003), stress, physical stimulation, and environmental pollution (Aoki et al. 2003; Batchelor 2001). However, the exact mechanism of hair loss is not known exactly (Kaufman and Dawber 1999; Price 1999).

To date, only two peptides, finasteride (Propecia) and minoxidil (Rogain), have been approved by the Food and peptide Administration (FDA) for use. Finasteride and minoxidil were developed for the treatment of benign prostatic hyperplasia and hypertension, respectively, but both peptides are used as hair growth agents after their efficacy in promoting hair growth was found (Kaufman and Dawber 1999; Messenger and Rundegren 2004). Finasteride is known to improve androgenetic alopecia by inhibiting the activity of type II 5α-reductase and inhibiting the conversion of testosterone (T) to dihydrotestosterone (DHT) (Kaufman and Dawber 1999). Minoxidil is reported to promote cell proliferation by suppressing apoptosis of dermal papilla cells (Han et al. 2004), and to induce hair growth effects by opening ATP-sensitive potassium channels (Hamaoka et al. 1997) and activating Wnt/β-catenin pathway (Kwack et al. 2011).

Studies on many regulatory factors (fibroblast growth factor-7, Sonic hedgehog, and transforming growth factor-β) involved in the hair loss mechanism are being actively conducted. In particular, it has been continuously reported that the hair growth effect is regulated by factors related to the hair cycle of the growth phase, catagen phase, and telogen phase and signal transduction by their receptors (Cotsarelis and Millar 2001). In particular, it is known that the TGF-β signaling pathway plays an important role in the regulation of hair follicle formation and hair cycle (Paus et al 1997). Early in anagen, DHT stimulates the synthesis of TGF-β2 in dermal papilla cells. TGF-β2 inhibits epithelial cell proliferation and stimulates the synthesis of specific caspases. Then it triggers the intrinsic caspase network and consequently epithelial cells are eliminated through apoptosis (Tsuji et al. 2003).

Peptide is a molecule composed of two or more amino acids. It is composed of the same monomer as protein, but is involved in metabolism in the human body in a similar or different way to protein. Therefore, it is a compound that is used in various ways (Bergmann and Zervas 1932; Carpino and Han 1970). In particular, unlike proteins for which 3D structure is important, properties and physiological activities are influenced by the primary structure by sequence, so it has the advantage of being easy to fuse and mix with various compounds. In addition, peptides synthesized with the sequence of the active site of a protein show activity similar to that of the protein. In addition, peptides can be easily obtained compared to proteins and are advantageous in storage and activity maintenance, so they can be used in various fields (Kim et al. 2011; Moh et al. 2011).

Ginseng berry is a fruit of ginseng that can be seen in ginseng that is more than 3 years old and has been known to help improve skin. However, it has a disadvantage that it is difficult to store because it starts to bear fruit in mid-July of the year and falls off by itself after about 4 to 7 days and withers within a day. Ginseng berry is known to have the highest content of ginsenoside, the indicator component of ginseng, in the fruit of ginseng, 4-6 times more than in the root, and among them, ginsenoside Re is known to have the highest content. It is reported to be effective in anti-diabetes, sexual function improvement, skin improvement, and blood circulation improvement as it contains abundant natural ingredients (Choi et al. 2013; Kang and Park 2019; Kim et al. 2017). In the case of ginseng berry, various existing effects have been verified and it can show various pharmacological activities, so it seems to have a very high utilization value as a functional material.

Therefore, in this study, the cell proliferation rate and antioxidant effect were confirmed using dermal papilla cells and keratinocytes using ginseng berry-derived peptides and sequence-modified peptides confirmed in previous studies (Kang and Park 2019), and TGF-β2 antagonists it was intended to be used as basic data as a functional material for hair loss improvement in the future by verifying the possibility through 3D modeling.

