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Undergraduate Theses Undergraduate Works
4-24-2020
Screening a Library of Chalcone Derivatives for Antibacterial Properties via Kirby Bauer Disk Diffusion
Breena Z. Frazier Bellarmine University, [email protected]
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Recommended Citation Frazier, Breena Z., "Screening a Library of Chalcone Derivatives for Antibacterial Properties via Kirby Bauer Disk Diffusion" (2020). Undergraduate Theses. 44. https://scholarworks.bellarmine.edu/ugrad_theses/44
This Honors Thesis is brought to you for free and open access by the Undergraduate Works at ScholarWorks@Bellarmine. It has been accepted for inclusion in Undergraduate Theses by an authorized administrator of ScholarWorks@Bellarmine. For more information, please contact [email protected], [email protected]. Frazier 1
Screening a Library of Chalcone Derivatives for Antibacterial
Properties via Kirby Bauer Disk Diffusion
Breena Frazier
Spring 2020
A Senior Honors Thesis Presented in Partial Fulfillment of the Requirements
of the Bellarmine University Honors Program
Advisor: Dr. Amanda Krzysiak Reader: Dr. Savita Chaurasia Reader: Dr. Mary Huff
Frazier 2
TABLE OF CONTENTS LIST OF FIGURES ...... 4 LIST OF TABLES ...... 4 ABSTRACT ...... 6 INTRODUCTION ...... 7 Natural Products...... 7 Classification...... 8 Chalcones ...... 9 Chalcone Biosynthesis and Modification ...... 10 Biological and Pharmaceutical Properties ...... 10 Antimicrobials...... 11 Antibiotics ...... 11 Mechanisms of Action ...... 11 Antibiotic Resistance ...... 13 Antimicrobial Properties of Chalcone Derivatives ...... 15 METHODS ...... 17 RESULTS ...... 20 Gram Positive Strains ...... 23 Gram Negative Strains ...... 23 DISCUSSION ...... 25 ACKNOWLEDGEMENT ...... 29 LITERATURE CITED ...... 30 APPENDIX A – ZONE OF INHIBITION MEASUREMENTS ...... 34 TABLE 4. Zone of Inhibition Measurements for S. pneumoniae ...... 34 TABLE 5. Zone of Inhibition Measurements for S. enteritidis ...... 37 TABLE 6. Zone of Inhibition Measurements for B. subtilis ...... 40 TABLE 7. Zone of Inhibition Measurements for E. coli ...... 44 APPENDIX B – PHOTOS OF AGAR PLATES POST-INCUBATION SHOWING ZONE OF INHIBITION ...... 47 Frazier 3
TABLE 8. Post-chalcone diffusion photographs of S. pneumoniae grown on Tryptic Soy Agar with 5% Sheep Blood after 24-36-hour Incubation at 37° C ...... 47 TABLE 9. Post-chalcone diffusion photographs of S. enteritidis grown on LB agar after 24- 36-hour incubation at 37° C ...... 51 TABLE 10. Post-chalcone diffusion photographs of B. subtilis grown on LB agar after 24-36- hour incubation at 30° C ...... 55 TABLE 11. Post-chalcone diffusion photographs of E. coli grown on LB agar after 24-36-hour incubation at 37° C ...... 60
Frazier 4
LIST OF FIGURES
Figure 1. 1,3-Diphenylprop-2-en-1-one. Figure taken from Das, et al...... 9
Figure 2. Reaction Scheme for the Synthesis of Chalcones via Claisen-Schmidt Condensation. Figure taken from Durairaj M., et al...... 10
Figure 3. Full library of chalcone compounds synthesized by Tucker, et al...... 17
Figure 4. Disk Placement for Disk Diffusion (Anterior view) ...... 19
Figure 5. Gram-positive bacteria growing on agar plates with chalcones showing mild bacterial inhibition...... 23
Figure 6. Gram-negative bacteria growing on agar plates with chalcones showing mild bacterial inhibition...... 23
Figure 7. Licochalcone A...... 27
LIST OF TABLES
Table 1. Bacterial Growth Conditions...... 18
Table 2. Results from SciFinder Literature Survey ...... 20
Table 3. Antibacterial activity of chalcones against three bacterial strains ...... 21
Table 4. Zones of Inhibition for S. pneumoniae ...... 34
Table 5. Zones of Inhibition for S. enteritidis ...... 37
Table 6. Zones of Inhibition for B. subtilis ...... 40
Table 7. Zones of Inhibition for E. coli ...... 44
Table 8. Post-chalcone diffusion photographs of S. pneumoniae grown on Tryptic Soy Agar with
5% Sheep Blood after 24-36-hour Incubation at 37° C ...... 47
Table 9. Post-chalcone diffusion photographs of S. enteritidis grown on LB agar after 24-36-hour incubation at 37° C ...... 51 Frazier 5
Table 10. Post-chalcone diffusion photographs of B. subtilis grown on LB agar after 24-36-hour incubation at 30° C ...... 55
Table 11. Post-chalcone diffusion photographs of E. coli grown on LB agar after 24-36-hour incubation at 37° C ...... 60 Frazier 6
ABSTRACT
As antibiotic resistance emerges stronger than ever, novel antimicrobial compounds are necessary to continue the infectious fight against disease and prevent millions of deaths by multidrug-resistant bacteria. One compound thought to be a natural source of antimicrobial activity is the chalcone. Abundant in fruits and vegetables, chalcones are versatile compounds that are easily synthesized via Claisen-Schmidt condensation and have demonstrated a wide- range of biological activity. Previous studies have found that the addition of a free hydroxyl group to either the A or B ring of the chalcone skeleton may contribute to mild to moderate antimicrobial activity. Halogen substituents, such as bromine, chlorine, and fluorine, were also reported to aid in antibacterial activity. In this study, twenty chalcone compounds, fourteen of which had never been screened, were assessed for their antibacterial potential against two Gram- negative and two Gram-positive strains of bacteria. This was assessed via Kirby-Bauer disk diffusion in which filter paper disks were impregnated with chalcone derivatives of varying concentrations, ranging from 1 µM to 1 mM, arranged on an agar plate swabbed with bacteria, and then incubated overnight. The potential for antimicrobial activity was quantified by measuring the zone of inhibition (ZOI) of the bacteria after disk diffusion. Similar to findings in prior studies, the results from this study suggest that electron donating groups (e.g. hydroxyl, ethoxy, and phenoxy groups) on the A ring may contribute to weak inhibition of both Gram- positive and Gram-negative bacteria. Further studies must be conducted to determine a possible mechanism by which chalcones work on these microorganisms. Frazier 7
INTRODUCTION
Natural Products
Natural products can be defined and viewed differently depending on the scientific field or context. While natural products can be broadly defined as any substance produced by a living organism, the field of organic chemistry narrowly defines them as organic compounds produced as products or byproducts of metabolism, isolated from natural sources, and purified for industrial uses such as in medicine, for energy development, and for the production of consumer products such as food, health, and beauty products.1 In medicinal chemistry, the category of natural products is restricted even further to only include secondary metabolites.
