THE EFFECTS OF GINKGO BILOBA ON MEMORY, REACTION TIME, AND MOOD. Emily Katherine Qualls

Transcription

1 THE EFFECTS OF GINKGO BILOBA ON MEMORY, REACTION TIME, AND MOOD by Emily Katherine Qualls B.A., The University of West Florida, 2004 A thesis submitted to the Department of Psychology College of Arts and Sciences The University of West Florida In partial fulfillment of the requirements for the degree of Master of Arts 2008

2 2008 Emily Katherine Qualls ii

3 The thesis of Emily Katherine Qualls is approved: Samuel R. Mathews, Ph.D., Committee Member Date James E. Arruda, Ph.D., Committee Chair Date Accepted for the Department/Division: Laura L. Koppes, Ph.D., Chair Date Accepted for the University: Richard S. Podemski, Ph.D., Dean of Graduate Studies Date iii

4 ACKNOWLEDGMENTS I would like to thank my advisor and Committee Chair, James Arruda, for his dedication and perseverance during the planning and preparation of this thesis. Furthermore, I would like to thank James Arruda for his assistance in the recruitment of participants for this study. I would like to express appreciation for Erica House and Harlen Lawrence and their assistance during data collection. Finally, I thank my husband and family for their encouragement throughout this process. iv

5 TABLE OF CONTENTS ACKNOWLEDGMENTS...iv LIST OF TABLES... vii LIST OF FIGURES... viii ABSTRACT...ix CHAPTER I. INTRODUCTION...1 A. Research Review Human Studies Non-human Studies...8 B. The Current Study...10 CHAPTER II. METHODS...11 A. Participants...11 B. Materials Demographic Questionnaire Visual Analog Mood Scales Verbal Memory Spatial Memory Reaction Time Stanford Sleepiness Scale Break Activity Checklist Ginkgo biloba...15 C. Procedure...16 D. Design and Analysis...18 CHAPTER III. RESULTS...19 A. Spatial Memory...19 B. Verbal Memory...20 C. Reaction Time...21 D. Mood...23 v

6 CHAPTER IV. DISCUSSION...27 REFERENCES...34 APPENDIXES...37 A. Institutional Review Board Approval Letter...38 B. Informed Consent...42 C. Demographic Questionnaire...45 D. Visual Analog Mood Scales...47 E. Stanford Sleepiness Scale...49 F. Break Activity Checklist...51 vi

7 LIST OF TABLES 1. Tests of Between-Subjects Effects on Cognitive Tasks Tests of Between-Subjects Effects on the VAMS Statistical Power on the VAMS...25 vii

8 LIST OF FIGURES 1. Data trends for afraid, tense, and tired...31 viii

9 ABSTRACT THE EFFECTS OF GINKGO BILOBA ON MEMORY, REACTION TIME, AND MOOD Emily Katherine Qualls The present study investigated the effects of Ginkgo biloba on verbal memory, spatial memory, simple reaction time, choice reaction time, and mood. Pretest and posttest measures of memory and reaction time were assessed using the Stim2 computer software designed by Neuroscan. Pretest and Posttest measures of mood were assessed using the Visual Analog Mood Scales (VAMS). Following the pretest measure, participants received one of three treatments: placebo, 120mg Ginkgo biloba, or 360mg Ginkgo biloba. Posttest measures were taken 2.5 hours after administration of treatment. Statistical analyses showed no significant differences between any of the three treatments in terms of cognitive performance or mood. Implications of the study s findings are discussed. ix

10 CHAPTER I INTRODUCTION Ginkgo biloba is a plant that dates back at least 200 million years. The Ginkgo tree, also called the maidenhair tree, produces fan shaped leaves, the components of which are commonly used as an herbal supplement. Various processing techniques are used to withdraw the leaves constituents, which are thought to benefit the human body. The active chemicals in Ginkgo biloba are flavonoid glycosides, biflavones, and terpene lactones (Spinella, 2001). Products comprised of these chemicals are distributed in the form of dried leaf, tincture, and various extracts. The most common extract is EGb 761; this extract has been standardized across manufacturers to ensure product quality (Spinella, 2001). The use of Ginkgo biloba as an herbal supplement dates back many centuries to China where it was used to treat memory loss, breathing problems, and even ailments of the skin and of the digestive system (Spinella, 2001). Today, Ginkgo biloba is also commonly used as an herbal supplement. Its use has many indications. Ginkgo biloba is commonly touted for improving overall cognitive functioning and memory. Ginkgo biloba has also been used in the treatment of symptoms that are commonly associated with Alzheimer s disease, vascular dementia, and other disorders of cerebro-vascular origin (Sierpina, Wollschlaeger, & Blumenthal, 2003). 1

11 Research Review Human Studies Research has demonstrated that Ginkgo biloba consumption increases the circulation of blood in the peripheral nervous system (Boles Ponto & Schultz, 2003). However, increased cerebral blood flow as a result of Ginkgo biloba treatment has not been clearly demonstrated in humans, but has been demonstrated in non-human animal studies (Krieglstein, Beck, & Seibert, 1986). The hypothesis that Ginkgo biloba induces an increase in cerebral blood flow is based on the following assumptions: if peripheral blood flow increases, then cerebral blood flow must also increase and, if cognitive improvements are identified, then there must be an increase in cerebral blood flow (Boles Ponto & Schultz, 2003). Ginkgo biloba has also been suggested to have antioxidant properties and to function as a scavenger for free radicals (Maclennan, Darlington, & Smith, 2002). Several studies have demonstrated Ginkgo biloba to aid in cell protection and to prevent apoptosis related to various physiological injury and dysfunction. Ginkgo biloba has also been shown to have neuroprotective qualities including protection from the neurotoxin linked to Parkinson s disease and possible modulation of monoamine oxidase activity (Boles Ponto & Schultz, 2003). Ginkgo biloba can be purchased over the counter in pharmacies, grocery stores, and other retail stores. The supplement is available in a variety of forms and dosages. Once-daily vitamins including Ginkgo biloba extracts are also now available. Advertisers market the new vitamins as a means for improving cognitive functioning. However, herbal supplements are not regulated by the Food and Drug Administration in 2

