Gastroprotective activity of Cenostigma macrophyllum Tul. var. acuminata Teles Freire leaves on experimental ulcer models

Ethnopharmacological relevance

Cenostigma macrophyllum Tul. var. acuminata Teles Freire (Leguminosae-Caesalpinioideae), popularly known in Brazil as “caneleiro”, is widely used in folk medicine against gastrointestinal diseases. In previous studies, the ethanol extract of leaves from Cenostigma macrophyllum Tul. var. acuminata Teles Freire had shown antinociceptive, anti-inflammatory, antibacterial, antioxidant and antiulcerogenic activities.

Aim of the study

The aim of this study was to assess the gastroprotective effect of the hydroalcoholic fraction of leaves of Cenostigma macrophyllum Tul. var. acuminata Teles Freire (Cm-FHA), as well as to elucidate the possible underlying mechanisms of action.

Materials and methods

Mice were used for the evaluation of the acute toxicity, and mice and rats to study the gastroprotective activity. The potential gastroprotective of Cm-FHA was assessed on different gastric ulcer models in rodents, such as absolute ethanol, HCl/ethanol, ischemia-reperfusion, cold restraint stress and indomethacin. The participation of prostaglandins, NO-synthase pathway and ATP-sensitive potassium channels (K_ATP) in gastroprotective activity of Cm-FHA were evaluated after treatment with a cyclooxygenase inhibitor (indomethacin), a NO-synthase inhibitor (L-NAME) and a K_ATP channel blocker (glibenclamide 5 mg/kg), respectively. Likewise, the catalase activity was determinated in order to assess the possible participation of antioxidant mechanisms.

Results

No signs of acute toxicity was observed after oral acute administration of Cm-FHA, considering the analyzed parameters. Likewise, Cm-FHA promoted a protective effect against gastric ulcers induced by absolute ethanol (lesion inhibition by 40% at both 100 and 200 mg/kg), HCl/ethanol (lesion inhibition by 50 or 48% at 100 or 200 mg/kg, respectively), ischemia-reperfusion (lesion inhibition by 49 or 90% at 100 or 200 mg/kg, respectively) and cold restraint stress (lesion inhibition by 63 or 76% at 100 or 200 mg/kg, respectively), as well as a increase of catalase activity was observed. Otherwise, Cm-FHA was not able to protect gastric mucosa against indomethacin-induced lesions. Nitric oxide release, the of K_ATP channels opening and antioxidant activity are the possibly involved in the Cm-FHA-induced gastroprotective activity.

Conclusion

This study corroborates the folk medicine use of Cenostigma macrophyllum for treatment of gastric ulcers, as well as reinforces this species as a valuable source of promising natural drugs with gastroprotective activity.     

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.     

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.     

Effects of leaf extracts from Croton zambesicus Müell. Arg. on hemostasis

Ethnopharmacological relevance

The leaf decoction of Croton zambesicus Müell. Arg. (Euphorbiaceae; syn. Croton amabilis Müell. Arg., Croton gratissimus Burch) is traditionally used in Benin to treat hypertension.

Aim of the study

As hypertension and thromboembolism are often associated in several cardiovascular diseases, we studied the potential effects of leaf extracts from Croton zambesicus on hemostasis.

Materials and methods

We prepared the dichloromethane and aqueous extracts from the air-dried leaves of Croton zambesicus and separated the aqueous extract in its aqueous and dichloromethane fractions. The potential effects of these four extracts/fractions were investigated on red blood cells integrity using spectrophotometric lysis assays, on primary hemostasis using platelet aggregation studies and on secondary hemostasis using calibrated automated thrombin generation assays and coagulation factors inhibition tests.

Results

In the in vitro testing, we found that none of the tested extracts/fractions exhibit hemolytic or antiplatelet activity. However, they display a moderate but significant anticoagulant activity which would be mediated through the direct inhibition of thrombin, FXa and TF/FVIIa. The active anticoagulant compound(s) seem to be mainly in the aqueous extract and especially in its aqueous fraction.

