Mitsue Haraguchi

Colangiopatia associada a cristais em ovinos alimentados com Brachiaria decumbens que contém a saponina protodioscina

An outbreak of hepatogenous photosensitization is reported in a flock of 28 sheep grazing Brachiaria decumbens in Mato Grosso do Sul State, Central-Western Brazil. Seven lambs and an adult sheep were affected and 6 of them died. Two surviving affected lambs and one lamb without clinical signs had increased serum values of gamma glutamyltransferase, bilirubin, and cholesterol. In two adult unaffected sheep those parameters were within normal values. An adult sheep submitted to necropsy presented moderate body condition, unilateral corneal opacity, drying of the muzzle, moderate jaundice, increased lobular pattern of the liver, and a distended gallbladder. Histological lesions were epithelial degeneration, necrosis, and hyperplasia of small bile ducts. Mild amounts of foamy macrophages were observed, mainly in the centroacinar zone. Diffuse swelling and vacuolation were observed in hepatocytes. Crystal negative images were found within bile ducts, foamy macrophages, and the lumen of some renal tubules. The heart showed multifocal areas of degeneration and necrosis of the muscle fibers. Pasture samples (Brachiaria decumbens) contained 2.36% of protodioscin. No Pithomyces chartarum spores were found in the pasture. Samples from a similar neighboring B. decumbens pasture grazed by cattle without photosensitization contained 1.63% of protodioscin isomers. Outbreaks of photosensitization caused by Brachiaria spp. are common in cattle in the Brazilian Cerrado (savanna) with about 51 million hectares of Brachiaria spp pastures. Sheep farming has been recently developed in this region, and the number of sheep is increasing significantly. Because sheep are more susceptible than cattle to lithogenic saponins, poisoning by Brachiaria should be an important limiting factor for the sheep industry.

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.

Steroidal saponin concentrations in Brachiaria decumbens and B. brizantha at different developmental stages

Brachiaria species contain steroidal saponins and are involved in outbreaks of hepatogenous photosensitization. This research presents the levels of a steroidal saponin, protodioscin, in the seeds and aerial parts of B. brizantha and B. decumbens during different developmental stages (growth, bloom, fructification and seed fall). The butanolic fraction of the ethanolic extract of each stage was submitted to thin layer chromatography (TLC) and spectrophotometric analysis through the Ehrlich reagent in 515nm. The chromatograms in TLC of the butanolic fraction of B. brizantha and B. decumbens showed similar spots as the protodioscin standard. The estimated level of protodioscin isomers in B. brizantha and B. decumbens ranged from 0.5% to 2.1%, having the highest level at the end of their developmental stages during seed falling comparison with the previous one. Protodioscin was not detected in the seeds. Outbreaks of Brachiaria spp. poisoning in central Western Brazil are frequently observed in pastures that had been more than 30 days without animals grazing, and also during the growing or blooming stage of the pastures. Other saponin determinations in toxic and non toxic pastures are necessary to determine the saponin concentrations that cause intoxication.

