Limonene: Aroma of innovation in health and disease.

Natural products obtained in dietary components may aid the prevention and treatment of a variety of diseases. Reports in the scientific literature have demonstrated that the consumption of terpenes is a successful alternative in the treatment of several diseases, triggering beneficial biological effects in clinical and preclinical studies. The monoterpene limonene is largely used in alimentary items, cleaning products, and it is one of the most frequent fragrances used in cosmetics formulation. The therapeutic effects of limonene have been extensively studied, proving anti-inflammatory, antioxidant, antinociceptive, anticancer, antidiabetic, antihyperalgesic, antiviral, and gastroprotective effects, among other beneficial effects in health. In this review, we collected, presented, and analyzed evidence from the scientific literature regarding the usage of limonene and its activities and underlying mechanisms involved in combating diseases. The highlighting of limonene applications could develop a useful targeting of innovative research in this field as well as the development of a limonene-based phytomedicine which could be used in a variety of conditions of health and disease.

Antiulcerogenic Activity and Toxicity of Bauhinia holophylla Hydroalcoholic Extract.

Several species of Bauhinia are used in traditional medicine for the treatment of gastrointestinal diseases, diabetes, and inflammation, among other conditions. The aim of this study was to investigate the antiulcer effect of a hydroalcoholic extract from the leaves of B. holophylla. The chemical profile of the extract was determined by HPLC-PAD-ESI-IT-MS. A dose-effect relation was constructed using the ethanol-induced gastric ulcer model in male Wistar rats. Histological analyses and studies of antioxidant and anti-inflammatory activities were performed in stomach samples. The involvement of SH compounds, NO, K(+) ATP channels, and α 2-adrenergic receptors in the gastroprotective effect was evaluated. A toxicity study was performed with a single oral dose of 5000 mg/kg. The extract was composed mainly of cyanoglucoside and flavonol-O-glycosides derivatives of quercetin and myricetin. SH compounds, NO release, K(+) ATP channel activation, and presynaptic α 2-adrenergic receptor stimulation each proved to be involved in the antiulcer effect. The levels of GSH and activity of GR and GPx were increased, and the levels of TNF-α, IL-6 and IL-10 were modulated. There was an antidiarrheal effect and there were no signs of toxicity. B. holophylla presents antiulcer activity mainly by decreasing oxidative stress and attenuating the inflammatory response, without inducing side effects.

Effect of menthol in experimentally induced ulcers: pathways of gastroprotection.

Based on ethnopharmacological indications that Mentha species may be used in the treatment of gastrointestinal diseases, this study aimed to characterize the gastroprotective mechanisms of menthol (ME), the major compound of the essential oil from species of the genus Mentha. The gastroprotective action of ME was analyzed in gastric ulcers that were induced by ethanol or indomethacin in Wistar male rats. The mechanisms responsible for the gastroprotective effect were assessed by analyzing the amount of mucus secreted, involvement of non-protein sulfhydryl (NP-SH) compounds, involvement of calcium ion channels and NO/cGMP/K(+)ATP pathway, gastric antisecretory activity and the prostaglandin E2 (PGE2) production. The anti-diarrheal activity and acute toxicity of ME were also evaluated. Oral treatment with ME (50mg/kg) offered 88.62% and 72.62% of gastroprotection against ethanol and indomethacin, respectively. There was an increased amount of mucus and PGE2 production. The gastroprotective activity of ME involved NP-SH compounds and the stimulation of K(+)ATP channels, but not the activation of calcium ion channels or the production of NO. The oral administration of ME induced an antisecretory effect as it decreased the H(+) concentration in gastric juice. ME displayed anti-diarrheal and antiperistaltic activity. There were no signs of toxicity in the biochemical analyses performed in the rats' serum. These results demonstrated that ME provides gastroprotective and anti-diarrheal activities with no toxicity in rats.

Morphologic and pharmacological investigations in the epicatechin gastroprotective effect.

