Cucumis sativus L. Seeds Ameliorate Muscular Spasm-Induced Gastrointestinal and Respiratory Disorders by Simultaneously Inhibiting Calcium Mediated Signaling Pathway

Abstract

Cucumis sativus L. is globally cultivated as an edible vegetable. Besides its nutritional benefits, it is used in traditional medicines against various ailments. The current study was designed to elucidate the multi-target mechanisms of a C. sativus seeds extract against asthma and diarrhea using network pharmacology along with a molecular docking approach. Furthermore, in-vitro and in-vivo experiments were conducted to verify the mechanistic insight of in silico studies. LC-ESI-MS/MS was performed to identify the bioactive compounds in the extract; later, some compounds were quantified by HPLC. C. sativus seed. EtOH has kaempferol in higher concentration 783.02 µg/g, followed by quercetin (693.83 µg/g) and luteolin (617.17 µg/g). In silico studies showed that bioactive compounds interfered with asthma and diarrhea-associated target genes, which are members of calcium-mediated signaling to exert a calcium channel blocker activity. The seeds extract exerted a concentration-dependent spasmolytic response on isolated jejunum, trachea, and urinary bladder preparations and caused relaxation of spastic contraction of K⁺ (80 mM) with suppressed calcium concentration-response curves at dose 0.3 and 1 mg/mL. It also showed antiperistalsis, antidiarrheal and antisecretory activity in animal models. Thus, C. sativus seeds have therapeutic effects by regulating the contractile response through a calcium-mediated signaling pathway.

Keywords: Cucumis sativus; HPLC; LC-ESI-MS/MS; antidiarrheal; asthma; bronchodilator; cucumber.

Figure 1

ESI-MS/MS spectra in negative and positive mode for tentative compounds of a hydroethanolic extract of C. sativus seeds. The tentative compounds are quercetin, kaempferol, stigmasterol, epicatechin, ferulic acid, 1,4-dicaffeoylquinic acid, apigenin, luteolin, kaempferol-3-O-glucoside, ellagic acid, scopoletin, and β-sitosterol.

Figure 1

ESI-MS/MS spectra in negative and positive mode for tentative compounds of a hydroethanolic extract of C. sativus seeds. The tentative compounds are quercetin, kaempferol, stigmasterol, epicatechin, ferulic acid, 1,4-dicaffeoylquinic acid, apigenin, luteolin, kaempferol-3-O-glucoside, ellagic acid, scopoletin, and β-sitosterol.

Figure 2

HPLC DAD-UV/Vis chromatograms of a hydroethanolic extract of C. sativus seeds at different wavelengths and standard addition validation chromatograms of bioactive compounds. HPLC chromatogram of (A) scopoletin, 1,4-dicaffeoylquinic acid, luteolin, kaempferol, and ferulic acid wavelength 280 nm; (B) epicatechin, ellagic acid, kaempferol-3-O-glucoside, quercetin, and apigenin at wavelength 320 nm; (C) Stigmasterol and β–sitosterol at wavelength 250 nm. The standard addition chromatograms of (D) quercetin; (E) kaempferol-3-O-Glucoside; (F) 1,4 dicaffeoylqunic acid; (G) ellagic acid; (H) kaempferol; (I) luteolin (J) apigenin.

Figure 2

HPLC DAD-UV/Vis chromatograms of a hydroethanolic extract of C. sativus seeds at different wavelengths and standard addition validation chromatograms of bioactive compounds. HPLC chromatogram of (A) scopoletin, 1,4-dicaffeoylquinic acid, luteolin, kaempferol, and ferulic acid wavelength 280 nm; (B) epicatechin, ellagic acid, kaempferol-3-O-glucoside, quercetin, and apigenin at wavelength 320 nm; (C) Stigmasterol and β–sitosterol at wavelength 250 nm. The standard addition chromatograms of (D) quercetin; (E) kaempferol-3-O-Glucoside; (F) 1,4 dicaffeoylqunic acid; (G) ellagic acid; (H) kaempferol; (I) luteolin (J) apigenin.

Figure 3

KEGG and GO biological analysis of bioactive compounds of C. sativus seeds EtOH extract. (A) Dot plot (B) Map enrichment and (C) Chord plot of KEGG and GO biological analysis.

Figure 3

KEGG and GO biological analysis of bioactive compounds of C. sativus seeds EtOH extract. (A) Dot plot (B) Map enrichment and (C) Chord plot of KEGG and GO biological analysis.

Figure 4

Network interaction analysis of bioactive compounds of C. sativus seeds with target disease proteins (C-T-D), compound target pathway (C-T-P) network interaction KEGG and GO biological process pathways.

Figure 5

KEGG pathway for calcium mediates signaling and cholinergic synapse. The red boxes indicate the possible proteins targets of compounds.

