Diclofenac in Gyps vultures : a molecular mechanism of toxicity

Over the last decade, three species of Gyps vultures on the Asian subcontinent have declined dramatically in population numbers, some as much as 97 to 99%. Although the initial cause was believed to be infectious, it was later shown to be due to an inadvertent exposure to diclofenac via the food chain. In order to protect the remaining wild vultures, diclofenac needed to be removed from the food chain. Unfortunately the Indian government was reluctant to ban diclofenac until an alternate veterinary non-steroidal anti-inflammatory drug (NSAID) that was both safe in vultures and effective in cattle could be identified. Although meloxicam was tentatively identified as this drug, toxicity testing still needed to be undertaken. Using a previously validated model, two studies were undertaken to determine the acute toxic effect of diclofenac in vulture as well as to ascertain if the drug had the potential to accumulate. In the first study, meloxicam in formulation was shown to be safe as a single oral dose up to 2mg/kg in African White Backed-Vultures (Gyps africanus). To further demonstrate the safety of food borne meloxicam, vultures were exposed to meat rich in meloxicam residues, with once again no signs of toxicity being evident. In the second study the drugs ability to accumulate was evaluated pharmacokinetically in Cape Griffon Vultures (Gyps corprotheres). From this study meloxicam was shown to have a very short half-life of elimination, making it unlikely that the drug could be a cumulative toxin. This was subsequently confirmed clinically by the absence of toxicity in birds receiving repeated doses of meloxicam. Although meloxicam was shown to be adequately safe, the safety of other veterinary NSAIDs still required elucidation. While further testing in vultures would have been possible, the small population size of the various vulture species made this unethical. Therefore a surrogate species needed to be identified. With the domestic chicken (Gallus domesticus) being commonly available, attempts were made to validate the chicken as a model. Although the dosed chickens did show similar toxicity patterns from clinical pathology to histopathology, a major problem was their higher tolerance making it impossible to use them as a surrogate. It was, however, concluded that the domestic chicken may be used in mechanistic studies in an attempt to establish an in vitro model. From the mechanistic studies both diclofenac and meloxicam were directly toxic to chicken and vulture renal tubular epithelial cells following 48h of incubation. It was later shown that this toxicity was associated with an increased production of reactive oxygen species (ROS), which could be temporarily ameliorated by pre-incubation with uric acid due to its anti-oxidant activity. When cultures were incubated with either drug for only two hours, meloxicam showed no toxicity in contrast to the cellular toxicity present for diclofenac. In both cases no increase in ROS production was evident. In addition diclofenac influenced the excretion of uric acid by interfering with p-amino-hippuric acid channels. The effect on uric acid excretion persisted after the removal of the diclofenac. It was therefore concluded that vulture susceptibility to diclofenac results from a combination of an increase in cellular ROS, a depletion of intracellular uric acid concentration and most importantly the drug’s long half-life in the vulture. Unfortunately the importance of the drug’s half-life in the toxicodynamics makes it unlikely that in vitro testing will be possible.Thesis (PhD (Paraclinical Sciences))--University of Pretoria, 2007.Paraclinical Sciencesunrestricte

Screening of four plants commonly used in ethnoveterinary medicine for antimicrobial, antiprotozoal and anti-oxidant activity

