Cucumis sativus L. has also been used in the traditional medicine of Iran, Pakistan, and India. Its seeds are used by herbalists to treat gastrointestinal, respiratory, and urinary problems. However, more investigations are required to explain its mechanisms for treating GI, respiratory, and urinary diseases. Accordingly, the aim of the present work was to investigate the antispasmodic, bronchodilator, and antidiarrheal activities of C. sativus seeds extracts and the underlying mechanisms of action. For this purpose, sequential extracts of C. sativus seeds were prepared in n-hexane, dichloromethane, ethanol, and water. Bioactive compounds in C. sativus seed extracts were identified and quantified by utilizing LC ESI–MS/MS and HPLC. Moreover, network pharmacology and molecular docking were employed to examine the antispasmodic and bronchodilator effects of the bioactive substances in the extracts. In vitro and in vivo experiments were also conducted to validate the mechanistic insights gained from the in silico analysis. Results indicated the presence of kaempferol with a concentration of 813.74 µg/g (highest concentration) in the seed extract of C. sativus, followed by quercetin (713.83 µg/g), narcissin (681.87 µg/g), and orientin (676.19 µg/g). In silico investigations demonstrated that the bioactive chemicals in C. sativus seeds inhibited the expression of the target genes involved in smooth muscle contraction and calcium-mediated signaling. Sequential seed extracts of C. sativus caused a dose-dependent relaxant response for spasmolytic reaction and resulted in a relaxation of K+ (80 mM) spastic contraction. In animal models, C. sativus seed extracts exhibited partial or complete antiperistalsis, antidiarrheal, and antisecretory actions. By modulating the contractile response through calcium-mediated signaling target proteins, C. sativus seeds generated bronchodilator, antispasmodic, and antidiarrheal therapeutic effects.Apart from the nutritional value,  Cucumis sativus L., often called cucumber, is a member of the Cucurbitaceae family extensively cultivated as a culinary vegetable in tropical Asia, South America, and Africa. [C. sativus seeds to treat intestinal, respiratory [C. sativus seeds [C. sativus exhibit anti-depression and anti-ulcer activities [C. sativus seeds are employed in Unani formulations: Sada zarooni jawarish [Sharbat–e–bazoori mutadil [Banadiq ul buzoor [Qurs zarishk [Qurs sartan kafoori [Laooq badam (dry cough and tuberculosis); and Laboob barid [ Africa. ,2. Becau Africa. . On the piratory ,4, and upiratory ,6. Its fpiratory , stomachpiratory , antacidpiratory  qualitiepiratory . Furtherpiratory . It exhipiratory , cytotoxpiratory , antihyppiratory , anti-inpiratory . Diabetepiratory , hyperlipiratory , intermius seeds ,18. Resetivities . Furtherjawarish ; Sharbat mutadil ; Banadiql buzoor ; Qurs za zarishk ; Qurs sa kafoori ; Laooq bob barid ,24 for uC. sativus contain various sterols, including β–sitosterol, stigmasterol, and campesterol [C. sativus seeds [O–dirhamnoside, ferulic acid, quercetin, cinnamic acid, p-coumaric acid, caffeic acid, and chlorogenic acid in the seeds of C. sativus [C. sativus seeds and fruits [From a phytochemical point of view, the seeds of pesterol ,25,26; hus seeds . In thisus seeds  reported sativus ,29,30. I sativus ,31, are d fruits ,25,30. Td fruits .C. sativus seeds; (2) determine the bioactive compounds present in successive extracts of C. sativus seeds; (3) assess its potential bronchodilator, antispasmodic, anti-asthmatic, and antidiarrheal properties; and (4) based on the results, explore the pharmacodynamic mechanisms of C. sativus extracts employing in vitro, in vivo, and in silico models. In light of the preceding discussion, the primary objectives of this study were to: (1) prepare sequential extracts of n-hexane, dichloromethane (DCM), ethanol, and the aqueous seed extracts of C. sativus. The data for these detected compounds are summarized in The LC ESI–MS/MS study revealed the presence of 42 bioactive constituents in C. sativus seed extracts were compared with commercially available external standards using retention time (Rt) of separation peaks at various wavelengths (λ) 320, 280, 270, and 250 nm to increase the visibility of the chromatographic spikes  for respiratory and gastrointestinal illnesses using different pathways.In contrast, for the DCM extract, the numerous GO terms exhibited a predominance of target genes for bioactive compounds, with a particular emphasis on the regulation of neurotransmitter levels, rhythmic process, peptidyl-serine phosphorylation, peptidyl-serine modification, peptidyl-threonine phosphorylation, circadian rhythm, neurotransmitter transport, placenta development, peptidyl-threonine modification, neurotransmitter metabolic process, modulation of chemical synaptic transmission, regulation of DNA-binding transcription factor activity, neurotransmitter secretion, and cellular response to dopamine. Cholinergic synapse, chemokine, GnRH, ErbB, neurotrophin, HIF-1, the synthesis, secretion, and action of growth hormones, relaxin, prolactin, glioma, EGFR tyrosine kinase inhibitor resistance, adrenergic signaling in cardiomyocytes, dopaminergic synapse, and the calcium signaling pathway were some of the KEGG signaling pathways impacted by active compounds. The target genes of the ethanol extract’s bioactive compounds were significantly portrayed in numerous GO pathways, with a particular emphasis on neurotransmitter regulation, blood circulation regulation, chemical synaptic transmission modulation, trans-synaptic signaling control, homeostasis of cellular calcium ions, homeostasis of cellular divalent inorganic cations, peptidyl threonine phosphorylation, and concentration regulation of cytosolic calcium ions. In addition, the ethanol bioactive compounds modulate the following KEGG signaling pathways: cholinergic synapse; calcium signaling pathway; GnRH signaling system; dopaminergic synapse; lipid and atherosclerosis, and ErbB signaling; contraction of vascular smooth muscle; the production, secretion and action of growth hormones; cardiomyocyte adrenergic signaling; gastric acid secretion; oxytocin signaling; diabetic cardiomyopathy; cancer proteoglycans; and cAMP signaling. Similarly, the numerous GO terms exhibited a predominance of target genes for aqueous extracts’ bioactive compounds clearly emphasizing chemical synaptic transmission modification, trans-synaptic signaling, muscular system regulation, metal ion transport regulation, neurotransmitter levels control, amyloid–beta response, cellular response to chemical stress, and peptidyl-threonine. The route of calcium signaling, cholinergic synapse, proteoglycans in cancer, ErbB cascade pathways, GnRH cascade systems, HIF-1 cascade systems, oxytocin cascade systems, lipids and atherosclerosis, cAMP signaling routes, cGMP–PKG signaling pathways, dopaminergic synapses, vascular smooth muscle contractions, cardiomyocyte adrenergic signaling, and leukocyte transendothelial migration are the key KEGG signaling pathways affected by active substances.C. sativus and disease target genes, a C–T–D network with 103 nodes and 265 edges was built in Cytoscape. According to network analysis, kaempferol has the most favorable impact on the target genes with a degree of 45, after quercetin (degree = 43), luteolin (degree = 38), apigenin (degree = 38), and linoleic acid (degree = 23) (C. sativus seed extract (All retained pathogenic targets were subjected to the STRING plugin of Cytoscape for protein–protein interaction (PPI). The target genes for the gastrointestinal and respiratory tracts contained 28 nodes and 79 edges. Between the bioactive chemicals from ee = 23) . Based oee = 23)  and KEGG extract .n-hexane extract had 41 nodes and 154 edges. The network study of n-hexane revealed that linoleic acid and palmitic acid, with degrees of 33.6 and 16.8, had stronger, more positive impacts on the disease target genes. The following GO biological process terms had a strong positive interaction with n-hexane bioactive constituents: protein-coupled receptor signaling pathway coupled to cyclic nucleotide second messenger (degree = 76.8); trans-synaptic signaling (degree = 18.9); modulation of chemical synaptic transmission (degree = 18.9); regulation of neurotransmitter levels (degree = 18.9); adenylate cyclase-modulating G protein-coupled receptor signaling pathway (degree = 16.8); and neurotransmitter transport (degree = 16.8). The n-Hexane had 45 nodes and 133 edges in the KEGG pathways C–T–P. The network analysis revealed that linoleic and palmitic acids, with degrees of 44.8 and 11.2, had more powerful and positive impacts on the disease target genes. The following KEGG pathways had a strong positive interaction with n-hexane bioactive constituents: cholinergic synapse (degree = 47.6); neuroactive ligand–receptor interaction (degree = 44.4); signaling route in cAMP (degree = 36); calcium-dependent signaling (degree = 11.2); cancer proteoglycans (degree = 9.6); estrogen signaling pathway (degree = 9.6); and signaling route for relaxin (degree = 9.6).In the C–T–P of GO biological process terms, DCM extract had 39 nodes and 134 edges in the C–T–P of GO biological process terms. β-Sitosterol (degree = 21), umbelliferone (degree = 18.9), and stigmasterol (degree = 18.9) exhibited more powerful therapeutic impacts on the disease targeted genes, according to network analysis. Peptidyl-serine modification (degree = 18.9), peptidyl-serine phosphorylation (degree = 18.9), regulation of neurotransmitter levels (degree = 18.9), regulation of DNA-binding transcription factor activity (degree = 16.8), and modulation of chemical synaptic transmission (degree = 16.8) were the biological processes with the most target genes and compounds. There were 36 nodes and 121 edges in the KEGG pathways C–T–P. β-sitosterol (degree = 12.8), umbelliferone (degree = 12.8), and stigmasterol (degree = 11.2) exhibited more powerful positive impacts on disease target genes, according to the network analysis. Cholinergic synapse (degree = 56), adrenergic signaling in cardiomyocytes (degree = 29.6), the chemokine signaling route (degree = 21.2), and the calcium signaling system (degree = 11.2) were the KEGG pathways that had the highest target genes and chemicals. The ethanol extract had 62 nodes and 253 edges in the C–T–P of GO biological process terms. Quercetin (degree = 90.3), narcissin (degree = 43.1), luteolin (degree = 25.2), and apigenin (degree = 25.2) exhibited more significant therapeutic impacts on disease targeted genes, according to the network analysis. Cellular calcium ion homeostasis (degree = 87.3), regulation of neurotransmitter levels (degree = 53.1), muscle system process (degree = 45.2), calcium ion homeostasis (degree = 27.3), and regulation of trans-synaptic signaling (degree = 27.3) were the biological processes with the most target genes and compounds. The KEGG pathways C–T–P included 59 nodes and 254 edges. Quercetin (degree = 65.6), luteolin (degree = 19.2), apigenin (degree = 17.6), and narcissin (degree = 16) had more powerful positive impacts on the disease targeted genes, according to the network analysis. Cholinergic synapse (degree = 60.8), calcium signaling route (degree = 56.5), adrenergic signaling in cardiomyocytes (degree = 56), cAMP signaling pathway (degree = 47.6), and cGMP–PKG signaling system (degree = 37.6) were the KEGG pathways with the highest target genes and chemicals. Aqueous extract had 56 nodes and 199 edges in the C–T–P of GO biological process terms. Kaempferol (degree = 94.5), kaempferol 3–O–glucoside (degree = 23.1), and ferulic acid (degree = 21) exhibited more powerful therapeutic impacts on disease-targeted genes, according to the network analysis. The muscular system process (degree = 25.2), control of trans-synaptic signaling (degree = 25.2), modulation of chemical synaptic transmission (degree = 25.2), and regulation of metal ion transport (degree = 23.1) were the biological processes with the most target genes and chemicals. The KEGG pathways C–T–P has 52 nodes and 199 edges. According to the results of the network analysis, kaempferol (degree = 65.6), kaempferol 3–O–glucoside (degree = 16), and ferulic acid (degree = 16) have more potent therapeutic effects on disease target genes. The calcium signaling route (degree = 54.4), cholinergic synapse (degree = 36), proteoglycans in cancer (degree = 19.2), and cAMP signaling pathway (degree = 17.6) were the KEGG pathways with the highest target genes and chemicals.The protein homology model of voltage-gated calcium ion channel (P22002), muscarinic M3 acetylcholine receptor (P08483), myosin light chain kinase (P20689), and phospholipase C–gamma–1 (P10686) were built with sequences obtained from UniProt.