Antibacterial and antibiofilm activity of selected medicinal plant leaf extracts against pathogens implicated in poultry diseases

Antimicrobial resistant poultry pathogens are responsible for treatment failure and economic losses, and can also be a source of resistant zoonotic infections representing a risk to human health. In 2006 the European Union banned the use of antibiotics as growth promoters in farm animals and other regions are likely to follow suit. Alternative products and strategies are sought to help maintain animal gut health to reduce the prevalence of pathogens in the food chain. The minimum inhibitory concentration (MIC) of organic and aqueous leaf extracts of Alchornea laxiflora, Ficus exasperata, Morinda lucida, Jatropha gossypiifolia, Ocimum gratissimum, and Acalypha wilkesiana were tested against bacterial poultry pathogens including Staphylococcus aureus, Enterococcus faecalis, Salmonella spp., Escherichia coli, Campylobacter spp., and fungal species (Aspergillus fumigatus, Aspergillus flavus, and Candida albicans) using a 2-fold serial microdilution method. Activity of extracts against biofilms of the pathogens was done using a modified crystal violet staining in vitro assay. The safety of extracts was determined against Vero and Caco-2 cells using a tetrazolium-based in vitro assay. Acetone and cold water extracts of M. lucida had the best activity against three bacteria (MIC = 0.05–0.07 mg/ml) and two fungal (MIC = 0.03–0.15 mg/ml) organisms, respectively. The E. coli isolate and A. flavus were the most susceptible bacteria and fungi, respectively. Caco-2 cells generally displayed higher selectivity index (SI) values compared to Vero cells and average SI values against Vero and Caco-2 cells for both bacteria and fungi ranged from 0.01 to 4.48 and 0.005 to 16.41, respectively. All plant extracts had good anti-biofilm activity (>50%) against at least one organism. The disruption of established biofilm growth by the plant samples proved to be more difficult to achieve than efficacy against planktonic forms of bacteria. This study shows that some of the plant species are potential candidates as alternative feed additives in poultry production. In the future, a poultry feed trial to evaluate their in vivo efficacy as herbal feed additives will be conducted.The National Research Foundation (NRF) of South Africa and the University of Pretoria.https://www.frontiersin.org/journals/veterinary-sciencedm2022Paraclinical Science

Biofilm expression and antimicrobial resistance patterns of Streptococcus uberis isolated from milk samples of dairy cows in South Africa

The research described in this Research Communication addresses the hypothesis that intramammary infections with Streptococcus uberis (S. uberis) are associated with biofilm formation, which limits antibiotic efficacy. This retrospective study investigated biofilm expression and antimicrobial resistance (AMR) patterns of 172 S. uberis infections. Isolates were recovered from milk samples of subclinical, clinical, and intramammary infection cases on 30 commercial dairy herds. We determined the presence and intensity of biofilm expression of S. uberis isolates in vitro in three somatic cell count categories to recognise their AMR patterns. An automated minimum inhibitory concentration system with a commercially available panel of 23 antimicrobial agents evaluated AMR, while biofilm determination was conducted using a microplate method. The study established that all the S. uberis isolates assessed expressed biofilm with the following varying degrees of intensities: 30 (17.8%) strong, 59 (34.9%) medium and 80 (47.3%) weak biofilms. The newly registered UBAC mastitis vaccine containing biofilm adhesion components may, therefore, be a viable option for proactive mastitis management under field conditions. No differences were identified between biofilm intensity and the three somatic cell count groups. Most S. uberis isolates indicated a high-level sensitivity to the antimicrobial agents tested. Resistances were present in 8.7, 8.1 and 7.0% cases to rifampin, minocycline and tetracycline, respectively. Multidrug resistance was observed in 6.4%, emphasising AMR to antibiotics used in human medicine only. The low overall resistance suggests that farmers adhere to the prudent use of antimicrobials in the dairy industry.The National Research Foundation and Milk South Africa.https://www.cambridge.org/core/journals/journal-of-dairy-researchhj2023Paraclinical SciencesProduction Animal Studie

Selenium Enrichment of Green and Red Lettuce and the Induction of Radical Scavenging Potential

Selenium (Se)-enriched vegetables are promising dietary sources of Se, which provides beneficial biological effects in humans. In this study, we investigated the effects of foliar application of Se on hydroponically grown multi-leaf green (V1) and red (V2) lettuce plants. Three selenate (SeIV) amendment levels were evaluated for their influence on plant growth, elemental composition and radical scavenging capacity. Lettuce heads biofortified with 0.598 mg Se plant−1 accumulated 19.6–23.6 and 14.9–17.6 μg Se g−1 DM in the multi-leaf green (V1) and red (V2) lettuce plants, respectively. The accumulated Se levels can contribute significantly to the recommended dietary allowance of 70 µg day−1 for adult men and 60 µg day−1 for adult women. Accordingly, both V1 and V2 lettuce cultivars grown under the Se3 foliar application condition can cover the daily requirement for adult men by approximately 100% and 85% to 100%, respectively, by consuming 75–90 g or 100 g fresh weight from V1 or V2, respectively. The ABTS radical scavenging potential of green lettuce was induced at Se2 and Se3 foliar application levels, where the IC50 was 1.124 ± 0.09 μg mL−1 at Se0 and improved to 0.795 ± 0.03 and 0.697 ± 0.01 μg mL−1, respectively. There was no cytotoxicity against Vero kidney cells among all treated lettuce plants at the highest concentration tested of 1 mg/mL. Finally, a further focused investigation of the metabolic profile of lettuce plants under varied Se levels needs to be investigated in future studie

