In vitro antibacterial activity of seven plants used traditionally to treat wound myiasis in animals in southern Africa

In the extreme situation of subsistence farming where insecticides and other veterinary medicines are either unavailable or unaffordable, the use of plants in the treatment of wound myiasis in livestock has been reported worldwide. However, the exact effect of these plants on myiatic wounds has not been established. This study was therefore undertaken to establish the biological activity of seven species of plants which are used traditionally and are claimed to be effective in the treatment of wound myiasis. Plants that have a wide distribution in southern Africa were selected. This paper focuses on the antibacterial activity of these plants on bacteria known to be among the common contaminants of wounds. It has been shown that bacterial action on wounds produce compounds which have an odour that serve as an attractant of myiasis-causing flies. The antibacterial activity of the plants was investigated using a microdilution assay and bioautography methods. All the tested plants had inhibitory activity against the test bacteria. Inhibiting bacterial activity reduces the attractants of myiasis-causing flies to the wound. Thus, inhibiting bacteria action on wounds will interfere with the development of wound myiasis. This could be one of the mechanism through which the plants that are used traditionally in the treatment of wound myiasis work.The University of Pretoria and the National Research Foundation. L. Mukandiwa gratefully acknowledges the financial support from German Academic Exchange Service, DAAD, during the period of this study.http://www.academicjournals.org/JMPRam2013mn201

Larvicidal activity of leaf extracts and seselin from Clausena anisata (Rutaceae) against Aedes aegypti

The Aedes aegypti mosquito is a vector of various diseases in both humans and livestock.Mosquito control focuses on reducing the longevity as well as the population of mosquitoes to lessen their damage on human and animal health. It entails several strategies such as environmental management, insecticide treatments, and molecular entomological approaches. Environmental management centres on elimination of breeding sites, however mosquitoes can breed in sites that cannot be eliminated. Resultantly, focus is turned onto mosquito larvae control. The objective of this study was to evaluate the larvicidal activity of extracts and compounds from Clausena anisata against A. aegypti. The World Health Organization guidelines for testing of mosquito larvicides were used. The acetone, dichloromethane and hexane crude leaf extracts were evaluated in a preliminary screening for larvicidal activity at the concentrations of 12.5, 25, 50, 100 and 200 ppm. Batches of 25 third-instar larvae were transferred into cups each containing test solutions and larval mortality was recorded 24 h and 48 h after exposure. Acetone was used as the solvent control whilst permethrin was used as a positive control. Only the n-hexane extract caused mortality at the tested concentrations, thus it was further tested at 40, 60, 80, 100, and 120 ppm and had LC50 values of 68.30 and 59.65 ppm after 24 h and 48 h respectively. A stored hexane extract, of 2months,was also evaluated under simulated field conditions to establish stability of extract. It caused about 90% mortality when tested at 100 ppm. The n-hexane extract was subjected to open column chromatography on silica gel to isolate the active compound. The isolated compound was identified as the pyranocoumarin, seselin. Dose dependentmortality was observed in the larvae exposed to seselin. The LC50 values at 24 and 48 h were 13.90 and 9.96 ppmrespectively. Results obtained from this study indicate a potential of the incorporation of C. anisata extracts into the control of mosquito populations.University of Pretoria (UP Postdoctoral Fellowship Fund) and the National Research Foundation (Grant number: Eloff 95991).http://www.elsevier.com/locate/sajb2016-09-30hb201

The use of Clausena anisata in insect pest control in Africa : a review

ETHNOPHARMACOLOGICAL RELEVANCE : Clausena anisata is used traditionally by various communities across Africa against pests such as mosquitoes, flies and weevils among others. Pests are a major cause of disease and production losses in various crop and livestock production systems in Africa. This review discusses the available information on the occurrence, chemistry, biological activity and possible commercialization of Clausena anisata with a view to see the plant species being integrated in pest management. MATERIALS AND METHODS : Information on the ethnomedical use, chemistry and biological activity of C. anisata published between 1980 and 2016 was accessed from various databases namely Science Direct, Springer Link and Wiley Online Library. In addition various relevant books were also consulted. RESULTS : The crude extracts as well as different fractions of C. anisata have been evaluated for activity against various insect pests and have been shown to be active. Furthermore, close to 50 compounds have been isolated and identified from C. anisata, which include coumarins, carbazole alkaloids, limonoids and essential oils (monoterpenes). Some of these compounds have been proven to exhibit pesticidal properties in both laboratory and field studies against various pests including mosquitoes, flies and weevils. The possible mechanisms of action of these compounds have been explored in this review. CONCLUSION : The results of pesticidal and hytochemical screening of C. anisata strongly indicate that the species is endowed with pesticidal properties that can be harnessed into commercial products. However, one glaring challenge in the evaluation of this plant species for pesticidal activity has been the non-availability of standard testing systems. Researchers have used various methods which they developed based on their own circumstances and resources. Formulation, standard appropriate testing systems and agronomic research are key in unlocking the potential of this important African species.The National Research Foundation of South Africa (NRF) (Grant No. 95408)http://www.elsevier.com/locate/jethpharm2017-12-31hb2017Paraclinical Science