−1cm−1 for the Cys, Trp, and Tyr concentrations. This coefficient helps quantify the protein–protein and protein–ligand interactions in solution.ProtParam was employed to determine the physicochemical characteristics of voltage-gated calcium channel sequences, muscarinic acetylcholine receptor, myosin light chain kinase, and phospholipase C–gamma–1. The amino acid profile of the voltage-gated calcium channel revealed a molecular weight of 243,482.46 and 2169 total number of amino acids with a maximum of 10.3% of Leu, 7.7% of Ser, and 7.5% of Ala residues. The negatively charged (Asp + Glu) residues were 229, whereas the positively charged residues (Arg + Lys) were 95. The PI value of 6.60 indicates that the protein is almost neutral, while the instability index of 50.36 shows that it is more unstable than 40. This instability was anticipated owing to the presence of specific dipeptides that are absent from stable proteins. A higher aliphatic index (91.34) indicates that the protein’s thermostability is significant, whereas a lower GRAVY score (−0.059) indicates that the protein might interact better with water. At 280 nm, the extinction coefficient is 230,910 M−1cm−1 for the Cys, Trp, and Tyr concentrations.The amino acid profile of the muscarinic acetylcholine receptor revealed a molecular weight of 66065.76 and 589 total number of amino acids with a maximum of 12.8% Ser, 7.1% Ala, and 5.3% Asn residues. The negatively charged (Asp + Glu) residues were 45 and positively charged (Arg + Lys) 62. The PI value of 9.22 indicates that the protein is alkaline, while the instability index is 52.69. A high aliphatic index (86.45) shows that the protein is thermostable, whereas a lower GRAVY score (−0.129) indicates that the protein might interact better with water. At 280 nm, the extinction coefficient is 105,310 M−1cm−1 for the Cys, Trp, and Tyr concentrations.The amino acid profile of myosin light chain kinase revealed a molecular weight of 65,816.22 and 610 total number of amino acids with a maximum of 9.2 percent Ala residues, followed by 8.7 percent Leu residues, and 8.5 percent Glu residues. The negatively charged (Asp + Glu) residues were 89, and the positively charged (Arg + Lys) residues were 67. The PI value of 5.05 indicates that the protein is acidic, while the instability index is 48.27. A higher aliphatic index (73.00) showed that the protein is thermostable, whereas a lower GRAVY score (−0.497) indicated that the protein might interact better with water. At 280nm, the extinction coefficient is 33,920 M−1cm−1 for the Cys, Trp, and Tyr concentrations. The amino acid profile of phospholipase C–gamma–1 revealed a molecular weight of 148,548.09 and 1290 total number of amino acids with a maximum of 9.2% Leu, followed by 8.8% Glu, and 7.4% Ser residues. The negatively charged (Asp + Glu) residues were 187, and the positively charged (Arg + Lys) residues were 158. The PI value of 5.67 indicates that the protein is acidic, and the instability index is 45.12. A high aliphatic index (74.47) shows that the protein is thermostable, whereas a low GRAVY score (−0.549) indicates that the protein might interact better with water. At 280 nm, the extinction coefficient is 179000 MWe employed the homology BLAST search to find templates for homology modeling voltage-gated calcium channel, muscarinic acetylcholine receptor, myosin light chain kinase, and phospholipase C–gamma–1; the sequence alignment is shown in The Ramachandran plot was used to verify the model when it was checked using PROCHECK, and this plot reflected the amino acid distribution in phi and psi angles. B. The caMolecular docking calculations may be used to determine the position of the ligand inside the binding pockets of the target proteins. The physical energy terms such as solvation energy are combined with a suitable force field for docking calculation accuracy ,34. The Our findings showed the following: Hesperidin  scored the first rank for VGCC and formed conventional H-bonds interaction with Thr734 (1.96 Å), Leu733 (1.73 Å), Thr1443 (2.20 Å), Thr1142 (2.60 Å), Gly1444 (2.90 Å), Gly1444 (1.75 Å), Leu298 (2.26 Å), Carbon H-Bonds interaction with Leu733 (2.44 Å), Leu733 (2.63 Å), Gly1444 (2.34 Å), Thr391 (2.77 Å), Pi–Pi T-Shaped with Phe767 (5.17 Å), alkyl interaction with Ala302 (4.28 Å), Met392 (4.91 Å), Met295 (3.90 Å), Pi–alkyl interaction with Ala302 (5.23 Å), Val430 (5.00 Å), Ile768 (5.25 Å). Rutin  ranked second position in the docking scores. It formed conventional H-bonds interaction with Tyr1489 (2.19 Å), Ala1183 (1.89 Å), Ala1183 (1.88 Å), Thr1443 (1.85 Å), Ala1442 (2.44 Å), Ile390 (1.97 Å), Thr1142 (2.86 Å), Gly1444 (2.21 Å), Gly1444 (1.76 Å), carbon H-bonds interaction with Thr1443 (2.56 Å), Amide–Pi Stacked:Gly735; C, O; Glu736 (5.21 Å), Alkyl interaction with Ala1493 (3.37 Å), Pi–Alkyl interaction with Tyr1489 (3.97 Å), Met392 (5.35 Å), Met392 (5.36 Å), Leu427 (5.26 Å), Met392 (5.45 Å). Narcissin  formed conventional H-bonds interaction with Tyr1489 (2.03 Å), Ala1442 (1.75 Å), Thr1142 (2.10 Å), Ser1141 (2.52 Å), Ala1183 (1.70 Å), carbon H-bonds interaction with Ala1442 (3.05 Å), Thr1142 (2.66 Å), Thr1443 (2.39 Å), Ala1442 (3.01 Å), Thr1443 (2.46 Å), Pi–Sulfur interaction with Met1187 (5.56 Å), Pi–Pi T-Shaped:Phe1190 (5.18 Å), Tyr1489 (5.92 Å), alkyl interaction with Met1187 (3.81 Å), Pi–Alkyl interaction with Phe1190 (4.04 Å), Val1191 (4.78 Å), Ala1493 (4.19 Å).Quercetin  formed Conventional H-Bonds interaction with Ile390 (2.07 Å), Leu298 (1.94 Å), Pi–donor hydrogen bonds interaction with Tyr772 (2.52 Å), Pi–Alkyl interaction with Val430 (4.70 Å), Ile768 (5.00 Å), Leu298 (5.02 Å), Ala302 (4.93 Å), Val430 (5.16 Å). Orientin  formed conventional H-bonds interaction with Tyr1489 (2.15 Å), Tyr1489 (2.71 Å), Leu733 (2.09 Å), Leu733 (1.93 Å), Asn771 (2.42 Å), Thr391 (1.86 Å), Thr1142 (1.81 Å), Asn1188 (1.72 Å), Asn1188 (1.78 Å), carbon H-bonds interaction with Leu733 (2.72 Å), Thr734 (2.94 Å), Pi–Pi T-Shaped with Phe1143 (4.75 Å), Phe1143 (4.87 Å). Kaempferol  formed conventional H-bonds interaction with Ile390 (2.08 Å), Leu298 (1.96 Å), Pi–Alkyl interaction with Val430 (4.74 Å), Ile768 (4.95 Å), Leu298 (5.05 Å), Ala302 (4.90 Å), Val430 (5.16 Å). Verapamil  formed carbon H-bonds interaction with Leu733 (2.65 Å), Leu733 (2.49 Å), Asn771 (2.92 Å), Leu733 (3.08 Å), Thr734 (2.78 Å), Thr391 (2.69 Å), Ile390 (2.51 Å), Pi–Pi T–Shaped interaction with Phe767 (5.41 Å), Alkyl interaction with Leu427 (5.06 Å), Ile1497 (5.33 Å), Leu431 (5.22 Å), Val1191 (3.69 Å), Met392 (3.89 Å), Pi–Alkyl interaction with Val1191 (5.46 Å). Our results revealed that Orientin  was ranked first in the docking scores, and formed stable hydrogen and hydrophobic bonds with the M3 receptor. It formed conventional H-bonds with Phe312 (1.78 Å), Val390 (2.09 Å), Glu393 (2.95 Å), Asp355 (1.82 Å), Asp355 (1.80 Å), carbon H-bonds with Phe312 (2.66 Å), Glu393 (2.67 Å), Glu393 (2.28 Å), Glu393 (2.63 Å), Pi–cation interaction with His311 (3.89 Å). Rutin  scored the second rank in the docking scores and formed conventional H-bonds with Phe312 (2.94 Å), Asp329 (1.95 Å), Asp355 (2.04 Å), Asp355 (1.96 Å), carbon H-bonds with Trp313 (2.81 Å), Trp313 (2.74 Å), Trp313 (2.49 Å), Pi–Anion interaction with Glu393 (3.00 Å), Glu393 (3.61 Å), Pi–Sigma: GLU393 (2.81 Å), Pi–alkyl interaction with Val390 (5.02 Å). Next, narcissin  scored the third position. It formed conventional H-bonds with Gln389 (2.52 Å), Glu393 (1.90 Å), Gln389 (2.81 Å), Asp394 (1.91 Å), Asp329 (1.90 Å), carbon H-bonds with Val390 (2.48 Å), Glu393 (2.37 Å), Gln389 (2.90 Å), Glu393 (2.43 Å), Asp394 (2.97 Å), Glu393 (3.07 Å), Glu393 (2.64 Å), Phe312 (2.51 Å), Glu393 (2.84 Å), Pi–Cation interaction with His311 (4.67 Å), Pi–Anion interaction with Asp329 (4.78 Å), Pi–Lone Pair interaction with TRP313 (2.94 Å), Pi–Pi T-shaped bond with His311 (4.73 Å). Hesperidin  formed conventional H-bonds with Asp329 (1.81 Å), Glu393 (1.82 Å), Gln389 (1.85 Å), Glu393 (3.04 Å), Phe312 (1.66 Å), carbon H-bonds with Arg304 (3.05 Å), Arg304 (2.66 Å), Arg304 (2.74 Å), His311 (2.50 Å), Val390 (2.63 Å), Glu393 (2.70 Å), Asn392 (2.75 Å), Pi–Anion interaction with GLU393 (3.87 Å), alkyl interaction with ALA362 (3.86 Å). Quercetin , a significant flavonoid responsible for many biological activities, formed conventional H-bonds with Val390 (1.68 Å), Pi–cation interaction with His311 (4.68 Å), Pi–Anion interaction with Asp355 (5.00 Å), Pi–anion interaction with Glu393 (3.39 Å). Kaempferol  formed conventional H-bonds with VAL390 (1.86 Å), Asp355 (1.75 Å), Carbon H-bonds with His311 (2.91 Å), Pi–anion interaction with Glu393 (3.54 Å), Pi–Anion interaction with Glu393 (4.04 Å), Pi–Lone Pair with Phe312 (3.00 Å), Pi–alkyl interaction with Val390 (5.49 Å). Verapamil  formed a salt bridge and electrostatic attractive charge with Glu393 (1.93 Å), conventional H-bonds with Arg361 (2.04 Å), carbon H-bonds with Ser358 (2.96 Å), Phe312 (2.79 Å), Phe312 (2.72 Å), Gln389 (2.85 Å), Glu393 (2.83 Å), Asn392 (2.66 Å), Pi–Sigma: Glu393 (2.39 Å), Alkyl interaction with Ala362 (4.18 Å), Arg361 (4.40 Å). Rutin  ranked in the first position MYLCK and formed conventional H-bonds with Gln333 (2.43 Å), Lys422 (3.09 Å), Gln333 (1.96 Å), Gln333 (2.01 Å), Gly458 (3.00 Å), Asp337 (2.53 Å), Gly442 (2.67 Å), Gly458 (1.77 Å), carbon H-bonds with Lys309 (2.58 Å), Gly458 (2.92 Å), Pi–sulfur interaction with Met340 (4.28 Å), Pi–Pi stacked interaction with Phe310 (4.35 Å), Phe310 (3.84 Å), Phe310 (5.74 Å), Pi–alkyl interaction with Val455 (5.07 Å). Hesperidin  scored the second rank and formed conventional H-bonds with Gln333 (1.96 Å), Lys422 (2.48 Å), Thr459 (2.09 Å), Asn456 (1.87 Å), Gly458 (1.81 Å), Thr459 (2.90 Å), Carbon H-bonds with Asn456 (3.01 Å), Gly458 (2.56 Å), Gly458 (2.87 Å), Asp501 (2.91 Å), Pi–Anion interaction with Asp501 (3.60 Å), alkyl interaction with Met340 (5.38 Å), Val455 (4.85 Å). 1,4–Dicaffeoylquinic acid  formed an attractive charge with Lys336 (4.92 Å), conventional H-bonds with Gln333 (2.87 Å), Gln333 (2.65 Å), Lys336 (1.78 Å), Lys422 (2.63 Å), Met340 (2.67 Å), Asp337 (1.73 Å), Asp420 (2.30 Å), Gly458 (2.29 Å), Pi–alkyl interaction with Val455 (5.34 Å). Narcissin  formed conventional H-bonds with Lys336 (2.93 Å), Lys336 (2.30 Å), Lys422 (2.30 Å), Met340 (2.52 Å), Tyr470 (1.81 Å), Gly458 (1.71 Å), carbon H-bonds with Lys331 (2.84 Å).Orientin  formed conventional H-bonds with Asp420 (2.06 Å), Gly442 (1.98 Å), Gly458 (2.86 Å), carbon H-bonds with Thr459 (2.69 Å), Pi–cation interaction with Lys422 (3.61 Å), Pi–cation and Pi–donor hydrogen bonds with Lys422 (2.79 Å), Pi–anion interaction with Asp420 (4.22 Å), amide–Pi stacked interaction with Lys309 C–O and Phe310 (3.65 Å). Quercetin  formed conventional H-bonds with Gln333 (2.85 Å), Gly442 (2.88 Å), Gly458 (1.72 Å), Asp420 (2.24 Å), Pi–anion interaction with Asp420 (4.09 Å), Pi–Pi stacked interaction with Phe310 (4.57 Å), Phe310 (5.76 Å), Pi–alkyl interaction with Leu463 (5.45 Å). Verapamil  formed an attractive charge with Asp420 (3.54 Å), conventional H-bonds with Lys336 (2.74 Å), carbon H-bonds with Lys309 (3.02 Å), Lys309 (2.61 Å), Asp420 (2.79 Å), Asn456 (2.40 Å), Pi–donor hydrogen bonds with Asn456 (2.92 Å), alkyl interaction with Met340 (4.71 Å), Pi–alkyl interaction with Val455 (5.07 Å), Lys309 (5.17 Å). Rutin  had the first position for PLCG–1. It formed an attractive charge with Lys941 (5.01 Å), carbon H-bonds with Ser981 (2.85 Å), Ser981 (2.29 Å), Ser982 (2.73 Å), Glu473 (2.63 Å), Glu414 (2.28 Å), conventional H-bonds with Lys464 (2.44 Å), Arg945 (2.59 Å), Arg946 (1.77 Å), Ser982 (2.06 Å), Tyr1012 (2.52 Å), Glu365 (1.77 Å), Ser982 (1.72 Å), Pi–alkyl interaction with Tyr1012 (5.03 Å), Arg945 (5.14 Å), Pro964 (4.98 Å), Met480 (5.06 Å), Arg945 (5.31 Å), Met480 (4.13 Å), Pro964 (3.89 Å), Pi–cation interaction with Lys941 (4.61 Å), Pi–cation, and Pi–donor hydrogen bonds with Arg946 (3.03 Å), Pi–Sulfur interaction with Met480 (4.46 Å). 