马尾松松针提取物对人毛乳头细胞的保护作用及机制研究

doi:10.3969/j.issn.1001-1803.2023.01.009

摘要/Abstract

摘要：

研究了马尾松松针提取物对人毛乳头细胞（Human dermal papilla cells，HDP）的保护作用及机制。通过制备并分析不同溶剂松针提取物中主要活性成分含量；建立二氢睾酮（Dihydrotestosterone，DHT）诱导HDP细胞损伤模型，采用CCK8法检测细胞相对活力，qPCR检测细胞中血管内皮生长因子（Vascular endothelial growth factor，VEGF）、转化生长因子-β2（Transforming growth factor-β2，TGF-β2）、β-连环蛋白（β-Catenin）和蓬乱蛋白1（Dishevelled 1，DVL1）基因相对表达量，探究松针提取物对Wnt/β-Catenin信号通路的影响；最后采用UPLC-QTOF/MS分析鉴定提取物成分。结果表明，松针50%醇提物可显著提高HDP细胞相对活力，最高达88.3%（P<0.05）；显著提高VEGF mRNA表达量（P<0.05），降低TGF-β2 mRNA相对表达量（P<0.01）；并显著促进Wnt/β-Catenin信号中DVL1 mRNA和β-Catenin mRNA表达（P<0.05，P<0.01）。从松针50%醇提物中共鉴定出36种成分，包括黄酮类、木脂素类、生物碱和萜类等化合物。马尾松松针提取物对人毛乳头细胞具有保护作用，其机制可能与Wnt/β-Catenin信号传导途径有关。

关键词: 马尾松松针, 二氢睾酮, 人毛乳头细胞, 雄激素性脱发

Abstract:

The effect and mechanism of Pinus massoniana pine needle extract on the activity of protecting human dermal papilla cells （HDP） were explored in this paper. The different extracts from pine needle were prepared with different solvents, and the content of active components was determined and analyzed by the corresponding determination method. HDP cells were induced by dihydrotestosterone（DHT）to establish androgenic alopecia cell model. CCK8 method was used to analyze the effect of pine needle extracts on the relative viability of the cells, and qPCR was adopted to investigate the effect of pine needle extract on regulating Wnt/β-Catenin signaling pathway by detecting the relative expression of VEGF, TGF-β2, β-Catenin, and DVL1 genes. UPLC-QTOF/MS was used to identify the components of the pine needle extract. The results show that the 50% alcohol extract of pine needle can significantly improve the relative activity of HDP cells （P<0.05）, increase the expression of VEGF mRNA （P<0.05）, decrease TGF-β2 mRNA relative expression （P<0.01）, and promote the expression of DVL1 mRNA and β-Catenin mRNA expression in the Wnt/β-Catenin signaling pathway（P<0.05, P<0.01）. In addition, a total of 36 components are identified from the 50% alcohol extract of pine needle, including flavonoids, lignans, alkaloids, and terpenes. Pinus massoniana pine needle extract has a protective effect on HDP cells, and its mechanism may be related to Wnt/β-Catenin signaling pathway.

Key words: Pinus massoniana pine needle, dihydrotestosterone, human dermal papilla cells, androgenetic alopecia

中图分类号:

TQ658

王建成, 邢岩, 徐婷, 王玥, 李永强, 张丽梅. 马尾松松针提取物对人毛乳头细胞的保护作用及机制研究[J]. 日用化学工业（中英文）, 2023, 53(1): 62-70.

Wang Jiancheng, Xing Yan, Xu Ting, Wang Yue, Li Yongqiang, Zhang Limei. Protective effect and mechanism of Pinus massoniana pine needle extract in human dermal papilla cells[J]. China Surfactant Detergent & Cosmetics, 2023, 53(1): 62-70.

