Giulia Di Carlo

FLAVONOIDS : OLD AND NEW ASPECTS OF A CLASS OF NATURAL THERAPEUTIC DRUGS

Flavonoids are natural products widely distributed in the vegetable kingdom and currently consumed in large amounts in the daily diet. Flavonoids are capable of modulating the activity of enzymes and affect the behaviour of many cell systems, suggesting that the compounds may possess significant antihepatotoxic, antiallergic, anti-inflammatory, antiosteoporotic and even antitumor activities. This review summarizes available data on these beneficial effects of flavonoids.

Inhibition of inducible nitric oxide synthase and cyclooxygenase-2 expression by flavonoids in macrophage J774A.1.

The present study focuses on the effect of various naturally occurring flavonoids (apigenin, galangin, morin, naringenin, quercetin, and silymarin) on nitric oxide (NO) and prostaglandin E2 (PGE2) production induced by lipopolysaccharide (LPS) in the macrophage cell line J774A.1. Moreover, we evaluated flavonoid modulation of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) enzyme expression by western blot analysis. Apigenin and quercetin (0.5-50 microM) were the most potent inhibitors of NO production and this effect was concentration-dependent and significant at 5 and 50 microM. These data were consistent with the modulation of iNOS enzyme expression. A similar pattern was observed considering the inhibitory effect of flavonoids on LPS-induced PGE2 release and COX-2 expression. Quercetin, galangin, apigenin, and naringenin markedly decreased PGE2 release and COX-2 expression in a concentration-dependent manner. This study suggests that inhibition of iNOS and COX-2 expression by flavonoids may be one of the mechanisms responsible for their anti-inflammatory effects.

St John's wort: Prozac from the plant kingdom

Conventional antidepressants are associated with a range of adverse drug reactions. The herb Hypericum perforatum (St Johns wort) might offer another approach to the treatment of depression. Biochemical and animal studies suggest that the phloroglucinol derivative hyperforin is the main active ingredient of St Johns wort, and inhibits the synaptosomal uptake of 5-HT, noradrenaline, dopamine, glutamate and GABA. St Johns wort has been shown to alleviate symptoms of mild to moderate depression, and seems to offer significant advantages over conventional antidepressants because it is associated with fewer adverse reactions. However, important herb--drug interactions have been described. In view of its efficacy and safety records, St Johns wort should be considered for the first-line treatment of mild to moderate depression.

Cannabinoids for gastrointestinal diseases: potential therapeutic applications

Δ9-Tetrahydrocannabinol (the active ingredient of marijuana), as well as endogenous and synthetic cannabinoids, exert many biological functions by activating two types of cannabinoid receptors, CB1 and CB2 receptors. CB1 receptors have been detected on enteric nerves, and pharmacological effects of their activation include gastroprotection, reduction of gastric and intestinal motility and reduction of intestinal secretion. The digestive tract also contains endogenous cannabinoids (i.e., the endocannabinoids anandamide and 2-aracidonylglycerol) and mechanisms for endocannabinoid inactivation (i.e., endocannabinoids uptake and enzymatic degradation). Cannabinoid receptors, endocannabinoids and the proteins involved in endocannabinoids inactivation are collectively referred as the ‘endogenous cannabinoid system’. A pharmacological modulation of the endogenous cannabinoid system could provide new therapeutics for the treatment of a number of gastrointestinal diseases, including nausea and vomiting, gastric ulcers, irritable bowel syndrome, Crohn’s disease, secretory diarrhoea, paralytic ileus and gastroesophageal reflux disease. Some cannabinoids are already in use clinically, for example, nabilone and Δ9-tetrahydrocannabinol are used as antiemetics.

In-vivo and in-vitro anti-inflammatory effect of echinacea purpurea and Hypericum perforatum

Echinacea purpurea (L.) Moench and Hypericum perforatum (L.) were evaluated for their anti‐inflammatory activity against carrageenan‐induced paw oedema in mice. Each drug was administered orally to mice at 30 and 100 mg kg−1, twice daily. Only the higher dose significantly inhibited, time dependently, the formation of oedema, evaluated as area under the curve (echinacea P < 0.01; hypericum P < 0.05). Western blot analysis showed that in‐vivo treatment with these extracts could modulate lipopolysaccharide (LPS) and interferon‐γ induced cyclooxygenase‐2 (COX‐2) and inducible nitric oxide synthase (iNOS) expression in peritoneal macrophages. In particular, treatment with 100 mg kg−1 hypericum inhibited both iNOS and COX‐2 expression, whereas treatment with 100 mg kg−1 echinacea down‐regulated only COX‐2 expression. The present study suggests that the anti‐inflammatory effect of these extracts could be in part related to their modulation of COX‐2 expression.

Effect of vanilloid drugs on gastrointestinal transit in mice

We have studied the effect of capsaicin, piperine and anandamide, drugs which activate vanilloid receptors and capsazepine, a vanilloid receptor antagonist, on upper gastrointestinal motility in mice. Piperine (0.5 – 20 mg kg−1 i.p.) and anandamide (0.5 – 20 mg kg−1 i.p.), dose‐dependently delayed gastrointestinal motility, while capsaicin (up to 3 mg kg−1 i.p.) was without effect. Capsazepine (15 mg kg−1 i.p.) neither per se affected gastrointestinal motility nor did it counteract the inhibitory effect of both piperine (10 mg kg−1) and anandamide (10 mg kg−1). A per se non effective dose of SR141716A (0.3 mg kg−1 i.p.), a cannabinoid CB1 receptor antagonist, counteracted the inhibitory effect of anandamide (10 mg kg−1) but not of piperine (10 mg kg−1). By contrast, the inhibitory effect of piperine (10 mg kg−1) but not of anandamide (10 mg kg−1) was strongly attenuated in capsaicin (75 mg kg−1 in total, s.c.)‐treated mice. Pretreatment of mice with NG‐nitro‐L‐arginine methyl ester (25 mg kg−1 i.p.), yohimbine (1 mg kg−1, i.p.), naloxone (2 mg kg−1 i.p.), or hexamethonium (1 mg kg−1 i.p.) did not modify the inhibitory effect of both piperine (10 mg kg−1) and anandamide (10 mg kg−1). The present study indicates that the vanilloid ligands anandamide and piperine, but not capsaicin, can reduce upper gastrointestinal motility. The effect of piperine involves capsaicin‐sensitive neurones, but not vanilloid receptors, while the effect of anandamide involves cannabinoid CB1, but not vanilloid receptors.

