Toxicity study about a medicinal plant Casearia sylvestris: A contribution to the Brazilian Unified Health System (SUS).

ETHNOPHARMACOLOGICAL RELEVANCE: Casearia sylvestris S.w (Salicaceae) is catalogued by the Brazilian Unified Health System as a plant of interest for the Brazilian population with the purpose of treating inflammatory disorders, such as pain and gastrointestinal disorders based on the folk use and some literature about efficacy; however, no toxicological studies concerned the safety of extract fluid of this plant have been reported. AIM OF THE STUDY: The present study was carried out to evaluate the acute and subchronic toxicity of the hydroethanolic extract fluid (FE) obtained from leaves of C. sylvestris in Wistar rats. MATERIALS AND METHODS: In the acute toxicity test three female Wistar rats were treated with a single dose of FE (2000 mg/kg) administered by oral gavage and observed for 14 days in order to identify signs of toxicity or death. In subchronic toxicity study animals received, by daily gavage three doses 60, 120 and 240 mg/kg of the FE of the plant for 28 and 90 days. The animals were observed daily for clinical signs and mortality. Body weight and food consumption were measured weekly and at the end of treatment were analysed hematological, biochemical and histopathological parameters. Also was analysed the cellularity of bone marrow and spleen. Moreover, phytochemical analysis by HPLC-PDA-ESI(+)/MS and CG/MS/EI was carried out to qualify the constituents of the extract. RESULTS: The results of acute study indicated that the LD50 is higher than 2000 mg/kg and at 28 and 90 day oral toxicity showed that there were no toxic effects detected in any of the parameters evaluated: body weight and relative organ weight, general behavioral changes, haematological and biochemical parameters and histopathological examination. The analysis by HPLC-PDA-ESI(+)/MS and CG/MS/EI identified the flavonoids rutin, quercetin and luteolin and also chlorogenic on the extract. CONCLUSION: Based on this study the hydroethanolic fluid extract of C. sylvestris could be safe even when used over a long period for therapeutic uses proposed by the Brazilian Unified Health System.

Selenium reverses Pteridium aquilinum-induced immunotoxic effects.

We have previously shown that bracken fern (Pteridium aquilinum) has immunomodulatory effects on mouse natural killer (NK) cells by reducing cytotoxicity. Alternatively, it has been demonstrated that selenium can enhance NK cell activity. Therefore, the aims of the present study were to evaluate if ptaquiloside, the main toxic component found in P. aquilinum, is responsible for the immunotoxic effects observed in mice, and if selenium supplementation could prevent or even reverse these effects. Male C57BL/6 mice were administered the P. aquilinum extract by daily gavage for 30 days, and histological analyses revealed a significant reduction in splenic white pulp area that was fully reversed by selenium treatment. In addition, mice administered ptaquiloside by daily gavage for 14 days demonstrated the same reduction of NK cell activity as the P. aquilinum extract, and this reduction was prevented by selenium co-administration. Lastly, non-adherent splenic cells treated in vitro with an RPMI extract of P. aquilinum also showed diminished NK cell activity that was not only prevented by selenium co-treatment but also fully reversed by selenium post-treatment. The results of this study clearly show that the immunosuppressive effects of P. aquilinum are induced by ptaquiloside and that selenium supplementation can prevent as well as reverse these effects.

Toxic effects of prenatal Ipomoea carnea administration to rats.

Chronic exposure of livestock to Ipomoea carnea, a toxic plant, promotes toxicosis characterized by lysosomal vacuolization of different organs, and is clinically manifested by CNS signs, abnormal endocrine and gastrointestinal functions, alteration of the immune system, and abnormal embryogenesis. The present study evaluated the effects of different doses of the plant extract on pregnant rats and their offspring after oral administration to the dams from day 6 to day 20 of gestation. Histopathology of thyroid, pancreas, liver and kidneys of dams on gestational day 21 showed characteristic vacuolization promoted by I. carnea toxicosis in these organs; the same was observed in the organs of 7-d-old pups. On the other hand, no alteration was found in these same organs of dams the 7th d after parturition. Although the lesions were reversed in the dams, the same did not occur in their pups. I. carnea administration also promoted decreased body weight, thymus atrophy and spleen enlargement in pups. The toxic principle of I. carnea (swainsonine) seems to pass through the placenta.

