M3 muscarinic receptor- and Ca influx-mediated muscle contractions induced by croton oil in isolated rabbit jejunum

Aim of study

Croton oil is the fruit oil of Croton tiglium L., which is well known in folk medicine for the treatment of gastrointestinal (GI) diseases, including constipation, abdominal pain, peptic ulcer, and intestinal inflammation for a long period. This study was to investigate the pharmacological effect of croton oil on GI tract.

Materials and methods

The effect of croton oil on the smooth muscle contractions was investigated in vitro using the isolated rabbit jejunum model.

Results

Croton oil has a biphasic action contracting and relaxing intestinal tissue. At the concentrations of 20–80 μg/mL, croton oil produced a concentration-dependent increase in the amplitude and tension of muscle contractions, whereas at high concentrations (>200 μg/mL) it decreased the contractile amplitude and had no impact on the tension. Moreover, croton oil was less effective in increasing muscle amplitude and tension than Ach, confirming that the effect of croton oil on muscle contractions is not a simply stimulatory or inhibitory action, but a unique modulatory process depending on the concentration of croton oil. In addition, croton oil concentration-dependently suppressed the frequency of muscle contractions. On the other hand, atropine (10 μM) and 4-DAMP (10 μM) produced a significant inhibition of contractions caused by croton oil, while either hexamethonium (10 μM) or methoctramine (10 μM) was inactive, implying that the regulatory effects of croton oil on GI motility are mediated via the activation of M3 muscarinic receptor. Furthermore, muscle contractions induced by croton oil were dramatically reduced by verapamil (0.1 μM) but not by NE (1 μM), suggesting that the action of croton oil on GI motility is also mediated by Ca²⁺ influx through L-type Ca²⁺ channel.

Conclusions

The results suggest that croton oil possesses spasmogenic and spasmolytic properties and the regulatory effects of croton oil on GI motility are mediated via the activation of M3 muscarinic receptor and Ca²⁺ influx through L-type Ca²⁺ channel.     

The spasmolytic activity of extracts and some isolated compounds from the leaves of Morinda morindoides (Baker) Milne-Redh. (Rubiaceae)

Aim

The study was aimed to evaluate the in vitro antispasmodic activity of Morinda morindoides leaves aqueous extract, its soluble fractions and isolated compounds to provide the pharmacological basis for its use for the treatment of constipation and diarrhoea in traditional medicine.

Methods

The antispasmodic activity of each sample was evaluated on acetylcholine (ACh) and the depolarized KCl solution induced contractions on guinea-pig isolated ileum suspended in Tyrode's solution.

Results

At a test concentration of 40 μg/ml in organ bath, the aqueous extract and its petroleum ether soluble fraction showed a spasmogenic effect on both agonists. The diethylether, ethyl acetate, n-butanol and residual aqueous phase soluble fractions from the partition of the aqueous extract exhibited spasmolytic activity producing 47–100% inhibition of contractions induced by both agonists with IC₅₀ values ranged from 6 to 15 μg/ml according to the case. In addition, the n-butanol and residual aqueous phase soluble fractions showed an inhibitory effect of 75 and 66% respectively on low high [K⁺] (25 mM) and 65 and 60% respectively on high [K+] (80 mM). Crude flavonoids showed spasmolytic on both agonists while crude saponins only showed spasmolytic activity on ACh and displayed spasmogenic effect on KCl. Quercetin, quercitrin and rutin exhibited significant antispasmodic effect with IC₅₀ values <0.1 μg/ml. Epoxygaertneroside and gaertneroside showed good antispasmodic activity on both agonists (4 < IC₅₀ < 7 μg/ml).

Conclusion

Morinda morindoides leaves possess spasmogenic and spasmolytic properties that can at least explain and support its traditional use against constipation and diarrhoea respectively.     

Antispasmodic and antioxidant activities of fractions and bioactive constituent davidigenin isolated from Mascarenhasia arborescens

Ethnopharmacological relevance

Mascarenhasia arborescens A. DC. (Apocynaceae) is used in traditional medicine in the North of Madagascar to treat intestinal disorders, intestinal spasms and diarrhoea.

