José Roberto Santin

Gastroprotective activity of essential oil of the Syzygium aromaticum and its major component eugenol in different animal models

Syzygium aromaticum, a medicinal plant commonly known as clove, is used to treat toothache, respiratory disorders, inflammation, and gastrointestinal disorders. From the flower buds of S. aromaticum, it is possible to obtain an essential oil comprised of a mixture of aliphatic and cyclic volatile terpenes and phenylpropanoids, being eugenol as the main component. The aims of this study were: (1) to extract the essential oil of the flower buds of S. aromaticum, (2) to identify and quantify the main component of the essential oil, and (3) to evaluate its antiulcer activity using different animal models. Assays were performed using the following protocols in rats: indomethacin-induced and ethanol/HCl-induced ulcer model. Both essential oils from S. aromaticum and eugenol displayed antiulcer activities in the rat models of indomethacin- and ethanol-induced ulcer. Studies focusing on the possible mechanisms of gastroprotection were also undertaken using the following experiments: evaluation of gastric secretion by the pylorus-ligated model, determination of mucus in gastric content, participation of nitric oxide (NO) and endogenous sulfhydryl in gastric protection. The results show that there was no significant effect on the volume of gastric juice and total acidity. However, the quantification of free gastric mucus showed that the clove oil and eugenol were capable of significantly enhancing mucus production. With regard to the NO and endogenous sulfhydryls, the results demonstrated that the gastroprotection induced by clove oil and eugenol are not related to the activities of the nitric oxide and endogenous sulfhydryls. No sign of toxicity was observed in the acute toxicity study. In conclusion, the results of this study show that essential oil of S. aromaticum, as well as its main component (eugenol), possesses antiulcer activity. The data suggest that the effectiveness of the essential oil and eugenol is based on its ability to stimulate the synthesis of mucus, an important gastroprotective factor. However, further pharmacological and toxicological investigations are required to enable its use for the treatment of gastric ulcer.

Gastroprotective activity of methanol extract and marrubiin obtained from leaves of Marrubium vulgare L. (Lamiaceae)

Objectives  The purpose of this study was to assess the gastroprotective properties of the methanol extract and the diterpene marrubiin obtained from the leaves of M. vulgare.

Gastroprotective activity of hydroalcoholic extract obtained from the leaves of Brassica oleracea var. acephala DC in different animal models

ETHNOPHARMACOLOGICAL RELEVANCE Brassica oleracea var. acephala DC has been extensively used in Brazilian traditional medicine to treat gastric ulcer. AIM OF THE STUDY This study was conducted to evaluate the antiulcerogenic property of hydroalcoholic extract obtained from the leaves of Brassica oleracea. MATERIALS AND METHODS Antiulcer assays were performed using the protocol of ulcer induced by ethanol/HCl, and non-steroidal anti-inflammatory drugs (NSAIDs). Parameters of gastric secretion (volume, pH and [H(+)]) were determined by the pylorus ligation model and mucus in gastric contents. RESULTS In the ethanol-induced ulcer model, we observed a significant reduction in all the parameters analyzed, obtaining curative ratios of 58.8 ± 11.5, 86.2 ± 12.2 and 42.8 ± 6.6% for the groups treated with 50 and 100mg/kg of extract and omeprazole (30 mg/kg), respectively. The dose of 25mg/kg of hydroalcoholic extract of Brassica oleracea showed no significant results. In the indomethacin-induced ulcer, the percentages of ulcer inhibition were 64.3 ± 9.9, 66.4 ± 12.3 and 81.2 ± 7.5% for the groups treated with 50 and 100mg/kg extract and positive control (cimetidine, 100mg/kg), respectively. The results showed a significant increase in pH and mucus production in the groups treated with Brassica oleracea when compared with the control group. No sign of toxicity was observed in the acute toxicity study. CONCLUSIONS The results of the present study show that hydroalcoholic extract of Brassica oleracea displays antiulcer activity, as demonstrated by the significant inhibition of ulcer formation induced using different models. The data suggest that the effectiveness of the extract is based on its ability to stimulate the synthesis of mucus, increase pH and decrease H(+) ions in the stomach. This work corroborates the ethnopharmacology use of Brassica oleracea preparations, contributing to its pharmacological validation by suggesting that preparations obtained from Brassica oleracea could be used for the development of new phytopharmaceuticals for the treatment of gastric ulcer.

Evidence of gastric ulcer healing activity of Maytenus robusta Reissek: In vitro and in vivo studies.