Effects of Ipomoea carnea aqueous fraction intake by dams during pregnancy on the physical and neurobehavioral development of rat offspring.

The effects of daily prenatal exposure to 0.0, 0.7, 3.0 and 15.0 mg/kg of the aqueous extract (AQE) of Ipomoea carnea dried leaves on gestational days 5-21 were studied in rat pups and adult offspring. The physical and reflex developmental parameters, open-field, plus-maze, social interaction, forced swimming, catalepsy and stereotyped behaviors, as well as striatal, cortical and hypothalamic monoamine levels (at 140 days of age) were measured. Maternal and offspring body weights were unaffected by exposure to the different doses of the AQE. High postnatal mortality, smaller size at Day 1 of life, reversible hyperflexion of the carpal joints and delay in the opening of both ears and in negative geotaxis were observed in the offspring exposed to the higher dose of AQE. At 60 and 90 days of age, open-field locomotion frequency was quite different between 0.0 and animals treated with 0.7 and 3.0 mg/kg AQE. No changes were observed in the plus-maze, social interaction, forced swimming, catalepsy, stereotyped behavior and central nervous system monoamines concentrations. Dams treated with the higher AQE dose showed severe cytoplasmic vacuolation in liver, kidney, pancreas and thyroid tissues, in contrast to the mild vacuolation observed in the other experimental groups. No alterations were observed in the histopathological study of the offspring of all experimental groups at 140 days of age. During adulthood, behavior was not modified in offspring exposed to the higher dose of AQE as well as no changes occurred in central nervous system neurotransmitters. The present data show that the offspring development alterations were not severe enough to produce behavioral and central monoamine level changes.

Evaluation of immunomodulatory activity of Ipomoea carnea on peritoneal cells of rats.

In the present study, animals of the experimental groups were treated with an aqueous fraction (AF) of Ipomoea carnea diluted in drinking water in order to obtain daily doses of 3gdryleaves/kg/body weight (bw) and 15g/kg/bw for 14 and 21 days, or by gavage 15g/kg/bw administered for 14 days, respectively. Peritoneal macrophages were collected and submitted to the spreading, phagocytosis, and hydrogen peroxide release tests. AF administration in drinking water for 14 and 21 days promoted increased macrophage phagocytosis activity and hydrogen peroxide release. However, the administration of 15g/kg/bw of AF by gavage for 14 days resulted in no alteration in macrophage activity. These results suggest that low dosages of Ipomoea carnea induced enhanced phagocytosis activity and hydrogen peroxide production by macrophages.

Lack of protective action of cysteine against the fetotoxic effect of monocrotaline.

Monocrotaline (MCT), a pyrrolizidine alkaloid present in Crotalaria species, has hepatotoxic, nephrotoxic, pneumotoxic and fetotoxic effects. However, the toxic effects of exposure to MCT in adult rats can be prevented by cysteine. Thus, the present study was conducted to evaluate the possible prevention by cysteine of the toxic effects of MCT on pregnant rats. Thirty-six pregnant rats were used. The females in the experimental groups were fed ration containing 0.02% MCT, 0.02% MCT + 1% cysteine, or 1% cysteine from day 6 to day 21 of pregnancy; the control group was fed only common ration for the same period of time. All rats were killed on day 21 of pregnancy and their blood was collected for determination of liver and kidney function. General toxicity to pregnant dams was assessed. Fetuses were removed by caesarian section and embryofetotoxic parameters were examined. Results showed impaired body weight gain in rats fed MCT, with or without cysteine supplementation. Plasma levels of AST, ALT, LDH, GGT, urea and creatinine were increased in MCT animals compared to controls. The pathology study revealed lesions only in dams from the MCT group. The weights of the placentas and fetuses of the MCT and MCT + cysteine groups were significantly lower than those of the control group. Thus, the present data suggests some protective action of 1% of cysteine in ration against the toxic effects of MCT on the dams but not on the litter.