Previous studies of the gastroprotective activity of plants have highlighted the importance of the polyphenolic compound epicatechin (EC) in the treatment of gastric ulcers. This paper aimed to evaluate and characterize the gastroprotective mechanism of action of EC using male rats. The gastroprotective action of EC was analyzed in gastric ulcers induced by ethanol or indomethacin. The involvement of sulfhydryl (SH) groups, K(+) (ATP) channels, α(2) adrenoceptors, gastric antisecretory activity, and the amount of mucus in the development of gastric ulcers were investigated. The lowest effective dose of EC providing gastroprotective effects was 50 mg/kg in the ethanol-induced gastric ulcers and 25 mg/kg in the indomethacin-induced gastric ulcers. The gastroprotection seen upon treatment with EC was significantly decreased in rats pretreated with a SH compound reagent or an α(2)-receptor antagonist, but not with a K(+) (ATP) channel blocker. Furthermore, oral treatment with EC increased mucus production and decreased H(+) secretion. Immunohistochemistry demonstrated the involvement of superoxide dismutase (SOD), nitric oxide (NO), and heat shock protein-70 (HSP-70) in the gastroprotection. These results demonstrate that EC provides gastroprotection through reinforcement of the mucus barrier and neutralization of gastric juice and this protection occurs through the involvement of SH compounds, α(2)-adrenoceptors, NO, SOD, and HSP-70.

Acute cerebral focal ischaemia alters the adrenergic and NANC responses in the bisected rat vas deferens.

1. Disturbances of the autonomic nervous system are common in right hemisphere stroke patients, including a marked decline in male sexual functions. There is a lack of information on the influence of stroke on male secondary sex organs such as the vas deferens. 2. This study investigates the effect of right brain focal ischaemia on the adrenergic and purinergic responses in isolated epididymal and prostatic portions of rat vas deferens. 3. In both epididymal and prostatic portions the concentration-response curves to noradrenaline are flattened resulting in a reduction (up to 67 - 76%) of the maximum contractile response in the tissue from ischaemic rats compared to the controls. In the prostatic portion from ischaemic rats the concentration-response curve to alpha,beta-methylene ATP was also depressed. 4. The first purinergic and the second delayed adrenergic phase to single pulse was not modified by brain ischaemia. In contrast both phasic and tonic components of the electrically induced contractions by trains of stimuli at high frequencies (2 - 30 Hz) were significantly depressed in the epididymal and prostatic portions from ischaemic rats. 5. These results demonstrate an autonomic imbalance at the level of male sexual secondary organs which may contribute to sexual impairment after stroke.

The Cycloxygenase-2 inhibitor SC58236 is neuroprotective in an in vivo model of focal ischemia in the rat.

Focal ischemia was induced in the fronto-parietal region of rat brain, by injection of Rose Bengal, followed by light activation. Focal ischemia was accompanied by formation of PGD(2) peaking 60-90 min post irradiation and declining thereafter. Increased Cycloxygenase-2 (COX-2) expression was also observed. Control ischemic rats showed distinct morphological alterations with necrosis of neurons, glial cells and blood vessels, surrounded by a halo with pyknotic cells with cytoplasm swelling and vacuolization. Compound SC58236, a selective COX-2 inhibitor, dose-dependently prevented, ischemia-induced eicosanoid formation (area under the curve (AUC) of controls: 3.11 +/- 0.87; AUC of 20 mg/kg SC58236: 0.39 +/- 0.24), and caused significant reduction of damaged area (30.7 and 18.9% at SC58236 20 and 6.6 mg/kg), suggesting that selective inhibitors of COX-2 are neuroprotective.

Influence of different anaesthetics on extracellular aminoacids in rat brain.