图/表 11

表 1

表 2

表 3

图 1

图 2

图 3

图 4

图 5

图 6

图 7

表 4

松针提取物中化合物鉴定结果表"

序号	保留时间/ min	中文名称	英文名称	分子式	[M-H] ^-	Score
1	1.06	杜仲甙Ⅱ	Eucommioside-Ⅱ	C₁₅H₂₆O₉	349.158 6	97.04
2	1.128	（-）-表儿茶素	（-）-Epicatechin	C₁₅H₁₄O₆	289.079 1	99.55
3	1.465	3，4-二羟基烯丙基苯-4-O-β-D-吡喃葡萄糖苷	3, 4-Dihydroxy-allylbenzene-4-O-beta-D-glucopyranoside	C₁₅H₂₀O₇	311.121 7	97.25
4	2.004	丁香苷	Syringafghanoside	C₂₅H₃₂O₁₁	507.194 7	98.64
5	2.273	柚皮素-7-O-葡萄糖苷	Naringenin-7-O-glucoside	C₂₁H₂₂O₁₀	433.122 7	98.23
6	2.812	1-（2’-γ-吡喃酮）-6-咖啡酰-α-D-吡喃葡萄糖	1-（2’-gamma-Pyranone）-6-caffeoyl-alpha-D-pyranoglucose	C₂₀H₂₀O₁₁	435.100 5	98.83
7	3.621	女贞果苷A	Lucidumoside A	C₂₅H₃₄O₁₂	525.205 5	99
8	3.823	花旗松素-3’-O-葡萄糖苷	Taxifolin-3’-O-glucoside	C₂₁H₂₂O₁₂	465.103 8	98.23
9	5.171	（-）-5’-甲氧基异戊烯雌酚-2a-O-β-D-木吡喃糖苷	（-）-5’-Methoxyisolariciresinol-2a-O-beta-D-xylopyranoside	C₂₆H₃₄O₁₁	521.210 9	99.13
10	5.845	6-O-E-阿魏基胡桃醇	6-O-E-Feruloylajugol	C₂₅H₃₂O₁₂	523.189 9	98.47
11	9.82	（-）-异紫杉醇-2a-O-β-D-木吡喃糖苷	（-）-Isolariciresinol-2a-O-beta-D-xylopyranoside	C₂₅H₃₂O₁₀	491.200 4	97.54
12	10.292	左旋马尾松树脂醇	Massoniresil	C₂₀H₂₄O₈	391.144 9	98.23
13	11.101	淫羊藿苷H1	Icariside H1	C₁₈H₂₆O₉	385.157 8	99.73
14	11.438	3，4’，5，7-四羟基黄酮-3-L-鼠李糖苷	3, 4’, 5, 7-Tetrahydroxyflavone-3-L-rhamnoside	C₂₁H₂₀O₁₀	431.106 1	95.52
15	11.707	（+）-1-羟基松脂醇-4’’-O-β-D-吡喃葡萄糖苷	（+）-1-Hydroxypinoresinol-4’’-O-beta-D-glucopyranoside	C₂₆H₃₂O₁₂	535.191	97.77
16	11.91	淫羊藿苷E6	Icariside E6	C₂₆H₃₆O₁₁	523.225 7	99.77
17	11.977	马尾松糖苷C	Massonianoside C	C₂₆H₃₄O₁₀	505.216	97.07
18	12.449	细叶皂苷D	Tenuifoliside D	C₁₈H₂₄O₉	383.142	99.54