Repellent and mosquitocidal effects of leaf extracts of Clausena anisata against the Aedes aegypti mosquito (Diptera : Culicidae)

Mosquitoes are rapidly developing resistance to insecticides that millions of people relied on to protect themselves from the diseases they carry, thereby creating a need to develop new insecticides. Clausena anisata is used traditionally as an insect repellent by various communities in Africa and Asia. For this study, the repellency and adulticidal activities of leaf extracts and compounds isolated from this plant species were evaluated against the yellow fever mosquito, Aedes aegypti. In the topical application assays, using total bites as an indicator, repellency was dose dependent, with the acetone crude extract (15 %) having 93 % repellence and the hexane fraction (7.5 %) 67 % repellence after 3 h. Fractionation resulted in a loss of total repellence. As mosquito-net treating agents, the acetone and hexane extracts of C. anisata, both at 15 %, had average repellences of 46.89 ± 2.95 and 50.13 ± 2.02 %, respectively, 3 h after exposure. The C. anisata acetone extract and its hexane fraction caused mosquito knockdown and eventually death when nebulised into the testing chamber, with an EC50 of 78.9 mg/ml (7.89 %) and 71.6 mg/ml (7.16 %) in the first 15 min after spraying. C. anisata leaf extracts have potential to be included in protection products against mosquitoes due to the repellent and cidal compounds contained therein.The University of Pretoria and the National Research Foundation.http://link.springer.com/journal/113562017-06-30hb2016Paraclinical Science

Isolation of seselin from Clausena anisata (Rutaceae) leaves and its effects on the feeding and development of Lucilia cuprina larvae may explain its use in ethnoveterinary medicine

ETHNOPHARMACOLOGICAL RELEVANCE : The leaves of Clausena anisata are used traditionally to expel maggots from wounds of animals in Zimbabwe. We have previously proved in the laboratory that the plant certainly affects the behaviour and growth of blowfly larvae. The objective of this study was to isolate and identify the active compounds responsible for this activity.MATERIALS AND METHODS : The acetone extract of C. anisata leaf powder was separated by solvent-solvent partition into five fractions. The n-hexane fraction was the most active in the larvicidal assay and therefore subjected to open column chromatography on silica gel. RESULTS : The isolated compound was identified by nuclear magnetic resonance (NMR) and mass spectroscopy (MS) as the pyranocoumarin, seselin, chemically known as 2',2'- dimethylpyranocoumarin . It inhibited feed intake in the first and second instars of blowflylarvae at the minimum concentration tested of 1 ppm resulting in significant lower mass pupae (13.5 ± 0.5 mg and 22.4 ± 0.4 mg for the first and second instar larvae respectively) compared to the solvent control group (26.19 ± 0.8 mg) (p < 0.05). CONCLUSIONS : This is the first report of the isolation of seselin from the leaves of C. anisata and the first report of the compound having an effect against blow fly larvae.The University of Pretoria, the National Research Foundation and German Academic Exchange Service, DAAD.http://www.elsevier.com/locate/jephb201

Percentage of faecal excretion of meloxicam in the Cape vultures (Gyps corprotheres)

Asian Gyps vulture species are gradually recovering from the devastating effect of diclofenac being present in contaminated carcasses. This drug was responsible for the death of over 10 million vultures in India, Nepal and Pakistan. To prevent the extinction of vultures, meloxicam was introduced after the ban of veterinary diclofenac. Meloxicam's safety in vultures was attributed to its short elimination half-life in contrast with diclofenac. The reason for the rapid elimination of meloxicam is yet to be explained. The aim of this study was to evaluate the role of biotransformation in the elimination of meloxicam. Six Cape griffon vultures (Gyps coprotheres) were treated with 2 mg/kg meloxicam intramuscularly for faecal and plasma quantification of meloxicam concentration over time. In the plasma meloxicam was characterised by a half-life, mean residence time, clearance and volume of distribution at steady state of 0.37 ± 0.10 h, 0.90 ± 0.12 h, 0.02 ± 0.00 l/h kg and 0.02 ± 0.00 l/kg respectively (presented as geometric mean). Over the 24 h monitoring period, the total non-metabolised meloxicam in the faeces was 1.35 ± 0.71% of the total concentration in the plasma. Based on the short meloxicam elimination half-life and low cumulative concentration of total faecal meloxicam over a period in excess of 10 half-lives, this study indicates that Cape griffon vultures are efficient metaboliser of meloxicam, which is suggestive of different set of cytochrome enzymes being involved in the metabolism to that for diclofenac in this species. Identification of orthologous human CYP2C9 and CYP3A4 enzyme families in vultures will be an important further step in explaining the differences in the metabolic pathway(s) of meloxicam and diclofenac for the species.The National Research Foundation (NRF) of South Africa (Grant no 87772).http://www.elsevier.com/locate/cbpc2020-01-01hj2018Paraclinical Science