Figure 6

3D protein–ligand interaction between bioactive compounds (verapamil, kaempferol, quercetin, ellagic acid, luteolin and kaempferol-3-O-glucoside) and proteins; (A) voltage-gated calcium channel β2a (VGCC, PDB:1T0J); (B) calcium/calmodulin-dependent protein kinase IIB (CAMK2B, PDB: 3BHH); (C) myosin light chain kinase-1 (MLCK-1, PDB:6C6M); and (D) phosphoinositide phospholipase C-gamma-1 (PLCγ-1, PDB: 4EY0). (E) Heatmap of binding energies (kcal/mol), of bioactive compounds (verapamil, kaempferol, quercetin, ellagic acid, luteolin and kaempferol-3-O-glucoside) and proteins. (F) Heatmap of p-adjusted values of binding energies when compared to verapamil. (p < 0.05 vs. verapamil).

Figure 7

2D protein–ligand interaction between bioactive compounds (verapamil, kaempferol, quercetin, ellagic acid, luteolin and kaempferol-3-O-glucoside) and proteins; (A) voltage-gated calcium channel β2a (VGCC, PDB:1T0J); (B) calcium/calmodulin-dependent protein kinase IIB (CAMK2B, PDB: 3BHH); (C) myosin light chain kinase-1 (MLCK-1, PDB:6C6M); and (D) phosphoinositide phospholipase C-gamma-1 (PLCγ-1, PDB: 4EY0).

Figure 8

Effect of C. sativus seeds extracts (Cu.EtOH) and verapamil on jejunum preparation in respect of spontaneous, K⁺ (80 mM), carbachol, and K⁺ (25 mM) contraction concentration-response curves. (A) Physiological responses of Cu.EtOH (B) sigmoidal dose-response curve of Cu.EtOH on jejunum preparations (Values are expressed as Mean ± SD, data was analyzed by sigmoidal dose-response curve).

Figure 8

Effect of C. sativus seeds extracts (Cu.EtOH) and verapamil on jejunum preparation in respect of spontaneous, K⁺ (80 mM), carbachol, and K⁺ (25 mM) contraction concentration-response curves. (A) Physiological responses of Cu.EtOH (B) sigmoidal dose-response curve of Cu.EtOH on jejunum preparations (Values are expressed as Mean ± SD, data was analyzed by sigmoidal dose-response curve).

Figure 9

Effect of C. sativus seeds extracts (Cu.EtOH) and verapamil on tracheal preparations in respect of spontaneous, K⁺ (80 mM), carbachol, and K⁺ (25 mM) contraction concentration-response curves. (A) Physiological responses of Cu.EtOH (B) sigmoidal dose-response curve of Cu.EtOH on tracheal bladder preparations (Values are expressed as Mean ± SD, data was analyzed by sigmoidal dose-response curve).

Figure 9

Effect of C. sativus seeds extracts (Cu.EtOH) and verapamil on tracheal preparations in respect of spontaneous, K⁺ (80 mM), carbachol, and K⁺ (25 mM) contraction concentration-response curves. (A) Physiological responses of Cu.EtOH (B) sigmoidal dose-response curve of Cu.EtOH on tracheal bladder preparations (Values are expressed as Mean ± SD, data was analyzed by sigmoidal dose-response curve).

Figure 10

Effect of C. sativus seeds extracts (Cu.EtOH) and verapamil on urinary bladder preparations in respect of spontaneous, K⁺ (80 mM), carbachol, and K⁺ (25 mM) contraction concentration-response curves. (A) Physiological responses of Cu.EtOH (B) sigmoidal dose-response curve of Cu.EtOH on urinary bladder preparations (Values are expressed as Mean ± SD, data was analyzed by sigmoidal dose-response curve).

Figure 10

Effect of C. sativus seeds extracts (Cu.EtOH) and verapamil on urinary bladder preparations in respect of spontaneous, K⁺ (80 mM), carbachol, and K⁺ (25 mM) contraction concentration-response curves. (A) Physiological responses of Cu.EtOH (B) sigmoidal dose-response curve of Cu.EtOH on urinary bladder preparations (Values are expressed as Mean ± SD, data was analyzed by sigmoidal dose-response curve).

Figure 11

In vivo studies; (A) antiperistalsis; (B) castor oil induces fluid accumulation inhibition; and (C) antidiarrheal effect of seeds extract and verapamil. (Values are expressed as Mean ± SD, data was analyzed by one-way ANOVA followed by Dunnett’s test for in vivo compared to control (normal saline or castor oil group) and p < 0.05 was considered significant (* p < 0.05, ** p > 0.01, *** p < 0.001, ****p < 0.0001), N.S: Normal Saline (10 mL/kg).

Figure 12

Schematic diagram of proposed mechanism of C. sativus seeds extracts on l type voltage gated calcium ion channel, M₃ muscarinic receptor and phosphoinositide phospholipase C (PLC). (IP3: inositol 1, 4, 5-trisphosphate, DAG: Diacylglycerol MLCK: Myosin Light Chain Kinase).