n-butanol fraction of ethanol extract of Periploca forrestii Schltr.: its active components, targets and pathways for treating Alcheimer's disease in rats

doi:10.12122/j.issn.1673-4254.2025.04.14

Abstract

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

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.

Figures/Tables 17

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

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]

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

Fig.3 Mass spectrometry and fragmentation pathway of wogonin.

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

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.

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.

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.

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.

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.

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.

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.

Fig.12 Drug-disease intersection targets.

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

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

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

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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