Antibacterial activity and mode of action of acetone crude leaf extracts of underinvestigated Syzygium and Eugenia (Myrtaceae) species on multidrug resistant porcine diarrhoeagenic Escherichia coli

BACKGROUND : Diarrhoea, a global economically important disease burden affecting swine and, especially piglets, is commonly caused by infection with entero-toxigenic E. coli (ETEC). Adherence of ETEC to porcine intestinal epithelial cells following infection, is necessary for its pathogenesis. While antimicrobials are commonly given as therapy or as feed additives for prophylaxis against microbial infections, the concern over increased levels of antimicrobial resistance necessitate the search for safe and effective alternatives in livestock feed. Attention is shifting to natural products including plants as suitable alternatives to antimicrobials. The activity of acetone crude leaf extracts of nine under-explored South African endemic plants from the Myrtaceae family with good antimicrobial activity were tested against pathogenic E. coli of porcine origin using a microplate serial dilution method. Bioautography, also with p-iodonitrotetrazolium violet as growth indicator was used to view the number of bioactive compounds in each extract. In vitro toxicity of extracts was determined against Caco-2 cells using the 3-(4,5-dimethythiazolyl-2)-2,5-diphenyltetrazolium bromide reduction assay. The antimicrobial susceptibility of E. coli isolates was tested on a panel of antimicrobials using the Kirby-Bauer agar diffusion method while the anti-adherence mechanism was evaluated using a Caco-2 cell enterocyte anti-adhesion model. RESULTS : The MIC of the extracts ranged from 0.07–0.14 mg/mL with S. legatii having the best mean MIC (0.05 mg/ mL). Bioautography revealed at least two active bands in each plant extract. The 50% lethal concentration (LC50) values ranged between 0.03–0.66 mg/mL. Eugenia zeyheri least cytotoxic (LC50 = 0.66 mg/ml) while E. natalitia had the highest cytotoxicity (LC50 = 0.03 mg/mL). All the bacteria were completely resistant to doxycycline and colistin sulphate and many of the plant extracts significantly reduced adhesion of E. coli to Caco-2 cells. CONCLUSIONS : The extracts of the plants had good antibacterial activity as well as a protective role on intestinal epithelial cells against enterotoxigenic E. coli bacterial adhesion. This supports the potential use of these species in limiting infection causes by E. coli. Some of these plants or extracts may be useful as phytogenic feed additives but it has to be investigated by animal feed trials.The University of Pretoria and the South African National Research Foundation/The World Academy of Science (NRF/ TWAS).https://bmcvetres.biomedcentral.comam2020Paraclinical SciencesVeterinary Tropical Disease

In vitro potential of the acetone leaf extract and fractions of Psychotria capensis (Eckl.) Vatke (Rubiaceae) to combat co-infection of tuberculosis and helminthiasis

Tuberculosis (TB) is a disease of global importance that affects millions of people. Approximately a quarter of the world’s population is currently infected with M. tuberculosis, and about 10% of those infected will develop into active disease, particularly immune compromised individuals. Helminthiasis is of global health importance, affecting over 2 billion people mostly in resource-poor countries. Coinfection with tuberculosis (TB) and helminths (worms) is an emerging global public health concern with both affecting about one-third of the global population. Chronic infection with helminths can result in impaired immune responses to TB as well as enhancing failure to TB therapy and BCG vaccination. Antimycobacterial and anthelmintic activities of the acetone extract and fractions of Psychotria capensis were evaluated, including their in vitro safety. In addition, the anti-inflammatory and immunomodulatory effect of the fractions and crude extract of P. capensis were assessed. Antimycobacterial activity of the extract and fractions was tested against four non-tuberculous mycobacteria (Mycobacterium smegmatis, M. fortuitum, M. aurum, M. bovis BCG) and pathogenic M. tuberculosis H37Rv while the Egg Hatch Assay (EHA) was used for the anthelmintic test on eggs of Haemonchus contortus. Cytotoxicity was determined against Vero kidney cells while in vitro immune modulation via cytokine production was determined on activated macrophages. The minimum inhibitory concentration (MIC) values of the Psychotria capensis acetone extract and fractions ranged from 39 to 1,250 μg/ml with the crude extract and hexane fraction having the best MIC values (both 39 μg/ml). In the EHA, the inhibitory concentration (IC50) ranged from 160 to 630 μg/ml with the hexane fraction having the best activity. The hexane and chloroform fractions were relatively non-toxic with LC50 values of 290 and 248 μg/ml respectively, while the acetone crude extract (64 μg/ml) and n-butanol fraction (71 μg/ml) were moderately toxic. The SI values (LC50/MIC) ranged from 0.1 to 7.4 with the hexane fraction having the highest value against M. smegmatis (7.4). The hexane fraction had the best dual anthelmintic and antimycobacterial activity. This fraction had the best NO inhibitory activity and was the least cytotoxic, indicating that its activity was not due to general metabolic toxicity, with 96.54% cell viability. Pro-inflammatory cytokines such as IL-12p70 were upregulated while IL-10 expression was inhibited by the extracts. Compounds were detected using GC-MS analysis, and in both the crude acetone extract and the hexane fraction was the diterpene neophytadiene, which has antiinflammatory and antimicrobial activity. Finding alternative or complementary approaches to dealing with TB infections by, amongst other things, reducing the incidence of helminth infestations may lessen the burden of TB, contributing to slowing the spread of multi-drug resistance.The Women in Research Fund, South African Medical Research Council (MRC), the National Research Foundation, the University of South Africa and University of Pretoria.https://www.frontiersin.org/journals/pharmacologydm2022Veterinary Tropical Disease

