黑骨藤正丁醇萃取成分治疗大鼠阿尔茨海默病的药效学及作用机制

doi:10.12122/j.issn.1673-4254.2025.04.14

摘要/Abstract

摘要：

目的探讨黑骨藤正丁醇萃取部位对阿尔茨海默病（AD）的药效学研究及潜在作用机制预测。方法采用超高效液相-四级杆-静电场轨道阱高分辨质谱（UPLC-QE-MS）技术对黑骨藤正丁醇部位的化学成分进行分析鉴定，建立三氯化铝（AlCl₃）和D-半乳糖（D-gal）联合诱导50只SPF级雄性AD大鼠模型，使用酶联免疫吸附试验（ELISA）、苏木精-伊红染色（HE）和尼氏染色（Nissl）、免疫组化染色（ICH）、Western blotting等实验为基础，通过网络药理学及分子对接技术预测抗AD潜在作用机制。结果黑骨藤正丁醇萃取部位鉴定出17个化学成分，主要包括苯丙素类、黄酮类、蒽醌类、三萜类、甾体类以及挥发油类。药效学实验结果显示经过黑骨藤正丁醇萃取部位组处理后，大鼠海马中乙酰胆碱酯酶（AChE）含量降低（P<0.05），而乙酰胆碱（ACh）、超氧化物歧化酶（SOD）、过氧化氢酶（CAT）和谷胱甘肽过氧化物酶（GSH-Px）的含量增加（P<0.05），在Western blotting实验中神经细胞凋亡因子B淋巴细胞瘤-2（Bcl-2）、磷脂酰肌醇-3激酶（PI3K）、蛋白激酶B（Akt）、磷酸化磷脂酰肌醇-3激酶（p-PI3K）和磷酸化蛋白激酶B（p-Akt）表达上升（P<0.05），而Bax蛋白、半胱氨酸天冬氨酸蛋白酶-3（Caspase-3）表达下降（P<0.01），氧化应激因子核因子红细胞系2相关因子2（Nrf-2）和血红素氧合酶1（HO-1）蛋白表达水平上调（P<0.01），氧化应激反应转录因子（Keap-1）蛋白表达水平下调（P<0.01），脑源性神经营养因子BDNF表达水平均升高（P<0.01），以及β-淀粉样蛋白Aβ、Tau蛋白表达水平均下调（P<0.01），且表现出一定的剂量依赖性。黑骨藤正丁醇萃取部位活性成分与AD共获得TNF、AKT1和ESR1等14个关键靶点。结论初步阐明了黑骨藤正丁醇萃取成分的药效物质基础，并证实了其具有较好的抗AD效果。预测黑骨藤正丁醇萃取部位可通过多组分、多靶点、多途径和多通路发挥抗AD作用。

关键词: UPLC-QE-MS, 黑骨藤, 正丁醇萃取部位, 化学成分, 阿尔茨海默病, 网络药理学

Abstract:

Objective To investigate the active components and possible mechanisms of n-butanol fraction of Periploca forrestii Schltr. ethanol extract for treating Alzheimer's disease (AD). Methods The active components of n-butanol fraction of Periploca forrestii Schltr. ethanol extract were analyzed using UPLC-QE-MS technique. In a SD rat model of AD induced by treatment with AlCl₃ and D-gal, the therapeutic effects of low, moderate and high doses of the n-butanol fraction, saline, and donepezil hydrochloride were evaluated using ELISA, HE and Nissl staining, immunohistochemistry and Western blotting. The therapeutic mechanisms of the n-butanol fraction were explored using network pharmacology and molecular docking. Results Seventeen active components were identified from the n-butanol fraction of Periploca forrestii Schltr. ethanol extract, including phenylpropanoids, flavonoids, anthraquinones, triterpenoids, steroids, and volatile oils. In the rat models of AD, treatment with the n-butanol fraction significantly lowed AChE content in the hippocampus, increased the contents of ACh, SOD, CAT, and GSH-Px, enhanced the expressions of neuronal apoptotic factors Bcl-2, PI3K, Akt, p-PI3K, and p-Akt, and reduced the expressions of Bax and caspase-3 proteins. The treatment also dose-dependently up-regulated hippocampal expressions of Nrf-2, HO-1 and BDNF and down-regulated Keap-1, Aβ and Tau expressions. Bioinformatics analysis identified 14 key intersected targets (including TNF, AKT1 and ESR1) between the n-butanol fraction and AD. Conclusion The therapeutic effect of n-butanol fraction of Periploca forrestii Schltr. ethanol extract in AD mice is mediated by its multiple active components that regulate multiple targets and pathways.