Urginea sanguinea, Aloe marlothii, Elephantorrhiza elephantina and Rhoicissus tridentate are all plants utilized for the management of tick borne diseases in the Madikwe area of North-west province. These plants, in certain concoctions, are believed to be effective against “seme”, “gala” and “Bolwetsi jwa mothlapo o moshibidu” which we have assumed to represent heartwater, gallsickness and redwater from circumstantial epidemiological data available. To obtain a representative extract, which would be indicative of the general activity of the plant, only acetone or methanol extracts were tested for the presence of antimicrobial, antiparasitic or anti-oxidant activity within that specific plant. Activity in all cases made use of either an in vitro biological assay or more specific chemical tests, which were validated in all cases. Ehrlichia ruminantium, Babesia caballi and Theileria equi, all grown in specific cell cultures, were used as a model for evaluating the efficacy against the common protozoan and rickettsial diseases caused by these organisms in livestock. Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa and Escherichia coli, four human nosocomial infectious agents, were used as an indicator for the presence of antibacterial activity against these common animal bacterial pathogens. Diphenyl-picrylhydrazyl and the trolox equivalent anti-oxidant chemical assays were used to determine anti-oxidant activity, which although not curative, may aid in the recovery from an infection by stimulating the immune system. The activities demonstrated among the various plants and organisms were not consistent. E. elephantine extracts were the most effective, with activity demonstrable in all biological and chemical screening assays. Although R. tridentate demonstrated poor activity (> 100 ìg/ml) against the tick-borne parasites, the plant extract did demonstrate significant anti-oxidant activity. U. sanguinea extracts showed good activity in both the antibacterial and anti-rickettsial assays (EC50 = 44.49 ng/ml), which may be due to the presence of the toxic bufadienolides present within the plant. A. marlothii possessed significant anti-rickettsial activity (EC50= 111.4 µg/ml) and to a lesser degree antibacterial activity. The results of the study support the use of these plants against heartwater, gallsickness and redwater, which gives credence for the traditional use against “Seme, Gala, and Bolwetsi jwa mothlapo o moshibidu”. Further studies are required to isolate and determine the structure of the active compounds of these plants as well as to confirm the safety and efficacy of the extracts against disease conditions in livestock. CopyrightUrginea sanguinea, Aloe marlothii, Elephantorrhiza elephantina and Rhoicissus tridentata word tradisioneel gebruik vir die bekamping van siektes deur bosluise oorgedra in die Madikwe gebied van die Noordwes provinsie. Ekstrakte van hierdie species word gebruik teen “seme”, “gala” en “Bolwetsi jwa mothlapo o moshibidu” wat waarskynlik op hartwater, galsiekte and rooiwater dui volgens die beskikbare epidemiologiese data. Asetoon en metanol ekstrakte is gebruik vir die bepaling van antimikrobiese, antiparasitiese en antioksidant aktiwiteite in verskillende species deur gevalideerde in vitro metodes. Selkulture van Ehrlichia ruminantium, Babesia caballi en Theileria equi, , is in ‘n model gebruik om die doeltreffendheid van ekstrakte teen algemene siektes deur protozoa en ricketsias te bepaal. Vier algemene menslike nosokomiale patogene Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa and Escherichia coli, is gebruik om antibakteriese aktiwiteit van ekstrakte te bepaal. Difeniel-pikrielhidrasiel en die trolox ekwivalente anti-oksidant essajeermetode is gebruik om anti-oksidantaktiwiteit te bepaal. Antioksidante mag herstel na infeksies bespoedig deur stimulering van die immuunstelsel. Daar was ‘n groot verskil in die aktiwiteite tussen die verskillende ekstrakte en organismes. E. elephantina ekstrakte was die mees doeltreffende met die biologiese and chemiese bepalings. R. tridentata het sterk anti-oksidantaktiwiteit gehad, maar het lae aktiwiteit (> 100 µg/ml) teen bosluis-oorgedraagde parasiete gehad. U. sanguinea ekstrakte was aktief in beide die antibakteriese en anti-riketsiale bepalings (EC50 = 44.49 ng/ml), wat moontlik toegeskryf kan word aan die giftige bufadienoliede teenwoordig in hierdie species. A. marlothii ekstrakte het betekenisbolle anti-riketsiale aktiwiteit (EC50 = 111.4 µg/ml) maar slegs geringe antibakteriese aktiwiteit gehad. Hierdie resultate bevestig die moontlike waarde van hierdie species teen hartwater, galsiekte and rooiwater, en ondersteun die tradisionele etnoveterinêre gebruik teen “Seme, Gala, and Bolwetsi jwa mothlapo o moshibidu”. Verdere studies word benodig om die 5 aktiewe verbindings te isoleer en te karakteriseer en om die veiligheid en doeltreffendheid van ekstrakte teen hierdie siektes in vee te bevestig.Dissertation (MSc (Veterinary Science))--University of Pretoria, 2004.Paraclinical Sciencesunrestricte

Cytotoxic, antimicrobial, antioxidant, antilipoxygenase activities and phenolic composition of Ozoroa and Searsia species (Anacardiaceae) used in South African traditional medicine for treating diarrhoea