12 the United States. Therefore, the production and the usage of Ginkgo biloba supplements and other products containing Ginkgo biloba is not controlled by United States law. This means that manufacturers may make claims about a product with or without the evidence to support such claims. Despite this, a number of independent investigators have examined the effects of Ginkgo biloba on cognitive functioning. Unfortunately, the results of those investigations have been inconclusive. There are several reasons for the ambiguity that exists in the body of research concerning Ginkgo biloba. First, the experimental designs employed in Ginkgo biloba research have been extremely diverse. For example, researchers have tested the effects of Ginkgo biloba using a variety of dosages and a variety of administration procedures (i.e., acute or chronic administration). Researchers have also failed to systematically examine the effects of Ginkgo biloba in a variety of sub-populations, including the elderly. Experimental studies have also employed a wide variety of dependent measures on which to test the effects of Ginkgo biloba. Constructs of memory, reaction time, and even mood are often measured in studies of Ginkgo biloba efficacy; however, the instruments used to measure these domains are not consistent across studies. Additionally, many of these studies employ limited sample sizes. All of these factors contribute to the difficulty of comparing results and drawing conclusions based upon such research. There are many studies that suggest that Ginkgo biloba has no effect on cognitive functioning. Other studies suggest that any effect seen is no greater than the improvements that are associated with a placebo (Gold, Cahill, & Wenk, 2002). In an experiment examining the effects of Ginkgo biloba on cognitive functioning in healthy 3

13 older adults, Ginkgo biloba was found to have no effect when administered acutely (Nathan et al., 2002). The researchers utilized a within-subjects design, exposing participants to two conditions, a placebo condition and a 120mg of Ginkgo biloba condition. Participants were tested prior to receiving treatment and were tested again 90 minutes after treatment. A test battery derived from the cognitive drug research computerized assessment was used to examine working memory, spatial working memory, picture recognition, simple and choice reaction time, and auditory verbal learning. No significant effect of Ginkgo biloba was found between pretest and posttest measures on any of the cognitive modalities tested (Nathan et al., 2002). However, it is worth noting that this study had a limited sample size consisting of only 11 participants a limitation that was not addressed by the authors. The small sample size may have reduced the statistical power of the investigation, making it impossible to detect an actual effect if it existed. A different group of researchers investigated the effects of Ginkgo biloba on memory and reaction time after a chronic administration of Ginkgo biloba (Moulton, Boyko, Fitzpatrick, & Petros, 2001). Participants in this study consisted of 60 young healthy males who were given either placebo or 120mg of Ginkgo biloba daily for five consecutive days. Baseline measures of memory and reaction time were taken on the first day of the study using the Sternberg Memory Scanning Test and tasks of digit span and reading span. On the fifth day, participants were again tested on memory and reaction time. No significant differences were found between the placebo and treatment groups on any measures (Moulton et al., 2001). 4

14 Burns, Bryan, and Nettelbeck (2006) also investigated the chronic effects of Ginkgo biloba. However, this research extended the period of Ginkgo biloba administration to 12 weeks while maintaining the once-daily dosage of 120mg. This study also encompassed a larger battery of cognitive testing that included measurements of mood, speed of processing, and various subsets of memory. Mood was measured using the Profile of Mood States (POMS) and cognitive factors were measured using subsets of the Woodcock-Johnson Psycho-Educational Battery-Revised (WJ-R). The participants in this study consisted of both younger and older healthy adults. The group of young participants consisted of 104 males and the group of older participants included both males and females totaling 93 individuals. The researchers found no significant differences between pretest and posttest measures in the young adult group. The older adult group demonstrated improved performance on a test of long-term memory after being treated with Ginkgo biloba; no other differences were observed between the older adult placebo and treatment groups (Burns et al., 2006). Contrary to research suggesting the absence of a cognitive effect, many studies have demonstrated a positive cognitive effect. For example, Rigney, Kimber, and Hindmarch (1999) examined the acute effects of Ginkgo biloba on a subset of memory and reaction time tests. In this investigation, participants were randomly assigned to one of five experimental conditions. Six hour intervals were observed between the multiple doses (Rigney et al., 1999). Participants consisted of 31 healthy adults with age ranging from 30 to 59 years. Participants completed the test battery before the first dose of Ginkgo biloba and again at various time intervals up to 11 hours after the administration of the assigned treatment. The experimental conditions were as follows: placebo, 120mg 5

15 Ginkgo biloba administered once daily, 240mg Ginkgo biloba administered once daily, 150mg Ginkgo biloba divided into three 50mg doses per day, or 300mg Ginkgo biloba divided into three 100mg doses per day. The results of this study suggested an improvement in memory performance as well as a decrease in reaction time after treatment with Ginkgo biloba. Treatment related improvements were most evident after the once daily dosage of Ginkgo biloba at 120mg; however, the positive effects of Ginkgo biloba were not observed to increase linearly as the dosage increased. Furthermore, this study demonstrated the positive effects associated with 120mg of Ginkgo biloba to be most evident in older participants ranging from ages years (Rigney et al., 1999). These findings are similar to those of Burns et al. (2006) in that the positive effects associated with Ginkgo biloba were demonstrated in older participants. Kennedy, Scholey, and Wesnes (2000) also tested the acute effects of Ginkgo biloba by administering 120 mg, 240 mg, or 360 mg of Ginkgo biloba to participants while they assessed the speed and the accuracy of attention, the speed and the quality of memory, and the subjective internal mood state of participants. Ginkgo biloba was not found to have any significant effect on mood at any dosage level. Speed of attention improved significantly in participant groups receiving 240mg or 360mg of Ginkgo biloba; accuracy of attention did not improve in any of the treatment groups. Significant improvements for quality of memory were observed only in the group receiving 120mg of Ginkgo biloba. Finally, the speed of memory significantly improved under treatment with 360mg Ginkgo biloba. 6