Aspectos epidemiológicos da seneciose na região sul do Rio Grande do Sul

Seneciosis is the main cause of livestock mortality due to poisonous plants in the State of Rio Grande do Sul, Brazil. This paper presents epidemiological data of 24 outbreaks in cattle and one in horses, diagnosed by the Regional Diagnostic Laboratory at Pelotas University in southern Rio Grande do Sul, from 1998 to 2000. Additionally, data of 54 outbreaks which occurred in 1978-1997 were analyzed. The farms where outbreaks occurred in 1998-2000 were visited to obtain clinical and epidemiological data and to verify the presence of Senecio spp. Eleven outbreaks (45.83%) affected cattle up to 3 years of age, and 13 (54.16%) cattle over 3 years. Nine (37.5%) outbreaks affected females and 15 (62.5%) males. Ten (41.66%) outbreaks occurred in spring, 4 (16.66%) in summer, 5 (20.83%) in autumn, and 5 (20.83%) in winter. Morbidity rate was estimated with 4.92% and case fatality with 95.59%. The predomionating Senecio species were S. brasiliensis on 12 farms (57.14%), S. selloi on 10 (47.61%), S. oxyphyllus on 6 (28.57%), S. heterotrichius on 3 (14.28%), and S. leptolobus on 1 farm (4.76%). The main clinical signs were progressive emaciation, incoordination, diarrhea, tenesmus, rectal prolapse and aggressiveness. The clinical manifestation periods of affected cattle observed during the farm visits, or of cattle sent for post-mortem examination,were 24-96 hours in 4 outbreaks (16.66%), 4-7 days in 7 (29.16%), 1-2 weeks in 4 (16.66%), 2-3 weeks in 2 (8.33%), 1-2 months in 2 (8.33%), and 2-3 months in 1 outbreak (4.16%). In 4 outbreaks (16.66%) the clinical manifestation period was not established. In outbreaks with longer clinical manifestation periods some animals showed photosensitization. The main necropsy findings were a hard and enlarged liver, distended gall bladder, edema of the mesenterium and abomasum, and increased amount of liquid in the cavities. Histopathological findings were fibroplasia, megalocytosis and biliary ductal proliferation of the liver, and spongy degeneration of the cerebral white matter. Samples of different Senecio species, in different seasons, were analyzed for detection of pyrrolizidine alkaloids (PAs) by thin layer chromatography. Retrorsine was found in Senecio brasiliensis, S. heterotrichius, S. selloi and S. oxyphyllus. In S. brasiliensis and S. heterotrichius one and two more non-identified PAs were detected, respectively. The total PAs concentration by spectrophotometric method was 0.25% for S. brasiliensis, 0.19% for S. heterotrichius, 0.03% for S. oxyphyllus, and 0.03% for S. selloi. The highest PAs concentration occurred in winter (June/July). No alkaloids were found in samples of S. leptolobus. These results show that S. brasiliensis is the most important cause of seneciosis in southern Rio Grande do Sul. Additional data obtained dealt with 54 outbreaks of PAs poisoning in 1978-1997. During this period, 7 outbreaks (12.96%) affected cattle up to 3 years of age, 39 (72.22%) cattle over 3 years, and 3 outbreaks (5.55%) affected cattle of different ages. In 5 outbreaks (9.25%) the age was not informed. Seven outbreaks (12.96%) affected males, 39 (72.22%) females, 3 (5.55%) both sexes, and in 5 outbreaks (9.25%) the sex was not informed. Twenty-three outbreaks (42.59%) occurred in spring, 9 (16.66%) in summer, 9 (16.66%) in autumn, and 13 (24.07%) in winter. The greater number of outbreaks during 1998-2000 (24 outbreaks in 3 years) in regard to 1978-1997 (54 outbreaks in 20 years) is probably due to a decrease of more than 50% in the number of sheep in the region.

Enterolosaponins A and B, novel triterpene bisdesmosides from Enterolobium contortisiliquum, and evaluation for their macrophage-oriented cytotoxic activity.

Two novel triterpene bisdesmosides, designated as enterolosaponin A (1) and B (2), were isolated from Enterolobium contortisiliquum. The chemical structures of 1 and 2 were determined by analysis of their extensive spectroscopic data, as well as hydrolysis followed by chromatographic study. Enterolosaponins have a 2-amino-2-deoxy-D-glucosyl unit (D-glucosamine) as one of the monosaccharides constituting their oligosaccharide moieties, which have been rarely found in natural product research. Enterolosaponin A (1) exhibited a highly selective cytotoxicity against BAC1.2F5 mouse macrophages, and it should be notable that the macrophage death caused by 1 was shown to be neither necrotic nor due to induction of apoptosis from morphology of the died cells, whose cytosol occurred in vacuolation.

