Catechins: natural free-radical scavengers against ochratoxin A-induced cell damage in a pig kidney cell line (LLC-PK1).

Besides aflatoxin B1, recent findings suggested that oxidative stress plays an important role in the toxicity of an other mycotoxin: ochratoxin A (OTA). The protective effect of two catechins (epigallocatechin gallate, EGCG, and epicatechin gallate, ECG) against OTA-induced cytotoxicity was investigated in a pig kidney cell line (LLC-PK1). The ability of the catechins to reduce ROS production and DNA fragmentation induced by OTA was also investigated. Our experiments proved the significant cytoprotective effects of the molecules in vitro from OTA-induced cell damage. In particular a 24h pre-treatment with EGCG or ECG restored cell viability with respect to OTA alone. Pre-treatment with EGCG at low concentration for 8 days protected cells from OTA-induced cell death. Moreover both catechins reduced OTA-induced ROS production. A reduction of OTA-induced DNA fragmentation was found for LLC-PK1 cells pre-treated with EGCG and ECG. The free-radical scavenging capacity of both catechins was tested with the Briggs-Rauscher oscillating method (pH approximately 2) and the TEAC assay (pH 7.4). The results show a good scavenging power according with inhibition of ROS production. Catechins could be useful to develop alimentary strategies for both humans and animals to prevent OTA-induced cytotoxicity.

Anti-inflammatory, anti-nociceptive and antioxidant activities of Balanites aegyptiaca (L.) Delile.

The anti-inflammatory and anti-nociceptive activities of methanol (ME), butanol (BE) extracts and of two new saponins isolated from Balanites aegyptiaca bark were evaluated. The study was carried out in vivo and in vitro. The samples, extracts and pure substances, were intra-gastrically administered to animals. Two different animal models, the carrageenin-induced edema, in the rat, and acetic acid-induced writhing test in mice, were adopted. Moreover, the antioxidant power of extracts, fractions and individual constituents from Balanites aegyptiaca has been evaluated in vitro, using a method based on the Briggs-Rauscher (BR) oscillating reaction. Results obtained demonstrate that both ME or BE have a significant effect at the highest dose on the number of abdominal writhes induced by acetic acid, with a 38 and 54% inhibition respectively, but no significant difference was observed for extracts at the lowest dose and for the pure compounds compared with control animals. The same extracts exhibit a significant reduction on the rat paw edema. The inhibition produced by ME is about the same (28+/-3% lowest dose, 32+/-3% highest dose) after administration. A more evident effect is obtained by BE (41+/-3% and 68+/-6% respectively) and single saponins B1 and B2 (62+/-5% and 59+/-6% respectively) after oral administration. The antioxidant activity obtained seems to be in good accordance with the pharmacological results. The histological sections of rat paw confirm the antiflogistic activity of the plant extracts.

Possible mediation of catecholaminergic pathways in the antinociceptive effect of an extract of Cannabis sativa L.

An extract of cannabis (5 and 15 mg/kg expressed as delta 9-THC) orally administered to rats caused an elevation of the nociceptive threshold (tail-flick latency and vocalization tests). Naloxone and naltrexone (blockers of mu-type opiate receptors) as well as MR 1452 (blocker of kappa opiate receptors) did not prevent the antinociceptive effect of cannabis when used at the dose of 2 mg/kg SC; only a high dose (10 mg/kg SC) of these narcotic antagonists partially blocked cannabis antinociception. ICI 154, 129, an antagonist of delta-type opiate receptors, failed to prevent the cannabis-induced rise in nociceptive threshold when used at a dose of 2 mg/kg SC but produced a significant effect at 10 mg/kg SC. While the role of opiate receptors does not seem fundamental to cannabis antinociception, the clear-cut effectiveness shown by 6-hydroxydopamine (a neurotoxin which causes a degeneration of catecholamine-containing terminals) in reducing cannabis antinociception is indicative of a participation of catecholamines in the phenomenon.

Investigations on behavioral effects of an extract of Cannabis sativa L. in the rat.