16 This study also addressed the lag time associated with the effectiveness of Ginkgo biloba consumption and found the supplement produced positive effects from 2.5 hours post administration to 6 hours post administration (Kennedy et al., 2000). These same researchers conducted a follow-up study where the acute effects of 360mg Ginkgo biloba on attention, memory, and mood were again tested (Kennedy, Scholey, & Wesnes, 2002). As before, improved performance on tasks of memory and attention were observed. Furthermore, the follow-up study demonstrated positive effects on subjective mood ratings for those participants receiving the 360mg Ginkgo biloba treatment. Following treatment with Ginkgo biloba, participants rated themselves significantly higher on factors alert and content (Kennedy et al., 2002). In a study addressing the effects of Ginkgo biloba taken chronically, Mix and Crews (2000) provide further support for cognitive benefits associated with Ginkgo biloba consumption. However, these researchers were only able to provide evidence for positive effects on reaction time; no significant effects on memory performance were observed. In this study, healthy older adults were given a daily dose of either placebo or 180mg Ginkgo biloba over a six week time period. Pretest and posttest measures were administered in order to assess changes in the capacities of memory and speed of processing. The group receiving the Ginkgo biloba treatment demonstrated significant improvement on tasks involving speed of processing. While not significant, the Ginkgo biloba group did exhibit a trend toward improved performance on recall memory tasks after the six week treatment period (Mix & Crews, 2000). Another study also found positive effects associated with Ginkgo biloba taken chronically (Stough, Clarke, Lloyd, & Nathan, 2001). This research tested the effects of 7

17 Ginkgo biloba on young, healthy adults over a 30-day period. Participants were given either 120mg Ginkgo biloba or placebo once daily for 30 consecutive days. Pretest and posttest measures of attention, memory, and reaction time were administered for each participant. Results of this study demonstrated significant improvements in the Ginkgo biloba treatment group on the following tasks: digit span, working memory, and the Rey Auditory Verbal Learning Test (Stough et al., 2001). Furthermore, this study also examined the participants subjective opinions about the effect of the treatment they had received. Participants who had received the Ginkgo biloba rated themselves significantly higher than participants in the placebo group on self-reported measures of cognitive clarity, memory, and attention (Stough et al., 2001). Non-human Studies Non-human, animal research has demonstrated beneficial effects associated with Ginkgo biloba consumption. In rats, Ginkgo biloba was shown to improve short-term retention of spatial memory (Hoffman, Donato, & Robbins, 2004). This study tested the effects of four week exposure to a Ginkgo biloba dosage of 10mg/kg of body weight (n=40). Rats exposed to the four week Ginkgo biloba treatment performed better than rats in the placebo group on a task of short-term spatial memory (Hoffman et al., 2004). Zhang and Cai (2005) also tested the effects of Ginkgo biloba on spatial memory in rats. This study aimed to examine the ability of Ginkgo biloba to reverse cognitive deficits in spatial memory that were pharmacologically induced by the researchers. Rats in this study (n=11) were exposed to a drug known to produce deficits in spatial working memory. After deficits in spatial working memory had been confirmed, the rats were 8

18 then treated with Ginkgo biloba at 4 different dosage levels: 25, 50, 100, and 200mg/kg body weight. Treatment with Ginkgo biloba using the dosage of 50, 100 and 200mg/kg body weight was demonstrated to eliminate the deficits in spatial working memory that had been previously induced (Zhang & Cai, 2005). The dosage of 25mg/kg did not produce significant effects on spatial working memory. It is important to note that the dosages used in these two studies are much higher than the dosages used in human research and suggested by the dietary supplement manufacturers. A dosage of 20mg/kg in a rat would be comparable to a dosage of 1360mg Ginkgo biloba for a human weighing 150 pounds. Non-human animal research has also addressed the interaction between Ginkgo biloba and mood. In a comprehensive review of Ginkgo biloba, Gold et al. (2002) review research on the effects of Ginkgo biloba on mood related neurotransmitters, including serotonin, norepinephrine, and gamma-amino butyric acid (GABA). For instance, the number of serotonin receptors has been known to decrease with long-term exposure to stress. The chronic use of Ginkgo biloba can slow the loss of serotonin receptors (Gold et al., 2002). Chronic use of Ginkgo biloba has also been demonstrated to increase norepinephrine levels and reduce depression (Gold et al., 2002). Interestingly, the effects of Ginkgo biloba on serotonin and norepinephrine are greater in older rats. Finally, Ginkgo biloba is thought to increase levels of GABA in the brain, thus promoting a decrease in anxiety (Gold et al., 2002). 9

19 The Current Study The purpose of the present investigation was to test the effects of Ginkgo biloba on verbal and spatial working memory, simple and choice reaction time, and subjective ratings of mood state. It was hypothesized that the acute administration of Ginkgo biloba would affect memory performance, reaction time, and mood. It was also predicted that Ginkgo biloba would improve performance on measures of memory and reaction time, as well as increased ratings of positive mood state. 10

20 CHAPTER II METHODS Participants Participants in the study consisted of 56 undergraduate and graduate students, 47 female and 9 male. Two participants were not included in the statistical analysis (1 male, 1 female) due to incidences of traumatic brain injury and drug use in their personal history. Participants were recruited from undergraduate and graduate level psychology courses. All participants signed an informed consent form (Appendix A) before participating in the study. Upon completion of the study, participants were given course credit. Approval was obtained from the Institutional Review Board (Appendix B) of the responsible institution. Materials All participants completed a series of pen and paper questionnaires requesting demographic and medical history information. Mood was assessed using the Visual Analog Mood Scales developed by Robert A. Stern. Participants completed a series of tasks in a battery assessing verbal memory, spatial memory, and reaction time (RT). All tasks were presented on a 17-inch computer monitor. Participants were seated approximately 18 inches from the computer screen. A computer keyboard and mouse 11

