Jejunum Preparations Research Articles

Sodium oligomannate activates the enteroendocrine-vagal afferent pathways in APP/PS1 mice.

Enteroendocrine cells (EECs) and vagal afferent neurons constitute functional sensory units of the gut, which have been implicated in bottom-up modulation of brain functions. Sodium oligomannate (GV-971) has been shown to improve cognitive functions in murine models of Alzheimer's disease (AD) and recently approved for the treatment of AD patients in China. In this study, we explored whether activation of the EECs-vagal afferent pathways was involved in the therapeutic effects of GV-971. We found that an enteroendocrine cell line RIN-14B displayed spontaneous calcium oscillations due to TRPA1-mediated calcium entry; perfusion of GV-971 (50, 100 mg/L) concentration-dependently enhanced the calcium oscillations in EECs. In ex vivo murine jejunum preparation, intraluminal infusion of GV-971 (500 mg/L) significantly increased the spontaneous and distension-induced discharge rate of the vagal afferent nerves. In wild-type mice, administration of GV-971 (100 mg· kg-1 ·d-1, i.g. for 7 days) significantly elevated serum serotonin and CCK levels and increased jejunal afferent nerve activity. In 7-month-old APP/PS1 mice, administration of GV-971 for 12 weeks significantly increased jejunal afferent nerve activity and improved the cognitive deficits in behavioral tests. Sweet taste receptor inhibitor Lactisole (0.5 mM) and the TRPA1 channel blocker HC-030031 (10 µM) negated the effects of GV-971 on calcium oscillations in RIN-14B cells as well as on jejunal afferent nerve activity. In APP/PS1 mice, co-administration of Lactisole (30 mg ·kg-1 ·d-1, i.g. for 12 weeks) attenuated the effects of GV-971 on serum serotonin and CCK levels, vagal afferent firing, and cognitive behaviors. We conclude that GV-971 activates sweet taste receptors and TRPA1, either directly or indirectly, to enhance calcium entry in enteroendocrine cells, resulting in increased CCK and 5-HT release and consequent increase of vagal afferent activity. GV-971 might activate the EECs-vagal afferent pathways to modulate cognitive functions.

Medical Constituents of Ajwain (Trachyspermum ammi) for Human Benefits

Ajwain seed analysis has revealed it to contain fiber (11.9%), carbohydrates (38.6%), tannins, glycosides, moisture (8.9%), protein (15.4%), fat (18.1%), saponins, flavone and mineral matter (7.1%) containing calcium, phosphorous, iron and nicotinic acid. Ajwain fruits yield 2% to 4% brownish essential oil, with thymol as the major constituent (35% to 60%). The non-thymol fraction (thymine) contains para-cymene, γ-terpenine, α- and β-pinenes, diketene, α-terpinene, and carvacrol. Minute amounts of camphene, myrcene, and α-3-carene also have been found in the plant. Alcoholic extracts contain a highly hygroscopic saponin. From the fruits, a yellow, crystalline flavone and a steroid-like substance has been isolated and it also contains 6-O-β-glucopyranosyloxythymol, glucoside and yields 25% oleoresin containing 12% volatile oil (thymol, γ-terpinene, para-cymene, and α- and β-pinene). The principal oil constituents of T. Ammi are carvone (46%), limonene (38%), and dillapiole (9%). Ajwain is administered for curing stomach disorders, a paste of crushed fruits is applied externally for relieving colic pains; and a hot and dry fomentation of the fruits is applied on chest for asthma. Therapeutic uses of T. Ammi fruits include; stomachic, carminative and expectorant, antiseptic and amoebiasis, antimicrobial. Seeds soaked in lemon juice with P runs Amygdalus (badan) are given in curing amenorrhea and it is also used as antipyretic, febrifugal and in the treatment of typhoid fever. The antihypertensive effect of T. Ammi administered intravenously in vivo, and the antispasmodic and bronchodilation actions in vitro showed that calcium channel blockade has been found to mediate the spasmolytic effects of plant materials and it is being considered that this mechanism contributed to their observed result and supported the traditional use of T. Ammi in hyperactive disease states of the gut such as colic and diarrhea as well as in hypertension. Ajwain seeds revealed significant reduction of cough number which may be a result of its potent antitussive effect. The bronchodilator effect of decocted extract of Ajwain on the asthmatic patients’ airways was examined in a subsequent trial study. According to the results, the extract has a relatively bronchodilator effect on asthmatic airways compared to the effect of Theophylline at concentrations used. Ajwain was attributed to have diuretic and antilithiatic activity in ethnopharmacological reports. Accordingly, a human study was performed and in which, seeds of Ajwain were decocted in milk and given orally to volunteers suffering from urinary stone for a nine days period. The results were reported satisfactory against pure ca-oxalate stone. Another activity which has been proved for Ajwain is the antihyperlipidemic property. An in vivo study revealed that Ajwain seeds powder is extensively effective on lipid profile and can decrease total cholesterol, LDL-cholesterol, triglycerides and total lipids. Moreover, organic extract of seeds reduced atherogenic index and increased the level of HDL-cholesterol in albino rabbits. Ajwain was evaluated for the potentiality of antihypertensive and antispasmodic activity. In the related investigation, the aqueous-methanolic extract of the seeds caused a dose dependent decrease in arterial blood pressure in anaesthetized animal models. Furthermore, inhibitory effect on the K+-induced contractions was seen in isolated rabbit aorta and jejunum preparations during the application of Ajwain extract. These findings prove the potential antihypertensive and antispasmodic activity of Ajwain.

