Francisca Beatriz M. Sousa

Phytol, a diterpene alcohol, inhibits the inflammatory response by reducing cytokine production and oxidative stress

Studies have shown that diterpenes have anti‐inflammatory and redox‐protective pharmacological activities. The present study aimed to investigate the anti‐inflammatory properties of phytol, a diterpene alcohol, in a mouse model of acute inflammation, and phytol effect on leukocyte recruitment, cytokines levels, and oxidative stress. The anti‐inflammatory activities of phytol were assessed by measuring paw edema induced by different inflammatory agents (e.g., λ‐carrageenan, compound 48/80, histamine, serotonin, bradykinin, and prostaglandin E2 [PGE2]), myeloperoxidase (MPO) activity, peritonitis model and cytokine levels. Further, oxidative stress was evaluated by determining glutathione (GSH) levels and malondialdehyde (MDA) concentration. The results showed that phytol (7.5, 25, 50, and 75 mg/kg) significantly reduced carrageenan‐induced paw edema, in a dose‐dependent manner. In addition, phytol (75 mg/kg) inhibited compound 48/80‐, histamine‐, serotonin‐, bradykinin‐ and PGE2‐induced paw edema. It also inhibited the recruitment of total leukocytes and neutrophils; decreased MPO activity, tumor necrosis factor‐α (TNF‐α) and interleukin‐1β (IL‐1β) levels, and MDA concentration; and increased GSH levels during carrageenan‐induced acute inflammation. These results suggest that phytol attenuates the inflammatory response by inhibiting neutrophil migration that is partly caused by reduction in IL‐1β and TNF‐α levels and oxidative stress.

Carvacryl acetate, a derivative of carvacrol, reduces nociceptive and inflammatory response in mice

AIMS The present study aimed to investigate the potential anti-inflammatory and anti-nociceptive effects of carvacryl acetate, a derivative of carvacrol, in mice. MAIN METHODS The anti-inflammatory activity was evaluated using various phlogistic agents that induce paw edema, peritonitis model, myeloperoxidase (MPO) activity, pro and anti-inflammatory cytokine levels. Evaluation of antinociceptive activity was conducted through acetic acid-induced writhing, hot plate test, formalin test, capsaicin and glutamate tests, as well as evaluation of motor performance on rotarod test. KEY FINDINGS Pretreatment of mice with carvacryl acetate (75 mg/kg) significantly reduced carrageenan-induced paw edema (P<0.05) when compared to vehicle-treated group. Likewise, carvacryl acetate (75 mg/kg) strongly inhibited edema induced by histamine, serotonin, prostaglandin E2 and compound 48/80. In the peritonitis model, carvacryl acetate significantly decreased total and differential leukocyte counts, and reduced levels of myeloperoxidase and interleukin-1 beta (IL-1β) in the peritoneal exudate. The levels of IL-10, an anti-inflammatory cytokine, were enhanced by carvacryl acetate. Pretreatment with carvacryl acetate also decreased the number of acetic acid-induced writhing, increased the latency time of the animals on the hot plate and decreased paw licking time in the formalin, capsaicin and glutamate tests. The pretreatment with naloxone did not reverse the carvacryl acetate-mediated nociceptive effect. SIGNIFICANCE In conclusion, the current study demonstrated that carvacryl acetate exhibited anti-inflammatory activity in mice by reducing inflammatory mediators, neutrophil migration and cytokine concentration, and anti-nociceptive activity due to the involvement of capsaicin and glutamate pathways.

Antidiarrheal activity of cashew GUM, a complex heteropolysaccharide extracted from exudate of Anacardium occidentale L. in rodents.

ETHNOPHARMACOLOGICAL RELEVANCE Anacardium occidentale L. (Anacardiaceae) is commonly known as the cashew tree. It is native to tropical America and extracts of the leaves, bark, roots, chestnut net and exudate have been traditionally used in northeast Brazil for the treatment of various diseases. The exudate of the cashew tree (cashew gum) has been exploited by locals since ancient times for multiple applications, including the treatment of diarrheal diseases. AIM OF THE STUDY The primary aim of the present study is to evaluate the antidiarrheal activity of cashew gum (CG), a complex heteropolysaccharide from the exudate of the cashew tree, using various models. MATERIALS AND METHODS The antidiarrheal activity of cashew gum (CG) against acute diarrhea was investigated using the castor oil-induced diarrhea model. The effects of CG on gastrointestinal transit and castor oil- and PGE2- induced enteropooling were also examined in rodents. In addition, the effect of CG against secretory diarrhea was investigated using a model of fluid secretion in cholera toxin-treated intestinal closed loops in live mice. RESULTS Cashew gum (30, 60, and 90 mg/kg, p.o.) showed a significant (P<0.05-0.01) antidiarrheal effect in rats with castor oil-induced diarrhea, inhibiting the total amount of stool and diarrheal stools. The 60 mg/kg dose of CG exhibited excellent antidiarrheal activity and significantly reduced the severity of diarrhea (diarrhea scores) in rats. CG (60 mg/kg) significantly (P<0.05) decreased the volume of castor oil- and PGE2-induced intestinal fluid secretion (enteropooling). In addition, similar to loperamide (standard drug, 5 mg/kg, p.o.), CG treatment reduced the distance traveled by a charcoal meal in the 30-min gastrointestinal transit model by interacting with opioid receptors. In cholera toxin-induced secretory diarrhea, CG (60 mg/kg) significantly inhibited the intestinal fluid secretion and decreased Cl(-) ion loss in the cholera toxin(-)treated isolated loops model of live mice by competitively binding to cholera toxin-GM1 receptors. CONCLUSIONS In conclusion, our results indicate that a complex heteropolysaccharide extracted from the exudate of A. occidentale L. has antidiarrheal activity in acute, inflammatory, and secretory diarrhea models, which could justify its traditional use in the treatment of diarrhea in northeast Brazil. The antidiarrheal activity might be explained by the capacity of CG to inhibit gastrointestinal motility and thereby reduce the accumulation of intestinal fluid and the secretion of water and chloride ions in the lumen of the intestine.