Biosurfactants produced by Bacillus subtilis A1 and Pseudomonas stutzeri NA3 reduce longevity and fecundity of Anopheles stephensi and show high toxicity against young instars

Anopheles stephensi acts as vector of Plasmodium parasites, which are responsible for malaria in tropical and subtropical areas worldwide. Currently, malaria management is a big challenge due to the presence of insecticide-resistant strains as well as to the development of Plasmodium species highly resistant to major antimalarial drugs. Therefore, the present study focused on biosurfactant produced by two bacteria Bacillus subtilis A1 and Pseudomonas stutzeri NA3, evaluating them for insecticidal applications against malaria mosquitoes. The produced biosurfactants were characterized using FT-IR spectroscopy and gas chromatography-mass spectrometry (GC-MS), which confirmed that biosurfactants had a lipopeptidic nature. Both biosurfactants were tested against larvae and pupae of A. stephensi. LC50 values were 3.58 (larva I), 4.92 (II), 5.73 (III), 7.10 (IV), and 7.99 (pupae) and 2.61 (I), 3.68 (II), 4.48 (III), 5.55 (IV), and 6.99 (pupa) for biosurfactants produced by B. subtilis A1 and P. stutzeri NA3, respectively. Treatments with bacterial surfactants led to various physiological changes including longer pupal duration, shorter adult oviposition period, and reduced longevity and fecundity. To the best of our knowledge, there are really limited reports on the mosquitocidal and physiological effects due to biosurfactant produced by bacterial strains. Overall, the toxic activity of these biosurfactant on all young instars of A. stephensi, as well as their major impact on adult longevity and fecundity, allows their further consideration for the development of insecticides in the fight against malaria mosquitoes

The efficacy of seven southern African ethnoveterinary medicinal plants in the mitigation of cutaneous myiasis

Myiasis, the infestation of the skin of mammals by larvae of a variety of fly species, has been recognised as a major disease from ancient times. Despite being well known as a disease, it remains poorly controlled in the animal production industry with severe economic losses resulting thereof. The control of the myiasis-causing flies relies heavily on the use of pharmaceutical chemicals such as the organophosphates, pyrethroids or insect growth regulators. Unfortunately these chemicals are characterised by a high potential to cause human or animal toxicity in addition to being environmental contaminants. Another problem resulting from their continued use has been the development of resistance in the treated flies. Newer products need to be discovered. One source of these compounds could be the ethnoveterinary plants already in use by subsistence farmers where insecticides are either unavailable or unaffordable. This study focused on seven plant species used as such in South Africa and Zimbabwe: Aloe marlothii A. Berger, Aloe zebrina Baker, Calpurnia aurea (Aiton) Benth, Psydrax livida (Hiern) Bridson (Canthium huillense), Clausena anisata (Willd), Erythrina lysistemon Hutch, and Spirostachys africana Sond. In the first step of evaluation, the selected plant species were screened for their activity against selected wound contaminating bacteria, which are recognised as the inciting factor attracting myiasis-causing flies due to the characteristic odour they produce. Using the microdilution and bioautography methods, all plants had inhibitory activity with the acetone extracts being superior with MICs ranging from 0.04 to 0.6 mg/ml. Using the surface area of a general wound and the density of the extract, it was demonstrated that wounds treated with these extracts could reach these MIC levels. It was concluded that these plants could be beneficial in managing myiasis, in part, by reducing secondary infections and fly attraction. Two studies were undertaken to determine the in vitro effects of these plant species on the behaviour and development of blowfly larvae. For the first study, larvicidal activity of the seven plant species was determined on third instar larvae fed on a combination of meat (30 g) baited with the acetone leaf extracts (1 ml at 10 mg/ml). Aloe zebrina, C. anisata, E. lysistemon and S.africana, induced developmental anomalies in the larvae including paralysis, prolongation of the prepuparium stage, reduced pupation rates, pupae malformation and reduced adult emergence. The results suggested that the plant extracts interfered with the neuroendocrine control in the blowfly. For the second study the four active plant species from the first study were further evaluated at 10, 25, 50, 75, 100 and 150 mg/ml. Larval behaviour, larval development and emergence of adult flies were assessed after exposure to the baited meat. The increasing concentrations of the acetone plant extracts decreased ingestion of the meat by the larvae, pupae mass and adult emergence rates. For C. anisata and S. africana extracts the increase in the concentration was also associated with larvae circling on top of the testing cups, as far away as possible from the meat, possibly indicating repellency and the emerging adult flies being smaller. Clausena anisata was selected for field evaluation on populations of blowflies on two farms (one control and one test site) in Mpumalanga (South Africa) over 12 weeks when fly populations were highest. Larvae exposed to liver baits treated with C. anisata showed slow development, prolonged larval period, smaller body size, sluggish behaviour, delayed pupation and reduced eclosion rates in comparison to the controls. No significant differences were present between the numbers and sizes of flies on the treated and on the control farm. A difference in fly species was noted on the baits before and after treatment, which may be indicatory of a repellent effect. It is concluded that C. anisata could be beneficial in an overall control strategy through its ability to decrease blowfly populations in the long term by reducing the total number of life cycles completed in a year and perhaps as a surface repellent agent. From the beneficial in vitro and in field presence of efficacy, isolation of the active compound(s) from C. anisata was attempted. As a first step, due to the potential repellent effect on the farm and the in vitro result suggesting a similar effect, the plant was screened for the presence of pyrethrins which are known natural repellents. None of the pyrethrins were present although a terpene compound with an Rf value close to the pyrethrin II compounds was present. Using bio-guided fractionation and column chromatography, seselin was isolated and identified from the n-hexane fraction which was most active. This study therefore adds support for the use of the selected plants in myiasis and more importantly demonstrates that C. anisata may be valuable as a new agent in the control of farm fly populations.Thesis (PhD)--University of Pretoria, 2012.Paraclinical SciencesPhDUnrestricte