Medical Constituents of Ajwain (Trachyspermum ammi) for Human Benefits

Ajwain seed analysis has revealed it to contain fiber (11.9%), carbohydrates (38.6%), tannins, glycosides, moisture (8.9%), protein (15.4%), fat (18.1%), saponins, flavone and mineral matter (7.1%) containing calcium, phosphorous, iron and nicotinic acid. Ajwain fruits yield 2% to 4% brownish essential oil, with thymol as the major constituent (35% to 60%). The non-thymol fraction (thymene) contains para-cymene, γ-terpenine, α- and β-pinenes, dipentene, α-terpinene, and carvacrol. Minute amounts of camphene, myrcene, and α-3-carene also have been found in the plant. Alcoholic extracts contain a highly hygroscopic saponin. From the fruits, an yellow, crystalline flavone and a steroid-like substance has been isolated and it also contains 6-O-β-glucopyranosyloxythymol, glucoside and yields 25% oleoresin containing 12% volatile oil (thymol, γ-terpinene, para-cymene, and α- and β-pinene). The principal oil constituents of T. ammi are carvone (46%), limonene (38%), and dillapiole (9%). Ajwain is administered for curing stomach disorders, a paste of crushed fruits is applied externally for relieving colic pains; and a hot and dry fomentation of the fruits is applied on chest for asthma. Therapeutic uses of T. ammi fruits include; stomachic, carminative and expectorant, antiseptic and amoebiasis, antimicrobial. Seeds soaked in lemon juice with P runus amygdalus (badam) are given in curing amenhorroea and it is also used as antipyretic, febrifugal and in the treatment of typhoid fever. The antihypertensive effect of T. ammi administered intravenously in vivo, and the antispasmodic and bronchodilating actions in vitro showed that calcium channel blockade has been found to mediate the spasmolytic effects of plant materials and it is being considered that this mechanism contributed to their observed result and supported the traditional use of T. ammi in hyperactive disease states of the gut such as colic and diarrhea as well as in hypertension. Ajwain seeds revealed significant reduction of cough number which may be a result of its potent antitussive effect. The bronchodilatory effect of decocted extract of Ajwain on the asthmatic patients’ airways was examined in a subsequent trial study. According to the results, the extract has a relatively bronchodilatory effect on asthmatic airways compared to the effect of Theophylline at concentrations used. Ajwain was attributed to have diuretic and antilithiasis activity in ethnopharmacological reports. Accordingly, a human study was performed and in which, seeds of Ajwain were decocted in milk and given orally to volunteers suffering from urinary stone for a nine days period. The results were reported satisfactory against pure ca-oxalate stone. Another activity which has been proved for Ajwain is the antihyperlipidemic property. An in vivo study revealed that Ajwain seeds powder is extensively effective on lipid profile and can decrease total cholesterol, LDL-cholesterol, triglycerides and total lipids. Moreover, organic extract of seeds reduced atherogenic index and increased the level of HDL-cholesterol in albino rabbits. Ajwain was evaluated for the potentiality of antihypertensive and antispasmodic activity. In the related investigation, the aqueous-methanolic extract of the seeds caused a dose dependent decrease in arterial blood pressure in anaesthetized animal models. Furthermore, inhibitory effect on the K+-induced contractions was seen in isolated rabbit aorta and jejunum preparations during the application of Ajwain extract. These findings prove the potential antihypertensive and antispasmodic activity of Ajwain.

Role of nitric oxide and calcium ions in the effects of hydrogen sulfide on contractile activity of rat jejunum

This study was performed to explore the role of nitric oxide, intracellular and extracellular calcium in the effects of hydrogen sulfide on spontaneous and carbachol-induced contractions of a rat jejunum preparation during a isometric contraction. Application of H2S donor, sodium hydrosulfide, led to a decrease in tonic tension, the amplitude and frequency of spontaneous contractions, as well as in the amplitude induced by carbachol, a nonspecific acetylcholine receptor agonist. Inhibiting the production of endogenous NO synthesis by with L-NAME, the effect of H2S donor remained unchanged, while in the presence of SNAP, a NO donor, the effects of NaHS on the amplitude of spontaneous and carbachol-induced contractions were less pronounced. Dantrolene, a ryanodine receptor inhibitor was used to stop a decrease in tonic tension in the presence of NaHS. The calcium-free solution reduced the inhibitory effect of NaHS on carbachol-induced contractions. This suggests that the inhibitory effect of H2S is associated with the dynamics of the intracellular concentration of calcium ions, and the interaction between NO and H2S occurs at the level of common targets of two gases.

Modulation of chicken gut contractility by Melissa officinalis—ex vivo study

Melissa officinalis (lemon balm) has a long history of being used in traditional medicine for the treatment of gastrointestinal tract disorders in human thanks to its spasmolytic and stress reducing effects. These pharmacological properties have been confirmed in laboratory animals. Unfortunately, in the case of veterinary medicine, the effect of lemon balm on gut contractility has been never subjected to a detailed investigation. On the other hand, there is urgent need of new drugs that could be safely used in animals for both, causative and symptomatic treatment. In broiler chicken, one of the major health concerns includes gastrointestinal disorders with gut hypermotility. Thus, it is crucial to verify the potential utility of Melissa officinalis extract in gastrointestinal dysmotilities. The aim of the study was to analyze the effect of lemon balm extract and some of its active ingredients on chicken intestine motility. The study was performed on isolated proximal and distal jejunum preparations collected from broiler chicken which underwent routine slaughter procedure. The effect of lemon balm and 3 phenolic acids (rosmarinic, chlorogenic, and lithospermic) was verified under isometric conditions, toward spontaneous and acetylcholine (ACh)-induced smooth muscle activity. Surprisingly, M. officinalis turned out to be rather a myocontractile agent as it increased ACh-provoked contractility of proximal and distal jejunum strips and also intensified the spontaneous activity of distal jejunum. Only in the case of proximal intestine lemon balm extract diminished the force of spontaneous motoric activity up to approx. Sixty-seven percent of the control conditions. None of the tested phenolic acids displayed analog effect with the whole plant extract. In fact in the case of ACh-induced contractility, the acids had the opposite, that is, myorelaxant, effect than the extract, with a small exception of lithospermic acid in distal jejunum. Thus, it is impossible to assign one or more individual constituents to the effect of the whole Melissa officinalis extract. The obtained results do not support the use of lemon balm extract in broiler diseases which are accompanied by gut motility disturbances, including diarrhea.

