Gynostemma pentaphyllum Makino extract induces hair growth and exhibits an anti‐graying effect via multiple mechanisms

In traditional Asian medicine, Gynostemma pentaphyllum Makino leaf extract (Gp) is used to treat aging, metabolic syndrome, diabetes, and neurodegenerative diseases. Hair loss and hair‐graying are common phenomena that haunt everyone. However, whether Gp activities on inhibition of hair loss and getting gray have been rarely studied.


| INTRODUC TI ON
In general, having thick and dark hair indicate good health and youth.
Different forms of hair loss, including androgenetic alopecia and alopecia areata, are considered serious psychological and cosmetic issues.Data from the American Hair Loss Association have indicated that the market for hair loss treatment is >$3.5 billion each year in the United States. 1,2The growth cycle of human hair comprises anagen (growth), catagen (degeneration), and telogen (rest). 3During hair follicle differentiation, proliferation, and cycle control, human dermal papilla cells (hDPCs) play an important role. 4nasteride and minoxidil (MXD) are well-known medications used to treat hair loss.Finasteride, a 5α-reductase inhibitor, is prescribed for the treatment of alopecia; it acts by suppressing testosterone.However, studies have reported that it leads to sexual dysfunction in some users. 5,6Although MXD is also widely used, its exact mechanism of action remains unclear. 7Recently, natural products that promote hair growth are being continuously discovered. 8rious methods, including the use of herbal topical formulations for hair growth promotion or hair loss treatment, have long been anticipated.These natural substances are low cost, easily available, exhibit fewer side effects, and function via multiple biochemical pathways. 9,10 East and Southeast Asia, Gynostemma pentaphyllum Makino (Gp, Jiao-Gu-Lan in Chinese) leaves as perennial creepers are widely distributed and used as herbal medicines for treating diabetes mellitus, neurodegenerative diseases, gastricism, and inflammation for centuries.In China, it is traditionally known as southern ginseng and an "immortality herb". 11,12However, no studies have reported the stimulating effects of Gp extract on hair growth.Therefore, in the present study, we investigated the effects of Gp on hair loss in vitro and in vivo using hDPCs cultures and the C57BL/6 mouse model.
In addition to hair loss, loss of melanin in the hair and skin, resulting in graying of hair, is a point of concern for everyone. 13,14Natural hair color is considered a symbol of health and youth, whereas hair graying indicates advanced age; at present, hair repigmentation cannot be medically treated. 15During the anagen phase, mature melanocytes that differentiate from melanocyte stem cells in the hair follicle bulge synthesize and secrete melanin, deepening hair color. 1,16Microphthalmia-associated transcription factor (MITF) functions as a critical melanogenesis regulator, and tyrosinase (TYR) and tyrosine-related protein-2 (TRP-2) are central enzymes that affect the quantity and quality of melanin. 17TYR oxidizes tyrosine into DOPA and then converts it into DOPA quinone and dopachrome. 18By interacting with TRP-2, dopachrome is converted to 5,6-dihydroxyindole carboxylic acid (DHICA); finally, DHICA is converted to eumelanin via tyrosine-related protein-1 (TRP-1).At the final stage, pheomelanin is synthesized when DOPA quinone binds to cysteine, and hair color is determined based on their ratio. 17,18cently, age-related hair graying has been considered as a symptom resulting from DNA damage and melanocyte apoptosis in hair follicles and reactive oxygen species-induced cell damage around hair follicles, resulting in a decrease in the number of melanocytes and inhibition of melanin production. 191][22] People in modern society face all kinds of life pressures; therefore, we also determined the ability of Gp to protect melanocyte viability from norepinephrine stress by measuring melanocyte viability, melanin content, intracellular TYR activity, and mRNA expression of TYR, TRP-1, TRP-2, and MITF in B16 cells.
In the present study, we used Gp in the field of hair health and investigated its mechanism of action.We noted that the hair lossrelated gene TGF-β1 was downregulated, whereas β-catenin, an essential gene in the canonical Wnt signaling pathway, 23 was upregulated.Further, Wnt 5a, which activates the noncanonical Wnt signaling pathway, 24 was upregulated, suggesting that Gp promotes the expression of signature genes in hDPCs.Next, we explored Gp applications in a C57BL/6 mice model; histological differences revealed that entry into the catagen phase was delayed after Gp treatment.Moreover, in B16 melanocytes, Gp exhibited a protective effect against the deleterious effects of exposure to norepinephrine stress, demonstrating the utility of Gp as a potential active ingredient to protect melanocytes.Taken together, these findings indicate that Gp plays an important role in promoting hair growth and inhibiting hair graying.

