Activation of M3 muscarinic receptor and Ca²⁺ influx by crude fraction from Crotonis Fructus in isolated rabbit jejunum

Ethnopharmacological relevance

Crotonis Fructus is the mature fruit of Croton tiglium L. (Euphorbiaceae), which has been used in traditional Chinese medicine for the treatment of gastrointestinal (GI) diseases, such as constipation, abdominal pain, peptic ulcer, and intestinal inflammation for thousands of years. The aim of this study was to investigate the pharmacological effect of extracts and fractions from Crotonis Fructus on GI tract.

Materials and methods

The activities of methanol extract and fractions from Crotonis Fructus on the smooth muscle contractions were evaluated using isolated rabbit jejunum model.

Results

The results suggest that the n-BuOH and H₂O fractions showed spasmolytic activity, while the MeOH extract, PE and EtOAc fractions exerted spasmogenic effect. Moreover, bioassay-guided fractionation verified that the EtOAc fraction was more potent than others, followed by PE fraction and methanol extract. Additionally, atropine (10 μM), 4-DAMP (10 μM) and verapamil (0.1 μM) produced a significant inhibition of contractions caused by EtOAc fraction, while either hexamethonium (10 μM) or methoctramine (10 μM) was inactive. Additionally, a HPLC fingerprint of EtOAc fraction was appraised to ensure its chemical consistency and the main component has been identified as phorbol 12-acetate-13-tiglate.

Conclusions

These data indicate that the regulatory effect of EtOAc fraction on GI motility are medicated via the activation of M3 muscarinic receptor and Ca²⁺ influx through L-type Ca²⁺ channel. These provide a scientific basis for the traditional use of Crotonis Fructus in GI disorders.     

Relaxant effect of flavonoid naringenin on contractile activity of rat colonic smooth muscle

Ethnopharmacological relevance

Disturbed gastrointestinal (GI) motility can be associated with smooth muscle abnormalities and dysfunction. Exploring innovative approaches that can modulate the disturbed colonic motility are of great importance for clinical therapeutics. Naringenin, a flavonoid presented in many traditional Chinese herbal medicines, has been shown to have a relaxant effect on different smooth muscles. The aim of the present study was to investigate the effect of naringenin on regulation of GI motility.

Material and methods

Mechanical recording was used to investigate the effect of naringenin on isolated rat colonic smooth muscle spontaneous contractions. Whole cell patch clamp, intracellular [Ca²⁺] concentration ([Ca²⁺]_i) and membrane potential measurements were examined on primary cultures of colonic smooth muscle cells (SMCs). A neostigmine-stimulated rat model was utilized to investigate the effect of naringenin in vivo.

Results

Naringenin induced a concentration-dependent inhibition (1–1000 μM) on rat colonic spontaneous contraction, which was reversible after wash out. The external Ca²⁺ influx induced contraction and [Ca²⁺]_i increase were inhibited by naringenin (100 μM). In rat colonic SMCs, naringenin-induced membrane potential hyperpolarization was sensitive to TEA and selective large-conductance calcium-activated K⁺ (BK_Ca) channel inhibitor iberiotoxin. Under whole cell patch-clamp condition, naringenin stimulated an iberiotoxin-sensitive BK_Ca current, which was insensitive to changes in the [Ca²⁺]_i concentration. Furthermore, naringenin significantly suppressed neostigmine-enhanced rat colon transit in vivo.

Conclusion

Our results for the first time demonstrated the relaxant effect of flavonoid naringenin on colon smooth muscle both in vitro and in vivo. The relaxant effect of naringenin was attributed to direct activation of BK_Ca channels, which subsequently hyperpolarized the colonic SMCs and decreased Ca²⁺ influx through VDCC. Naringenin might be of therapeutic value in the treatment of GI motility disorders.     

Antispasmodic effect of Mentha piperita essential oil on tracheal smooth muscle of rats

Aim of the study

Mentha piperita is a plant popularly known in Brazil as “hortelã-pimenta” whose essential oil is used in folk medicine for its anti-inflammatory, antispasmodic, expectorant actions and anti-congestive. Here, it was investigated the effect of Mentha piperita essential oil (peppermint oil) in rat tracheal rings along with its mechanism of action.

Materials and methods

Tracheal tissue from Male Wistar rats (250–300 g) were used. Peppermint oil was added in cumulative concentrations [1–300 μg/ml] to the tissue basal tonus or pre-contracted by carbachol [10 μM] at 10 min intervals, incubated or not with indomethacin [10 μM], l-N-metyl-nitro-arginine [100 μM], hexamethonium [500 μM], or tetraethylammonium [5mM].