Conclusions

This experimental work reported for the first time the anticoagulant effect of leaf extracts from Croton zambesicus. These findings are of particular interest as the leaves from Croton zambesicus are commonly used in infusion by local population and may provide a new natural source for the development of original anticoagulant agents. Furthermore, this activity, associated with the vasorelaxant properties of some of its diterpenes may prove to be interesting for the prevention of cardiovascular diseases in traditional medicine.     

Evidence of the anti-Helicobacter pylori, gastroprotective and anti-inflammatory activities of Cuphea aequipetala infusion

Ethnopharmacological relevance

Cuphea aequipetala (Lythraceae) is a medicinal plant highly appreciated in Mexico to treat stomach ailments such as pain and burning sensation, stomach infections, ulcers, diarrhea, dysentery, and different types of tumors and bruises. In this work, the infusion of aerial parts of this plant (CAI) was investigated for its polypharmacological potential.

Materials and methods

In vitro anti-Helicobacter pylori activity was assessed by broth dilution method. Pharmacological studies included acute toxicity in mice using Lorke´s model, anti-inflammatory activity by xylene and TPA induced ear edema assay, as well as gastroprotection with ethanol-induced gastric ulcer model. DPPH and ABTS assays were used to determine antioxidant capacity. Polyphenols and flavonoid contents were determined by Folin–Ciocalteu method and AlCl₃ reaction, respectively.

Results

CAI showed good anti-Helicobacter pylori activity with a MIC of 125 μg/mL. The infusion was not toxic according to Lorke's model with a LD₅₀ greater than 5 g/kg. CAI exhibited low anti-edematogenic action in the models assayed. Oral administration of 300 mg/kg CAI significantly reduced gastric lesions by 87.9%. The effect was reversed only by indomethacin and N-ethylmaleimide demonstrating the role of endogenous prostaglandins and sulfhydryl compounds in gastroprotection. Total phenolic and flavonoid contents of CAI were 109.9 mg GAE/g DW and 28.1 mg QE/g DW, respectively, and the infusion exhibited a good antioxidant activity that is thought to play a role in its biological activity. The analysis of a preliminary fractionation of the infusion indicates that the complete extract conserves all its pharmacological activities in contrast to fractionated extracts.

Conclusions

Cuphea aequipetala is a promising native herb in an integral therapy for the treatment of bacterial or non-bacterial gastric ulcer because it possesses some anti-inflammatory properties, as well as exhibits good gastroprotective and antibacterial effects. It represents an important source for the isolation of anti-Helicobacter pylori compounds. This work provides ethnopharmacological evidence that supports the traditional use of this species.     

The essential oil of Croton zehntneri and trans-anethole improves cutaneous wound healing

Ethnopharmacology relevance

Croton zehntneri is a Euphorbiaceae species native to northeastern Brazil, where teas and beverages made from Croton zehntneri leaves are used as healing agents. To our knowledge, there is no experimental study supporting this claim of pharmacological activity.

Materials and methods

Full-thickness excisional wounds were made in the left and right sides of the dorsum of anesthetised Swiss mice, and a topical pharmaceutical formulation, developed by including essential oil extracted from the leaves of Croton zehntneri (2% and 20% EOCz) in pluronic-127 (PF-127), was administered to mice twice daily for 15 days post-wounding. To evaluate the contribution of trans-anethole, the major constituent of EOCz (85.7%), in the wound healing activity of EOCz, the effect of the topical administration of trans-AT on wound tissue repair was also evaluated and compared to other groups. A macroscopic analysis of swelling and exudates was performed and scored as 0 (missing), 1 (light), 2 (moderate) and 3 (intense). The number of capillaries and leukocytes was counted in hematoxylin and eosin (HE)-stained sections of the injured tissue. For extracellular matrix remodelling analysis, fibroblasts and collagen fibres present in the photomicrography of the Masson’s Trichrome (MT)-stained sections were counted. Each experimental group comprised six mice.

