基于网络药理学探究桑黄多酚的体外抗肿瘤作用及其机制

doi:10.13560/j.cnki.biotech.bull.1985.2024-0732

摘要/Abstract

摘要：

【目的】 分离、筛选瓦尼桑黄（Sanghuangporus vaninii）子实体中具有良好抗肿瘤活性的多酚组分，并对其抗肿瘤活性和潜在作用机制进行解析。【方法】 采用超声辅助提取法提取乙酸乙酯部位的组分，通过对肝癌细胞HepG-2的抑制活性筛选出具有良好抗肿瘤作用的多酚组分，基于超高效液相色谱-四极杆飞行时间质谱（UPLC-Q-TOF-MS）技术鉴定其主要成分，利用网络药理学策略阐释其潜在抗肿瘤作用机制。【结果】 分离制备获得了5种（SVa-SVe）多酚组分，其中SVe多酚组分表现出独特的抗肿瘤活性，100 µg/mL浓度下对HepG-2细胞抑制率为76.54%。SVe可以显著地促进HepG-2细胞凋亡和诱导其细胞周期阻滞，并呈现剂量依赖性。成分分析显示，SVe主要由31种化合物组成，基于网络药理学分析表明，SVe中的osmundacetone、hispolon、phellibaumin A、davallialactone等关键化合物通过与多个靶点蛋白作用（STAT3、mTOR、VEGFA、SRC、ERBB2和HSP90AA），发挥抗肿瘤活性。【结论】 来源于桑黄的多酚提取物SVe通过诱导细胞凋亡和细胞周期阻滞对HepG-2细胞具有独特的抑制活性，其中的多酚化合物通过多靶点发挥作用。

关键词: 瓦尼桑黄, 多酚, 肝癌, 网络药理学

Abstract:

【Objective】 The polyphenolic components with excellent anti-cancer activity from the fruiting body of Sanghuangporus vaninii were isolated and screened, and their anti-cancer activity and potential mechanisms of action were analyzed. 【Method】 Ultrasound-assisted extraction was used to extract components from the ethyl acetate fraction. Polyphenolic components with excellent anti-cancer effects were screened for their inhibitory activity on liver cancer cell HepG-2. The main components were identified using ultra-high performance liquid chromatography quadrupole time-of-flight mass spectrometry（UPLC-Q-TOF-MS）technology, and the potential mechanism of anti-cancer was elucidated using network pharmacology strategies. 【Result】 Five polyphenolic components（SVa-SVe）were isolated and prepared, among which SVe polyphenolic component demonstrated unique anti-cancer activity, with an inhibition rate of 76.54% on HepG-2 cells at a concentration of 100 µg/mL. SVe significantly promoted apoptosis and induced cell cycle arrest in HepG-2 cells, showing a dose-dependent effect. Ingredient analysis showed that SVe was mainly composed of 31 compounds. Based on network pharmacology analysis, key compounds such as osmundacetone, hispolo, phellibaumin A, davallialactone in SVe exerted anti-cancer activity by interacting with multiple target proteins（STAT3, mTOR, VEGFA, SRC, ERBB2, and HSP90AA）. 【Conclusion】 The extract SVe derived from S. vaninii possessed unique inhibitory activity on HepG-2 cells by inducing apoptosis and cell cycle arrest, which was related to the presence of polyphenolic substances.

Key words: Sanghuangporus vaninii, polyphenols, liver cancer, network pharmacology

王雨阳, 刘朋, 张忠, 陈万超, 吴迪, 李文, 杨焱. 基于网络药理学探究桑黄多酚的体外抗肿瘤作用及其机制[J]. 生物技术通报, 2024, 40(11): 68-77.

WANG Yu-yang, LIU Peng, ZHANG Zhong, CHEN Wan-chao, WU Di, LI Wen, YANG Yan. In Vitro Anti-tumor Effect and Its Mechanism of Polyphenols from Sanghuangporus vaninii Based on Network Pharmacology[J]. Biotechnology Bulletin, 2024, 40(11): 68-77.