Secondary metabolites are organic compounds produced during metabolism which are not necessary for survival but may provide organisms with certain evolutionary advantages.
Secondary metabolites play an important role in plants, serving as defense mechanisms against herbivores and other predators as well as serving as signaling compounds to attract pollinators and other seed dispersing animals.2 Many years of natural selection and evolutionary processes have created compounds with high structural diversity, some exceeding the capabilities of synthetic organic chemists. These diverse compounds also possess many unique medicinal and biological properrties.1 While these compounds may have been synthesized over time to keep plants alive, they also have potential as drug scaffolds. It is these unique properties and complex structures that interest medicinal chemists, thus leading to the isolation, organic synthesis, and modification of these secondary metabolites. Frazier 8
Records from ancient Mesopotamia reveal that humans have been utilizing natural products for medicinal purposes as far back as 2600 BCE, when it was documented that healers used oils from the Cypress tree – from which compounds are still found useful today– to treat cough, colds, and inflammation.3 Additionally, Papaver somniferum, the original source of morphine and codeine, was used as early as 4000 BCE by the Sumerians, who nicknamed the plant, “hul gil” or joy-plant.4 The two compounds, morphine and codeine, likely evolved within the genus Papaver as a defensive mechanism against herbivores. In the 19th century, early organic chemists became interested in modifying the structure of morphine, taken from P. somniferum, to create a nonaddictive form of the drug. Instead, they acetylated morphine to produce heroin, which was actually more additive than its predecessor. Similarly, most modern medicinal chemists use natural products as basic scaffolds for drug design and employ synthetic changes to the base compounds to reduce side effects and increase therapeutic effect and overall bioavailability.
In recent years, the pharmaceutical industry has seen a decline in the use of these compounds as drug developers focus more on synthetically derived drugs.5 However, with less than 10% of the world’s biodiversity currently being evaluated for biological properties, there are a multitude of compounds of great chemical diversity and unique biological and pharmaceutical functionality yet to be studied.3
Classification
Medicinal chemists often classify natural products into four main classes based broadly on their structure. These classes are terpenoids and steroids, alkaloids, fatty acid-derived substances and polyketides, and phenylpropanoids. Terpenoids are structurally diverse Frazier 9 compounds derived from 5-carbon monomers called isoprene. Alkaloids are compounds containing a basic amine group that are derived from amino acids.6 Fatty acid-derived substances and polyketides are synthesized from acyl precursors, or the building blocks of acetate and malonate. Phenylpropanoids are synthesized from the amino acids, phenylalanine and tyrosine.1
Chalcones
Chalcones are a member of the polyketide class of natural products. They are small compounds composed of two aromatic rings joined by a three carbon α, β-unsaturated carbonyl system.7
Figure 1. 1,3-Diphenylprop-2-en-1-one. Figure taken from Das, et al. (2018)
It is the conjugation of the double bond with the carbonyl group of the chalcone that is thought to contribute to its biological activity, as removal of this conjugated system makes them inactive.
Chalcones are found abundantly in edible plants and serve as intermediates in the flavonoid metabolic pathway.8 Frazier 10
Chalcone Biosynthesis and Modification
Figure 2. Reaction Scheme for the Synthesis of Chalcones via Claisen-Schmidt Condensation. Figure taken from Durairaj M., et
al. (2018)9
In the laboratory setting, chalcones are synthesized by Classen-Smith Condensation
(Figure 2). They can be modified by reacting substituted acetophenones and benzaldehydes to produce novel chalcone derivatives with substitutions to either the A or B ring.10 By making substitutions to either ring, chemists hope to alter the biological properties to discover a novel use of the compound or improve a known property through modification of a base structure.
Biological and Pharmaceutical Properties
Chalcones have been studied for an extensive range of biological and pharmaceutical properties. Researchers have evaluated chalcones and their analogues as anti-cancer, antiprotozoal, anti-HIV, and antioxidant agents as well as tested them for anti-malarial, antimicrobial, and anti-inflammatory properties.8 In addition to their versatility, clinical studies have also demonstrated excellent bioavailability and maximum tolerance in vivo. In fact, there are currently several drugs on the market or in clinical trials that contain the chalcone skeleton in their structures. For instance, metochalcone was approved as a choleretic drug, used to treat liver and gallbladder disease, and sofalcone was approved as an anti-ulcer medication.11 Frazier 11
Antimicrobials
Antibiotics
Antibiotics are drugs used to fight bacterial infections. They are designed to selectively target bacterial cells without affecting animal cells.12 This is accomplished by taking into consideration the differences between the two cell types and targeting specific properties of bacterial cells. Moreover, most species of bacteria fall into one of two groups based on a staining technique, which reveals unique characteristics about the cell. Gram-positive cells have a thick cell wall (20-40 nm) and stain purple. Gram-negative cells have a thinner cell wall (2-7 units) and an outer membrane composed of lipopolysaccharides; these bacteria stain pink. Furthermore, there are some bacteria called mycobacteria that are impermeable to gram staining due to the presence of mycolic acid in their cell walls. These bacteria can appear as either Gram-positive or
Gram-negative because they share characteristics of both groups.14 Due to these structural differences, some classes of antibiotics work to treat Gram-negative or Gram-positive infections more effectively due to their mechanism of action.
Mechanisms of Action
Most antibiotics can be grouped into one of five main mechanisms of action (MOA).12
The first antibiotic mechanism works by inhibiting the cell’s metabolism. These agents are termed antimetabolites. The primary mechanism by which this is accomplished is by inhibiting an enzyme-catalyzed reaction which is present in bacterial cells but not in mammalian cells. This is the mechanism of action of the sulphonamide class of antibiotics. They work as competitive enzyme inhibitors of the bacterial enzyme, dihydropteroate synthetase, resulting in the disruption Frazier 12 of tetrahydrofolate biosynthesis in the cell. Tetrahydrofolate is an enzyme cofactor involves in pyrimidine nucleic acid base synthesis. Mammals receive folic acid, the precursor to tetrahydrofolate, in their diet; therefore, they do not possess this enzyme.13 By blocking production of these nucleic acids, DNA synthesis is interrupted, and the bacterial cells can no longer grow and multiply.