Results

Peppermint oil [100 and 300 μg/ml] inhibited the contractions induced by carbachol, which was reversed by indomethacin, l-N-metyl-nitro-arginine and hexamethonium, but not by tetraethylammonium. These data suggest the participation of prostaglandin E₂, nitric oxide and autonomic ganglions in the peppermint oil relaxant effect and may be correlated with its popular use in respiratory diseases.

Conclusions

Peppermint oil exhibited antispasmodic activity on rat trachea involving prostaglandins and nitric oxide synthase.     

Croton lechleri sap and isolated alkaloid taspine exhibit inhibition against human melanoma SK23 and colon cancer HT29 cell lines

Ethnopharmacological relevance

Croton lechleri Mull. Arg. (Euphorbiaceae) is a traditional medicinal plant which produces a red sap, traditionally known as “Sangre de Drago”; it is used in folk medicine externally for wounds, fractures, and haemorrhoids, internally for intestinal and stomach ulcers and also for the empirical cure of cancers.

Materials and methods

We investigated the effects of Croton lechleri sap and taspine in comparison with taxol and vinblastine on the growth of human cancer cell lines of SK23 (melanoma), LoVo and HT29 (colorectal cancer) using MTT and Trypan blue assays. Further, we studied cell cycle by flow cytometry and detected acetylated-α-tubulin by confocal microscope.

Results

Croton lechleri inhibited cell proliferation starting from 1 μg/mL in SK23 cells, whereas 10 times higher concentrations were required for growth inhibition of HT-29 and LoVo cell lines. Also taspine (0.1 μg/mL) inhibited the SK23 and HT29 cell proliferation. Further, assay was assessed on SK23 and HT29 cell lines with 24–48 h treatment with sap and taspine. Both sap and taspine inhibited cancer cell proliferation; taspine showed higher activity on SK23 cells, which was significantly increased after 48 h of SK23 treatment. Using confocal microscopy we observed that Croton lechleri (1 μg/mL) caused a loss of microtubule structure, whereas taspine (0.5 μg/mL) caused an increase in acetylated α-tubulin and a modification of cellular morphology, mainly in SK23 cells. Croton lechleri sap 10 and 50 μg/mL influence cell cycle; 50 μg/mL sap caused a dramatic reduction of cells in G₁/G₀ and S phases with a great increase of subG₀ cells.

Conclusions

The data showed that Croton lechleri and taspine could inhibit cell proliferation with higher potency against melanoma SK23 cells, supporting the empirical use of the sap as anticancer in ethnomedicine and taspine as a possible anticancer agent.     

Relaxant effect of 1-butanol fraction from Elaeagnus pungens leaf through inhibiting l-type Ca channel on guinea pig tracheal smooth muscle

Ethnopharmacological relevance

The leaf of Elaeagnus pungens thunb. (Family Elaeagnaceae) has been documented as an effective herb for the treatment of asthma and chronic bronchitis in traditional Chinese medicine. In the past years, only a few of preliminary studies reported the chemical constituents and pharmacology effects of the herb, but their action on the tracheal relaxation has not been investigated.

Aim of the study

To investigate the relaxing effect and mechanism of the extracts from Elaeagnus pungens leaves on guinea pig tracheal smooth muscle and bronchi smooth muscle cells.

Materials and methods

Four fractions of different polarities from Elaeagnus pungens leaves were tested to the tracheal strips on the resting tension or pre-contracted by histamine (20 μM) and acetylcholine (20 μM). Inhibitory effects of the 1-butanol fraction (400 mg/ml) on cumulative histamine and acetylcholine (0.2–20 μM) induced contraction were measured. In order to determine the mediators on the 1-butanol fraction effect, the relaxing effect of the 1-butanol fraction was evaluated in the absence and presence of β-adrenoceptor antagonists (1 μM propranolol), K⁺ channels-blockers (4-aminopyridine (2 mM), tetraethylammonium chloride (5 mM) or glibenclamide (10 μM)), the cyclooxygenase inhibitor (indomethacin, 10 μM), nitric oxide synthase inhibitor (Nω-nitro-l-arginine methyl ester, 100 μM) or l-type Ca²⁺ channel inhibitor (nifedipine, 1 μM). Moreover, [Ca²⁺]_i in bronchi smooth muscle cells was analyzed by measuring the fluorescence intensity with confocal system.