图/表 9

参考文献 34

[1]	陈万超, 杨焱, 张劲松, 等. 桑黄类真菌活性代谢产物的研究进展[J]. 食用菌学报, 2020, 27(4): 188-201.
	Chen WC, Yang Y, Zhang JS, et al. Recent advances in bioactive metabolites from ‘Sanghuang’ mushrooms[J]. Acta Edulis Fungi, 2020, 27(4): 188-201.
[2]	霍进喜, 李有贵, 孙雨晴, 等. 桑黄水提物诱导黑色素瘤细胞系B16-F10 G₀/G₁期阻滞的研究[J]. 蚕业科学, 2023, 49(6): 544-550.
	Huo JX, Li YG, Sun YQ, et al. Study on the induction of G₀/G₁ phase arrest in melanoma cell line B16-F10 by aqueous extracts of Sanghuang[J]. Acta Sericologica Sin, 2023, 49(6): 544-550.
[3]	轩贵平, 李天平, 李云贵, 等. 桑黄有效成分抗肿瘤的作用及研究进展[J]. 临床医学进展, 2024, 14(5):2489-2494.
	Xuan GP, Li TP, Li YG, et al. The anti-tumor effect and research progress of active ingredients in Sanghuang[J]. Advances in Clinical Medicine, 2024, 14(5):2489-2494.
[4]	宋吉玲, 陆娜, 闫静, 等. 桑枝屑对瓦尼桑黄主要活性成分含量和体外抗氧化活性的影响[J]. 菌物学报, 2023, 42(4): 949-960.
	Song JL, Lu N, Yan J, et al. Effects of mulberry sawdust on the content of main active components and antioxidant activities in Sanghuangporus vaninii[J]. Mycosystema, 2023, 42(4): 949-960.
[5]	闫帅帅, 郭辛茹, 徐建国, 等. 桑黄裂蹄针层孔菌提取物生物活性成分及功能特性分析[J]. 食品研究与开发, 2023, 44(23): 22-28.
	Yan SS, Guo XR, Xu JG, et al. Analysis of bioactive components and functional properties of extract from Phellinus linteus[J]. Food Res Dev, 2023, 44(23): 22-28.
[6]	李志军, 包海鹰. 中药桑黄粗毛纤孔菌的化学成分与药理作用研究进展[J]. 菌物研究, 2022, 20(3): 203-213.
	Li ZJ, Bao HY. Research advances on chemical constituents and pharmacological effects of traditional Chinese medicine Sanghuang—Inonotus hispidus[J]. J Fungal Res, 2022, 20(3): 203-213.
[7]	昝立峰, 郭海燕, 包海鹰, 等. 鲍姆桑黄子实体提取物的体外细胞毒活性及其化学成分分析[J]. 菌物学报, 2023, 42(4): 961-972.
	Zan LF, Guo HY, Bao HY, et al. Characterization of cytotoxicity and chemical constituents of extracts of Sanghuangporus baumii basidiomata[J]. Mycosystema, 2023, 42(4): 961-972.
[8]	孙雨晴, 吴伟杰, 钟石, 等. 桑黄子实体中多酚含量测定的方法学研究[J]. 浙江农业科学, 2022, 63(9): 2117-2120.
	Sun YQ, Wu WJ, Zhong S, et al. Methodological study on determination of polyphenol content in Phellinus igniarius fruiting body[J]. J Zhejiang Agric Sci, 2022, 63(9): 2117-2120.
[9]	张俊峰, 张忠, 汪雯翰, 等. 桑黄菌丝体和子实体中次级代谢产物及其活性的比较[J]. 菌物学报, 2020, 39(2): 398-408.
	Zhang JF, Zhang Z, Wang WH, et al. Comparison of active secondary metabolites between mycelia and fruiting bodies of Sanghuangporous Sanghuang[J]. Mycosystema, 2020, 39(2): 398-408.
[10]	于荣利, 贾薇, 张劲松, 等. 八种食药用菌子实体醇提物对乳腺癌细胞MCF-7的体外影响[J]. 食用菌学报, 2017, 24(2): 88-92.
	Yu RL, Jia W, Zhang JS, et al. Effect of ethanolic extracts from eight kinds of medicinal and edible fungi on breast cancer cell MCF-7 in vitro[J]. Acta Edulis Fungi, 2017, 24(2): 88-92.
[11]	Zhang XQ, Li Z, Yang P, et al. Polyphenol scaffolds in tissue engineering[J]. Mater Horiz, 2021, 8(1): 145-167.
[12]	Messaoudene M, Pidgeon R, Richard C, et al. A natural polyphenol exerts antitumor activity and circumvents anti-PD-1 resistance through effects on the gut microbiota[J]. Cancer Discov, 2022, 12(4): 1070-1087.
[13]	胡一凡, 高晓余, 苏俊宇, 等. 多酚、黄酮功能性成分导向的草果种质资源评价[J]. 热带作物学报, 2024, 45(5): 915-927.
	Hu YF, Gao XY, Su JY, et al. Evaluation of Amomum tsao-ko germplasm resources oriented by functional components of polyphenols and flavonoids[J]. Chin J Trop Crops, 2024, 45(5): 915-927.
[14]	Luca SV, Macovei I, Bujor A, et al. Bioactivity of dietary polyphenols: The role of metabolites[J]. Crit Rev Food Sci Nutr, 2020, 60(4): 626-659.
[15]	Guo SS, Duan WW, Wang YX, et al. Component analysis and Anti-Colorectal cancer mechanism via AKT/mTOR signalling pathway of Sanghuangporus vaninii extracts[J]. Molecules, 2022, 27(4): 1153.
[16]	李天, 王婷, 邢宝娟, 等. 女贞苷通过阻滞G1/S期抑制非小细胞肺癌奥西替尼耐药细胞增殖[J]. 药学学报, 2023, 58(11): 3349-3353.