Secondly, antibiotics can work by inhibiting bacterial cell wall synthesis causing cell lysis and death.12 Penicillins, cephalosporins, and glycopeptides such as vancomycin act by this
MOA. Penicillins work by inhibiting the enzyme, transpeptidase, which is responsible for the final cross-linking reaction in bacterial cell wall synthesis. Without the interlinking of the sugar backbone, the framework for the bacteria’s cell wall becomes compromised, making it more fragile and susceptible to swelling and eventual cell lysis.
Other antibiotics work by interacting with the plasma membrane, thereby affecting membrane permeability and resulting in eventual cell death. Both polymyxins and tyrothricin work in this manner. Polymyxin B, a commonly used polypeptide antibiotic, works by acting selectively on the lipopolysaccharide outer membrane of Gram-negative bacteria. It works to displace the calcium and magnesium bridges that stabilize the lipopolysaccharides causing an uncontrolled leakage of small molecules like nucleosides out of the cell.15 The mechanism of selectivity by which this antibiotic works is not fully understood.
Antibiotics can also work to disrupt protein synthesis and halt the production of essential proteins and enzymes.12 Agents working in this way include the rifamycins, aminoglycosides, tetracyclines, and chloramphenicol. Rifamycins work by inhibiting RNA synthesis in bacteria by blocking the prokaryotic DNA-dependent RNA polymerase. Rifamycins have a 100-10,000-fold Frazier 13 greater affinity for prokaryotic RNA polymerase than eukaryotic enzyme.16 Aminoglycosides, like streptomycin, neomycin, and tobramycin, work effectively on Gram-negative organisms.10
In slightly alkaline conditions, they carry a positive charge which interacts with various negatively charged groups on the outer surface of the cell membrane allowing more ready absorption into the bacterial cell. Once in the cell, they bind to specific bacterial proteins in the
30S ribosomal subunit and restrict movement of the ribosome along mRNA, making it impossible to read the code and synthesis proteins. Tetracyclines also bind to bacterial ribosomes and prevent transcriptions, but they are more broad-spectrum allowing them to kill both Gram- negative and Gram-positive cells. Chloramphenicol works similarly to tetracyclines by diffusing through the bacteria cell wall and binding to the bacterial 50S ribosomal subunit, thus interfering with peptide elongation and blocking protein synthesis.17
Lastly, antibiotics can work to inhibit nucleic acid replication and transcription, thereby preventing cell division or halting the production of essential proteins.12 Both nalidixic acid and proflavine work by this MOA. Nalidixic acid was the first agent discovered in the class later known as the quinolones and fluroquinolones. They inhibit the replication and transcription of bacterial DNA by stabilizing the interaction between topoisomerases and DNA resulting in an enzyme with no activity. Proflavine works by intercalating DNA, thereby distorting the structure such that it can no longer by replicated. This disruption of DNA synthesis prevents bacterial reproduction.18
Antibiotic Resistance
Antibiotic resistance occurs when bacteria develop the ability to survive exposure to antibiotics.19 The mechanism by which this resistance develops is either by genetic mutation of Frazier 14 the bacterial DNA which in turn provides the bacteria an evolutionary advantage or by acquisition of an antibiotic resistance gene from other bacteria stains via horizontal gene transfer.
As these antibiotic resistant bacteria grow and multiply within the host, they remain unaffected by the antibiotics used to kill them or stop their growth. Infections caused by antibiotic resistant bacteria can be very difficult to treat, often requiring costly and toxic alternatives to traditional antibiotics. Unfortunately, antibiotic resistance has been an increasingly more urgent issue over the past few decades. In 2014, the prime minister of United Kingdom commissioned a “Review on Antimicrobial Resistance,” the purpose of which was to take a look at the global issue of rising antimicrobial resistance and proposed reasonable plans to tackle the issue in the long term.20 In this report, they predicted that by 2050, 10 million people will die each year due to multidrug resistant pathogens. The global cost due to these infections was projected to reach 100 trillion US dollars, if no action is taken. Moreover, the rapid growth of antibiotic resistance threatens advances in modern healthcare.19 The advent of antibiotics has allowed many procedures that typically have a high risk of infection to become common place. However, as effective antibiotics become unavailable, healthcare providers will no longer be able to safely perform common surgeries, like hip replacements, or administer chemotherapeutics, which lower the recipient’s immune system.
This steady increase in antibiotic resistance can be attributed to two main factors, (1) the prescription of antibiotics to treat infections of a nonbacterial nature, such as viral infections, and
(2) lack of patient compliance.21 For instance, when patients do not complete their full course of antibiotic therapy because they begin to feel better and feel they no longer need it, the patient becomes more susceptible to the growth of antibiotic-resistant bacteria. This occurs because patients take a sublethal dose, and as a result, do not completely kill off the infectious bacteria. Frazier 15
While nationwide efforts promoting better drug adherence and more effective diagnostic tools can help slow the development of antibiotic resistance, it cannot be stopped.22 As bacteria evolves, growth in antibiotic resistance is inevitable. There will always be a need for new antibiotics to keep up with resistant bacteria. However, despite the need for new therapies, the antibiotic drug pipeline is currently scarcely populated.3 In fact, the number of major pharmaceutical companies invested in the production new antibiotic therapies has decreased from 18 in 1990 to only 4 in 2013.20
Antimicrobial Properties of Chalcone Derivatives
Historically, natural products have been pivotal in the discovery and development of antibiotics.5 The first modern antibiotic, penicillin, was discovered because it is naturally synthesized by Penicillium mold. Furthermore, of the nine classes of antibiotics, six are based on natural products, while only three classes (sulfonamides, fluoroquinolones, and oxazolidinones) rely entirely on synthetic chemistry.21 As antibiotic resistance continues to emerge as a threat to global health, researchers are beginning to gravitate back to where it all began – finding natural products with antibiotic activity.7 Among the many other pharmacological activities of these molecules, the antimicrobial effects of chalcone derivatives have been a hot topic of interest amongst many medicinal chemists.
