References
Introduction
Results
Future Directions
Spruce Gordon, Denzil Bilson, Kayleigh Vanderwerff, Ashley Agyepong
The Effects of Chlorogenic Acid & Quercetin on Salmonella Typhimurium Growth
Abstract
Hypothesis
Acknowledgments
The negative control, DMSO, produced an average absorbance value of 0.2586, indicated by the blue line. Calculations were made for two standard deviations above and below the mean, indicated by the two purple dotted lines.
As expected, the absorbance value of Ampicillin, the positive control, fell well below under two standard deviations with a value of 0.0408. Although the first test for Quercetin and Chlorogenic in a 1:1 ratio produced an absorbance value of 0.0443, well below 2 standard deviations from the mean, the second test had an absorbance of 0.5118, two standard deviations above the mean, contradicting with the first test.
Methods
Conclusions
Chlorogenic acid and Quercetin found within the ingredients of “Mampong Herbal Enterica” may individual properties that are bacteriostatic or bactericidal. If these compounds are hits individually or become hits when combined, the trials that we run will show the halted growth of Salmonella Typhimurium
4 experimental procedures were conducted. These included a Max Dose test for Chlorogenic acid and Quercetin individually, a dose response test of varying ratios between the two compounds( Chlorogenic acid: Quercetin), a dose Response Curve Quercetin & Chlorogenic Acid only using a 1:1 ratio, and a Bactericidal/Bacteriostatic test. The duration of testing all methods was 5 weeks. Used compounds from Selleckchem (Chlorogenic acid) and Chem-IMPEX International Inc (Quercetin dihydrate); Kept at room temperature. Both compounds were solid prior to creating solutions in DMSO. Given the short lifespan of the chlorogenic acid stock solution, the stock was remade at the top of every week to ensure that shelf-life would not interfere with experimentation. Positive control was 10X ampicillin; negative control was 10X DMSO.
Zhang, F., Zhai, T., Haider, S., Liu, Y., & Huang, Z. J. (2020). Synergistic Effect of Chlorogenic Acid and Caffeic Acid with Fosfomycin on Growth Inhibition of a Resistant Listeria monocytogenes Strain. ACS Omega, 5(13), 7537–7544. https://doi.org/10.1021/acsomega.0c00352
Lou, Z., Wang, H., Zhu, S., Ma, C., & Wang, Z. (2011). Antibacterial activity and mechanism of action of chlorogenic acid. Journal of Food Science, 76(6), M398-403. https://doi.org/10.1111/j.1750-3841.2011.02213.x
Staedler, D., Idrizi, E., Kenzaoui, B. H., & Juillerat-Jeanneret, L. (2011). Drug combinations with quercetin: doxorubicin plus quercetin in human breast cancer cells. Cancer Chemotherapy and Pharmacology, 68(5), 1161–1172. https://doi.org/10.1007/s00280-011-1596-x
Department of Molecular, Cellular, and Developmental Biology
University of Colorado Boulder
We would like to acknowledge Dr. Pamela Harvey and Delwin Maben for their direct mentorship. We also extend our special thanks to the Howard Hughes Medical Institute and the CU Boulder Department of Molecular, Cellular, and Developmental Biology for their funding, without which this research would not be possible. We are grateful for the advocacy and support of scientific research provided by Dr. Corrie Detweiler, the principal investigator of our lab, and Lee Niswander, PhD, the Chair of the MCDB Department.
The tests had very contradictory results. The max dosage test for Chlorogenic Acid and Quercetin individually showed no difference in the absorbance value from the negative control of DMSO, showing a lack of antibiotic properties. The first combined test showed statistically significant stoppage of bacterial growth, with no difference between concentrations, but the second and third replications of these tests showed the opposite result, a statistically significant growth of bacteria. There is some significant risk of error, as there was over-dilution in the combined trials, so at this time there is no conclusive proof that the compounds have any statistically significant effect in combination with one another. The Mampong Herbal Enterica was also tested, and showed no statistically significant results. This indicates that the compound does not display antibiotic properties in a 96 well plate, which may be due to the natural sugars present.
While we have about 10 different antibiotic classes in use today, bacteria regularly develop antibiotic resistance via random mutation that makes entire classes ineffective treatment options. By the year 2050, antibiotic resistant bacteria will account for more deaths than cancer. This is a public health crisis, and requires mass testing of a variety of compounds, particularly including naturopathic remedies from non-western cultures, as these herbal remedies used for centuries have not been tested in a lab environment.
A member of our group was treated with Mampong Herbal Enterica when they contracted Typhoid fever living in Ghana. The compound selections, Chlorogenic acid(CA) and Quercetin(QU) were the available compounds in the laboratory that were within the herbal remedy. On investigating CA, there was some research finding that CA was found to induce leakage of nucleotides(Lou et al. 2011). CA had been investigated by a previous semester where it showed some promise in combination with other compounds against our model organism, Salmonella Typhimurium, selected for its applicability in testing on rodents. According to the previous semester, CA does not replicate that nucleotide leakage death in a 96 well plate.
Because the herbal remedy was a curative antibiotic at such a small dosage, we wanted to investigate if CA may be found to have synergistic antibiotic effects on Salmonella Typhimurium. One article noted successful use of CA in combination with another compound and antibiotics for treatment of a resistant Listeria monocytogenes Strain, likely due to the competitive inhibitory properties of the compounds (Zhang et al. 2020). QU has also shown synergistic effects with developed antibiotics in the treatment of multidrug-resistant clinical strains of Pseudomonas aeruginosa due to its quorum sensing inhibiting properties (Vipin et al. 2020). It has also been shown to both increase the effectiveness of one type of chemotherapy in one study on breast cancer(Staedler et al. 2011). While CA and QU work on vastly different systems within their use as synergistic compounds for the treatment of infections, this may be their strength and the strength of the herbal remedy as a whole.
Antibiotic resistance is the current rising public health crisis in this century. By 2050, the number of deaths caused by antibiotic-resistant bacteria will be greater than the number of deaths caused by all forms of cancer. To make the situation even more dire, there has not been a new class of antibiotics since the 80s and all of the current classes are simple in design. This is why our research is necessary. Time is running out to find new, complex methods to combat antibiotic-resistant bacteria.
This research has not only the potential to offer new ways to design drugs that target antibiotic-resistant bacteria, but also has implications for the pharmaceutical industry, as findings could lead to the development of new, more effective antibiotics. This would have a positive impact on global health, as well as the global economy.
MAX DOSE VARYING
RATIOS
DOSE RESPONSE (1:1 ratio)
Solution | |
1:1 Chlorogenic: Quercetin | 50µL: 50µL (10µM) |
2:1 Chlorogenic: Quercetin | 100µL: 50µL (10µM) |
1:2 Chlorogenic/Quercetin | 50µL: 100µL (10µM) |
Ampicillin (Pos. Control) | |
DMSO (Neg. Control) | |
Dose (stock: DMSO) | Dose 1 10µM 0.5μM of each Q and CA (MAX DOSE) | Dose 2 1:2 500µL:500 | Dose 3 1:2 500µL:500 | Dose 4 1:2 500µL:500 |
While the first test of the chlorogenic acid and quercetin solution showed signs of being bacteriostatic, subsequent testing had bacterial growth promotion. Mampong Enterica medicine in 1:1 solution with DMSO was unsuccessful in decreasing bacterial concentrations.
Quercetin Dihydrate
Chlorogenic Acid