21 were used by the participant to input responses to the tasks presented on the computerscreen. All computerized tests were created using the Stim2 computer software program developed by Neuroscan. The Stim2 system allows for the administration of stimuli as well as the recording of responses in millisecond units. A pen and paper version of the Stanford Sleepiness Scale was also administered before both the pretest and the posttest. Demographic Questionnaire The questionnaire (Appendix C) served to gather common demographic information about the participants, including the age, sex, and race of participants. Additionally, participants were asked to report if they were taking any prescription or over-the-counter medications. Participants were also asked to report any medical history involving traumatic brain injury, neurological diagnosis, psychological diagnosis, or psychiatric diagnosis. Finally, participants were asked to report the frequency of their usage of the following substances: blood thinners (including but not limited to: Heparin, Coumadin, and Aspirin) caffeine, and nicotine. Visual Analog Mood Scales The Visual Analog Mood Scales (VAMS; Appendix D) have been established as a reliable means for assessing internal mood state in a variety of clinical and non-clinical populations (Arruda, Stern, & Legendre, 1996; Arruda, Stern, & Somerville, 1999; Nyenhuis, Stern, Yamamoto, Luchetta, & Arruda, 1997; Stern, Arruda, Hooper, Wolfner, & Morey, 1997). The VAMS consist of eight visual analog scales representing eight internal mood states: afraid, confused, sad, angry, energetic, tired, happy, and 12

22 tense. Each participant was asked to place a mark along a 100 millimeter vertical line that best represented his/her current internal mood state. The top pole of each scale was labeled with the word Neutral, while the bottom pole was labeled with one of eight mood state words (e.g., Sad ). Scores were obtained by measuring the distance in millimeters (i.e., 0-100) that each mark was placed from the neutral pole. This test was administered in pencil and paper format. Verbal Memory The verbal memory task assessed the ability of the participant to recognize words which had been previously presented. This task consisted of the presentation of 20 words on a computer screen. Each word was presented on the screen individually, and in random order. Each participant was instructed to study each word as it appeared on the screen. After a brief pause, a second set of 20 words was presented on the screen. For this set of words the participant was instructed to indicate if each word, which was presented individually, had been presented in the study list. This was a task of recognition memory. The instructions were to press the left mouse button if the word was in the study list and to press the right mouse button if the word was not in the study list. All words used in this task were nouns. Nouns selected were deemed equivalent in ratings of abstractness-concreteness, imagery, meaningfulness, and frequency of use (Paivio, Yuille, & Madigan, 1968). Two scores were generated from this task; the percentage of correct responses and the reaction time in milliseconds associated with those correct responses. 13

23 Spatial Memory In the spatial memory task, a 3x3 matrix was presented on the computer screen. The participant was instructed to study the screen as a series of rectangular shapes appeared one at a time within one of the 9 locations of the matrix. Eight rectangles appeared in each trial in random order. After the series had been presented, a blank 3x3 matrix appeared on the screen. This task required free recall of the previously presented information. The participant was instructed to recall the order and the location each rectangle appeared in the first matrix. The participant used the mouse to log responses by clicking within the matrix to repeat the location and order of the series which had been presented. Percent correct was calculated based on accuracy of recall for both order and location of the rectangles as they appeared in the matrix. Reaction Time The reaction time tasks assessed the amount of time, in milliseconds (ms), required to respond to a stimulus presented on the computer screen. Both reaction time tasks involved the presentation of circles and squares. The shapes appeared one at a time with randomly assigned inter-stimulus intervals ranging from ms. There were 20 stimuli in each task. The simple reaction time task required a participant to press the left mouse button as soon as the participant saw any shape appear on the computer screen. The choice reaction time task required a participant to press the left mouse button for a circle appearing on the screen and to press the right mouse button for a square appearing on the screen. Simple and choice reaction times were generated by averaging all of the simple and choice reaction time responses, respectively. 14

24 Stanford Sleepiness Scale The Stanford Sleepiness Scale (SSS; Appendix E) has been demonstrated to be a reliable means for assessing subjective sleepiness (Maclean, Fekken, Saskin, & Knowles, 1992). This scale required the participant to rate his/her current degree of sleepiness. Seven different rating options were presented wherein a number, 1-7, corresponded to a description of sleepiness ranging from, feeling active, vital, alert (1) to, no longer fighting sleep, sleep onset soon (7). This test was used in order to control for any variables that might be introduced as a result of abnormal sleepiness. Any participant rating themselves at a 6 or 7 was not included in the final analysis (Maclean et al., 1992). Break Activity Checklist The break activity checklist (Appendix F) was designed to account for the behavior of the participant during the 2.5 hour break between the pretest and posttest sessions. Participants were asked specifically about the use of caffeine or nicotine during the break. Participants were also asked if they had slept or eaten during the break. Next, participants were asked to write a brief description of their activities during the break. The last item on the break activity checklist asked the participant to guess which treatment he/she had received with the option to check a box preceding one of the three treatments: placebo, 120mg Ginkgo biloba, or 360mg Ginkgo biloba. Ginkgo biloba The Ginkgo biloba used in this study was purchased from Vitamin World. This particular brand of Ginkgo biloba was selected because it is standardized to contain 24% 15

25 Ginkgo flavone glycosides; the same Ginkgo biloba standard used elsewhere in academic research. Furthermore, Vitamin World adheres to the Food and Drug Administration s Good Manufacturing Practices standard set forth under section 520 of the Federal Food, Drug and Cosmetic (FD&C) Act (2004). All Ginkgo biloba capsules contained 120mg of Ginkgo biloba extract. Placebo capsules contained unbleached-all purpose flour and were prepared under sanitary conditions using a capsule filling machine from Capsuline. Capsules were made from clear gelatin, also acquired from Capsuline. Procedure Each participant in the study was tested on an individual basis using a placebocontrolled, double-blind research design. The study consisted of two 30 minute sessions; a pretest session in the morning followed by a posttest session in the afternoon, approximately 3 hours after arrival for the pretest. Daily testing sessions began with the first participant arriving at 9:00 a.m. for the pretest session; additional participants continued to arrive at 30 minute intervals. No more than six participants were tested in one day. Participants were asked to return for the posttest session 3 hours after the scheduled time of the pretest session, with the participant arriving at 9:00 a.m. returning at 12:00 p.m. Again, participants returned one at a time at 30 minute intervals beginning at 12:00 p.m. Upon arrival for the pretest session, participants were asked if they had eaten a morning meal. All participants who reported not eating were offered a breakfast bar in order to prevent any stomach symptoms that might arise from taking Ginkgo biloba on 16