Materials and Methods

Materials

The ginseng berry-derived peptides and sequence-modified peptides used in this study were the peptides provided by A&PEP Co., Ltd. (Ochang, Chungcheongbuk-do, Korea), and the peptide information is shown in Table 1. Dimethyl sulfoxide (DMSO), 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazol-ium Bromide (MTT), and 2’,7’-Dichlorodihydrofluorescein diacetate (DCFH-DA) were manufactured by Sigma Aldrich Chemical Co. (St. Louis, MO, USA). As reagents used for cell culture, fetal bovine serum (FBS), penicillin, and Dulbecco’s Modified Eagle Medium (DMEM; Gibco BRL, Gaithersberg, USA) were purchased by Gibco BRL Co. (Grand Island, NY, USA). The cell line used in the experiment, human dermal papilla (DP) cells, was used by Promocell GmbH (Heidelberg, Germany), and the HaCaT cell line, a human keratiocyte, was purchased from the Korea Cell Line Bank (Seoul, Korea).

Table 1 . Original and modified peptides of ginseng berry-derived peptides.

Peptides nameModificationAmino acid sequenceChemical formulaMolecular weight (g/mol)
GB-1OriginalMAGHC16H26N6O5S414.48
GB-2Sequence modificationMAHG
GB-3Sequence modificationMHGA
GB-4Sequence modificationMHAG
GB-5Sequence modificationMGHA
GB-6Sequence modificationMGAH


Cell culture

DP cells and HaCaT cells were used as a culture medium by adding 10% FBS and 1% penicillin-streptomycin to DMEM medium, and subcultured once every 2 to 3 days in an incubator at 37°C and 5% CO2.

MTT assay

DP cells were divided into 1 × 104 cells in 48 well-plate and cultured for 24 h. Cultured cells were treated with peptides at 50 and 100 μg/mL and cultured for 72 h. HaCaT cells were divided into 1 × 104 in 48 well-plate, cultured for 24 h, and then cultured cells were treated with peptides at 50 and 100 μg/mL and cultured for 24 h. After culturing for each cell line, 10 μL of 5 mg/mL MTT (Sigma) reagent was added and cultured for 4 h. After removing the supernatant, the formed formazan was dissolved in 100 μL of DMSO, and the absorbance was measured at 550 nm using an ELISA reader.

Measurement of reactive oxygen species (ROS) in cells

Intracellular ROS were measured using the principle that when intracellular ROS react with DCFH-DA (Sigma), they are oxidized to fluorescent DCF. As a control group, a control treated with 500 µM H2O2 without any treatment and a blank not treated with both the sample and 500 µM H2O2 were used. The cultured HaCaT cells were dispensed into a 96-well black plate so that the number of 1 × 104 cells per well was incubated for 24 h in a 37°C, 5% CO2 incubator, and then used for experiments. The cell line was treated with the peptide prepared at a concentration of 100 μg/mL, and pretreated for 3 h in a 37°C, 5% CO2 incubator. Thereafter, 500 μM H2O2 was treated for 3 h to induce ROS production. After 3 h, 25 μM DCFH-DA was treated for 30 min to stain. After that, fluorescence was measured with a fluorescence analyzer at excitation 485 nm and emission 528 nm. The results were expressed as the ROS production rate (%) by converting the fluorescence measurement value to 100% when H2O2 was not treated (Okimoto et al. 2000).

Virtual screening and ligand preparation

Peptides identified from Ginseng Berry is used for the virtual screening using default settings and 6 best hits has been obtained. These 10 compounds were subjected to site specific docking to finally select MAGH compounds with the best docking score for further analysis.

Molecular docking studies

The protein-ligand binding mechanism of the chosen protein ligand complexes was performed using Autodock 4.2 (Bikadi and Hazai 2009; Forli et al. 2016). The docking analyses were performed using semi-flexible docking approach. In this study proteins are kept rigid and ligands were kept flexible. The allowed degrees of freedom for ligand molecules are 10. The steps involving conversion of molecules into format, box type, grid box generation, etc. are specified by AutoDock. The docking poses with the least energy were analyzed using Discovery studio visualizer (Biovia 2017).

Stastical analysis

All experiments were repeated three times and expressed as mean ± SD. Statistical significance was verified by calculating the p-value using the student’s t-test of Sigma Plot (San Jose, CA, USA). Significance was indicated by *p < 0.05, **p < 0.01, and ***p < 0.001.