Aim of the study

The main objective of this work was to evaluate the antispasmodic activity of the crude methanolic extract of Mascarenhasia arborescens and of its four partitions and to identify the effective compound responsible for this effect.

Materials and methods

Isolation and structure elucidation techniques were performed in order to identify the bioactive constituent of Mascarenhasia arborescens and HPLC analysis was used for its quantification. Total phenolic content (TPC) of crude extracts and partitions were determined using the Folin–Ciocalteu method. Crude methanolic extract, partitions and the bioactive compound were investigated for their spasmolytic activity on several isolated organs. Their antiradical activity was also investigated by the DPPH test.

Results

Bioassay-guided fractionation using isolated guinea pig ileum pre-contracted with histamine 3 × 10⁻⁶ M led to the isolation of davidigenin (DG), a dihydrochalcone, as the main active constituent from the most promising methylene chloride partition (McP). This partition was effective on isolated guinea pig ileum pre-contracted with 3 × 10⁻⁶ M histamine, with a median effective concentration (EC₅₀) of 41.19 ± 3.74 μg/ml. The DG content of this partition was shown to be 26.5% by HPLC. DG induced a concentration-dependent relaxation of the histamine pre-contracted guinea pig ileum with an EC₅₀ of 8.04 ± 0.81 μg/ml and a concentration-dependent relaxation of the acetylcholine pre-contracted rat duodenum with an EC₅₀ of 9.35 ± 0.30 μg/ml. It inhibited in a non-competitive manner histamine-induced isolated ileum contraction and the acetylcholine-induced isolated duodenum contraction. Moreover, DG does not have any antiradical activity.

Conclusions

We demonstrated for the first time antispasmodic and antioxidant effects of Mascarenhasia arborescens. This study supports its use in traditional medicine. Furthermore, we highlighted the crucial role of davidigenin in the antispasmodic activity of this plant.     

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.     

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.     

Anti-Helicobacter pylori activity of plants used in Mexican traditional medicine for gastrointestinal disorders

Aim of the study

Helicobacter pylori is the major etiological agent of chronic active gastritis and peptic ulcer disease and is linked to gastric carcinoma. Treatment to eradicate the bacteria failed in many cases, mainly due to antibiotic resistance, hence the necessity of developing better therapeutic regimens. Mexico has an enormous unexplored potential of medicinal plants. This work evaluates the in vitro anti-H. pylori activity of 53 plants used in Mexican traditional medicine for gastrointestinal disorders.

Materials and methods

To test the in vitro antibacterial activity, agar dilution and broth dilution methods were used for aqueous and methanolic extracts, respectively.

Results

Aqueous extracts of Artemisia ludoviciana subsp. mexicana, Cuphea aequipetala, Ludwigia repens,and Mentha × piperita (MIC 125 to <250 μg/ml) as well as methanolic extracts of Persea americana, Annona cherimola, Guaiacum coulteri, and Moussonia deppeana (MIC <7.5 to 15.6 μg/ml) showed the highest inhibitory effect.

Conclusions

The results contribute to understanding the mode of action of the studied medicinal plants and for detecting plants with high anti-Helicobacter pylori activity.     

Spasmolytic activity of Rosmarinus officinalis L. involves calcium channels in the guinea pig ileum

Ethnopharmacological relevance

Rosmarinus officinalis L. is a plant used around the world for its properties to cure pain in several conditions, such as arthritic and abdominal pain or as an antispasmodic; however, there are no scientific studies demonstrating its spasmolytic activity. Therefore, the aim of the present study was to investigate the effect of an ethanol extract from Rosmarinus officinalis aerial parts and the possible mechanism involved by using rings from the isolated guinea pig ileum (IGPI).