Nitric oxide involvement in sodium choleate-induced fluid secretion and diarrhoea in rats

Bile salt-induced diarrhoea, net water and electrolyte secretion, gastrointestinal transit and nitric oxide (NO) synthase activity were studied in rats. NG-Nitro-L-arginine methyl ester (2.5-25 mg/kg i.p.), an inhibitor of NO synthase, and dexamethasone (0.03-0.3 mg/kg i.p.), an inhibitor of the inducible isoform of NO synthase, antagonized the diarrhoeal response. The NO precursor, L-arginine and isosorbide-5-mononitrate (an NO donor), reversed the inhibitory effect of NG-nitro-L-arginine methyl ester. The bile salt-stimulated fluid secretion, transit through the gut and NO synthase all were inhibited by NG-nitro-L-arginine methyl ester (but not NG-nitro-D-arginine methyl ester). NO synthase activity also was inhibited by dexamethasone. The results are consistent with bile salt induction of epithelial cell injury and concomitant synthesis of NO, mainly through activation of the inducible form of the enzyme. We believe that in this study NO is a mediator of intestinal secretion and motility changes that enhance transit of luminal contents through the gut, resulting in diarrhoea.

NG-nitro-L-arginine methyl ester modulates intestinal secretion and motility produced by carbachol

The effects of the nitric oxide (NO) synthesis inhibitor, NG-nitro-L-arginine methyl ester, on carbachol-induced diarrhoea, fluid accumulation and motility changes were studied. Pretreatment of mice with NG-nitro-L-arginine methyl ester (1-25 mg/kg i.p.) and NG-nitro-L-arginine (2.5-50 mg/kg i.p.) but not NG-nitro-D-arginine methyl ester (25 mg/kg i.p.) prevented in a dose-related manner the carbachol (0.5 mg/kg i.p.)-induced diarrhoea in mice. L-Arginine (150-1500 mg/kg i.p.) administered to mice pretreated with NG-nitro-L-arginine methyl ester counteracted the antidiarrhoeal activity of NG-nitro-L-arginine methyl ester in a dose-related manner. Pretreatment of rats with NG-nitro-L-arginine methyl ester (2.5-25 mg/kg i.p.) decreased the intestinal fluid accumulation induced by carbachol in rats. NG-Nitro-D-arginine methyl ester was without effect. Intraperitoneal pretreatment of rats with NG-nitro-L-arginine methyl ester (2.5-25 mg/kg) reduced the increase in small intestinal transit induced by carbachol. NG-nitro-L-arginine methyl ester had no effect. These results provide evidence that nitric oxide may play a role in diarrhoea, intraluminal fluid accumulation and motility changes induced by carbachol.

Effect of fumonisin B1 on structure and function of macrophage plasma membrane

Fumonisin B1 (FB1), a mycotoxin produced by Fusarium moniliforme and related fungi, is nephrotoxic, neurotoxic, hepatotoxic, carcinogenic and immunosuppressive in animals and man. In this study we evaluate the modifications of fluidity, endocytosis and peroxidative damage of plasma membrane induced by FB1 in macrophage cell line J774A.1. In these immune cells FB1 (1-10 microM) enhances membrane fluidity and increases, time-dependently, the horseradish peroxidase (HRP) endocytosis. This effect is concentration-dependent, significant at 10 microM, and reverted by IFN-gamma (100 U/ml). Moreover, FB1 (1-10 microM) induces a membrane peroxidative damage as evident by the increase of malondialdehyde (MDA) production. All these mycotoxin effects provide additional insight into potential mechanism by which FB1, in macrophages, might enhance membrane damage and oxidative stress contributing to the pathogenesis of mycotoxin induced diseases.

Modulation of apoptosis in mice treated with Echinacea and St. John's wort.

Apoptosis, or programmed cell death (PCD), is a physiological active cellular suicide process that occurs in non-contiguous cells, and is usually not associated with inflammation. The apoptotic process can be modulated by various stimuli, including hormones, cytokines, growth factors, and some chemotherapeutic agents. To determine whether Echinacea purpurea and Hypericum perforatum are able to regulate the process of apoptosis in vivo and to define the role of the Fas-Ag and Bcl-2 signal transduction cascade, we have orally treated groups of mice with these vegetable drugs for 14 days. The splenic lymphocytes from mice treated with E. purpurea and H. perforatum at the two dose levels used (30 and 100 mg kg(-1) per day) were shown to be significantly more resistant to apoptosis than those from mice treated only with the vehicle. In addition, mice treated with the natural substances showed a decrease in Fas-Ag expression and an increase in Bcl-2 expression. In conclusion, our results shown that in vivo the studied drugs modulate apoptosis in mice splenic lymphocytes and that this action could be mediated in part by a decrease in Fas-Ag expression and in part by an increase in Bcl-2 expression.