Results

DP cells and HaCaT cells proliferation promoting effect

In order to confirm the effect of ginseng berry-derived peptides and sequence-modified peptides on the proliferation promoting effect of DP cells and HaCaT cells, peptides were treated with 50 and 100 μg/mL, and after 72 and 24 h, respectively, using MTT assay measured. As a result of the experiment, in DP cells, it was found that cell proliferation was promoted from a concentration of 50 μg/mL compared to the control group in all groups after 72 h (Fig. 1A). HaCaT cells also showed that cell proliferation was promoted by more than 100% in all groups. In particular, in the GB-6 group, treatment with 50 and 100 μg/mL showed the highest cell proliferation promoting effect at 125.61 ± 2.13 (p < 0.001) and 138.56 ± 0.90% (p < 0.001), respectively (Fig. 1B).

Figure 1. Effect of ginseng berry-derived peptides and sequence-modified peptides on the proliferation of DP and HaCaT cells. (A) DP cells were treated with peptides (50 and 100 μg/mL) for 72 h. (B) HaCaT cells were treated with peptides (50 and 100 μg/mL) for 24 h. The proportion of living cells was measured via the MTT assay. All experiments were conducted in triplicate and the results are expressed as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001

Effects of ginseng berry-derived peptides and sequence-modified peptides on ROS generation induced by H2O2-induced oxidative stress

To investigate the effect of ginseng berry-derived peptides and sequence-modified peptides on the production of ROS, the peptides were pre-treated with 100 μg/mL for 3 h and then treated with 500 μM H2O2 for 3 h. Thereafter, the cells were stained with DCFH-DA to confirm the production of ROS. As a result, ROS generation was significantly (p < 0.001) increased (157.21 ± 4.70%) in the H2O2 (500 μM) treated group than in the H2O2 untreated group. However, in the groups treated with GB-1, GB-2, and GB-3, ROS generation was suppressed significantly (p < 0.01) with 127.55 ± 13.60, 131.81 ± 8.93, and 107.59 ± 16.03%, respectively (Fig. 2).

Figure 2. Effects of ginseng berry-derived peptides and sequence-modified peptides on H2O2-induced ROS production in human HaCaT keratinocytes. The cells were pre-treated with or without peptides (100 μg/mL) for 3 h, after which they were induced with or without H2O2 (500 μM). After 3 h, the cells were harvested for ROS detection. ROS production was then measured as DCFH-DA-positive cell percentages. Similar results were obtained from three additional experiments. ###p < 0.001 compared to the cells without H2O2 treatment; **p < 0.01 and ***p < 0.001 compared to the cells treated with H2O2 alone

Protein modeling

The protein modeling for the TGF-β2 performed using Phyre2 webserver (Kelley et al. 2015) yielded a high confidence full length model. Fig. 3 shows the representation and of the TGF-β2 protein and MAGH ligand. Virtual screening and ligand selection. Initial virtual screening of peptides obtained from Ginsaeng berry yielded 6 best compounds. Top 6 hits obtained after virtual screening with respective binding energies in kcal/mol. These MAGH best hits were then subjected to site specific molecular docking Fig. 4 and 5. β-sheet formed TYR323, ILE324 and ASP325, α-helix fomed ALA305, ALA307, TYR 308 and CYS309. The compounds screened were mostly phytochemicals from natural sources which showed capability of biological activities such as anti-oxidants and anti-inflammatory.

Figure 3. Three-dimensional structure of the TGF-β2 (A) and MAGH peptide (B)

Figure 4. Docking pose and ligand interaction diagram of the TGF-β2 protein and the MAGH ligand (gray balls and sticks). The interacting residues are labeled and shown in pink

Figure 5. Docking pose and ligand interaction diagram of the TGF-β2 protein and the MAGH ligand (A), close and extended TGF-β2 protein-MAGH ligand interaction (B), interaction group and interacting receptor group (C), and hydrophobic interaction (D)

Molecular docking studies

Molecular docking performed with AutoDock 4.2 (Bikadi and Hazai 2009) further strengthened our study in finding an effective peptide against this deadly disease. The peptide binding scores in the form of kcal/mol for all peptides.