Results

1-Butanol fraction induced the highest relaxant effect among four fractions of different polarities from Elaeagnus pungens leaves, and significantly relaxed the tracheal strip in the concentration-dependent manner on the resting tension and pre-contracted by histamine phosphate and acetylcholine. It also produced an unparallel rightward shift of the cumulative concentration-response curve of histamine or acetylcholine. Furthermore, the relaxant effect of 1-butanol fraction was not affected by propranolol, glibenclamide, tetraethylammonium chloride, 4-aminopyridine, indomethacin and Nω-nitro-l-arginine methyl ester. However, 1-butanol fraction-induced relaxation decreased after adding nifedipine. It also concentration-dependently inhibited CaCl₂-induced contraction in the Ca²⁺-free, 60 mM K⁺-containing solution. Additionally, [Ca²⁺]_i in the BSMCs significantly reduced after administration of the 1-butanol fraction.

Conclusions

The 1-butanol fraction from Elaeagnus pungens leaves resulted in a relaxation in the non-precontracted and pre-contracted tracheal strips. The relaxant effect was not related to K⁺ channels, NO, cGMP or β-adrenoceptors, but related to the inhibition of Ca²⁺ influx through l-type Ca²⁺ channels.     

Antispasmodic and bronchodilator activities of Artemisia vulgaris are mediated through dual blockade of muscarinic receptors and calcium influx

Aim of the study

The present study describes antispasmodic, antidiarrheal, bronchodilatory and tracheo-relaxant activities of Artemisia vulgaris to rationalize some of its traditional uses.

Materials and methods

Crude extract of Artemisia vulgaris (Av.Cr) was studied in the isolated tissue preparations of rabbit jejunum and guinea-pig trachea, as well as in the in vivo castor oil-induced diarrhea and bronchodilatory techniques.

Results

Av.Cr which tested positive for alkaloids, coumarins, flavonoids, saponins, sterols, tannins and terpenes caused concentration-dependent (0.03–10 mg/mL) relaxation of jejunum spontaneous contractions. Av.Cr inhibited the carbachol (CCh, 1 μM) and K⁺ (80 mM)-induced contractions in a pattern, similar to that of dicyclomine. Av.Cr shifted the Ca²⁺ concentration–response curves to right, like that caused by verapamil and dicyclomine. Av.Cr produced rightward parallel shift in CCh-curves, followed by non-parallel shift at higher concentration with the suppression of the maximum response, similar to that caused by dicyclomine. It exhibited protective effect against castor oil-induced diarrhea and CCh-mediated bronchoconstriction in rodents. In trachea, Av.Cr relaxed the CCh (1 μM) and K⁺ (80 mM)-induced contractions and shifted the CCh-curves to right.

Conclusion

These results indicate that Artemisia vulgaris exhibits combination of anticholinergic and Ca²⁺ antagonist mechanisms, which provides pharmacological basis for its folkloric use in the hyperactive gut and airways disorders, such as abdominal colic, diarrhea and asthma.     

Screening of the topical anti-inflammatory activity of the bark of Acacia cornigera Willdenow, Byrsonima crassifolia Kunth, Sweetia panamensis Yakovlev and the leaves of Sphagneticola trilobata Hitchcock

Ethnopharmacological relevance

An investigation of topical anti-inflammatory activity was undertaken on plants used in Central America traditional medicine.

Aim of study

Four herbal drugs used in the folk medicine of Central America to treat inflammatory skin affections (Acacia cornigera bark, Byrsonima crassifolia bark, Sphagneticola trilobata leaves and Sweetia panamensis bark) were evaluated for their topical anti-inflammatory activity.

Materials and methods

Petroleum ether, chloroform and methanol extracts were obtained for herbal medicines and then extracts were tested on Croton oil-induced ear dermatitis model in mice.

Results

Almost all the extracts reduced the Croton oil-induced ear dermatitis in mice and the chloroform ones showed the highest activity, with ID₅₀ (dose giving 50% oedema inhibition) values ranging from 112 μg/cm² (Byrsonima crassifolia) to 183 μg/cm² (Sphagneticola trilobata). As reference, ID₅₀ of the non-steroidal anti-inflammatory drug indomethacin was 93 μg/cm².

Conclusions

Lipophilic extracts from these species can be regarded as potential sources of anti-inflammatory principles.     

Hypotensive and endothelium-independent vasorelaxant effects of methanolic extract from Curcuma longa L. in rats

Ethnopharmacological relevance

Curcuma longa L. (CL) is a yellow rhizome that is used in African traditional medicine to treat palpitation, hypertension or other related blood circulation disorders.

Aim of the study

To justify the use of CL in ethnomedicine, we investigated the vasorelaxant effect of methanolic extract of CL (CLME) and its underlying mechanisms in isolated rat mesenteric artery.