ETHNOPHARMACOLOGICAL RELEVANCE Maytenus robusta Reissek (Celastraceae) is traditionally used in Brazilian folk medicine to treat gastric ulcer, as a substitute for M. ilicifolia, which is almost extinct. The gastroprotective properties of M. robusta were demonstrated previously using only preventive approaches, such as acute gastric ulcer models. However, the healing effect of M. robusta in gastric ulcers remains unclear. AIM OF THE STUDY The current study was carried out to investigate the healing effectiveness of M. robusta hydroalcoholic extract (HEMR) from aerial parts in the acetic acid-induced chronic ulcer model and to determine its effect on cell proliferation, scavenging free radicals, and inflammatory and oxidative damage. MATERIAL AND METHODS To evaluate the healing properties of HEMR in vivo, chronic gastric ulcer was induced in rats by 80% acid acetic. Next, different groups of animals (n=6) were treated orally with vehicle (water plus 1% tween, 1 ml/kg), omeprazole (20mg/kg), or HEMR (1-10mg/kg), twice daily for 7 days. At the end of the treatment, the total ulcer area (mm(2)) was measured and a sample of gastric tissue was taken for histological and histochemical analysis. Evaluation of GSH and LOOH levels, GST, SOD, CAT and MPO activity was also performed at the site of the lesion. In parallel, radical scavenging activity, cytoprotective effect, and cell proliferation activity in fibroblasts (L929 cells) were determined by in vitro trials. The antisecretory properties were evaluated using the pylorus ligature model in rats, and the anti-Helicobacter pylori activity was determined in vitro. Acute toxicity was evaluated by relative organ weight and biochemical parameters in serum. The prokinetic properties were also evaluated in mice. RESULTS Oral administration of HEMR (10mg/kg) reduced the gastric ulcer area by 53%, compared to the vehicle group (120.0 ± 8.3mm(2)), the regeneration of gastric mucosa was evidenced in histological analysis. Moreover, HEMR treatment increased gastric mucin content and reduced oxidative stress and inflammatory parameters at the site of the ulcer. In vitro, HEMR (1-1000 µg/ml) was able to scavenge free radical DPPH and promote cytoprotection against H2O2 in fibroblasts at 0.1-100 µg/ml. Moreover, HEMR healing properties also were confirmed by enhancement of proliferation and coverage of scratched wounds in fibroblast monolayer. However, HEMR (10mg/kg) by the intraduodenal route did not promote changes in volume, pH, total acidity or pepsin activity in the pylorus ligature model, and HEMR up to 2000 µg/ml also did not present considerable activity against H. pylori. In relation to gastrointestinal motility, HEMR (10mg/kg, p.o) did not provoke alterations. It is also important to mention that oral administration of HEMR did not produce any sign of acute toxicity in animals. CONCLUSIONS The data here obtained show that M. robusta has evident ulcer healing potential, mainly through the strengthening of protective factors of gastric mucosa, such as mucus layer, antioxidant defenses and cell proliferation. Taking into account the advantages of cultivation and harvesting of M. robusta compared to M. ilicifolia, and the evidence presented here, it is plausible to conclude that hydroalcoholic extract obtained from aerial parts of M. robusta is an interesting source for the development of a phytotherapeutic formulation to treat gastric ulcer.

Role of an Indole-Thiazolidine Molecule PPAR Pan-Agonist and COX Inhibitor on Inflammation and Microcirculatory Damage in Acute Gastric Lesions

The present study aimed to show the in vivo mechanisms of action of an indole-thiazolidine molecule peroxisome-proliferator activated receptor pan-agonist (PPAR pan) and cyclooxygenase (COX) inhibitor, LYSO-7, in an ethanol/HCl-induced (Et/HCl) gastric lesion model. Swiss male mice were treated with vehicle, LYSO-7 or Bezafibrate (p.o.) 1 hour before oral administration of Et/HCl (60%/0.03M). In another set of assays, animals were injected i.p. with an anti-granulocyte antibody, GW9962 or L-NG-nitroarginine methyl ester (L-NAME) before treatment. One hour after Et/HCl administration, neutrophils were quantified in the blood and bone marrow and the gastric microcirculatory network was studied in situ. The gastric tissue was used to quantify the percentage of damaged area, as well as myeloperoxidase (MPO), inducible nitric oxide synthase (iNOS), endothelial nitric oxide synthase (eNOS) protein and PPARγ protein and gene expression. Acid secretion was evaluated by the pylorus ligation model. LYSO-7 or Bezafibrate treatment reduced the necrotic area. LYSO-7 treatment enhanced PPARγ gene and protein expression in the stomach, and impaired local neutrophil influx and stasis of the microcirculatory network caused by Et/HCl administration. The effect seemed to be due to PPARγ agonist activity, as the LYSO-7 effect was abolished in GW9962 pre-treated mice. The reversal of microcirculatory stasis, but not neutrophil influx, was mediated by nitric oxide (NO), as L-NAME pre-treatment abolished the LYSO-7-mediated reestablishment of microcirculatory blood flow. This effect may depend on enhanced eNOS protein expression in injured gastric tissue. The pH and concentration of H+ in the stomach were not modified by LYSO-7 treatment. In addition, LYSO-7 may induce less toxicity, as 28 days of oral treatment did not induce weight loss, as detected in pioglitazone treated mice. Thus, we show that LYSO-7 may be an effective treatment for gastric lesions by controlling neutrophil influx and microcirculatory blood flow mediated by NO.