Results

At day 3 post-wounding, it was observed that treatment with 20% EOCz greatly reduced the swelling and exudates with a similar magnitude to the dexamethasone treatment. The inflammatory cell infiltration and angiogenesis were not altered by either the EOCz- or trans-AT treatments. In contrast, an acceleration of the wound closure was observed, with an enhanced number of fibroblasts and collagen fibres in both the 20% EOCz- and trans-AT-treated mice.

Conclusion

Our data indicate that EOCz exerts significant wound healing activity, demonstrating its relevant therapeutic potential.     

芸薹子化学成分研究

对芸薹子95%乙醇提取物中醋酸乙酯萃取部位进行化学成分的分离鉴定。利用硅胶柱色谱,凝胶柱色谱,半制备液相等色谱技术共得到14个化合物,根据理化性质和波谱数据鉴定化合物的结构分别为（5Z,7E）-4,4-二甲基-5-乙酮基-5,7-壬二烯酸（1）,吲哚-3-甲醛（2）,blumenol A（3）,vinylsyringol（4）,反式芥子酸（5）,反式芥子酸乙酯（6）,原儿茶酸（7）,crinosterol （8）,菜油甾醇（9）,7-羰基豆甾醇（10）,山柰酚（11）,龙胆酸（12）,丁香酸（13）,胡萝卜苷（14）。其中,化合物 1 为新化合物,除化合物 4,5,13 外,其他化合物均为首次从该植物中分离得到。     

Pro-sexual effects of Turnera diffusa Wild (Turneraceae) in male rats involves the nitric oxide pathway

Ethnopharmacological relevance

Turnera diffusa Wild has been used in folk medicine by its aphrodisiac and tranquilizing properties. Previously we experimentally showed the aphrodisiac effect of a chemically characterized aqueous extract of Turnera diffusa in male rats. However, the mechanism of action underlying such effects has not been studied.

Study aims

As part of our systematic studies of pharmacological properties of Turnera diffusa, we aimed to analyze whether the increased sexual motivation and the augmented sexual performance of sexually sluggish (SL) male rats treated with Turnera diffusa involves the NO pathway. Additionally we analyzed whether such effects were exerted at the level of the brain or the spinal cord. Finally, anxiety levels and ambulatory activity were also evaluated.

Material and methods

Turnera diffusa (10–40 mg/kg) and sildenafil citrate (10 mg/kg) with or without a nonspecific inhibitor of NO synthase, Nω-nitro-l-arginine methyl esther (l-NAME, 12.5 mg/kg) were evaluated in SL rats, in a standard sexual behavior test and in the fictive ejaculation model in spinal cord transected and urethane-anaesthetized SL rats. Anxiety levels or ambulation were assessed in the burying behavior and open-field tests.

Results

Turnera diffusa and sildenafil (both at 10 mg/kg) facilitated expression of male sexual behavior by shortening mainly ejaculation latency. Treatments also facilitated the number of discharges in the ejaculatory motor pattern as well as the number of ejaculatory motor patterns and its associated penile erections. l-NAME prevented the pro-sexual effects of treatments on both experimental models. Besides, the extract of Turnera diffusa (10 mg/kg) produced an anxiolytic-like effect in male rats without affecting ambulation.

Conclusions

Findings from the present work support the notion that pro-sexual effect of the aqueous extract of Turnera diffusa in rats involves the participation of NO pathway, mainly at central level. The anxiolytic-like effect of Turnera diffusa is an advantage to its use for improving sexual performance.     