Materials and methods

The effect of CLME on the mean arterial pressure (MAP) and heart rate (HR) (pulse interval) were determined in vivo in non-anaesthetized rats. Superior mesenteric rings were isolated, suspended in organ baths containing Tyrode solution at 37 °C and gassed with 95% O₂ + 5% CO₂, under a resting tension of 0.75 g. The vasorelaxant effects of CLME were studied by means of isometric tension recording experiments.

Results

In normotensive rats, CLME (10, 20 and 30 mg/kg, i.v.) induced dose-dependent hypotension (2.0 ± 0.5%; 27.1 ± 5.0% and 26.7 ± 4.6%, respectively), and pronounced bradycardia (5.8 ± 1.2%, 19.3 ± 3.2% and 22.9 ± 4.6%, respectively). CLME (1–1000 μg/mL) induced concentration-dependent relaxation of tonic contractions evoked by phenylephrine (Phe) (10 μM) and KCl (80 mM) in rings with intact-endothelium (E_max = 82.3 ± 3.2% and 97.7 ± 0.7%) or denuded-endothelium (E_max = 91.4 ± 1.0% and 97.8 ± 1.1%). Also, in a depolarized, Ca²⁺ free medium, CLME inhibited CaCl₂ (1 μM–30 mM)-induced contractions and caused a concentration-dependent rightward shift of the response curves, indicating that CLME inhibited the contractile mechanisms involving extracellular Ca²⁺ influx. In addition, in Ca²⁺ free media containing EGTA (1 mM), CLME inhibited the transient contraction of denuded rings constricted with Phe, but not those evoked by caffeine (20 mM). In contrast, neither glibenclamide, BaCl₂, tetraethylammonium nor 4-aminopyridine affected CLME-induced relaxation.

Conclusions

These results demonstrate the hypotensive and bradycardic effects of CLME, as well as its potent vasodilation of rat mesenteric arteries. These effects, may in part, be due to the inhibition of extracellular Ca²⁺ influx and/or inhibition of intracellular Ca²⁺ mobilization from Phe-sensitive stores.     

Antiplasmodial activity of root extract and fractions of Croton zambesicus

Aim of the study

Antiplasmodial activity of root extract and fractions of Croton zambesicus were evaluated to ascertain the folkloric claim of its antimalarial activity and elucidate its antiplasmodial mechanism of action.

Material and method

The crude ethanolic root extract (27–81 mg/kg) and gradient fractions ( n- hexane, chloroform, ethyl acetate and methanol; 54 mg/kg) of Croton zambesicus were investigated for antiplasmodial activity against chloroquine - sensitive Plasmodium berghei infections in mice. The antiplasmodial activity during early and established infections as well as the prophylactic activity were investigated. Chloroquine (5 mg/kg) and pyrimethamine (1.2 mg/kg) were used as positive controls. Thin films made from tail blood of each mouse were used to assess the level of parasitaemia of the mice. Oxidant generation potentials of the crude extract and fractions was also evaluated to elucidate their mechanism of action.

Results

The crude root extract (27 – 81 mg/kg) demonstrated significant (P < 0.01–0.001) schizonticidal activity during early and established infections and also had prophylactic activity. The activity was comparable to that of the standard drug used (chloroquine 5 mg/kg, pyrimethamine 1.2 mg/kg). Methanol, ethyl acetate and chloroform fractions had comparative in vivo antiplasmodial activity and oxidant generation potentials.

Conclusion

The antiplasmodial activity of this root extract and fractions which is likely to be through peroxidation confirms the folkloric use of this plant.     

Hexane extract of Poncirus trifoliata (L.) Raf. stimulates the motility of rat distal colon

Aim of the study

Poncirus trifoliata (L.) Raf. (Rutaceae, PT) has been commonly used for treating gastrointestinal (GI) disorders in Korean traditional medicine, but its pharmacological roles in the regulation of colonic motility have not been clarified. This study investigated the regulatory effects of PT on the colonic motility.

Materials and methods

Immature fruits of PT were sequentially partitioned with MeOH, n-hexane, CHCl₃, EtOAc, n-BuOH and H₂O, and the effects of PT extracts on the contractility of colonic strips and colonic luminal transit in rats were measured in vitro and in vivo, respectively.