Ethnopharmacological basis and pharmacodynamics prospectives for folkloric claims of Rosa webbiana wall. Ex. Royle in diarrhea and asthma via In vitro, In vivo and In silico techniques

Ethnopharmacological relevanceRosa webbiana (Family: Rosaceae) is used by South Asian herbalists to treat gastrointestinal and respiratory disorders. Aim of the studyThis research aimed at multiple targets to verify R. webbiana for treating diarrhea and asthma. In vitro, in vivo, and in silico experiments were planned to demonstrate the antispasmodic and bronchodilator potential of R. webbiana. Materials and methodsThe bioactive compounds of R. webbiana were identified and quantified through LC ESI-MS/MS and HPLC. These compounds were predicted for muti-mechanisms of bronchodilator and antispasmodic potential in network pharmacology and molecular docking. In vitro methods (isolated rabbit trachea, bladder, and jejunum tissues) confirmed these multi-mechanisms for antispasmodic and bronchodilator effects. Antiperistalsis, antidiarrheal, and antisecretory experiments were conducted in in-vivo experiments. ResultsThe phytochemical analysis indicates the presence of rutin (742.91 μg/g), kaempferol (726.32 μg/g), and quercitrin (688.20 μg/g) in Rw. EtOH. These bioactive compounds in network pharmacology interfere with the pathogenic genes of diarrhea and asthma, which are the members of calcium-mediated signaling pathways and showed the stronger binding affinity towards voltage-gated L-type calcium channels, myosin light chain-kinase, Calcium calmodulin-dependent-kinase, Phosphodiesterase-4, and phosphoinositide phospholipase-C in molecular docking. Rw. EtOH elicited a spasmolytic response in isolated jejunum, trachea, and urine preparations by relaxing K+ (80 mM) and CCh (1 μM) spastic contractions. Additionally, it suppressed calcium concentration-response curves to the right, like verapamil. Like dicyclomine, it caused a rightward parallel shift of the CCh curves, followed by a non-parallel shift at higher concentrations with suppression of the maximal response. Like papaverine, it also caused isoprenaline-induced inhibitory CRCs to shift to the left. Verapamil did not potentiate isoprenaline-induced inhibitory CRCs, although it was more efficacious against K+ (80 mM) than CCh (1 μM)-induced contractions. R. webbiana EtOH extract exhibited complete antiperistalsis (21.55%), antidiarrheal (80.33%), and antisecretory (82.59±0.60) activities in vivo experiments at the dose of 300 mg/kg. ConclusionThus, Rw. EtOH modulated multiple pathways, produced calcium antagonistic, anticholinergic, and phosphodiesterase inhibitory actions, and had antidiarrheal and bronchodilator effects.

Atorvastatin and Fluvastatin Potentiate Blood Pressure Lowering Effect of Amlodipine through Vasorelaxant Phenomenon.

Background and Objectives: We have recently reported that stains have calcium channel blocking activity in isolated jejunal preparations. In this study, we examined the effects of atorvastatin and fluvastatin on blood vessels for a possible vasorelaxant effect. We also studied the possible additional vasorelaxant effect of atorvastatin and fluvastatin, in the presence of amlodipine, to quantify its effects on the systolic blood pressure of experimental animals. Materials and Methods: Atorvastatin and fluvastatin were tested in isolated rabbits' aortic strip preparations using 80mM Potassium Chloride (KCl) induced contractions and 1 micro molar Norepinephrine (NE) induced contractions. A positive relaxing effect on 80 mM KCl induced contractions were further confirmed in the absence and presence of atorvastatin and fluvastatin by constructing calcium concentration response curves (CCRCs) while using verapamil as a standard calcium channel blocker. In another series of experiments, hypertension was induced in Wistar rats and different test concentrations of atorvastatin and fluvastatin were administered in their respective EC50 values to the test animals. A fall in their systolic blood pressure was noted using amlodipine as a standard vasorelaxant drug. Results: The results show that fluvastatin is more potent than amlodipine as it relaxed NE induced contractions where the amplitude reached 10% of its control in denuded aortae. Atorvastatin relaxed KCL induced contractions with an amplitude reaching 34.4% of control response as compared to the amlodipine response, i.e., 39.1%. A right shift in the EC50 (Log Ca++ M) of Calcium Concentration Response Curves (CCRCs) implies that statins have calcium channel blocking activity. A right shift in the EC50 of fluvastatin with relatively less EC50 value (-2.8 Log Ca++ M) in the presence of test concentration (1.2 × 10-7 M) of fluvastatin implies that fluvastatin is more potent than atorvastatin. The shift in EC50 resembles the shift of Verapamil, a standard calcium channel blocker (-1.41 Log Ca++ M). Conclusions: Atorvastatin and fluvastatin relax the aortic strip preparations predominantly through the inhibition of voltage gated calcium channels in high molar KCL induced contractions. These statins also inhibit the effects of NE induced contractions. The study also confirms that atorvastatin and fluvastatin potentiate blood pressure lowering effects in hypertensive rats.

