The Cognitive Effects of Ginkgo biloba on Mice

ABSTRACT Ginkgo biloba is one of many natural herbal remedies that have been used to avail several different sicknesses. There is already over the counter medication made from Ginkgo biloba that is given to humans, yet any conclusive evidence that Ginkgo biloba is beneficial to its applied illnesses has yet to be conclusively proven. Our goal is to find definitive results in whether Ginkgo biloba has an effect on improving cognitive abilities by analyzing a mouse model. In order to test their cognitive advancements we utilized a maze for them to run through. If the mice ran faster times it would be indicative to an increase in cognitive ability. The experiment was run over a period of 13 days with daily injections of Ginkgo biloba given interperitoneally and mazes were run three times a week. Ultimately, in the end the null hypothesis was correct and we were not able to measure a significant increase in cognitive ability due to the injections of Ginkgo biloba.

INTRODUCTION The Ginkgo biloba tree represents the only remaining species of the order Ginkgoales of class Gymnospermae that existed during the time of the dinosaurs a little more than 200 million years ago, and is one of the oldest thriving trees on the planet which is why it is known as a “living fossil”(DeFeudis & Drieu, 2000; Smith & Lou, 2003). The first writing that was publicized covering the internal use of the Ginkgo biloba tree leaves for medical purposes dates back to 1505 A.D (DeFeudis & Drieu, 2000; Smith & Lou, 2003). The Ginkgo bilboa extract, EGb 761, consist of 24% flavonols glycosides (kaempferol, Quercetin and isoharmnetin), and 6%terpene lactones: ginkgolide A, ginkgolide B, ginkgolide C, ginkgolide M, ginkgolide J, and bilobalide B (DeFeudis & Drieu, 2000; Smith & Lou, 2003). In today’s society EGB 761 is

widely used in clinical trials (DeFeudis & Drieu, 2000; Smith & Lou, 2003). In the United States the extract is used as a dietary supplement due to the possibility for a better memory (Smith & Lou, 2003). In the European countries the extract is being prescribed by doctors to help with peripheral and cerebral insufficiency (Smith & Lou, 2003). Lastly in China pharmacopoeias have and still use the EGb 761 for the treatment of dysfunctions of the heart and lungs (DeFeudis & Drieu, 2000). The extract has been said to hinder coagulation, aid in attenuates occurring agerelated deterioration of cognitive functions in rats, and improve degenerative dementias of the Alzheimer’s and multi-infarct type (Smith & Lou, 2003).

Previous experiments have been conducted to test the benefits of other “traditional treatments,” such as alfalfa, blackberry, celandine, eucalyptus, among others (Swantston-Flatt, et al. 1990) for illnesses like diabetes, Alzheimer’s, or other common ailments (Howes & Houghton 2003).

Ginkgo biloba has also been seen in the laboratory to test if its traditional uses are truly beneficial. These benefits are believed to give relief for blood disorders memory-loss diseases, and brain injuries (DeFuedis & Drieu 2000). Ginkgo biloba has already been tested on humans with Alzheimer’s disease and multi-farct dementia (Le Bars, et al. 1997 & Dongen, et al. 2000), yet the results were contradicting and therefore non-conclusive. There have been many experiments on Ginkgo biloba that are considered non-conclusive because their methods were non-standardized or contradicted another publication (Mix and Crews 2000 & 2002, Soloman, et al. 2002). Not unlike the present experiment, mice and Gingko biloba have been paired together for many types of tests. Some focus more on the anatomical effects, such as the effects of Ginkgo biloba on the cortex of the brain and the hippocampus (Watanabe, et al. 2001). Some focus on the behavioral aspect, looking at memory and cognitive functions (Satvat and Mallet

2009). The wide variety of different possible approached to test the effectiveness of Ginkgo biloba could be the cause for its non-conclusive stature.