Flavonoid-rich fraction of Maytenus ilicifolia Mart. ex. Reiss protects the gastric mucosa of rodents through inhibition of both H+,K+ -ATPase activity and formation of nitric oxide

Maytenus ilicifolia Mart. ex. Reissek (Celastraceae), a medicinal plant known in Brazil as "espinheira-santa" is commonly used to treat gastric disorders. The effect of the flavonoid-rich fraction separated from the leaves was evaluated for its gastroprotective properties and the mechanism(s) involved in this activity. Intraperitoneal administration of the flavonoid-rich fraction potently protected rats from experimentally induced chronic (ED(50)=79 mg/kg) and acute gastric lesions by ethanol (ED(50)=25mg/kg) and indomethacin (ED(50)=4 mg/kg) without altering the decreased amount of cytoprotective glutathione and mucus amount in the injured gastric mucosa. A potent reduction of gastric acid hypersecretion (ED(50)=7 mg/kg, i.p.) was accompanied by a reduction of nitric oxide release (ED(50)=1.6 mg/kg, i.p.) in the gastric secretion of 2h pylorus ligated rats which suggests an important role for nitric oxide-dependent mechanisms. Inhibition of gastric acid secretion in vivo was correlated with the in vitro inhibition of rabbit gastric H(+),K(+)-ATPase activity (IC(50)=41 microg/mL). Chemical investigation of the fraction showed galactitol (25%), epicatechin (3.1%) and catechin (2%) as the majoritary components. Collectively, the results show that the flavonoid-rich fraction of Maytenus ilicifolia potently protects animals from gastric lesions with high potency through inhibition of gastric acid secretion.     

Byrsonima intermedia A. Juss.: gastric and duodenal anti-ulcer, antimicrobial and antidiarrheal effects in experimental rodent models

Ethnopharmacological relevance

An ethnopharmacological survey indicated that the leaves of Byrsonima intermedia A. Juss. (Malpighiaceae), a medicinal species commonly found in the Brazilian Cerrado, can be used against gastroduodenal disorders, such as gastric ulcers and diarrhea.

Aim of the study

The objective of this study was to evaluate the effects of a methanolic extract of Byrsonima intermedia (MBI) leaves on gastric and duodenal ulcers and to assess the antimicrobial and antidiarrheal effects of this extract.

Material and methods

The anti-ulcerogenic effect of MBI was investigated with different ulcerogenic agents in rodents (mice and rats), including non-steroidal anti-inflammatory drug (NSAID), HCl/ethanol, pyloric ligature, absolute ethanol, cysteamine and ischemia–reperfusion. The gastroprotective effect of MBI was assessed by analysing the volume of gastric juice, pH, total acidity, mucus, NO, sulfhydryl compound, vanilloid receptor, glutathione (GSH) levels, and myeloperoxidase (MPO) activity in the gastric and duodenal mucosa. The gastric and duodenal healing effects of MBI were also evaluated during 7 or 14 days of treatment. The antidiarrheal action (measured by intestinal motility and diarrhea induced by castor oil) and anti-bacterial action of MBI against Staphylococcus aureus, Escherichia coli and Helicobacter pylori were also evaluated by microdilution methods.

Results

The phytochemical profile from MBI indicated the presence of phenolic acids, flavan-3-ols, oligomeric proanthocyanidins, and flavonoids. MBI (500 mg/kg, p.o.) significantly inhibited totally gastric and duodenal lesions (69%) and healed gastric (49% on 14 days) and duodenal lesions (45% on 7 and 14 days). The MBI exert gastroprotective action by participation of endogenous sulfhydryl compounds, vanilloid receptors and increase in GSH level to effective gastric and duodenal protection. MBI also displayed curative (42%) and preventive (49%) antidiarrheal effects by involvement of opiate receptors and also antimicrobial effects in vitro.

Conclusions

Byrsonima intermedia leaves present gastroprotective, healing and antidiarrheal activities, supporting previous claims that its traditional use can treat gastrointestinal disorders.     