Investigations of plausible pharmacodynamics supporting the antispasmodic, bronchodilator, and antidiarrheal activities of Berberis lycium Royle. Via in silico, in vitro, and in vivo studies

Ethnopharmacological relevanceBerberis lycium Royle, a member of the Berberidaceae family, is a high-value medicinal plant with a documented history of usage in traditional medicine and has demonstrated significant therapeutic results among local populations throughout the globe. It is used traditionally in many parts of Pakistan to treat diarrhea, abdominal spasms, coughs, and chest problems. Aim of the studyTo investigate the antispasmodic, bronchodilator, and antidiarrheal effects of B. lycium and its possible underlying mechanisms through in silico, in vitro, and in vivo studies. Materials and methodsLC ESI–MS/MS analysis was used to identify bioactive components within the hydromethanolic extract of B. lycium. In silico studies, including network pharmacology and molecular docking, were utilized to investigate the antispasmodic and bronchodilator properties of the extract's bioactive components. In vitro pharmacological studies were conducted using isolated rabbit jejunum, trachea, urinary bladder, and rat ileum preparations. In vivo antidiarrheal activities were conducted in mice, including castor oil-induced diarrhea, intestinal transit, and castor oil-induced enteropooling. ResultsThe LC ESI–MS/MS analysis of the hydromethanolic extract of B. lycium identified 38 bioactive compounds. Network pharmacology study demonstrated that the mechanism of BLR for the treatment of diarrhea might involve IL1B, TLR4, PIK3R1, TNF, PTPRC, IL2, PIK3CD, and ABCB1, whereas, for respiratory ailments, it may involve PIK3CG, TRPV1, STAT3, ICAM1, ACE, PTGER2, PTGS2, TNF, MMP9, NOS2, IL2, CCR5, HRH1, and VDR. Molecular docking research revealed that chlorogenic acid, epigallocatechin, isorhamnetin, quinic acid, gallic acid, camptothecin, formononetin-7-O-glucoside, velutin, caffeic acid, and (S)-luteanine exhibited a higher docking score than dicyclomine with validated proteins of smooth muscle contractions such as CACB2_HUMAN, ACM3_HUMAN, MYLK_HUMAN, and PLCG1_HUMAN. In vitro investigations demonstrated that Blr.Cr, Blr.EtOAc, and Blr.Aq relaxed spontaneously contracting jejunum preparations; carbachol (1 μM)-induced and K+ (80 mM)-induced jejunum, trachea, and urinary bladder contractions in a concentration-dependent manner, similar to dicyclomine. Moreover, Blr.Cr, Blr.EtOAc, and Blr.Aq exhibited a rightward shift in Ca+2 and carbachol cumulative response curves, similar to dicyclomine, demonstrating the coexistence of antimuscarinic and Ca+2 antagonistic mechanisms due to the presence of alkaloids and flavonoids. In vivo antidiarrheal activities showed that the hydromethanolic extract was significantly effective against castor oil-induced diarrhea and castor oil-induced enteropooling, similar to loperamide, and charcoal meal intestinal transit, similar to atropine, in mice at doses of 50, 100, and 200 mg/kg body weight, which supports its traditional use in diarrhea. ConclusionThe dual blocking mechanism of muscarinic receptors and Ca+2 channels behind the smooth muscle relaxing activity reveals the therapeutic relevance of B. lycium in diarrhea, abdominal spasms, coughs, and chest problems.

Pharmacological Basis of Rumex hastatus D. Don in Gastrointestinal Diseases with Focusing Effects on H+/K+-ATPase, Calcium Channels Inhibition and PDE Mediated Signaling: Toxicological Evaluation on Vital Organs.

This present study aimed to delineate Rumex hastatus D. Don crude extract (Rh.Cr), n-Hexane, ethyl acetate, aqueous fractions (Rh.n-Hex, Rh.ETAC, Rh.Aq) and rutin for antidiarrheal, antisecretory effects, anti-spasmodic, gastrointestinal transient time, anti H. pylori, antiulcer effects, and toxicology. The preliminary phytochemical analysis of Rumex hastatus showed different phytoconstituents and shows different peaks in GC-MC chromatogram. Rumex hastatus crude extract (Rh.Cr), fractions, and rutin attributed dose-dependent (50–300 mg/kg) protection (0–100%) against castor oil-induced diarrhea and dose-dependently inhibited intestinal fluid secretions in mice. They decreased the distance traversed by charcoal in the gastrointestinal transit model in rats. In rabbit jejunum preparations, Rh.Cr and Rh.ETAC caused a concentration-dependent relaxation of both spontaneous and K+ (80 mM)-induced contractions at a similar concentration range, whereas Rh.n-Hex, rutin, and verapamil were relatively potent against K+-induced contractions and shifted the Ca2+ concentration–response curves (CRCs) to the right, Rh.Cr (0.3–1 mg/mL) and Rh.ETAC (0.1–0.3 mg/mL) shifted the isoprenaline-induced inhibitory CRCs to the left. Rh.n-Hex, Rh.ETAC and rutin showed anti-H. pylori effect, also shows an inhibitory effect against H+/K+-ATPase. Rumex hastatus showed gastroprotective and antioxidant effects. Histopathological evaluation showed improvement in cellular architecture and a decrease in the expression of inflammatory markers such as, cyclooxygenase (COX-2), tumor necrosis factor (TN,F-α) and phosphorylated nuclear factor kappa B (p-NFƙB), validated through immunohistochemistry and ELISA techniques. In RT-PCR it decreases H+/K+-ATPase mRNA levels. Rumex hastatus was found to be safe to consume up to a dose of 2000 mg/kg in a comprehensive toxicity profile. Docking studies revealed that rutin against H+/K+-ATPase pump and voltage-gated L-type calcium channel showed E-values of −8.7 and −9.4 Kcal/mol, respectively. MD simulations Molecular Mechanics Poisson Boltzmann surface area and molecular mechanics Generalized Born surface area (MMPBSA/GBSA) findings are consistent with the in-vitro, in-vivo and docking results.