26 an empty stomach. Next, the participant was given an informed consent form to read and to sign. Any questions regarding the informed consent form were addressed by the investigator. Following the informed consent, the participant was given a packet containing a demographic questionnaire, two copies of the Stanford Sleepiness Scale, and a break activity checklist inquiring about participant activity during the break between pretest and posttest sessions. The participant was then asked to complete the demographic questionnaire and one copy of the Stanford Sleepiness Scale. The additional copy of the Stanford Sleepiness Scale and the break activity checklist were retained to be completed at the posttest session. Once all forms had been completed, the investigator then directed the participant into a room where all computer tasks as well as the Visual Analog Mood Scales would be administered. In total, four computerized tasks were administered during each session. These tasks consisted of verbal memory, spatial memory, simple reaction time, and choice reaction time. During the pretest session, each task was preceded by a practice session. The participant was allowed to ask questions at the end of each practice example if necessary. The administration of all tasks, including verbal memory, spatial memory, simple reaction time, choice reaction time, and the Visual Analog Mood Scales, were counterbalanced to prevent carry-over or practice effects. Following the completion of all five tasks, the participant was given a sealed packet containing three capsules and an 8 ounce packaged bottle of water. The participant was instructed to swallow all three capsules and drink the entire 8 ounces of water. Each participant ingested the capsules in the presence of an investigator. Capsules contained either 120mg Ginkgo biloba, 360mg Ginkgo biloba, or placebo. The 17

27 participant was then asked to refrain from caffeine use during the break. The participant was directed to go about his or her normal routine during the break and reminded of the return time for the posttest before being dismissed. Upon returning for the posttest session, the participant was first asked to complete the Stanford Sleepiness Scale. Next, the participant was directed back into the room where all tasks were readministered. Here the participant completed all five tasks again, in the same order as in the pretest session. However, practice examples were not administered during the posttest session. After all tasks had been completed, the participant was asked to fill out the break activity checklist inquiring about any activities that the participant engaged in during the break between pretest and posttest sessions. At this point, the participant was allowed time to ask any questions he or she might have regarding the study, thanked for his or her time, and dismissed. Design and Analysis The independent variable in this study was the Ginkgo biloba treatment. There were three levels of the independent variable: placebo, 120mg Ginkgo biloba, and 360mg Ginkgo biloba. Dependent variables were the performance scores from the posttest session. An analysis of covariance (ANCOVA) was performed for each posttest measure using its corresponding pretest measure as a covariate. 18

28 CHAPTER III RESULTS The demographic questionnaire indicated that the participant sample was comprised of 85% females (n = 46) and 15% males (n = 8). The sample was also comprised of 79% Caucasian (n = 43), 13% African American (n = 7), 4% Asian (n = 2), and 4% Hispanic (n = 2). The age of participants ranged from 18 to 47 years, with 72% of participants being between the ages of 18 and 25 years (n = 39), 19% of participants being between the ages of 26 and 35 years (n = 10), and 9% of participants being 35 years of age or older. The break activity checklist revealed that 89% of participants (n = 47) followed the researcher s request to abstain from caffeine during the break. Ninety-four percent of participants (n = 50) did not use nicotine during the break. Sleeping during the break was only reported by 4% of participants (n = 2) and eating during the break was reported by 69% of participants (n = 36). Spatial Memory Results of a one-way, between-subjects ANCOVA revealed no statistically significant difference in spatial memory performance between 0mg, 120mg, and 360mg of Gingko biloba, F(2, 48) =.38, p >.05 (Table 1). The effect size, as measured by a partial eta-squared, was.02, indicating a small treatment effect. Post hoc examination of statistical power indicated that the analysis had low statistical power (power =.11). 19

29 A sample size of 477 (n = 159) would have been necessary to detect such a small effect (power =.80) Verbal Memory As can be seen below, one-way, between-subjects ANCOVAs revealed no statistically significant differences in verbal memory performance (i.e., percent correct or reaction time) between the 0mg, 120mg, and 360mg Gingko biloba conditions. Verbal memory performance was operationally defined as the percentage of words a participant correctly recalled and how long, on average, it took the participant to respond. There were no significant differences in percent correct associated with verbal memory, F(2, 50) =.85, p >.05 (Table 1). The effect size, as measured by a partial etasquared, was.03, indicating a small treatment effect. Post hoc examination of statistical power indicated that the analysis had low statistical power (power =.19). A sample size of 315 (n = 105) would have been necessary to detect any effect on this dependent variable (power =.80). There were also no significant differences in reaction time (RT) associated with verbal memory, F(2, 50) =.48, p >.05 (Table 1). The effect size, as measured by a partial eta-squared, was.02, indicating a small treatment effect. Post hoc examination of statistical power indicated that the analysis had low statistical power (power =.12). A sample size of 477 (n = 159) would have been necessary to demonstrate an effect (power =.80). 20

30 Reaction Time As can be seen below, results of separate, one-way, between-subjects ANCOVAs revealed no statistically significant difference in either simple or choice reaction time (RT). Simple and choice reaction time tasks place different cognitive demands on a test taker, with the choice reaction time task being widely recognized as the more cognitively complex of the two. There were no significant differences in simple reaction time between the three experimental conditions (0 mg, 120 mg, and 360 mg), F(2, 49) =.56, p >.05 (Table 1). The effect size, as measured by a partial eta-squared, was.02, indicating a small treatment effect. Post hoc examination of statistical power indicated that the analysis had low statistical power (power =.14). A sample size of 477 (n = 159) would have been necessary to detect an effect (power =.80). There were also no significant differences in choice reaction time between the three experimental conditions (0 mg, 120 mg, and 360 mg), F(2, 50) =.04, p >.05 (Table 1). The effect size, as measured by a partial eta-squared, was.002, indicating a small treatment effect. Post hoc examination of statistical power indicated that the analysis had very low statistical power (power =.06). A sample size of 4812 (n = 1604) would have been necessary to detect such a small effect (power =.80) 21

31 Table 1 Tests of Between-Subjects Effects on Cognitive Tasks Posttest Placebo* 120mg* 360mg* F Measure M (SD) M (SD) M (SD) Spatial Memory (14.82) 47.27(11.63) (19.14).38 % Correct Verbal Memory (10.45) (81.88) (9.22).85 % Correct Verbal Memory (158.39) (204.37) (167.67).48 Correct RT Simple RT (21.13) (40.04) (23.64).56 Choice RT (40.47) (41.39) (43.05).04 Note. *p >.05.