Key words: UPLC-QE-MS, Periploca forrestii Schltr., n-butanol extract, chemical constituents, Alzheimer's disease, network pharmacology

冉念东, 刘杰, 徐剑, 张永萍, 郭江涛. 黑骨藤正丁醇萃取成分治疗大鼠阿尔茨海默病的药效学及作用机制[J]. 南方医科大学学报, 2025, 45(4): 785-798.

Niandong RAN, Jie LIU, Jian XU, Yongping ZHANG, Jiangtao GUO. n-butanol fraction of ethanol extract of Periploca forrestii Schltr.: its active components, targets and pathways for treating Alcheimer's disease in rats[J]. Journal of Southern Medical University, 2025, 45(4): 785-798.

图/表 17

图1 黑骨藤正丁醇萃取部位的正负总离子流图

Fig.1 Positive (A) and negative (B) total ion flow diagrams of n-butanol fraction of Periploca forrestii Schltr. ethanol extract.

表1 黑骨藤正丁醇部位中鉴定出的17个化合物信息

Tab.1 Information of the 17 compounds identified in n-butanol fraction of Periploca forrestii Schltr. ethanol extract

Serial number	Retention time	Molecular formula	Measured value	Error value/ppm	Feature fragment(m/z)	Compound name	References
1	1.68	C₁₆H₁₈O₉	353.08691[M-H]^-	-3.45	192.05919, 191.05542, 179.03412,173.04466, 93.03349	Chlorogenic acid	[9]
2	1.73	C₁₆H₁₈O₉	353.40290[M-H]^-	-2.56	191.05544, 179.03429, 135.04407, 91.98469,85.02808, 57.03342	Neochlorogenic acid	[9]
3	9.18	C₁₇H₂₀O₉	369.11716[M+H]⁺	2.12	145.09915, 149.02530, 179.06972, 164.83426	3-O-Caffeoylquinic acid methyl ester	[10]
4	11.15	C₁₀H₈O₄	193.07759[M+H]⁺	4.73	178.24461, 161.94366, 150.83627	Scopoletin	[11]
5	11.98	C₃₅H₆₀O₆	577.98993[M+H]⁺	1.98	413.25134, 319.17458, 295.77209	Daucosterol	[12]
6	13.67	C₁₅H₁₀O₆	287.47942[M+H]⁺	4.34	164.8902, 152.63197	Kaempferol	[13]
7	14.33	C₉H₁₀O₃	167.98975[M+H]⁺	1.09	167.10678, 165.98264, 148.96109,121.96677, 125.96402, 91.05489	Paeonol	[14]
8	14.55	C₂₉H₅₀O	415.21033[M+H]⁺	1.21	119.08556, 91.05432	Beta-Sitosterol	[15]
9	14.76	C₁₅H₁₀O₇	303.23000[M+H]⁺	3.06	301.29196, 257.51855, 151.59630, 107.08958	Quercetin	[16]
10	16.55	C₃₀H₄₈O₃	457.53849[M+H]⁺	1.84	439.68873, 411.50410, 393.35737, 191.18426	Ursolic Acid	[17]
11	18.87	C₂₁H₂₀O₁₁	449.10769[M+H]⁺	1.63	287.16988, 153.36526, 135.96421	Kaempferol 3-O-β- D-glucopyranoside	[18]
12	21.57	C₁₅H₁₂O₄	257.26276[M+H]⁺	3.94	257.26599, 210.88354, 120.98715	Isoliquiritigenin	[19]
13	23.67	C₁₆H₁₂O₅	285.28204[M+H]⁺	3.58	240.88678, 282.27838	Questin	[20]
14	24.41	C₁₆H₁₂O₅	283.05704[M-H]^-	-3.41	267.04141, 239.87805	Wogonin	[21]
15	27.46	C₁₆H₁₂O₅	284.29410[M+H]⁺	1.19	285.29733, 238.87862, 164.61707	Physcion	[22]
16	35.12	C₉H₈O₃	165.11798[M+H]⁺	5.85	165.12164, 148.08638	Trans-4- Hydroxycinnamic acid	[23]
17	35.37	C₁₈H₃₄O₂	283.04980[M+H]⁺	2.08	281.04965, 265.01825	Oleic acid	[21]

