Blog Post #3

Results

After recording all of my trials, I used the footage to time the seizure recovery of each individual fly in every treatment group. I made sure to limit inconsistencies with the visual assay by timing each fly 3 separate times. The average of these times were calculated and documented. I then calculated the overall average time for each sample group and created a bar graph to compare the data. Finally, I tested for statistical significance by performing two two-sample T-tests to compare my licorice root data to both the negative control and the positive control. 

Average Recovery Times

There were 32 flies collected in the 3 trials performed for my negative control. Overall, the no-treatment vials had a mean value of 24.3475 seconds. There were 38 flies collected in the 3 trials performed for my positive control. The avobenzone had a mean value of 13.58760 seconds for the flies’ seizure recovery time. There were 39 flies collected in the 3 trials performed for my experimental group. The overall average recovery time for licorice root was 15.262260 seconds. From this data it can be concluded that licorice root was more effective than the negative control but not as effective as the positive control. (Fig 1). 

Statistical Analysis

I performed two two-sample T-tests to compare licorice root to both avobenzone and the negative control. Tests were evaluated against a P-value of 0.05. When comparing licorice root and the negative control, a P-value of 1.25116x10-5 was obtained. Since this value was below the alpha value of 0.05, the statistical test shows that the data is statistically significant and the conclusion is not due to chance. When comparing licorice root and avobenzone, a P-value of 0.80884 was obtained. This number is higher than the alpha value of 0.05 showing that the data is statistically insignificant and that licorice root was not statistically more effective than avobenzone, but rather as effective as avobenzone. (Fig 3.)

Discussion

Epilepsy affects millions of people all over the world, especially those in low income countries, as they cannot afford or access scientifically validated AEDs. Due to this, many alternative treatments have been used to combat epilepsy, however; these have not been scientifically validated to be effective in reducing epilepsy. One such example is licorice root. 

I evaluated the effect of licorice root on seizure recovery time in Drosophila melanogaster and hypothesized that licorice root would lower seizure recovery time as much as avobenzone, my positive control. I found that the average seizure recovery time in Julius seizure bs Drosophila treated with no treatment was 24.3475 secs, in those treated with avobenzone it was 13.58760 secs, and in those treated with licorice root it was 15.262260 secs. Licorice root lowered seizure recovery time by 37% in comparison to the negative control, and was statistically significant with a P-value of 1.25116x10-5.  Licorice root was also statistically as effective as avobenzone in treating epilepsy in Drosophila (P-value of 0.80884). These results support my hypothesis, because licorice root did lower seizure recovery time as much as avobenzone, a drug shown to reduce seizure recovery time in Drosophila in previous studies (Lin et al., 2017). My  results indicate that licorice root was effective in treating epilepsy in Drosophila with the PUM-2 gene under-expression mutation. With this research, future studies are now justified to increase the complexity of their model organism to attempt and replicate similar results.

Other cases of licorice root being used in epileptic studies has shown that licorice root has not only helped reduce neurological degradation caused by seizures, it can also help reduce other types of seizures, not only temporal lobe epilepsy. Research from Shahed University concluded that licorice root can not only attenuate the occurrence of seizures in mice, but also has neuroprotective potential. The results of this study show that licorice root significantly lowered oxidative stress markers in the mice after a seizure was induced and prevented hippocampal neuronal loss which often occurs after several types of seizures. This study connects with my research in supporting the potential of a licorice root-based seizure treatment for human seizures. While my research mostly focuses on the seizure recovery time, this study indicates that the long-term implications and degradation of seizures can also be somewhat prevented in mice by the right dosage of licorice root (Kiasalari et al., 2013). Similarly, research from Fundamental Clinical Pharmacology tested the liposomal formulation of licorice root in different types of epilepsy. They found that licorice root in doses 25 and 50 mg/kg demonstrated a significant increase in seizure threshold current in mice (the likelihood of the mice having seizures decreased). Additionally, they tested the acute effect of liposome-entrapped licorice root in three different types of epilepsy in mice: current electroshock seizures (ICES), pentylenetetrazole (PTZ)-induced seizures, and status epilepticus. Overall, their findings also suggest that licorice root decreases the strength and duration of these different types of seizures and possesses anticonvulsant activity in mice (Agarwal et al., 2013). While their research does not specifically look at temporal lobe epilepsy and the PUM-2 gene, it connects to my findings similarly to the previously mentioned study.

Potential errors in my research methods could have resulted from issues with sterilization, time, and resource limitations. One source of error is that I used a visual assay, so the timing may have been slightly off due to human error. This was controlled for by taking the averages of three timing attempts; however, it could have introduced millisecond-level errors in my data. Another source of error could have been the age of the Drosophila. Although I euthanized the adults in a vial 36 hours before administering treatment, the flies could have hatched at any point during those 36 hours. Since the Drosophila needed to be at least 2 days old for seizure induction to work properly, there may have been some flies that were simply not old enough to seize properly. This likely accounts for any outliers in my data. A third source of error could be that the measurement of licorice root may have varied slightly due to measurement errors at a measurement threshold of 5 mg. Finally, I had no way to quantify the amount of treatment that the Drosophila ingested for each trial. This means I am unable to conclude that the dosages I observed producing the results of my experiment were consistent in each Drosophila.

       There are a few next steps that could be taken to further this research, including performing RNA synthesis, testing different dosages of licorice root, testing other alternative treatments to AEDs, and using higher level model organisms. I was only able to use a visual assay to analyze my data due to both time and budget restraints; however, using RNA synthesis in the future could allow for data analysis on the actual pumilio levels in the Drosophila’s brain to see if licorice root really did increase PUM-2 gene expression. In terms of furthering this research within Drosophila, future research could be done to test other alternatives to AEDs, such as St. John’s Wort, as well as testing different dosages of licorice root to see if more or less has a different effect on seizure recovery time. Another step that could be taken is moving the research to higher level model organisms and test licorice root on specifically mice and rats with mutations in the PUM-2 expression to further bridge the gap to testing licorice root on epilepsy in humans.