Toxicological implications of amplifying the antibacterial activity of gallic acid by immobilisation on silica particles: A study on C. elegans

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Highlights

  • Bactericidal properties of free and immobilised gallic acid was studied.

  • The minimum bactericidal concentration for L. innocua was obtained.

  • Toxicological properties of those concentrations were tested on C.elegans.

  • The amplification of activity was evidenced with immobilization.

  • Immobilisation reduced mortality from 28 % to 6 % with the same reduction of bacteria.

Abstract

Immobilisation of natural compounds on solid supports to amplify antimicrobial properties has reported successful results, but modifications to physico-chemical properties can also imply modifications from a toxicological viewpoint. This work aimed to study the immobilising process of gallic acid in the antibacterial activity of L. innocua and its toxicological properties in vivo using Caenorhabditis elegans. The experiment was based on obtaining the minimum bactericidal concentration for free and immobilised gallic acid by comparing lethality, locomotion behaviour, chemotaxis and thermal stress resistance on C.elegans at those concentrations. The results showed a lowering minimum bactericidal concentration and modifications to nematode responses. Increased lethality and velocity of movements was observed. Immobilisation increased the repellent effect of gallic acid with a negative chemotaxis index. Thermal stress resistance was also affected, with higher mortality for immobilised gallic acid compared to bare particles and free gallic acid. Thus despite evidencing a generalised increase in the toxicity of gallic acid in vivo, lowering the minimum bactericidal concentration allowed a bacterial reduction of 99 % with less than one third of mortality for the nematodes exposed to free gallic acid.

Introduction

Emerging problems of the negative impact of some synthetic antimicrobials on consumer health, abuse of antimicrobial substances due to inadequate traditional food-conservation methods and a rise in antibiotic-resistant bacteria and fungi strains all render the development of new strategies to prevent food spoilage and contamination necessary (Pisoschi et al., 2018). One of the main approaches in this area is to use naturally-occurring antimicrobial chemicals and to focus on employing natural compounds from sources like plants, bacteria, animals and fungi to ensure food-product safety given their antimicrobial activity produced against given pathogens (Saleem, 2014).

Specifically for plant compounds, properties such as antioxidants, antimicrobials, anti-diabetics, anti-carcinogens, flavourings, beverages and repellents have been demonstrated for several herb and plant extracts, which represent wide applicability in food manufacturing (Hygreeva et al., 2014). Although the origin of these compounds is natural, observations generate requirements for specific systemic research to test the toxicity and mechanisms of action in order to devise a complete safety mode of use and regulations. Thus not only should original compounds be tested, but also their possible physico-chemical modified versions. Research about modifications to natural compounds has focused on the change in their properties, such as solubility, dispersion across food matrices, avoiding volatility, etc., to amplify the antimicrobial effect and to cushion the impact on products’ organoleptic properties (Weiss et al., 2009).

Immobilisation of compounds on solid matrices offers higher potential in relation to the above-mentioned aim. This process provides increased antimicrobial capacity from a small amount of compound compared to its free version. This modification allows higher local compound concentrations and, thus, enhances membrane disruption mechanisms. Based on this approach, silica particles have been used as a solid matrix to be coated by certain natural compounds with successful results. Some of those results have been reported by Li and Wang (2013), who worked with lysozyme-coated silica nanoparticles, and showed the efficient amplification of antibacterial activity against Escherichia coli. Qi et al. (2013) demonstrated how silica nanoparticles reduced gram-positive bacteria by coatings with vancomycin. Pȩdziwiatr-Werbicka et al. (2014) synthesised fatty acid-functionalised mesoporous silica particles with antimicrobial activity.

One of the groups with a potential for this aim is phenolic compounds, which form one of the most numerous and ubiquitously distributed groups of plant secondary metabolites commonly found in diverse dietary products, particularly vegetables, fruit, chocolate and beverages (Soobrattee et al., 2005). Natural phenolic products can exhibit a wide range of biological effects, which include antibacterial, antifungal, antiviral. Gallic acid (GA) is one of the main compounds in that group that possesses antimicrobial properties in human pathogens, such as E. coli, L. monocitogenes, S. aureus, etc., according to several reported experiments (Borges et al., 2013; Jayaraman et al., 2010). These antimicrobial properties have also been evidenced in in vivo systems such the nematode C. elegans by Singulani et al. (2017), where GA protected against C. albicans infection. Saul et al. (2011) reported improvements to the life span and thermal stress resistance of these nematodes by exposure to GA. Hence the immobilisation of GA onto particles could modify these properties, as reported by Abdel-Wahhab et al. (2016), who evidenced enhanced antioxidant and antimicrobial activities in vitro and in vivo by immobilising GA onto chitosan and silica nanoparticles. Therefore, possible toxicological effects must be taken into account when making these modifications.

The aim of this work was to study the immobilising process of GA onto silica mesoporous particles in the antibacterial activity of Listeria innocua and its toxicological properties in vivo using Caenorhabditis elegans

Section snippets

Reagents

The chemicals N-cetyltrimethylammonium bromide (CTABr), tetraethylorthosilicate (TEOS), NaOH, triethanolamine (TEAH3), (3-Aminopropyl)triethoxysilane (APTES), gallic acid (GA), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were supplied by Sigma-Aldrich (Madrid, Spain). Acetonitrile, ethanol and microbiological media grade were provided by Scharlab (Barcelona, Spain).

Synthesis of MCM-41 microparticles

The synthesis of microparticulated MCM-41 particles was carried out following

Synthesising and characterising the GA-functionalised silica particles

The characterisation results, including particle size distribution, zeta-potential and GA content of the bare and functionalised materials, are shown in Table 1. P and PGA displayed a size distribution within the microscale size range with non-significant differences between samples. As regards the zeta-potential, P fell within ca. −30 mV due to the presence of silanol groups on the support’s surface. The zeta-potential of the PGA particles significantly differed from the non-modified silica

Conclusion

Amplifying the antimicrobial effect of GA by immobilisation on silica particles led to significant changes in the toxicological response of C. elegans compared to the same free compound. PGA increased mortality and its theoretical LC50 was half that of GA. The locomotion behaviour of all the parameters was also affected. Thus we conclude that stress increased as a result of combining rising total displacement, high velocity and the effective area, compared to lesser equivalent GA

CRediT authorship contribution statement

Samuel Verdú: Conceptualization, Methodology, Formal analysis, Investigation, Writing - original draft, Supervision, Visualization. María Ruiz-Rico: Conceptualization, Methodology, Formal analysis, Investigation, Writing - original draft. Alberto J. Perez: Software, Data curation, Writing - original draft. José M. Barat: Project administration, Funding acquisition, Supervision. Pau Talens: Project administration, Funding acquisition, Supervision. Raúl Grau: Conceptualization, Writing - review &

Declaration of Competing Interest

The authors report no declarations of interest.

Acknowledgements

The authors gratefully acknowledge financial support from the University Polytechnic of Valencia by programme “Ayudas para la Contratación de Doctores para el Acceso al Sistema Español de Ciencia, Tecnología e Innovación, en Estructuras de Investigación de la UPV (PAID-10-17)” and the Ministerio de Ciencia, Innovación y Universidades, the Agencia Estatal de Investigación and FEDER-EU (Project RTI2018-101599-B-C21).

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