Potential biomedical applications of Araucaria araucana as an antispasmodic, bronchodilator, vasodilator, and antiemetic: Involvement of calcium channels

Ethnopharmacological relevanceSince pre-Columbian era, the resin of Araucaria araucana tree has been used traditionally for the treatment of ulcers and wounds. Araucaria species have also been used to treat inflammation, respiratory problems, viral infections, ulcers, and rheumatoid, cardiovascular, and neurological disorders. Aims and objectiveDue to its popular use, the authors aimed to scrutinize the potential of this plant as an antispasmodic and an antiemetic agent. Furthermore broncho- and vasodilatory effects of this plant was explored to rationalize its folkloric uses. Materials and methodsAraucaria araucana crude extract (Aa.Cr) was evaluated in isolated preparations of rabbit jejunum, trachea, aorta, and atria to investigate the antispasmodic, bronchodilator, and vasodilator effects. The potential mechanistic approaches were compared with the standard drug ‘verapamil’. The antiemetic activity was determined and compared with the standard drug ‘domperidone’ via chick emesis model. ResultsAa.Cr dose-dependently relaxed both spontaneous and K+-induced contractions in the isolated jejunum preparations of rabbits. In concentration–response curves of calcium (Ca++), Aa.Cr also triggered the rightward shift like verapamil. Applying carbachol and phenylephrine (1 μM) and K+ (80 mM) to the isolated tracheal and aortic tissue preparation, respectively, resulted in broncho- and vasodilatory activities, respectively which may be due to the inhibition of Ca++ channels. Aa.Cr inhibited atrial force and spontaneous contractions in the rabbit's right atria. Aa.Cr exhibited significant antiemetic activity (P < 0.001 vs. saline) in dose-dependent (50–150 mg/kg) manner like domperidone. In silico molecular docking was performed to investigate the biological targets of purified components of Aa.Cr which revealed that cadinol dominantly targets β2 receptors to cause bronchodilation, however, eudesmin binds non-specifically to all the selected targets, while secoisolariciresinol mediated high hydrogen bonding with muscarinic receptors (M1 and M3) and Ca++ channels, thus shows the suggested mechanistic pathways of targeted activities. ConclusionsThe results of this study indicates that Aa.Cr may exhibit antispasmodic activity, bronchodilation, and vasodilation by inhibiting voltage-dependent Ca++ channels and release of subcellular calcium. This explains its folkloric use in hypertension, bronchospasms, gastrointestinal spasms, and emesis.

Effect of Rumex dentatus on Gastrointestinal Protection and Toxicology in Rodents via Investigating H+/K+-ATPase, Calcium Channels, and PDE Mediated Signaling

This present study aims to delineate Rumex dentatus crude extract (Rd.Cr), n-Hexane, ethyl acetate, aqueous fractions (Rd.n-Hex, Rd.ETAC, and Rd.Aq), and emodin for antidiarrheal, antisecretory effects, anti-spasmodic, gastrointestinal transient time, anti-H. pylori, antiulcer effects, and toxicology. Plant extracts attributed dose-dependent protection against castor oil-induced diarrhea and dose-dependently inhibited intestinal fluid secretions in mice. They decreased the distance transverse by charcoal in the gastrointestinal transit model in rats. In rabbit jejunum preparations, it causes a concentration-dependent relaxation of both spontaneous and K+ (80 mM)-induced contraction, Rd.n-Hex and verapamil were relatively potent against K+-induced contractions and shifted the Ca2+ concentration-response curves (CRCs) to the right, Rd.Cr and Rd.ETAC shifted the isoprenaline-induced inhibitory CRCs to the left, showing potentiating effect similar to papaverine. Rd.n-Hex showed anti-H. pylori effect. Extracts and emodin also show an inhibitory effect against H+/K+-ATPase. Rumex dentatus showed a gastroprotective and antioxidant effect. Histopathological evaluation showed improvement in cellular architecture and decrease in the expression of inflammatory markers such as cyclooxygenase (COX2), tumor necrosis factor (TNF-α), and phosphorylated nuclear factor kappa B (p-NFƙB), validated through immunohistochemistry, ELISA, and western blot techniques. In RT-PCR, it decreases H+/K+-ATPase mRNA levels. Rumex dentatus was analyzed for certain safety aspects and exhibited a relative safety profile as no impairment was observed in kidneys, heart, liver, and brain further assisted by biochemical and hematological analysis. Docking studies revealed that emodin against H+/K+-ATPase pump and voltage gated L-type calcium channel showed E-value of −7.9 and −7.4 kcal/mol, respectively. MD simulations and molecular mechanics Poisson Boltzmann surface area and molecular mechanics Generalized Born surface area MMPBSA/GBSA findings are consistent with the in-vitro, in-vivo, and docking results. In conclusion, Rumex dentatus extracts and its phytoconstituent could be considered a potent antioxidant and anti-inflammatory drug candidates that possess anti-diarrheal, anti-secretary, antispasmodic, anti-H. pylori, and anti-ulcer potential. Toxicity studies were done according to OECD standards 425. It belongs to group 5 (LD50 > 2000 mg/kg), which suggests that it is in the lower toxicity class.

LPS transport during fat absorption is inhibited in caveolin‐1 knockout murine jejunum

Lipopolysaccharides (LPS) entry from the intestinal lumen to the circulation is associated with systemic inflammation. We have found that LPS is transcellularly transported to portal vein during long‐chain fatty acid (LCFA) exposure via CD36‐ and lipid raft‐mediated pathways in rats, suggesting the involvement of caveolae‐mediated endocytosis. We thus examined LPS transport in caveolin‐1 (Cav1) knockout (KO) murine jejunum.FITC‐LPS was applied to the mucosal bath of Ussing chambered muscle‐stripped jejunal mucosa‐submucosa preparations of Cav1 KO and wild type (WT) mice. Serosal appearance of FITC‐LPS was measured with or without luminal application of oleic acid (OA, 30 mM) with taurocholic acid (TCA, 0.1 mM), or medium‐chain fatty acid sodium caprate (C10, 10 mM).Luminal application of OA/TCA increased FITC‐LPS m‐to‐s transport in WT jejunum, inhibited by the CD36 inhibitor sulfosuccinimidyl oleate (SSO, 0.1 mM) or the lipid raft inhibitor methyl‐β‐cyclodextrin (MβCD, 0.1 mM), not by the clathrin inhibitor chlorpromazine (0.1 mM) or Pitstop2 (30 µM), suggesting that LCFA‐induced LPS transport is mediated by caveolae‐mediated endocytosis, not by clathrin‐mediated endocytosis. In contrast, OA/TCA‐induced FITC‐LPS transport was abolished in Cav1 KO jejunum. Nevertheless, luminal application of C10 increased FITC‐LPS transport both in WT and Cav1 KO jejuna without transepithelial electrical resistance changes, suggesting that C10 enhances transcellular LPS transport via caveolin‐independent endocytosis in the jejunum.These results suggest that LPS transport during LCFA exposure is mediated by Cav1‐mediated endocytosis, whereas MCFA‐induced LPS transport is likely via clathrin‐mediated endocytosis. Modulation of epithelial endocytosis may be a new therapeutic target for LPS‐associated diseases mediated by dietary fatty acids, including metabolic syndrome.