Gastroprotective effect of Cissus sicyoides (Vitaceae): involvement of microcirculation, endogenous sulfhydryls and nitric oxide

AIM OF THE STUDY: Cissus sicyoides L. is a medicinal plant popularly known in Brazil against various diseases and the research interest in this plant is justifiable because of its potential medicinal value in stomachache and gastric ulcer. MATERIALS AND METHODS: The methanolic extract obtained from the leaves of Cissus sicyoides (Cc) was evaluated for the ability to protect the gastric mucosa against injuries caused by necrotizing agents (0.3 M HCl/60% EtOH, absolute ethanol, piroxicam and pylorus ligature) in rodents. We also evaluated microcirculation, antioxidant action and participation of NO (nitric oxide) and sulfhydryls (SH) groups in the Cc gastroprotective action. RESULTS: Administration of Cc significantly reduced gastric lesions induced by different ulcerogenic agents in rodents. This extract administered by oral route significantly increased gastric volume without exerting antisecretory effect. The Cc effect involved an increase of the defense mechanism of the gastrointestinal mucosa such as NO and SH groups that prevent and attenuate the ulcer process. The Cc also has antioxidant property against oxidative stress but does not modify microcirculation response in gastric mucosa. CONCLUSIONS: These results confirmed the traditional use of Cissus sicyoides for the treatment of gastric ulcer.     

Protective effect of ethyl acetate fraction of Acacia ferruginea DC. against ethanol-induced gastric ulcer in rats

Ethnopharmacological relevance

In traditional systems of medicine, stem bark of Acacia ferruginea DC. is used for the treatment of itching, leucoderma, ulcers, stomatitis and diseases of the blood. In the present study, we determined antioxidant and anti-ulcerogenic activities of Acacia ferruginea stem bark.

Materials and methods

Acetone extract and its sub-fractions of Acacia ferruginea stem bark were subjected to assess their antioxidant potential using various in vitro systems such as DPPH^•, ABTS^•+ scavenging, FRAP and phosphomolybdenum reduction activities. Based on the antioxidant potential, the ethyl acetate fraction was used to evaluate the protective effect of ethanol-induced gastric damage in rat model. Enzyme activities such as superoxide dismutase, glutathione, catalase and lipid peroxidation were also determined in the stomach tissues.

Results

Ethyl acetate fraction (AFE) of Acacia ferruginea stem bark registered higher antioxidant and free radical scavenging activities than the crude acetone extract and other fractions. In addition, AFE exhibited that the IC₅₀ values of DPPH (2.5 µg/ml) and ABTS (1.8 µg/ml) were lower when compared to the standard quercetin (12.4 µg/ml and 4.7 µg/ml, respectively). In ethanol induced gastric ulcer, administration of AFE at doses of 10 mg/kg, 50 mg/kg and 100 mg/kg body weight prior to ethanol ingestion significantly protected the stomach ulceration. Consequently significant changes were observed in enzyme activities such as SOD, CAT, GSH and LPO in the stomach tissues when compared with ethanol control group.

Conclusion

It is concluded that the ethyl acetate fraction of Acacia ferruginea stem bark possessed higher antioxidant and anti-ulcerogenic activities. Based on the results, we suggest that Acacia ferruginea stem bark has potential to provide a therapeutic approach to ethanol mediated ulcer as an effective anti-ulcer agent.     

Hymenaea stigonocarpa Mart. ex Hayne: A Brazilian medicinal plant with gastric and duodenal anti-ulcer and antidiarrheal effects in experimental rodent models

Ethnopharmacological relevance

Hymenaea stigonocarpa Mart. ex Hayne (Fabaceae) is a medicinal species commonly found in the Brazilian savannah. The stem bark of this medicinal plant, popularly known as “jatobá-do-cerrado”, is widely used in tea form to treat gastric pain, ulcers, diarrhoea and inflammation, whereas its fruits pulp is edible.

Aim of the study

The aim of this study was to investigate the antidiarrheal and anti-ulcer effects of a methanolic extract derived from the stem bark (MHs) and diet with fruit pulp of H. stigonocarpa.