Docking analyses and peptide interactions

The best compound obtained after docking which binds effectively with TGF-β2 is MAGH (the highest-ranked bound compound in all poses made as a cluster at the ligand binding sites of target proteins). The analyses were performed target by target to check the efficiency of the ligand and the state of interaction using Discovery studio visualizer (Biovia 2017) as mentioned here below: The docking TGF-β2 gave us the best hit with MAGH. Analysis of docking results revealed that the peptide binds in the active site of the protein, interacting with important residues with polar and non-polar interactions. Key interactions involved the presence of H-bond with Ser356, Ser357, Ser382, hydrophobic interactions with Leu385. Five different hydrophobic Interactions and two charge Interactions observed. The docking pose and ligand interaction diagram are displayed in Fig. 6. The minimum binding energy for this interaction was found to be 6.5 kcal/mol. The interaction inhibiting the TGF-β2 protein will help in the crucial and important step in the hair loss mechanism.

Figure 6. In silico molecular 3D docking pattern of the TGF-β2 protein and molecular surface shape feature of the MAGH ligand

Ligand interaction site were A:GLN383:CG - A:DG3: O1P, A:GLN383:CD - A:DG3:P, A:GLN383:CD - A:DG3: O1P, A:GLN383:OE1 - A:DG3:P, A:GLN383:OE1 - A:DG3: O1P, A:LYS412:CB - A:DG3:O2P, A:LYS412:CG - A:DG3: O2P

A:LYS412:CD - A:DA2:C2’, A:LYS412:CD - A:DA2:H2’2, A:LYS412:CD - A:DG3:O2P

A:LYS412:CD - A:DG3:H8, A:LYS412:CE - A:DA2:C2’, A:LYS412:CE - A:DA2:C1’

A:LYS412:CE - A:DA2:H2’1, A:LYS412:CE - A:DA2:H2’2, A:LYS412:NZ - A:DA2:C2’

A:LYS412:NZ - A:DA2:C1’, A:LYS412:NZ - A:DA2:N9, A:LYS412:NZ - A:DA2:C8

A:LYS412:NZ - A:DA2:H2’1, A:LYS412:NZ - A:DA2:H8, A:LYS412:HZ2 - A:DA2:C8

A:DA2:H5’1 - A:GLN383:O, A:LYS412:NZ - A:DA2:O2P, A:LYS412:NZ - A:DG3:O2P

A:SER356:OG - A:ACT4:O2P, A:SER356:OG - A:ACT4:O5’, A:LYS412:NZ - A:DA2:O5’

A:ACX1:H3’ - A:SER382:OG, A:DA2:H4’ - A:GLN383:O, A:DA2:H8 - A:DA2:05’