Differentiation of Tarchonanthus camphoratus L. and Tarchonanthus parvicapitulatus P.P.J. Herman (Asteraceae) using electron microscopy, and comparison of their biological activities

ETHNOPHARMACOLOGICAL RELEVANCE : Tarchonanthus camphoratus L. complex has numerous medicinal uses amongst the sub-Saharan African populace, including treatment for bronchospasm. This study focused on providing scientific rationale for the traditional use of the extracts of T. camphoratus and T. parvicapitulatus. T. camphoratus L. complex has been published under diverse names by various taxonomists. Tarchonanthus parvicapitulatus was one of the newly described taxa, leaving Tarchonanthus camphoratus L. sens. strict. as a homogenous taxon. However, some of the morphological characters used tend to overlap, making it difficult to identify the different taxa. AIMS : The aim of this study was to evaluate the bronchodilatory, antioxidant and toxicological properties of the leaves of T. camphoratus L. and T. parvicapitulatus. This study also aimed to use scanning electron microscopy (SEM) to assess the differences between T. camphoratus L. and T. parvicapitulatus. MATERIALS AND METHODS : Thin layer chromatography (TLC) with vanillin as visualizing agent was used to qualitatively compare the phytoconstituents of the plant acetone extracts. The free radical scavenging antioxidant qualitative assay was done by spraying TLC plates with DPPH free radical. The bronchodilatory effects of the aqueous extracts were assessed using pre-contracted guinea pig trachea. The effects of the extracts of T. camphoratus L. and T. parvicapitulatus on superoxide and ATP production was also investigated on isolated human neutrophils. A micromorphology study was done using scanning electron microscopy to study the leaves. RESULTS : Different compounds were visualized on the TLC plates with more than 40 compounds of intermediate polarity. The TLC plates sprayed with DPPH revealed the presence of 20 and 23 antioxidant compounds for T. camphoratus and T. parvicapitulatus respectively. Upon pre-contraction of the tracheal smooth muscles, the aqueous extracts of T. parvicapitulatus significantly relaxed the trachea while the relaxation observed for T. camphoratus was not significant. All the tested concentrations had a dose dependent inhibitory effect on superoxide production. The crude extract of T. parvicapitulatus at the highest concentration (10 mg/ml) significantly decreased ATP production while a non-significant increase in ATP production was observed for T. camphoratus at the highest concentration (10 mg/ml) when compared with the control. The micromorphology study was useful in revealing the presence of trichomes on the upper leaf surface of the studied taxa. CONCLUSIONS : The results obtained from this study showed that the studied plant extracts had bronchodilatory effects on contracted guinea pig trachea and could also inhibit the production of free radicals including superoxide anions. To the best of our knowledge, this is the first report on the bronchodilatory activity of T. camphoratus and T. parvicapitulatus. The micromorphological studies were useful in distinguishing between the two species, confirming that T. camphoratus L. and T. parvicapitulatus are different taxa. This study provides evidence to support the traditional use of T. camphoratus and T. parvicapitulatus in managing bronchospasm.The University of South Africa; Sefako Makgatho University of Health Sciences, Ga-rankuwa; the University of Pretoria and Tshwane University of Technology.http://www.elsevier.com/locate/jethpharmhj2022Paraclinical Science

Anthelmintic and antimycobacterial activity of fractions and compounds isolated from Cissampelos mucronata