Ultrastructure changes induced by the phloroglucinol derivative agrimol G isolated from Leucosidea sericea in Haemonchus contortus

Plant extracts used for the treatment of helminth infections in sheep are an alternative to chemical anthelmintic drugs. Previous studies have reported the anthelmintic activity of acetone leaf extracts of Leucosidea sericea. For this study, we evaluate the ultrastructure changes induced by the acetone leaf extract of L. sericea and the component agrimol G (AG) that was isolated for the first time on adult haemonchus parasites. Adult haemonchus parasites harvested from sheep were incubated with the plant extract and AG for 3 h and evaluated by both scanning and transmission electron microscopy in comparison and in combination with albendazole or ivermectin. In all cases the method of evaluation shows ultrastructural changes, with albendazole inducing mitochondrial damage and ivermectin inducing muscle degeneration, both as previously described. Incubation with the plant extract and AG resulted in the formation of numerous non-membrane bound multi-vesicular like bodies and evenly spread disruptions/erosion in the epicuticle. Combining AG with ivermectin or albendazole resulted in an absence of effect of AG. Based on the structural changes induced by AG, together with the absence of an effect in combination with ivermectin and albendazole would suggest a disrupted microtubular network. The latter does however require biochemical confirmation.The National Research Foundation of South Africahttp://www.elsevier.com/locate/yexpr2020-12-01hj2020Paraclinical Science

The use of liver slices from the Cape vulture (Gyps coprotheres) to better understand the role of liver toxicity of non-steroidal anti-inflammatory drugs (NSAIDs) in vultures

Diclofenac, a non-steroidal anti-inflammatory drug (NSAID) was responsible for the death of millions of vultures on the Asian subcontinent, following the consumption of diclofenac contaminated carcasses. The aim of this research was to establish if liver slices could serve as an alternate means of predicting the toxicity of NSAIDs in Gyps vultures. The Cape vulture liver slices was prepared and incubated with four NSAIDs for 6 h. A percent clearance of 1.0 ± 0.253, 0.58 ± 0.153, 0.961 ± 0.312 and 1.242 ± 0.406 (%/h*g) was attained for diclofenac, carprofen, ketoprofen and meloxicam respectively. Both meloxicam and diclofenac exerted toxic effects on the hepatic cells. Protein content indicated that the vulture tissue had lower enzyme levels than expected for an animal of its size. The poor distinction between the ex vivo hepatic percent clearance of meloxicam and diclofenac indicates that liver slices is not an ideal model to investigate NSAIDs toxicity in Cape vulture.The National Research Foundation (NRF) of South Africa (Grant no 87772 ).http://www.elsevier.com/locate/etap2019-09-01hj2018Paraclinical Science