We used different anaesthetic procedures to study the possible effects of anaesthesia on extracellular aminoacid concentration in rat brain. Glutamate, aspartate and glycine concentrations were determined by HPLC in samples collected from the right fronto-parietal region of the rat brain cortex by transcerebral microdialysis before and up to 2 h following anaesthesia induction. Anaesthesia induced by ketamine, alone or in association with xylazine, caused a significant decrease in the levels of glutamate, aspartate and glycine, compared to before anaesthesia values (range: 27-72% according to the time of sampling and to the anaesthetic used). Inhalation anaesthesia with halothane (3%) in N2O/O2 mixture produced no significant effects on aminoacid levels. Equitensine (pentobarbital in association with chloral hydrate and ethanol) and pentobarbital also had no significant effect on glutamate, aspartate and glycine levels during anaesthesia. This demonstrates that some anaesthetics alter excitatory aminoacid release and suggests that Equitensine may represent an easy and reliable method to induce a long lasting anaesthesia associated without changes in excitatory aminoacid extracellular concentration.

Effect of acute alcohol on ischemia-induced glutamate release and brain damage.

Epidemiological studies show that chronic ethanol consumption at high doses enhances the risk of cerebral stroke. The mechanisms responsible for the greater vulnerability of alcoholics' brains to stroke have to be completely understood, but a role for excitatory amino acids has been suggested. In order to study the interaction between alcohol and ischemia, we investigated the effect of acute alcohol administration in a model of focal cerebral ischemia. In particular, we evaluated the release of glutamate and aspartate from the cerebral frontal cortex by a transdialysis technique. Alcohol was acutely administered at 1.5 and 3.0 g/kg ip. During the period of maximal alcoholemia, ethanol almost abolished the ischemia-induced release of glutamate leading to glutamate values around or below the basal. Aspartate levels were unaltered both following ischemia and alcohol+ischemia. The decrease in glutamate release, however, was not accompanied by a significant reduction of the extension of the damaged area assessed by histological analysis.

[The evaluation by video capillaroscopy of the efficacy of a Ginkgo biloba extract with L-arginine and magnesium in the treatment of trophic lesions in patients with stage-IV chronic obliterating arteriopathy]. / Valutazione mediante videocapillaroscopia dell'efficacia dell'estratto di Ginkgo biloba con L-arginina e magnesio nel trattamento delle lesioni trofiche nei pazienti con AOC IV stadio.

BACKGROUND AND AIMS: This study aimed to evaluate the influence of Ginkgo biloba extract with arginine and magnesium used for the treatment of trophic lesions in the lower limbs caused by both diabetic and non-diabetic microangiopathy. METHODS: A comparative study was carried out in 20 patients who were divided into two groups: 10 were treated with ASA plus Ginkgo biloba extract with arginine and magnesium and 10 with ASA plus conventional hemorheology. The observation time was extended to 6 months, taking into consideration patients with trophic lesions to the lower limbs suffering from diabetic and non-diabetic peripheral arterial occlusive disease. The evaluation was performed by clinical, ultrasonographic and perilesional video capillaroscopy monitoring. Ultrasonographic and video capillaroscopy instrumental methods were used because they provide a full picture of macro and microcirculatory conditions around lesions. RESULTS: The study showed the undoubted efficacy of Ginkgo biloba extract with magnesium and arginine in relation to the following points: reduced healing times for the trophic lesion compared to the control group, improved painful symptoms, increased perilesional neoangiogenesis. No significant differences were observed from a Doppler ultrasonographic point of view or with regard to the claudication free interval. CONCLUSIONS: Ginkgo biloba extract with arginine and magnesium can improve the dynamics of cutaneous trophism in lesions caused by diabetic and non-diabetic microangiopathy.

Inhibition of prostanoid synthesis protects against neuronal damage induced by focal ischemia in rat brain.

Changes in prostanoids concentration and effects of the non-specific COX inhibitor indomethacin on prostanoids levels and extension of tissue damage were studied following focal ischemia induction in the fronto-parietal region of rat brain. Ischemia was induced in animals bearing a transcerebral microdialysis probe by injection of Rose Bengal, a photosensitive dye, followed by light activation. Prostanoid levels were determined in the dialysate using immunoenzymatic techniques. PGD2 levels rose significantly up to 237+/-22 pg/ml compared to a basal level measured before ischemia induction which was below the detection limit. TXB2 changes were smaller and had a different time course. Treatment with indomethacin abolished the ischemia-induced PGD2 release and reduced the extent of injury to the area by 43+/-3.7%. These results suggest that prostanoid release may play an important role in neurodegenerative processes and that cyclooxygenase inhibitors may contribute to protect against cerebral tissue damage.

