The extract of Cyperus rotundus rhizome promotes hair growth and modulates hair cycle in vivo and in vitro

Abstract

Hair loss, a multifactorial disorder characterized by follicular miniaturization and excessive shedding, significantly impairs psychological well-being and quality of life. Cyperus rotundus rhizome (CR), a traditional Chinese medicine used for various ailments, has not been evaluated for efficacy in treating hair loss. This study presents the first comprehensive assessment of the hair growth-promoting effects of ethanol extract from CR on mouse primary dermal papilla cells (MDPCs) and human immortalized hair DPCs (IHHDPCs), employing cell counting kit-8 (CCK-8), scratch assay, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and Western blot (WB). CR treatment activated the Wnt/β-Catenin signaling pathway by upregulating Wnt10b, increasing β-Catenin protein levels and promoting its nuclear translocation, while simultaneously downregulating transforming growth factor-beta 1 (TGF-β1), BMP4, and dickkopf-related protein 1 (DKK1) in MDPCs. These molecular changes enhanced cell proliferation and increased secretion of key growth factors—insulin-like growth factor 1 (IGF1), keratinocyte growth factor (KGF), and vascular endothelial growth factor (VEGF)—thereby stimulating hair growth and prolonging the anagen phase, which was confirmed in an ex vivo hair follicle (HF) organ culture model. Chromatographic analysis identified the petroleum ether fraction (CRP), enriched in sesquiterpenes, as the primary bioactive component. Both CR and CRP promoted IHHDPC proliferation, migration, and growth factor expression through activation of the Wnt/β-Catenin pathway, with CRP exhibiting superior bioactivity. Furthermore, both treatments stimulated HF cycling, increased follicular density, and upregulated Ki67 and β-Catenin expression in the dorsal skin of C57BL/6 mice. Collectively, these findings demonstrate that CR and CRP promote hair growth and modulate the hair cycle via enhancement of Wnt/β-Catenin signaling, providing a scientific basis for the potential clinical application of C. rotundus rhizomes in hair loss therapy and the development of related pharmaceuticals or cosmetics.

Keywords

Cyperus rotundus / Hair growth / Hair cycle / Wnt/β-Catenin

Fundign

This work was supported by the Start-up Fund of China Pharmaceutical University (No. 3150020057).

Supporting information

Supporting information of this paper, including the schematic diagram of the isolation process of primary MDPCs (Fig. S1), the identification of them (Fig. S2), the TIC of CR analysis by LC-TOF-MS/MS (Fig. S3), the primers sequences of RT-qPCR analyses in this study (Table S1), the LC-Q-TOF-MS/MS ion fragment data (Tables S2 and S3), the assay method, methodology, and result of cyperenone, α-cyperone, and nootkatone (Fig. S4 and Table S4), the TLC analysis of CRP-1−CRP-5 (Fig. S5), can be requested by sending E-mail to the corresponding author.

Declaration of competing interest

The authors declare no competing financial interest.

References

[1] Park S, Lee J. Modulation of hair growth promoting effect by natural products. Pharmaceutics. 2021; 13(12):2163-2187. https://doi.org/10.3390/pharmaceutics13122163.

[2] Lin X, Zhu L, He J. Morphogenesis, growth cycle and molecular regulation of hair follicles. Front Cell Dev Biol. 2022; 10:899095. https://doi.org/10.3389/fcell.2022.899095.

[3] Guerrero-Juarez CF, Plikus MV. Emerging nonmetabolic functions of skin fat. Nat Rev Endocrinol. 2018; 14(3):163-173. https://doi.org/10.1038/nrendo.2017.162.

[4] Paus R, Muller-Rover S, van der Veen C, et al. A comprehensive guide for the recognition and classification of distinct stages of hair follicle morphogenesis. J Invest Dermatol. 1999; 113(4):523-532. https://doi.org/10.1046/j.1523-1747.1999.00740.x.

[5] Natarelli N, Gahoonia N, Sivamani RK. Integrative and mechanistic approach to the hair growth cycle and hair loss. J Clin Med. 2023; 12(3):893. https://doi.org/10.3390/jcm12030893.

[6] Dong TR, Li YJ, Jin SY, et al. Progress on mitochondria and hair follicle development in androgenetic alopecia: relationships and therapeutic perspectives. Stem Cell Res Ther. 2025; 16(1):44. https://doi.org/10.1186/s13287-025-04182-z.

[7] Deng Z, Chen M, Liu F, et al. Androgen receptor-mediated paracrine signaling induces regression of blood vessels in the dermal papilla in androgenetic alopecia. J Invest Dermatol. 2022; 142(8):2088-2099. https://doi.org/10.1016/j.jid.2022.01.003.