| Reagents
Gynostemma pentaphyllum Makino leaf extract was purchased from Nanjing DASF Biotechnology Co. Ltd.The voucher specimen (WUK0401501) was deposited in the Herbarium of Northwest A&F University.hDPCs and B16 cells were obtained from ScienCell Research Laboratories, Inc.

| hDPCs growth test and melanocyte viability assay
Cell Counting Kit-8 (CCK-8, Shanghai Hanheng Biotechnology Co. Ltd.) was used to measure cell proliferation and viability.hDPCs or B16 cells were seeded into 96-well plates at a density of 3000 cells/ well and incubated under 5% CO 2 and humidified air at 37°C.The cultured hDPCs cells were treated with different concentrations of Gp (0.025, 0.05, 0.1, 0.2, and 0.4 mg/mL) for 48 h.On the other hand, cultured B16 cells were treated with norepinephrine (1 μM) for 24 h and then with different concentrations of Gp (0.1, 0.2, 0.4, 0.8, 1.6, 3.2, and 6.4 mg/mL) for 48 h.The CCK-8 solution was added, and the cells were incubated for 4 h.Absorbance was measured at 450 nm using the TECAN Enzyme standard instrument Spark 10M.Cell proliferation or viability rates were calculated and compared with the OD percentages of the control samples.

| Measurement of melanin content and intracellular TYR activity
In B16 cells, intracellular melanin content was measured according to the method described by Tsuboi et al. 25 Notably, in this study, B16 cells were pretreated with norepinephrine (1 μM) before adding different concentrations of Gp (0.4, 0.8, and 1.6 mg/mL) and treating the cells for 48 h.The treated cells were solubilized in 1 M NaOH for 60 min at 60°C.Then, melanin content was determined by measuring the absorbance at 475 nm.
In B16 cells, intracellular TYR activity was measured according to the steps described by Yang et al. 26 The cells were treated with norepinephrine (1 μM) and different concentrations of Gp (0.4, 0.8, and 1.6 mg/mL) for 48 h.Freshly prepared 0.1% L-DOPA solution in phosphate-buffered saline (PBS) was mixed with 100 μL of the cells.
Then, the cells were incubated at 37°C for 30 min, followed by absorbance measurements at 492 nm.

| Quantitative real-time polymerase chain reaction (RT-PCR)
The mRNA expression of TGF-β1 and CTNNB1 in hDPCs and that of MITF, TYR, TRP-1, and TRP-2 in B16 cells were measured using quantitative RT-PCR after treating the cells with various concentrations of Gp for 48 h.On the other hand, B16 cells were exposed to norepinephrine (1 μM) before treating the cells with various concentrations of Gp.The negative control (NC) was cultured in a medium containing norepinephrine.Monzol™ Reagent (Monad Biotech Co., Ltd) was used to isolate total RNA according to the manufacturer's instructions.