Materials and methods

The IGPI rings were pre-contracted with potassium chloride (KCl; 60 mM), acetylcholine (ACh; 1 × 10⁻⁹ to 1 × 10⁻⁵ M) or electrical field stimulation (EFS; 0.3 Hz of frequency, 3.0 ms of duration and 14 V intensity) and tested in the presence of the Rosmarinus officinalis ethanol extract (150, 300, 600 and 1 200 μg/mL) or a referenced smooth muscle relaxant (papaverine, 30 μM). In addition, the possible mechanism of action was analyzed in the presence of hexametonium (a ganglionic blocker), indomethacine (an inhibitor of prostaglandins), l-NAME (a selective inhibitor of the nitric oxide synthase) and nifedipine (a calcium channel blocker).

Results

Rosmarinus officinalis ethanol extract exhibited a significant and concentration-dependent spasmolytic activity on the contractions induced by KCl (CI₅₀ = 661.06 ± 155.91 μg/mL); ACh (CI₅₀ = 464.05 ± 16.85 μg/mL) and EFS (CI₅₀ = 513.72 ± 34.13 μg/mL). Spasmolytic response of Rosmarinus officinalis (600 μg/mL) was reverted in the presence of nifedipine 1 μM, but not in the presence of hexamethonium 0.5 mM, indomethacine 1 μM or l-NAME 100 μM.

Conclusion

The present results reinforce the use of Rosmarinus officinalis as antispasmodic in folk medicine. Moreover, it is demonstrated the involvement of calcium channels in this activity, but not the participation of nicotinic receptors, prostaglandins or nitric oxide.     

Antisecretory activity from the flowers of Chiranthodendron pentadactylon and its flavonoids on intestinal fluid accumulation induced by Vibrio cholerae toxin in rats

Ethnopharmacological relevance

The flowers of Chiranthodendron pentadactylon Larreat. (Sterculiaceae) has been traditionally used as folk medicine in Mexico for the treatment of gastrointestinal disorders such as diarrhea and dysentery.

Aim of the study

This study aimed to assess the antisecretory activity which supports the therapeutic use of Chiranthodendron pentadactylon and its flavonoids to treat diarrhea.

Materials and methods

The methanol extract of Chiranthodendron pentadactylon, subsequent fractions, and flavonoids were evaluated on cholera toxin-induced intestinal secretion in rat jejunal loops model.

Results

Three antisecretory flavonoids were isolated by bioassay-guided purification, namely, isoquercitrin 3, (+)-catechin 4 and (−)-epicatechin 5. Among them, epicatechin exhibited the most potent antisecretory activity with ID₅₀ of 8.3 μM/kg. Its potency was close that of to loperamide (ID₅₀ 6.1 μM/kg), drug used as control. Isoquercitrin (ID₅₀ 19.2 μM/kg) and catechin (ID₅₀ 51.7 μM/kg) showed moderate and weak activity, respectively.

Conclusion

The results of the present study lend some support to the anecdotal report for the traditional use of the flowers of Chiranthodendron pentadactylon in the control of dysentery.     

Relaxant effects of Artemisia ludoviciana on isolated rat smooth muscle tissues

Ethnopharmacological relevance

Artemisia ludoviciana spp. mexicana (Willd. Ex.) Spring D.D. Keck (Asteraceae), known as “estafiate” is employed for the treatment of diarrhea, dysentery, parasites, abdominal pain, vomiting, stomach ache, and also as antispasmodic agent. The aim of the present study was to evaluate the relaxant effect of hexanic (HEAl), dichloromethanic (DEAl) and methanolic (MEAl) extracts on isolated trachea, ileum and aorta rat rings, and to establish the tracheo-relaxant mode of action of DEAl.

Materials and methods

All extracts were investigated based on their capacity of to inhibit the rat ileum spontaneous contraction, to relax contraction induced by noradrenaline (0.1 μM) on endothelium-intact and endothelium-denuded thoracic aorta rat rings, and also to inhibit contraction provoked by carbachol (1 μM) on rat trachea.