The behavioral responses of the rat to an extract of Cannabis sativa were examined after IP injection of 5, 15 and 30 mg/kg (expressed as delta 9 tetrahydrocannabinol). The lowest dose of the extract induced stereotyped behavior (rhythmic head movements, intermittent gnawing and sniffing) together with hypersensitivity to stimuli and hyperthermia. The administration of higher doses of the extract resulted, initially, in similar behavioral effects but of greater intensity, followed by a cataleptic state alternating with atonic muscular prostration; rectal temperature was decreased. Pre-treatment with 6-hydoxydopamine (6-OHDA, which produces degeneration of catecholamine-containing nerve terminals)or pimozide (blocker of dopamine receptors) significantly reduced both stereotype and hyperreactivity. Thermic effects were also antagonized by 6-OHDA pre-treatment. Cannabis-induced catalepsy was enhanced by pimozide but reduced by atropine (3 mg/kg SC). These results support the hypothesis that catecholamines play an important role in the complex behavioral effects of cannabis.

The opioid peptide DAMME modulates histamine's content in gastric mucosa of the rat.

The effects of a synthetic Met-enkephalin analogue D-Ala2, MePhe4Met(O)5-ol]enkephalin (DAMME) (1 mg/kg. IP) on gastric damage produced by necrotizing agents (0.6 N HCl, ethanol 1 ml/rat, PO) were evaluated, and the correlation between histamine and opioids in stomach was investigated, Rats pretreated with DAMME bad significantly less severe lesions and lower histamine content in gastric tissue. The histamine level, expressed in mg/g of gastric tissue, changed from 0.41 +/- 0.10 of control animals to 1.33 +/- 0.12 for HCl and 1.51 +/- 0.20 for ethanol treatment, whereas in animals pretreated with DAMME the values were significantly reduced to 0.55 +/- 0.13 and 0.65 +/- 0.15. These results confirm a link between the gastroprotection produced by opioids and the modulation of histaminergic activity in the rat stomach.

Dynorphin B-like immunoreactivity in gastroduodenal biopsy specimens from gallstone patients.

Dynorphin B-like immunoreactivity (ir-dyn B) was measured by a validated radio-immunoassay in gastroduodenal biopsy specimens from control and gallstone patients. Levels were significantly lower in acetic acid extracts of specimens of the transverse portion of the duodenum from gallstone patients. Gel permeation chromatography showed that almost all ir-dyn B in duodenal samples corresponded to a molecular form co-eluting with authentic dyn B. Duodenal extracts from gallstone patients had less of this form. Reverse-phase high performance liquid chromatography of the pooled gel chromatography fractions showed up a molecular form with the same retention time as synthetic dyn B which was significantly less in fractions from duodenal extracts of gallstone patients. These results indicate the occurrence of dyn B in the human gastrointestinal tract; however, at this stage of our understanding, no causal relationship can be demonstrated with functional alterations of the biliary tree.

Effect of omega-conotoxin and verapamil on antinociceptive, behavioural and thermoregulatory responses to opioids in the rat.

This study with the rat evaluated the contribution of omega-conotoxin GVIA-(omega-CgTx) and verapamil-sensitive Ca2+ channels in behavioural, antinociceptive and thermoregulatory responses to intracerebroventricular (i.c.v.) injection of [D-Ala2,NMePhe4,Gly-ol5]enkephalin (DAMGO), [D-Pen2,D-Pen5]enkephalin (DPDPE) and dynorphin A-(1-17), which are selective agonists for putative mu, delta and kappa-opioid receptors, respectively. The rats treated with omega-CgTx (8-32 pmol i.c.v.) showed transient, dose-dependent shaking behaviour, hyperalgesia and hypothermia which gradually disappeared within 4 h. The behaviour of the rats was normal by 24 h. Histological examination of brain sections showed morphological alterations of neurons in the hippocampus, medial-basal hypothalamus and pyriform cortex. antinociception, catalepsy and thermoregulatory responses elicited by DAMGO (0.4 and 2.0 nmol) were significantly prolonged and potentiated by verapamil (20 pmol i.c.v. 15 min before) or omega-CgTx (8 pmol 24 h before). Antinociception and hypothermia induced by DPDPE were antagonized by verapamil and omega-CgTx, whereas only omega-CgTx prevented the behavioural arousal observed after DPDPE. Similarly, hypothermia induced by dynorphin A-(1-17) (5.0 nmol) and by the kappa-opioid receptor agonist U50,488H (215 nmol) was antagonized by the two Ca2+ channel blockers but only omega-CgTx prevented the barrel rolling and bizarre postures caused by the opioid peptide.

