New Plant Extracts Exert Complementary Anti-Hair Loss Properties in Human In Vitro and Ex Vivo Models

Abstract

Background: Hair loss is linked to dysfunction of the growth (anagen), regression (catagen) and rest (telogen) phases of the hair follicle (HF) cycle.

Aims: To evaluate the effects of a Silybum marianum extract (SME), manganese PCA (MnPCA), and a Lespedeza capitata extract (LCE) on markers of hair growth and anchorage in human follicle dermal papilla cells (HFDPCs), and to investigate the ability of a topical serum containing these active ingredients to improve HF growth in an ex vivo human scalp skin model.

Methods: In HFDPCs, we assessed receptor tyrosine kinase phosphorylation and Wnt/β-catenin pathway activation; quantified versican, vascular endothelial growth factor (VEGF) and Dickkopf-1 (DDK1) secretion; and evaluated 5α-reductase (5αR) activity. Using scalp skin biopsies from two female donors, we measured hair shaft elongation, analyzed hair matrix keratinocyte proliferation and apoptosis, and determined HF cycle stage and score.

Results: Compared to untreated HFDPCs, SME upregulated phosphorylation of growth factor receptors (EGFR:1.9 × and PDGFR: 2.8 ×) and their downstream effectors (ERK, GSK3, Akt, and STAT: 1.2-2.0 ×); MnPCA enhanced versican (33.0 ×) and VEGF (3.3 ×) secretion, and stimulated the Wnt/β-catenin pathway (+80%); and LCE reduced DKK1 secretion (-72%) and 5αR activity (dihydrotestosterone/testosterone ratio: -60%). Compared to untreated scalp skin biopsies, the serum enhanced hair shaft elongation (+102%), and significantly prolonged the anagen phase by improving hair cycle scores and stimulating hair matrix keratinocyte proliferation (+58%).

Conclusions: SME, MnPCA, and LCE displayed complementary anti-hair loss properties. The serum combining these active ingredients may be useful in hair loss treatment.

Keywords: active ingredients; anchorage; dermal papilla; hair growth; hair loss; human hair follicle.

FIGURE 1

Phosphorylation of receptor tyrosine kinases (RTKs) and their downstream effectors in human follicle dermal papilla cells treated with a Silybum marianum extract (SME, 30 μg/mL, dissolved in dimethylsulfoxide [DMSO]) or DMSO for 1 h (n = 2 donors, tests performed in duplicate for DMSO‐treated and SME‐treated cells from each donor). (a) EGFR and PDGFRβ, and (b) the downstream phosphokinases ERK1/2, GSK3α/β, Akt1/2/3, STAT5α and STAT5α/β. Data were obtained using the Human Proteome Profiler Phospho‐RTK and Phospho‐Kinase array kits, respectively. Each bar represents the mean ± SEM of SME/DMSO ratio.

FIGURE 2

Effects of manganese PCA (MnPCA) on human follicle dermal papilla cells (HFDPCs). (a) Activation of the Wnt/β‐catenin pathway in untreated and MnPCA‐treated HFDPCs. Treated HFDPCs were incubated with 0.009% MnPCA for 24 h. Pathway activation was quantified by measuring the luminescence of HFDPCs transfected with a luciferase reporter gene under the control of a Wnt/β‐catenin promoter (n = 3 donors, tests performed in triplicate on untreated and treated cells from each donor). (b, c) The levels of versican and VEGF released into the culture medium of untreated and MnPCA‐treated HFDPCs. Versican and VEGF were quantified by immunoassays, 48 h and 24 h, respectively, after the addition of 0.002%–0.009% MnPCA to the treated HFDPCs (n = 3 donors, tests performed in triplicate on untreated and treated cells from each donor). Results show the mean ± SEM of donor triplicates. Percentage and fold increases versus untreated controls are also indicated. *p < 0.05; **p < 0.01 (one‐way ANOVA with a Dunnett's multiple comparison test on data normalized to the untreated controls). RLU, relative luminescence unit.

FIGURE 3

Effect of Lespedeza capitata extract (LCE) on Dickkopf‐1 (DDK1) production and 5α‐reductase (5αR) activity in human follicle dermal papilla cells (HFDPCs). (a) Levels of DDK1 in the culture medium of untreated and LCE‐treated HFDPCs. The levels of DDK1 released into the culture medium were quantified by immunoassay 24 h after the addition of LCE to the treated HFDPCs. (b) Dihydrotestosterone (DHT)/testosterone ratio reflecting 5αR activity in untreated, 10 μM finasteride‐treated (positive control) and 0.01% LCE‐treated HFDPCs. The levels of [¹⁴C]‐DHT and [¹⁴C]‐testosterone in the culture medium were quantified 24 h after addition of finasteride or LCE to the treated HFDPCs by autoradiography on radioactive thin layer chromatograms. Results show the mean ± SEM of tests performed in triplicate on HFDPCs from three donors (a) and two donors (b); the percentage decreases versus the untreated controls are also indicated. *p < 0.05; **p < 0.01 (one‐way ANOVA with a Dunnett's multiple comparison test on data normalized to the untreated controls).

FIGURE 4

Effect of the study serum on hair shaft elongation after 5 days of ex vivo culture of 6 mm‐scalp skin samples in a minimal medium ± topical treatment applied daily from Day 1 to Day 4. Each bar represents the mean ± SEM of the change in length of the hair follicles (HFs) from Day 1 to Day 5 (n = 21–34 HFs) from duplicate cultures from two donors. Percentage increases versus untreated controls are also indicated. **p < 0.01; ***p < 0.001 (unpaired t‐test).

FIGURE 5

(a) Hair cycle stages, (b) associated hair cycle scores [35], and (c, d) keratin 75 (K75) expression in hair follicles (HFs) after 5 days of ex vivo culture of 6‐mm scalp skin samples in a minimal medium ± topical treatment applied daily from Day 1 to Day 4. (a, b, c) Each bar represents the mean ± SEM of analyses performed on duplicate cultures from samples obtained from two donors, and the percentage increase versus the untreated control is also indicated; (a, b) n = 30–36 HFs and (c) n = 65–82 HFs. (d) Representative confocal microscopy image of K75 immunostaining (in red) and DAPI counterstaining (in blue) of a HF from one donor. *p < 0.05; **p < 0.01 (unpaired t‐test).

FIGURE 6

Hair matrix keratinocyte proliferation and apoptosis in untreated and treated ex vivo skin samples cultures. (a) Hair matrix keratinocyte proliferation assessed by quantifying the percentage of Ki‐67 + keratinocytes. (b) Hair matrix keratinocyte apoptosis assessed by quantifying the percentage of TUNEL+ keratinocytes. Ex vivo cultures of 6‐mm scalp skin samples were grown in a minimal medium ± topical treatment applied daily from Day 1 to Day 4. Data shown are the mean ± SEM fold changes in the percentages of Ki‐67+ or TUNEL+ keratinocytes in the treated cultures relative to the untreated cultures at Day 5, with data for the untreated cultures normalized to 1. Cultures were obtained from two donors and all tests were performed in duplicate. ns, not significant; *p < 0.05; **p < 0.01 (unpaired t‐test).