Results

Organic extracts had no spasmolytic action on ileum strips compared to positive control (papaverine, p < 0.05). On the other hand, all extracts induced a significant concentration- and partial endothelium-dependent vasorelaxant activity. Extracts also showed significant relaxant effect on pre-contracted tracheal tissue in a concentration-dependent manner. In last two experiments, DEAl was the most potent and efficient extract; however, it was less potent than papaverine and theophylline, used as positive controls (p < 0.05). In tracheal preparation, DEAl shifted to the right, in a parallel manner, the concentration-response curves induced by carbachol (p < 0.05). Also, DEAl induced a significant relaxant effect on the contraction produced by potassium chloride (KCl, 80 mM). Pre-incubation with 1-H-[1,2,4]-oxadiazolo-[4,3a]-quinoxalin-1-one (ODQ, 10 μM), indomethacin (10 μM), N_ω-nitro-l-arginine methyl ester (l-NAME, 10 μM), glibenclamide (10 μM) and 2-aminopyridine (2-AP, 100 μM) did not modify the DEAl-relaxant curves.

Conclusions

Functional experiments suggest that the most active extract, DEAl, induced its relaxant effect by possible muscarinic receptors antagonism and calcium channel blockade in tracheal rings. On the other hand, significant vasorelaxant activity showed by DEAl is partially endothelium-dependent. Finally, spasmolytic activity induced by the extracts in the rat ileum was not significant, which suggests that the antidiarrheic effect of the plant is related to antimicrobial and antiparasitic properties previously described.     

Protein tyrosine phosphatase 1B inhibitory by dammaranes from Vietnamese Giao-Co-Lam tea

Ethnopharmacological relevance

Gynostemma pentaphyllum (Thunb.) tea was used in Vietnamese folk medicine as anti-diabetic agent.

Aim of the study

This study was aimed to investigate the inhibitory activities of fractions and constituents isolated from Gynostemma pentaphyllum on protein tyrosine phosphatase 1B (PTP1B) since it has been proposed as a treatment therapy for type 2 diabetes and obesity.

Materials and methods

The 70% EtOH extract, CHCl₃ fraction, EtOAc fraction, BuOH fraction, and seven isolated dammarane triterpenes were evaluated for their inhibitory activity in protein phosphatase enzymes (PTP1B and VHR).

Results

CHCl₃-soluble fraction showed a dose-dependent inhibitory activity of the PTP1B enzyme with the IC₅₀ value of 30.5 μg/mL. Among seven tested compounds, compounds 6 showed the most potent PTP1B inhibitory activity with IC₅₀ value of 5.3 ± 0.4 μM compared to a range 15.7–28.5 μM for the other six compounds. The inhibition mode of 6 was competitive toward p-NPP with a K_i value of 2.8 μM.

Conclusion

These study results suggested that the PTP1B inhibitory activity of these dammaranes may enable this plant to play an important role in the treatment of diabetes.     

Anticonvulsant effect of aqueous extract of Valeriana officinalis in amygdala-kindled rats: Possible involvement of adenosine

Ethnopharmacological relevance

Valeriana officinalis L. (valerian) root extract has been used as an antiepileptic herbal medicine in Iran.

Aim of this study

In the present study the effect of valerian extracts on an experimental model of temporal lobe epilepsy (TLE) was evaluated. Moreover, the involvement of adenosine system in the actions of aqueous extract of valerian was evaluated.

Materials and methods

Bipolar stimulating and monopolar recording electrodes were implanted stereotaxically in the right basolateral amygdala of male Sprague–Dawley rats. After kindling, the effect of aqueous (200, 500 and 800 mg/kg; intraperitoneal) and petroleum ether (PE; 50 and 100 mg/kg; intraperitoneal) extracts of valerian and CPT (selective A₁ receptor antagonist; 10 and 20 μM; intracerebroventricular) on afterdischarge duration (ADD), duration of stage 5 seizure (S5D) and latency to the onset of bilateral forelimb clonuses (S4L) were measured. The effect of CPT (10 μM) on the response of aqueous extract of valerian (500 mg/kg) was also determined.