Fetotoxicity and reproductive effects of monocrotaline in pregnant rats.

Four groups of 12 pregnant Wistar rats each were fed with rations containing 0, 0.01, 0.015 and 0.02% of monocrotaline (MCT) from day 6 to 21 of gestation. Liver weights of the dams from the three experimental groups were significantly lower than those from the control group. Serum levels of aspartate aminotransferase; alkaline phosphatase; lactate dehydrogenase; gamma glutamyltransferase, urea and creatinine were significantly higher in dams from MCT 0.02% group. The weights of the placenta, fetuses and fetal lungs of the 0.02% MCT group were significantly lower than those of the control group. A mild to moderate interstitial pneumonia and liver lesions were observed in dams ingesting 0.02% of MCT. These results showed the toxicity of MCT to the females that ingested 0.02% and their fetuses. Because there was no differences on the weight gains and food and water consumption of the dams it is suggested that this toxic effects in the fetuses was caused by the diffusion of MCT through the placenta. No significant differences were observed in the frequency of skeletal and visceral malformation or anomalies between the control and treated groups suggesting that MCT had no teratogenic effect.

Toxicity testing of Senna occidentalis seed in rabbits.

The effect was investigated of administering ground Senna occidentalis seeds to rabbits in different concentrations (1%, 2%, 3% and 4%) in the ration. The experiment lasted 30 days and the toxic effects of the plant were evaluated on the basis of weight gain, histopathological, biochemical and morphometric parameters, as well as histochemistry and electron microscopy. Animals that received the ration containing 4% ground S. occidentalis seeds gained less weight (p < 0.05) and died in the third week. Histopathology revealed that the heart and liver were the main organs affected, with myocardial necrosis and centrolobular degeneration. There was a reduction in cytochrome oxidase activity in the glycogenolytic fibres, together with muscle atrophy, confirmed by the morphometric studies. Electron microscopy of the liver cells revealed dilated mitochondria, with destruction of the internal cristae.

Evaluation in rabbits of the fetal effects of maternal ingestion of Solanum malacoxylon.

A study was performed to determine the possible toxic effects on the young of does that had been fed during the gestational period on a ration containing Solanum malacoxylon (Sm), a calcinogenic plant that contains a vitamin D3-glycoside conjugate. Experimental animals received a ration containing 0.03% or 0.04% of Sm leaves on days 6 to 30 of gestation. The levels of calcium, phosphorus and alkaline phosphatase in their sera, as well as their feed intake and body weight, were evaluated weekly. The does were euthanized 3 days after parturition and paraffin sections stained with haematoxylin and eosin were prepared from their heart, lungs, kidneys and aorta for histopathological examination. The young from does in the Sm 0.03% group were euthanized 3 days after birth and biochemical and histopathological determinations were performed, as described for the does. The does in both experimental groups showed decreased feed consumption and those in the Sm 0.04% group showed lower body weights throughout their gestation. Animals treated with Sm 0.04% presented a high incidence of abortion and stillbirth. There were biochemical and histopathological alterations in both experimental groups, which were more prominent in the does in the Sm 0.04% group. Litters from does treated with Sm 0.03% showed mineralization of soft tissue and an increase in phosphorus and calcium levels. These findings indicate that the vitamin D3-glycoside passes through the placental barrier to the fetus.

Muscle atrophy induced in broiler chicks by parts of Senna occidentalis seeds.