Results

Among six different extracts, only hexane extract of PT (PTHE) dose-dependently increased the low frequency contraction of longitudinal muscle in distal colonic strips, and the ED₅₀ value was revealed to be 0.71 μg/ml. The contractile patterns induced by PTHE were remarkably different from those caused by acetylcholine (ACh) and serotonin (5-HT). The stimulatory effects of PTHE on the whole distal colonic strips were more prominent than on the mucosa/submucosa-denuded segments. The M₂ receptor-preferring, methoctramine (0.5 μM), and M₃ receptor-preferring antagonist, 4-DAMP (0.5 μM) significantly blocked the PTHE (1 μg/ml)-induced contraction of distal colon longitudinal muscles, whereas the 5-HT receptor antagonists (1.0 μM, alone or in combination) selective for 5-HT₃ (ondansetron), 5-HT₄ (GR113808) and 5-HT_{1, 2, 5–7} (methysergide) receptors did not change the PTHE (1 μg/ml)-induced contractility of distal colon longitudinal muscles. SNAP (0.1 mM), a NO donor, enhanced the stimulatory effects of PTHE on the longitudinal muscle of distal colon, but l-NAME (0.1 mM), a NO synthesis inhibitor, had no effects. PTHE (10–100 mg/kg) caused a dose-dependent increase of colonic luminal transit.

Conclusions

Collectively, these findings suggest that PTHE specifically acts on the longitudinal muscle of distal colon in rats, and these stimulatory effects are likely mediated, at least, by activation of acetylcholinergic M₂ and M₃ receptors.     

Antiarrhythmic effects of dehydroevodiamine in isolated human myocardium and cardiomyocytes

Ethnopharmacological relevance

Dehydroevodiamine alkaloid (DeHE), a bioactive component of the Chinese herbal medicine Wu-Chu-Yu (Evodiae frutus), exerted antiarrhythmic effect in guinea-pig ventricular myocytes. We further characterize the electromechanical effects of DeHE in the human atrial and ventricular tissues obtained from hearts of patients undergoing corrective cardiac surgery or heart transplantation.

Materials and methods

The transmembrane potentials of human myocardia were recorded with a traditional microelectrode technique while sarcolemmal Na⁺ and Ca²⁺ currents in single human cardiomyocytes were measured by a whole-cell patch-clamp technique. The intracellular pH (pH_i) and Na⁺–H⁺ exchanger (NHE) activity were determined using BCECF-fluorescence in human atria.

Results

In human atria, DeHE (0.1–0.3 μM) depressed upstroke velocity, amplitude of action potential, and contractile force, both in slow and fast response action potential. Moreover, the similar depressant effects of DeHE were found in human ventricular myocardium. Both in isolated human atrial and ventricular myocytes, DeHE (0.1–1 μM) reversibly, concentration-dependently decreased the Na⁺ and Ca²⁺currents. Moreover, DeHE (0.1 and 0.3 μM) suppressed delayed afterdepolarizations and aftercontractions, induced by epinephrine and high [Ca²⁺]_o in atria. In human ventricular myocardium, the strophanthidin-induced triggered activities were attenuated by pretreating DeHE (0.3 μM). The resting pH_i and NHE activity were also significantly increased by DeHE (0.1–0.3 μM).

Conclusions

We concluded for the first time that, in the human hearts, DeHE could antagonize triggered arrhythmias induced by cardiotonic agents through a general reduction of the Na⁺ and Ca²⁺ inward currents, while increase of resting pH_i and NHE activity.     

Vasorelaxant effects of extracts of the stem bark of Terminalia superba Engler & Diels (Combretaceae)

Aim of the study

The stem bark of Terminalia superba (Combretaceae) (TS) is used in traditional Cameroonian medicine as antihypertensive remedy. In the present study, we investigated the vasorelaxant properties of different extracts of TS and their underlying mechanisms.

Materials and methods

Activities of aqueous (AQU), methanolic (MET), methylene chloride (MC), and methylene chloride–methanol (MCM) extracts of TS were evaluated on isolated rat aortic rings precontracted with phenylephrine (PE) or high KCl.

Results

All extracts induced a vasodilating effect both on KCl- and PE-induced contractions. The effects of MC and MCM extracts were greater than those of AQU or MET extracts (P < 0.05). MC had an endothelium-independent effect and reduced Ca⁺⁺-induced contraction following PE or KCl challenge (P < 0.05). After incubation with verapamil, MC induced a relaxation in rings precontracted by PE (P < 0.001). By contrast, the effect of MCM was endothelium-dependent and decreased by the nitric oxide synthase inhibitor N_W-nitro-l-arginine methyl ester (P < 0.05).

Conclusions

These data demonstrate that the MC extract exhibits vasorelaxant effects that are partly due to inhibition of extracellular Ca⁺⁺ influx and/or inhibition of intracellular Ca⁺⁺ release in vascular smooth muscle cells. By contrast, the effect of the MCM extract was found to be endothelium- and nitric oxide dependent.     