In previous studies, researchers have found that the bactericidal effects of chalcone derivatives may be linked to the tendency of the α,β-unsaturated ketone of the chalcone to react with a nucleophilic group, like a thiol group in a protein, and undergo conjugated addition. In addition to proposing a mechanism for their activity, researchers are also interested in better understanding the structure-activity relationship (SAR) between chalcones and their antibacterial Frazier 16 properties. The purpose of SAR analysis is to determine possible pharmacophores, or chemical structures responsible for evoking certain biological effects. Many studies have found that electron-releasing groups, such as methoxy and hydroxy groups, may contribute to antibacterial activity, as chalcones with these type of additions have demonstrated mild to moderate inhibition in vivo.23, 24, 25, 26 Compounds containing halogen substituents (e.g. chlorine, bromine, fluorine) have also shown potential as possible antimicrobial agents.27, 28, 29, 30 The purpose of this thesis is two-fold: (i) evaluate an existing chalcone library for antimicrobial activity and (ii) analyze findings from the study to determine what type of substituents may contribute to bacterial inhibition. Frazier 17
METHODS
Kirby-Bauer Disk Diffusion
Figure 3. Full library of chalcone compounds synthesized by Tucker, et al. (2017)
Chalcones were obtained from the Krzysiak lab at Bellarmine University (Louisville,
KY) (Figure 3). They were synthesized by Claisen-Schmidt Condensation10 and stored in glass vials at room temperature. GE Healthcare Whatman™ Antibiotic Assay Discs were purchased from Fisher Scientific (Hampton, NH). Chalcones were diluted in dimethyl sulfoxide (DMSO) to four different doses (1 mM, 100 µM, 10 µM, 1 µM) for use in disk diffusion. Disks were Frazier 18 prepared by applying 25 µL of the diluted chalcone/DMSO solution to an individual 6 mm blank disc. The solution was allowed to soak into the disk for 15-20 minutes prior to placement to allow for complete absorption. DMSO (25 uL) was applied to blank discs for use as a negative control. Ciprofloxacin discs were used as a positive control for all four bacterial cultures.
Four MicroKwik cultures ® (Streptococcus pneumoniae, Salmonella enteritidis, Bacillus subtilis, and Escherichia coli) were purchased from Carolina Biological Supply (Burlington, NC) and reactivated according to the manufacturer’s instructions. After initial incubation, each bacterial culture was then initiated via heavy inoculum and grown at the optimal growth temperature on the suggested growth media for 24-48 hours (Table 1). These plates were used as stock plates; colonies were taken from them as needed for broth cultures.
Table 1. Bacterial Growth Conditions
Bacterial Strain Growth Medium Optical Growth
Temperature (°C)
Streptococcus Tryptic Soy Agar with 37 pneumoniae 5% Sheep Blood
Salmonella enteritidis LB Agar 37
Bacillus subtilis LB Agar 30
Escherichia coli LB Agar 37
Two to three colonies were plucked from the stock plate via a sterile inoculation loop and transferred into LB broth; the LB broth was then incubated for 24-72 hours at the optimal growth temperature. The optical density of the broth was measured at 600 nm, and the broth cultures Frazier 19 were diluted to 0.7 before being plated. This method of standardizing the turbidity was used as opposed to the McFarland standard protocol.31
Each chalcone was tested against all four organisms, and every assay was run in triplicate. Plates were inoculated using a sterile cotton swab. The cotton swab was dipped in the broth culture, and the surface of each plate was swabbed over the entire surface three times – with a 60-degree rotation between each to ensure proper distribution. Disks were applied according to the diagram below.
Figure 4. Disk Placement for Disk Diffusion (Anterior view). Graphic created by author.
The plates were incubated at optimal growth temperature and stored upside down to prevent collection of water droplets. After 24-36 hours, plates were visually assessed, and zones of inhibition were measured for each disk. Frazier 20
RESULTS
To determine if chalcone derivatives synthesized at Bellarmine University have antibacterial activity, each compound was screened via a Kirby-Bauer disk diffusion assay. This is a method used to quantify bacterial inhibition due to the presence of a specific compound. At the start of this study, the full Tucker, et al. library was reviewed for antimicrobial properties using the SciFinder database (Figure 3). This database allows users to search for literature related to a specific chemical structure and certain biological activities. The selected chalcones were chosen based on available stock and the number of “hits” in the database relating to how many studies looked at the same structure in the same context. It is important to note that chalcone
1A1B had the greatest number of hits because it is typically cited as a basic scaffold structure in most studies looking at chalcone derivative activity. In this case, the number of hits on the database does not correlate directly with increased biological activity, it simply correlates with use of the compound in prior studies. Chalcones with the fewest hits in the literature were selected for this study. Out of the chalcones available for testing, twenty were chosen for disk diffusion (Table 2).
TABLE 2. Results from SciFinder Literature Survey – Chalcones chosen for antibacterial screening.
Previous publication(s) according to Chalcones
SciFinder database
Not previously studied (No “hits” on 2A1B, 3A2B, 5A2B, 4A3B, 5A3B,
SciFinder) 2A4B,5A4B, 2A5B, 3A5B, 3A’5B, 4A5B,
3A6B, 3A’6B, 5A6B Frazier 21
Not assessed against the same organisms 5A1B, 1A3B
Structure only assessed in one prior study 4A2B 23, 1A4B24, 4A4B 24, 2A6B 25 using similar organisms
Each chalcone that was identified to have potential antimicrobial activity was tested against four different bacterial strains (Streptococcus pneumoniae, Salmonella enteritidis,
Bacillus subtilis, and Escherichia coli) using the Kirby Bauer disk diffusion method. In order to quantify antibacterial activity, zones of inhibition were measured for each chalcone compound and the controls for each plate. Zone of inhibition was measured by taking the diameter of the circular area around the disc of interest in which the bacteria did not grow. Since the discs used in the assay were 6 mm in diameter, ZOIs greater than 6 mm demonstrated some antimicrobial activity. Appendix A shows the raw data collected from the disk diffusion assay. Photographs of each plate can be found in Appendix B. The results indicate that none of the chalcone derivatives tested showed significant inhibition of bacterial growth across any of the four strains they were tested against. Some compounds, however, did show weak inhibition of growth against specific bacterial strains. These results are summarized in Table 2.
TABLE 3. Antibacterial activity of chalcones* against three bacterial strains.
Chalcone Zone of ID and Test Strain Strength effective inhibition Substituent properties structure (mm) Gram-Positive A Ring: Electron-donating 3A2B S. pneumoniae 10 µM 6.5 mm B Ring: Electron-donating
4A2B** S. pneumoniae 10 µM 6.5 mm Frazier 22
A Ring: Electron-donating 9.25 mm group B. subtilis 1 mM B Ring: Electron- withdrawing group A Ring: Electron-donating 5A3B S. pneumoniae 1 µM 6.1 mm group B Ring: Hydrophobic
Gram-Negative
7.5 1 µM mm
10 µM 7 mm A Ring: Electron-donating 5A2B S. enteritidis group 100 µM 8 mm B Ring: Hydrophobic
1 mM 9 mm
A Ring: Electron-donating 1A4B** S. enteritidis 1 µM 6.75 mm group B Ring: N/A
1 µM 7 mm
A Ring: Hydrophobic 2A6B** S. enteritidis 1 µM 7 mm B Ring: Hydrophobic
10 µM 7.75 mm Frazier 23
Gram Positive Strains
Figure 5. Gram-positive bacteria growing on agar plates with chalcones showing mild bacterial inhibition. Photos taken by
author.