Antinociceptive activity of salmon calcitonin injected intrathecally in the rat.

Salmon calcitonin injected intrathecally in unanesthetized rats produced long-lasting, dose-dependent elevations of nociceptive threshold as measured in the hot plate test. This antinociceptive action was nonopiate in nature as it was uninfluenced by the narcotic antagonists naloxone and MR 1452; moreover, the peptide was still able to raise the nociceptive threshold in morphine-tolerant rats. It is suggested that the spinal cord may represent one of the sites of action for calcitonin-induced antinociception.

Comparison between chinese medical herb Pueraria lobata crude extract and its main isoflavone puerarin antioxidant properties and effects on rat liver CYP-catalysed drug metabolism.

Ge-gen (Radix Puerariae; RP) is used in traditional oriental medicine for various medicinal purposes. The drug is the root of a wild leguminous creeper, Pueraria lobata (Willd) Ohwi. It possesses a high content of flavonoid derivatives, the most abundant of which is puerarin (PU). Here, using the enhanced chemiluminescence technique based on horseradish peroxidase and a luminol-oxidant-enhancer reagent, we evaluated in vitro the antioxidant activity of PU and RP crude extract. Both biological samples inhibited the steady-state chemiluminescent reaction in a dose-dependent fashion. However, different inhibition mechanism were postulated, since only RP behaved like conventional antioxidants. This activity was supposed to be due the presence of compounds other than PU in the crude extract. Using each of the specific substrates to different cytochrome P450 (CYP) isoforms or the regio- and stereo-selective hydroxylation of testosterone as polyfunctional probe we found that when intragastrically administered in male Wistar rats, PU (100 or 200 mg/kg b.w.) and RP (700 or 1,400 mg/kg b.w.) significantly altered hepatic CYP-linked monooxygenases. While both CYP content and NADPH-(CYP)-c-reductase activity were significantly increased in all situations, a complex pattern of CYP modulation was observed, including both induction (PU: CYP2A1, 1A1/2, 3A1, 2C11; RP: CYP1A2, 3A1, 2B1) and inactivation (PU and RP: CYP3A, 2E1, 2B1), the latter being due to either parental agents or metabolites, as demonstrated by in vitro studies. Overall, these findings indicate that RP contains compounds with potent antioxidant activity and that both PU and RP impairs CYP-catalysed drug metabolism.

Mechanism for the prevention of cholestasis involving cytochrome P4503A overexpression.

BACKGROUND: To clarify the preventive effect of taurohyodeoxycholic acid on liver cholestasis induced by toxic bile acids in rats, we evaluated whether modulation of cytochrome P4503A-linked oxidases is involved in the hepatic bile acid retention and secretion mechanism. We investigated whether the safe or the toxic taurochenodeoxycholic acid, administered singly or together, affects cytochrome P450-catalyzed drug metabolism or biliary parameters. We also considered whether the inhibition of the P-glycoprotein export pump by vinblastine might be related to cytochrome P4503A overexpression. METHODS: Hydroxylation of testosterone and N-demethylation of aminopyrine were studied in subcellular rat liver preparations after intravenous infusion of hepatoprotective and toxic bile acids administered singly or together. Bile flow, calcium secretion, biliary enzymes activity, and secretion rates of the endogenous and administrated bile acids were determined. CYP3A-dependent monooxygenases were also measured in the same coinfusion model in the presence of vinblastine. RESULTS: Although wide modulation of the activities of different P450 subfamily of isoenzymes was seen, P4503A-associated monooxygenases showed similar patterns in the various situations, i.e., induction by taurohyodeoxycholic acid, reduction by taurochenodeoxycholic acid, and protection (intermediate induction) in the coinfusion experiments. This correlates well with biliary parameters demonstrating the hepatoprotective ability of taurohyodeoxycholic acid. Coadministration of bile acids and vinblastine significantly modifies CYP3A-linked activities. CONCLUSIONS: Bile acid structure seems to be linked with hepatotoxicity/hepatoprotection and P4503A modulation. Taurohyodeoxycholic acid could be therapeutic in cholestatic liver disease by inducing P4503A; we can hypothesize that an associated P-glycoprotein expression might facilitate biliary excretion of toxic taurochenodeoxycholic acid accumulated in the liver during cholestasis.