Results

The results showed that aqueous extract of valerian had anticonvulsant effect. However, PE extract and CPT (20 μM) had proconvulsant effect. Administration of CPT (10 μM) before the administration of aqueous extract decreased the anticonvulsant effect of valerian.

Conclusions

The results showed significant anticonvulsant effect for aqueous but not PE extract of valerian. Moreover, CPT as a selective adenosine A₁ receptor antagonist decreased the anticonvulsant effect of valerian aqueous extract. Therefore, we concluded that part of anticonvulsant effect of valerian probably is mediated through activation of adenosine system.     

Pharmacological mechanisms underlying the vascular activities of Loranthus ferrugineus Roxb. in rat thoracic aorta

Aim of the study

The present study was aimed to investigate the pharmacological basis for the use of Loranthus ferrugineus in hypertension.

Materials and methods

Loranthus ferrugineus methanol extract (LFME) was obtained using Soxhelt extractor and then successively fractionated using chloroform, ethyl acetate and n-butanol. The n-butanol fraction of LFME (NBF-LFME) was studied using isolated rat thoracic aorta.

Results

NBF-LFME (1.0 × 10⁻⁵ to 3.0 mg/ml) was found to be the most potent to concentration-dependently relax the endothelium-intact phenyephrine (PE, 1 μM)- and high K⁺ (80 mM)-precontracted rat aortic rings. Removal of the endothelium completely abolished the vascular relaxing properties of NBF-LFME. Pretreatment with atropine (1 μM), l-NAME (10 μM), indomethacin (10 μM) and methylene blue (10 μM) significantly blocked NBF-LFME-mediated relaxation. Endothelium-dependent and -independent relaxations induced by acetylcholine (ACh) and sodium nitroprusside (SNP), respectively, were significantly enhanced in aortic rings pretreated with NBF-LFME when compared to those observed in control aortic rings. On the contrary, glibenclamide (10 μM), propranolol (1 μM) and prazosin (0.01 μM) did not alter NBF-LFME-induced relaxation.

Conclusions

The results suggest that NBF-LFME induced vascular relaxation by stimulating muscarinic receptors, activating the endothelium-derived nitric oxide-cGMP-relaxant pathway, promoting prostacyclin release and/or possibly through its ability to lengthen the released nitric oxide half-life. The present data further supports previous in vivo findings and explain the traditional use of Loranthus ferrugineus as an anti-hypertensive agent.     

In vitro biological activities of niloticane,a new bioactive cassane diterpene from the bark of Acacia nilotica subsp. kraussiana

Ethnopharmacological relevance

Acacia nilotica subsp. kraussiana was reported in African traditional medicine for the treatment of various ailments. Isolation of an active compound in this study from the bark extract may lead to the validation of its efficiency as a traditional crude drug.

Aims of the study

This study aimed to isolate active compound(s) from an ethyl acetate bark extract of Acacia nilotica subsp. kraussiana and to investigate some of its biological activity.

Materials and methods

The isolation process was carried out using bioassay-guided fractionation. The isolated compound was tested for antibacterial activity using the micro-dilution assay; anti-inflammatory activity using the COX-1 and COX-2 assays and investigated for inhibitory effect against acetylcholinesterase using the microplate assay.

Results

A new bioactive compound was isolated and identified as a cassane diterpene, niloticane. Niloticane showed antibacterial activity against Gram-positive bacteria Bacillus subtilis and Staphylococcus aureus with MIC values of 4 and 8 μg/mL, respectively. With Gram-negative bacteria, niloticane showed weak activity. MIC values obtained were 16 and 33 μg/mL against Klebsiella pneumonia and Escherichia coli, respectively. In the cyclooxygenase test, niloticane possessed activity with IC₅₀ values of 28 and 210 μM against COX-1 and COX-2, respectively. IC₅₀ values observed with indomethacin (positive control) were 3.6 μM for COX-1 and 189 μM for COX-2. In the acetylcholinesterase test, niloticane showed anti-cholinesterase activity with an IC₅₀ value of 4 μM. IC₅₀ values obtained by the galanthamine (positive control) was 2.0 μM.

