Evaluation of the genotoxic potential of water impacted by acid mine drainage from a coal mine in Mpumalanga, South Africa, using the Ames test and Comet assay

Several potential genotoxins found in water samples arise from anthropogenic activities. Acid mine effluent resulting from coal mining poses serious environment concerns all over the world. The use of toxicity tests to evaluate the quality of streams add value by providing site-specific toxicological data. Treatment systems such as the use of natural wetlands (passive) or conventional physical and chemical pH-neutralised processes (active) are employed mainly to meet certain water quality guidelines. Nonetheless, potential genotoxins or residues remain which influence the quality of discharged effluents. The objective of this study was to evaluate the genotoxic potential of acid mine drainage (AMD) released into a natural stream following treatment by passive and active methods. This study aimed to identify the extent of AMD mutagenicity and genotoxicity to African Vero monkey kidney cell line and a fish gill cell line (RTgill-W1) using two assays, the Ames test, and the comet assay, as a rapid and effective screening tool. The Ames test performed without metabolic activation using Salmonella typhimurium TA98 and TA100 strains showed no indication of mutagenicity in the water samples tested. Differing results were however obtained for the comet assay using the African Vero monkey kidney cell line and a fish gill cell line (RTgill-W1), which revealed DNA fragmentation and variations in morphologies indicative of genotoxicity in the water samples following the two treatment processes. A significant reduction in DNA damage was observed in water samples following active treatment of the AMD, evidenced by reduced damage frequency and a lowered comet score. This bioassay confirms the urgency of integrating high-throughput screening in aquatic toxicity assessment at genetic levels, giving further evidence that in-vitro bioassays can be incorporated for use in short-term genotoxicity assays. The result suggests that the comet assay proved sensitive at detecting genotoxicity, supporting the integration of this into environmental monitoring frameworks targeted at AMD-contaminated sites

Anti-inflammatory and antioxidant properties of leaf extracts of eleven South African medicinal plants used traditionally to treat inflammation

ETHNOPHARMACOLOGICAL RELEVANCE : Inflammation is a complex mechanism employed by the body to promote healing and restoration to normal function in the event of injury. Eleven plant species were selected in this study based on their use in traditional medicine against inflammation in South Africa. METHODS : Hexane, acetone, ethanol, methanol and water extracts of the powdered plants were prepared and a total of fifty-five extracts were tested for their anti-inflammatory and antioxidant activities. The anti-inflammatory activity of extracts was evaluated via the 15-lipoxygenase (15-LOX) inhibitory and the nitric oxide (NO) inhibition assays using lipopolysaccharide (LPS)-activated RAW 264.7 murine macrophages. Total flavonoid and total phenolic contents were determined. The antioxidant activity of the extracts was performed using radical scavenging DPPH (2, 2-diphenyl-1-picrylhydrazyl) and electron reducing ABTS (2, 2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) assays. RESULTS : The hexane extract of Typha capensis (TC) had good lipoxygenase inhibitory activity with IC50 of 4.65 µg/mL, significantly (p < 0.05) higher than that of the positive control quercetin (IC50 = 24.60). The same extract also had good nitric oxide inhibitory activity with 86% NO inhibition and cell viability of 97% at 50 µg/mL. The TC acetone extract had the best antioxidant activity with IC50 of 7.11 and 1.91 µg/mL respectively in the DPPH and ABTS assays. Following fractionation of the TC plant material, the ethyl acetate fraction had interesting antioxidant activity and the methanol/water (35%) and hexane fractions had good 15-LOX inhibitory activity. The antioxidant and anti-inflammatory activities therefore resided in both polar and more non-polar fractions. CONCLUSION : The acetone extract of Typha capensis and its fractions had good anti-inflammatory and antioxidant activities, supporting the medicinal use of this species against inflammation. Other species including Ficus elastica, Carpobrotus edulis, Cotyledon orbiculata and Senna italica also had good activity worthy of further investigation.The National Research Foundation , South Africa (grant number 105993 ) is thanked for providing research funding. The NRF and University of Pretoria , South Africa are acknowledged for doctoral fellowships to OM.http://www.elsevier.com/locate/jethpharm2020-04-24hj2019Paraclinical Science