The safety and effectiveness of many of the medicinal plants used in traditional medicine by rural people with little or no access to allopathic drugs is yet to be evaluated. With this in mind, Ozoroa and Searsia (previously known as Rhus) species traditionally used in South Africa to treat microbial infections and gastrointestinal disorders were selected for in vitro evaluation of biological activities and cytotoxicity. Phenolic-enriched leaf extracts were prepared using mixture of 1% HCl acidified 70% acetone and n-hexane. The crude extractwas further fractionated with solvent of different polarities. Crude extracts and fractionswere tested against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Enterococcus faecalis, Aspergillus fumigatus, Candida albicans and Cryptococcus neoformans. In addition, the antioxidant potentialswere determined by DPPH, ABTS, hydroxyl radical scavenging and linoleic acid peroxidation inhibition. The cytotoxic activity of the crude extracts was assayed against Vero cells. The crude extract and the various fractions had good biological activities. The most noteworthy activity is the growth inhibition of the hexane and dichloromethane fractions with MIC values as low as 19 μg/ml. The ethyl acetate and butanol fractions had moderate to low antimicrobial activities with MICs ranging from 39 to 2500 μg/ml. The polar fractions were more active against the fungal pathogens compared with the non-polar fraction. In the DPPH antioxidant assays, the active compounds were concentrated in the polar fractions (IC50 of the crude extract ranged between 0.90 and 15.82 μg/ml). The ethyl acetate fraction was the most active (IC50 ranging between 0.84 and 7.92 μg/ml). Although thewater fraction was the most polar, the antioxidant activities were low due to the transfer of the active components into the ethyl acetate and butanol fractions. The crude extracts also had good linoleic acid peroxidation inhibition (LC50 ranging between 13.99 and 40.45 μg/ml). Crude extracts and fractions of the Ozoroa mucronata, O. paniculosa, Searsia leptodictya, S. pendulina, and S. pentheri species tested in this study had good activities relating to diarrhoea mechanisms of pharmacological relevance. However, use of phenolic-enriched crude extracts from these plants for diarrhoeal treatment or any other diseases need to be applied with caution as most of the plant extracts were reasonably toxic against Vero cell line. A next step in the possible application of these extracts to treat diarrhoea would be to identify the bioactive and toxic compounds.South African Medical Research Council (MRC).http;//www.elsevier.com/locate/sajb2015-11-30hb201

Polarity of extracts and fractions of four Combretum (Combretaceae) species used to treat infections and gastrointestinal disorders in southern African traditional medicine has a major effect on different relevant in vitro activities

ETHNOPHARMACOLOGICAL IMPORTANCE : Infections and gastrointestinal (GIT) disorders such as diarrhoea causes many problems in human health and animal production. Many Combretum species are used in traditional medicine to treat various diseases by rural people in Africa and Asia. Much of the work done to date on some species to validate their ethnopharmacological use was on the non-polar or intermediate polarity components. Many species are yet to be studied against relevant disease parameters using more polar extracts. AIMS : The polar components were extracted and fractionated by solvent-solvent fractionation to yield fractions of different polarities. The activity of these fractions on different parameters that could be involved in infectious and gastrointestinal track (GIT) disorders was investigated. The cytotoxic activities of the extracts were also determined to evaluate the potential of these extracts to combat diarrhoea in production animals. MATERIALS AND METHODS : Phenolic-enriched leaf extracts of Combretum bracteosum (Cob), Combretum padoides (Cop), Combretum vendae (Cov) and Combretum woodii (Cow) were obtained by extracting with a mixture of 70% acetone acidified with 1% HCl and n-hexane. The extract was sequentially treated by solvent-solvent fractionation with dichloromethane, ethyl acetate, and butanol to yield fractions with a large variation in polarity. The phenolic constituents of the extracts and fractions were determined using standard procedures. The antioxidant activities were determined using various standard methods. The minimum inhibitory concentrations (MICs) of the crude extracts and fractions against four bacterial and three fungal strains were assessed with a microplate serial dilution method. Cyclooxygenase (COX) and lipoxygenase (LOX) enzyme inhibitory assays and cytotoxicity studies against Vero cells were also carried out. RESULT : Some of the fractions had much higher antioxidant activity than the positive controls. The average EC50 values of the extracts for the DPPH and ABTS antioxidant assays were 0.21-12 μg/ml (Cop), 0.25-16 μg/ml (Cov), 0.33-9.41 μg/ml (Cow) and 4.97-85 μg/ml (Cob) respectively while the mean EC50 values for the positive controls ascorbic acid and trolox were 1.28-1.51 and 1.02-1.19 μg/ml respectively. All the crude extracts inhibited lipid peroxidation of linoleic acid by more than 80% at a concentration of 64 μg/ml. Even though some crude extracts had relatively low antimicrobial activity, fractions from these had high activity. Cop had the highest antibacterial activity with MICs ranging between 19-2500 μg/ml,followed by Cov with MICs ranging between 39-625 μg/ml. Cop also had the highest antifungal activity with MICs between 19-625 μg/ml. The MIC for Cow and Cov ranged from 19 to 1250 μg/ml. The extracts had no activity against COX 1 and 2 enzymes in the anti-inflammatory assay but had good lipoxygenase inhibition. The crude extracts had high concentrations of hydrolysable tannin (gallotannin). A good correlation (R2= 0.99) was found between the antioxidant activity and total tannin content indicating that, gallotannins may be responsible for the antioxidant activity. CONCLUSION : The results obtained in this study provided a scientific basis for the use of leaf extracts from these plant species to treatinfectious and GIT disorders. In general non-polar fractions had a high antimicrobial activity and polar fractions had a high antioxidant activity.South African Medical Research Council and the National Research Foundation (NRF), South Africa.http://www.elsevier.com/locate/jephb201