| Western blotting
After rinsing the cultured cells two times with precooled (at 4°C) PBS, they were collected and lysed with RIPA buffer (Beyotime) containing protease inhibitors (Beyotime).Protein content was quantified using the BCA protein concentration determination kit (Beyotime).Then, they were separated via SDS-PAGE.The blots were electroblotted onto polyvinylidene fluoride membranes.Primary antibodies and horseradish peroxidase-conjugated secondary antibodies were added to the membranes after blocking with 5% nonfat milk at 25°C for 2 h.Rabbit antiβ-catenin, rabbit anti-Wnt5a, rabbit anti-TGF-β1, and rabbit anti-tubulin (1:1000, Proteintech Group, Inc.) were used as the primary antibodies.BeyoECL Star (Beyotime) reagent was used for protein detection.

| Animals and histological analysis of depilated mice
All animal experiments were approved by the Academic Ethics Committee of Anhui Normal University.During the experiments, the animals were housed in a temperature-controlled room with a 12-h light/dark cycle and provided ad libitum access to food and water.Twenty-five 4-week-old male C57BL/6 mice (Henan Sikebas Biotechnology Co. Ltd.) were randomly divided into five groups (five in each group).Approximately 2 cm wide and 2 cm long hair on the dorsal areas of the mouse in the telogen phase was shaved 24 h before treatment.Gp solutions of different concentrations were prepared in water.Five groups with five randomized mice were used.
The animals in Group 1 served as NC (water), and Group 2, which received 20 mg/mL MXD, was the positive control group.The animals in Groups 3, 4, and 5 were treated with different doses of Gp (5, 10, and 20 mg/mL).A 0.2 mL dose was applied to the test area every day for 28 days.The back skin of the mice was observed and photographed to assess the hair regrowth ability of Gp.
On Day 29, all mice were euthanized via cervical dislocation, followed by excision of the dorsal skin tissues of each mouse.These tissues were fixed with 4% neutral paraformaldehyde for 24 h and then embedded in paraffin.Using the standard hematoxylin and eosin staining protocol, 5μm tissue sections were stained to histologically evaluate hair follicles. 32Hematoxylin dyes nucleic acids purple, whereas eosin dyes the cytoplasm and extracellular matrix pink.The histological morphology of hair follicles was evaluated by qualitatively comparing the observations and photographs.

| Statistical analysis
SPSS WIN (v25.0)software was used to statistically analyze the data.For comparison among groups, Student's t-test was used.The experiments were performed in triplicate.p-values < 0.05 were considered statistically significant.

| Proliferation assay of hDPCs
The CCK-8 assay revealed that Gp (0, 0.025, 0.05, 0.1, 0.2, and 0.4 mg/mL) dose-dependently exhibited proliferative effects on hDPCs (Figure 1).The number of hDPCs was markedly higher in respectively.However, no significant difference (105.1%) was observed at 0.025 mg/mL.Taken together, these results suggest that Gp promotes the proliferation of hDPCs and improves their ability to induce hair growth.

| Quantitative RT-PCR results for hDPCs
Previous studies have reported that hDPCs play a vital role in the reconstruction of hair follicles. 33,34To explore the specific mechanism of action by which Gp activates hDPCs, we evaluated the effects of Gp on Wnt signaling pathway activity and TGF-β1 expression.
In hair follicles, the noncanonical Wnt family member Wnt5a and canonical Wnt genes such as LEF1, CTNNB1, and DKK1 were expressed in cells in the telogen stage and were demonstrated to be important for controlling hair cell fate. 35The Wnt signaling pathway has been implicated in the transcriptional regulation of hair growth 32,[36][37][38] ; therefore, our results suggest that the Wnt A study has reported androgen-inducible TGF-β1 as a hair growth suppression-related factor. 39In other words, TGF-β1 decreased the growth of hDPCs.However, the addition of Gp to the culture counteracted the effect of TGF-β1.Gp at concentrations of 0.05, 0.1, 0.2, and 0.4 mg/mL exhibited 24% (**), 55% (**), 37% (**), and 11% (*) lower TGF-β1 mRNA expression compared with the NC, respectively (Figure 2).
However, it is vital to study other related factors; moreover, DPC proliferation and hair growth are still governed by various mechanisms.Therefore, the detailed mechanisms by which Gp promotes the growth and proliferation of hDPCs in vivo should be studied further.