Anti-diarrhoeal activity of methanol extract of Santalum album L. in mice and gastrointestinal effect on the contraction of isolated jejunum in rats

Ethnopharmacological relevance

Santalum album L., namely Sandalwood, honored as “Green Gold”, is a traditional Chinese herb which has the effects of anti-diarrhoeal and antibacterial activity. But there is limit scientific study on its activity and mechanism in gastrointestinal disorders.

Materials and methods

in vivo, after intragastric administration, the methanol extract of Sandalwood (SE) (200, 400 and 800 mg/kg) were studied in castor oil-induced diarrhoea mice. By the test of small intestinal hyperfunction induced by neostigmine, SE was studied on gastrointestinal transit including gastric emptying and small intestinal motility. Meanwhile, in vitro, the effects of SE (0.02, 0.05, 0.1, 0.2, 0.3, 0.4 mg/mL) on the isolated tissue preparations of rat jejunum were also investigated. The rat jejunum strips were pre-contracted with acetylcholine (Ach; 10⁻⁶ M), 5-hydroxytryptamine (5-HT, 200 μM) or potassium chloride (KCl; 60 mM) and tested in the presence of SE. In addition, the possible myogenic effect was analyzed in the pretreatment of the jejunum preparations with SE or verapamil in Ca²⁺-free high-K⁺ (60 mM) solution containing EDTA.

Results

At doses of 200, 400 and 800 mg/kg, SE showed significant anti-diarrhoeal activity against castor oil-induced diarrhoea as compared with the control. At the same doses, it also inhibited the gastric emptying and small intestinal motility in the mice of which small intestinal hyperfunction induced by neostigmine. It caused inhibitory effects on the spontaneous contraction of rat-isolated jejunum in dose-dependent manner ranging from 0.02 to 0.4 mg/mL, and it also relaxed the Ach-induced, 5-HT-induced and K⁺-induced contractions. SE shifted the Ca²⁺ concentration–response curves to right, similar to that caused by verapamil (0.025 mM).

Conclusions

These findings indicated that SE played a spasmolytic role in gastrointestinal motility which was probably mediated through inhibition of muscarinic receptors, 5-HT receptors and calcium influx. All these results provide pharmacological basis for its clinical use in gastrointestinal tract.     

Protective effects of Croton campestris A. St-Hill in different ulcer models in rodents: Evidence for the involvement of nitric oxide and prostaglandins

Ethnopharmacological relevance

Croton campestris A. St.-Hill., popularly known as “velame do campo”, is a species native from savannah area of Northeast Brazil, which is used by traditional communities in folk medicine for a variety of health problems, especially detoxification, inflammation and gastritis. The present study investigates the possible gastric antiulcer activity of Croton campestris root extract (CCRE) and mechanisms of action underlying this effect.

Materials and methods

Gastric lesions were induced in mice by ethanol, acidified ethanol and indomethacin. CCRE was previously administered orally in doses ranging from 50 to 750 mg/kg. Stomach lesions were measured. The involvement of Nitric Oxide (NO), prostaglandins (PGEs), ATP-dependent K+ channel and adrenergic receptor was investigated through specific inhibitors.

Results

CCRE produced significant antiulcer activity against absolute ethanol, acidified ethanol and indomethacin induced gastric lesions. The pretreatment with L-NAME (10 mg/kg, p.o.), an inhibitor of nitric oxide synthesis and indomethacin (10 mg/kg, s.c.), an inhibitor of prostaglandin production, reversed the antiulcer action of CCRE.

Conclusion

Taking together, these results suggest that the antiulcer activity of CCRE is dependent of NO and prostaglandin pathways possibly due to its ability to stimulate the synthesis of NO, and activation of endogenous prostaglandin production. Therefore, the use of CCRE in traditional Brazilian medicine against gastric disorders has a scientific basis.     

Simo decoction promotes contraction of antral circular smooth muscle mainly via muscarinic M3 receptor

Ethnopharmacological relevance

Simo Decoction (SMD), a traditional Chinese medicine, included four elements, such as Fructus aurantii, Radix aucklandiae, Semen arecae and Radix linderae. It has been used to improve gastrointestinal dysmotility in clinical practice for a long history in China. However, the explicit mechanisms are unclear. The aim of this study was to investigate the effect of SMD on contractions of antral circular smooth muscle strips of Sprague-Dawley (SD) rats and its underlying mechanism.