In the Kirby-Bauer disk diffusion assay, the selected chalcones were evaluated for antimicrobial activity against two strains of gram-positive bacteria, S. pneumoniae and B. subtilis. The results of the assay suggest that chalcones 3A2B, 4A2B, and 5A3B may have potential as antimicrobial agents effective against gram-positive bacterial strains. In terms of structural similarity, all three compounds have electron-donating substituents on the A ring, whereas additions to the B ring vary among the compounds.
Gram Negative Strains
Figure 6. Gram-negative bacteria growing on agar plates with chalcones showing mild bacterial inhibition. Photos taken by
author. Frazier 24
The selected chalcones were also evaluated against two gram-negative strains of bacteria,
S. enteritidis and E. coli. None of the selected chalcones showed activity against E. coli.
Chalcones 5A2B, 1A4B, and 2A6B demonstrated weak inhibition of S. enteritidis. Among the three compounds, two have electron-donating additions to the A ring, while the other had a hydrophobic addition. As for additions to the B ring, both compounds with substituents to the B ring had hydrophobic substituents. In summary, the results for the gram-negative strains are generally inconclusive. However, when results from all four strains are considered, it seems that electron-donating additions to the A ring may be important for activity against both gram- negative and gram-positive strains as five out of six active compounds possess this type of addition. Frazier 25
DISCUSSION
In this study, twenty chalcone derivatives were evaluated for antibiotic potential via
Kirby Bauer disk diffusion. The results of the assay were quantified by measuring the zone of inhibition for each compound as well as positive and negative controls. Chalcone derivatives showing ZOIs greater than 6 mm demonstrated potential as antimicrobial agents. Out of the six compounds that were found to be active in this study, three of the chalcones (4A2B, 1A4B, and
2A6B) have been evaluated in prior studies assessing antimicrobial activity (Table 1).
Chalcone 4A2B was assessed by Gasha, et al. (1972) in a study looking at antimicrobial activity of a specific chalcone library.32 Instead of evaluating antimicrobial potential by disk diffusion, they found the minimum inhibitory concentration (MIC) of each compound they tested. MIC is the minimum concentration of a compound needed to inhibit viable growth of an organism. The lower the MIC, the more effective the compound is at inhibiting growth. The MIC of 4A2B was greater than 12.5 µg/mL for all ten strains it was tested against, including B. subtilis and E. coli. When compared to other compounds in the study, chalcone 4A2B exhibited weak inhibition of bacterial and fungal growth. In the study conducted in this thesis, chalcone
4A2B only exhibited weak inhibition of the gram-positive strains of bacteria with no inhibition of E. coli.
A study conducted by Kalyanasundaram, et al. (2014) tested chalcone 1A4B, referred to in the study as “entry #1,” against B. subtilis and E. coli via Kirby Bauer disk diffusion. They found that the compound weakly inhibited the growth of the B. subtilis with a zone of inhibition of 8 mm, while no inhibition was seen against the E. coli.33 In this study, however, chalcone Frazier 26
1A4B had the opposite effect. The compound was ineffective against B. subtilis, a gram-positive strain, and instead weakly inhibited growth of S. enteritidis, a gram-negative strain of bacteria.
Lastly, Budhiraja, et al. (2012) assessed 2A6B for antimicrobial properties and found that the compound was not effective against any of the organisms used, including B. subtilis and E. coli.34 2A6B showed a MIC of 125 µg/mL against both strains demonstrating very weak inhibition of growth. In this study, 2A6B weakly inhibited growth of S. enteritidis in two out of three trials. It is important to note that none of these compounds were tested against S. pneumoniae or S. enteritidis in prior studies.
Chalcone 4A4B was also previous assessed in the study by Kalyanasundaram, et al.
(2014). They found 4A4B to be active against E. coli with a zone of inhibition of 8 mm, whereas in this study, the compound was not active against any bacterial strain, including E. coli.33 Based on the results of this assay, there may have been an experimental error in the growth of the E. coli culture because none of the chalcone compounds that were active against E. coli in prior studies were active in this study. Moreover, it was the only strain whose growth was not inhibited by any of the compounds tested. This could be attributed to potential contamination of the culture, leading to growth of other bacterial strain(s) that were only susceptible to ciprofloxacin, the broad-spectrum antibiotic utilized as the positive control.
Among the six compounds with antibacterial potential, five had electron-donating substituents to the A ring, whereas substituents to the B ring varied among the group. This suggests that antibacterial activity may be associated with electron-donating additions to the A ring. This finding has been supported by other studies. For instance, Silva et al. (2013) did a similar study looking at the effect of certain chalcone derivatives on bacterial growth. They Frazier 27 found that the best activity was achieved when a hydroxyl group, an electron-donating group, was added in the ortho-position to the A-ring of the compound.26 The absence of a hydroxyl group to the A-ring resulted in no antibacterial activity.
There has been debate regarding whether the position of the electron-donating substituents, A versus B-ring, matters when it comes to antibacterial activity. Many studies have found that electron-donating substituents to the B ring show better activity. In a review looking at anti-infective and anti-inflammatory properties of chalcones, Nowakowska (2007) noted that analogues of licochalcone A (Figure 5), a chalcone isolated from licorice root, were found to be inactive without the free hydroxyl group, an electron-donating group, in the 4’-position of ring
B.7 Removal of the free-hydroxyl from the A-ring, on the other hand, had no effect on the activity. Moreover, a study completed by Hasan, et al looked at a series of six chalcone derivatives and evaluated their activity against both bacteria and fungi.35 They found that electron-donating groups, specifically methoxy and hydroxy groups, on the B ring were most effective at inhibiting bacterial growth. They did not, however, test the same substituents to the
A ring.
Figure 7.. Licochalcone A - a compound isolated from Glycyrrhiza inflata, or licorice root that has shown potent antibacterial
activity against Bacillus subtilis,Staphylococcus aureus and Micrococcus luteus. Figure taken from Nowakowska. (2007) Frazier 28
Prior evidence also supports the addition of hydrophobic substituents to the chalcone scaffold to increase antibacterial activity. Chalcone 2A6B possesses two hydrophobic substituents, one to the A-ring and one to the B-ring. In her review, Nowakowska (2007) also cited that removal of the lipophilic prenyl group of licochalcone A analogues results in total loss of activity in these compounds.7 Therefore, hydrophobicity, also referred to as lipophilicity, plays an essential role in the antibacterial effect of lipochalcone A analogues.