Evaluation of the genotoxic potential of water impacted by acid mine drainage from a coal mine in Mpumalanga, South Africa, using the Ames test and comet assay

Several potential genotoxins found in water samples arise from anthropogenic activities. Acid mine effluent resulting from coal mining poses serious environment concerns all over the world. The use of toxicity tests to evaluate the quality of streams add value by providing site-specific toxicological data. Treatment systems such as the use of natural wetlands (passive) or conventional physical and chemical pH-neutralised processes (active) are employed mainly to meet certain water quality guidelines. Nonetheless, potential genotoxins or residues remain which influence the quality of discharged effluents. The objective of this study was to evaluate the genotoxic potential of acid mine drainage (AMD) released into a natural stream following treatment by passive and active methods. This study aimed to identify the extent of AMD mutagenicity and genotoxicity to African Vero monkey kidney cell line and a fish gill cell line (RTgill-W1) using two assays, the Ames test, and the comet assay, as a rapid and effective screening tool. The Ames test performed without metabolic activation using Salmonella typhimurium TA98 and TA100 strains showed no indication of mutagenicity in the water samples tested. Differing results were however obtained for the comet assay using the African Vero monkey kidney cell line and a fish gill cell line (RTgill-W1), which revealed DNA fragmentation and variations in morphologies indicative of genotoxicity in the water samples following the two treatment processes. A significant reduction in DNA damage was observed in water samples following active treatment of the AMD, evidenced by reduced damage frequency and a lowered comet score. This bioassay confirms the urgency of integrating high-throughput screening in aquatic toxicity assessment at genetic levels, giving further evidence that in-vitro bioassays can be incorporated for use in short-term genotoxicity assays. The result suggests that the comet assay proved sensitive at detecting genotoxicity, supporting the integration of this into environmental monitoring frameworks targeted at AMD-contaminated sites.The Department of Paraclinical Sciences (University of Pretoria), the National Research Foundation (NRF), South Africa : Incentive Funding for Rated Researchers) and the Schlumberger Stitching Fund, Netherlands.https://watersa.netParaclinical Science

In vitro antioxidant activity of crude extracts of Harpagophytum zeyheri and their anti-inflammatory and cytotoxicity activity compared with diclofenac

BACKGROUND : This study evaluated the in vitro antioxidant activity and comparison of anti-inflammatory and cytotoxic activity of Harpagopytum zeyheri with diclofenac. METHODS : In vitro assays were conducted using water, ethanol, and ethyl acetate extracts of H.zeyheri. The antioxidant activity was evaluated using the 2,2′-diphenyl-1-picrylhydrazy (DPPH) and 2,2′- azino-bis (3- ethylbenzothiazoline-6-sulphonic acid) (ABTS) assays. The anti-inflammatory activity was determined by measuring the inhibition of nitric oxide (NO) on lipopolysaccharide (LPS)-induced RAW 264.7 mouse macrophages as well as cytokine (TNF-α and IL-10) expression on LPS-induced U937 human macrophages. For cytotoxicity, cell viability was determined using the 3-(4, 5-dimethylthiazol- 2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. RESULTS : The ethyl acetate extract had the lowest IC50 values in the DPPH (5.91 μg/ml) and ABTS (20.5 μg/ml) assay compared to other extracts. Furthermore, the ethyl acetate extracts effectively inhibited NO and TNF-α and proved to be comparable to diclofenac at some concentrations. All extracts of H. zeyheri displayed dose-dependent activity and were associated with low levels of human-IL-10 expression compared to quercetin. Furthermore, all extracts displayed low toxicity relative to diclofenac. CONCLUSIONS : These findings show that H. zeyheri has significant antioxidant activity. Additionally, similarities exist in the inflammatory activity of H. zeyheri to diclofenac at some concentrations as well as low toxicity in comparison to diclofenac.This manuscript is part of a PhD project supported by a grant (Grant: RB/58/ 16) from the Research Board of the National University of Science and Technology, Zimbabwe and Higherlife Foundation Delta Philanthropies Doctoral Fellowship (NCU56422B).https://bmccomplementmedtherapies.biomedcentral.com/am2022Paraclinical Science

