Toxicological Effects of Aqueous Extract From African Walnut ( Tetracarpidium conophorum) Leaves in Rats.

Tetracarpidium conophorum leaves are used in traditional medicine for the treatment of male infertility, without considering its toxicity and side effects. In this study, we investigated the effects of T conophorum leaves on some biochemical parameters such as alanine aminotransferase, aspartate aminotransferase, bilirubin, albumin, creatinine, and uric acid. Histology of the liver and kidney were also assessed. The result revealed that the alanine aminotransferase and aspartate aminotransferase levels of the control group were not significantly different from the experimental groups. There was no significant difference in the albumin and bilirubin levels of the control and experimental groups. Similarly, the uric acid and creatinine levels of the experimental rats were not significantly different from the control. The examination of liver and kidney sections did not show any morphological changes and inflammatory cell infiltrations. These findings suggest that the leaves did not induce any pathological changes at the doses tested.

Detection of calprotectin and apoptotic activity within the equine colon from horses with black walnut extract-induced laminitis.

The black walnut extract (BWE) model of equine laminitis is associated with a systemic inflammatory response manifest by increased expression of inflammatory cytokines in the lungs and liver as well as the laminae. The specific role of the gastrointestinal tract in development of this response is unclear and is of utmost importance, as gastrointestinal disease and laminitis are intimately related. We investigated calprotectin expression and epithelial and endothelial apoptosis in the colon of horses exposed to orally administered BWE. Sections of colon from 19 horses including 7 controls not exposed to BWE, 6 horses at the developmental time-point of leukopenia (DTP) and 6 at the onset of Obel grade 1 laminitis (LAM) after BWE-administration were histologically examined. Immunohistochemical evaluation for calprotectin expression with MAC 387 antibody was performed along with assessment of epithelial and endothelial apoptosis with caspase-3 active antibody. Calprotectin expression and percentage of apoptotic cells were compared between controls and the two treatment groups and presence of a correlation between calprotectin expression and apoptosis was evaluated. Histological findings from BWE-treated horses included eosinophil and lymphocyte epitheliotropism. The DTP group had a higher (p<0.01) calprotectin score with respect to the control group, while there was no significant difference in percentage of epithelial and endothelial apoptotic cells between groups (p=0.08 and p=0.48 respectively). No significant correlation was found between calprotectin score and epithelial or endothelial apoptosis (p=0.69 and p=0.29 respectively). There is preliminary evidence that exposure of horses to BWE results in an early inflammatory response in the colon. Further studies are needed to characterize the nature of the colonic injury in BWE-exposed horses and the link to the development of laminitis.

Presence of mononuclear cells in normal and affected laminae from the black walnut extract model of laminitis.

REASONS FOR PERFORMING STUDY: There is increasing evidence of involvement of inflammatory cells in acute laminitis. OBJECTIVE: To immunolocalise monocytes/macrophages and B and T lymphocytes in the laminar tissue of normal horses and those with black walnut extract (BWE)-induced laminitis. METHODS: Immunohistochemistry was used in archived laminar tissue samples from 20 horses divided equally into 4 groups: control animals (CON), and those administered BWE at 1.5 h (1.5H DTP group), at the onset of leucopenia (3H DTP group) and at the onset of lameness (LAM group). Antibodies against CD3, CD20 and CD163 were used to recognise lymphocytes (T and B) and monocytes/macrophages, respectively. RESULTS: Mononuclear cells were present in laminar tissue of normal horses. The majority of CD3- and CD20-positive lymphocytes were localised around the deep dermal vessels but were also evident around vessels of the primary dermal laminae. CD163-positive macrophages were primarily perivascular in deep dermis or in dermal laminae. No changes in the number of laminar B or T lymphocytes occurred at any time point post BWE administration. However, increases (P=0.0016) in laminar CD163-positive cells occurred in the secondary dermal laminae (SDL) in the 1.5H DTP and 3H DTP groups, returning to basal values in LAM group. CONCLUSIONS: Lymphocyte and macrophage populations are present in the laminar tissue of clinically normal horses and BWE administration induces an increase in CD163-positive macrophages in SDL. POTENTIAL RELEVANCE: Both the host tissue population of mononuclear cells and the influx of monocytes may play an important role in the pathophysiological changes leading to laminar injury.