A:DA2:H2 - A:ASP357:OD2, A:DG3:H5’1 - A:GLN383:OE1, A:DG3:H1 - A:SER356:OG

A:DG3:H8 - A:DG3:O5’, A:ACX1:N9 - A:DA2, A:SER356:O - A:DG3, A:DA2 - A:DG3

A:DA2 - A:DG3, A:DA2 - A:DG3, A:DA2 - A:DG2, A:DA2 - A:LYS412, A:ACX1:HTER

A:ACX1:O1P, A:ACX1:OP2, A:ACX1:O5’, A:ACX1:O4’, A:ACX1:O3, A:DA2:O4’, A:DA2:O3’, A:DA2:N7, A:DA2: N1, A:DA2:N3, A:DA2:H61, A:DA2:H62, A:DG3:O1P, A:DG3:O4’, A:DG3:O3’, A:DG3:N7, A:DG3:O6, A:DG3:N3, A:DG3:H1, A:DG3:H21, A:DG3:H22, A:ACT4:O1P, A:ACT4: O4’, A:ACT4:N9, A:ACT4:HACP, A:SER382:C - A:DA2: O1P, A:SER382:O - A:DA2:P, A:SER382:O - A:DA2:O1P, A:SER382:O - A:DA2:O2P, A:SER382:O - A:DA2:O5’, A:SER382:CB - A:DA2:O1P, A:SER382:CB - A:DA2:O1P, A:GLN383:N - A:DA2:O1P, A:GLN383:CA - A:DA2:C3’, A:GLN383:CA - A:DA2:H3’, A:GLN383:CB - A:DA2:H3’, A:GLN383:CG - A:DA2:C3’, A:GLN383:CG - A:DA2:O3’, A:GLN383:CG - A:DG3:P, A:GLN383:CG - A:DG3:O1P, A:GLN383:CD - A:DG3:P, A:GLN383:CD - A:DG3:O1P, A:GLN383:OE1 - A:DG3:P, A:GLN383:OE1 - A:DG3:O1P, A:LYS412:CB - A:DG3:O2P, A:LYS412:CG - A:DG3:O2P, A:LYS412:CD - A:DA2:C2’, A:LYS412:CD - A:DA2:H2’2, A:LYS412:CD - A:DG3:O2P, A:LYS412:CD - A:DA2:H8, A:LYS412:CE - A:DA2:C2’, A:LYS412:CE - A:DA2:C1’, A:LYS412:CE - A:DA2:H2’1, A:LYS412:CE - A:DA2:H2’2, A:LYS412:NZ - A:DA2:C2’, A:LYS412:NZ - A:DA2:C1’, A:LYS412:NZ - A:DA2:N9, A:LYS412:NZ - A:DA2:C8, A:LYS412:NZ - A:DA2:H2’1, A:LYS412:NZ - A:DA2:H8, A:LYS412:HZ2 - A:DA2:C8, A:DA2:H5’1 - A:GLN383:O, A:LYN412:NA - A:DA2:O2P, A:LYN412:NA - A:DG3:O2P, A:SER356:OG - A:ACT4:O2P, A:SER356:OG - A:ACT4:O5’, A:LYN412:NZ - A:DA2:O5’, A:ACX1:H3’ - A:SER382:OG, A:DA2:H4’ - A:GLN383:O, A:DA2:H8 - A:DA2:O5’, A:DA2:H2 - A:ASP357:OD2, A:SER356:CA - A:ACT4:N9, A:SER356:C - A:ACT4:N9, A:SER356:CB - A:ACT4:C2’, A:SER356:CB - A:ACT4:C1’, A:SER356:CB - A:ACT4:N9, A:SER356:CB - A:ACT4:H2’1, A:ASP357:N - A:ACT4:O4’, A:ASP357:N - A:ACT4:C1’, A:ASP357:N - A:ACT4:N9, A:ASP357:CA - A:DG3:C2, A:ASP357:CA - A:DG3:N2, A:ASP357:CA - A:DG3:H21, A:ASP357:C - A:DG3:C2, A:ASP357:C - A:DG3:N2, A:ASP357:C - A:DG3:H21, A:ASP357:C - A:DG3:H22, A:ASP357:C - A:DG3:C2, A:ASP357:O - A:DG3:N2, A:ASP357:O - A:DG3:H21, A:ASP357:O - A:DG3:H22, A:ASP357:CB - A:DG3:C2, A:ASP357:CB - A:DG3:N2, A:ASP357:CB - A:DG3:N3, A:ASP357:CB - A:DG3:H21, A:ASP357:HN - A:ACT4:O4’, A:THR358:N - A:DG3:N2, A:THR358:N - A:DG3:H22, A:SER382:CA - A:DA2:O1P, A:SER382:C - A:DA2:P, A:SER382:C - A:DA2:O1P, A:SER382:O - A:DA2:P, A:SER382:O - A:DA2:O1P, A:SER382:O - A:DA2:O2P. From this, TGF-β2 : MGAH key interacting residues are GLN383, LYS412, SER356 SER382, ASP357 and THR358.

Discussion

In recent years, the number of people suffering from hair loss has increased, and the number of women suffering from hair loss has also increased. It is known that hair growth and hair cycle control occur by various mechanisms, such as the action of various growth factors and their receptors, and the action of hormones. In particular, hair matrix cells of hair follicles composed of epithelial cells and dermal papilla cells composed of mesenchymal cells act as pivotal elements in the formation and growth of hair (Ellis et al. 2002). In the anagen phase of the hair follicle, proliferation of constituent cells of the hair follicle, including keratinocytes of the matrix surrounding the dermal papilla cells, occurs (Elliott et al. 1999).

In this study, to find new peptides that can promote hair growth, the cell proliferation effects of ginseng berry-derived peptides and sequence-modified peptides on DP cells and HaCaT cells were investigated. As a result, DP cells and HaCaT cells showed cell proliferation promoting effects in all groups (Fig. 1).