Materials and methods

The antidiarrheal action of MHs was measured against the intestinal motility and diarrhoea induced by castor oil in mice. The preventive action of MHs (50, 100, 150 and 200 mg/Kg, by oral route (p.o.)) against peptic ulcers was evaluated in experimental rodent models challenged with absolute ethanol, non-steroidal anti-inflammatory drugs (NSAIDs), ischemia–reperfusion (I/R) (200 mg/Kg, p.o.) and cysteamine (200 mg/Kg, p.o.). The main anti-ulcer mechanisms of action of MHs were analysed as follows: evaluation of the gastric juice parameters, assessment of mucus adherence to the gastric wall, determination of the role of nitric oxide (NO) and sulfhydryl compounds (SH), glutathione (GSH) levels and myeloperoxidase (MPO) activity. The healing effects from MHs (200 mg/Kg) and diet with fruit pulp (10%) against gastric and duodenal ulcers induced by acetic acid were also evaluated by treating rats over 7 or 14 consecutive days of treatment.

Results

The phytochemical profile of MHs and fruit pulp indicated the presence of phenolic compounds (mainly flavonoids and condensed tannins). MHs (200 mg/Kg, p.o.) displayed an antidiarrheal effect and were able to protect gastric mucosa against absolute ethanol (68% protection) and also against the injurious effect of NSAIDs (86% protection) when compared to the group treated with vehicle. These results were accompanied by the prevention of GSH depletion and an inhibition of MPO activity when compared to animals treated with vehicle (P<0.05). MHs markedly protected duodenal mucosa against injuries caused by cysteamine (98%) and also against I/R induced gastric ulceration (80%) when compared to the group treated with vehicle. Furthermore, MHs also prevented the GSH depletion of gastric mucosa relative to the control group treated with vehicle. NO appeared to be involved in this gastroprotective effect. MHs and diet with fruit pulp clearly demonstrated gastric healing actions after treatment for 7 (MHs – 53% inhibition) or 14 days (MHs – 60% inhibition and fruit pulp – 61% inhibition). Treatment with diet with fruit pulp for 7 days demonstrates a significant duodenal healing effect (71% inhibition) without any signs of toxicity.

Conclusions

MHs clearly demonstrate antidiarrheal, gastroprotective and cicatrising effects in experimental gastric and duodenal ulcers, and the diet with fruit pulp displays duodenal healing effects. The observed effects may be associated with the antioxidant effect, which may be due the presence of condensed tannins and flavonoids in the bark and fruit of H. stigonocarpa.     

Evaluation of the antinociceptive effect of Rosmarinus officinalis L. using three different experimental models in rodents

The rationale of this investigation was to examine the antinociceptive effect of an ethanol extract of Rosmarinus officinalis (RO) aerial parts, using three different experimental models: acetic acid-induced writhing test and formalin test in mice; and a model of arthritic pain: “pain-induced functional impairment model in the rat (PIFIR model)”. The antinociceptive efficacies were evaluated using several dose–response curves and time courses. The antinociceptive effects from RO extract were compared with the antinociceptive effect of either tramadol (TR: 3.16–50 mg/kg, i.p. in mice, and 1.0–31.62 mg/kg, i.p. in rats) or acetylsalicylic acid (AA: 31.62–562.32 mg/kg, p.o.). RO extract (10–300 mg/kg, p.o.) significantly (P < 0.001) reduced the number of writhing movement induced by the i.p. administration of acetic acid solution in a dose-dependent way (ED₅₀ = 108.84 mg/kg, whereas, TR showed an ED₅₀ = 12.38 mg/kg). In addition, RO extract (30–300 mg/kg) significantly (P < 0.001) inhibited licking and shaking behaviours in both early (neurogenic pain) and in the late (inflammatory pain) phases of the formalin test. These effects were like those produced by TR. Concerning the results using the PIFIR model, RO extract (30–3000 mg/kg, p.o.) like either TR or AA, produced a significant (P < 0.001) and dose-dependent antinociceptive response in rats (RO: ED₅₀ = 222.78 mg/kg versus TR: ED₅₀ = 11.06 mg/kg and AA: ED₅₀ = 206.13 mg/kg). These results strongly suggest that aerial parts of RO possess antinociceptive and anti-inflammatory activity, and reinforce the use of this plant in folk medicine.     