Nitrite/nitrate balance during photoinduced cerebral ischemia in the rat determined by high-performance liquid chromatography with UV and electrochemical detection.

A specific and simple method for the direct simultaneous detection of extracellular nitrite (NO2-) and nitrate (NO3-) has been developed, using high-performance liquid chromatography separation with UV and electrochemical detection in series. These stable endproducts of nitric oxide (NO.) were determined in dialysis perfusate obtained through in vivo brain microdialysis during and after experimental photoinduced cerebral ischemia in rats. The chromatographic conditions were optimized with a reversed-phase column (250 x 46 mm) using 10 mM n-octylamine pH 6.0 as a mobile phase. Absorbance was measured at 220 nm for NO3- detection; electrochemical detection was performed at +0.7 V for NO2- evaluation. This assay system holds the advantages of in vivo consecutive measurements, high precision, good reproducibility, technical simplicity, fast response (about 7 min), and wide availability.

Effect of idebenone on in vivo serotonin release and serotonergic receptors in young and aged rats.

The effect of idebenone on the serotonergic system was evaluated in the aging rat by measuring the kinetic constants of 3H-5HT and 3H-ketanserin binding sites in the cerebral cortex of rats at 3, 15 and 24 months of age following acute and subchronic administration of the drug. Idebenone displayed no in vitro affinity toward any population of serotonin receptors and did not modify their kinetic parameters after a single dose of 100 mg/kg, at any age tested. A subchronic treatment with the drug for 21 days at the dose of 30 mg/kg did not induce any relevant change in 3- and 15-month-old rats, whereas it significantly increased the density of both 3H-5HT and 3H-ketanserin binding sites in 24-month-old rats, where a lower number of receptors is detected as a consequence of aging. This effect was rather specific, since under the same experimental conditions no changes were detected in the density of cortical beta-adrenergic receptors in aged animals. In microdialysis studies, acute administration with idebenone did not affect 5HT and 5HIAA release at any age. Conversely, the pattern of serotonin metabolism was significantly modified in aged rats following repeated treatment with idebenone and was partially restored to a value similar to the one observed in young animals. These results suggest that idebenone, a putative neuroprotective agent which has been shown to improve brain metabolism in ischemic conditions, might also attenuate age-associated neuronal damage, acting probably on several neurotransmitter systems which undergo selective modification during aging.

Aminoacid recovery via microdialysis and photoinduced focal cerebral ischemia in brain cortex of rats.

A photochemical method using the Rose Bengal dye as thrombogenic agent was employed to induce focal cerebral ischemia in frontoparietal cortex of rats. A transcerebral microdialysis probe was used to collect samples from ischemic cortical area. An increase in glutamate (6-fold) and in taurine (4-fold) within the first hour occurred. Neuropathological investigations demonstrate a reproducible damaged area surrounded by a thin peripheral area showing neuronal apoptotic phenomena. The method represents a reproducible model of focal cerebral ischemia with neuropathological aspects superimposable to those characteristic of thrombogenic stroke in man. This method could also be relevant in the study of neurotransmitters during the evolution of ischemia. Furthermore, the presence of apoptotic phenomena in the perilesional halo confirms an ischemic penumbra suggesting the significance of preclinical pharmacological trials.

K-opioid receptor changes in experimental models of cerebral ischaemia and atherosclerosis in the rabbit.

Thromboembolic phenomena and transient ischaemic attacks (TIA) are considered the basis of ischaemic pathologies. The aim of the present research is to investigate the involvement of k-opioid receptors in cerebral blood flow (CBF) impairment which results in experimental stroke or dietary atherosclerosis in rabbits. CBF measurement showed a significant decrease in rabbits submitted to embolization and/or atherosclerosis. Binding studies showed that massive cerebral ischaemia and atherosclerosis produced a significant increase in the number of k-opioid receptors (Bmax), without changing (KD) affinity values. In conclusion, the results obtained seem to indicate that the increase in k-opioid receptors might play a crucial role in a common cerebral biochemical mechanism both in ischaemic and atherosclerotic pathologies.