Immunomodulatory effects of Pteridium aquilinum on natural killer cell activity and select aspects of the cellular immune response of mice

Pteridium aquilinum (bracken fern) is one of the most common plants. Epidemiological studies have revealed a higher risk of certain types of cancers (i.e., esophageal, gastric) in people who consume bracken fern directly (as crosiers or rhizomes) or indirectly through the consumption of milk from livestock that fed on the plant. In animals, evidence exists regarding the associations between chronic bracken fern intoxication, papilloma virus infection, and the development of carcinomas. While it is possible that some carcinogens in bracken fern could be responsible for these cancers in both humans and animals, it is equally plausible that the observed increases in cancers could be related to induction of an overall immunosuppression by the plant/its various constituents. Under the latter scenario, normal tumor surveillance responses against nascent (non-bracken-induced) cancers or responses against viral infections (specifically those linked to induction of cancers) might be adversely impacted by continuous dietary exposure to this plant. Therefore, the overall objective of this study was to evaluate the immunomodulatory effects of bracken fern following daily ingestion of its extract by a murine host over a period of 14 (or up to 30) days. In C57BL/6 mice administered (by gavage) the extract, histological analyses revealed a significant reduction in splenic white pulp area. Among a variety of immune response parameters/functions assessed in these hosts and isolated cells, both delayed-type hypersensitivity (DTH) analysis and evaluation of IFNγ production by NK cells during TH1 priming were also reduced. Lastly, the innate response in these hosts—assessed by analysis of NK cell cytotoxic functionality—was also diminished. The results here clearly showed the immunosuppressive effects of P. aquilinum and that many of the functions that were modulated could contribute to the increased risk of cancer formation in exposed hosts.

Cytotoxic effects of butanolic extract from Pfaffia paniculata (Brazilian ginseng) on cultured human breast cancer cell line MCF-7.

Roots of Pfaffia paniculata have been well documented for multifarious therapeutic values and have also been used for cancer therapy in folk medicine. This study has been performed in a human breast tumor cell line, the MCF-7 cells. These are the most commonly used model of estrogen-positive breast cancer, and it has been originally established in 1973 at the Michigan Cancer Foundation from a pleural effusion taken from a woman with metastatic breast cancer. Butanolic extract of the roots of P. paniculata showed cytotoxic effect MCF-7 cell line, as determined with crystal violet assay, cellular death with acridine orange/ethidium bromide staining, and cell proliferation with immunocytochemistry of bromodeoxyuridine (BrdU). Subcellular alterations were evaluated by electron microscopy. Cells treated with butanolic extract showed degeneration of cytoplasmic components and profound morphological and nuclear alterations. The results show that this butanolic extract indeed presents cytotoxic substances, and its fractions merit further investigations.

Steroidal saponins from the leaves of Cestrum sendtenerianum.

Five steroidal saponins were isolated from the EtOH extract of Cestrium sendtenerianum (Solanaceae), as confirmed by detailed analysis of their 1H, 13C, and two-dimensional NMR spectral data, and by the results of hydrolytic cleavage. The saponins were revealed to contain three hydroxyl groups at the C-1beta, C-2alpha, and C-3beta positions in the spirostanol skeleton, and to bear a di- or triglycoside at C-3 as the common structural features. One of the compounds, a spirostanol triglycoside, showed weak cytotoxic activity on HL-60 human promyelocytic leukemia cells, with an IC50 value of 7.7 microg/ml.

Stryphnosides A–F, six new triterpene glycosides from the pericarps of Stryphnodendron fissuratum

Abstract Six new triterpene glycosides stryphnosides A–F ( 1 – 6 ) were isolated from the pericarps of Stryphnodendron fissuratum (Leguminosae). The structures of 1 – 6 were determined on the basis of extensive spectroscopic analysis, including two-dimensional (2D) NMR data, and the results of hydrolytic cleavage. The sugar moieties of 3 – 6 are very unique in structure having not only novel sugar sequences but also the terminal α- l -arabinopyranosyl unit with a 1 C 4 conformation. Stryphnosides C ( 3 )–F ( 6 ) are the first representative of naturally occurring glycosides with the 1 C 4 terminal α- l -arabinopyranosyl group.

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.