Isolation and characterization of two acaricidal compounds from Calpurnia aurea subsp. aurea (Fabaceae) leaf extract

The menace caused by ticks and tick-borne diseases is a major limitation to the livestock industry in Africa. The high costs and non-availability of synthetic, chemical acaricides to resource-limited farmers, resistance of ticks to available acaricides and residue problems in meat and milk consumed by humans further complicate matters. The use of plant extracts as a possible source of new acaricides has received much interest in the last decade. In our endeavour to discover natural acaricidal compounds, tick toxicant bioassays were conducted and the chloroform fraction of Calpurnia aurea ethanol leaf extract had good acaricidal activity. Further purification of the fraction revealed two flavonoids, isolated from C. aurea for the first time. These flavonoids were characterized as apigenin-7-O-β-d-glycoside and isorhoifolin by means of NMR spectroscopic and mass spectrometry analysis. Isorhoifolin was the most potent compound (LC50 = 0.65 mg/ml), was not cytotoxic and should be further investigated for its potential as an acaricidal agent.The Technology Innovation Agency in conjunction with the Council for Scientific and Industrial Research, University of Pretoria and the Schlumberger Faculty for the Future Foundation.http://link.springer.com/journal/104932019-07-01hj2018Paraclinical Science

Natural compounds isolated from African mistletoes (loranthaceae) exert anti-inflammatory and acetylcholinesterase inhibitory potentials : in vitro and in silico studies

DATA AVAILABILITY STATEMENT: All data generated or analysed during this study are included in this published article.Please read abstract in article.The Central University of Technology operational expenses and the National Research Foundation (NRF) of South Africa.https://www.mdpi.com/journal/applsciParaclinical Science

Natural Compounds Isolated from African Mistletoes (Loranthaceae) Exert Anti-Inflammatory and Acetylcholinesterase Inhibitory Potentials: In Vitro and In Silico Studies

Despite the medicinal uses of Phragmanthera capitata, Globimetula dinklagei and Tapinanthus bangwensis against memory loss, convulsions and pain, their efficacy against brain-dysfunction diseases and inflammation models has not yet been studied. Therefore, this study aims to investigate the anti-inflammatory and acetylcholinesterase (AChE) inhibitory potentials of their crude extracts and isolated natural compounds by combining in vitro and in silico experiments. Crude extracts and isolated compounds were tested in vitro for their AChE inhibitory activity by using Ellman&rsquo;s method. Additionally, their anti-inflammatory activity was determined by evaluating the nitric oxide (NO) production inhibitory activity in lipopolysaccharide-stimulated RAW 264.7 macrophage cells and the 15-lipoxygenase (15-LOX) inhibitory activity by using the ferrous oxidation xylenol orange assay. Furthermore, the in silico efficacy of natural compounds was investigated against ten putative target enzymes relevant in Alzheimer&rsquo;s disease (AD) pathogenesis and inflammation. It was found that the crude extracts had weak to moderate inhibitory potential against AChE, with the extract of T. bangwensis being the most active (50% inhibitory concentration (IC50) = 48.97 &micro;g/mL). Six natural compounds, namely, 3-O-&beta;-D-glucopyranosyl-28-hydroxy-&alpha;-amyrin (2), apigenin- 8-C-&beta;-D-glucopyranoside (3), globimetulin B (5), globimetulin C (6), bangwaursene B (8) and 3&beta;-acetoxy-11,12-epoxytaraxerol (9), were identified for the first time as having anti-AChE potential, among which (9) had the highest efficacy with an IC50 of 13.89 &micro;M. Among the anti-AChE compounds, (5) was also efficient against NO production and 15-LOX, and the data are in agreement with the docking score. In summary, compounds (5) and (9) are the most prominent lead compounds that should be further tested experimentally against molecular targets of AD and inflammation

Croton gratissimus leaf extracts inhibit cancer cell growth by inducing caspase 3/7 activation with additional anti-inflammatory and antioxidant activities