Conclusion

The results obtained support the traditional uses of the bark of Acacia nilotica subsp. kraussiana in African traditional medicine for the treatment of some ailments that relate to microbial diseases, inflammation and central nervous system disorders.     

Vascular effects and antioxidant activity of two Combretum species from Democratic Republic of Congo

Ethnopharmacological relevance

Combretum racemosum P. Beauv (Combretaceae) leaves (CrLv) and root bark (CrRB) and Combretum celastroides subsp. laxiflorum Welw (Combretaceae) leaves (ClLv) are used in Congolese traditional medicine for several therapeutic purposes, notably for the treatment of conditions consistent with hypertension. The present study aims to investigate the vasorelaxant and in vitro antioxidant activities of these plants polar extracts and to examine the in vivo antihypertensive effect of the extract which displays the most potent vasorelaxant effect.

Material and methods

The vasorelaxant effect of CrLv, CrRB and ClLv methanolic extracts was studied on rat aorta rings pre-contracted with phenylephrine (PE, 1 μM) in the presence or absence of the endothelium. In some experiments, prior to the addition of the extract, rings were incubated for 30 min with either L-N^G-nitroarginine methyl ester (L-NAME; 100 μM), a nitric oxide synthase (NOS) inhibitor, indomethacin (10 μM), a cyclooxygenase inhibitor, or 1 H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10 μM), a guanylate cyclase inhibitor. The antioxidant activity was determined by the measurement of the scavenging ability of extracts towards the stable free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH). Blood pressure was measured on normotensive Wistar rats and spontaneously hypertensive rats (SHR) treated orally with a daily dose (40 mg/kg) of the CILv extract for 5 weeks. Tested extracts have been characterised by TLC profiles targeted at flavonoids.

Results

All tested extracts showed an important DPPH scavenging activity, ranging from 0.6 to 1.1 quercetin-equivalents. They caused a concentration-dependent vasorelaxation on intact aortic rings pre-contracted with PE (1 μM). The responses to CrRB and CrLv methanolic extracts reached 74.0±5.1% and 62.2±8.6% at a cumulative concentration of 50 μg/ml, respectively. The ClLv (10 μg/ml) extract was more active and, in the same conditions, relaxed aortic rings by 90.3±5.8%. The vasorelaxant activity of all extracts disappeared or was significantly attenuated by removal of the endothelium or after pretreatment with L-NAME or ODQ. Indomethacin only inhibited the activity of CrLv and CrRB extracts. The ClLv extract was able to lower the systolic blood pressure in SHR rats by 7% after a 5-week treatment.

Conclusions

The present study shows that methanolic extracts from ClLv, CrRB and CrLv have an antioxidant activity and an endothelium-dependent vasorelaxant effect. ClLv induces the vasorelaxant effect through the NO-cGMP pathway while CrLv and CrRB extracts also act via a prostanoid pathway. ClLv extract demonstrated a modest but significant antihypertensive activity in SHR rats.     

Antibacterial and antifungal activity of Flindersine isolated from the traditional medicinal plant, Toddalia asiatica (L.) Lam.

Aim of the study

The leaves and root of Toddalia asiatica (L.) Lam. (Rutaceae) are widely used as a folk medicine in India. Hexane, chloroform, ethyl acetate, methanol and water extracts of Toddalia asiatica leaves and isolated compound Flindersine were tested against bacteria and fungi.

Materials and methods

Antibacterial and antifungal activities were tested against bacteria and fungi using disc-diffusion method and minimum inhibitory concentrations (MICs). The compound was confirmed using X-ray crystallography technique.