Achyrocline satureioides (Lam.) D.C. Hydroalcoholic Extract Inhibits Neutrophil Functions Related to Innate Host Defense

Achyrocline satureioides (Lam.) D.C. is a herb native to South America, and its inflorescences are popularly employed to treat inflammatory diseases. Here, the effects of the in vivo actions of the hydroalcoholic extract obtained from inflorescences of A. satureioides on neutrophil trafficking into inflamed tissue were investigated. Male Wistar rats were orally treated with A. satureioides extract, and inflammation was induced one hour later by lipopolysaccharide injection into the subcutaneous tissue. The number of leukocytes and the amount of chemotactic mediators were quantified in the inflammatory exudate, and adhesion molecule and toll-like receptor 4 (TLR-4) expressions and phorbol-myristate-acetate- (PMA-) stimulated oxidative burst were quantified in circulating neutrophils. Leukocyte-endothelial interactions were quantified in the mesentery tissue. Enzymes and tissue morphology of the liver and kidney were evaluated. Treatment with A. satureioides extract reduced neutrophil influx and secretion of leukotriene B4 and CINC-1 in the exudates, the number of rolling and adhered leukocytes in the mesentery postcapillary venules, neutrophil L-selectin, β2-integrin and TLR-4 expression, and oxidative burst, but did not cause an alteration in the morphology and activities of liver and kidney. Together, the data show that A. satureioides extract inhibits neutrophil functions related to the innate response and does not cause systemic toxicity.

The therapeutic lead potential of metabolites obtained from natural sources for the treatment of peptic ulcer

For over a century, ulcer has been a major cause of morbidity and mortality. Its treatment has progressed from vagotomy to proton pump inhibitors. However, the drugs used produce many adverse effects and are less effective than they ought to be. Therefore, there is a growing interest in alternative therapies and the use of natural products. This review emphasizes recent studies involving naturally occurring antiulcer metabolites, categorized according to their chemical structure. Both terrestrial and marine sources are included. More than a hundred and fifty different compounds are presented, and where possible, their main mechanisms of action are summarized. Considering that Helicobacter pylori is an important causal factor in the pathogenesis of ulcer disease, an overview of some natural compounds with anti-H. pylori activity is presented.

Chemical synthesis, docking studies and biological effects of a pan peroxisome proliferator-activated receptor agonist and cyclooxygenase inhibitor

The compound (5Z)-5-[(5-bromo-1H-indol-3-yl)methylene]-3-(4-chlorobenzyl)-thiazolidine-2,4-dione (LYSO-7) was synthesised in order to obtain a new type of anti-inflammatory drug, designed with hybrid features to inhibit cyclooxygenase (COX) and also to activate peroxisome proliferator-activated receptor (PPAR). Results obtained from docking (in silico) studies corroborated with experimental data, showing the potential affinity between the studied ligand and targets. The specificity of LYSO-7 for COX-enzymes was detected by the inhibition of COX-1 and COX-2 activities by 30% and 20%, respectively. In transactivation reporter gene assays LYSO-07 showed a pan partial agonist effect on the three PPAR subtypes (PPARγ, PPARα and PPARβ/δ). The agonist action on PPARγ was also observed by a pharmacological approach, as the reduction in the Escherichia coli lipopolysaccharide (LPS)-induced interleukin 1 beta (IL-1β) secretion and nitric oxide (NO) production by mouse neutrophils was blocked by GW9962, a specific PPARγ antagonist. Additionally, the in vivo effect was measured by reduced carrageenan-induced neutrophil influx into the subcutaneous tissue of mice. Taken together, these data show that LYSO-7 displays a potent in vivo anti-inflammatory effect during the innate acute response, which is dependent on its associated COX inhibitory activities and PPAR activation.