	Li T, Wang T, Xing BJ, et al. Ligustroflavone mediates the resistance of non-small cell lung cancer to osimertinib by arresting G1/S phase[J]. Acta Pharm Sin, 2023, 58(11): 3349-3353.
[17]	孙雨晴, 伏瑶, 纪藕霄, 等. 重楼皂苷VII对弥漫大B细胞淋巴瘤细胞增殖、凋亡及细胞周期的影响[J]. 中华血液学杂志, 2024, 45(4): 391-395.
	Sun YQ, Fu Y, Ji OX, et al. Effects of polyphyllin VII on proliferation, apoptosis and cell cycle of diffuse large B-cell lymphoma cells[J]. Chin J Hematol, 2024, 45(4): 391-395.
[18]	Jamasbi E, Hamelian M, Hossain MA, et al. The cell cycle, cancer development and therapy[J]. Mol Biol Rep, 2022, 49(11): 10875-10883.
[19]	Weston WA, Barr AR. A cell cycle centric view of tumour dormancy[J]. Br J Cancer, 2023, 129(10): 1535-1545.
[20]	赵宁, 黄永吉, 马广斌, 等. 鞣花酸对骨髓瘤SP2/0细胞的作用[J]. 医药导报, 2014, 33(10): 1321-1325.
	Zhao N, Huang YJ, Ma GB, et al. Effect of ellagic acid on myeloma SP2/0 cells[J]. Her Med, 2014, 33(10): 1321-1325.
[21]	冯甜, 刘盟, 程路峰, 等. 基于凋亡和自噬途径的石榴皮多酚对人前列腺癌PC3细胞的抑制作用机制研究[J]. 中国药房, 2020, 31(16): 1978-1983.
	Feng T, Liu M, Cheng LF, et al. Study on the inhibitory mechanism of pomegranate peel polyphenols on human prostate cancer PC3Cells based on apoptosis and autophagy pathway[J]. China Pharm, 2020, 31(16): 1978-1983.
[22]	伊娟娟, 王振宇, 周丽萍, 等. 红松松塔多酚对S180荷瘤小鼠抗肿瘤及免疫活性[J]. 食品工业科技, 2017, 38(6): 345-349.
	Yi JJ, Wang ZY, Zhou LP, et al. Antitumor and immunomodulation activities of polyphenols from pinecone of Pinus koraiensis in cancer-bearing S180 mice[J]. Sci Technol Food Ind, 2017, 38(6): 345-349.
[23]	Gao H, Yin CM, Li C, et al. Phenolic profile, antioxidation and anti-proliferation activity of phenolic-rich extracts from Sanghuangporusvaninii[J]. Curr Res Food Sci, 2023, 6: 100519.
[24]	Chen WH, Tan HY, Liu Q, et al. A review: the bioactivities and pharmacological applications of Phellinus linteus[J]. Molecules, 2019, 24(10): 1888.
[25]	Lee YS, Kang YH, Jung JY, et al. Inhibitory constituents of aldose reductase in the fruiting body of Phellinus linteus[J]. Biol Pharm Bull, 2008, 31(4): 765-768.
[26]	Sarfraz A, Rasul A, Sarfraz I, et al. Hispolon: a natural polyphenol and emerging cancer killer by multiple cellular signaling pathways[J]. Environ Res, 2020, 190: 110017.
[27]	Islam MT, Ali ES, Khan IN, et al. Anticancer perspectives on the fungal-derived polyphenolic hispolon[J]. Anticancer Agents Med Chem, 2020, 20(14): 1636-1647.
[28]	Yang Y, He PY, Hou YH, et al. Osmundacetone modulates mitochondrial metabolism in non-small cell lung cancer cells by hijacking the glutamine/glutamate/α-KG metabolic axis[J]. Phytomedicine, 2022, 100: 154075.
[29]	Ke C, Chen CH, Yang M, et al. Osmundacetone inhibits angiogenesis of infantile hemangiomas through inducing caspases and reducing VEGFR2/MMP9[J]. Anticancer Agents Med Chem, 2024, 24(2): 125-131.
[30]	Zan LF, Xin JC, Guo HY, et al. Systematic characterization of active antitumor constituents from the shaggy bracket medicinal mushroom Inonotus hispidus(Agaricomycetes)by UPLC-Q-TOF/MS and network pharmacology[J]. Int J Med Mushrooms, 2023, 25(3): 47-62.
[31]	纪红燕, 吴佳妮, 吴欣圆, 等. 内生真菌诱导子对火木层孔菌次级代谢的影响[J]. 西北药学杂志, 2021, 36(6): 871-875.
	Ji HY, Wu JN, Wu XY, et al. Effects of endophytic fungus elicitors on secondary metabolism of Phellinus igniarius[J]. Northwest Pharm J, 2021, 36(6): 871-875.
[32]	Soosanabadi M, Ghahfarokhi AM, Pourghazi F, et al. Expression of ERBB gene family in females with breast cancer and its correlation with clinicopathological characteristics of the disease[J]. Mol Biol Rep, 2022, 49(9): 8547-8553.
[33]	He Y, Sun MM, Zhang GG, et al. Targeting PI3K/Akt signal transduction for cancer therapy[J]. Signal Transduct Target Ther, 2021, 6(1): 425.
[34]	Gill BS, Navgeet, Mehra R, et al. Ganoderic acid, lanostanoid triterpene: a key player in apoptosis[J]. Invest New Drugs, 2018, 36(1): 136-143.