Gastroprotective Properties of Cashew Gum, a Complex Heteropolysaccharide of Anacardium occidentale, in Naproxen-Induced Gastrointestinal Damage in Rats

Preclinical Research

The efficacy of a sulphated polysaccharide fraction from Hypnea musciformis against diarrhea in rodents

Seaweeds are sources of diverse bioactive compounds, such as sulphated polysaccharides. This study was designed to evaluate the chemical composition and anti-diarrheal activity of a fraction of sulphated polysaccharide (PLS) obtained from the red seaweed Hypnea musciformis in different animal models, and to elucidate the underlying mechanisms. PLS was obtained by aqueous extraction, with a yield of 31.8% of the seaweed dry weight. The total carbohydrate content accounted for 99% of the sample. The sulfate content of the polysaccharide was 5.08% and the percentage of carbon was 25.98%. Pretreatment with all doses of PLS inhibited castor oil-induced diarrhea, with reduction of the total amount of stool, diarrheal stools, and the severity of diarrhea. PLS (90 mg/Kg) decreased castor oil- and PGE2-induced enteropooling. In addition, PLS (90 mg/Kg) increased the Na(+)/K(+)-ATPase activity in the small intestine and reduced gastrointestinal transit, possibly via activation of cholinergic receptors. Interestingly, the cholera toxin-induced fluid secretion and Cl(-) ion levels decreased in the intestinal contents of the animals pretreated with PLS (90 mg/kg), probably via reduction of toxin-GM1 receptor binding. In conclusion, PLS exerts anti-diarrheal activity by increasing Na(+)/K(+)-ATPase activity, inhibiting gastrointestinal motility, and blocking the toxin-GM1 receptor binding.

Diminazene aceturate, an angiotensin-converting enzyme II activator, prevents gastric mucosal damage in mice: Role of the angiotensin-(1–7)/Mas receptor axis

The angiotensin (Ang) II converting enzyme (ACE II) pathway has recently been shown to be associated with several beneficial effects in various organisms, including gastroprotection. ACE II is responsible for converting Ang II into an active peptide, Ang-(1-7), which in turn binds the Mas receptor. Recent studies have shown that diminazene aceturate (Dize) a trypanocidal used in animals, activates ACE II. Thus, in this study, we aimed to evaluate the gastroprotective effects of Dize via the ACE II/Ang-(1-7)/Mas receptor pathway against gastric lesions induced by ethanol and acetic acid in mice. The results showed that Dize could promote gastric protection via several mechanisms, including increased levels of antioxidants and anti-inflammatory factors (e.g., decreasing tumor necrosis factor and interleukin-6 expression and reducing myeloperoxidase activity), maturation of collagen fibers, and promotion of re-epithelialization and regeneration of gastric tissue in different injury models. Thus, Dize represents a novel potential gastroprotective agent.

Sulphated Polysaccharide Isolated from the Seaweed Gracilaria caudata Exerts an Antidiarrhoeal Effect in Rodents

Diarrhoea is a significant health problem for children in developing countries that causes more than 1 million deaths annually. This study aimed to evaluate the antidiarrhoeal effect of sulphated polysaccharide (PLS) from the alga Gracilaria caudata in rodents. For the evaluation, acute diarrhoea was induced in Wistar rats (150–200 g) by administration of castor oil (10 mg/kg). Then, different parameters, including enteropooling and gastrointestinal transit and its pharmacological modulation by opioid and cholinergic pathways, were assessed using activated charcoal in Swiss Mice (25–30 g). Secretory diarrhoea was examined using cholera toxin (CT) (1 mg/loop)‐treated, isolated intestinal loops from Swiss mice (25–30 g), which were also used to examine fluid secretion, loss of chloride ions into the intestinal lumen and absorption. In addition, a GM1‐dependent ELISA was used to evaluate the interaction between PLS, CT and the GM1 receptor. Pre‐treatment with PLS (10, 30 and 90 mg/kg) reduced faecal mass, diarrhoeal faeces and enteropooling. However, 90 mg/kg more effectively reduced these symptoms; therefore, it was used as the standard dose in subsequent experiments. Gastrointestinal transit was also reduced by PLS treatment via a cholinergic mechanism. Regarding the diarrhoea caused by CT, PLS reduced all study parameters, and the ELISA showed that PLS can interact with both the GM1 receptor and CT. These results show that PLS from G. caudata effectively improved the parameters observed in acute and secretory diarrhoea, which affects millions of people, and may lead to the development of a new alternative therapy for this disease.