Equine neutrophil elastase in plasma, laminar tissue, and skin of horses administered black walnut heartwood extract.

Laminitis is a local manifestation of a systemic inflammatory response that is characterized by neutrophil activation and movement of neutrophils into the laminar tissues. Given the evidence for the involvement of neutrophils in the development of laminitis, we measured concentrations of neutrophil elastase, a serine protease released from the azurophilic granules of neutrophils, in plasma, skin and laminar tissues obtained from control horses and horses given black walnut heartwood extract (BWHE) to induce laminitis. Healthy horses (5-15 years old) were randomly assigned to 4 groups: 3 experimental groups given BWHE via nasogastric tube, and a control group given an equal volume of water. The experimental groups consisted of horses euthanized 1.5h (n=5), 3h (n=6) or 12h (n=10) after BWHE administration. Control horses (n=7) were euthanized 12h after intragastric administration of water. Plasma samples were collected in all horses of the control and 12h BWHE groups at 0, 1, 2, 3, 4, 6, 8, 10, and 12h after treatment, and laminar tissue and skin from the middle region of the neck were harvested at the time of euthanasia in all 1.5 and 3h BWHE horses, in 6 of the 12h BWHE horses and in 5 of the control horses. Plasma and tissue concentrations of neutrophil elastase were determined using an equine specific ELISA, and statistical significance was set at p<0.05. Plasma concentrations of neutrophil elastase in the BWHE group were significantly higher at 6 and 8h compared to the control group and at 8 and 10h compared to time 0. Concentrations of neutrophil elastase in skin and laminar tissue were significantly higher in the 3 and 12h BWHE groups compared to the control group. Concentrations of neutrophil elastase were significantly higher in the skin than in the lamina in the 12h BWHE horses. The administration of BWHE thus results in significant increases in the concentration of neutrophil elastase in the circulation, skin and laminar tissue. These results confirm a role for neutrophils in the developmental phase of laminitis, and the systemic nature of the inflammatory process. Furthermore, neutrophil elastase may play a key role in the disintegration of the hoof basal membrane and be a target for the development of new treatments for laminitis.

Evaluation of genotoxicity of medicinal plant extracts by the comet and VITOTOX tests.

We report the results of our genotoxic evaluation of extracts from three medicinal plants Acacia nilotica, Juglans regia, and Terminalia chebula and the herbal drug Triphala employing the VITOTOX and comet tests.These tests detect DNA damage in prokaryotic and eukaryotic test systems, respectively. In the VITOTOX test, none of the extracts were identified as genotoxic. In the comet assay, extracts of Acacia nilotica showed statistically significant DNA damage only in a concentration of 2500 ppm (highest tested dose), whereas extracts from Juglans regia showed significant damage in concentrations above 250 ppm and more. Extracts from Terminalia chebula and Tripahala significantly increased DNA damage in a concentration above 500 ppm. This is not considered contradictory, because DNA damage in the alkaline comet assay may not be permanent and hence may not necessarily result in mutations. All the extracts were previously found in the Ames assay to have potent antimutagenic effects against the direct acting mutagens NPD, sodium azide, and the S9-dependent mutagen 2-AF. The results of the previous study using the Ames assay are in conformity with those of the VITOTOX test. It was found that the extracts were safe in concentrations of up to 1000 microg/0.1 mL and 2500 microg/0.1 mL. A literature survey also showed that plant extracts can be mutagenic as well as antimutagenic depending on the test system used. This indicates that a battery of assays is needed before any conclusion can be reached.