19	15.212	（+）-环氧树脂醇	（+）-Epipinoresinol	C₂₀H₂₂O₆	357.142	98.99
20	16.761	槲皮素	Quercetin	C₁₅H₁₀O₇	301.042 5	98.54
21	21.276	鸢尾甲黄素B	Iristectorigenin B	C₂₉H₃₄O₁₇	653.180 2	98.97
22	25.454	6-乙酰基呋喃醇	6-Acetylfuranofukinol	C₁₇H₂₄O₄	291.168	98.55
23	27.341	5-羟基-6，7，3-三甲氧基黄酮-8- O-β-D-葡萄糖苷	5-Hydroxy-6, 7, 3-trimethoxyflavone-8-O-beta-D-glucoside	C₂₄H₂₆O₁₂	505.142 7	99.56
24	28.352	14-脱氧-11，12-二氢穿心莲内酯	14-Deoxy-11, 12-didehydroandrographolide	C₂₀H₂₈O₄	331.199 3	97.94
25	31.789	芹菜素-7-O-β-D-吡喃葡萄糖苷	Apigenin-7-O-β-D-glucopyranoside	C₃₉H₃₂O₁₄	723.179 5	99.69
26	33.541	10-姜二酮	10-Gingerdione	C₂₁H₃₂O₄	347.23	98.54
27	35.293	6-甲基二乙酸姜酯	6-Methylgingediacetate	C₂₂H₃₄O₆	393.235 8	98.91
28	36.978	12-甲氧基-8，11，13-枞三烯-7β，11-二醇	12-Methoxy-8, 11, 13-abietatriene-7beta, 11-diol	C₂₁H₃₂O₃	331.234 9	98.24
29	40.212	（-）-17-羟基-16β-考兰-19-油酸	（-）-17-Hydroxy-16beta-kauran-19-oicacid	C₂₀H₃₂O₃	319.235 1	99.53
30	42.908	高三尖杉酯碱	Homoharringtonine	C₂₉H₃₉NO₉	544.261 8	96.98
31	43.986	7β，12-二甲氧基-8，11，13-枞三烯-11-醇	7beta, 12-Dimethoxy-8, 11, 13-abietatrien-11-ol	C₂₂H₃₄O₃	345.250 5	99.27
32	47.962	6-甲基隐乙酰内酯	6-Methylcryptoacetalide	C₁₉H₂₄O₃	299.173 2	97.17
33	49.242	α，β-二半乳糖基-α’-亚麻酸甘油酯	alpha, beta-Digalactosyl-alpha’-linolenic-glyceride	C₃₃H₅₆O₁₄	675.367 8	98.55
34	49.646	9-D-羟基顺式-12-十八烯酸	9-D-Hydroxy-cis-12-octadecenoicacid	C₁₈H₃₄O₃	297.251	99.61
35	52.072	1-（3，4-亚甲基二氧苯基）-1E-十四烯	1-（3, 4-Methylenedioxyphenyl）-1E-tetradecene	C₂₁H₃₂O₂	315.240 7	99.18
36	53.892	补骨脂酚	Backuchiol	C₁₈H₂₄O	255.183 1	99.45