Senna occidentalis (formerly Cassia occidentalis) is a common contaminant of agricultural commodities. It is toxic to cattle and poultry, reportedly being responsible for skeletal myodegeneration in these animals. All parts of the plant present toxicity, but the seeds are the most toxic. The toxin(s) responsible for the myodegeneration have not been definitively identified, nor is it known which part of the seeds is most toxic. Intoxication by this plant leads to weight loss with considerable economic repercussions. The effects of the whole seed and of parts of S. occidentalis seeds (1% in commercial feed) were compared on the pectoralis major muscle of broiler chicks intoxicated from birth until 22 days of life. There were severe clinical signals and reduced body weight in birds that received the external tegment of the seed, whereas no adverse effects were observed in birds that received the whole seed or other parts of the seed. Histological and morphometric studies showed an intense muscle fibre atrophy (both type 1 and type 2 fibres were affected) in the group that received 1% external tegment. This study may be the first step to identifying the substance(s) involved in this pathological process.

Toxic peripheral neuropathy of chicks fed Senna occidentalis seeds.

Plants of the genus Senna (formerly Cassia) are poisonous to livestock and other laboratory animals, leading to a syndrome of a widespread muscle degeneration, incoordination, recumbence, and death. The main histologic lesion is necrosis of skeletal muscle fibers. Recently, a mitochondrial myopathy with ragged-red and cytochrome oxidase (COX)-negative muscle fibers was recognized in hens chronically intoxicated with parts of seeds of S. occidentalis. The purpose of the present work was to investigate if there was peripheral nerve involvement in the acute intoxication of chicks with S. occidentalis seeds. Teasing of individual fibers revealed signs of extensive axonal damage with myelin ovoids. Ultrathin sections confirmed the axonal damage. Axons were filled with membranes, some residual disorganized filaments, and enlarged mitochondria. In some instances the axon disappeared and there was secondary degeneration of the myelin sheath. The present work is the first description of the neurotoxic effect of S. occidentalis intoxication. Future work should attempt to determine the mechanisms involved in this neuropathy.

Mitochondrial myopathy in Senna occidentalis-seed-fed chicken.

Plants of the genus Senna (formerly Cassia) have been recognized as the cause of a natural and experimental syndrome of muscle degeneration frequently leading to death in animals. Histologically, it demonstrated skeletal and cardiac muscle necrosis, with floccular degeneration and proliferation of sarcolemmal nuclei. Recently, it was described as an experimental model of mitochondrial myopathy in hens chronically treated with Senna occidentalis. Currently, skeletal muscles of chicks intoxicated with seeds of the poisonous plant S. occidentalis were studied by histochemistry and electron microscopy. Since birth, the birds were fed ground dried seeds of this plant with a regular chicken ration at a dose of 4% for 11 days. Microscopic examination revealed, besides muscle-fiber atrophy, lipid storage in most fibers and a moderate amount of cytochrome oxidase-negative fibers. By electron microscopy, enlarged mitochondria with disrupted or excessively branched cristae were seen. This picture was characteristic of mitochondrial myopathy. These findings have hitherto remained unnoticed in skeletal muscle of young birds treated with S. occidentalis.

Experimental mitochondrial myopathy induced by chronic intoxication by Senna occidentalis seeds.

Histochemical and electron microscopic studies of biceps femoris, pectoralis major and rectus femoris of chronically treated birds with seeds of the poisonous plant Senna occidentalis (0.2% external/internal tegment), were performed. The muscles had similar features of human mitochondrial myopathy as ragged-red fibers, cytochrome-oxidase negative fibers, and weak activity of the oxidative enzymes. Fibers with lipid storage were also present. Acid phosphatase activity in rare muscle fibers was also detected, and represents probably a secondary degenerative process. By electron microscopy, enlarged mitochondria with disrupted or excessively branched cristae were seen. The present study presents a new experimental model of mitochondrial myopathy that may be useful for the best knowledge of this group of diseases and for experimental trials of drugs that could reverse the mitochondrial impairment in the mitochondrial myopathies.