[8] Madaan A, Verma R, Singh AT, et al. Review of hair follicle dermal papilla cells as in vitro screening model for hair growth. Int J Cosmetic Sci. 2018; 40(5):429-450. https://doi.org/10.1111/ics.12489.

[9] Gentile P, Garcovich S. Advances in regenerative stem cell therapy in androgenic alopecia and hair loss: Wnt pathway, growth-factor, and mesenchymal stem cell signaling impact analysis on cell growth and hair follicle development. Cells-Basel. 2019; 8(5):466-487. https://doi.org/10.3390/cells8050466.

[10] Huang P, Yan R, Zhang X, et al. Activating Wnt/β-Catenin signaling pathway for disease therapy: challenges and opportunities. Pharmacol Therapeut. 2019; 196:79-90. https://doi.org/10.1016/j.pharmthera.2018.11.008.

[11] Lim X, Nusse R.Wnt signaling in skin development, homeostasis, and disease. Csh Perspect Biol. 2013; 5(2):a8029. https://doi.org/10.1101/cshperspect.a008029.

[12] Liu J, Mu Q, Liu Z, et al. Melatonin regulates the periodic growth of cashmere by upregulating the expression of Wnt10b and β-Catenin in inner mongolia cashmere goats. Front Genet. 2021; 12:665834. https://doi.org/10.3389/fgene.2021.665834.

[13] Lei M, Lai X, Bai X, et al. Prolonged overexpression of Wnt10b induces epidermal keratinocyte transformation through activating EGF pathway. Histochem Cell Biol. 2015; 144(3):209-221. https://doi.org/10.1007/s00418-015-1330-6.

[14] Zhao B, Li J, Zhang X, et al. Exosomal miRNA-181a-5p from the cells of the hair follicle dermal papilla promotes the hair follicle growth and development via the Wnt/β-Catenin signaling pathway. Int J Biol Macromol. 2022; 207:110-120. https://doi.org/10.1016/j.ijbiomac.2022.02.177.

[15] Enshell-Seijffers D, Lindon C, Kashiwagi M, et al. β-Catenin activity in the dermal papilla regulates morphogenesis and regeneration of hair. Dev Cell. 2010; 18(4):633-642. https://doi.org/10.1016/j.devcel.2010.01.016.

[16] Rishikaysh P, Dev K, Diaz D, et al. Signaling involved in hair follicle morphogenesis and development. Int J Mol Sci. 2014; 15(1):1647-1670. https://doi.org/10.3390/ijms15011647.

[17] Dou J, Zhang Z, Xu X, et al. Exploring the effects of Chinese herbal ingredients on the signaling pathway of alopecia and the screening of effective Chinese herbal compounds. J Ethnopharmacol. 2022; 294(10):115320. https://doi.org/10.1016/j.jep.2022.115320.

[18] Plikus MV, Mayer JA, de la CD, et al. Cyclic dermal BMP signalling regulates stem cell activation during hair regeneration. Nature. 2008; 451(7176):340-344. https://doi.org/10.1038/nature06457.

[19] Greco V, Chen T, Rendl M, et al. A two-step mechanism for stem cell activation during hair regeneration. Cell Stem Cell. 2009; 4(2):155-169. https://doi.org/10.1016/j.stem.2008.12.009.

[20] Vasserot AP, Geyfman M, Poloso NJ. Androgenetic alopecia: combing the hair follicle signaling pathways for new therapeutic targets and more effective treatment options. Expert Opin Ther Tar. 2019; 23(9):755-771. https://doi.org/10.1080/14728222.2019.1659779.

[21] Bayati P, Taherian M, Soleimani M, et al. Induced pluripotent stem cells modulate the Wnt pathway in the bleomycin-induced model of idiopathic pulmonary fibrosis. Stem Cell Res Ther. 2023; 14(1):343. https://doi.org/10.1186/s13287-023-03581-4.

[22] Kamiya N. The role of BMPs in bone anabolism and their potential targets sost and DKK1. Curr Mol Pharmacol. 2012; 5(2):153-163. https://doi.org/10.2174/1874467211205020153.

[23] Alessandrini A, Bruni F, Piraccini BM, et al. Common causes of hair loss-clinical manifestations, trichoscopy and therapy. J Eur Acad Dermatol. 2021; 35(3):629-640. https://doi.org/10.1111/jdv.17079.

[24] Devjani S, Ezemma O, Kelley KJ, et al.Androgenetic alopecia: therapy update. Drugs. 2023; 83(8):701-715. https://doi.org/10.1007/s40265-023-01880-x.