Results

Antibacterial and antifungal activities were observed in ethyl acetate extract. One active principle Flindersine (2,6-dihydro-2,2-dimethyl-5H-pyrano [3,2-c] quinoline-5-one-9cl) was isolated from the ethyl acetate extract. The MIC values of the compound against bacteria Bacillus subtilis (31.25 μg/ml), Staphylococcus aureus (62.5 μg/ml), Staphylococcus epidermidis (62.5 μg/ml), Enterococcus faecalis (31.25 μg/ml), Pseudomonas aeruginosa (250 μg/ml), Acinetobacter baumannii (125 μg/ml) and fungi Trichophyton rubrum 57 (62.5 μg/ml), Trichophyton mentagrophytes (62.5 μg/ml), Trichophyton simii (62.5 μg/ml), Epidermophyton floccosum (62.5 μg/ml), Magnaporthe grisea (250 μg/ml) and Candida albicans (250 μg/ml) were determined.

Conclusions

Ethyl acetate extract showed promising antibacterial and antifungal activity and isolated compound Flindersine showed moderate activity against bacteria and fungi.     

Anti-inflammatory and antinociceptive activities of Campomanesia adamantium

Ethnopharmacological relevance

Campomanesia species are used in folk medicine as anti-inflammatory, anti-rheumatic, anti-diarrheal and hypocholesterolemic.

Aim of the study

The present study investigated the in vivo anti-inflammatory and antinociceptive properties of ethyl acetate (AE) and aqueous (Aq) extracts from leaves of Campomanesia adamantium and in vitro anti-inflammatory activity of AE and its isolated flavonols, myricitrin and myricetin.

Materials and methods

The antinociceptive activity of AE and Aq was evaluated using acetic acid-induced writhing and formalin methods. The in vivo anti-inflammatory effect of AE and Aq was evaluated using carrageenan-induced paw oedema in mice. AE, myricitrin and myricetin were evaluated for their abilities to modulate the production of NO, TNF-α and IL-10 in LPS/IFN-γ stimulated J774.A1 macrophages.

Results

It was found that orally administrated AE and Aq (125 and 250 mg/kg) inhibited carrageenan-induced paw oedema in mice. AE (125 and 250 mg/kg) and Aq (125 mg/kg) reduced the time to licking at the second phase of the formalin method in vivo in mice. AE (250 mg/kg) and Aq (125 mg/kg) also reduced the number of writhes. AE, myricitrin and myricetin inhibited NO (320 μg/mL and 6.25–100 μM, respectively) and TNF-α production by macrophages (320 μg/mL for AE, 100 μM for myricitrin and 25–100 μM for myricetin). AE (160 and 320 μg/mL), myricitrin (50 and 100 μM) and myricetin (25–100 μM) increased IL-10 production by macrophages.

Conclusions

The ethyl acetate and aqueous extracts from Campomanesia adamantium showed antinociceptive and anti-inflammatory effects supporting the use of the plant in folk medicine. The results suggest that anti-oedematogenic effect promoted by aqueous extract involves several anti-inflammatory mechanisms of action. The antinociceptive effect shown by aqueous extract can be due to the modulation of release of inflammatory mediators involved in nociception. The anti-inflammatory effects of AE and of its isolated flavonols may be attributed to inhibition of pro-inflammatory cytokines production, TNF-α and NO and to the increased of IL-10 production.     

Cochlospermum vitifolium induces vasorelaxant and antihypertensive effects mainly by activation of NO/cGMP signaling pathway

Aim of the study

Cochlospermum vitifolium is a medicinal plant used for the treatment of diabetes, hepatobilary and cardiovascular illnesses. The aim of current study was to determine the in vivo antihypertensive and in vitro functional vasorelaxant mechanism of methanol extract of Cochlospermum vitifolium (MECv) and naringenin (NG).

Materials and methods

Test material was assayed on rat isolated aorta rings test with- and without-endothelium to determine their vasorelaxant mechanism. Also, the in vivo antihypertensive effect was evaluated on spontaneously hypertensive rat (SHR) model. In addition, presence of NG into the extract was confirmed by reverse phase high performance liquid chromatography (RP-HPLC) analysis.