While no new trends in data or unique SAR were noted in this study, the results of this study contribute to the growing knowledge of what we currently know about chalcones and their antibacterial activity. This study serves to further show that electron-donating substituents, like methoxy and hydroxy groups, play an important role in antibacterial activity. Additionally, this study assessed fourteen new structures as possible antibacterial agents and found two new compounds with antibiotic potential. Modification of these compounds in future studies may allow for synthesis of structures with even greater antibiotic activity. This study was limited because it relied on a previously synthesized library of compounds and it only evaluated chalcones against four common strains of bacteria. Further research could certainly be conducted evaluating more potentially harmful bacterial strains, such as multidrug resistant bacteria or mycobacterium. Overall, this study provides further evidence that chalcones show potential as antibacterial agents. In addition to their versatility, chalcones are also easily synthesized and cost-effective, making them attractive as potential drug scaffolds. Furthermore, six out of nine classes of antibiotics currently used today come from natural products. Considering the amount of biodiversity not yet evaluated for medicinal purposes, there are many compounds yet to be assessed for use in antibacterial therapy. In a world of growing antibiotic resistance, natural products, like chalcones, should be considered as possible agents in the fight against disease. Frazier 29
ACKNOWLEDGEMENT
I would like to thank my advisor, Dr. Amanda Krzysiak, for all her support and encouragement throughout this process. I would not be the researcher I am today without her guidance. Thank you to my readers, Dr. Savita Chaurasia and Dr. Mary Huff, for taking time out of their business schedules to read drafts of my thesis throughout this semester. Their feedback was instrumental in getting this to publication. Thank you to my amazing, supportive friends who pushed me to work hard, encouraged me when things did not go according to schedule, and spent countless hours keeping me company in the lab throughout this long research process.
Lastly, a special thank you to Dr. Jon Blandford and the Honors Program for the opportunity to complete this thesis research. Frazier 30
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Frazier 34
APPENDIX A – ZONE OF INHIBITION MEASUREMENTS
TABLE 4. Zone of Inhibition Measurements for S. pneumoniae Chalcone Strength/Control Zone of Inhibition* (mm) –Two diameter measurements were taken for each disk, and the two measurements were averaged. Plate 1 Plate 2 Plate 3 Average average average average across all plates 2A1B Positive 29.75 29 28.75 29.17 Negative - - - - 1 µM - - - - 10 µM - - - - 100 µM - - - - 1 mM - - - - 5A1B Positive 31 28.5 29.5 29.67 Negative - - - - 1 µM - - - - 10 µM - - - - 100 µM - - - - 1 mM - - - - 3A2B Positive 30.75 29.75 29.75 30.08 Negative - - - - 1 µM - - - - 10 µM - 6.5 - 6.17 100 µM - - - - 1 mM - - - - 4A2B Positive 31.75 30.75 30 30.83 Negative - - - - 1 µM - - - - 10 µM 6.5 - - 6.17 100 µM - - - - 1 mM - - - - 5A2B Positive 32 31.25 29.5 30.92 Negative - - - - 1 µM - - - - 10 µM - - - - 100 µM - - - - 1 mM - - - - 1A3B Positive 30.75 31 29.5 30.42 Frazier 35
Negative - - - - 1 µM - - - - 10 µM - - - - 100 µM - - - - 1 mM - - - - 4A3B Positive 31 30.25 30 30.41 Negative - - - - 1 µM - - - - 10 µM - - - - 100 µM - - - - 1 mM - - - - 5A3B Positive 30.25 30 31.75 30.67 Negative - - - - 1 µM - - - - 10 µM - - - - 100 µM - - - - 1 mM - - - - 1A4B Positive 30.5 30 30.25 30.75 Negative - - - - 1 µM - - - - 10 µM - - - - 100 µM - - - - 1 mM - - - - 2A4B Positive 30 30.75 39.5 30.08 Negative - - - - 1 µM - - - - 10 µM - - - - 100 µM - - - - 1 mM - - - - 4A4B Positive 29.5 27 28.5 28.33 Negative - - - - 1 µM - - - - 10 µM - - - - 100 µM - - - - 1 mM - - - - 5A4B Positive 28.5 30 30 29.5 Negative - - - - 1 µM - - - - 10 µM - - - - Frazier 36
100 µM - - - - 1 mM - - - - 2A5B Positive 30 30 31 30.33 Negative - - - - 1 µM - - - - 10 µM - - - - 100 µM - - - - 1 mM - - - - 3A’5B Positive 31 28 29 29.33 Negative - - - - 1 µM - - - - 10 µM - - - - 100 µM - - - - 1 mM - - - - 3A5B Positive 30.5 28 29 29.17 Negative - - - - 1 µM - - - - 10 µM - - - - 100 µM - - - - 1 mM - - - - 4A5B Positive ND 29 29.5 29.25 Negative - - - - 1 µM - - - - 10 µM - - - - 100 µM - - - - 1 mM - - - - 2A6B Positive 30 28.5 27.5 28.67 Negative - - - - 1 µM - - - - 10 µM - - - - 100 µM - - - - 1 mM - - - - 3A’6B Positive 27.5 29.5 29.6 28.33 Negative - - - - 1 µM - - - - 10 µM - - - - 100 µM - - - - 1 mM - - - - 3A6B Positive 26 30 29 28.33 Frazier 37
Negative - - - - 1 µM - - - - 10 µM - - - - 100 µM - - - - 1 mM - - - - 5A6B Positive 30 29 28 29 Negative 6.25 - - 6.08 1 µM - - - - 10 µM - - - - 100 µM - - - - 1 mM - - - - *The dash (-) represents 6 mm, or no inhibition. ND means “not determined;” this designation was used for ZOI that were disrupted by either other ZOI or cracks in the agar.