1,4–Dicaffeoylquinic acid  ranked the second position for PLCG–1 and formed conventional H-bonds with Gly940 (2.53 Å), Glu483 (1.85 Å), Ser478 (1.75 Å), Ile413 (1.91 Å), Ile413 (3.00 Å), Arg946 (1.83 Å), Arg946 (2.05 Å), Arg946 (2.33 Å), Lys941 (2.52 Å), Pi–Alkyl interaction with Val479 (5.36 Å), Lys464 (5.41 Å), Pi–anion interaction with Glu483 (4.48 Å). Quercetin  formed conventional H-bonds with Lys462 (2.75 Å), Lys462 (2.44 Å), Lys464 (2.73 Å), Arg945 (2.35 Å), Arg945 (2.62 Å), Glu414 (2.54 Å), Ile413 (2.17 Å), Lys462 (2.67 Å), His380 (1.83 Å), Carbon H-bonds with Lys464 (2.63 Å), Ser482 (2.97 Å), Arg945 (2.43 Å), Gly1015 (2.94 Å), Pi–anion interaction with Glu414 (3.56 Å), Pi–Pi T-Shaped: Tyr1012 (4.77 Å). Narcissin  formed conventional H-bonds with Ser478 (2.32 Å), Arg945 (2.43 Å), Arg946 (2.74 Å), Ser478 (1.81 Å), Arg946 (2.92 Å), carbon H-bonds with Thr477 (2.68 Å), Pi–Sigma: Glu944 (2.85 Å), Pi–alkyl interaction with Lys941 (4.76 Å), Arg945 (5.34 Å), Val479 (5.33 Å), Lys941 (3.74 Å).Orientin  formed conventional H-bonds with Met480 (2.53 Å), Arg945 (2.27 Å), Arg946 (2.80 Å), Lys941 (1.66 Å), Ser478 (1.58 Å), Ser478 (1.65 Å), carbon H-bonds with Glu944 (2.65 Å), Glu944 (2.40 Å), Pi–cation interaction with Arg946 (3.32 Å), Pi–sulfur: Met480 (5.89 Å), Pi–alkyl interaction with Arg945 (5.38 Å), Lys941 (5.31 Å), Arg945 (5.44 Å). Hesperidin  formed conventional H-bonds with Lys464 (2.22 Å), Met480 (1.80 Å), Arg945 (2.63 Å), Arg946 (1.91 Å), Arg946 (3.04 Å), Arg946 (2.12 Å), Ser478 (2.32 Å), carbon H-bonds with Val479 (2.73 Å), Glu944 (2.74 Å), Glu944 (2.77 Å), Pi–cation, and Pi–donor hydrogen bonds with Arg946 (3.83 Å), Pi–sulfur: Met480 (4.26 Å), alkyl interaction with Val479 (4.84 Å), Pro962 (4.89 Å), Pi–Alkyl interaction with Lys941 (5.11 Å), Arg945 (4.52 Å), Pro964 (4.43 Å). Verapamil  formed a salt bridge and attractive charge with Glu473 (3.00 Å), conventional H-bonds with Met480 (2.53 Å), Arg945 (2.57 Å), carbon H-bonds with Glu473 (2.41 Å), Asp415 (2.94 Å), Glu414 (2.42 Å), Ser478 (2.76 Å), Pi–cation interaction with Arg946 (3.05 Å), Pi–anion interaction with Glu414 (3.44 Å), alkyl interaction with Arg945 (3.74 Å), Arg945 (4.43 Å), Arg946 (4.41 Å), Pi–alkyl interaction with His416 (4.21 Å), Arg945 (5.35 Å), Met480 (4.24 Å), Arg945 (5.12 Å), Pro964 (5.24 Å). n-hexane, DCM, ethanol, and aqueous seed extracts of C. sativus were studied. All extracts exhibited dose-dependent spasmogenic and spasmolytic effects, and a dose-dependent partial or complete relaxation of K+ (80 mM) and K+ (25 mM) elicited contractions. When both n-hexane and DCM were exposed to rhythmic or periodic jejunal contractions, they displayed dose-dependent contractile responses (n-hexane and DCM extracts were attenuated and a dose-dependent spasmolytic effect was established in an atropine (1 µM) pretreatment jejunum preparation, with EC50 values of 0.3572 mg/mL  and 0.8343 mg/mL . On the other hand, ethanol and aqueous seed extracts diminished the spontaneous rhythmic contraction in a dose-dependent pattern, with EC50 values of 0.3313 mg/mL  and 0.5625 mg/mL , respectively.In spontaneously contracting jejunum preparations, the antispasmodic action of esponses . The span-hexane, DCM, ethanol, and aqueous seed extracts of C. sativus were evaluated in jejunum preparations against contractions of K+ (80 mM) and K+ (25 mM). The n-hexane extract showed a dose-dependent partial inhibition towards the sustained contractions of K+ (80 mM) with an EC50 of 15.0 mg/mL . It also inhibited the K+ (25 mM)-induced contraction in a dose-dependent manner with an EC50 of 0.5396 mg/mL , hence it facilitated the opening of potassium ion channels for relaxation. In jejunum preparation for K+ (80 mM) and K+ (25 mM) elicited persistent contractions. On the other hand, the DCM, ethanol, and aqueous seed extracts showed dose-dependent relaxing effects. The respective EC50 values for K+ (80 mM) were 6.365 mg/mL (4.823–8.591), 0.3538 mg/mL (0.2708–0.4704), and 0.5647 mg/mL (0.4430–0.7249), respectively. The EC50 values for K+ (25 mM) were 0.6536 mg/mL (0.5408–0.7937), 0.3141 mg/mL (0.2484–0.4016), and 0.3976 mg/mL (0.3200–0.5007), respectively  pretreated tissues. They caused a non-parallel rightward shift of calcium CRCs and suppressed them in atropinized tissue, suggesting that the cholinomimetic components mediated the contractile response in the absence of atropine  and K+ (25 mM) provoked contractions in jejunal preparations with EC50 values of 0.05872 µM , 0.1163 µM , and 0.09815 µM , respectively . In the pretreatment of atropine (1 µM), the contractile response of n-hexane and DCM was significantly (p < 0.001 vs. Ach 1 µM) reduced, equivalent to cholinergic contraction , CCh (1 µM), and K+ (25 mM) , with EC50 values of 10.61 mg/mL . However, it inhibited the spastic contractions of CCh (1 µM) and K+ (25 mM) in a dose-dependent manner, with EC50 values of 0.6294 mg/mL  and 0.7957 mg/mL , respectively. On the other hand, DCM, ethanol, and aqueous extracts inhibited K+ (80 mM) elicited contractions in a dose-dependent fashion, with EC50 values of 4.975 mg/mL (3.536–7.158), 0.1915 mg/mL (0.1544–0.2382), and 0.8502 mg/mL (0.6443–1.137), respectively. These extracts demonstrated that a dose-dependent inhibition of CCh (1 µM) and K+ (25 mM) elicited contractions. The EC50 values for CCh (1 μM)-induced contractions were 0.2386 mg/mL (0.1856–0.3090), 0.1141 mg/mL (0.08161–0.1634), and 0.2637 mg/mL (0.1853–0.4044), respectively. The EC50 values for the K+ (25 mM)-elicited contractions were 0.6538 mg/mL (0.5303–0.8107), 0.2713 mg/mL (0.2103–0.3536), and 0.4938 mg/mL (0.3887–0.6405).Our findings revealed that the  (25 mM) . The n-hC. sativus extracts (1 and 3 mg/mL). These extracts inhibited carbachol CRCs in a noncompetitive rightward shift , K+ (80 mM), and K+ (25 mM) in a dose-dependent manner, with EC50 values of 0.1864 µM , 0.1212 µM , and 0.1214 µM , respectively. The CRCs for CCh were also produced for verapamil, which resulted in the CRCs for CCh being suppressed at a dose equivalent to that of C. sativus extracts , K+ (80 mM), and K+ (25 mM)  persistent contraction were 0.8711 mg/mL (0.6984–1.096), 0.6436 mg/mL (0.5122–0.8114), 0.1378 mg/mL (0.06979–0.2638mg/mL), and 0.2378 mg/mL (0.1834–0.3110 mg/mL), respectively. The EC50 for K+ (80 mM)-induced contractions were 2.279 mg/mL (1.725–3.039), 2.086 mg/mL (1.564–2.858), 0.1262 mg/mL (0.09527–0.167), and 0.1680 mg/mL (0.1208–0.2351), respectively. The EC50 for K+ (25 mM)-evoked contractions were 2.556 mg/mL (1.837–3.708), 1.098 mg/mL (0.9157–1.328), 0.1343 mg/mL (0.1048–0.1720), and 0.1958 mg/mL (0.1453–0.2657), respectively.The  (25 mM) . The EC5C. sativus extracts (1 and 3 mg/mL) on rabbit urinary bladder preparation, and C. sativus seed extracts caused a non-competitive rightward shift with suppression of calcium CRCs (+ (80 mM), and K+ (25 mM) in a dose-dependent manner, with respective EC50 values of 0.05390 µM , 0.1425 µM , and 0.1653 µM , respectively  reduction in charcoal meal propulsive movement. The peristalsis index of the ethanol extract was 22.6% at 150 mg/kg and 16.30% at 300 mg/kg, whereas the peristalsis index of the aqueous extract was 32.1% at 150 mg/kg and 18.4% at 300 mg/kg. Loperamide and verapamil were used as the positive controls and showed similar results. On the other hand, the n-hexane and DCM extracts failed to reduce peristaltic motions, which differs somewhat from the negative control (71.4%). At 150 mg/kg, the peristalsis index for n-hexane and DCM extracts was 49.6% and 38.6%, respectively, and at 300 mg/kg, it was 64.6% and 52.1%, respectively (Pretreatment with ethanol and aqueous ectively . C. sativus seed extracts causes a significant (p < 0.001 vs. the negative control) inhibitory effect on castor oil-induced diarrhea. At 150 mg/kg, the ethanol and aqueous extracts showed 66.6% and 60.4% protection against castor oil-induced diarrhea, respectively, and caused 81.3% and 77.6% protection at 300 mg/kg. That was equivalent to the positive controls of loperamide and verapamil in terms of diarrhea prevention (p < 0.001 vs. the negative control). However, unlike the negative control, the n-hexane and DCM extracts failed to suppress diarrhea (4.3%). At 150 mg/kg, the n-hexane and DCM extracts showed 25.5% and 52% protection, respectively, while at 300 mg/kg, they exhibited 11% and 32.3% protection, which is non-significant against diarrhea  in fluid accumulation (136 ± 1.9 g) in the intestinal fluid accumulation assay (112 ± 1.8 g). When mice were pretreated with ethanol and aqueous C. sativus seed extracts, the intestinal secretions were decreased considerably (p < 0.001 vs. castor oil group). At 150 mg/kg, the weights of fluid for ethanol and aqueous extracts were 96.6 ± 1.15 and 101.3 ± 2.06 g, respectively, whereas, at 300 mg/kg, the weights were 83.7 ± 1.2 and 88.1 ± 0.93 g, respectively, which were similar to the positive control groups treated with loperamide (75.4 ± 1.2 g) and verapamil (78.0 ± 0.13 g). In contrast, the n-hexane and DCM extracts failed to cause an antisecretory effect. At a dosage of 150 mg/kg, the n-hexane and DCM extracts raised fluid accumulation to 109 ± 2.6 g and 105 ± 2.1 g, respectively, whereas at 300 mg/kg, these extracts increased fluid accumulation to 122 ± 1.7 g and 118 ± 2.3 g, respectively , alkaloids, flavones, and flavonoids in the seed extracts of C. sativus [C. sativus seeds are rich in polyphenols such as quercitrin, rutin, and orientin [Herbal therapy has been globally recognized as a safe and effective approach for treating a wide range of chronic disorders. Consumption of non-nutrient secondary metabolites  from a hdiseases ,16,25,41 sativus ,15,38,42orientin , along worientin ,44.C. sativus seed extracts anticipated that these compounds would have antispasmodic action via interfering with calcium-mediated signaling and cholinergic synapses , and muscarinic M3 receptor. Thus, it can be assumed that the strong antispasmodic activity of C. sativus seed extracts was due to the high affinity of compounds for target proteins, which may inhibit signal transduction responsible for contraction. In silico analyses of bioactive chemicals isolated from synapses . The GO C. sativus seed extracts were tested against spontaneous contractions of jejunal preparations, and the findings of the polar and nonpolar extracts were opposite. The ethanol and aqueous C. sativus seed extracts reduced involuntary contractions by preventing calcium entry into the cells. However, n-hexane and DCM extracts revealed a contractile response at low dosages and a spasmolytic reaction at higher concentrations. As previously stated, physiological mediators elicit contractile response binding with their receptors to speed up cytosolic calcium influx and trigger depolarization. The spasmogenic response of n-hexane and DCM extracts was reduced by atropine (1 µM), demonstrating the existence of cholinergic components in both extracts [The gastrointestinal motor tone is regulated by several physiological mediators . These pextracts .C. sativus seed extracts were evaluated for their spasmolytic activity in jejunum preparations to see whether it was mediated by calcium channel blockage or potassium channel opening [+ (80 mM) promotes depolarization in the cell through calcium influx, thus increasing the calcium ions. In jejunum preparations, this intense depolarization of the membrane action potential induced a long-lasting sustained contractile response [C. sativus seed extracts relaxed K+ (80 mM), elicited contractions, inhibited the provoked depolarization, and repolarized the membrane potential by inhibiting the calcium current inflow. In addition, the following series of events were inhibited [ opening . Resultsresponse ; the smonhibited ,51: (1) nhibited .n-hexane exhibited partial relaxation in response to contractions caused by K+ (80 mM). Similar to verapamil, pretreatment with ethanol and aqueous C. sativus seed extracts resulted in the suppression and rightward shift of calcium ion channel CRCs on jejunum preparations [C. sativus seed extracts acted as calcium ion channel blockers and repolarized the membrane action potential [n-hexane and DCM extracts failed to diminish the calcium CRCs, although both extracts showed a shift to