表 4

参考文献 21

[1]	Fan W X. Pathogenesis of androgenic alopecia and treatment progress[J]. Journal of Clinical Dermatology, 2001, 30 (6) : 401-403.
[2]	Hamideh M F, Meshkat R G, Ghalam K F, et al. Evaluation of association between androgenic alopecia and coronary artery diseases in men[J]. Pejouhandeh, 2007, 12: 153-160.
[3]	N S V, K A A, Govind S. Male pattern androgenetic alopecia[J]. SKINmed, 2006, 5 (3) : 128-135. pmid: 16687981
[4]	Lolli F, Pallotti F, Rossi A, et al. Androgenetic alopecia: a review[J]. Endocrine, 2017, 57 (1) : 9-17. doi: 10.1007/s12020-017-1280-y pmid: 28349362
[5]	Premanand A, Reena Rajkumari B. Androgen modulation of Wnt/β-catenin signaling in androgenetic alopecia[J]. Archives of Dermatological Research, 2018, 310 (5) : 391-399. doi: 10.1007/s00403-018-1826-8 pmid: 29549490
[6]	Hu Z Q, Miao Y. Guidelines for the diagnosis and treatment of androgenic alopecia in China[J]. Chinese Journal of Aesthetic and Plastic Surgery, 2019, 30 (1) : 8-12.
[7]	Zhang N N, Park D K, Park H J. Hair growth-promoting activity of hot water extract of Thuja orientalis[J]. BMC Complementary and Alternative Medicine, 2013, 13 (1) : 9-21. doi: 10.1186/1472-6882-13-9
[8]	Deng W J, Deng C J, Han L, et al. Differential expression of miR-133b in human hair follicles treated with DHT in vitro and its functional analysis in human dermal papille cells[J]. Journal of Sun Yat sen University (Medical Science Edition), 2021, 42 (2) : 202-208.
[9]	Luo J, Chen M, Liu Y, et al. Nature-derived lignan compound VB-1 exerts hair growth-promoting effects by augmenting Wnt/β-catenin signaling in human dermal papilla cells[J]. PeerJ, 2018, 6: e4737. doi: 10.7717/peerj.4737
[10]	Yano K, Brown L F, Detmar M. Control of hair growth and follicle size by VEGF-mediated angiogenesis[J]. Journal of Clinical Investigation, 2001, 107 (4) : 409-417. doi: 10.1172/JCI11317 pmid: 11181640
[11]	Keita, Inoue, Noriyuki, et al. TGF-β2 is specifically expressed in human dermal papilla cells and modulates hair folliculogenesis[J]. Journal of Cellular and Molecular Medicine, 2009, 13 (11) : 4643-4656. doi: 10.1111/j.1582-4934.2009.00739.x
[12]	Meng Y H, Min J H, Shang Q. Analysis of chemical constituents of Pinus massoniana needles[J]. Asia-Pacific Traditional Medicine, 2021, 17 (3) : 60-62.
[13]	Li C Y, Xu S Y, Wang Z. Determination of proanthocyanidins in grape seeds and stems by vanillin hydrochloric acid method[J]. Food Industry Technology, 2004, 25 (6) : 157-161.
[14]	Guo Y H. Study on extraction and purification of total lignans from Pinus massoniana needles[D]. Zhengzhou: Henan College of Traditional Chinese Medicine, 2010.
[15]	Dastan M, Najafzadeh N, Abedelahi A, et al. Human platelet lysate versus minoxidil stimulates hair growth by activating anagen promoting signaling pathways[J]. Biomedicine & Pharmacotherapy, 2016, 84: 979-986. doi: 10.1016/j.biopha.2016.10.019
[16]	Chen S L, Sun J F. Pharmacological and clinical study of minoxidil in the treatment of alopecia[J]. Foreign Medical Sciences (Section of Dermatology and Venereology), 2004 (5) : 276-278.
[17]	Abreu C M, Cerqueira M T, Pirraco R P, et al. Rescuing key native traits in cultured dermal papilla cells for human hair regeneration[J]. Journal of Advanced Research, 2021, 30: 103-112. doi: 10.1016/j.jare.2020.10.006 pmid: 34026290
[18]	Wang Z D, Feng Y, Sun L, et al. Anti-androgenetic alopecia effect of policosanol from Chinese wax by regulating abnormal hormone levels to suppress premature hair follicle entry into the regression phase[J]. Biomedicine & Pharmacotherapy, 2021, 136: 111241. doi: 10.1016/j.biopha.2021.111241
[19]	Xu L L, Xuan M, Li T, et al. Role of Wnt/β-catenin signaling pathway in seborrheic alopecia and its intervention with traditional Chinese medicine: A Review[J]. Chinese Journal of Experimental Traditional Medical Formulae, 2022, 28 (4) : 254-261.
[20]	Lien W H, Polak L, Lin M, et al. In vivo transcriptional governance of hair follicle stem cells bycanonical Wnt regulators[J]. Nature Cell Biology, 2014, 16 (2) : 179-190. doi: 10.1038/ncb2903
[21]	Wang W, Li X, Lee M, et al. FOXKs Promote Wnt/β-catenin signaling by translocating DVL into the nucleus[J]. Developmental Cell, 2015, 32 (6) : 707-718. doi: 10.1016/j.devcel.2015.01.031 pmid: 25805136

基因	上游引物	下游引物
VEGF	ATCGAGTACATCTTCAAGCCAT	GTGAGGTTTGATCCGCATAATC
TGFβ2	GCAAAGTTGTGAAAACAAGAGC	ATCCCAGGTTCCTGTCTTTATG
DVL1	TTTGAGAACATGAGCAATGACG	GGGACGGTGAAGTAGCTTC
β-Catenin	TGGATTGATTCGAAATCTTGCC	GAACAAGCAACTGAACTAGTCG
GAPDH	GGTGA AGGTC GGAGT CAACG	CAAAG TTGTC ATGGA TGHACC

提取物	总黄酮含量/ （mg/g，以芦丁计）	粗多糖含量/ （mg/g，以葡萄糖计）	原花青素含量/ （mg/g，以儿茶素计）	木脂素含量/ （mg/g，以新木脂素A计）
水提取物	55.9	148.3	69.5	183.0
30%乙醇提取物	76.2	117.2	97.8	200.4
50%乙醇提取物	91.2	63.8	102.4	228.3