Gastroprotective effects of the essential oil of Hyptis crenata Pohl ex Benth. on gastric ulcer models

Ethnopharmacological relevance

Hyptis crenata Pohl ex Benth (Lamiaceae), popularly known as “hortelã-brava” or “hortelã do campo”, is widely distributed in the northeast of Brazil. In Brazil, the leaves of this plant have been used in traditional medicine for the treatment of gastrointestinal disturbances, including gastric ulcers. In an attempt to experimentally validate this claimed antiulcerogenic activity, the gastroprotective effects of the essential oil extracted from the leaves of the Hyptis crenata Pohl ex Benth (EOHc) were evaluated in recognized gastric ulcer models in mice.

Materials and methods

EOHc was obtained by hydrodistillation and analyzed using gas chromatography–mass spectrometry (GC–MS). Swiss male mice (25–30 g) were used for the studies. The gastric ulcers were induced by oral administration of absolute ethanol or indomethacin 45 min after oral pretreatment with EOHc, vehicle and positive control drugs. One hour after the ulcerative challenges, the stomachs were removed and the area of the lesions was measured. The volume, pH and total acidity of the gastric secretions were determined using the pylorus ligature model. The gastrointestinal motility was measured using gastric emptying and intestinal transit. The ethanol-induced gastric mucus depletion and lipid peroxidation were also analyzed.

Results

Our findings are as follows: A significant inhibition of gastric lesions induced by absolute ethanol was observed in the mice pre-treated with EOHc, at a dose of 30 and 100 and 300 mg/kg (5.56±1.51, 2.88±0.82 and 1.71±0.54 mm², respectively) compared to control group (118.03±35.4 mm²). Also, EOHc (300 mg/kg) produced a gastroprotective effect against the gastric lesions induced by indomethacin (16.07±4.68 mm²) compared to control group (38.64±6.1 mm²). EOHc pretreatment produced a reduction in the ethanol-induced lipid peroxidation from 3.9±0.22 to 2.4±0.1 μmol/mg tissue (EOHc-300 mg/kg and control group, respectively). We also observed that EOHc pretreatment decreased the gastric emptying, but did not alter the intestinal transit ratio, ethanol-induced depletion of the gastric wall mucus or secretion parameters (volume, pH and [H⁺]).

Conclusion

Our data indicate that EOHc exerts a gastroprotective effect, indicated by its significant inhibition of gastric lesions in ethanol- and indomethacin-induced ulcer models, which may be associated with its accelerating effect on gastric emptying and reduction in oxidative damages. Our data suggest a potential therapeutic application for EOHc in the treatment of gastric ulcers.     

Gastroprotective activity of Zanthoxylum rhoifolium Lam. in animal models

Ethnopharmacological relevance

The stem barks of Zanthoxylum rhoifolium Lam. (Rutaceae), locally known as “mamica de cadela”, are popularly used in dyspepsies, stomachic, tonic, antitumoral, antipyretic and are used in treating flatulence and colic. The objective of this study was to evaluate the gastroprotective effect of the ethanolic extract of Zanthoxylum rhoifolium (EEZR) stem barks in acute gastric lesion models, investigating their possible mechanisms.

Materials and methods

Mice were used for the evaluation of the acute toxicity, and mice and rats to study the gastroprotective activity. The gastroprotective action of EEZR was analyzed in the absolute ethanol, HCl/ethanol and indomethacin-induced gastric lesion models in mice, hypothermic-restraint stress, and ischemia/reperfusion in rats. In the investigation of the gastroprotective mechanisms of EEZR, the participation of the NO-synthase pathway, ATP-sensitive potassium channels (K_ATP), the levels of the non-protein sulfhydril groups (NP-SH) and the catalase activity using the ethanol-induced gastric mucosa lesion model and the quantification of the gastric mucus and the antisecretory activity through pylorus ligature model in rats were analyzed.