Materials and methods

The antral circular strips were prepared in the organ bath under baseline or to be incubated with muscarinic receptor antagonist atropine (10⁻⁶ M), muscarinic M3 receptor antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) (0.4×10⁻⁶ M), muscarinic M2 receptor antagonist gallamine (10⁻⁶ M), adrenergic receptor agonist adrenaline (10⁻⁷ M), exogenous nitric oxide (NO) donor l-arginine (10⁻⁴ M), nicotinic receptor antagonist hexamethonium chloride (10⁻⁴ M) and Ca²⁺ channel antagonist nifedipine (30 nM), and consecutive concentrations of SMD were added to the bath to observe the strip responses. As a control, the responses of strips after administration with the same volume of Krebs solution as SMD were also noted. The strip responses to acetylcholine (10⁻⁷–10⁻³ M) were also noted in organ bath to compare with SMD-induced contraction.

Results

SMD dose-dependently evoked hypercontractility of antral circular strips, and the maximal contractile effect of circular smooth muscle induced by SMD was significantly higher than that induced by acetylcholine (10⁻³ M). The responses of antral circular strips to SMD were completely antagonized by atropine, 4-DAMP or 4-DAMP+gallamine, but partly inhibited by gallamine and partly suppressed by adrenaline, l-arginine, hexamethonium chloride and nifedipine.

Conclusions

SMD promotes contractions of antral circular strips in rats mainly via activation of muscarinic M3 receptor, but partly via activation of muscarinic M2 receptor, Ca²⁺ channel and nicotinic receptor, inhibition of adrenergic receptor and releasing of NO.     

Herbal formula SYJN protect PC12 cells from neurotoxicity induced by corticosterone

Aim of the study

SYJN is a Chinese herbal formula, containing four herbs: Bupleurum chinense DC., Curcuma aromatica Salisb., Perilla frutescens (Linn.) Britt. and Acorus tatarinowii Schott. Previous studies on the formula in our laboratory revealed an antidepressant-like effect on animal models of behavioral despair. However,the mechanisms underlying such antidepressant-like effect are yet to be understood. The aim of this work was to verify the previously established antidepressant-like effects on cell level using corticosterone-induced neurotoxicity in rat pheochromocytoma (PC12) cells to see if SYJN possesses any neuroprotective properties.

Materials and methods

PC12 cells were treated with 200 μM corticosterone in the absence or the presence of various concentrations of SYJN for 48 h. Then, cell viability, apoptosis, intracellular Ca²⁺ ([Ca²⁺]i) concentration and caspase-3 activity were determined.

Results

Following the exposure of PC12 cells to 200 μM corticosterone for 48 h, there were reductions in cell survival rate but increases in lactate dehydrogenase (LDH) release. In parallel, corticosterone caused significant elevations in DNA fragmentation, [Ca²⁺]i concentration and caspase-3 activity. However, when the PC12 cells were incubated with SYJN at different concentrations (10, 50 and 100 mg/L) in the presence of 200 μM corticosterone for 48 h, the above effects were evidently alleviated in a dose-dependent manner.

Conclusion

SYJN could generate a neuroprotective effect on corticosterone-induced neurotoxicity in PC12 cells, suggesting a possible action pathway of SYJN in vivo by decreasing the [Ca²⁺]i concentration and caspase-3 activity.     

Extracts of bark from the traditional Chinese herb Phellodendron amurense inhibit contractility of the isolated rat prostate gland

Aim of the study

To assess the effectiveness of the traditional Chinese herb Phellodendron amurense in treating urological disorders.

Materials and methods

Prostate smooth muscle relaxant activity of an extract from the bark of Phellodendron amurense was tested on contractions of isolated rat prostate gland induced by electrical nerve stimulation and direct muscle stimulation.

Results

Electrical field stimulation (0.5 ms, 60 V, 1–20 Hz) induced nerve mediated contractions of isolated rat prostate were inhibited by Phellodendron amurense extract dissolved in either dimethylsulfoxide (DMSO), acetic acid or water (P ≤ 0.022, n = 6 for each) but not boiling water (P = 0.619, n = 6). Phellodendron amurense extract also inhibited contractions of isolated rat prostates elicited by exogenous administration of noradrenaline (10 nM to 100 μM), acetylcholine (10 nM to 100 μM) or adenosine 5′-triphosphate (ATP, 100 nM to 100 μM) (P ≤ 0.004, n = 6–8 for each).

Conclusion

Phellodendron amurense is able to inhibit prostatic contractility suggesting that it may be useful in the treatment of urological disorders caused by prostatic urethral obstruction such as in the case of benign prostatic hyperplasia (BPH).     

Coriander fruit exhibits gut modulatory,blood pressure lowering and diuretic activities

Aim of the study

Coriander (Coriandrum sativum) is traditionally used for various gastrointestinal and cardiovascular disorders and this study was designed to rationalize its use in dyspepsia, abdominal colic, diarrhea, hypertension and as diuretic.