the right of the CRCs with the reduction in atropine pretreated tissues. Findings showed that the n-hexane and DCM extracts contain cholinomimetic and CCBs compounds [n-hexane and DCM extracts to be impotent [+ (25 mM) provoke contractions and are referred to as K+ channel openers, CCBs have an equal affinity to inhibit both K+ (80 mM) and K+ (25 mM) provoked contractions [C. sativus seed extracts and verapamil inhibited K+ (80 mM) entirely, while K+ (25 mM) induced contractions [n-hexane and DCM extracts was investigated using rat ileum, which is advantageous for assessing spasmogenic response due to its quiescent nature [n-hexane or DCM extract was administered in bolus dosages, it induced a dose-dependent contractile response similar to acetylcholine. Atropine (1 µM) inhibited the contractile response of both n-hexane and DCM extracts, suggesting the presence of cholinomimetic drugs capable of initiating the activation of M3 muscarinic receptors [At a concentration of 10 mg/mL, the DCM extract relaxed, while the arations , which motential . On the ompounds . Cholinoimpotent ,36. Althractions . Thus, Cractions . The spat nature . When n-C. sativus seed extracts were tested for antispasmodic activity by depolarizing tracheal and urinary bladder preparations with persistent contractions of CCh (1 µM) and K+ (80 mM) [C. sativus seed extracts relaxed the CCh (1 µM) induced contractions. However, C. sativus seed extracts demonstrated the same findings for K+ (80 mM)-induced contractions on the tracheal and urinary bladder, as previously found in jejunal preparations, that ethanol and aqueous extracts relaxed the contractions. However, the DCM extract relaxed the contractions at 10 mg/mL, whereas the n-hexane extract could not attain complete relaxation. Based on the EC50 value of CCh (1 µM), C. sativus seed extracts act on the muscarinic receptors in tracheal and urinary bladder preparations. A rightward shift in carbachol CRCs on tracheal preparations similar to verapamil revealed the anticholinergic effect of C. sativus seed extracts [3) and diacylglycerol (DAG). IP3 stimulates inositol 1,4,5-trisphosphate receptors (IP3R) on the sarcoplasmic reticulum to release calcium ions, increasing the cytosolic calcium ion levels. Diacylglycerol and calcium ions activate a regulatory protein phosphokinase C (PKC), through which phosphorylation of calmodulin occurs to form a calcium/calmodulin complex. This calcium/calmodulin complex activates MLCK, which causes the phosphorylation of MLCs. The phosphorylated MLCs and actin form an interaction network to produce a contractile response [C. sativus seed extracts was confirmed by a rightward shift in the carbachol CRCs on tracheal preparations such as verapamil [C. sativus extracts behaved as bronchodilators to reduce cytosolic calcium release from the sarcoplasmic reticulum, by inhibiting the muscarinic receptor path of contractile response. (80 mM) . By estaextracts . The actresponse . The anterapamil . Consequp < 0.05 vs. control), whereas the n-hexane and DCM analogs failed to suppress them. Moreover, the ethanol and aqueous seed extracts produced an antispasmodic response, thus preventing the charcoal meal from traveling the distance and blocking the gut’s rhythmic contractions. Castor oil hydrolysis in the gut releases an active ingredient called ricinoleic acid [n-hexane and DCM seed extracts showed no substantial suppression of the peristaltic movements, castor oil-induced fluid secretion, and castor oil-induced diarrhea. These findings suggest the existence of certain constituents that prevent the n-hexane and DCM extracts from inhibiting spasms.On the other hand, the ethanol and aqueous extracts significantly inhibited peristalsis, diarrhea, and electrolyte imbalance  was deposited at the herbarium located at the same institute. After removing the husks, the inner part of the seeds was ground with an herbal grinder into a granular powder. Approximately 120 g of the pulverized material was subjected to successive extraction (fractions) in the Soxhlet apparatus with solvents of varying polarity. Solvents were evaporated at a reduced pressure using rotary evaporation to yield oils (n-hexane and DCM) and thick brownish yellowish tinted residues (ethanol and aqueous) of C. sativus seed extracts. The overall yield of C. sativus seeds extracts was 53.9%, whereas the n-hexane, DCM, ethanol, and aqueous yields were 28 g, 16.1 g, 12 g, and 8.6 g, respectively. The extracts of C. sativus seed were placed in an amber-colored container and kept at a temperature of −20 ± 3 °C. The n-hexane and DCM extracts were freshly prepared in 1.5 percent v/v Tween 80 on the experiment day, whereas the ethanol or aqueous extract was dissolved in distilled water or 0.9% normal saline solution. For Tween 80, we first dissolved both n-hexane and DCM extracts in respective solvents n-hexane and dichloromethane, then added the 1.5 percent v/v Tween 80 and thoroughly mixed it on the ultra-sonicator. Then we evaporated the respective solvent at 30 ± two °C and then dissolved it in distilled water or 0.9 percent normal saline solution. All preparations were diluted to the desired concentration. The seeds of p-coumaric acid, luteolin 7–O–glucoside, kaempferol, ferulic acid, kaempferol–3–O–glucoside, hesperidin, sodium chloride (NaCl), potassium dihydrogen phosphate (KH2PO4), glucose (C6H12O6), calcium chloride (CaCl2), sodium biphosphate (NaH2PO4), potassium chloride (KCl), magnesium sulfate (MgSO4), magnesium chloride (MgCl2), sodium bicarbonate (NaHCO3), and ethylene tetra acetic acid (EDTA), acetylcholine chloride (CID: 6060), atropine sulfate (CID: 5927), loperamide hydrochloride (CID: 71420), verapamil hydrochloride (CID: 62969), carbachol chloride (CID: 5831). The following chemicals and materials were obtained from Sigma–Aldrich Co.  and used as received without further purification: Analytical grade solvents such as methanol (MeOH), ethanol (Et), acetonitrile (ACN), formic acid (FA), trifluoroacetic acid (TFA), umbelliferone, stigmasterol, campesterol, β-sitosterol, epicatechin, scopoletin, ellagic acid, luteolin, apigenin, orientin, 1,4–dicaffeoylquinic acid, narcissin, quercetin, naringenin, C. sativus seed extracts were diluted in 1 mL of 100% methanol for HPLC and LC–ESI–MS/MS analysis, and the supernatant was collected after centrifugation for 12 min at 14,000 rpm. The supernatant was filtered using a 0.22 µm syringe filter. The sequential polarity-based C. sativus seed extracts to identify bioactive constituents [LC ESI–MS/MS analyses were performed on tituents ,61. We eBinary Eluent system: Different solvent systems were employed for C. sativus seed extracts due to the different nature of extracts. The mobile phase for separating the constituents of the ethanol and aqueous extracts was solvent A, made up of 0.1 percent v/v formic acid (FA) in methanol, and solvent B, which was made up of 0.1 percent v/v FA in aqueous and ACN (80:20). Similarly, the mobile phase was used to separate the constituents of Cu.Hexane and Cu.DCM was solvent A, which comprised 0.1 percent v/v FA in methanol, and solvent B, which comprised 0.1 percent v/v FA in ACN. For solvent A (v/v), the elution gradient profile was modified as follows: 98%; 98–80%; 80–70%; 70–10%; and 10–98% from 0–4.0; 4.0–8.0; 8.0–15.0; 15.0–17.0; and 17.0–20.0 min, respectively [ectively .Optimal chromatographic conditions: The optimal chromatographic parameters for separation of the constituents of C. sativus seed extracts were: the flow-rate of the mobile phase was 0.4 mL/min; the temperature of the column was 25 °C; the volume of sample-injection was 8 µL; the range for mass-spectrum (m/z) was 50 to 1000; and the size of the column (C–18) was 2.1 mm × 100 mm, 3.0 µm. The optimal mass-spectrometry conditions were: the positive and negative mode ion spray voltage was 5500 V and −5500 V, respectively; the pressure of curtain gas (CUR), nebulizer gas (GS1), auxiliary gas (GS2) were 25, 55, 55 psi, respectively; the ion source temperature for mass-spectrometry was 400 °C. Nitrogen was used as a CUR and GS2. A collision-induced dissociation (CID) energy range of 10 to 45 was used for ion fragmentation based on parent ion stability. The ESI–MS/MS data were collected and processed using XCalibur 3.0.Identification of Compounds: Bioactive constituents of C. sativus seed extracts were identified by comparing their mass spectral and chromatographic data to those previously published and reference libraries [ibraries . ChemDraWe performed RP-HPLC experiments to establish the validity and quantification of bioactive compounds ,49. Binary Eluent system: The mobile phase in HPLC for separating the constituents of ethanol and aqueous extracts was solvent A, made up of 0.1 percent v/v trifluoroacetic acid (TFA) in methanol, and solvent B, comprised of 0.1 percent v/v TFA in aqueous and ACN (80:20). Similarly, the mobile phase used to separate the n-hexane and DCM extracts constituents was solvent A, which comprised 0.1 percent v/v TFA in methanol, and solvent B, which comprised 0.1 percent v/v TFA in ACN. For solvent A (v/v), the elution gradient profile was modified as follows: 95%; 95–90%; 90–80%; 80–10%; and 10–95% from 0–4.0; 4.0–8.0; 8.0–14.0; 14.0–20.0; and 20.0–30.0 min, respectively. Optimal chromatographic conditions: The optimal chromatographic parameters for separation of constituents of C. sativus seed extracts were: the flow-rate of the mobile phase was 0.8 mL/min; the temperature of the column was 25 °C; the volume of sample-injection was 8 µL; the size of the column (C–18) was 4.6 mm × 150 mm, 5.0 µm; and the appropriate wavelengths for peak detection were 250; 270; 280; and 320 nm. The results of the C. sativus seed extracts were compared with the peaks and retention time (Rt) of external standards. A validation method was developed under the International Conference on Harmonization (ICH) principles, as outlined in our prior work . Preparation of external standards: A total of 150 µg of external standards  were dissolved in 1 mL of 100 percent methanol to prepare the stock solution of external standards, then centrifuged at 14,000 rpm for 10 min and the supernatant was collected and stored at −20 °C. On the experiment day, the stock solution of external standards was further diluted as per the experiment method. A 0.22 µm syringe filter was used to collect and filter the supernatant.Linearity, limits of detection, and quantification: The dilutions (7.81–500 µg/mL) of each external standard were prepared for linearity validation of HPLC, and a linear regression standard was developed for each external standard, based on the concentration and peak area of each standard. The detection (LOD) and quantification (LOQ) limits were calculated by using the following formula:Specificity: An injection volume of 8 µL of C. sativus seed extracts and external standards samples were injected at a flow rate of 0.8 mL/min into RP-HPLC using the above ideal chromatographic settings. To measure the peak purity of the C. sativus seed extracts, the peaks and retention time (Rt) of the C. sativus seed extracts were compared with external standards. The peaks were quantified using a linear regression standard for each external standard. Precision and repeatability: Under the above-mentioned appropriate chromatographic parameters, the precision and repeatability of this method was studied for each external standard on inter-day (n = 4) and intra-day (n= 4 for 3 days), and the relative standard deviation (RSD) was determined.Accuracy and recovery: The external standards were recovered from the C. sativus seed extracts to assess the accuracy and recovery of this method. Three different concentrations  of external standards were used for this approach. Furthermore, 100 mg of C. sativus seed extract and 50, 100, and 150 µg of the external standards were mixed thoroughly in 0.5 mL of methanol and then centrifuged at 14,000 rpm for 10 min, and the supernatant was collected, and the volume was made up to 1 mL with methanol, to get a final concentration of external standards of 50, 100, and 150 µg/mL. An injection volume of 0.8 µL of this mixture was injected into the RP-HPLC utilizing the optimal chromatographic parameters, as described above. The sample recovery percentage of an external standard