Removing the Threat of Diclofenac to Critically Endangered Asian Vultures

Veterinary use of the nonsteroidal anti-inflammatory (NSAID) drug diclofenac in South Asia has resulted in the collapse of populations of three vulture species of the genusGyps to the most severe category of global extinction risk. Vultures are exposed to diclofenac when scavenging on livestock treated with the drug shortly before death. Diclofenac causes kidney damage, increased serum uric acid concentrations, visceral gout, and death. Concern about this issue led the Indian Government to announce its intention to ban the veterinary use of diclofenac by September 2005. Implementation of a ban is still in progress late in 2005, and to facilitate this we sought potential alternative NSAIDs by obtaining information from captive bird collections worldwide. We found that the NSAID meloxicam had been administered to 35 captiveGyps vultures with no apparent ill effects. We then undertook a phased programme of safety testing of meloxicam on the African white-backed vultureGyps africanus, which we had previously established to be as susceptible to diclofenac poisoning as the endangered AsianGyps vultures. We estimated the likely maximum level of exposure (MLE) of wild vultures and dosed birds by gavage (oral administration) with increasing quantities of the drug until the likely MLE was exceeded in a sample of 40G. africanus. Subsequently, sixG. africanus were fed tissues from cattle which had been treated with a higher than standard veterinary course of meloxicam prior to death. In the final phase, ten Asian vultures of two of the endangered species(Gyps bengalensis,Gyps indicus) were dosed with meloxicam by gavage; five of them at more than the likely MLE dosage. All meloxicam-treated birds survived all treatments, and none suffered any obvious clinical effects. Serum uric acid concentrations remained within the normal limits throughout, and were significantly lower than those from birds treated with diclofenac in other studies. We conclude that meloxicam is of low toxicity toGyps vultures and that its use in place of diclofenac would reduce vulture mortality substantially in the Indian subcontinent. Meloxicam is already available for veterinary use in India

Relationship of Uric Acid in Serum to the Dose of Meloxicam and Diclofenac Administered and to the Administration Method

<p>Serum concentration of uric acid inGyps africanus 48 h (turquoise) and 96 h (blue) after treatment, in relation to the dose of meloxicam administered per kg of vulture body weight. For comparison, the geometric mean uric acid level (central horizontal line) and 95% range (upper and lower horizontal lines) of the experimental birds 24 h before treatment are shown. Also shown are serum concentrations of uric acid 24 h after treatment inG. africanus (red squares),G. bengalensis (red diamonds), andG. fulvus (red triangles), to which diclofenac was administered by various methods. The red line shows the regression model fitted to these data. Panels show results for different methods of administration of meloxicam to<i>G. africanus:</i> (A) gavage, (B) by feeding liver from meloxicam treated cattle, (C) by feeding muscle from meloxicam-treated cattle. Data from diclofenac experiments were taken from references [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040066#pbio-0040066-b001" target="_blank">1</a>] and [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040066#pbio-0040066-b007" target="_blank">7</a>].</p