| Topical Gp treatment induced hair regeneration
Hair regeneration was tested using the C57BL/6 mouse dorsal skin model.During the telogen stage of the hair follicles of the dorsal skin, the back hair of 44-day-old male mice was shaved.Treatments were topically applied every day.In NC mice, only a few scattered pigmented spots were observed at least until Day 14.After 7 days, the hair on the mice in the Gp (5, 10, and 20 mg/mL) and 20 mg/mL MXD groups regrew and became remarkably gray (Figure 4).Histological analysis of Gp-treated mice revealed hair follicle formation and differentiation.
F I G U R E 3 Different concentrations of Gp affect the expression levels of β-catenin, Wnt 5a, and TGF-β1 protein in hDPCs.Cells were treated with Gp from 0.05 to 0.4 mg/mL for 48 h.Protein extracts were prepared from each treatment groups and analyzed by western blotting analysis and hybridized with specific antibodies indicated in Panels (A).β-tubulin was used as a protein-loading control.Densitometric quantitations were showed in Panels (B).*p < 0.05, **p < 0.01, compared with the negative control.
F I G U R E 4 C57BL/6 male mouse model demonstrated the stimulatory effects of Gp on hair growth.On postnatal day 44, mice were shaved and topically treated with MXD, NC or Gp (dissolved in water at 5, 10 and 20 mg/mL final) every day over 28 days.Photographs shown were taken on Day 0, 7, 14, 21 and 28 posttreatment.Melanin pigmentation was visible in the skin of Gp and MXD treated animals by Day 7 indicating the induction of anagen by the treatment.NC mice did not show significant pigmentation for at least 14 days; However, in Day 28, all mice in NC, Gp, or MXD treatments resulted in almost full hair growth.

| Histological analysis of depilated mice
The morphology of hair follicles was histologically evaluated to determine the effects of Gp on hair growth in vitro.Histological images were used to qualitatively analyze follicle number, size, and depth in the NC, MXD, and Gp groups (Figure 5).The 10 and 20 mg/ mL Gp groups exhibited deeper hair follicles, thicker dermal layers, and longer hair follicular shafts than the NC and MXD groups.
These findings suggest that Gp maintains the anagen phase of the hair cycle and prevents the entry of hair follicles into the catagen phase.

| Quantitative RT-PCR analysis of B16 cells
B16 cells were exposed to Gp (0.4, 0.8, and 1.6 mg/mL) to determine how Gp treatment contributes to the upregulation of melanogenesisrelated genes.The mRNA expression of melanogenesis marker genes such as MITF, TYR, TRP-1, and TRP-2 was markedly increased by Gp treatment in a dose-dependent manner (Figure 8).

| D ISCUSS I ON AND CON CLUS I ON S
Gynostemma pentaphyllum Makino leaf extract is a naturally brewed tea and herbal medicine in China with several physiological and pharmacological activities. 12Recent study has reported that the saponins in G. pentaphyllum induce melanogenesis and activate the cAMP/PKA and Wnt signaling pathways in B16 and B16F10 melanocytes. 11Furthermore, a study reported that stress accelerates hair graying 43
Taken together, these findings indicate that Gp can promote hDPCs activity at the genetic level.Next, we performed western blotting to analyze if Gp promotes β-catenin, Wnt5a, and TGF-β1 at the protein level.F I G U R E 1 Gp proliferative effects on hDPCs were assessed with different concentrations of Gp (0, 0.025, 0.05, 0.1, 0.2 and 0.4 mg/mL).The CCK-8 assay was used to determine cell viability on the second day.Data are reported as mean + SEM.Student's t-test was used to compare data.*p < 0.05, **p < 0.01.F I G U R E 2 Expression of DKK1, LEF1, Wnt 5a, β-catenin, which were Wnt-signaling pathway-related genes, and TGF-β1 gene in hDPCs.The hDPCs (2 × 10 5 cells/well) were cultured with Gp at 0.05, 0.1, 0.2, and 0.4 mg/mL concentrations for 48 h.Values are mean SD of triplicate experiments.*p < 0.05, **p < 0.01 compared with control.