Antispasmodic activity of the ethanol extract of Citrullus lanatus seeds: Justifying ethnomedicinal use in Pakistan to treat asthma and diarrhea

Ethnopharmacological relevanceCitrullus lanatus (Thunb.) belongs to the ground family, Cucurbitaceae, known for edible fruit. Besides nutritional benefits, the traditional herbal practitioners in Pakistan and India used their seeds to treat gastrointestinal, respiratory, and urinary disorders. In Northern Sudan, its seeds are often used as a laxative. Its root is laxative and emetic at a high dose. Its seeds are also used to treat bedwetting and urinary tract obstruction. Aim of the studyThis study aimed to elucidate the multi-target mechanisms of Citrullus lanatus seeds to treat asthma and diarrhea. The pharmacological experiments were designed and conducted, along with the pharmacology network and molecular docking predictions, to verify the seeds biopotency for antispasmodic and bronchodilator properties. MethodsLC ESI-MS/MS were performed to identify the potentially active compounds in hydroethanolic extract of Citrullus lanatus seeds, then to quantify them by HPLC. The quantified bioactive compounds of Citrullus lanatus, i.e., stigmasterol, quinic acid, malic acid, epicatechin, caffeic acid, rutin, p-coumaric acid, quercetin, ferulic acid, scopoletin, apigenin, and kaempferol were subjected to in silico studies for molecular docking. The hydroethanolic extract of Citrullus lanatus seeds was examined on isolated rabbit tissue, i.e., jejunum, trachea, and urinary bladder. The antiperistalsis, antidiarrheal and antisecretory studies were also performed in animal models. ResultsIn silico studies revealed that bioactive compounds of C. lanatus seeds interfere with asthma and diarrhea-associated target genes, which are a member of calcium mediate signaling, regulation of cytosolic calcium concentration, smooth muscle contraction, and inflammatory responses. It was also found that rutin, quercetin, kaempferol, and scopoletin were stronger binding to voltage-gated calcium channels, calcium/calmodulin-dependent protein kinase, myosin light chain kinase, and phosphoinositide phospholipase C, thus, exerting calcium channel blocker activity. The hydroethanolic extract of C. lanatus seeds exerted a concentration-dependent relaxant response for the spasmolytic response on isolated jejunum and trachea preparations and caused relaxation of spastic contraction of K+ (80 mM). Furthermore, it caused a non-parallel rightward shift with suppression of calcium concentration-response curves. In animal models, the Cl.EtOH showed antiperistalsis, antidiarrheal and antisecretory response. ConclusionThus, we confirm Citrullus lanatus seeds have some medicinal effects by regulating the contractile response through target proteins of calcium mediates signaling and can be a promising component in the medical treatment for asthma and diarrhea.

Scientific basis for medicinal use of Citrullus lanatus (Thunb.) in diarrhea and asthma: In vitro, in vivo and in silico studies

BackgroundCitrullus lanatus (Thunb.) is a member of the Cucurbitaceae family, commonly farmed as an edible vegetable around the globe. It has been used in traditional therapies in addition to nutritional advantages. Traditional herbal practitioners employ C. lanatus seeds to treat gastrointestinal, respiratory, and urinary diseases in Pakistan and India. However, more investigation is needed to understand the effect of C. lanatus seeds on treating gastrointestinal, respiratory, and urinary disorders. PurposeThis research aimed to use network pharmacology and molecular docking to understand multi-target mechanisms of C. lanatus seeds against asthma and diarrhea and to validate its effects using biological tests to investigate antispasmodic and bronchodilator capabilities. MethodsThe ground seeds of C. lanatus were extracted in hexane, dichloromethane, ethanol, and aqueous for sequential extracts. The bioactive components in sequential extracts of C. lanatus seeds were identified using LC ESI-MS/MS, and specific compounds were quantified using HPLC. The quantified bioactive compounds of C. lanatus were subjected to in silico studies for network pharmacology and molecular docking to elucidate their role in antispasmodic and bronchodilator properties. The sequential extracts were tested on isolated rabbit tissue, i.e., jejunum, trachea, and urinary bladder. The antiperistalsis, antidiarrheal and antisecretory studies were also performed in animal models. ResultsIn silico studies indicate that bioactive chemicals from sequential extracts of C. lanatus seeds interfere with asthma and diarrhea-associated pathogenic genes. Those are members of calcium mediate signaling, cholinergic synapse, regulation of cytosolic calcium concentration, smooth muscle contraction, and inflammatory responses. It was also found that rutin, quercitrin, stearic acid, umbelliferone, and kaempferol were stronger binding to voltage-gated calcium channels and muscarinic M3 receptor, thus exerting calcium channel blocker activity and cholinergic receptor stimulant response. On isolated jejunum, trachea, and urinary preparations, sequential extracts of C. lanatus seeds elicited the spasmolytic response and showed the relaxation of spastic contractions of K+ (80 mM) and carbachol (1 µM). Furthermore, it induced a non-parallel rightward shift in calcium concentration-response curves with suppression. In animal models, C. lanatus seed extracts exhibited partially or completely antiperistalsis, antidiarrheal, and antisecretory effects. ConclusionThus, Citrullus lanatus had therapeutic benefits by modulating the contractile response through calcium-mediated signaling target proteins, hence exerting bronchodilator and antidiarrheal properties. The current study provides evidence for further mechanistic studies and the development of C. lanatus seeds as a potential therapeutic intervention for patients with gastrointestinal, respiratory, and urinary disorders.