[The effect of superoxide dismutase and catalase on the delayed toxicity of doxorubicin]. / Effetto della superossido-dismutasi e della catalasi sulla tossicità tardiva da doxorubicina.

The production of oxygen-free radicals has been proposed as a determinant of the delayed toxicity of doxorubicin. The aim of the present investigation was to evaluate the potential cardioprotective effect of superoxide dismutase (SOD) and catalase (CAT) against the delayed cardiomyopathy induced by doxorubicin (DXR) in a rat model. Female Sprague Dawley rats received 3 mg/kg of DXR intravenously weekly for 4 weeks. SOD or CAT were administered intravenously at the dose of 10000 U/kg 2 min before and 30 min after each DXR administration. Cardiac toxicity was monitored by means of electrocardiography (QaT interval) and by light and electron microscopy evaluation of left ventricle fragments. DXR treated rats showed, in comparison with control animals, a decrease of body weight gain, a progressive and irreversible prolongation of QaT and significant morphologic lesions consisting in myocyte vacuolization and myofibrillar loss. SOD significantly prevented the impairment of body weight gain and QaT prolongation. Moreover, morphologic lesions were significantly reduced in rats receiving DXR + SOD. On the contrary, CAT seems to be completely devoid of protective effect.

[The prevention of the myocardial toxicity of doxorubicin with superoxide dismutase]. / Prevenzione della tossicità miocardica da doxorubicina con superossido dismutasi.

The production of oxygen free radicals during anthracycline therapy has been proposed as a determinant of the toxicity of anthracyclines. Oxygen radical generation might specifically affect the myocardium because of the low antioxidant defense systems in cardiac tissue. The aim of the present investigations was to evaluate the potential cardioprotective effect of superoxide dismutase (SOD) against the delayed cardiomyopathy induced by doxorubicin (DXR) in a rat model. Female Sprague Dawley rats received 3 mg/kg of DXR intravenously weekly for 4 weeks. SOD was administered intravenously at the dose of 10,000 U/Kg one minute before and 30 minutes after each DXR administration. Cardiac toxicity was monitored in vivo by means of electrocardiography (QaT interval), by determining the contractile properties of isolated atria, and by light and electron microscopy evaluation of left ventricle fragments excised 5 weeks after the last DXR administration. The degree of morphologic lesions was quantitated according to the score system proposed by E. Billingham. DXR treated rats showed, in comparison with control animals treated with saline a decrease of body weight gain, a progressive and irreversible prolongation of QaT, decrease of contractility of isolated atria, and significant morphologic lesions consisting in myocyte vacuolization and myofibrillar loss. SOD significantly prevented the impairment of body weight gain, QaT prolongation and the impairment of myocardial contractility. Moreover morphologic lesions were significantly reduced in rats receiving DXR + SOD. The present data indicate that SOD could represent an important issue in myocardial protection against DXR cardiotoxicity.

Ischemic cerebral pathologies and K opioid receptors in rabbits.

Recently it has been suggested that endogenous k-opioid receptors may have a physiopathological role in ischemia induced neurodegeneration. The aim of this research is to show that in experimental thromboembolic (obtained mechanically using microspheres injected in the carotid) and atherosclerotic pathologies (obtained through a special diet) there is a common mechanism which involves mediation by dynorphine and the receptor compartment considered. The results, obtained using receptor binding techniques, showed a statistically significant increase in the number of k-opioid receptors (Bmax) without variations in the affinity (Kd) for the 3H dynorphine. We can therefore support the hypothesis that these changes in the modulation of the dynorphinergenic system may be part of a mechanism causing early cerebrovascular damage which results from embolic insults and is a consequence of such metabolic risk factors as are activated by atherogenesis.

Effect of flunarizine on the delayed cardiotoxicity of doxorubicin in rats.