Most of the research on Ginkgo biloba has been performed on older specimens. For example, in (Wang et. al 2006) adult and aged adult rats were used to test the possibility of cognitive enhancements due to gingko biloba administration. Not much research has been performed one, on mice and Gingko biloba (rats seem to be the animal of choice), and two, young mice for a change in cognitive behavior. In identifying several types of methodologies to perform this experiment, one paper seemed to mirror the objectives that we would like to test. In (Satvat & Mallet 2009), the efficacy of Ginkgo biloba was evaluated in comparison to several other experiments. The methodology, while similar, will be different to test cognitive learning abilities of young adult mice. Because such contradictory findings of Gingko biloba exist, further justification is needed to conclude definitive results and whether Gingko biloba does have an effect of cognitive behavior in young specimen. The conclusion can then be applied medicinally for people. Our null hypothesis is that the Ginkgo biloba will have no cognitive effect on the juvenile aged rats. The tested hypothesis is that the Ginkgo biloba will have a cognitive effect on the juvenile rats such that it improves memory which will be measured by the time it takes for them to individually finish the maze.

METHOD: Mice We provided our mice with simple class cages under a controlled temperature and controlled twelve hour light-dark cycle (Zhou, et al. 2006 & Satvat, et al. 2009 & Shenoy, et al. 2001 & Schindowski, et al. 2001). Our mice were all bought from the same source like other research

has done, at a local pet store (Zhou, et al. 2006 & Satvat, et al. 2009). These mice were meant to be feeding mice and it must be noted that their quality of life is lower than that of a bred lab mouse. As for the diet of our mice they will be provided tap water that will be freely available to them at all times (Zhou, et al. 2006 & Satvat, et al. 2009, Schindowski, et al. 2001), and will be feeding them mouse chow and checking on food levels every day (Zhou,e t al. 2006 & Shenoy, et al. 2001 & Stackman, et al. 2003). We are using 15 mice total, 5 for the untreated control group, and five for each of the two different concentrations of Ginkgo biloba injections. The groups of five will all be kept in separate cages, resulting in a total of three cages.

Gingko biloba We used the extract of dried Ginkgo biloba leaves called EGb 761, which includes all of the active ingredients that are believed to improve memory and thin blood. This extract contains these active ingredients: 24% ginkgoflavonolglycosides, 6% terpene lactones (Clostre 1999).

EGb761 is not a liquid; therefore we had to stray from the pure EGb 761 and buy “Whole Leaf Ginkgo biloba, Immune System� to be able to inject it into the mice interperitoneally. This method has been successful in distributing the Ginkgo evenly throughout every mouse (Satvat 2009). Once we obtained the Ginkgo biloba we made serial dilutions of 3 mg/ kg and 5 mg/kg (DeFeudis, et al. 2003 & Liu, et al. 2008) and used these to inject these into the mice (Ilhan, et al.

2006 & Stoll, et al. 1996 & Schindowski, et al. 2001 & Al-Attar 2012). Since the volume of the injections must be large enough to successfully be injected, we increased the overall volume that every mouse was injected to 0.3 mL by adding the difference of the volumes with distilled water.

The dilutions are as follows: the control group was injected 0.3 mL of distilled water, the 3 mg/kg group was injected 0.76 mL of Ginkgo biloba and 0.224 mL of distilled water and the 5 mg/kg group was injected with 0.126 mL of Ginkgo biloba and 0.174 mL of Di water. The

Ginkgo biloba and the distilled water were mixed together prior to the injections. We injected the mice every day at 9 in the morning.

Procedure Once the mice were obtained we separated them according to the serial dilutions we gave them, five mice per variable. We used CADIA organic Mild Cheddar Cheese as the reinforcement, which was placed at the end of the maze. Then one after the other we timed the mice as they ran through the maze. At the end of the trial we injected the mice with their first interperitoneal injections of Gingko biloba. The mice were injected every day at 9 am with the same concentration of Gingko biloba according to their dilutions until the experiment was completed. The maze trials were performed every Sunday, Wednesday, and Friday at 2 pm. The cages were cleaned once a week on Fridays. At the end of the experiment, the data and observations that were collected were extrapolated to establish a conclusion to the effects of Gingko biloba.

RESULTS The results were based on the mean maze times of each variable group through five maze test days. A line graph was made to illustrate the improvements of the mice over the duration of the experiment (Figure 1). It can be seen from the graph that there was slightly more improvement in the mice group that was injected with 3 mg/kg of Ginkgo biloba than the control and the 5 mg/kg group. A One-Way ANOVA Test was run on the collected data of maze times.