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

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

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

LC–DAD–MS phenolic characterisation of six invasive plant species in Croatia and determination of their antimicrobial and cytotoxic activity

Invasive plants’ phytochemicals are important for their invasiveness, enabling them to spread in new environments. However, these chemicals could offer many pharmaceutical compounds or active ingredients for herbal preparations. This study provides the first LC–MS phytochemical screening of six invasive alien plant species (IAPS) in the Istria region (Croatia): Ailanthus altissima, Ambrosia artemisiifolia, Conyza canadensis, Dittrichia viscosa, Erigeron annuus, and Xanthium strumarium. The study aims to identify and quantify the phenolic content of their leaf extracts and assess their antimicrobial and cytotoxic potential. A total of 32 species-specific compounds were recorded. Neochlorogenic, chlorogenic, and 5-p-coumaroylquinic acids, quercetin-3-glucoside, and kaempferol hexoside were detected in all the tested IAPS. Hydroxycinnamic acid derivatives were the main components in all the tested IAPS, except in E. annuus, where flavanones dominated with a share of 70%. X. strumarium extract had the best activity against the tested bacteria, with an average MIC value of 0.11 mg/mL, while A. altissima and X. strumarium extracts had the best activity against the tested fungi, with an average MIC value of 0.21 mg/mL in both cases. All the plant extracts studied, except X. strumarium, were less cytotoxic than the positive control. The results provided additional information on the phytochemical properties of IAPS and their potential for use as antimicrobial agents.SUPPLEMENTARY MATERIALS : Figure S1: Heat map presenting the representations of phenolic groups in different invasive plants; Figure S2: Chromatogram of the acetone extracts of the plant species leaves developed in ethyl acetate/methanol/water (EMW) solvent system sprayed with vanillin– sulphuric acid and TLC bioautograms; Table S1: Spectrum, mass-to-charge ratio (m/z) values of the molecular masses, and main fragments (MS2—second-generation product ion, MS3—thirdgeneration product ion) in negative ion mode ((M-H)−) identified with ESI–MS and the distribution of individual compounds in different invasive plants.The Croatian Science Foundation, the Slovenian Research Agency (ARRS) and the University of Pretoria, South Africa.https://www.mdpi.com/journal/plantsdm2022Paraclinical Science

Synthesis and anticancer activity evaluation of some new 1,2,3,5-tetrazine derivatives attached to benzothiazole moiety

A series of novel tetrazine derivatives, containing benzothiazole framework, were prepared during the coupling reactions of some diazotized 2-aminobenzo[d]thiazole derivatives with p-acetaminophen. Their structures were elucidated based on NMR and MS spectrometry. The anticancer activity and the safety of the synthesized compounds along with the entire precursors were assessed against three human cancer cell lines and a normal cell line. All the synthesized compounds showed selective cytotoxic activity against the cancer cell lines used in comparison to the normal Vero cell line. Their IC50 values varied from 2.02 to 171.67 μM.The Cameroonian Ministry of Higher Education special research allocation, the German Academic Exchange Service (DAAD) and the University of Pretoria.https://www.arkat-usa.org/arkivoc-journalam2023Paraclinical Science

Ethnoveterinary botanical medicine in South Africa : a review of research from the last decade (2009 to 2019)

ETHNOPHARMACOLOGICAL RELEVANCE : Ethnoveterinary medicine (EVM) practices remain a common feature of South African animal husbandry, particularly in rural livestock healthcare. This review provides an update of research undertaken on South African EVM from 2009 until 2019. AIM OF THE STUDY : This review collates information and investigates trends in the increasing field of EVM research in South Africa over the last decade. MATERIALS AND METHODS : A literature search was conducted using available databases including ScienceDirect, PubMed, Scopus and Google Scholar. Dissertations, theses, books and technical reports were also searched. RESULTS : In the past decade, ethnoveterinary surveys conducted in South Africa report the use of 139 plants from 50 families used against 21 animal diseases and conditions. Leaves, roots and bark have remained popular plant parts used for EVM. In terms of livestock species reported, the major focus was on cattle, goats and poultry. Only four of the nine provinces in the country have been surveyed. CONCLUSIONS : Relatively few publications reporting on ethnoveterinary surveys have originated from South Africa. These papers refer to many plants used for a variety of commonly encountered animal diseases and afflictions. With reference to recently published guidelines on conducting ethnobotanical surveys, several recommendations can be made to improve the robustness of surveys documenting the use of plants for EVM in South Africa.The National Research Foundation (NRF) of South Africa and the University of Pretoria.http://www.elsevier.com/locate/jethpharm2021-07-15hj2020Paraclinical Science