Effect of Administration of Meloxicam and Diclofenac by Gavage on Uric Acid in the Serum of Vultures

<p>Blue symbols show the ratio of the geometric mean serum concentration of uric acid for a group ofGyps africanus treated with meloxicam by gavage to that for a control group treated with water and sampled at the same time. Vertical lines show 95% confidence limits for the ratio. The dashed horizontal line indicates a ratio of 1; i.e., no effect of treatment. For each of six samplings after treatment, results are shown for experiments in which different doses of drug were used. The fill colour of the blue symbols indicates the meloxicam dose for the treated group: white = 0.5 mg kg⁻¹ (Phase I); light blue = 1.0 mg kg⁻¹ (Phase II); dark blue = 2.0 mg kg⁻¹ (squares = Phase III, diamonds = Phase IV-2). Red vertical bars show the maximum and minimum values of the equivalent ratio for two groups of<i>G. africanus,</i> one group treated with 0.8 mg kg⁻¹ of diclofenac by gavage and another group treated with water and sampled at the same time. Open red symbols show the ratio of the serum concentration after treatment to that at the time of treatment for three individualG. fulvus given 0.8 mg kg⁻¹ of diclofenac by gavage. Filled red symbols show the ratio of the serum concentration 24 h post-treatment to that 1 h post-treatment for three individualG. bengalensis given 0.25 mg kg⁻¹ (squares) and 2.5 mg kg⁻¹ (diamond) of diclofenac by gavage. Data from diclofenac experiments were taken from references [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040066#pbio-0040066-b001" target="_blank">1</a>] and [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040066#pbio-0040066-b007" target="_blank">7</a>].</p

Some southern African plant species used to treat helminth infections in ethnoveterinary medicine have excellent antifungal activities

<p>Abstract</p> <p>Background</p> <p>Diseases caused by microorganisms and parasites remain a major challenge globally and particularly in sub-Saharan Africa to man and livestock. Resistance to available antimicrobials and the high cost or unavailability of antimicrobials complicates matters. Many rural people use plants to treat these infections. Because some anthelmintics e.g. benzimidazoles also have good antifungal activity we examined the antifungal activity of extracts of 13 plant species used in southern Africa to treat gastrointestinal helminth infections in livestock and in man.</p> <p>Methods</p> <p>Antifungal activity of acetone leaf extracts was determined by serial microdilution with tetrazolium violet as growth indicator against <it>Aspergillus fumigatus, Cryptococcus neoformans</it> and <it>Candida albicans</it>. These pathogens play an important role in opportunistic infections of immune compromised patients. Cytotoxicity was determined by MTT cellular assay. Therapeutic indices were calculated and selectivity for different pathogens determined. We proposed a method to calculate the relation between microbicidal and microbistatic activities. Total activities for different plant species were calculated.</p> <p>Results</p> <p>On the whole, all 13 extracts had good antifungal activities with MIC values as low as 0.02 mg/mL for extracts of <it>Clausena anisata</it> against <it>Aspergillus fumigatus a</it>nd 0.04 mg/mL for extracts of <it>Zanthoxylum capense, Clerodendrum glabrum,</it> and <it>Milletia grandis</it>, against <it>A. fumigatus. Clausena anisata</it> extracts had the lowest cytotoxicity (LC₅₀) of 0.17 mg/mL, a reasonable therapeutic index (2.65) against <it>A. fumigatus</it>. It also had selective activity against <it>A. fumigatus</it>, an overall fungicidal activity of 98% and a total activity of 3395 mL/g against <it>A. fumigatus</it>. This means that 1 g of acetone leaf extract can be diluted to 3.4 litres and it would still inhibit the growth. <it>Clerodendrum glabrum, Zanthoxylum capense</it> and <it>Milletia grandis</it> extracts also yielded promising results.</p> <p>Conclusions</p> <p>Some plant extracts used for treatment of parasitic infections also have good antifungal activity. Because it is much easier to isolate antifungal compounds by bioassay guided fractionation, this approach may facilitate the isolation of anthelmintic compounds from active plant extracts. The viability of this approach can be tested by isolating the antifungal compounds and then determining its anthelmintic activity. Some of these plant extracts may also be useful in combating fungal infections.</p