Anticariogenic properties of dodonaea viscosa var, angustifolia derived flavone stabilized nanoparticles

Semelemetja, Mpho Audrey
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Introduction Dental caries is the most important oral infection. It is caused by Streptococcus mutans due to its ability to form biofilm and the production of acids in the oral cavity. Many oral hygiene products containing antimicrobial chemicals have been used to control and prevent dental caries. Medicinal plants have also been investigated for their ability to prevent dental caries. Dodonaea viscosa var. angustifolia, has been found to have this property. However, beneficial concentrations are difficult to maintain in the oral cavity due to continual saliva flow which can be overcome using nanoparticles. The aim of this study was to investigate the anti-cariogenic properties of Dodonaea viscosa var. angustifolia (DVA) derived flavone stabilized nanoparticles. Methods and materials Stock cultures of Streptococcus mutans were obtained from the Oral microbiology laboratory and the DVA plant was collected from Mkhunyane Eco Reserve in Mpumalanga, South Africa. The leaves were dried and milled to powder. The crude extract was prepared from DVA dried leaves using methanol, which were fractionated to produce six fractions. Fraction F 5 was further fractionated to subfraction F 5.1 and F 5.2. A flavone containing subfraction (subfraction F 5.1) was selected. Subfraction F5.1 was chosen as the main agent due to its antimicrobial activities. Subfraction F5.1 was found to have anti-streptococcus. mutans activity, anti-acidogenic and anti-aciduric properties (Patel et al., 2009; Naidoo et al., 2012 and Ngabaza et al., 2017). Poly lactic-co-glycolic acid (PLGA) and poly ethylene glycol (PEG) were polymerized to form diblock copolymers. These copolymers were then used to synthesize PLGA-PEG nanoparticles, stabilized with the DVA derived subfraction and crude extract. Ten nanoparticles were prepared using PLGA-PEG copolymers. The synthesized products were then used to screen for antimicrobial activity to obtain minimum inhibitory concentrations (MIC) and minimum bactericidal concentration (MBC) using a mictrotitre broth dilution technique. The most active nanoparticles were chosen and characterized. Further tests were done using the best active nanoparticle mixture (PLGA-PEG subfraction stabilized nanoparticles), subfraction F 5.1 (flavone) and unattached PLGA-PEG nanoparticles (blank). The effect of acid production and biofilm production of the chosen nanoparticles were determined using acid production assay and biofilm production assay respectively. The subfraction release profile (substantivity) from the nanoparticle’s matrix was determined at physiological pH (7.4) and cariogenic pH (5.5) over a period of 12 hours. The cytotoxicity of PLGA-PEG subfraction stabilized nanoparticles was determined using human hepatic cells. Results were analyzed using Wilcoxon sum test (Mann-Whitney) and student T-test. Results The ten nanoparticles were divided into two groups depending on the molecular weight: Five nanoparticles were prepared using higher molecular weight (two surface adsorbed nanoparticles, two surface/ encapsulated nanoparticles and one blank nanoparticles) and five nanoparticles were prepared from lower molecular weight (two surface adsorbed nanoparticles, two surface/ encapsulated nanoparticles and one blank nanoparticles). Each group consisted of crude nanoparticles and flavone nanoparticles. The results showed that the ten groups of synthesized nanoparticles had a degree of antimicrobial activity. The median MIC/MBC values of the ten nanoparticles ranged from 25 mg/ml to 1.56 mg/ml. Subfraction stabilized nanoparticles were chosen, and MIC/MBC was repeated. The nanoparticles retained the known anti-streptococcus. mutans property of flavone. PLGAPEG Subfraction F 5.1 stabilized nanoparticles and blank nanoparticles had a median MIC value of 1.56 mg/ml and 6.25 mg/ml respectively. Sub-inhibitory concentrations of the flavone (P=0.02), subfraction stabilized nanoparticles (P=0.02) and blank nanoparticles (P=0.03) significantly reduced the acid production in S. mutans. Biofilm formation was reduced in all tests with the highest reduction in F 5.1 and lowest reduction in blank nanoparticles. The lowest sub-inhibitory concentration of subfraction F 5.1 stabilized nanoparticles (0.20 mg/ml) reduced biofilm formation by 84.0% after 6 hours of incubation and 85.8% after 24 hours. The lowest sub-inhibitory concentration of blank nanoparticles (0.78 mg/ml) reduced biofilm formation by 35.5% after 6 hours of incubation and 33.8% after 24 hours. The retention and slow release profiles of subfraction F 5.1 stabilized nanoparticles showed good substantivity, with 60% and 40% of the flavone being released over a period of 12 hours at pH of 5.5 and 7.4 respectively. The cytotoxicity profile of subfraction F 5.1 stabilized nanoparticles showed that the nanoparticles are safe to use. Conclusion Nanoparticles stabilized with subfraction F 5.1 from the plant DVA which is well known to have anti-streptococcus. mutans properties have shown improved potency against S. mutans due to the slow release of the subfraction drug from the nanoparticles over a period of 12 hours at a physiological pH (7.4) and at cariogenic pH (5.5). The slow release of the flavone from the nanoparticles showed good substantivity. These results suggest that PLGA-PEG stabilized DVA flavone nanoparticles have the potential to be used as an anti-cariogenic agent.
Dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfillment of the requirements for the degree of Master of Science in Medicine Johannesburg 2019