TABLE 5. Zone of Inhibition Measurements for S. enteritidis Chalcone Strength/Control Zone of Inhibition* (mm) – Each ZOI measurement was taken twice and the two measurements were averaged. Plate 1 Plate 2 Plate 3 Average average average average across all plates 2A1B Positive 41 41 42 41.33 Negative ND 8 ND 8 1 µM - 7 - 6.33 10 µM - - - - 100 µM - - - - 1 mM ND ND ND ND 5A1B Positive 42.25 41.5 43.5 42.42 Negative - ND - - 1 µM - - - - 10 µM - - - - 100 µM - - - - 1 mM - - - 3A2B Positive 42 43.5 43 42.83 Negative - ND - - 1 µM - - - - 10 µM - - - - 100 µM - - - - 1 mM ND - ND - 4A2B Positive 43 40.5 3.9 29.13 Frazier 38
Negative ND ND ND ND 1 µM - - - - 10 µM - - - - 100 µM - - ND - 1 mM ND ND ND ND 5A2B Positive 39.5 40 38 39.17 Negative - - - - 1 µM - - 7.5 6.5 10 µM - - 7 6.33 100 µM - - 8 6.67 1 mM - - 9 7 1A3B Positive 37.5 39 40.5 39 Negative ND ND ND ND 1 µM - - - - 10 µM - - - - 100 µM - - - - 1 mM - - - - 4A3B Positive 39 38.5 39.5 39 Negative - - - - 1 µM - - - - 10 µM - - - - 100 µM - - - - 1 mM - - - - 5A3B Positive 40 39.5 39.5 39.67 Negative - - - - 1 µM - - - - 10 µM - - - - 100 µM - - - - 1 mM ND ND - - 1A4B Positive 38.5 41.5 40 40 Negative - - 7 6.33 1 µM - 6.75 7 6.58 10 µM - - 7 6.33 100 µM - - 7 6.33 1 mM - ND 7 6.5 2A4B Positive 41.5 40.5 39.5 40.5 Negative - - 7 6.33 1 µM - - 7 6.33 10 µM - - 7 6.33 Frazier 39
100 µM - - 7 6.33 1 mM - ND 7 6.5 4A4B Positive 39 38.5 43.5 40.33 Negative ND - - - 1 µM - - - - 10 µM - - - - 100 µM - - - - 1 mM ND - ND - 5A4B Positive 42.5 43.5 40.5 42.17 Negative - - - - 1 µM - - - - 10 µM - - - - 100 µM - - - - 1 mM ND ND ND ND 2A5B Positive 39.5 36.5 37.5 37.83 Negative - - - - 1 µM - - - - 10 µM - - - - 100 µM - - - - 1 mM - ND - - 3A’5B Positive 38.5 35 35 36.17 Negative - - - - 1 µM - - - - 10 µM - - - - 100 µM - - - - 1 mM - - - - 3A5B Positive 35 3.- 38 25.53 Negative - - ND - 1 µM - - - - 10 µM - - - - 100 µM - - - - 1 mM - - - - 4A5B Positive 35 3.- 36.5 25.03 Negative - - - - 1 µM - - - - 10 µM - - - - 100 µM - - - - 1 mM - - - - 2A6B Positive 39 3.75 36.5 26.42 Frazier 40
Negative - ND - - 1 µM 7 - 7 6.67 10 µM - - 7.75 6.58 100 µM - - - - 1 mM - - - - 3A’6B Positive 37 38 38.5 37.83 Negative - - - - 1 µM - - - - 10 µM - - - - 100 µM - - - - 1 mM - - - - 3A6B Positive 36.5 38 37 37.17 Negative - ND - - 1 µM - - - - 10 µM - - - - 100 µM - - - - 1 mM - - - - 5A6B Positive 40 38.25 39 39.08 Negative ND - - - 1 µM - - - - 10 µM - - - - 100 µM - - - - 1 mM - - - - *The dash (-) represents 6 mm, or no inhibition. ND means “not determined;” this designation was used for ZOI that were disrupted by either other ZOI or cracks in the agar.
TABLE 6. Zone of Inhibition Measurements for B. subtilis Chalcone Strength/Control Zone of Inhibition* (mm) – Each ZOI measurement was taken twice and the two measurements were averaged. Plate 1 Plate 2 Plate 3 Average average average average across all plates 2A1B Positive 35 32.5 32.25 33.25 Negative - - - - 1 µM - - - - 10 µM - - - - 100 µM - - - - 1 mM - - - - 5A1B Positive 36.75 34.5 32 34.42 Frazier 41
Negative - - - - 1 µM - - - - 10 µM - - - - 100 µM - - - - 1 mM - - - - 3A2B Positive 35.75 37 32.75 35.17 Negative - - - - 1 µM - - - - 10 µM - - - - 100 µM - - - - 1 mM - - - - 4A2B Positive 36 33.5 32 33.83 Negative - - - - 1 µM - - - - 10 µM - - - - 100 µM - - - - 1 mM - 9.25 - 7.08 5A2B Positive 36 32.5 34.25 34.25 Negative - - - - 1 µM - - - - 10 µM - - - - 100 µM - - - - 1 mM - - - - 1A3B Positive 34 33 34 33.67 Negative - - - - 1 µM - - - - 10 µM - - - - 100 µM - - - - 1 mM - - - - 4A3B Positive 35.75 34.5 33.5 34.58 Negative - - - - 1 µM - - - - 10 µM - - - - 100 µM - - - - 1 mM - - - - 5A3B Positive 36 34.5 33 34.5 Negative - - - - 1 µM - - - - 10 µM - - - - Frazier 42
100 µM - - - - 1 mM - - - - 1A4B Positive 34.5 34.5 33.5 34.17 Negative - - - - 1 µM - - - - 10 µM - - - - 100 µM - - - - 1 mM - - - - 2A4B Positive 34.5 33.5 36.5 34.83 Negative - - - - 1 µM - - - - 10 µM - - - - 100 µM - - - - 1 mM - - - - 4A4B Positive 35.25 33.75 31.5 33.5 Negative - - - - 1 µM - - - - 10 µM - - - - 100 µM - - - - 1 mM - - - - 5A4B Positive 33.25 34 31.5 32.92 Negative - - - - 1 µM - - - - 10 µM - - - - 100 µM - - - - 1 mM - - - - 2A5B Positive 34.75 32.5 31.5 32.92 Negative - - - - 1 µM - - - - 10 µM - - - - 100 µM - - - - 1 mM - - - - 3A’5B Positive 35.25 33.5 32.5 33.75 Negative - - - - 1 µM - - - - 10 µM - - - - 100 µM - - - - 1 mM - - - - 3A5B Positive 37 34.5 33.5 35 Frazier 43
Negative - - - - 1 µM - - - - 10 µM - - - - 100 µM - - - - 1 mM - - - - 4A5B Positive 34.25 34 33 33.75 Negative - - - - 1 µM - - - - 10 µM - - - - 100 µM - - - - 1 mM - - - - 2A6B Positive 36 32.5 31.5 33.33 Negative - - - - 1 µM - - - - 10 µM - - - - 100 µM - - - - 1 mM - - - - 3A’6B Positive 34 32.5 33 33.17 Negative - - - - 1 µM - - - - 10 µM - - - - 100 µM - - - - 1 mM - - - - 3A6B Positive 34 33.75 31 32.92 Negative - - - - 1 µM - - - - 10 µM - - - - 100 µM - - - - 1 mM - - - - 5A6B Positive 35 33.5 32.5 33.67 Negative - - - - 1 µM - - - - 10 µM - - - - 100 µM - - - - 1 mM - - - - *The dash (-) represents 6 mm, or no inhibition. ND means “not determined;” this designation was used for ZOI that were disrupted by either other ZOI or cracks in the agar.