Anti-diarrhoeal therapeutic potential and safety assessment of sulphated polysaccharide fraction from Gracilaria intermedia seaweed in mice

Sulphated polysaccharides extracted from algae have been extensively studied for their diverse biological activities. Thus, the purpose of this study was to evaluate the chemical composition, the anti-diarrhoeal effect and acute toxicity of a sulphated polysaccharide fraction obtained from Gracilaria intermedia (SP-Gi). Initially, the FT-IR of SP-Gi revealed to be an agaran with sulphation at C-6 of the l-galactosyl residues. The anti-diarrhoeal activity of SP-Gi was evaluated in a castor oil-induced diarrhoea model. The effects of SP-Gi on enteropooling, Na +-K +-ATPase activity, gastrointestinal transit, and gastric emptying were then examined. Subsequently, the effect of SP-Gi on diarrhoea induced by cholera toxin (CT) and Escherichia coli was examined. In addition, an acute toxicity test was conducted in accordance with OECD guideline 423. Pre-treatment with SP-Gi reduces the total faeces, total diarrhoeal faeces, and enteropooling. SP-Gi (30mg/kg p.o.) increased Na+/K+-ATPase activity and reduced gastrointestinal transit through anticholinergic mechanisms. ELISA demonstrated that SP-Gi can interact with GM1 receptors and CT. SP-Gi reduced diarrhoea induced by E. coli and prevented weight loss in the animals. Moreover, SP-Gi did not induce any toxicity signs. These results suggest that SP-Gi is a possible candidate for the treatment of diarrhoeal illnesses.

Antidiarrheal effects of water-soluble proteins from Plumeria pudica latex in mice

The water-soluble protein fraction obtained from Plumeria pudica (LPPp) latex has previously been demonstrated to have anti-inflammatory and antinociceptive effects. In the present study, LPPp was tested for activity against diarrhea induced by castor oil, prostaglandin E2 (PGE2) or cholera toxin. Different doses of LPPp (10, 20 or 40mg/kg) significantly inhibited the percentage of diarrheal stools (31.18%, 42.97% and 59.70%, respectively) induced by castor oil. This event was followed by significant reduction of both intestinal fluid accumulation (31.42%; LPPp 40mg/kg) and intestinal transit (68.4%; LPPp 40mg/kg). The pretreatment of animals with LPPp (40mg/kg) prevented glutathione and malondialdehyde alterations induced by castor oil. The effects of LPPp against diarrhea induced by castor oil were lost when the fraction was submitted to protein denaturing treatment with heat. LPPp (40mg/kg) also inhibited the average volume of intestinal fluid induced by PGE2 (inhibition of 46.0%). Furthermore, LPPp (40mg/kg) prevented intestinal fluid secretion accumulation (37.7%) and chloride ion concentration (50.2%) induced by cholera toxin. In parallel, colorimetric assays demonstrated that proteinases, chitinases and proteinase inhibitors were found in LPPp. Our data suggest that the antidiarrheal effect of LPPp is due to its protein content and is probably associated with its anti-inflammatory properties.

H2S is a key antisecretory molecule against cholera toxin-induced diarrhoea in mice: Evidence for non-involvement of the AC/cAMP/PKA pathway and AMPK

Hydrogen sulphide (H2S) is a gasotransmitter that participates in various physiological and pathophysiological processes within the gastrointestinal tract. We studied the effects and possible mechanism of action of H2S in secretory diarrhoea caused by cholera toxin (CT). The possible mechanisms of action of H2S were investigated using an intestinal fluid secretion model in isolated intestinal loops on anaesthetized mice treated with CT. NaHS and Lawessons reagent and l-cysteine showed antisecretory activity through reduction of intestinal fluid secretion and loss of Cl- induced by CT. Pretreatment with an inhibitor of cystathionine-γ-lyase (CSE), dl-propargylglycine (PAG), reversed the effect of l-cysteine and caused severe intestinal secretion. Co-treatment with PAG and a submaximal dose of CT increased intestinal fluid secretion, thus supporting the role of H2S in the pathophysiology of cholera. CT increased the expression of CSE and the production of H2S. Pretreatment with PAG did not reverse the effect of SQ 22536 (an AC inhibitor), bupivacaine (inhibitor of cAMP production), KT-5720 (a PKA inhibitor), and AICAR (an AMPK activator). The treatment with Forskolin does not reverse the effects of the H2S donors. Co-treatment with either NaHS or Lawessons reagent and dorsomorphin (an AMPK inhibitor) did not reverse the effect of the H2S donors. H2S has antisecretory activity and is an essential molecule for protection against the intestinal secretion induced by CT. Thus, H2S donor drugs are promising candidates for cholera therapy. However, more studies are needed to elucidate the possible mechanism of action.