32 Mood Results of one-way between-subjects ANCOVAs revealed no statistically significant differences in subjective mood ratings on the VAMS (Table 2). The effect sizes, as measured by a partial eta-squared, ranged from.001 ( Sad ) to.06 ( Tired), M eta-squared =.03, indicating that small treatment effects were present. Post hoc examination of statistical power (Table 3) indicated that each of the analysis had low statistical power (range power =.05 to.30). Sample sizes ranging from an N = 138 to an N = 9630 (n = 46 or n = 3210), respectively, would have been necessary to detect such small treatment effects (power =.80). 23

33 Table 2 Tests of Between-Subjects Effects on the VAMS Posttest Placebo* 120mg* 360mg* F Measure M (SD) M (SD) M (SD) Afraid 5.53 (6.51) 4.44 (5.12) 4.05 (5.38) 1.36 Confused (12.57) (24.92) (15.50) 1.00 Sad 8.89 (15.55) 9.56 (13.75) 9.58 (17.65).03 Angry 7.95 (10.08) 8.19 (16.45) 8.42 (12.95).59 Energetic (32.13) (28.89) (28.91).22 Tired (21.72) (28.81) (25.27) 1.48 Happy (23.70) (25.62) (27.26).48 Tense (22.94) (23.75) (17.86) 1.18 Note. *p >.05.

34 Table 3 Statistical Power on the VAMS Posttest Statistical N required for n required for Measure Power significant effect significant effect Afraid Confused Sad Angry Energetic Tired Happy Tense

35 When participants were asked to guess which treatment they had received, 47% (n = 24) guessed they had received placebo, 49% (n = 25) guessed that had received 120mg Ginkgo biloba, and 4% (n = 2) guessed they had received 360mg of Ginkgo biloba. A chi-square test of independence demonstrated that the actual treatment received by the participant and the treatment the participant had guessed they had received were not significantly associated, Pearson Chi-Square (2.487, 4), p >.05. No participants were excluded from the study as a result of scores on the SSS. As previously stated, any participant scoring a 6 or 7 on the pretest or posttest SSS would be excluded from the data analysis. The lowest score in this group of participants was 5. 26

36 CHAPTER IV DISCUSSION The purpose of this study was to test the effects of Ginkgo biloba on memory, reaction time, and subjective ratings of mood. It was hypothesized that the acute administration of Ginkgo biloba would affect memory performance, reaction time, and mood. It was predicted that Ginkgo biloba would improve performance on measures of memory and reaction time, as well as increased ratings of positive mood state. The hypothesis in the present investigation was not supported. Further, given there was no significant effect of Ginkgo biloba, the prediction that Ginkgo biloba would improve performance was not supported. The lack of statistically significant differences in memory and reaction time observed in the present investigation supports the results of several studies that also found no effect of Ginkgo biloba on these measures. For example, Moulton et al. (2001) found no effect of Ginkgo biloba (120mg) on memory or reaction time in young healthy males (N = 60) when administered chronically over a 5-day period (Moulton et al., 2001). Another study tested the chronic effects of 120mg Ginkgo biloba on young healthy males (N = 104) and also found no effects on memory, reaction time. Further, they also tested subjective rating of mood and found no significant effect of Ginkgo biloba (Burns et al., 2006). 27

37 It should be noted that both these investigations examined the effects of chronically administered ginkgo biloba and that each investigation possessed a sample size that was larger than the sample size used in the current study. However, others have tested the acute effects of ginkgo biloba (120mg) on healthy elderly (N = 11) and report similar findings (Nathan et al., 2002). Unfortunately, the lack of effect reported by Nathan et al. may have been due to a small sample size and low statistical power. That is, there may be an effect associated with the acute administration of gingko biloba in the elderly, but it may not have reached statistical significance because of low statistical power. The results of the current study are not in agreement with research that has demonstrated an effect of both chronic and acute Ginkgo biloba administration on young participants (Kennedy et al., 2000; Kennedy et al., 2002; Stough et al., 2001). For example, Kennedy and colleagues tested the acute effects of Ginkgo biloba and found improvements associated with measurements of memory and speed of processing (Kennedy et al., 2000; Kennedy et al., 2002). However, their findings regarding the effect of Ginkgo biloba on mood were inconsistent. Nevertheless, these findings, taken together with the aforementioned findings, suggest that gingko biloba may only have an effect when acutely administered and that age has no impact on the extract s effects. One potential explanation for the findings in the present investigation and for the findings of other investigations reporting similar findings may be low statistical power due to a small sample size. The sample size used in the current study was comparable to the sample sizes used in studies that reported no effect of Ginkgo biloba (Nathan et al., 2002). Unfortunately, none of the studies cited herein reported eta-squared values or 28

38 power estimates. However, inferences can be made from reported F statistics and p values to suggest that statistical power was low in these studies. Another potential explanation for the lack of statistically significant effects may be the age of the population receiving Ginkgo biloba treatment. Of the three studies reporting negative findings, two studies tested the effects of Ginkgo biloba on young, healthy participants (Burns et al., 2006; Moulton et al., 2001). The current study also tested a young healthy population; wherein the effect of Ginkgo biloba may be less pronounced than it would be for an older population due to the presumed optimal cognitive functioning of young healthy individuals. Therefore, one may be more likely to detect an effect of Ginkgo biloba within an elderly population. In the present study, the acute administration of Ginkgo biloba did not significantly affect memory performance, reaction time, or mood. If an effect of Ginkgo biloba was present, the sample size used in this study was not large enough to detect such a small effect. However, the sample size used in this study does not differ widely from other research that has reported differences. Larger research studies have consisted of sample sizes as large as 100 participants (Burns et al., 2006). Some smaller studies have included as few as 11 participants (Nathan et al., 2002). Sample size is not an issue that has been addressed by many of the authors of research cited within this study. Therefore, it is difficult to determine if the lack of a significant effect of Ginkgo biloba is due to low statistical power as a result of small sample size, or to another factor, such as age. It is apparent that it is possible to detect an effect of Ginkgo biloba with a relatively small sample size, as there are studies that have done so. It is likely that a small effect of 29