Results

MECv (120 mg/kg) and NG (50 and 160 mg/kg) showed acute antihypertensive effects on SHR when systolic and diastolic pressure were decreased at 1 h and 24 h after administration, respectively. Vasorelaxant effect of MECv and NG was shifted to the right when endothelium-intact aortic rings were pre-incubated with L-NAME (10 μM) and ODQ (1 μM). Also, NG relaxant curves were displaced to the right in the presence of tetraethylammonium (TEA, 1 mM) and 2-aminopyridine (2-AP, 100 μM) on endothelium-denuded aortic rings.

Conclusion

Experiments described above showed that MECv play an important role in hypertension regulation through NO synthesis and may be PGI₂ production and potassium channel activation on excessive endothelial dysfunction conditions. Unfortunately, presence of NG into the extract is not significant on bioactivity of the extract; however, this compound could be tested and evaluated as structural scaffold for future drug design for development of antihypertensive agents.     

Antinociceptive and anti-inflammatory kaempferol glycosides from Sedum dendroideum

Aim of the study

To identify the compounds responsible for the antinociceptive and anti-inflammatory effects previously described for Sedum dendroideum, through bioassay-guided fractionation procedures.

Materials and methods

Antinociceptive activity was evaluated through mouse acetic acid-induced writhing model. The anti-inflammatory activity was assessed through croton oil-induced mouse ear oedema and carrageenan-induced peritonitis.

Results

The Sedum dendroideum juice afforded seven known flavonoids identified with basis on NMR data. The oral administration of the major kaempferol glycosides kaempferitrin [1] (17.29 μmol/kg), kaempferol 3-O-β-glucopyranoside-7-O-α-rhamnopyranoside [2] (16.82 μmol/kg), kaempferol 3-O-neohesperidoside-7-O-α-rhamnopyranoside [3] (13.50 μmol/kg) or α-rhamnoisorobin [5] (23.13 μmol/kg) inhibited by 47.3%, 25.7%, 60.2% and 58.0%, respectively, the acetic acid-induced nociception (indomethacin: 27.95 μmol/kg, p.o.; 68.9%). Flavonoids 1, 2, 3 or 5, at the same doses, reduced by 39.5%, 46.5%, 35.6% and 33.3%, respectively, the croton oil-induced oedema (dexamethasone: 5.09 μmol/kg, s.c.; 83.7%) and impaired leukocyte migration by 42.9%, 46.3%, 50.4% and 49.6%, respectively (dexamethasone: 5.09 μmol/kg, s.c.; 66.1%).

Conclusions

Our findings show that the major kaempferol glycosides may account for the renowned medicinal use of Sedum dendroideum against pain and inflammatory troubles.     

Essential oils of Salvia bracteata and Salvia rubifolia from Lebanon: Chemical composition,antimicrobial activity and inhibitory effect on human melanoma cells

Aim of the study

Salvia bracteata Banks et Sol. and Salvia rubifolia Boiss. are known in folk medicine of Lebanon for the treatment of microbial infections, cancer, urinary and pulmonary problems. In the present study the chemical composition and antimicrobial activity of essential oils from aerial parts of Salvia bracteata and Salvia rubifolia collected in Lebanon were evaluated. The oils were also tested for their potential antiproliferative effects against M14 human melanoma cells.

Material and methods

The oils were studied by GC and GC–MS and their antibacterial activity (MIC and MBC) was tested against ten bacteria species using the broth dilution method. The inhibitory effect on human melanoma cells (measurement of cell vitality, cell membrane integrity and genomic DNA fragmentation) was studied using MTT assay, calculation of LDH release and COMET assay.

Results

The oils showed a good antibacterial activity (MIC = 50 μg/ml) against Gram+ bacteria. They besides exhibited an inhibitory effect on the human cancer cells examined inducing also apoptotic cell death, but the oil of Salvia rubifolia was significantly (p < 0.001) more active as compared to the oil of Salvia bracteata.

Conclusion

The results on the pharmacological activities of these Salvia species provide an in vitro scientific support for the use of these plants in traditional herbal preparations.