Anti-bacterial Potential of the Genus Rubus Público
Mendelsohn, Matthew A. (2017)
Abstract
With the decreased efficacy of many antibiotics in the face of rising drug resistance, there is an urgent need for the development of new and alternative therapeutics to combat this looming crisis. A novel approach to killing resistance bacteria utilizes active compounds to inhibit bacterial defense mechanisms, thereby increasing the efficacy of current antibiotics. Previously, an extract prepared from the roots of Rubus ulmifolius, was shown to inhibit biofilm formation in S. aureus, a major bacterial defense mechanism, and significantly improve biofilm clearance when used concomitantly with an antibiotic. The present study investigates the chemistry, bioactivity and anti-biofilm properties against S. aureus of eight different Rubus species and their various plant parts to see if the activity and chemistry are conserved across the genus.
Liquid extraction and partitioning techniques were employed to prepare 4 refined partitions and 1 crude extract for each of the 8 Rubus species and 11 plant parts tested. Broth dilution methods were employed to determine the minimum inhibitory concentrations (MIC) after 18 hours of growth using an optical density reading. Anti-biofilm effects were assessed by growing biofilms for 24 hours, then fixing and staining with crystal violet. After washing, the biofilms were eluted, optical density readings were taken, and minimum biofilm inhibitory concentrations (MBIC) were calculated. The presence of ellagic acid and two of its glycosidic derivatives was assessed using HPLC, matching retention times and UV spectrums of our extracts to the three standards prepared.
Ellagic acid was found to be present within every Rubus species; however, none of the species contained either of the two derivatives examined. Extracts prepared from each plant species exhibited an MIC50 at concentrations ranging from 32 - 256 µg/ml. Each species tested inhibited biofilm formation at a concentration below where you see growth inhibition. Two extracts in particular, 730 and 735D, potently inhibited biofilm formation at 8 µg/ml without inhibiting growth significantly and therefore represent promising candidates for the development of novel natural product inhibitors of biofilm formation. We recommend further studies and bioassay-guided fractionation be done to develop these compositions into antibiotic adjuvant therapeutics against S. aureus.
Table of Contents
CHAPTER 1: INTRODUCTION 1
My inspiration
Looming crisis of antimicrobial resistance in Staphylococcus aureus
Natural products as alternative treatments: an ethnobotanical approach to drug discovery
Project aims and research questions
CHAPTER 2: LITERATURE REVIEW 6
Why Blackberries?
Chemistry and bioactivity of the genus Rubus
An investigation of eight distinct Rubus species
Staphylococcus aureus and Antimicrobial resistance
Biofilms and Staphylococcus aureusCHAPTER 3: MATERIALS AND METHODS 38
Literature Review
Botanical Materials
Generation of Rubus Extracts
Minimum Inhibitory Concentration Assay (Growth Inhibition)
Assessment of Biofilm Formation
High Performance Liquid Chromatography
CHAPTER 4: RESULTS 44
Extraction
Liquid/Liquid Partitions
S. aureus growth and biofilm inhibition
MIC & MBIC
HPLC Analysis
CHAPTER 5: DISCUSSION 81
The inhibitory effects of various Rubus species against S. aureus
Anti-biofilm activity of Rubus against S. aureus
Ellagic acid, Ellagic Acid derivatives, and Rubus
The Potential of Biofilm Inhibitors
Limitations and Future Directions
Conclusion
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