多酚组分 Palyphenols components	样品浓度Sample concentration/（µg·mL^-1）
多酚组分 Palyphenols components	6.25	12.5	25.0	50.0	100.0
SVa	40.38±2.38 a	38.95±3.95 b	47.76±1.76 a	80.35±1.35 a	81.06±1.06 b
SVb	24.69±2.69 c	34.12±3.12 c	36.17±2.17 c	47.67±1.67 c	52.40±2.35 d
SVc	1.25±0.52 e	6.01±1.01 e	8.79±1.79 e	14.34±1.34 e	23.35±2.40 f
SVd	1.31±0.31 d	7.38±1.38 d	13.98±0.93 d	19.88±1.88 d	30.89±3.89 e
SVe	38.54±1.00 b	45.53±2.53 a	63.40±1.40 b	65.77±1.77 b	76.54±2.65 c

多酚组分 Palyphenols components	样品浓度Sample concentration/（µg·mL^-1）
多酚组分 Palyphenols components	6.25	12.5	25.0	50.0	100.0
SVa	40.38±2.38 a	38.95±3.95 b	47.76±1.76 a	80.35±1.35 a	81.06±1.06 b
SVb	24.69±2.69 c	34.12±3.12 c	36.17±2.17 c	47.67±1.67 c	52.40±2.35 d
SVc	1.25±0.52 e	6.01±1.01 e	8.79±1.79 e	14.34±1.34 e	23.35±2.40 f
SVd	1.31±0.31 d	7.38±1.38 d	13.98±0.93 d	19.88±1.88 d	30.89±3.89 e
SVe	38.54±1.00 b	45.53±2.53 a	63.40±1.40 b	65.77±1.77 b	76.54±2.65 c

序号 No.	化合物Component	分子式 Molecular formula	保留时间 RT/min
1	4-（4-hydroxyphenyl）-3-buten-2-one	C₁₀H₁₀O₂	19.6
2	Osmundacetone	C₁₀H₁₀O₃	16.75
3	Hispolon	C₁₂H₁₂O₄	20.08
4	7-Acetoxycoumarin-3-carboxylic acid	C₁₂H₈O₆	15.95
5	Hispidin	C₁₃H₁₀O₅	18.82
6	Citrinin	C₁₃H₁₄O₅	22.18
7	Phelligridin_J	C₁₃H₆O₈	74.18
8	7-O-methyleriodictyol	C₁₆H₁₄O₆	19.49
9	Palmitic acid	C₁₆H₃₂O₂	59.4
10	Linoleic acid	C₁₈H₃₂O₂	39.35
11	phellibaumin A	C₁₉H₁₂O₇	26.2
12	Phelligridin_C	C₂₀H₁₂O₇	29.18
13	Phelligridin_D	C₂₀H₁₂O₈	26.46
14	inoscavin_D	C₂₁H₁₆O₈	28.71
15	Phellibaumin_B	C₂₂H₁₆O₉	25.3
16	Hydroxy-docosanoic acid	C₂₂H₄₄O₃	68.69
17	inoscavin_C	C₂₃H₁₆O₈	28.62
18	interfungin_B	C₂₃H₂₀O₈	34.9
19	Kielcorin	C₂₄H₂₀O₈	15.81
20	Hydroxy-tetracosanoic acid	C₂₄H₄₈O₃	72.51
21	Inoscavin A	C₂₅H₁₈O₉	25.04
22	Davallialactone	C₂₅H₂₀O₉	21.06
23	Hypholomine B	C₂₆H₁₈O₁₀	23.25
24	Hypholomine A	C₂₆H₁₈O₉	29.22
25	SCHEMBL8676491	C₂₇H₂₀O₁₀	32.61
26	Phelligridin_I	C₃₃H₂₀O₁₃	27.33
27	Phelligridin_I_500	C₃₃H₂₀O₁₃	26.3
28	phelligridimer A	C₅₂H₃₂O₂₀	26.82
29	protocatechuic aldehyde	C₇H₆O₃	11.25
30	3-Hydroxycinnamic acid	C₉H₈O₃	13.47
31	Caffeic acid	C₉H₈O₄	13.58