was calculated using the following equationSprague Dawley rats (170 ± 20 g), mice (28 ± 3 g), and locally bred rabbits (1.2–1.4 kg) of either sex were used throughout this investigation. The temperature of these animals was maintained at 23–25 °C in dark/light cycled animal houses, with free access to a standard diet and water. They were maintained according to the Animals By-Laws, which were authorized by the Ethical Committee of the Department of Pharmacology (vide No. EC /04–PhDL/S2018), and the research was carried out in compliance with the Commission of Laboratory Animal Resources . RabbitsScreening of potential protein targets: The potential protein targets were retrieved from DrugBank  and Swiss target prediction  for the bioactive constituents of C. sativus seed extracts. The potential protein targets for diseases were obtained from GeneCards , DisGeNET , Pubmed , and Online Mendelian Inheritance in Man (OMIM)  with keywords “diarrhea”; “chronic diarrhea”; “chronic constipation”; “constipation”; “irritable bowel syndrome (IBS)”; “asthma”; “obstructive pulmonary disorder ”; and “coughing.” We adopted the approach provided by Xiao et al.  for netwhttps://ve.genecards.org/, accessed on 16 July 2021) was used to select the top 150 disease target genes for gastrointestinal and tracheal disorders under clinical phenotype and genetic association. Using the package “ggvenn” in RStudio, we conducted an intersection evaluation between the bioactive chemicals of C. sativus seed extracts and disease target genes, and a Venn Diagram was constructed to illustrate the relationship between the potential protein targets of bioactive chemicals and disease. Further, we evaluated the KEGG pathway and gene ontology (GO) enrichment for intersected potential protein targets. The VarElect tool  network, the Protein–Protein Interaction (PPI) network, and the Compounds–Targets–Pathways (C–T–P) network were all constructed and analyzed in Cytoscape 3.8.0. http://www.web.expasy.org/protparam, accessed on 23 July 2021) was used to identify different physical and chemical qualities [Homology modeling identifies the three-dimensional structure of a protein, based on UniProt sequence similarities to one or more PDB templates. This strategy is consistent with the notion that a target structural conformation is more conserved than its amino acid sequence. The ExPASy ProtParam tool  and the PDBSum server  [The following stages are involved in homology modeling : template identification from UniProt sequence; BLAST homology search (NCBI PDB Database); sequence alignment (ClustW); model construction; and then validation of the model. The protein model failed to pass the Protein Reliability report and the Ramachandran plot. We proceeded further for the refinement of the build model, which included loops refinement and energy reduction using the Prime Refine Loops module and the Prime minimize module, choosing OPLS3e force field and VSGB solvation model. The minimized protein model was then evaluated in PROCHECK for the Ramachandran plot on the SAVES version 6 server (ly 2021) ,65. https://pubchem.ncbi.nlm.nih.gov, accessed on 19 July 2021) was used to obtain three-dimensional structures of ligands in mol2 format. In addition, the force field of OPLS3e was utilized to optimize and minimize the energy of ligands to increase the precision and effectiveness of the protein-ligand model with the lowest energy conformer. The Epik tool was utilized for putative promoters, and ionization states were created at the desired pH (7.0 ± 2.0). The tautomeric states were created for each ionized ligand, and the ligands with the most atoms were kept. The stereoizer tool was used to generate stereoisomers of ligands, while retaining data on stereogenic chirality from the input file.We employed the method described by Sirous and colleagues  for the In addition, the protein preparation module was used to assign hydrogen bonds, minimize het states, ionize, and optimize the three-dimensional crystal structures of the built protein model. The built protein model structure was modified to eliminate duplicate binding sites and determine if the protein–ligand interaction is a dimer or a multimer. The chemical component dictionary (CCD) database was utilized to reorder the het groups and non-templated amino acid residues. The formal charges assigned to metal ions and surrounding atoms had been altered, and covalent bonds between metal ions and neighboring atoms had been reduced to zero—order. If two sulfur atoms are within 3.2 Å of each other in the protein structure, disulfide bonds are formed between them. Additionally, the prime tool had been used to validate and fill in the structural gaps in the protein, such as those caused by missing side-chain atoms. Finally, the Epik tool was used to produce the protonation and metal charge states of the het groups for cofactors and metals with a pH of (7.0 ± 2.0) within a range of 5.0 Å. The hydrogen atoms were substituted for water molecules and het groups in the protein structure within a range of 5.0 Å. The PROPKA tool had been used to optimize the assignment of hydrogen bonds in protein structures at a pH of 7.0 and the OPLS3e force field for constraint energy minimization and protein structural optimization.The binding pockets were identified for molecular docking and receptor grid generation modules. In this scenario, a literature search and sitemap module were used to identify the coordinates of the cubic grid box to facilitate the ligand binding in the protein pockets. The coordinates for muscarinic receptor M3 were , PLCG1 , VGCC , and MYLK . The length of the grid box was extended to 16 Å in each coordinate with a dock ligand length of 20 Å. Using Van der Waals radii of non-polar atoms of protein with a partial charge cutoff of 0.25, the potential of non-polar portions of the receptor was decreased by a factor of 1.0.The extra precision Glide was utilized to perform molecular docking with the previously prepared compounds, proteins, and receptor grid files. For the Van der Waals radii, the partial charge was adjusted to 0.15 with a scaling factor of 0.80. The ligand sampling was optimized for flexible docking and includes nitrogen (non-ring) inversions and ring conformations to generate conformers. Additionally, the torsions around the bond were sampled for bias. With the Epik tool, penalties were applied to the docking score. The ligand-protein complex binding energies were computed using the Prime MM–GBSA module with VSGB solvation and the force field OPLS3e.Inhibition Constant (Ki): The inhibition constant was derived using the ligand’s binding free energy, using the following equation:−1. K−1), and T is the room temperature (298 Kelvin). We have used the methods of Saqib and Janbaz , and Wah2 and 5% CO2). A tissue organ bath of 15 mL was used to fix each jejunum preparation filled with Tyrode’s solution, aerated with carbogen gas, and maintained at 37 ± 0.5 °C with a circulating thermoregulator. Before assessing the drug, a force of 1 ± 0.1 g was preloaded onto the jejunal preparation. Spontaneous rhythmic contractions were observed after allowing the jejunal preparation to be equilibrated for 25 ± 5 min with Tyrode’s solution changes every 8 min. The tissue physiological responses were recorded using an isotonic transducer (MLT0015), which were amplified by a data-collecting device, Power Lab® (4/25), and shown in Lab Chart Pro (Version 7). The physiological response was determined using the contraction percentage obtained immediately before the addition of the test substance, to test whether n-hexane and DCM, ethanol, and aqueous C. sativus seed extracts had spasmolytic and spasmogenic effects. A tissue organ bath materialized the jejunal preparation [Animals were sacrificed and dissected to open the abdomen. Tissues from the adjacent mesenteries were removed, and sections of jejunum tissue about 2 to 3 cm in length were produced. To keep the jejunum preparation alive and ready for use, they were removed and stored in a freshly prepared Tyrode’s solution, aerated with carbogen gas  or potassium ion channel activation with K+ (25 mM). The extracts were applied cumulatively to get a dose-dependent inhibitory response of C. sativus seed extracts. K+ (80 mM) may affect the smooth muscle contraction by depolarizing the cell and causing an influx of calcium ions. Due to the extract’s ability to inhibit contractions induced by K+ (80 mM), it was designated a calcium channel blocker [The antispasmodic activity of  blocker .C. sativus seed extracts were tested to see whether they have a calcium antagonistic effect. To diminish intracellular calcium concentrations, the jejunal preparation was incubated with K+ (80 mM) three times and then equilibrated in an EDTA containing calcium-free Tyrode solution for 25 ± 5 min, followed by 50 ± 5 min incubation in calcium-free and potassium-rich Tyrode’s solution. After incubation, calcium was added to the jejunum preparation in a cumulative fashion to produce calcium concentration–response curves (CRCs). When calcium CRCs were made as a control, jejunal preparations were washed and incubated with C. sativus seed extracts for 50 ± 5 min. These calcium CRCs were made with various doses of C. sativus seed extracts and compared to control calcium CRCs, to examine whether any calcium antagonistic activity was present [Further, the  present .n-hexane and DCM seed extracts [n-hexane and DCM extracts were used to assess the contractile response.The mesenteries encircling the ileum tissue of rats were removed, and 2 to 3 cm long ileum tissue segments were obtained. Each ileum preparation was processed in the same manner as previously described in the jejunum preparation section. The ileum preparation was incubated in a tissue organ bath to determine the cholinergic response of extracts . Bolus a® (4/25), and shown in Lab Chart Pro (Version 7). The physiological response was determined using the contraction percentage obtained immediately before the addition of the test substance. To test whether n-hexane and DCM, ethanol, and aqueous C. sativus seed extracts had bronchodilation activity [+ (80mM), and K+ (25mM).A rabbit tracheal tissue was dissected, and 3–4 mm length rings were formed by excising adjacent fatty and adhesion materials. Smooth muscles were positioned between cartilaginous segments after laterally incising the tracheal rings to produce a tracheal strip. A tissue organ bath of 15 mL was used to fix each tracheal preparation, filled with Krebs solution, aerated with carbogen gas, and maintained at 37 ± 0.5 °C with a circulating thermoregulator. Before assessing the drug, a force of 1 ± 0.1 g was preloaded onto the tracheal preparation after allowing the tracheal preparation to be equilibrated for 50 ± 5 min, with Krebs solution changes every 8 min. The physiological responses of the tissue were recorded using an isometric transducer (FORT100), which were amplified by a data-collecting device, Power Labactivity , they weC. sativus seed extracts were tested to see whether they have a muscarinic receptor antagonism effect. The tracheal preparation was incubated with CCh (1µM) three times and followed by 50 ± 5 min of incubation in Krebs solution. After incubation, carbachol was added to the tracheal preparation in a cumulative fashion to produce carbachol (CCh) CRCs. When the CCh CRCs were made as a control, tracheal preparations were washed and incubated with C. sativus seed extracts for 50 ± 5 min. These CCh CRCs were made with various doses of C. sativus seed extracts and compared to the control CCh CRCs to examine whether any muscarinic receptor antagonistic activity was present [Further,  present . ® (4/25), and shown in Lab Chart Pro (Version 7). The physiological response was determined using the contraction percentage obtained immediately before the addition of the test substance. To test whether n-hexane and DCM, ethanol, and aqueous C. sativus seed extracts had antispasmodic activity [+ (80 mM), and K+ (25 mM).The urinary bladder tissue of a rabbit was dissected, and the rings of the urinary bladder were incised laterally to form a urinary bladder strip. A tissue organ bath of 15 mL was used to fix each tracheal preparation, filled with Krebs solution, aerated with carbogen gas, and maintained