This test was used to find if there were any significant differences between the three groups average completion times. The statistical data in the ANOVA showed that there was no

significant data between either three of the groups (p-value = .342). This fails to reject our null hypothesis that the Ginkgo biloba will have no cognitive effect on the juvenile rats.

Figure 1: This maze illustrates the mean amount of time the three different groups of mice took to complete the maze. These maze tests were done five times over a period of 13 days.

a)

b)

c)

Table 1: a) These are the descriptives of the three mice groups. b) This test of Homogeneity of Variances is the Levene’s test, which is necessary to be able to run the ANOVA test. The p-value, or “Sig.” is supposed to be high, the null hypothesis is supposed to be rejected before the ANOVA test can be run. c) This is the actual ANOVA test

that compares the three means and sees if there are any significant differences between them. Since the “Sig.” or p-value is so high, it can be concluded that there is no significant difference between any of the means.

DISCUSSION Overall, this experiment was inconclusive. There were too many variables to try to control in such a short amount of time. We also ran into several problems that altered the experiment from controlled and precise to sloppy and unpredictable.

One of the first unexpected problems we ran into was the cage’s walls weren’t tall enough and mice were jumping out of them. On the first day four mice got out but they were all recovered; Covers were added to the cages. These lids proved to still be inadequate; therefore we had a few more escapes throughout the experiment. Another unexpected problem was the dilutions that we were injecting. The original injections were going to be DI water, 5 mg/kg, 10 mg/kg, and 15 mg/kg. We decided to inject this first amount because 5mg /kg was the concentration that (Liu et al., 2008) when testing on rodents. They did 5mg /kg injections for three consecutive days for a total of 15 mg/kg. This is how we decided on the low injection of 5mg/kg and the high of 15 mg/kg. This was a problem because about 15 seconds after being injected with the 10 mg/kg the mice started to look drunk. We proceeded to inject two of the mice in the 15 mg/kg group and they started to seize within 5 seconds. This first set of injections resulted in unexpected death.

There was an overall death count of four mice after the experiment was completed. Two died the day after they were injected with the 15 mg/kg, and two died that were originally in the 10 mg/kg testing group, and became the 3 mg/kg group. These last two died as the experiment progressed.

When we changed the dilutions, we kept the 5 mg/kg concentration, but let it be the high (Liu, et al. 2008). The dilutions were changed to DI water, 3 mg/kg, and 5 mg/kg. The controls stayed controls, the 10 mg/kg mice became our 3 mg/kg mice, and the 5 mg/kg stayed the same. The 15

mg/kg group was completely excluded from the experiment due to excessive stress. The mice seized up because the liquid “Whole Leaf Ginkgo” that we used for the i.p injections contained X%. The concentrations of alcohol in the liquid solution were more than enough to have a negative effect on the mice (Goldstein, 1972). So in theory, the mice died of alcohol poisoning.

Our data was more than likely affected by the loss and deaths of mice during this experiment.

The biggest factors that really hurt the experiment and its outcomes were the severe limitations of time and resources. The experiment was ran within a two week time period and the lab that we were working in had a limited budget and amount materials. The ideal amount of time that we would have 3liked to test them would be between two and three months (Satvat & Mallet, 2009).

That way we could have properly acclimated the mice, and had more time to run the mazes and see either more or less improvement. Also we would have more time to make injections a routine for the mice, giving their body times to fully adjust. This experiment seemed to have been stopped right as there was progress being made. If we had more resources we would have had higher quality cages that the mice wouldn’t have been able to escape. Also a combination of limited time and resources led to not being able to get the extract we would have preferred, the preferable extract would be a pure liquid EGb 761 with no alcohol in it.

Given that this experiment was indeterminate, this experiment should be duplicated again with better materials and more controlled variables. Once a more solid and conclusive experiment is completed, the next step would to apply this model to more sophisticated subjects, in progression to coming to an overall irrefutable conclusion for the cognitive effect of Ginkgo biloba. We learned from this experiment that the over the counter Ginkgo biloba supplements have more than just the “beneficial” EGb 761 extract. There may be other ingredients that could

potentially harm someone if they did not take the right dosage or they had blood coagulation problems to start.

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