Abstract Background Croton species (Euphorbiaceae) are distributed in different parts of the world, and are used in traditional medicine to treat various ailments including cancer, inflammation, parasitic infections and oxidative stress related diseases. The present study aimed to evaluate the antioxidant, anti-inflammatory and cytotoxic properties of different extracts from three Croton species. Methods Acetone, ethanol and water leaf extracts from C. gratissimus, C. pseudopulchellus, and C. sylvaticus were tested for their free radical scavenging activity. Anti-inflammatory activity was determined via the nitric oxide (NO) inhibitory assay on lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages, and the 15-lipoxygenase inhibitory assay using the ferrous oxidation-xylenol orange assay. The cytotoxicity of the extracts was determined on four cancerous cell lines (A549, Caco-2, HeLa, MCF-7), and a non-cancerous African green monkey (Vero) kidney cells using the tetrazolium-based colorimetric (MTT) assay. The potential mechanism of action of the active extracts was explored by quantifying the caspase-3/− 7 activity with the Caspase-Glo® 3/7 assay kit (Promega). Results The acetone and ethanol leaf extracts of C. pseudopulchellus and C. sylvaticus were highly cytotoxic to the non-cancerous cells with LC50 varying between 7.86 and 48.19 μg/mL. In contrast, the acetone and ethanol extracts of C. gratissimus were less cytotoxic to non-cancerous cells and more selective with LC50 varying between 152.30 and 462.88 μg/mL, and selectivity index (SI) ranging between 1.56 and 11.64. Regarding the anti-inflammatory activity, the acetone leaf extract of C. pseudopulchellus had the highest NO inhibitory potency with an IC50 of 34.64 μg/mL, while the ethanol leaf extract of the same plant was very active against 15-lipoxygenase with an IC50 of 0.57 μg/mL. A linear correlation (r<0.5) was found between phytochemical contents, antioxidant, anti-inflammatory and cytotoxic activities of active extracts. These extracts induced differentially the activation of caspases − 3 and − 7 enzymes in all the four cancerous cells with the highest induction (1.83-fold change) obtained on HeLa cells with the acetone leaf extract of C. gratissimus. Conclusion Based on their selective toxicity, good antioxidant and anti-inflammatory activities, the acetone and ethanol leaf extracts of C. gratissimus represent promising alternative sources of compounds against cancer and other oxidative stress related diseases

Antiproliferative effect of extracts and fractions of the root of Terminalia avicennioides (Combretaceae) Guill and Perr. on HepG2 and vero cell lines

BACKGROUND : Terminalia avicennioides Guill and Perr (Combretaceae) is an important West African medicinal plant. The plant is used locally against microbes and parasites in both humans and animals and studies have demonstrated its cytotoxicity potential. Thus, this study was carried out to test the cytotoxic effect of the extracts and fractions of the root of the medicinal plant Terminalia avicennioides Guill and Perr (Combretaceae) in two different cell lines. METHODS : Methanol, ethanol, 30 % ethanol, hot water and cold water extracts and ethylacetate, hexane, chloroform, butanol and residual water fractions, were evaluated at 1000, 750, 500, 250, 100 and 50 µg/mL concentrations, with doxorubicin as positive control. The cells were incubated with the extracts for 48 h at 37 °C in a 5 % CO2 humidified incubator. The inhibition of cell viability, determined with the methyl blue thiazole tetrazolium bromide (MTT) assay, was used to assess the anti-proliferative effect of the extracts, in normal Vero Monkey kidney and human liver cancer (HepG2) cell lines. RESULTS : There was a concentration-dependent inhibition of cell viability in both the HepG2 and Vero cell lines. For HepG2 cells, antiproliferative effect was highest for the hexane fraction (viability ranged from 19.63 ± 1.10 % to 70.30 ± 1.78 % for 1000 and 50 µg/mL, respectively. For Vero cells, the highest antiproliferative effect, at 1000 µg/mL, was with hexane fraction (cell viability 21.37 ± 3.50 %), while at 50 µg/mL the chloroform fraction demonstrated the highest effect (viability of 86.10 ± 1.95 %). CONCLUSIONS : The extracts and fractions from the root of Terminalia avicennioides have antiproliferative effect on the Vero and HepG2 cell lines tested. However, the extracts and fractions were not more toxic to the HepG2 than to the Vero cells. The cytotoxic effect of stem-bark and leaf extracts could be evaluated in the future to determine its anticancer potential.https://clinphytoscience.springeropen.comhj2022Paraclinical 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