Although Osha root extract has shown bacteria growth promotion and no immediate evidence of antibacterial properties, there are still tests that can be done.
-Research what compounds ligusticum porteri is made of and determine if they are viable to obtain with cost and availability.
Test these compounds individually in a max dose and dilution series to determine if any of the compounds have antibacterial properties.
References
Introduction
Future Directions
Department of Molecular, Cellular, and Developmental Biology
University of Colorado Boulder
Emnet Sima and Jacob Sharp
Ligusticum Porteri’s (Osha Root Extract) Effectiveness
of Anti-Microbial Properties against Salmonella Typhimurium.
Abstract
Cogle, C. (2022, June 15). Does osha root have anti-cancer properties? Retrieved October 13, 2022
https://addon.life/2021/01/23/osha-root-ligustilide-anti-cancer-properties/
McGrane, K. (2020, June 05). OSHA root: Benefits, uses, and side effects Retrieved October 13, 2022
https://www.healthline.com/nutrition/osha-root#benefits
Nguyen, Khanh. “Effects of Ligusticum porter (Osha) Root Extract on Human Promyelocytic Leukemia Cells”. National Library of Medicine, April 2017. DOI: 10.4103/0974-8490.204641
Hypothesis
Acknowledgments
Methods
Results
We would like to acknowledge Howard Hughes Medical Institute and the department (CU Boulder Molecular, Cellular, and Developmental Biology for funding our drug discovery lab materials and Dr. Pamela Harvey for teaching us about antibiotic resistance and we thank Shiau-Tau Ciecierska, Daniel Hassell and Anila Narayana, our wonderful TAs for guiding us through our several experimental trials. Lastly we would like to show gratitude to our Principal Investigator of our sponsor laboratory Dr. Corrie Detweiler.
Stock Solution Dilutions:
Dilution series of 1:10 was started by taking 10uM of the stock OSHA root extract and diluted 4 times creating a 5uM, 2.5uM, 1.25uM and .625uM dilutions.
Completed Dilution Placed in Wells:
From each individual dilution, 10uL was placed into individual wells in triplicates along with 90uL of Salmonella. For each dilution, in separate wells 10uL of the positive and negative control were added with the 90uL of Salmonella
Storage:
Before placing the completed 96 well plate into incubation, an initial reading from the spectrophotometer was taken due to the color of the Osha root extract. The plate was then placed into an incubator set at 37 degrees celsius for 24 hours.
Absorbance Values:
After the 24 hours the 96 well plates were read in a spectrophotometer with a wavelength set at 620 nm. These results were then adjusted for the initial readings done before incubation
Dilution series:
-Each dilution set started with the max dose of 10uM of Osha root extract from the source followed by a 1:10 dilution series placed into individual wells in triplicates.
-90uL of the Salmonella Typhimurium was added in each separate well containing 10uL of Ampicillin, 10uL of DMSO and the �dilution series of Osha root extract.
Conclusion
Results of the max dose of Osha root extract showed bacterial growth promotion in M9 minimal media which was calculated to be two standard deviations above of our negative control. The dilution series showed less growth than the negative control but was not considered a “hit” due to it being two standard deviations under the negative control. These results do not match with our original hypothesis but instead osha root extract as a whole does not appear to have antibacterial properties and instead can promote bacterial growth.
In most cases antibiotics are used to kill or inhibit bacteria but antibiotic resistance is continuing to increase and have acquired new resistance mechanisms. This allows them to survive and multiply which makes it impossible to treat common infections. By 2050 statistics show more people will die from antibiotic resistant bacteria than cancer in the U.S. Our compound of choice is Osha Root extract (Ligusticum porteri).
Our model organism is Salmonella Typhimurium; it causes typhoid fevers in mice but in humans causes food poisoning so it is safer for us to use. It also is beneficial because it is cost effective and efficient.
The compounds in consideration have had a long history of holistic medicine from those who were not as medically advanced at the time used. When researching, our TA Shiau-Tau mentioned a root known as “Osha root” that had been used by Native Americans for many medicinal uses and is still used today. There are promising peer-reviewed articles on Osha root (Ligusticum porteri) showing it’s antimicrobial activities against both Gram-positive and Gram-negative bacteria, but the studies we found have been mostly seeing the effects of Osha root against cancer cells and bacteria specifically. This led us the need for further testing with Osha root against Salmonella Typhimurium, a Gram-negative bacteria.
Salmonellosis is a disease caused by the bacteria Salmonella and is easily spread through contaminated food and water. Salmonella Typhi causes typhoid fever and can lead to death and Salmonella Typhimurium which causes gastroenteritis in humans and typhoid fever in mice. There are effective treatments for salmonellosis but antibiotic resistance has become a rapid emergence that affects people worldwide and extensive research is needed in order to resolve the issue. During the experiment, Ligusticum porteri (Osha Root extract) was tested against Salmonella Typhimurium to identify the compound's ability to inhibit bacterial growth. Various solutions with different concentrations were created for a dilution series of 10uM, 5uM, 2.5uM, 1.25uM and .625uM. The results showed the compound Osha root was ineffective in killing or inhibiting the growth of Salmonella Typhimurium and in fact grew Salmonella. The results discovered will need more testing by testing the compounds contained in Osha root such as alkaloids, sterols, saponins and more.
If Osha root extract has antimicrobial properties against gram negative bacteria considering the evidence, the compound will kill or inhibit Salmonella Typhimurium growth in vitro.
The graph shows the negative control (DMSO) in red, and the positive control (Ampicillin) in green. The darkest blue is the max dose of Osha root extract at 10uM, scaling down by color to each of the dilution series.
The results read by the spectrophotometer show the amount of light absorbed in the solution after incubation, the higher the number on the Y axis means more light was absorbed which means higher levels of bacteria are present in the solution.
Figure 2: Well plate consists of Salmonella with various solutions of concentrations and DMSO(red) and Ampicilian( green) with salmonella and incubated at 37C for 2 days
Figure 1
https://www.britannica.com/science/salmonellosis