39 Ginkgo biloba exists in nature especially in an elderly population but that the sampling error inherent in each investigation may make it difficult to detect. While there were no significant differences on measures of cognitive ability or mood between 0mg, 120mg, and 360mg of Gingko biloba, three measures of mood had treatment effects that were noticeably higher than the treatment effects associated with other dependent measures. The VAMS scales Tense and Afraid both demonstrated eta-squared values of.05; thus, an increase in sample size (N = 189) could result in a significant effect of Ginkgo biloba. Furthermore, the mean scores between the three treatment groups would suggest a decrease on ratings of Tense and Afraid as an effect of Ginkgo biloba treatment (Figure 1). The VAMS scale Tired demonstrated an eta-squared value of.06. Increasing the sample size to 138 participants could result in an effect of Ginkgo biloba on the VAMS scale Tired. The mean scores for Tired would suggest an increase in the subjective rating of Tired as an effect of Ginkgo biloba (Figure 1). 30

40 Figure 1. Data trends for afraid, tense, and tired. Of the 13 dependent variables measured, directional trends in the VAMS data were observed. While not statistically significant, scores of Tense and Afraid were lower for those participants receiving Ginkgo biloba. Such a result is consistent with previous findings indicating that Ginkgo biloba modulates neurotransmitters such as GABA, norepinephrine, and serotonin; all of which are associated with arousal and mood state (Gold et al., 2002). For instance, Ginkgo biloba is thought to increase levels of GABA in the brain, thus promoting a decrease in anxiety (Gold et al., 2002). These results are also consistent with research on subjective mood state, wherein, 31

41 subjects receiving Ginkgo biloba rated themselves significantly higher on factors of alert and content (Kennedy et al., 2002). The observed trend in the measurement of the scale Tired on the VAMS lends some confusion to what are thought to be the basic effects of Ginkgo biloba. In this study, participants who received Ginkgo biloba rated themselves as more tired than did those participants who received placebo. Furthermore, this effect increased respective to the dosage of Ginkgo biloba received, wherein, participants receiving 360mg Ginkgo biloba rated themselves more tired than those receiving 120mg Ginkgo biloba (Table 2). While this effect was not significant, the results of this dependent measure had the greatest statistical power out of all 13 dependent variables (power =.30). Some research would predict that participants would become more alert/less tired after taking Ginkgo biloba for multiple reasons including the increase in cerebral blood flow (Boles Ponto & Schultz, 2003) and the effects of Ginkgo biloba on neurotransmitters such as serotonin (Gold et al., 2002). However, there is also research that would support the effect this study found on the measure Tired, including the enhancing effects of Ginkgo biloba on the neurotransmitter GABA (Gold et al., 2002). GABA is an inhibitory neurotransmitter that is closely associated with central nervous system depressants such as alcohol and anti-anxiety medication. The inhibitory effect of GABA on the central nervous system would be in line with increased feelings of tiredness. The results of this study add further to the ambiguity that exists within research on the cognitive effects of Ginkgo biloba. Future research on the effects of Ginkgo biloba should employ larger sample sizes, this would increase the possibility of finding effects of Ginkgo biloba should they exist. Additionally, future studies should include 32

42 both young and elderly healthy volunteers. Furthermore, it would be beneficial to consider statistical power as an indication of the strength of Ginkgo biloba effects, and to report such statistics when publishing results. 33

43 REFERENCES Arruda, J.E., Stern, R.A., & Legendre, S.A. (1996). Assessment of mood state in patients undergoing electroconvulsive therapy: The utility of the Visual Analog Mood Scales designed for cognitively-impaired patients. Convulsive Therapy, 12, Arruda, J.E., Stern, R.A., & Somerville, J.A. (1999). Measurement of mood states in stroke patients: Validation of the Visual Analog Mood Scales. Archives of Physical Medicine and Rehabilitation, 80, Boles Ponto, L.L., & Schultz, S.K. (2003). Ginkgo biloba extract: Review of CNS effects. Annals of Clinical Psychiatry, 15(2), Burns, N.R., Bryan, J., & Nettelbeck, T. (2006). Ginkgo biloba: No robust effect on cognitive abilities or mood in healthy young or older adults. Human Psychopharmacology, 21, Federal Food, Drug, and Cosmetic Act, 21 U.S.C. 360j (2004). Gold, P.E., Cahill, L., & Wenk, G.L. (2002). Ginkgo biloba: A cognitive enhancer? Psychological Science in the Public Interest, 3(1), Hoffman, J.R., Donato, A., & Robbins, S.J. (2004). Ginkgo biloba promotes short-term retention of spatial memory in rats. Pharmacology, Biochemistry and Behavior, 77,