Behavioral effects of acute and long-term administration of catnip (Nepeta cataria) in mice.

Catnip or catmint (Nepeta cataria) is a plant used extensively to treat human diseases and in toys for pets. We investigated the effects of acute and long-term administration of the plant on some behaviors of mice. The plant was fed as 10% of the normal diet for 2 h/d for 1 or 7 d. Acute and long-term dosing increased both rearing and locomotion frequencies observed in an open field. Acute exposure to catnip increased stereotyped behavior and susceptibility to seizures, did not interfere with haloperidol-induced catalepsy, and decreased sleeping time after sodium pentobarbital administration. Long-term exposure induced tolerance to stereotypic behavior, catalepsy and sleeping time, and increased the susceptibility to seizures induced by picrotoxin and strychnine. An amphetamine-like effect of catnip was suggested to explain the acute effects, while dispositional and functional adaptative changes were considered involved with the long-term effects.

The acute toxicity of Riedeliella graciflora in laboratory animals.

Riedeliella graciflora is a plant mainly found in southeast South America and related to bovine mortality in Sao Paulo State, Brazil. Our laboratory showed that cattle intoxicated with 10 g R graciflora leaves/kg had organ congestion, edema and hemorrhage. The aim of this study was to determine which laboratory animal species would present the same lesions found in cattle. We also studied the effects of administering parts of the fruits or leaves po, sc, ip or im. All the laboratory species developed organ congestion, edema and hemorrhage and severe nephrosis regardless which plant part was given or the route of administration used. The effects were more intense in animals dosed with R graciflora fruits ip.

Effect of CNS depressant drugs on acute intoxication from Palicourea marcgravii St Hill in rats.

Palicourea marcgravii (Pm) is a highly toxic plant to cattle in Brazil. Previous experiments showed that Pm intoxication in rats is similar to that reported for cattle. The present study was undertaken to determine if central nervous system depressant drugs protected animals against Pm-induced seizures. Doses of 30.0 mg diazepan/kg or 2.5 or 5.0 mg xyalzine/kg increased the ET50 for seizures. Only 4.0 mg chloral hydrate/kg + 10.0 mg xylazine/kg protected animals from death. Rats treated with sodium pentobarbital, diazepan or chlorpromazine died in depression after acute Pm intoxication. The combination of 2.0 choral hydrate/kg + 10.0 mg xylazine/kg protected rats against the Pm toxic effect, since all treated animal survived.

The relationships between neuronal activity and behavioral seizures induced by Palicourea marcgravii in rats.

To characterize the convulsions induced by an aqueous extract of Palicourea marcgravii (Pm), male Wistar rats were injected sc with 3.75 g/kg of the extract, and electroencephalogram and behavior were observed for periods up to 180 min after the Pm injections. Sleep spindles and generalized convulsive seizures were observed following Pm administration. Spiking activity was detected in both cortical and hippocampus recordings. They lasted approximately 2 min and were followed by a period of postical depression and/or death. These results agreed with previous behavioral experiments which suggested the toxic effect of Pm aqueous extract was mainly at the central nervous system level.

Effects of a Palicourea marcgravii leaf extract on some dopamine-related behaviors of rats.

The effects of a Palicourea marcgravii leaf extract on some dopamine-related behaviors were studied in rats. The extract given subcutaneously decreased both spontaneous locomotion and rearing frequencies of rats observed in open-field studies and increased their periods of immobility. The extract was also able to produce a rightward displacement of the apomorphine dose-response curve for stereotyped behavior and decrease the maximum response possible. Although the extract (1.87 g/kg subcutaneously) was unable to produce true catalepsy by itself, it potentiated that induced by haloperidol. These results with the extract can be interpreted to be due to a direct blocking action for the extract on a mesostriatal dopamine receptor or to an indirect effect on dopamine pathways through central cholinergic activation.