Aqueous extract of Rosmarinus officinalis L. inhibits neutrophil influx and cytokine secretion.

Rosmarinus officinalis L. phenolic compounds have attracted considerable attention because of their antioxidant and antimicrobial properties, including its ability to treat inflammatory disorders. In this work, we investigated the in vivo and in vitro effects of R. officinalis aqueous extract on neutrophil trafficking from the blood into an inflamed tissue, on cell‐derived secretion of chemical mediators, and on oxidative stress. Anti‐inflammatory activity was investigated using carrageenan‐induced inflammation in the subcutaneous tissue of male Wistar rats orally treated with the R. officinalis extract (100, 200, or 400 mg/kg). The leukocyte influx (optical microscopy), secretion of chemical mediators (prostaglandin E2 (PGE2), TNF‐α, interleukin 6 (IL‐6), leukotriene B4 (LTB4), and cytokine‐induced neutrophil chemoattractant 1 by enzyme‐linked immunosorbent assay), and the anti‐oxidative profile (super oxide dismutase (SOD), glutathione peroxidase, and thiobarbituric acid reactive substance (TBARS) spectrophotometry) were quantified in the inflamed exudate. N‐Formyl‐methionine‐leucine‐phenylalanine‐induced chemotaxis, lipopolysaccharide‐induced NO2− production (Greiss reaction), and adhesion molecule expression (flow cytometry) were in vitro quantified using oyster glycogen recruited peritoneal neutrophils previous treated with the extract (1, 10, or 100 µg/mL). Animals orally treated with phosphate‐buffered saline and neutrophils incubated with Hanks balanced salt solution were used as control. R. officinalis extract oral treatment caused a dose‐dependent reduction in the neutrophil migration as well as decreased SOD, TBARS, LTB4, PGE2, IL‐6, and TNF‐α levels in the inflamed exudate. In vitro treatment with R. officinalis decreased neutrophil chemotaxis, NO2− production, and shedding of L‐selectin and β2 integrin expressions. Results here presented show that R. officinalis aqueous extract displays important in vivo and in vitro anti‐inflammatory actions by blocking pathways of neutrophil migration and secretion, suggesting its therapeutic application to acute inflammatory reactions. Copyright

Annexin A1 Is a Physiological Modulator of Neutrophil Maturation and Recirculation Acting on the CXCR4/CXCL12 Pathway

Neutrophil production and traffic in the body compartments is finely controlled, and the strong evidences support the role of CXCL12/CXCR4 pathway on neutrophil trafficking to and from the bone marrow (BM). We recently showed that the glucocorticoid‐regulated protein, Annexin A1 (AnxA1) modulates neutrophil homeostasis and here we address the effects of AnxA1 on steady‐state neutrophil maturation and trafficking. For this purpose, AnxA1−/− and Balb/C wild‐type mice (WT) were donors of BM granulocytes and mesenchymal stem cells and blood neutrophils. In vivo treatments with the pharmacological AnxA1 mimetic peptide (Ac2‐26) or the formyl peptide receptor (FPR) antagonist (Boc‐2) were used to elucidate the pathway of AnxA1 action, and with the cytosolic glucocorticoid antagonist receptor RU 38486. Accelerated maturation of BM granulocytes was detected in AnxA1−/− and Boc2‐treated WT mice, and was reversed by treatment with Ac2‐26 in AnxA1−/− mice. AnxA1 and FPR2 were constitutively expressed in bone marrow granulocytes, and their expressions were reduced by treatment with RU38486. Higher numbers of CXCR4+ neutrophils were detected in the circulation of AnxA1−/− or Boc2‐treated WT mice, and values were rescued in Ac2‐26‐treated AnxA1−/− mice. Although circulating neutrophils of AnxA1−/− animals were CXCR4+, they presented reduced CXCL12‐induced chemotaxis. Moreover, levels of CXCL12 were reduced in the bone marrow perfusate and in the mesenchymal stem cell supernatant from AnxA1−/− mice, and in vivo and in vitro CXCL12 expression was re‐established after Ac2‐26 treatment. Collectively, these data highlight AnxA1 as a novel determinant of neutrophil maturation and the mechanisms behind blood neutrophil homing to BM via the CXCL12/CXCR4 pathway. J. Cell. Physiol. 231: 2418–2427, 2016.