at 37 ± 0.5 °C with a circulating thermoregulator. Before assessing the drug, a force of 1 ± 0.1 g was preloaded onto the urinary bladder preparation after allowing the urinary bladder preparation to equilibrate for 50 ± 5 min with Krebs solution changes every 8 min. The physiological responses of tissue were recorded using an isometric transducer (FORT100), which were amplified by a data-collecting device, Power Labactivity , they weC. sativus seed extracts were tested to see whether they have a calcium antagonistic effect. To diminish intracellular calcium concentrations, the urinary bladder preparation was incubated with K+ (80 mM) three times and then equilibrated in an EDTA containing calcium-free Krebs solution for 25 ± 5 min, followed by 50 ± 5 min incubation in calcium-free and potassium-rich Krebs solution. After incubation, calcium was added to the jejunum preparation in a cumulative fashion to produce calcium CRCs. When calcium CRCs were made as a control, urinary bladder preparations were washed and incubated with C. sativus seed extracts for 50 ± 5 min. These calcium CRCs were made with various doses of C. sativus seed extracts and compared to control calcium CRCs to examine whether any calcium antagonistic activity was present [Further,  present . C. sativus seed extracts was determined by randomly dividing the rats into six groups (five rats of either gender per group). For 28 days, one group received saline, while the other groups received 50, 100, 150, 200, and 300 mg/kg of C. sativus seed extracts every day. The extract was injected into the stomach via the esophagus using a stainless-steel needle linked to a plastic syringe. Bodyweight, behavioral and mood changes, clinical symptoms of pain or discomfort, and animal mortality were all tracked for 28 days [The maximum tolerated dose of  28 days . C. sativus seed extracts was determined by randomly dividing the mice into 12 groups (five mice of either gender per group). Before the trial, all groups were allowed unrestricted access to water, but the food was removed for an overnight period.This test was conducted using the method described by Saqib and Janbaz . The antGroup I was marked as a negative control, orally given 10 mL/kg of 0.9% normal saline; Group II was marked as a positive control, orally given normal saline , then after 15 min, a charcoal meal (10 mL/kg) was given orally;Group III, as standard control, was orally given loperamide (10 mg/kg);Group IV, as standard control, was orally given verapamil (10 mg/kg);n-hexane C. sativus seed extract, respectively;Groups V and VI were orally given 150 and 300 mg/kg of C. sativus seed extract, respectively;Groups VII and VIII were orally given 150 and 300 mg/kg of DCM C. sativus seed extract, respectively;Groups IX and X were orally given 150 and 300 mg/kg of ethanol C. sativus seed extract, respectively.Groups XI and XII were orally given 150 and 300 mg/kg of aqueous After 15 min of treatment, all groups except Group I received an oral dose of the charcoal meal (10 mL/kg) comprising 20% starch, 10% vegetable charcoal, and 10% gum acacia in normal saline. After 30 min, mice were sacrificed by cervical dislocation, and their abdomens were dissected to remove the whole small intestine so that the distance traveled by the charcoal meal could be measured. The percentage of the peristaltic index was calculated using Dt/Li × 100, where Dt is the distance traveled by the charcoal meal, and Li is the length of an animal’s small intestine. C. sativus seed extracts was determined by randomly dividing the mice into 12 groups (five mice of either gender per group). Before the trial, all groups were allowed unrestricted access to water, but the food was removed for an overnight period.Castor oil-induced diarrhea was conducted using the method described by Wahid et al., . The antGroup I was marked as a negative control, orally given 10 mL/kg of 0.9% normal saline;Group II was marked as a positive control, orally given normal saline , then after 30 min, castor oil (10 mL/kg) was given orally;Group III, as standard control, was given loperamide (10 mg/kg) orally;Group IV, as standard control, was given verapamil (10 mg/kg) orally;n-hexane C. sativus seed extract, respectively;Groups V and VI were orally given 150 and 300 mg/kg of C. sativus seed extract, respectively;Groups VII and VIII were orally given 150 and 300 mg/kg of DCM C. sativus seed extract, respectively;Groups IX and X were orally given 150 and 300 mg/kg of ethanol C. sativus seed extract, respectively.Groups XI and XII were orally given 150 and 300 mg/kg of aqueous After 30 min of treatment, all groups except Group I received an oral castor oil (10 mL/kg) oral dose. Six hours later, wet diarrhea patches on white paper were observed in the cages, and the average quantity of feces in each group was calculated. The percentage inhibition was calculated using (Dc–Dt)/Dc × 100, where Dc is the mean defecation of the control group, and Dt is the mean defecation of the test group. C. sativus seed extracts was determined by randomly dividing the mice into 12 groups (five mice of either gender per group). Before the trial, all groups were allowed unrestricted access to water, but the food was removed for an overnight period.Castor oil-induced intestinal fluid accumulation was conducted using the method described by Wahid et al., . The antGroup I was marked as a negative control, orally given 10 mL/kg of 0.9% normal saline; Group II was marked as a positive control, orally given normal saline , then after 30 min, castor oil (10 mL/kg) was given orally;Group III, as standard control, was orally given loperamide (10 mg/kg);Group IV, as standard control, was orally given verapamil (10 mg/kg);n-hexane C. sativus seed extract, respectively;Groups V and VI were orally given 150 and 300 mg/kg of C. sativus seed extract, respectively;Groups VII and VIII were orally given 150 and 300 mg/kg of DCM C. sativus seed extract, respectively;Groups IX and X were orally given 150 and 300 mg/kg of ethanol C. sativus seed extract, respectively.Groups XI and XII were orally given 150 and 300 mg/kg of aqueous After 30 min of treatment, all groups except Group I received an oral dose of castor oil (10 mL/kg). After 30 min, mice were sacrificed by cervical dislocation, their abdomens were dissected, and both ends of the small intestine pylorus and caecum were ligated, then the whole small intestine was removed so that the weight of the small intestine with and without intestinal fluid could be calculated. The findings were determined using the formula (Pi/Pm) × 1000, where Pm represents the mouse’s body weight, and Pi represents the intestine’s weight. The weight of fluid collected in the intestine was used to calculate the amount of fluid accumulated (g). 50) values were calculated. Using logarithmic sigmoidal dose–response graphs, concentration–response curves were plotted. Data were subjected to one-way analysis of variance (ANOVA). Statistical analysis was performed with the aid of the Dunnett test for significance with the aid of GraphPad Prism–8 ; differences were considered significant at p ≤ 0.05.The results are expressed as the mean ± standard deviation (SD), and 95% confidence intervals (CI) for the half-maximal effective concentration (ECCucumis sativus L. seed extracts, mainly ethanol and aqueous, exhibited medicinal effects that could be used to treat disorders such as asthma and diarrhea by regulating the contractile response via the L voltage-gated calcium ion channel, M3 muscarinic receptor, or other proteins, such as PLC, to the repolarize membrane action potential. Our results demonstrated that the dichloromethane and n-hexane extracts elicited a spasmogenic reaction, with higher dosages eliciting a spasmolytic response. This might be due to the cholinomimetic phytoconstituents in jejunum preparation, which cause the spasmogenic reaction. In vitro and in silico studies of C. sativus seed extracts validate these suppression phenomena of phytoconstituents in the same extract. Finally, C. sativus seed extracts include several bioactive chemicals that might be utilized to cure diseases and illnesses. Furthermore, our results may explain why C. sativus is used as an antidiarrheal medicinal plant, which explains its usage in traditional medicine to treat diarrhea, dysuria, and urge incontinence.In conclusion,


Objective: Traditional Mongolian Medicine Qiqirigan-8 (MMQ-8) is a Chinese botanical drug with effective pharmacological properties in obesity. However, the pharmacological mechanism of MMQ-8 remains unclear. This study aimed to determine the active metabolites of MMQ-8 and its therapeutic effects on lipid metabolism and inflammation.Methods: The active metabolites of MMQ-8 were identified by ultrahigh-performance liquid chromatograph Q extractive mass spectrometry (UHPLC-QE-MS) assay and network analysis. An obesity rat model induced by high-fat diet was used in the study. Serum levels of lipids and inflammatory factors were detected using biochemical analysis and enzyme-linked immunosorbent assay (ELISA). Pathological analysis of liver tissues and arteries was conducted with hematoxylin and eosin (H&E) staining and immunohistochemistry. Protein expression of the tumor necrosis factor (TNF) signaling pathway was investigated by Western-blot. Simultaneously, bone marrow cells were used for RNA sequencing and relevant results were validated by cell culture and quantitative real-time polymerase chain reaction (RT-qPCR).Results: We identified 69 active metabolites and 551 target genes of MMQ-8. Of these, there are 65 active metabolites and 225 target genes closely related to obesity and inflammation. In vivo, we observed that MMQ-8 had general decreasing effects on body weight, white adipose tissue weight, and serum lipids. MMQ-8 treatment notably decreased the liver function markers and hepatic steatosis, and significantly decreased inflammation. In serum, it notably decreased TNF-α, interleukin (IL)-6, and inducible nitric oxide synthase (INOS), while elevating IL-10 levels. MMQ-8 treatment also significantly inhibited proteins phosphorylation of nuclear factor-kappa B inhibitor alpha (IκBα), mitogen-activated protein kinase (p38), extracellular regulated kinase 1/2(ERK1/2), and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), and decreased vascular endothelium damage and macrophage infiltration and polarization to M1. These findings coincide with the RNA-sequencing data of bone marrow cells and results of in vitro experiments.Conclusion: We determined the pharmacological actions and relevant metabolites of MMQ-8 in obesity for the first time. Our study revealed MMQ-8 can optimize lipid metabolism and reduce chronic inflammation in obesity. However, more in-depth research is needed, for example, to understand the principle of compound compatibility and the inhibition effects on hepatic steatosis, T cell differentiation, and inflammatory signal transduction. Obesity is a chronic metabolic disease that has become a major health problem worldwide. Furthermore, it is a significant risk factor for insulin resistance, Type 2 diabetes (T2D), non-alcoholic fatty liver disease (NAFLD), cancer, and cardiovascular diseases (CVD) . In the Numerous studies indicate that obesity is not only weight gain and abnormal lipid metabolism but also includes systemic chronic low-grade inflammation that has a critical role in the initiation and development of several human illnesses . In obesObesity causes great harm to the liver and cardiovascular system. The liver is a key site of metabolic homeostasis and its inflammation is closely related to the occurrence and development of obesity. Epidemiological investigations have found that 50%–90% of people with obesity have NAFLD . In the Kaempferia galanga L. , Inula helenium L. , Dolomiaea costus  Kasana and A.K. Pandey , Rheum palmatum L. , Hippophae rhamnoides L , Piper longum L. , Biancaea sappan (L.) Tod. , and Sus scrofa L. . In the clinic, MMQ-8 has great therapeutic effects on obesity, dyslipidemia, hypertension, AS, and coronary heart disease. Numerous studies have demonstrated that the botanical drugs such as K. galanga L., H. rhamnoides L., R. palmatum L., and P. longum L. have anti-inflammatory, antioxidant, and anti-hyperlipidemia effects . In Mongolian medicine, many classical compound preparations are especially effective in treating chronic metabolic diseases. The Traditional Mongolian Medicine Qiqirigan-8 (MMQ-8) composed by 8 botanical drugs including  effects . These b effects . TherefoIn this study, we first determined the active metabolites of MMQ-8 using ultrahigh-performance liquid chromatograph Q extractive mass spectrometry (UHPLC-QE-MS) and network analysis. Bioinformatics analysis and experimental validation were performed to examine the treatment effects of MMQ-8 on lipid metabolism and inflammation.Wistar rats  were purchased from SiPeiFu Co. . Rats were maintained in a normal environment on a 12-h light/dark cycle (lights on 06:00–18:00 h) with ad libitum access to food and water and were fed a standard diet in the control group. The other groups were fed an HFD  for 10 months. All animal experiments were permitted by the Institutional Animal Care and Use Committee of Inner Mongolia Medical University.n = 8): Control, Model, MMQ-8 (A&B), and Simvastatin (ST) (A&B). The model group was given saline (2 ml) daily for 10 months; the control group had no treatment. The MMQ-8(A) group was given MMQ-8 (312.5 mg/kg) daily for 10 months. The MMQ-8(B) group was given MMQ-8 (312.5 mg/kg) daily for 5 months after 5 months of modeling. The ST(A) group was given Simvastatin tablets (2.08 mg/kg) daily for 10 months. The ST(B) group was given Simvastatin tablets (2.08 mg/kg) daily for 5 months after 5 months of modeling.All rats were randomly divided into six groups  is 207 g; I. helenium L. is 66 g; D. costus  Kasana and A.K. Pandey  is 66 g; R. palmatum L.  is 41 g; H. rhamnoides L.  is 248 g; P. longum L.  is 165 g; B. sappan (L.) Tod  is 124 g; S. scrofa L.  is 83 g. The decoction pieces of these botanicals were mixed according to the ratio. Then, the mixture was smashed using a pulverizer and filtered with a 70-mesh sieve. If the decoction pieces could not be filtered, we continued to pulverize and sieve the mixture until all pass through the filter. Finally, it was carefully dried and stored in a cool, dry place. Clinical administration requires an MMQ-8 dose of 3 g each time with an adult body weight of 60 kg, which means the dose was 50 mg/kg body weight. The equivalent dose ratio of human to rat based on body surface area is 6.25. Therefore, the MMQ-8 dose given to rats per kg of body weight was 312.5 mg (Formula: MMQ-8 50 mg/kg*6.25 = 312.5 mg/kg). In addition, the simvastatin dose for hyperlipidemia is 20 mg, which means the adult dose was 0.33 mg/kg body weight. Thus, the dose given to rats per kg of body weight was 2.08 mg (formula: 0.33 mg/kg*6.25 = 2.08 mg/kg). Finally, after calculating the dose for each rat, the drugs were dissolved in 2 ml of pure water. All the treatments were administered orally once a day at a fixed time.Preparing drugs and dose calculations: MMQ-8 botanicals ratio are 2. The CTLL-2 cell treatment was supplemented with 1% MMQ-8 rat serum and normal rat serum, respectively, and cultured for 24 h at 37°C in a humidified atmosphere containing 5% CO2.Mouse T lymphocyte cells  were used in this study and routinely cultured in 1640 . CTLL-2 cells were supplemented with 10% fetal bovine serum  and 1% penicillin/streptomycin  and cultured at 37°C in a humidified atmosphere containing 5% COCTLL-2 cells were cultured in 96-well plates. Then cells were incubated with different concentrations of MMQ-8 serum: 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10%. After 24 h, CCK-8 was added and cultured for 4 h. Then absorption values were calculated using a microplate reader at 450 nm. Inhibition rate = [(Ac-As)/(Ac-Ab)] 100% .Metabolite extraction: 100 mg of TCM sample was added to 500 μl of the extracted solution containing 1 μg/ml of internal standard. The samples were then homogenized at 40 Hz for 4 min and sonicated for 1 h in an ice-water bath. After resting for 1 h at −20°C, the samples were centrifuged at 12,000 rpm  for 15 min at 4°C. Finally, 300 μl of the supernatant was carefully filtered through a 0.22 μm microporous membrane and placed in a fresh 2 ml tube for LC-MS/MS analysis.LC-MS/MS conditions: LC-MS/MS analysis was performed on an Agilent ultra-high performance liquid chromatography 1290 UPLC system with a Waters UPLC BEH C18 column (1.7 μm 2.1*100 mm). The flow rate was set at 0.4 ml/min and the sample injection volume was set at 3 μl. The mobile phase consisted of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). The multi-step linear elution gradient program was as follows: 0–3.5 min, 95%–85% A; 3.5–6 min, 85%–70% A; 6–6.5 min, 70%–70% A; 6.5–12 min, 70%–30% A; 12–12.5 min, 30%–30% A; 12.5–18 min, 30%–0% A; 18–25 min, 0%–0% A; 25–26 min, 0%–95% A; 26–30 min, 95%–95% A.An AQ Exactive Focus mass spectrometer coupled with Xcalibur software was used to obtain the MS and MS/MS data based on the IDA acquisition mode. During each acquisition cycle, the mass range was between 100 and 1,500. The top three of every cycle were screened and the corresponding MS/MS data were acquired. Specifications were as follows sheath gas flow rate: 45 Arb; Aux gas flow rate: 15 Arb; Capillary temperature: 400°C; Full ms resolution: 70,000; MS/MS resolution: 17,500; Collision energy: 15/30/45 in NCE mode; Spray Voltage: 4.0 kV (positive) or −3.6 kV (negative).https://www.ebi.ac.uk/chembl/). In the Gene Cards Database (https://www.genecards.org) and the Therapeutic Target Database (TTD) (https://db.idrblab.net/ttd/), the keywords “inflammation,” “obesity,” and “lipid metabolism” were input to obtain relevant target genes. Then the UniProtKB search function of the UniProt database (http://www.uniprot.org/) was used to standardize the names of those predicted target genes. Finally, Venn analysis was performed on MMQ-8 target genes, obesity-related genes, and inflammation-related genes to obtain common target genes. This took place using GO (http://www.geneontology.org/) and KEGG pathway (www.kegg.jp/kegg/pathway.html) analysis.The chemical compositions of MMQ-8 detected by UHPLC-QE-MS were screened according to the Percent Human Oral Absorption rate (≥30%) and drug likeness (DL) (≥0.185). Then chemical component targets of MMQ-8 were obtained from the CHEMBL database , total cholesterol (TC), low-density lipoprotein (LDL-C), high-density lipoprotein (HDL-C), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were assessed using commercially available diagnostic kits . The serum levels of inflammatory factors TNF-α, inducible nitric oxide synthase (INOS), IL-6, and IL-10 were detected using an enzyme-linked immunosorbent assay (ELISA) kit . While strictly following the ELISA kit instructions, detection was performed using a Multi-Plate Reader  with Gen5 software. Serum TNF-α, IL-1β, IL-6, and IL-10 contents were calculated according to the standard curve.Liver tissue sections from the same part of the liver lobe were cut, fixed in 4% buffered formalin at room temperature for 24 h, embedded in paraffin, and sliced into 4-μm sections. Sections were stained with hematoxylin and eosin (H&E). Histopathological changes were checked under a microscope. Furthermore, we assigned NAFLD activity scores (NAS) to liver H&E images. The scoring criteria are shown in Histological visualization of lipid deposition in rat hepatic tissues was performed using oil red O staining. Briefly, frozen sections of rat liver tissue were first incubated in distilled water for 2 min, then 60% propylene glycol for 2 min, and finally in oil red O solution for 5 min. Then the sections were soaked in 60% propylene glycol until the interstitial tissue was colorless. After rinsing for 2 min with distilled water, the slides were incubated at 37°C with hematoxylin for 1 min and mounted with gelatin mounting medium.Immunohistochemistry was performed on the arcus aorta sections. The sections were immersed in distilled water at room temperature for 25 min. The sections were blocked with 3% rabbit serum and then incubated overnight at 4°C with primary antibodies: anti-CD68  and anti-Mannose receptor CD206 . The sections were washed followed by incubation with corresponding goat anti-rabbit IgG H&L (HRP)  for 50 min at room temperature. They were then immersed in PBS for 5 min and stained with DAB and hematoxylin. The histopathological changes were checked under a microscope.p ˂ 0.01).Muscle tissue was removed from the femur and tibia and rinsed with PBS. Tissue scissors were used to cut off both ends of the bone, and marrow cells were flushed out using a syringe containing PBS. The cell suspension was centrifuged at 1000 rpm for 5 min to collect bone marrow cells. Then red blood cell lysis buffer was added in moderation to the bone marrow cells to remove all red blood cells. They were then cleaned with PBS and an appropriate amount of TRIZOL UP was added. Cells were then frozen at −80°C after complete mixing. Finally, the total RNA was extracted to synthesize cDNA sequenced using an Illumina platform and the information was analyzed. The genes that displayed a >1.5-fold change in expression—and based on at least two pairwise comparisons with the same trend—were selected for further examination in the test phase  according to the manufacturer’s instructions. The total RNA was directly used for RT-qPCR by Trans Script Green One-Step qRT-PCR Super Mix reagent  following the manufacturer’s protocol on an ABI Prism7500 instrument . Details of the mRNA—specific primers for GAPDH, TNF-α, interferon regulatory factor 1(IRF1), T-bet, signal transducer and activator of transcription (STAT)1, STAT3, STAT4, STAT6, GATA binding protein 3 (GATA3), IL-10, RORγt, nuclear factor-kappa B inhibitor alpha (IκBα), and IL-17A are shown in ® 800LT goat anti-rabbit  and anti-mouse  for 1 h at approximately 25°C. The primary antibody and corresponding secondary antibody were evaluated separately. After scanning, successive secondary rounds of incubation were performed for each membrane using seven different antibodies recognizing seven different proteins. Detection and quantification were performed using an Infrared Imaging System . Band intensities were determined using the median background method. The mitogen-activated protein kinase (p38), IκBα, extracellular regulated kinase 1/2(ERK1/2), stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), Phospho-p38, Phospho-IκBα, Phospho-SAPK/JNK, and Phospho-ERK1/2 levels were normalized to β-actin or GAPDH.The vascular and liver tissues that were frozen at −80°C were removed from the refrigerator and rinsed twice with PBS. Tissues were then cut into small pieces and put into a glass grinder with an appropriate amount of TPEB to be ground for half an hour on ice. Protein samples were obtained by centrifugation at 12,000 rpm for 10 min at 4°C. Protein concentrations were determined by BCA protein assays , and approximately 25 μg of total protein was used for SDS-PAGE through a 12% running and 5% stacking gel. Samples were transferred to a nitrocellulose transfer membrane  using the wet transfer method. Membranes were blocked with 5% non-fat dry milk and then incubated overnight at 4°C with primary antibodies and corresponding secondary antibodies: anti-p38 , anti-IκBα , anti-SAPK/JNK , anti-ERK1/2 , anti Phospho-p38 , anti Phospho-IκBα , anti Phospho-SAPK/JNK , anti Phospho-ERK1/2 , GAPDH , and anti β-Actin . Membranes were washed, followed by incubation with corresponding IRDyep-values < 0.05 were considered statistically significant.All data are represented as mean ± SEM. Significance was tested using one-way ANOVA, followed by Dunnett’s multiple comparisons, when appropriate . Results with TCM is characterized by multi-component, multi-target integration and regulation. Thus, it is important for research to determine the active metabolites of drugs . In thisin vivo and in vitro. Therefore, we performed further analysis on TNF signaling pathway, Th1 and 2 cell differentiation, and Th17 cell differentiation signaling pathways and obtained 37 active metabolites of MMQ-8 that directly participate in regulating these signaling pathways, as shown in The therapeutic effects of TCM involve complex processes that operate through the synergistic action of various compounds on different targets . To explBased on the clinical effects, components and network analysis results of