Frazier 44
TABLE 7. Zone of Inhibition Measurements for E. coli Chalcone Strength/Control Zone of Inhibition* (mm) – Each ZOI measurement was taken twice and the two measurements were averaged. Plate 1 Plate 2 Plate 3 Average average average average across all plates 2A1B Positive 43.5 44 39.5 42.33 Negative ND - - - 1 µM - - - - 10 µM - - - - 100 µM - - - - 1 mM - - - - 5A1B Positive 42 43.5 37 40.83 Negative - ND ND - 1 µM - - - - 10 µM - - - - 100 µM - - - - 1 mM - - - - 3A2B Positive 42.25 42.5 40.5 41.75 Negative ND - ND - 1 µM - - - - 10 µM - - - - 100 µM - - - - 1 mM - - - - 4A2B Positive 42 42.75 40.5 41.75 Negative ND - - - 1 µM - - - - 10 µM - - - - 100 µM - - - - 1 mM - - - - 5A2B Positive 43.75 42.75 42 42.83 Negative ND ND ND ND 1 µM - - - - 10 µM - - - - 100 µM - - - - 1 mM - - - - 1A3B Positive 44.5 43 40 42.5 Negative - ND - - 1 µM - - - - Frazier 45
10 µM - - - - 100 µM - - - - 1 mM - - - - 4A3B Positive 45.75 43.75 39.5 43 Negative - - ND - 1 µM - - - - 10 µM - - - - 100 µM - - - - 1 mM - - - - 5A3B Positive 44.75 44.5 40 43.08 Negative - - - - 1 µM - - - - 10 µM - - - - 100 µM - - - - 1 mM - - - - 1A4B Positive 43.25 40 40.5 41.25 Negative ND - ND - 1 µM - - - - 10 µM - - - - 100 µM - - - - 1 mM - - - - 2A4B Positive 43.75 40 3-.5 40.08 Negative ND ND ND ND 1 µM - - - - 10 µM - - - - 100 µM - - - - 1 mM - - - - 4A4B Positive 41.5 41 39 40.5 Negative - ND - - 1 µM - - - - 10 µM - - - - 100 µM - - - - 1 mM - - - - 5A4B Positive 43.5 43.75 39.5 42.25 Negative - - - - 1 µM - ND - - 10 µM - - - - 100 µM - - - - 1 mM - - - - Frazier 46
2A5B Positive 40.5 43.5 38.5 40.83 Negative ND - ND - 1 µM - - - - 10 µM - - - - 100 µM - - - - 1 mM - - - - 3A’5B Positive 43.5 4-.5 39.25 43.08 Negative - - ND - 1 µM - - - - 10 µM - - - - 100 µM - - - - 1 mM - - - - 3A5B Positive 42.5 42.5 39.75 41.58 Negative ND - - - 1 µM - - - - 10 µM - - - - 100 µM - - - - 1 mM - - - - 4A5B Positive 42.5 40.75 40 41.08 Negative ND - - - 1 µM - - - - 10 µM - - - - 100 µM - - - - 1 mM - - - - 2A6B Positive 43.5 43 39.5 42 Negative - ND - - 1 µM - - ND - 10 µM - - - - 100 µM - - - - 1 mM - - - - 3A’6B Positive 43.5 41.5 39.5 41.5 Negative - ND - - 1 µM ND - - - 10 µM - - - - 100 µM - - - - 1 mM - - - - 3A6B Positive 42 40.5 40 40.83 Negative - - - - 1 µM ND - - - Frazier 47
10 µM - - - - 100 µM - - - - 1 mM - - - - 5A6B Positive 40 40.25 39 39.75 Negative - - - - 1 µM - - - - 10 µM - - - - 100 µM - - - - 1 mM - - - - *The dash (-) represents 6 mm, or no inhibition. ND means “not determined;” this designation was used for ZOI that were disrupted by either other ZOI or cracks in the agar.
APPENDIX B – PHOTOS OF AGAR PLATES POST-INCUBATION SHOWING ZONE OF INHIBITION
TABLE 8. Post-chalcone diffusion photographs of S. pneumoniae grown on Tryptic Soy Agar with 5% Sheep Blood after 24-36-hour Incubation at 37° C CHALCONE PLATE 1 PLATE 2 PLATE 3 2A1B
5A1B
Frazier 48
3A2B
4A2B
5A2B
1A3B
4A3B
Frazier 49
5A3B
1A4B
2A4B
4A4B
5A4B
Frazier 50
2A5B
3A’5B
3A5B
4A5B
2A6B
Frazier 51
3A’6B
3A6B
5A6B
TABLE 9. Post-chalcone diffusion photographs of S. enteritidis grown on LB agar after 24- 36-hour incubation at 37° C CHALCONE PLATE 1 PLATE 2 PLATE 3 2A1B
Frazier 52
5A1B
3A2B
4A2B
5A2B
1A3B
Frazier 53
4A3B
5A3B
1A4B
2A4B
4A4B
Frazier 54
5A4B
2A5B
3A’5B
3A5B
4A5B
Frazier 55
2A6B
3A’6B
3A6B
5A6B
TABLE 10. Post-chalcone diffusion photographs of B. subtilis grown on LB agar after 24- 36-hour incubation at 30° C CHALCONE PLATE 1 PLATE 2 PLATE 3 Frazier 56
2A1B
5A1B
3A2B
4A2B
5A2B
Frazier 57
1A3B
4A3B
5A3B
1A4B
2A4B
Frazier 58
4A4B
5A4B
2A5B
3A’5B
3A5B
Frazier 59
4A5B
2A6B
3A’6B
3A6B
5A6B
Frazier 60
TABLE 11. Post-chalcone diffusion photographs of E. coli grown on LB agar after 24-36- hour incubation at 37° C CHALCONE PLATE 1 PLATE 2 PLATE 3 2A1B
5A1B
3A2B
4A2B
5A2B
Frazier 61
1A3B
4A3B
5A3B
1A4B
2A4B
Frazier 62
4A4B
5A4B
2A5B
3A’5B
3A5B
Frazier 63
4A5B
2A6B
3A’6B
3A6B
5A6B