序号 No.	化合物Component	分子式 Molecular formula	保留时间 RT/min
1	4-（4-hydroxyphenyl）-3-buten-2-one	C₁₀H₁₀O₂	19.6
2	Osmundacetone	C₁₀H₁₀O₃	16.75
3	Hispolon	C₁₂H₁₂O₄	20.08
4	7-Acetoxycoumarin-3-carboxylic acid	C₁₂H₈O₆	15.95
5	Hispidin	C₁₃H₁₀O₅	18.82
6	Citrinin	C₁₃H₁₄O₅	22.18
7	Phelligridin_J	C₁₃H₆O₈	74.18
8	7-O-methyleriodictyol	C₁₆H₁₄O₆	19.49
9	Palmitic acid	C₁₆H₃₂O₂	59.4
10	Linoleic acid	C₁₈H₃₂O₂	39.35
11	phellibaumin A	C₁₉H₁₂O₇	26.2
12	Phelligridin_C	C₂₀H₁₂O₇	29.18
13	Phelligridin_D	C₂₀H₁₂O₈	26.46
14	inoscavin_D	C₂₁H₁₆O₈	28.71
15	Phellibaumin_B	C₂₂H₁₆O₉	25.3
16	Hydroxy-docosanoic acid	C₂₂H₄₄O₃	68.69
17	inoscavin_C	C₂₃H₁₆O₈	28.62
18	interfungin_B	C₂₃H₂₀O₈	34.9
19	Kielcorin	C₂₄H₂₀O₈	15.81
20	Hydroxy-tetracosanoic acid	C₂₄H₄₈O₃	72.51
21	Inoscavin A	C₂₅H₁₈O₉	25.04
22	Davallialactone	C₂₅H₂₀O₉	21.06
23	Hypholomine B	C₂₆H₁₈O₁₀	23.25
24	Hypholomine A	C₂₆H₁₈O₉	29.22
25	SCHEMBL8676491	C₂₇H₂₀O₁₀	32.61
26	Phelligridin_I	C₃₃H₂₀O₁₃	27.33
27	Phelligridin_I_500	C₃₃H₂₀O₁₃	26.3
28	phelligridimer A	C₅₂H₃₂O₂₀	26.82
29	protocatechuic aldehyde	C₇H₆O₃	11.25
30	3-Hydroxycinnamic acid	C₉H₈O₃	13.47
31	Caffeic acid	C₉H₈O₄	13.58

名称 Name	等级值 Degree	介数中心度 Betweenness centrality	紧密中心度 Closeness centrality
STAT3	48	0.011951917	1
ERBB2	48	0.011951917	1
HSP90AA1	48	0.011951917	1
mTOR	48	0.011951917	1
SRC	48	0.011951917	1
VEGFA	48	0.011951917	1
MAPK1	46	0.009286467	0.96
BCL2L1	46	0.010953764	0.96
ESR1	46	0.010049717	0.96
EGFR	44	0.009743413	0.923076923
AR	40	0.006204746	0.857142857
EP300	40	0.00557225	0.857142857
CDK4	40	0.005307013	0.857142857
MMP9	40	0.005991339	0.857142857
CCNA2	38	0.003552283	0.827586207
STAT1	38	0.00459399	0.827586207
GSK3B	38	0.004853451	0.827586207
IGF1R	36	0.002201527	0.8
MAP2K1	36	0.001906972	0.8
TERT	36	0.004834839	0.8
ABL1	36	0.00292773	0.8
PPARG	34	0.002278796	0.774193548
PARP1	34	0.001530318	0.774193548
MET	30	0.00145824	0.727272727
KIT	30	0.001708309	0.727272727