Reactive oxygen species (ROS) is an unstable free radical, which is generated by oxidation and stress in cells. Due to various physical, chemical, and environmental factors, ROS are formed by superoxide radical, hydroxyl radical, hydrogen peroxide, or hydroxyl peroxide. When it is changed into very ROS such as hydroxyl peroxide and singlet oxygen, it acts on lipids, proteins, sugars, and DNA that make up the cells of our body. It is known to cause various diseases as well as aging. Excessive oxidative stress during the process of melanogenesis in growing hair causes hair follicle cell death, resulting in hair loss (Peters 2006), and mutations in nuclear and mitochondrial DNA, resulting in canities and alopecia. (Nixhimura et al. 2005; Tobin et al. 2001).

DCFH-DA was used to measure ROS level changes in HaCaT cells. HaCaT was first tested by dividing into control group, H2O2 treatment group, and ginseng berry-derived peptides and sequence-modified peptides + H2O2 treatment group. And as a result of measuring the ROS level by staining with DCFH-DA, ROS generation was significantly (p < 0.001) increased (157.21 ± 4.70%) in the H2O2 (500 μM) treated group than in the H2O2 untreated group. However, in the groups treated with GB-1, GB-2, and GB-3, ROS generation was suppressed significantly (p < 0.01) with 127.55 ± 13.60, 131.81 ± 8.93, and 107.59 ± 16.03%, respectively (Fig. 2). Here, increased ROS induce DNA damage (Kang et al. 2012). In addition, it was confirmed that ROS production was reduced by treatment with GB-1, GB-2, and GB-3, which showed the protective effect of damaged HaCaT.

In hair follicle cells, TGF-β2 inhibits epithelial cell proliferation and stimulates the synthesis of specific caspases. It then triggers the endogenous caspase network and consequently epithelial cells are eliminated through apoptosis (Tsuji et al. 2003). Therefore, we performed a computer-aided peptide discovery process for important proteins involved in the mechanism of action of TGF-β2 as a biomarker of hair loss.

TGF-β2 : MAGH complex form have strong H-bond D(H)..A(max dist) 3,4, salt bridge D(H)...A(max dist) 4, week H-bond D(H)..A(max dist) 3.8, acceptor-acceptor (max dist) 3, hydrogen-bong D-H 90, strong H-bond D(H)..A(max dist) 3,4, salt bridge D(H)...A(max dist) 4, week H-bond D(H)..A(max dist) 3.8, acceptor-acceptor (max dist) 3, hydrogen-bong D-H-A(min deg) 90, hydrogen-bong D-H-A(max deg) 180, hydrogen-bong D-H-Y(min deg) 90, hydrogen-bong D-H-Y(max deg) 180, hydrogen-bong X-D-A(min deg) 90, hydrogen-bong X-D-A(max deg) 90, hydrogen-bong D-A-Y(min deg) 90, hydrogen-bong D-A-Y(max deg) 180, acceptor-acceptor A-Lp-Lp-A torsion(max deg) 15, acceptor-acceptor A-Lp-A(min deg) 90, acceptor-acceptor A-Lp-A(max deg) 180, hydrogen bond with Sp donor X-D-A(min deg) 135, donor-donor H-H-D(min deg) 120, fluorine non-bond(max dist) 3.7, halogen (Cl,Br,I) VDW fraction(max) 1, halogen (Cl,Br,I) bond C-X-B(min deg) 120, halogen (Cl,Br,I) bond C-X-B(max deg) 180, halogen (Cl,Br,I) bond X-B-Y(min deg) 75, halogen (Cl,Br,I) bond X-B-Y(max deg) 180

Charge-charge(max dist) 5.6, Pi-Cation(max dist) 5, Pi-Cation(max deg) 40

Pi-Donor(max dist) 4.2 Pi-Donor(max deg) 45, Pi-Donor deviation(max deg) 40

Pi-Lone Pair(max dist) 3 Pi-Lone Pair(max deg) 45, Pi-Lone Pair devlation(max deg)40, Pi-Sigma(max dist) 4, Pi-Sigma(max deg) 45, Pi-Sulfur edge-on(min deg) 70

Pi-Sulfur face-on(max dist) 4.5, Pi-Sulfur face-on(max deg) 25

Pi-Pi centroid (max dist) 6, Pi-Pi closest atom(max dist) 4.5, Stacked Pi-Pi theta(max deg) 50, Stacked Pi-Pi gamma(max deg) 35, T-shaped Pi-Pi theta(max deg) 30, T-shaped Pi-Pi gamma(min deg) 55, Stacked Pi-Amide theta(max deg) 40

Stacked Pi-Amide gamma(max deg) 20, Alkyl centroid (max dist) 5.5, Sulface area scale factor 1 and X -7.20457, Y 14.0845, Z 8371292.