MMQ-8, we decided to establish obesity rat models through HFD and verify the effects of MMQ-8 on lipid metabolism and inflammation. HFD is an important factor that causes obesity. Long-time exposure to an HFD increases the amount of adipose tissue in humans, leading to obesity, weight gain, and steatosis of other tissues and organs. Moreover, studies have confirmed that expansion and anoxia of adipose tissue cause an inflammatory response that is a systemic chronic inflammation closely related to insulin resistance, T2D, AS, NAFLD, and some cancers .We observed that MMQ-8 could optimize lipid metabolism. As shown in In addition, blood lipid levels are an important target for observing lipid metabolism. Lipids exist in the blood in the form of TG, cholesterol, and lipoids binding with VLDL, LDL, and HDL. Blood lipid levels change with the absorption of lipids . In thisvia either ingestion or synthesis, surpasses fatty acid output, via oxidation or export. This is the case with an HFD. Histologically, when more than 50% of the liver cells have steatosis, this is termed NAFLD. In addition, hepatic steatosis is closely related to hepatic insulin resistance, endoplasmic reticulum, autistic autophagy, mitochondrial dysfunction, apoptosis, and inflammation  is highly expressed in M2-like macrophages that play an anti-inflammatory role during inflammation . Thus, tAS is a disease characterized by low-grade chronic inflammation of the arterial wall. Network analysis suggested that MMQ-8 significantly regulated inflammatory signaling pathways, including TNF, NF-κB, and MAPK. TNF is a classic, pleiotropic pro-inflammatory cytokine. It regulates cell survival, apoptosis, and differentiation, and promotes immune and inflammatory responses through the TNF receptor (TNFR1/2), activating multiple downstream signals transduction pathways, such as NF-κB, MAPK, and PI3K . In addiBone marrow is the main site for blood cell formation. It is a primary lymphoid organ that supports lymphoid development and a host of naive and memory immune cells, plasma cells, regulatory T cells, and myeloid immune cells. The cellular niches of bone marrow can regulate hematopoietic stem cells (HSCs) and immune cell behavior through direct cell contact, growth factors and cytokines, and components of the extracellular matrix . Studies+T cells exist in adipose tissue, NAFLD, and atherosclerotic plaques in obesity. These cells are implicated not only in inflammation but also in the development of insulin resistance and T2D. CD4+T cells mainly include Th1, Th2, Th17, and Treg cells. Of these, Th1 and Th17 cells are pro-inflammatory cells, while Th2 and Treg cells have the opposite effect. Th1 and Th17 cells promote macrophage migration and phenotypic differentiation to M1 by secreting inflammatory factors, while Th2 and Treg cells secrete anti-inflammatory factors such as IL-4, IL-10, and transforming growth factor-beta (TGF-β)  . In our in vitro . The MMQin vitro . Comparein vitro . However17 cells . In addi17 cells . These rCurrently, statins are the primary choice for treating hyperlipidemia and preventing secondary diseases caused by obesity. However, there are still no radical treatments available. Therefore, greater attention is being paid to botanical drug studies. In a holistic and synergistic way, Chinese botanical drugs possess superior pharmacological properties for treating obesity . Our stuin vivo in a holistic and synergistic way. The main sources of Mongolian medicines are botanical drugs , B cells, plasma cells, NKT cells, mesenchymal stem cells (MSCs), and myeloid-derived suppressor cells (MDSCs)  . MercierIn summary, we determined the pharmacological actions and relevant metabolites of MMQ-8 in obesity for the first time. Our study revealed that MMQ-8 can optimize lipid metabolism and reduce chronic inflammation in obesity. Its anti-inflammatory effect was accomplished by promoting macrophage polarization to M1, reducing macrophage infiltration, and inhibiting the signaling pathways regarding TNF, NF-κB, Th1, and Th2 cell and Th17 cell differentiation. However, we focused only on pharmacodynamic evaluation and relevant metabolites of MMQ-8. There remains a great deal of in-depth research to be done, for example, to understand the principle of compound compatibility and the inhibition effects on hepatic steatosis, T cell differentiation, and inflammatory signal transduction. We believe that it is necessary to continue exploring these potential mechanisms of MMQ-8.


Tumors exhibiting a Warburg effect rely on MCT activity for l-lactate release. Recently, high-resolution MCT structures revealed binding sites for anticancer drug candidates and the substrate. Three charged residues, Lys 38, Asp 309, and Arg 313 (MCT1 numbering) are essential for substrate binding and initiation of the alternating access conformational change. However, the mechanism by which the proton cosubstrate binds and traverses MCTs remained elusive. Here, we report that substitution of Lys 38 by neutral residues maintained MCT functionality in principle, yet required strongly acidic pH conditions for wildtype-like transport velocity. We determined pH-dependent biophysical transport properties, Michaelis–Menten kinetics, and heavy water effects for MCT1 wildtype and Lys 38 mutants. Our experimental data provide evidence for the bound substrate itself to accept and shuttle a proton from Lys 38 to Asp 309 initiating transport. We have shown before that substrate protonation is a pivotal step in the mechanisms of other MCT-unrelated weak acid translocating proteins. In connection with this study, we conclude that utilization of the proton binding and transfer capabilities of the transporter-bound substrate is probably a universal theme for weak acid anion/H+ cotransport.Human monocarboxylate/H Cells need to release l-lactate and the accompanying proton to maintain their energy metabolism. Secondary-active monocarboxylate/proton cotransporters, MCT, carry the main load of human l-lactate transport. MCT inhibitors are currently developed as anticancer drugs. Cryo-EM structures revealed binding sites for l-lactate and inhibitors. However, a fundamental question remained, namely how the cotransported proton binds to and traverses the transporter. We elucidated the proton transfer mechanism by comparing transport properties of MCT wildtype with point mutants. This way, we found that the bound l-lactate itself represents the missing proton binding and transfer site. Further examples of MCT-unrelated proteins suggest that substrate protonation is a universal principle in weak acid transport.a around 4 . Contrary to other expression systems, background transport is marginal in this strain, and the absence of l-lactate metabolizing enzymes allows for prolonged assay times to reach the transport equilibrium state  was determined to 3.97  supporting the view that MCT1 K38M equally uses neutral l-lactic acid as a substrate. Accordingly, at sufficiently acidic buffer, the l-lactate anion itself binds and carries the proton cosubstrate for MCT1 K38M.Next, we carried out a fine-grained titration of MCT1 wildtype and K38M transport rates against pH to determine the number of accessible protonation sites relevant to l-lactate/H+ transport functionality was shifted to neutral and mildly acidic physiological pH  with inflection points at pH 7.43 and pH 5.61  than at physiological pH conditions (Km 2.32 ± 0.36 mM)  is due to the shift of the l-lactate/lactic acid equilibrium towards the neutral species  vs. l-lactic acid (MCT1 K38M).To meet the acidic pH requirements of MCT1 K38M, we measured K3.8 Fig. E. The sul-lactamide, a permanently neutral mimic of l-lactic acid . A . A 12). a shift of monocarboxylic acids by approx. 0.5 . Other than that, the buffers were identically composed and contained the same concentrations of protons or deuterons, i.e. pH or pD. The height of the plateaus derived from the obtained or extrapolated uptake curves . Su. Su12). Currently, three types of membrane proteins have been functionally and structurally described that facilitate transmembrane translocation of a weak acid anion plus a proton. These are members of the ubiquitous AQP family , microbial formate/nitrate transporters, FNT , and MCT-type monocarboxylate transporters. All use electrostatic attraction of the weak acid anion by positively charged amino acid residues . The levLactobacillus AQPs attract l-lactate anions  and locally enhance the probability of substrate protonation for passing the lipophilic channel  was kindly provided by M. Casal. Cells were transformed using the PEG/LiAc/ss-carrier DNA-method (l-histidine (20 mg l−1), l-tryptophan (10 mg l−1), l-leucine (100 mg l−1), and adenine (25 mg l−1), lacking uracil for selection.A-method . Cells wg, 5 min, 4°C, OD600 of 1 ± 0.1), and washed with 50 ml water and 10 ml 25 mM TRIS, pH 7.5, 5 mM EDTA-buffer (TE). Pelleted cells were stored at −80°C. For disruption, cells were resuspended in 0.5 ml TE-buffer, repeatedly vortexed with an equal volume of acid-washed glass beads , and cleared . Microsomal proteins were prepared by removal of cell debris , followed by ultracentrifugation . Membrane pellets were resuspended in 100 mM Na2HPO4, pH 8.0, 50 mM NaCl. Total protein was quantified using Bio-Rad Protein Assay , separated by SDS-PAGE , and blotted on PVDF . Proteins were detected using monoclonal mouse-anti-His5 , secondary goat-anti-mouse antibodies , and Clarity ECL  in a ChemoStar Touch cabinet .Here 40 ml cultures were harvested , TRIS/MES , and TRIS/citric acid . For determining isotope effects, phosphate/citric acid buffers were used . 0.2 M Nlectrode . For ligl-lactate was assayed as previously , and washed with water. The cells were resuspended in pH/pD-adjusted buffer to an OD600 of 50 ± 5, and kept on ice for 30 min. For the assay, the cells were prewarmed to 19°C. Substrate uptake was initiated by adding 14C-spiked  l-lactate solution . Transport was stopped with 1 ml ice-cold water, and suction through GF/C glass microfiber filters . The washed filters were placed in 3 ml of ROTISZINTeco plus  overnight, and counted for 2 min . For Michaelis–Menten kinetics, substrate and radiolabel were increased up to 50 mM and 0.14 µCi. l-Lactamide competition was assayed at 4 mM l-lactate plus 4 mM or 40 mM l-lactamide. Batches in light and heavy water were prepared from the same yeast culture. Internalized l-lactate was normalized to 1 mg of yeast, and background radiolabel on non-expressing cells was subtracted. Uptake rates were determined from the initial linear phase of the curves . Michaelis–Menten parameters were calculated from exponential curve fittings (Km = ln(0.5)/rate constant; vmax = limit of the curve). Measurements were done in duplicates with two to six biological replicates.Uptake of radiolabeled eviously . Brieflyg, 5 min, 4°C, OD600 of 1 ± 0.1), washed and resuspended (2 ml) in 50 mM MOPS buffer, pH 7.2, with 0.2% of freshly added 2-mercaptoethanol. For protoplastation, zymolyase 20 T (400 U per gram of yeast cells), 100 mg of bovine serum albumin (fraction V), and 1.2 M sucrose  were added and incubated for 1 h at 30°C, 140 rpm. Protoplasts were collected , washed once in 5 ml of 20 mM MOPS buffer, pH 7.2, plus 1.2 M sucrose, 50 mM NaCl and 5 mM CaCl2, and diluted in this buffer to an OD600 of 2. For the assay, 75 µl of protoplast suspension were mixed using a stopped-flow device  with an equal volume of buffer supplemented with 600 mM l-lactate, chloroacetate, glycerol, l-lactamide, or l-proline (300 mM inward gradient). For an assay pH of 5.1 ± 0.3 the protoplast suspension was mixed with a pH 4.5 ± 0.3 buffer. Further parameters were: 20°C, dead time 2.7 ms, flow rate 8 ml s−1. The intensity of 90° light scattering (λ = 524 nm) was monitored. Nine traces were averaged, normalized, and done in three biological replicates. Substrate uptake velocity was derived from by linear fits (20–90 s range) of background-subtracted traces. Significance of was tested using one-tailed one-sample t-tests. Asterisks indicate P-values <0.05.The assay was adapted from previous work . Here, 50 ml transformed yeast were harvested (2,000 MCT1 protein structure displays were done with the Chimera software . Solvatipgad007_Supplementary_DataClick here for additional data file.