Materials and methods

Coriander crude extract (Cs.Cr) was evaluated through in vitro and in vivo techniques.

Results

Cs.Cr caused atropine sensitive stimulatory effect in isolated guinea-pig ileum (0.1–10 mg/ml). In rabbit jejunum preparations, Cs.Cr evoked a similar contractile response but in the presence of atropine, it exhibited relaxation against both spontaneous and high K⁺ (80 mM)-induced contractions as well as shifted the Ca²⁺ concentration–response curves to right, similar to that caused by verapamil. Cs.Cr (1–30 mg/ml) caused fall in arterial blood pressure of anesthetized animals, partially blocked by atropine. Cs.Cr produced vasodilatation against phenylephrine and K⁺ (80 mM)-induced contractions in rabbit aorta and cardio-depressant effect in guinea-pig atria. Cs.Cr produced diuresis in rats at 1–10 mg/kg. Bio-assay-directed fractionation revealed the separation of spasmogenic and spasmolytic components in the aqueous and organic fractions respectively.

Conclusions

These results indicate that coriander fruit exhibits gut stimulatory, inhibitory and hypotensive effects mediating possibly through cholinergic, Ca²⁺ antagonist and the combination of these mechanisms respectively. Diuretic activity adds value to its use in hypertension.     

Protective effects of aqueous extract from Acanthopanax senticosus against corticosterone-induced neurotoxicity in PC12 cells

Ethnopharmacological relevance

Acanthopanax senticosus, classified into the family of Araliaceae, has been known for thousands of years as a remedy and is used to treat various diseases in traditional Chinese medicine system including hypertension, ischemic heart disease and hepatitis.

Aim of the study

This study aimed to examine the protective effects of aqueous extract from Acanthopanax senticosus (ASE) on corticosterone-induced neurotoxicity and its possible mechanisms, using PC12 cells as a suitable in vitro model of depression.

Materials and methods

In this paper, PC12 cells were treated with 200 μM of corticosterone in the absence or presence of ASE in varying concentrations for 24 h. Then, cell viability was measured by MTT assay. The release amount of lactate dehydrogenase (LDH) was quantified using LDH assay kit. Apoptosis of PC12 cells was measured by Annexin V-FITC and PI labeling. The intracellular Ca²⁺ content was tested by fluorescent labeling. The mRNA level of brain-derived neurotrophic factor (BDNF) was examined by real-time RT-PCR, and the expression of cAMP response element binding protein (CREB) was determined by western blotting.

Results

The results showed that treatment with 200 μM of corticosterone could induce cytotoxicity in PC12 cells. However, different concentrations of ASE (50, 100, 200, and 400 μg/mL) significantly increased the cell viability, decreased the LDH release, suppressed the apoptosis of PC12 cells, attenuated the intracellular Ca²⁺ overloading, up-regulated the BDNF mRNA level and CREB protein expression compared with the corresponding corticosterone-treated group.

Conclusion

The present results suggest that ASE exerts a neuroprotective effect on corticosterone-induced neurotoxicity in PC12 cells, which may be one of the acting mechanisms that accounts for the in vivo antidepressant activity of ASE.     

Dragon's blood Croton palanostigma induces genotoxic effects in mice

Ethnopharmacological relevance

Dragon's blood is a dark-red sap produced by species from the genus Croton (Euphorbiaceae), which has been used as a famous traditional medicine since ancient times in many countries, with scarce data about its safe use in humans. In this research, we studied genotoxicity and clastogenicity of Croton palanostigma sap using the comet assay and micronucleus test in cells of mice submitted to acute treatment.

Material and methods

HPLC analysis was performed to identify the main components of the sap. The sap was administered by oral gavage at doses of 300 mg/kg, 1000 mg/kg and 2000 mg/kg. For the analysis, the comet assay was performed on the leukocytes and liver cells collected 24 h after treatment, and the micronucleus test (MN) on bone marrow cells. Cytotoxicity was assessed by scoring 200 consecutive polychromatic (PCE) and normochromatic (NCE) erythrocytes (PCE/NCE ratio).

Results and conclusion

The alkaloid taspine was the main compound indentified in the crude sap of Croton palanostigma. The results of the genotoxicity assessment show that all sap doses tested produced genotoxic effects in leukocytes and liver cells and also produced clastogenic/aneugenic effects in bone marrow cells of mice at the two higher doses tested. The PCE/NCE ratio indicated no cytotoxicity. The data obtained suggest caution in the use of Croton palanostigma sap by humans considering its risk of carcinogenesis.