Review on some plants of Indian traditional medicine with antioxidant activity.

A lot of medicinal plants, traditionally used for thousands of years, are present in a group of herbal preparations of the Indian traditional health care system (Ayurveda) named Rasayana proposed for their interesting antioxidant activities. Among the medicinal plants used in ayurvedic Rasayana for their therapeutic action, some of these have been throughly investigated. In the present paper seven plants (Emblica officinalis L., Curcuma longa L., Mangifera indica L., Momordica charantia L., Santalum album L., Swertia chirata Buch-Ham, Withania somnifera (L.) Dunal) are viewed for their historical, etymological, morphological, phytochemical and pharmacological aspects. The plants described contain antioxidant principles, that can explain and justify their use in traditional medicine in the past as well as the present. In order to identify the plants with antioxidant activity in Ayurveda, a formulation of some rasayanas with well defined antioxidant properties has been examinated. For this purpose, we have considered Sharma's work on the preparation MAK4, MAK5, MA631, MA 471, MA Raja's Cup, MA Student Rasayana, MA Ladies Rasayana.

Anti-inflammatory and cicatrizing activity of Echinacea pallida Nutt. root extract.

Among the different species belonging to the Echinacea family, largely used in traditional medicine, Echinacea pallida, Echinacea purpurea and Echinacea angustifolia were investigated. These different species, due to their difficult identification, were commonly confused in the past and probably used indifferently for the same therapeutic purposes. In fact, the three species have in common, some pharmacological activities, based on the presence of active compounds that act additively and synergistically. Nevertheless, the composition of each species has slight variation in the amount of each active component. In particular, echinacoside, a caffeoyl derivative, is present in E. pallida and only in traces in E. angustifolia. It seems to have protective effects on skin connective tissue and to enhance wound healing. The anti-inflammatory and wound healing activities of echinacoside, compared with the ones of the total root extract of E. pallida and E. angustifolia, were examined in rats, after topical application. The tissues of the treated animals were evaluated after 24, 48 and 72 h treatment and excised for histological observation at the end of the experiment. Results confirm the good anti-inflammatory and wound healing properties of E. pallida and of its constituent echinacoside, with respect to E. purpurea and control. This activity probably resides in the antihyaluronidase activity of echinacoside.

Efficacy of different Cynara scolymus preparations on liver complaints.

Cynara scolymus leaves extracts have long been used in folk medicine for their choleretic and hepatoprotective activities, that are often related to the cynarin content. These therapeutic properties are also attributed to mono- and di-caffeoylquinic acids and since commercial C. scolymus preparations can differ for their activities, we studied four extracts to evaluate, if present, a relationship between the hepatobiliary properties of the different preparations and their content in phenolics. The antioxidant activity of the commercial preparations examined was also considered in an in vitro system. The results showed that the extract with the highest content in phenolic derivatives (GAE) exerted the major effect on bile flow and liver protection. Also the results of the antioxidant capacity (BR) of the different preparations are in good agreement with the results obtained in vivo. On the contrary, administering rats with doses of chlorogenic acid, equivalent to those present in this extract, we did not observe any choleretic or protective action. An histopathological analysis of liver sections confirmed the biochemical results. Perhaps caffeoyl derivatives have a role in the therapeutic properties of C. scolymus extracts, as reported in literature for "in vitro" studies, but when administered alone, they are not so effective in exerting this action.

Analgesic and antiinflammatory activity of Cyclamen repandum S. et S.