ETHNOPHARMACOLOGICAL RELEVANCE : Cissampelos mucronata A. Rich., a perennial climber belonging to the family Menispermaceae, has been used traditionally to treat parasites and tuberculosis-related symptoms. Co-infection of helminth parasites and tuberculosis-causing pathogens heightens the risk of developing active tuberculosis. AIM OF THE STUDY : The aim was to isolate and characterize antimycobacterial compounds from Cissampelos mucronata and to investigate their antibiofilm and anthelmintic efficacy as well as cytotoxicity. MATERIALS AND METHODS : The acetone extract of C. mucronata leaves and stems was fractionated by vacuum liquid chromatography using hexane, ethyl acetate, acetone and methanol:chloroform (3:7). Separation of the active ethyl acetate fraction by column and preparative thin layer chromatography led to the isolation and identification of five compounds using NMR and LC-MS, as well as GC-MS for non-polar compounds. The anthelmintic, antimycobacterial, antibiofilm, antioxidant and anti-inflammatory effects as well as cytotoxicity of the fractions and compounds were determined. RESULTS : The ethyl acetate fraction had the best antimycobacterial activity (MIC = 0.015–0.08 mg/ml). The fractions were relatively non-toxic to Vero cells (0.03–0.79 mg/ml) and had good anti-inflammatory and antibiofilm effects. Five compounds were identified as stigmasterol, hentriacontane, simiarenol, nonacosene and carbonic acid. Nonacosene had moderate anthelmintic effects but poor antimycobacterial activity (MIC = 0.375 mg/ml). Nonacosene and hentriacontane had good biofilm inhibitory effect (90–100%). CONCLUSIONS : This study reveals that C. mucronata is a potential source of promising compounds with a range of useful bioactivities that support its use in traditional medicine. Development of plant-based remedies may assist in reducing the impact of co-infections with helminth parasites and tuberculosis-causing mycobacteria.The University of South Africa, the University of Pretoria, the South African Medical Research Council and the National Research Foundation.http://www.elsevier.com/locate/jethpharmhj2023Paraclinical Science

Antibacterial and antibiofilm activity of acetone leaf extracts of nine underinvestigated south African Eugenia and Syzygium (Myrtaceae) species and their selectivity indices

BACKGROUND : Antimicrobial resistance (AMR) remains an important global health issue but the gap between AMR and development of new antimicrobials is increasing. Plant extracts may have good activity per se or may be sources of effective antimicrobial compounds which can act against planktonic and/or biofilms of pathogens. We determined the antimicrobial efficacy and cytotoxicity of some under-investigated plants from the Myrtaceae family endemic to South Africa. The ability of the plant extracts to inhibit or destroy pre-formed bacterial biofilms was also determined. METHODS : Based on previous preliminary in vitro screening and on chemotaxonomy, nine species from the Myrtaceae family were selected. The antimicrobial activity of the crude acetone leaf extracts was determined against six common nosocomial pathogens, namely: Gram-positive bacteria (Bacillus cereus, Enterococcus faecalis, Staphylococcus aureus), Gramnegative bacteria (Escherichia coli, Pseudomonas aeruginosa, Salmonella Typhimurium) using a two-fold serial microdilution assay with p-iodonitrotetrazolium violet as growth indicator. The number of antimicrobial compounds present in extracts was determined by bioautography. Cytotoxicity of extracts was determined against Vero kidney cells using a colorimetric tetrazolium-based assay. The total antibacterial activity (TAA) in ml/g and selectivity index (LC50/MIC) of the plant extracts were calculated. A modified crystal violet assay was used to determine the antibiofilm activity of the extracts. RESULTS : Syzygium legatii, Syzygium masukuense, and Syzygium species A had the best activities against Gram-negative and Gram-positive bacteria (MIC) values ranging from 0.04–0.08 mg/ml. Eugenia erythrophylla had the best MIC (0.02 mg/ml) against Bacillus cereus. Many extracts had relatively low cytotoxicity (LC50>20 μg/ml) leading to reasonable selectivity indices. Three leaf extracts (Syzygium masukuense, Syzygium species A, and Eugenia natalitia) were moderately cytotoxic (20 μg/ml < LC50<100 μg/ml). The plant extracts had a good capacity to reduce biofilm formation and good to poor potential to destroy pre-formed biofilms. CONCLUSIONS : The plant species examined in this study had varying degrees of antibacterial activity against bacterial planktonic and biofilm forms with some having good activity against both forms. Several of these selected species may be potential candidates for further investigation to isolate antimicrobial compounds and to determine the mechanism of activity.The South African National Research Foundation, The World Academy of Science (Grant No. 99808) and the University of Pretoria.https://bmccomplementalternmed.biomedcentral.comam2020Paraclinical SciencesVeterinary Tropical Disease