The calcium antagonist flunarizine (FLN) was tested for its ability to prevent doxorubicin (DXR)-induced cardiotoxicity in the rat. A cumulative dose of 9.0 mg/kg of DXR was administered i.v. over a period of 1 week. FLN (10 mg/kg/day i.p., 6 days/week) was administered according to two different time schedules, covering respectively the first and last 4 weeks after the beginning of DXR treatment. The two schedules were adopted to assess whether early and/or delayed DXR-induced cardiotoxic effects were affected by FLN. The development of cardiac toxicity was monitored by ECG recordings. The animals were sacrificed 8 weeks after the beginning of DXR treatment. The contractile performance of isolated atria and the morphological pattern of left ventricular fragments were subsequently evaluated. The early administration schedule of FLN was shown to be ineffective in preventing DXR-induced cardiotoxicity and in some cases was actually found to potentiate the effects of DXR. In contrast, the histological evaluation of ventricular preparations from rats treated with DXR and FLN according to the delayed time schedule showed a significant improvement with respect to hearts from animals treated with DXR alone. An inhibition of the delayed calcium overload occurring after DXR administration has been proposed as a possible mechanism for this protective action.

Prevention of doxorubicin-induced cardiomyopathy by reduced glutathione.

The aim of the present investigation was to evaluate the potential cardioprotective effect of reduced glutathione (GSH) against the delayed cardiomyopathy induced by doxorubicin (DXR) in a well-documented rat model. DXR was administered i.v. at a weekly dose of 3 mg/kg for a total of 4 doses; 250 or 500 mg/kg of GSH was given i.v. 10 min before and 2 h after each DXR injection, resulting in a total weekly dose of 500 or 1000 mg/kg, respectively. The development of cardiotoxicity was monitored in vivo by means of electrocardiography (QaT duration), and was evaluated by measuring the contractile performance of isolated atria and by light and electron microscopy of left ventricular samples excised 5 weeks after the last DXR administration. DXR was found to impair body weight gain and to produce an irreversible and time-dependent prolongation of QaT, a decrease in myocardial contractility of isolated atria and typical morphologic alterations, including myocyte vacuolization and myofibrillar loss. Pretreatment with GSH at a dose of 500 mg/kg x 2, but not at 250 mg/kg x 2, partially prevented the impairment of body weight gain, QaT prolongation in ECG and the decrease in myocardial contractility of isolated atria induced by DXR. Alterations of the morphologic pattern were also significantly reduced in animals receiving the higher dose of GSH. Determinations of the cardiac non-protein sulfhydryl group content showed that GSH, at doses higher than or equal to 500 mg/kg, significantly increased this parameter, irrespective of the presence of DXR. In conclusion, the present data indirectly support the hypothesis that oxidative damage is involved in DXR cardiotoxicity and indicate that maintenance of the reduced thiol pool could be an important issue in myocardial protection.

Evaluation of the cardiovascular toxic effect of recombinant interleukin-2 in rats.

Cardiac and vascular toxicity of recombinant interleukin-2 (rIL-2) was evaluated by in vitro and in vivo experiments in rats. Using isolated spontaneously beating atria, no changes were detected in heart rate or myocardial contractility in response to rIL-2 treatment at concentrations ranging from 0.1-100 U/ml. Daily sc administration of rIL-2 for 7 consecutive days (at doses of 2.3 X 10(4) to 1.15 X 10(6) U/ml) produced a prolongation of the QaT interval and significant modifications in serum electrolyte concentrations. Ex vivo contractility of atria excised at the end of rIL-2 treatment showed no deterioration in myocardial contractility with increasing dosage. rIL-2, at concentrations of 0.1-1000 U/ml, did not induce any modification of perfusion pressure in isolated rat tail artery but produced a significant displacement to the left of the dose-response curves for norepinephrine compared to basal conditions. The relative potencies were not dose-dependent. Our results indicate that rIL-2 does not exert a direct cardiotoxic effect, and suggest an indirect action on vascular smooth muscle, i.e., an aspecific modulation of vascular response to mediators.