Antidiarrheal and Antioxidant Activities of the Aerial Parts of Caralluma dalzielii N. E. Brown

Pharmacognosy Research,2022,14,1,82-88.DOI:10.5530/pres.14.1.12Published:December 2021Type:Original ArticleAuthors:Chinenye J. Ugwah-Oguejiofor, Halilu E. Mshelia, Junaidu Bello, Isah Inuwa, and Abdullahi Sulaiman Author(s) affiliations:Chinenye J. Ugwah- Oguejiofor1,*, Halilu E. Mshelia2,3, Junaidu Bello1, Isah Inuwa1, Abdullahi Sulaiman1 1Department of Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences Usmanu Danfodiyo University Sokoto, Sokoto, NIGERIA. 2Department of Faculty of Pharmacy, Cyprus International University, Nicosia, Mersin 10, TURKEY. 3Department of Pharmacognosy and Ethnopharmacy, Faculty of Pharmaceutical Sciences, Usmanu Danfodiyo University, P.M.B. 2346 Sokoto, NIGERIA. Abstract:Background: Caralluma dalzielii N.E. Brown is commonly referred to as “Karan massalanci” in the Northwest Nigeria where it is used in treating several ailments including pain, diarrhea and stomach issues. Objectives: This study was aimed to investigate the antidiarrheal and antioxidant activities of aqueous aerial parts extract of Caralluma dalzielii. Materials and Methods: The aqueous aerial parts extract of Caralluma dalzielii (CDE) (100, 200 and 400 mg/kg) was evaluated for antidiarrheal properties against gastrointestinal motility, castor oil-induced and prostaglandin-E2 (PGE2) enteropooling models in Wistar rats. Its antioxidant properties were studied by determining the total phenolic content (TPC) using Folin-Ciocalteu reagent, its free radical scavenging activity using 1, 1-Diphenyl-2-Picrylhydrazyl (DPPH) and its ferric reducing antioxidant power (FRAP) assays using potassium ferrocyanide ferric chloride methods. Ascorbic acid was used as the reference. Spasmolytic effect was studied in isolated rabbit jejunum preparations in an organ bath experiment using acetylcholine (ACh). Results: CDE significantly (p<0.05) reduced gastrointestinal transit of charcoal meal and the total number of diarrheal feces in the animals. The total phenolic content was determined to be 36.67±3.33 mg GAE/g. IC50 value of 63.44 mg/mL was calculated in DPPH assay. The FRAP value was found to increase as the concentration of the extract increased. CDE inhibited acetylcholine-induced contraction of the rabbit jejunum in a concentration dependent manner with complete inhibition at 20.48 mg/mL of the extract. Conclusion: The extract of Caralluma dalzielii possesses antidiarrheal activities which may be related to its antimotility, antioxidant and antispasmodic properties. Keywords:Antioxidant, Antispasmodic, Caralluma dalzielii, Gastrointestinal motility, Rabbit jejunum.View:PDF (543.04 KB) Full Text

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

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

The Spasmolytic, Bronchodilator, and Vasodilator Activities of Parmotrema perlatum Are Explained by Anti-Muscarinic and Calcium Antagonistic Mechanisms.

Parmotremaperlatum is traditionally used in different areas of Pakistan to treat gastrointestinal, respiratory, and vascular diseases. This study evaluates the underlying mechanisms for traditional uses of P. perlatum in diarrhea, asthma, and hypertension. In vitro pharmacological studies were conducted using isolated jejunum, trachea, and aortic preparations, while the cytotoxic study was conducted in mice. Crude extract of P. perlatum(Pp.Cr), comprising appreciable quantities of alkaloids and flavonoids, relaxed spontaneously contracting jejunum preparation, K+ (80 mM)-induced, and carbachol (1 µM)-induced jejunum contractions in a concentration-dependent manner similar to dicyclomine and dantrolene. Pp.Cr showed a rightward parallel shift of concentration-response curves (CRCs) of Cch after a non-parallel shift similarto dicyclomine and shifted CRCs of Ca+2 to rightward much likeverapamil and dantrolene, demonstrating the coexistence of antimuscarinic and Ca+2 antagonistic mechanism. Furthermore, Pp.Cr, dicyclomine, and dantrolene relaxed K+ (80 mM)-induced and Cch (1 µM)-induced tracheal contractions and shifted rightward CRCs of Cch similar to dicyclomine, signifying the dual blockade. Additionally, Pp.Cr also relaxed the K+ (80 mM)-induced and phenylephrine (1 µM)-induced aortic contraction, similarly to verapamil and dantrolene, suggesting Ca+2 channel antagonism. Here, we explored for the first time thespasmolytic and bronchodilator effects of Pp.Crand whether they maybe due to the dual blockade of Ca+2 channels and muscarinic receptors, while the vasodilator effect might be owing to Ca+2 antagonism. Our results provide the pharmacological evidence that P. perlatum could be a new potential therapeutic option to treat gastrointestinal, respiratory, and vascular diseases. Hence, there is a need for further research to explore bioactive constituent of P. perlatum as well as further investigation by suitable experimental models are required to further confirm the importance and usefulness of P. perlatum in diarrhea, asthma, and hypertension treatment.