According to folk medicine some species belonging to the genus Cyclamen were used for their biological activities. Early investigation of the different species of the genus resulted in the isolation of triterpenic saponins. No phytochemical and biological data are available on C. repandum. As part of a series of phytochemical investigations for bioactive compounds from medicinal plants, Cyclamen repandum S. et S. was investigated. The present study sought to find the antiinflammatory and antinociceptive activities of C. repandum tubers in rats and mice. A preliminary screening was conducted with three different extracts in the tests used, particularly the paw edema and the writhing tests. Subsequently some saponins isolated from the ME extract, the more effective one, have been identified. This paper also describes the results of fractionation and bioassay guided chemical studies. Chemical investigation of the active extract afforded the isolation and characterization of six triterpenic saponins. The possible antiinflammatory and analgesic properties were investigated as the saponin content of the fractions allows to speculate on such aspect.

Effects of differential extraction of Verbena officinalis on rat models of inflammation, cicatrization and gastric damage.

Verbena officinalis L. is used in folk medicine for the treatment of inflammatory disorders, skin burns, abrasions, and gastric diseases. Extracts obtained with different solvents (methanol, VoME; enriched flavonoids, VoEF; supercritical CO2, VoCO2) were evaluated for anti-inflammatory, gastroprotective and cicatrizing activities. Additionally, the antioxidant capacity was determined in vitro. In order to confirm the activities investigated, histological observations were performed. All extracts induce a remarkable anti-inflammatory activity. The gastric damage is significantly reduced by all extracts administered, whereby the most pronounced protection is observed for the VoCO2 and VoEF extracts. Finally, a wound healing effect is obtained particularly by the CO2 extract, suggesting the presence of some lipophilic active principles. Histological evidence confirms the results evaluated with the animal procedures. The results obtained after oral administration of V. officinalis extracts are also in agreement with the antioxidant capacity evaluated in vitro, confirming the relationship between pharmacological activities and antiradical efficacy.

Cyanidin-3-O-beta-glucopyranoside, a natural free-radical scavenger against aflatoxin B1- and ochratoxin A-induced cell damage in a human hepatoma cell line (Hep G2) and a human colonic adenocarcinoma cell line (CaCo-2).

Recent findings have suggested that oxidative damage might contribute to the cytotoxicity and carcinogenicity of aflatoxin B1 (AFB1). Induction of oxidative stress also plays an important role in the toxicity of another mycotoxin, ochratoxin A (OTA). In the present study, the protective effect of cyanidin-3-O-beta-glucopyranoside (C-3-G; an anthocyanin contained in oranges, blackberries, strawberries and cranberries) against AFB1- and OTA-induced cytotoxicity was investigated in a human hepatoma-derived cell line (Hep G2) and a human colonic adenocarcinoma cell line (CaCo-2). The ability of C-3-G to reduce the production of reactive oxygen species (ROS), the inhibition of protein and DNA synthesis and the apoptosis caused by the two mycotoxins was also investigated in both cell lines. Our experiments proved the significant cytoprotective effect of C-3-G in vitro against OTA- and AFB1-induced cell damage. In particular, 24 h of pretreatment with 50 microm-C-3-G inhibited the cytotoxicity of 10 microm-AFB1 (by 35 %) and of 10 microm-OTA (by 25 %) in Hep G2 cells (P < 0.001) and of 10 microm-AFB1 (by 10 %, P < 0.01) and of 10 microm-OTA (by 14 %, P < 0.05) in CaCo-2 cells. Moreover, 50 microm-C-3-G attenuated ROS production induced by the two toxins in both cell lines (P < 0.05). Inhibition of DNA and protein synthesis induced by the mycotoxins was counteracted by pretreatment with the antioxidant at 50 microm. Similarly, apoptotic cell death was prevented as demonstrated by a reduction of DNA fragmentation and inhibition of caspase-3 activation. The in vitro free-radical scavenging capacity of the anthocyanin was tested with the Briggs-Rauscher oscillating reaction. This system works at pH approximately 2. The results showed good scavenging power, in accordance with the observed inhibition of ROS production.

An approach to the study of the biological activity of Eschscholtzia californica Cham.

Pharmacological activities of Eschscholtzia californica Cham. are not yet well known. The aim of this work is to verify the pharmacological properties and to get a first identification of the active principles.