Results

The animals did not present any signs of acute toxicity for the EEZR (up to the 4 g/kg dose, po), and it was not possible to calculate the DL₅₀. EEZR (125-500 mg/kg) exhibited a significant gastroprotective effect in absolute ethanol, HCl/ethanol, hypothermic-restraint stress, and ischemia/reperfusion-induced gastric lesion models. EEZR (250 and 500 mg/kg) exhibited still a gastroprotective activity in the indomethacin-induced ulcer model. Gastroprotection of EEZR was significantly decreased in pre-treated mice with l-NAME or glibenclamide, the respective nitric oxide synthase and K_ATP channels inhibitors. Our studies revealed that EEZR (500 mg/kg) prevented the decrease of the non-protein sulfhydril groups (NP-SH) and increased the catalase levels in ethanol-treated animals. Furthermore, the extract (500 mg/kg) significantly increased the mucus production, however, the gastric secretion parameters (volume, [H⁺], pH) did not show any alteration.

Conclusions

Our results indicate that the ethanolic extract of Zanthoxylum rhoifolium exhibits a significant gastroprotection, because it inhibits the formation of gastric lesions using different models. The release of the nitric oxide, the opening of the K_ATP channels, the participation of the non-protein sulfhydril groups (NP-SH), catalase and the increase of mucous secretion seem to be involved in the gastroprotection activity of the EEZR. Nevertheless, this activity does not seem to be related to antisecretory mechanisms.     

Involvement of heme oxygenase-1 in the anti-inflammatory activity of Chrysanthemum boreale Makino extracts on the expression of inducible nitric oxide synthase in RAW264.7 macrophages

Aim of the study

This study is to elucidate the involvement of anti-inflammatory heme oxygenase-1 (HO-1) in the inhibitory activity of a Chrysanthemum boreale Makino (CB) extract on nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages.

Materials and methods

Cell viability and NO assay were performed. In addition, iNOS expression was detected by Western blotting and real-time PCR. HO-1 expression was also evaluated by Western blotting, and blocking HO-1 activity on NO production was performed.

Results

The CB extract at the highest concentration (100 μg/ml) significantly inhibited NO production by approximately 90% and suppressed iNOS protein expression by approximately 84.8% compared to LPS-stimulated cells. Furthermore, the CB extract (100 μg/ml) inhibited iNOS mRNA expression in a concentration-dependant manner and suppressed iNOS mRNA expression by 94.8%. The CB extract induced the expression of HO-1 in a dose-dependent manner, and blocking HO-1 activity abolished the inhibitory effects of the CB extract. Moreover, the addition of carbon monoxide such as tricarbonyl dichlororuthenium (II) dimmer (RuCO), a byproduct derived from heme degradation, mimicked the inhibitory action of low concentrations of CB extract.

Conclusion

These results suggest that a CB extract has potent anti-inflammatory activity in RAW264.7 macrophages involving the induction of HO-1.     

iNOS在血管再狭窄大鼠的表达及黄芪当归的作用

目的:观察iNOS在血管再狭窄大鼠的表达变化及黄芪、当归的作用。方法:建立内皮剥脱后血管再狭窄动物模型,随机分为对照组、模型组、黄芪治疗组、当归治疗组、黄芪当归联合治疗组;治疗组在造模后给药,连续给药21 d,应用组织形态学、免疫组化的方法分别检测观察术后3,7,14,21 d iNOS在增生内膜中表达变化,并观察中药黄芪、当归注射液单独及联合应用对iNOS表达的影响。结果:正常主动脉内膜和中膜均检测到极少量iNOS表达;术后3 d大鼠主动脉壁内膜iNOS表达增强;7～21 d随着损伤时间延长及内膜的逐渐增厚,增生内膜iNOS阳性颗粒的数量增多,颜色变深,21 d达峰值;与模型组比较,黄芪、当归治疗21 d后,iNOS棕色颗粒明显减少,颜色变浅,以联合应用组作用更为明显。结论:iNOS表达参与内皮损伤后血管再狭窄过程,黄芪、当归可抑制血管内膜损伤后引起的血管内膜增生,其机制可能与抑制iNOS表达有关。     

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.