The effects of Tityus bahiensis scorpion venom on the contractility of jejunum, vas deferens, and the aorta is differentially affected by tetrodotoxin

The pharmacological effects of the crude venom of the scorpion Tityus serrulatus or its isolated toxins have been widely studied. However, few studies are available on Tityus bahiensis venom. We recently discovered that T. serrulaus venom leads to the release of tetrodotoxin-resistant acetylcholine. Thus, our objective was to verify whether T. bahiensis venom could have a similar action in the jejunum. Furthermore, we evaluated the possibility that this action occur in other tissues innervated by the autonomic nervous system. Thus, organ bath studies were conducted to evaluate the contractile and relaxant effects of venom on the jejunum, vas deferens and aorta of rats in the presence or absence of tetrodotoxin. We observed that jejunum, vas deferens and aorta contracted when the T. bahiensis venom was applied. In the jejunum, the venom reveals a contractile component resistant to tetrodotoxin. It also was able to relax pre-contracted preparations of jejunum and aorta but not vas deferens. Only in the aorta, the relaxation was resistant to tetrodotoxin. The effects of scorpion venoms are attributed to its action on ionic channels leading to neuronal depolarization and neurotransmitter release. Our results indicated that a similar mechanism is present in the observed effects of the venom. However, another mechanism must be present in the venom-induced contraction in the jejunum and relaxation in the aorta. Possible involvement of tetrodotoxin-resistant sodium channels or non-neuronal release of neurotransmitters is discussed. We emphasize that the study of the Tityus scorpion's venom, especially T. bahiensis, is of great importance because it can unveil unknown pharmacological and physiological mechanisms of excitable cells.

Antispasmodic activity of Warionia saharae Benthem ex Benth. &amp; Coss. on the rabbit and rat jejunums

Context: Warionia saharae Benthem ex Benth. &amp; Coss has been widely used for gastrointestinal problems in Morocco and Algeria. Aims: To explore the possible mechanism for its use to such ailments in order to rationalize some of these folkloric uses. We investigated the aqueous extract of this plant (AqWs) for antispasmodic activity on isolated rat and rabbit jejunums. Methods: To investigate the contractile activity of isolated jejunum preparations of rat and rabbit we had used in vitro techniques. Jejunums were suspended in tissue baths filled with KHB culture medium (37°C) and connected to force transducers. AqWs (0.1–3 mg/mL) was used with or without carbachol (10-6 M), KCl (25 mM), yohimbine (10-5 M), prazosin (10-5 M), propranolol (10-5 M), hexamethonium (10-4 M), atropine (10-6 M), methylene blue (10-5 M), L-NAME (10-4 M) and nifedipine (10-6 M). Results: AqWs at 0.1–3 mg/mL produced a relaxation on basal rabbit contractions with an IC50 = 1.55 ± 0.06 mg/mL. This effect was reversible and was not affected by pretreatment with the inhibitors of α and β adrenergic receptors yohimbine, prazosin and propranolol. The extract had an antispasmodic activity with an IC50 = 1.25 ± 0.02 mg/mL on rat jejunum precontracted with KCl rich medium (25 mM). This result suggests that the extract had a Ca2+ antagonist effect. This was fortified when pretreatment of the intestine with the extract induced a rightward shift in the Ca2+ concentration-response curves. AqWs exhibited also antispasmodic effect on carbachol (10-6 M)-induced contractions of rat jejunums with an IC50 = 1.53 ± 0.04 mg/mL. The extract at 3 mg/mL on rat jejunums pre-incubated with hexamethonium (10-4 M), atropine (10-6 M), and methylene blue (10-5 M), then contracted by KCl, decreased the maximum contraction but not totally like KCL alone without these inhibitors. For the jejunum pre-incubated with L-NAME (10-4 M), the spasmolytic effect of AqWs was comparable with KCL. Conclusions: These results suggest that the myorelaxant and antispasmodic effects are mediated possibly via Ca2+ antagonist, anticholinergic, and guanylate cyclase mechanisms but not by adrenergic and nitric oxide pathway.

Succinate‐Induced Tuft Cell Hyperplasia Enhances FFA2/5‐HT Mediated Anion Secretion in Mouse Duodenum

Tuft cells are one of luminal chemosensing components in the small intestine, while the role of tuft cell in mucosal secretory responses to luminal chemicals such as short-chain fatty acids (SCFA) has not been elucidated. We thus hypothesized that tuft cell hyperplasia may increase the secretory responses to luminal SCFA due to the increased cholinergic signals since tuft cells express choline acetyltransferase (ChAT). Tuft cell hyperplasia was induced by oral sodium succinate (NaSuc, 100 mM) in the drinking water for 7 days. Sodium propionate (NaP, 10 mM, m), the selective free fatty acid receptor 2 (FFA2) agonist 4-CMTB (10 µM, s), or the TRPA1 agonist allyl isothiocyanate (AITC, 1 mM, m) were applied to the mucosal (m) or serosal (s) baths of an Ussing chamber mounted with muscle-stripped mucosa-submucosa preparations of mouse proximal duodenum or jejunum. The density of tuft cells (doublecortin-like kinase 1; DCLK1), enteroendocrine L cells (proglucagon, glucagon-like peptide-2), and enterochromaffin (EC) cells (5-HT, tryptophan hydroxylase 1; TPH1) were assessed by real-time PCR and immunostaining. NaSuc treatment significantly increased the number of DCLK1-positive tuft cells in the duodenal and jejunal villi. mRNA expression of DCLK1 and FFA2 were increased in the duodenal and jejunal mucosa, whereas TPH1 was increased only in the duodenal mucosa, and ChAT was increased in the jejunal mucosa, but proglucagon was unchanged in both. 5-HT-positive mast cell density rather than EC cell density was increased in the duodenum. Isc in response to luminal NaP was enhanced in the jejunum, which was atropine sensitive. In contrast, Isc responses to serosal 4-CMTB and luminal AITC were enhanced in the duodenum, which were mediated by 5-HT3 and 5-HT4 receptors. These results suggest that NaSuc differentially enhances secretory responses, with 5-HT signals in the duodenum and with cholinergic signals in the jejunum. These data may be implicated in the pathogenesis of functional dyspepsia (FD), since increased duodenal mast cell density and enhanced 5-HT signals are present in FD patients.