[25] Choi JY, Boo MY, Boo YC. Can plant extracts help prevent hair loss or promote hair growth? A review comparing their therapeutic efficacies, phytochemical components, and modulatory targets. Molecules. 2024; 29(10):2288. https://doi.org/10.3390/molecules29102288.

[26] Hwang SB, Park HJ, Lee BH. Hair-growth-promoting effects of the fish collagen peptide in human dermal papilla cells and C57BL/ 6 mice modulating Wnt/β-Catenin and BMP signaling pathways. Int J Mol Sci. 2022; 23(19):11904. https://doi.org/10.3390/ijms231911904.

[27] Datta K, Singh AT, Mukherjee A, et al. Eclipta Alba extract with potential for hair growth promoting activity. J Ethnopharmacol. 2009; 124(3):450-456. https://doi.org/10.1016/j.jep.2009.05.023.

[28] Iwabuchi T, Ogura K, Hagiwara K, et al. Ginsenosides in Panax Ginseng extract promote anagen transition by suppressing BMP4 expression and promote human hair growth by stimulating follicle-cell proliferation. Biol Pharm Bull. 2024; 47(1):240-244. https://doi.org/10.1248/bpb.b23-00276.

[29] Peerzada AM, Ali HH, Naeem M, et al. Cyperus Rotundus L.: traditional uses, phytochemistry, and pharmacological activities. J Ethnopharmacol. 2015; 174(4):540-560. https://doi.org/10.1016/j.jep.2015.08.012.

[30] Xue BX, He RS, Lai JX, et al. Phytochemistry, data mining, pharmacology, toxicology and the analytical methods of Cyperus Rotundus L. (Cyperaceae): a comprehensive review. Phytochem Rev. 2023; 2023:1-46. https://doi.org/10.1007/s11101-023-09870-3.

[31] Zhang H, Shi Q, Nan W, et al. Ginkgolide B and bilobalide promote the growth and increase β-Catenin expression in hair follicle dermal papilla cells of American minks. Biofactors. 2019; 45(6):950-958. https://doi.org/10.1002/biof.1562.

[32] Topouzi H, Logan NJ, Williams G, et al. Methods for the isolation and 3D culture of dermal papilla cells from human hair follicles. Exp Dermatol. 2017; 26(6):491-496. https://doi.org/10.1111/exd.13368.

[33] Gan Y, Wang H, Du L, et al. Ficoll density gradient sedimentation isolation of pelage hair follicle mesenchymal stem cells from adult mouse back skin: a novel method for hair follicle mesenchymal stem cells isolation. Stem Cell Res Ther. 2022; 13(1):372-385. https://doi.org/10.1186/s13287-022-03051-3.

[34] Limbu S, Higgins CA. Isolating dermal papilla cells from human hair follicles using microdissection and enzyme digestion. Methods Mol Biol. 2020; 2154:91-103. https://doi.org/10.1007/978-1-0716-0648-3_8.

[35] Yang CC, Cotsarelis G. Review of hair follicle dermal cells. J Dermatol Sci. 2010; 57(1):2-11. https://doi.org/10.1016/j.jdermsci.2009.11.005.

[36] Ha EJ, Yun JH, Si C, et al. Application of ethanol extracts from Alnus Sibirica Fisch. Ex Turcz in hair growth promotion. Front Bioeng Biotech. 2021; 9:673314. https://doi.org/10.3389/fbioe.2021.673314.

[37] Fu D, Huang J, Li K, et al. Dihydrotestosterone-induced hair regrowth inhibition by activating androgen receptor in C57BL/6 mice simulates androgenetic alopecia. Biomed Pharmacother. 2021; 137:111247. https://doi.org/10.1016/j.biopha.2021.111247.

[38] Fu H, Li W, Weng Z, et al. Water extract of Cacumen platycladi promotes hair growth through the Akt/Gsk3β/β-Catenin signaling pathway. Front Pharmacol. 2023; 14:1038039. https://doi.org/10.3389/fphar.2023.1038039.

[39] Morgan BA. The dermal papilla: an instructive niche for epithelial stem and progenitor cells in development and regeneration of the hair follicle. Csh Perspect Med. 2014; 4(7):a15180. https://doi.org/10.1101/cshperspect.a015180.

[40] Junlatat J, Sripanidkulchai B. Hair growth-promoting effect of Carthamus tinctorius floret extract. Phytother Res. 2014; 28(7):1030-1036. https://doi.org/10.1002/ptr.5100.