3. 7 |
Effects of Gp on B16 melanin content and intracellular B16 TYR activityMelanin is widely distributed in the human skin; it determines hair color.40B16 cells were treated with norepinephrine to analyze the effect of Gp or IBMX on melanin production.Figure 7A demonstrates that melanin content was significantly increased in B16 cells after Gp treatment (melanin content was represented as a percentage of the control).Positive pigmentation standards were used with IBMX, whereas norepinephrine was used as the depigmenting standard agent.B16 cells without Gp or norepinephrine treatment were considered the NC group.After 48 h, at Gp concentrations of 0.4, 0.8, and 1.6 mg/mL, the melanin content was 113.96% (*), 129.67% (**), and 166.28% (**), respectively.Furthermore, the melanin content was 106.05% (*) after 48 h of IBMX (35 μM) treatment.Moreover, the melanin content decreased to 83.82% (**) after 24 h of norepinephrine (1 μM) treatment.The results suggest that 1.6 mg/ mL Gp exhibits a much higher stimulating effect on melanin formation than the positive control IBMX.

Figure
Figure 7B demonstrates that Gp treatment significantly increased the intracellular TYR activity of B16 cells in a dose-dependent manner after the cells were treated with norepinephrine (1 μM) for 24 h.After 48 h, at Gp concentrations of 0.4, 0.8, and 1.6 mg/mL and 35 μM IBMX (positive control), the intracellular TYR activity was ; however, Gp may be related to emotional relief in traditional Chinese medicine.The ethanol extract of G. pentaphyllum Makino leaves exhibited anxiolytic effects F I G U R E 6 As indicated, Gp proliferative effects on B16 cells were tested at different concentrations.CCK-8 assay was used to measure the viability of the cells, as well as the proliferation index after 48 h.NC: negative control (DMEM medium), Gp in various concentrations (0.1, 0.2, 0.4, 0.8, 1.6, 3.2, and 6.4 mg/mL) were applied to hDPCs.Data are reported as mean + SEM.Student's t-test was used to compare data.*p < 0.05, **p < 0.01.F I G U R E 7 (A) In B16 cells, the effects of Gp on melanin content are shown as a percentage of the NC, and the results are presented as the means with standard deviation of three different tests.Student's t-test was used to compare data.*p < 0.05, **p < 0.01.(B) In B16 cells, the effects of Gp on intracellular B16 tyrosinase activity are shown as a percentage of the NC, and the results are presented as the means with standard deviation of three different tests.Student's t-test was used to compare data.*p < 0.05, **p < 0.01. in a mouse model of chronic stress. 44By promoting the expression of TYR, MITF, TRP-1, and TRP-2, Gp enhances TYR activity and melanin production in B16 cells exposed to norepinephrine stress.Our findings suggest that Gp can be used to develop anti-graying hair products for individuals under the pressure of modern life.With a novel antiaging effect, Gp promotes hair growth and attenuates the effects of stress factors.We demonstrated that Gp promotes hair growth by acting on the TGF-β1 and Wnt signaling pathways.Although we proposed and confirmed the effect of Gp on hair growth at the cellular and animal levels, clinical trials involving humans are warranted; moreover, the activity of Gp in melanocytes should be studied in animal models and human clinical trials to explore its application potential.F I G U R E 8 RT-qPCR for MITF, TYR, TRP-1, and TRP-2 in Gp-treated B16 cells.B16 cells (3.0 × 10 4 cells/well) were cultured in serum-free DMEM for 24 h and then treated with Gp at concentrations of 0.4, 0.8, and 1.6 mg/mL for 48 h.*p < 0.05, **p < 0.01, compared with NC.