Structural biology approach which has been working with all diseases in the past has been found to be the fastest, cheapest and reliable to discover peptideagainst deadly diseases. We performed computer-aided peptide discovery process against the important proteins involved in the mechanism of action for TGF-β2. Our results show that MAGH, a herbal supplement and a plant based natural compound has the capability to inhibit TGF-β2 target proteins.

Acknowledgement

This research was supported by the Ministry of Trade, Industry & Energy (MOTIE), Korea Evaluation Institute of Industrial Technology (KEIT) through the Encouragement Proꠓgram for Bio industry core technology development project (Project NO: 20009105).

Conflict of Interest

The authors declare that they have no conflict of interest.

Fig 1.

Figure 1.Effect of ginseng berry-derived peptides and sequence-modified peptides on the proliferation of DP and HaCaT cells. (A) DP cells were treated with peptides (50 and 100 μg/mL) for 72 h. (B) HaCaT cells were treated with peptides (50 and 100 μg/mL) for 24 h. The proportion of living cells was measured via the MTT assay. All experiments were conducted in triplicate and the results are expressed as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001
Journal of Plant Biotechnology 2022; 49: 316-324https://doi.org/10.5010/JPB.2022.49.4.316

Fig 2.

Figure 2.Effects of ginseng berry-derived peptides and sequence-modified peptides on H2O2-induced ROS production in human HaCaT keratinocytes. The cells were pre-treated with or without peptides (100 μg/mL) for 3 h, after which they were induced with or without H2O2 (500 μM). After 3 h, the cells were harvested for ROS detection. ROS production was then measured as DCFH-DA-positive cell percentages. Similar results were obtained from three additional experiments. ###p < 0.001 compared to the cells without H2O2 treatment; **p < 0.01 and ***p < 0.001 compared to the cells treated with H2O2 alone
Journal of Plant Biotechnology 2022; 49: 316-324https://doi.org/10.5010/JPB.2022.49.4.316

Fig 3.

Figure 3.Three-dimensional structure of the TGF-β2 (A) and MAGH peptide (B)
Journal of Plant Biotechnology 2022; 49: 316-324https://doi.org/10.5010/JPB.2022.49.4.316

Fig 4.

Figure 4.Docking pose and ligand interaction diagram of the TGF-β2 protein and the MAGH ligand (gray balls and sticks). The interacting residues are labeled and shown in pink
Journal of Plant Biotechnology 2022; 49: 316-324https://doi.org/10.5010/JPB.2022.49.4.316

Fig 5.

Figure 5.Docking pose and ligand interaction diagram of the TGF-β2 protein and the MAGH ligand (A), close and extended TGF-β2 protein-MAGH ligand interaction (B), interaction group and interacting receptor group (C), and hydrophobic interaction (D)
Journal of Plant Biotechnology 2022; 49: 316-324https://doi.org/10.5010/JPB.2022.49.4.316

Fig 6.

Figure 6.In silico molecular 3D docking pattern of the TGF-β2 protein and molecular surface shape feature of the MAGH ligand
Journal of Plant Biotechnology 2022; 49: 316-324https://doi.org/10.5010/JPB.2022.49.4.316

Table 1 . Original and modified peptides of ginseng berry-derived peptides.

Peptides nameModificationAmino acid sequenceChemical formulaMolecular weight (g/mol)
GB-1OriginalMAGHC16H26N6O5S414.48
GB-2Sequence modificationMAHG
GB-3Sequence modificationMHGA
GB-4Sequence modificationMHAG
GB-5Sequence modificationMGHA
GB-6Sequence modificationMGAH

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