[41] Li Y, Sheng Y, Liu J, et al. Hair-growth promoting effect and anti-inflammatory mechanism of Ginkgo biloba polysaccharides. Carbohyd Polym. 2022; 278(15):118811. https://doi.org/10.1016/j.carbpol.2021.118811.

[42] Hyun J, Im J, Kim S, et al. Morus alba root extract induces the anagen phase in the human hair follicle dermal papilla cells. Pharmaceutics. 2021; 13(8):1155. https://doi.org/10.3390/pharmaceutics13081155.

[43] Lee E, Seo HD, Kim D, et al. Millet seed oil activates β-Catenin signaling and promotes hair growth. Front Pharmacol. 2023; 14:1172084. https://doi.org/10.3389/fphar.2023.1172084.

[44] Ryu YC, Lee DH, Shim J, et al. KY19382, a novel activator of Wnt/β-Catenin signaling, promotes hair regrowth and hair follicle neogenesis. Brit J Pharmacol. 2021; 178(12):2533-2546. https://doi.org/10.1111/bph.15438.

[45] Choi BY. Targeting Wnt/β-catenin pathway for developing therapies for hair loss. Int J Mol Sci. 2020; 21(14):4915-4931. https://doi.org/10.3390/ijms21144915.

[46] Papukashvili D, Rcheulishvili N, Liu C, et al. Perspectives on miRNAs targeting DKK1 for developing hair regeneration therapy. Cells-Basel. 2021; 10(11):2957-2993. https://doi.org/10.3390/cells10112957.

[47] Robinson M, Reynolds AJ, Jahoda CA. Hair cycle stage of the mouse vibrissa follicle determines subsequent fiber growth and follicle behavior in vitro. J Invest Dermatol. 1997; 108(4):495-500. https://doi.org/10.1111/1523-1747.ep12289730.

[48] Muller-Rover S, Handjiski B, van der Veen C, et al. A comprehensive guide for the accurate classification of murine hair follicles in distinct hair cycle stages. J Invest Dermatol. 2001; 117(1):3-15. https://doi.org/10.1046/j.0022-202x.2001.01377.x.

[49] Kim J, Kim SR, Choi YH, et al. Quercitrin stimulates hair growth with enhanced expression of growth factors via activation of MAPK/CREB signaling pathway. Molecules. 2020; 25(17):4004-4018. https://doi.org/10.3390/molecules25174004.

[50] Shin K, Choi H, Song SK, et al. Nanoemulsion vehicles as carriers for follicular delivery of luteolin. Acs Biomater Sci Eng. 2018; 4(5):1723-1729. https://doi.org/10.1021/acsbiomaterials.8b00220.

[51] Hwang SY, Deng X, Byun S, et al. Direct targeting of β-Catenin by a small molecule stimulates proteasomal degradation and suppresses oncogenic Wnt/β-Catenin signaling. Cell Rep. 2016; 16(1):28-36. https://doi.org/10.1016/j.celrep.2016.05.071.

[52] Ohn J, Kim KH, Kwon O. Evaluating hair growth promoting effects of candidate substance: a review of research methods. J Dermatol Sci. 2019; 93(3):144-149. https://doi.org/10.1016/j.jdermsci.2019.02.004.

[53] Williams R, Pawlus AD, Thornton MJ. Getting under the skin of hair aging: the impact of the hair follicle environment. Exp Dermatol. 2020; 29(7):588-597. https://doi.org/10.1111/exd.14109.

[54] Han M, Li C, Zhang C, et al. Single-cell transcriptomics reveals the natural product Shi-Bi-Man promotes hair regeneration by activating the FGF pathway in dermal papilla cells. Phytomedicine. 2022; 104:154260. https://doi.org/10.1016/j.phymed.2022.154260.

[55] Tang X, Zhang T, Wang B, et al. Biotransformation of Cacumen platycladi extract by Lactiplantibacillus plantarum CCFM1348 promotes hair growth in mice. J Agr Food Chem. 2024; 72(20):11493. https://doi.org/10.1021/acs.jafc.4c00807.

[56] Bertoli MJ, Sadoughifar R, Schwartz RA, et al. Female pattern hair loss: a comprehensive review. Dermatol Ther. 2020; 33(6):e14055. https://doi.org/10.1111/dth.14055.

[57] Shapiro J. Clinical practice.Hair loss in women. New Engl J Med. 2007; 357(16):1620-1630. https://doi.org/10.1056/NEJMcp072110.

[58] Cuevas-Diaz DR, Martinez-Ledesma E, Garcia-Garcia M, et al. The biology and genomics of human hair follicles: a focus on androgenetic alopecia. Int J Mol Sci. 2024; 25(5):2542. https://doi.org/10.3390/ijms25052542.