图2 绿原酸的质谱图及裂解途径

Fig.2 Mass spectrometry and fragmentation pathway of chlorogenic acid.

图3 汉黄芩素的质谱图及裂解途径

Fig.3 Mass spectrometry and fragmentation pathway of wogonin.

图4 各组大鼠海马HE染色显微图像

Fig.4 HE staining of rat hippocampus in each group. Nucleus (black arrow), cytoplasm (blue arrow), cell body (red arrow), irregular cell (green arrow).

图5 各组大鼠海马组织尼氏染色结果图

Fig.5 Nissl staining of rat hippocampal tissues (A) and statistics of the number of Nissl bodies (arrows)(B) in each group. ##P<0.01 vs Normal group; *P<0.05 vs Model group.

图6 各组大鼠海马内Tau蛋白结果图

Fig.6 Tau protein deposition in rat hippocampus detected by immunohistochemistry (A) and Tau optical density values in CA1 and CA3 regions (B) in each group. #P<0.05, ##P<0.01 vs Normal group; **P<0.01 vs Model group.

图7 ACh、AChE、SOD、CAT和GSH-Px活性检测结果

Fig.7 ACh, AChE, SOD, CAT and GSH-Px activities in rat hippocampus in each group. #P<0.05, ##P<0.01 vs Normal group; *P<0.05, **P<0.01 vs Model group.

图8 黑骨藤正丁醇部位各蛋白相对表达量及蛋白条带示意图

Fig.8 Western blotting for detecting protein expression levels in the hippocampus in each group. A-D: Bcl-2, Bax and caspase-3 protein bands and their relative expression levels. E-J: PI3K, Akt, p-PI3K and p-Akt protein bands and their relative expression levels. #P<0.05, ##P<0.01 vs Control group; *P<0.05, **P<0.01 vs Model group.

图9 黑骨藤正丁醇部位的BDNF蛋白条带示意图及相对表达量

Fig.9 Expression levels of BDNF protein in the hippocampus in each group detected by Western blotting. ##P<0.01 vs Control group; **P<0.01 vs Model group.

图10 黑骨藤正丁醇部位的Nrf-2、Keap-1、HO-1蛋白条带示意图及相对表达量

Fig.10 Expression levels of Nrf-2, Keap-1, and HO-1 in the hippocampus in each group detected by Western blotting. ##P<0.01 vs Control group; **P<0.01 vs Model group.

图11 黑骨藤正丁醇部位的Tau、Aβ蛋白条带示意图及相对表达量

Fig.11 Expression levels of Tau and Aβ protein bands in the hippocampus in each group detected by Western blotting. ##P<0.01 vs Control group; **P<0.01 vs Model group.

图12 药物-疾病交集靶点

Fig.12 Drug-disease intersection targets.

图13 药物-成分-交集靶点图

Fig.13 Drug-component-intersection target map. Red "V" shape: n-butanol fraction; Green rhombus: Active components; Blue circle: Targets of the components.

图14 黑骨藤抗AD潜在靶点PPI网络图

Fig.14 PPI network of the potential targets of n-butanol fraction of Periploca forrestii Schltr. ethanol extract for treating AD.

图15 GO富集分析柱状图和KEGG通路富集分析气泡图

Fig.15 Bar graph of GO enrichment analysis (A) and bubble graph of KEGG pathway enrichment analysis (B).

图16 活性成分与关键靶点分子对接结果图

Fig.16 Docking results of the active components with the key target molecules. A: Beta-Sitosterol-TNF. B: kaempferol-AKT1. C: Quercetin-AKT1. D: Wogonin-ESR1.

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