Petroleum ether fraction of Myrica nana roots relieves spasmodic abdominal pain in mice by inhibiting intestinal smooth muscle spasm

doi:10.12122/j.issn.1673-4254.2026.05.15

Abstract

Abstract:

Objective To investigate the effect of the petroleum ether fraction of Myrica nana roots (PEFM) for relieving spasmodic abdominal pain in mice and its underlying mechanism. Methods The chemical profile of PEFM was analyzed using GC-MS. Forty Kunming mice were randomized into 5 groups for daily gavage with solvent, dicyclomine, or low-, medium-, and high-dose PEFM for 5 consecutive days, and their effects against neostigmine-induced spasmodic abdominal pain were evaluated. Serum cGMP levels and ileal PKG and p-VASP expressions of the mice were measured. Acute toxicity of PEFM gavage at different doses was assessed in another 32 Kunming mice by monitoring behavioral changes and organ indices. An isolated ileal model of ACh-induced spasm was used to observe the antispasmodic effect of PEFM and its main constituent, stigmast-4-en-3-one (ST), and the mediating roles of the NO/sGC/cGMP/PKG pathway, potassium channels, and calcium channels were investigated using specific inhibitors and a high-potassium-induced spasm model. Molecular docking was used to predict the interactions between compounds and targets. Results PEFM contained mainly steroidal compounds, with ST as the most abundant component (31.51%). PEFM at the medium and high doses significantly reduced abdominal pain frequency, prolonged pain latency, and increased serum cGMP and ileal p-VASP expression without producing significant acute toxicity. Both PEFM and ST exhibited concentration-dependent antispasmodic effects against ACh-induced contractions (EC₅₀: 4.06 μg/mL and 0.53 μg/mL, respectively), which were inhibited by L-NAME, methylene blue, ODQ, and potassium channel blockers; they also strongly antagonized high potassium-induced contractions. Molecular docking confirmed stable binding of ST to PKG and p-VASP. Conclusion PEFM has spasmolytic and analgesic effects in mice with neostigmine-induced spastic abdominal pain, mediated primarily by its main active component ST, which upregulates cGMP, enhances ileal VASP phosphorylation, activates NO/sGC/cGMP/PKG pathway, promotes opening of voltage-gated potassium channels, and antagonizes L-type calcium channels.

Key words: roots of Myrica nana, spasmolysis, abdominal pain, K⁺ channels, NO/sGC/cGMP/PKG pathway, stigmast-4-en-3-one

Shenghai YANG, Wenting CHEN, Xueqin CHEN, Kai TIAN, Yong XIONG, Jingxian SUN, Lingling HUANG, Xiangzhong HUANG, Ziliang WANG. Petroleum ether fraction of Myrica nana roots relieves spasmodic abdominal pain in mice by inhibiting intestinal smooth muscle spasm[J]. Journal of Southern Medical University, 2026, 46(5): 1111-1120.

Figures/Tables 11

Fig.1 Total ion chromatogram of petroleum ether fraction of Myrica nana roots (PEFM).

Tab.1 Major chemical constituents of PEFM (relative content ≥1.5%)

Compounds	Mol. formula	RIexp	RIlit	Content (%)
Stigmast-4-en-3-one	C₂₉H₄₈O	3149.9	3150.2	31.51
24-Methylcholest-5-en-3β-ol acetate	C₂₉H₄₈O₂	3035.9	3035.5	8.96
Cholest-5-en-3β-ol	C₂₇H₄₆O	1968.4	1968.1	8.82
β-Sitosterol	C₂₉H₅₀O	2962.7	2962.8	6.04
γ-Tocopherol	C₂₈H₄₈O₂	2149.1	2149.8	5.41
Menaquinone-7	C₃₁H₄₆O₂	1974.4	1975.6	5.21
Stigmast-5-en-3β-ol	C₂₉H₅₀O	2557.2	2556.3	3.21
Hexadecanoic acid	C₁₆H₃₂O₂	1926.5	1926.0	2.94
3,4,5-Trimethoxybenzoic acid phenyl ester	C₂₀H₂₀O6	2787.4	2785.0	2.39
Cedrol	C₁₅H₂₆O	1544.7	1544.9	1.91

Tab.2 Organ indices of experimental mouse in each group (Mean±SD, n=8)

Group	Weight (g)	Heart index (%)	Liver index (%)	Splee index (%)	Lung index (%)	Kidney index (%)
Control	36.69±1.22	0.58±0.02	5.43±0.22	0.31±0.03	0.71±0.04	1.30±0.06
1 g/kg	36.14±1.8	0.58±0.08	5.41±0.38	0.29±0.05	0.69±0.04	1.31±0.08
2 g/kg	36.59±2.13	0.54±0.05	5.63±0.16	0.31±0.06	0.68±0.04	1.32±0.06
4 g/kg	35.96±1.71	0.56±0.05	5.55±0.27	0.29±0.03	0.68±0.06	1.27±0.05

Fig.2 Effects of PEFM on neostigmine-induced spastic abdominal pain in mice. A: Effect of PEFM on pain frequency. B: Effect of PEFM on painlatency. *P<0.05, **P<0.01, ***P<0.001 vs Control (Mean±SD, n=8).

Fig.3 Measurement of cGMP content in serum (A) and Western blot analysis of p-VASP in the ileum (B). *P<0.05, **P<0.01, ***P<0.001 vs control (n=8).

Fig.4 Effect of PEFM, ST and dicycloverine on Ach-induced spasm contraction of isolated rat ileal smooth muscle. A: PEFM group. B: ST group. C: Dicycloverine group. ***P<0.001 vs Vehicle control (n=6).

Fig.5 Effects of 5 and 10 μg/mL PEFM and 0.3 μg/mL atropine on dose-response curve of Ach. *P<0.05, ***P<0.001 vs Control (n=6).

Fig.6 Effects of NO/sGC/cGMP/PKG pathway blockers L-NAME, MB, and ODQ on antispasmodic activity of PEFM and ST. A: Concentration-response curve for PEFM. B: Concentration-response curve for ST. *P<0.05, **P<0.01, ***P<0.001 vs Control (n=6).

Fig.7 Effects of K+ channel blockers CsCl, TEA, 4-AP, and GLIB on antispasmodic activity of PEFM and ST. A: Concentration-response curve for PEFM. B: Concentration-response curve for ST. **P<0.01, ***P<0.001 vs Control (n=6).

Fig.8 Effects of PEFM, ST and verapamil on KCl-induced contraction of isolated rat smooth muscle. A: PEFM group. B: ST group. C: Verapamil group. ***P<0.001 vs Vehicle control (n=6).

Fig.9 Molecular docking results of ST with p-VASP (A) and PKG (B). The cyan stick in the figure represents small molecules, the blue cartoon represents proteins, and the gray dashed line indicates hydrophobic interactions.

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