44 Kennedy, D.O., Scholey, A.B., & Wesnes, K.A. (2000). The dose-dependent cognitive effects of acute administration of Ginkgo biloba to healthy young volunteers. Psychopharmacology, 151, Kennedy, D.O., Scholey, A.B., & Wesnes, K.A. (2002). Modulation of cognition and mood following administration of single doses of Ginkgo biloba, ginseng, and a ginkgo/ginseng combination to healthy young adults. Physiology & Behavior, 75, Krieglstein, J., Beck, T., & Seibert, A. (1986). Influence of an extract of Ginkgo biloba on cerebral blood flow and metabolism. Life Sciences, 39, Maclean, A.W., Fekken, G.C., Saskin, P., & Knowles, J.B. (1992). Psychometric evaluation of the Stanford Sleepiness Scale. Journal of Sleep Research, 1, Maclennan, K.M., Darlington, C.L., & Smith, P.F. (2002). The CNS effects of Ginkgo biloba extract and ginkgolide B. Progress in Neurobiology, 67, Mix, J.A. & Crews, W.D. (2000). An examination of the efficacy of Ginkgo biloba extract EGb 761 on the neuropsychological functioning of cognitively intact older adults. The Journal of Alternative and Complimentary Medicine, 6(3), Moulton, P.L., Boyko, L.N., Fitzpatrick, J.L., & Petros, T.V. (2001). The effect of Ginkgo biloba on memory in healthy male volunteers. Physiology & Behavior, 73, Nathan, P.J., Ricketts, E., Wesnes, K., Mrazek, L., Greville, W., & Stough, C. (2002). The acute nootropic effects of Ginkgo biloba in healthy older human subjects: A preliminary investigation. Human Psychopharmacology, 17,

45 Nyenhuis, D.L., Stern, R.A., Yamamoto, C., Luchetta, T., & Arruda, J.E. (1997). Standardization and validation of the Visual Analog Mood Scales. The Clinical Neuropsychologist, 11(4), Pavio, A., Yuille, J.C., & Madigan, S.A. (1968). Concreteness, imagery, and meaningfulness: Values for 925 nouns. Washington: American Psychological Association. Rigney, U., Kimber, S., & Hindmarch, I. (1999). The effects of acute doses of standardized Ginkgo biloba extract on memory and psychomotor performance in volunteers. Phytotherapy Research, 13, Sierpina, V.S., Wollschlaeger, B., & Blumenthal, M. (2003). Ginkgo biloba. American Family Physician, 68(5), Spinella, M. (2001). The psychopharmacology of herbal medicine: Plant drugs that alter mind, brain, and behavior. Cambridge, Mass: MIT Press. Stern, R.A., Arruda, J.E., Hooper, C.R., Wolfner, G.D., & Morey, C.E. (1997). Visual Analog Mood Scales to measure internal mood state in neurologically impaired patients: Description and initial validity evidence. Aphasiology, 11(1), Stough, C., Clarke, J., Lloyd, J., & Nathan, P.J. (2001). Neuropsychological changes after 30-day Ginkgo biloba administration in healthy participants. International Journal of Neuropsychopharmacology, 4, Zhang, M., & Cai, J. (2005). Extract of Ginkgo biloba leaves reverses yohimbineinduced spatial working memory deficits in rats. Behavioural Pharmacology, 16,

46 APPENDIXES 37

47 Appendix A Informed Consent 38

48 INFORMED CONSENT FORM I. Title of Research: a. The Effects of Ginkgo Biloba on Mood and Memory II. Overview: a. Federal and university regulations require us to obtain signed consent for participation in research involving human participants. After reading the statements in section III through VI below, please indicate your consent by signing and dating this form. III. Statement of Procedure: a. Once informed consent has been given, I will complete a demographics questionnaire containing questions pertaining to my age, sex, race, medical history, and previous caffeine, nicotine, alcohol, and drug use. I will then be asked to complete eight visual analogue mood scales that are designed to assess how afraid, confused, sad, angry, energetic, tired, happy, and tense I am. Next, I will be asked to perform three computerbased cognitive tasks involving memory and reaction time. Once I have completed the visual analogue mood scales and the cognitive tasks, I will be asked to orally ingest 3 gelatin capsules filled with either flour, 120mg of Ginkgo biloba, or 360 mg of Ginkgo biloba. I will then be asked to complete another set of visual analogue mood scales and cognitive tasks two hours and 30 minutes after my ingestion of the capsules. I will then be debriefed and thanked for my time. b. I understand that: i. The purpose of this research is to assess the effects of Ginkgo biloba on mood and memory. ii. My participation is entirely voluntary; I can withdraw my consent at any time without penalty and have the results of the participation, to the extent that it can be identified as mine, returned to me, removed from the research records, or destroyed. iii. No more than minimal discomforts or stresses are foreseen. 39

49 iv. Should you experience any discomfort subsequent to the administration of the Ginkgo biloba, we will escort you immediately to the Health Services for clinical help. v. Should I become disturbed by any of the materials, I can contact Dr. James Arruda ( ), Emily Qualls ( ), or Douglas Friedrich ( ) in the Department of Psychology for assistance. vi. The results of this participation will be confidential, and will not be released in any individually identifiable form without my prior consent, unless otherwise required by law. Identifying information I provide below will only be used for contact purposes and will be confidential. All other research materials (including the questionnaire responses) will be identified only by the participant code number. This informed consent form will be kept separate from all other research materials at all times. vii. If applicable, I will receive extra credit for a specified psychology course in return for my participation in this study regardless of my performance during participation viii. Financial compensation for such things as disability or discomfort due to injury are my responsibility. Compensation for wages, etc., is not available. Any medical expenses due to self-inflicted injury are my responsibility. Unless found to be liable in a court of law for medical damages, no other compensation for other damages is available from University of West Florida or the study investigators. ix. The investigator will answer any questions about the research, now or during the course of the project. A full debriefing about the purpose of the questionnaire will be available from the investigator following the completion of the study. 40

50 IV. Potential Risks of the Study: a. Possible side effects of Ginkgo biloba include stomach discomfort, headache, and ringing in the ears. V. Potential Benefits of the Study: a. The information obtained from this investigation may advance our understanding of the relationship between Ginkgo biloba, mood, and memory. VI. Statement of Consent: a. I certify that I have read and fully understood the Statement of Procedure provided above and agree to participate in the research project described therein. Permission is given voluntarily and without coercion or undue influence. It is understood that I may discontinue my participation at any time without penalty or loss of any benefits to which I may otherwise be entitled. I will be provided a copy of the informed consent form once I have signed and dated the form below. Signature of Participant Date Printed Name of Participant Emily K. Qualls Signature of Investigator Date Printed Name of Investigator Research at The University of West Florida that involves human participants is overseen by the Institutional Review Board. Questions or problems regarding your rights as a participant should be addressed to the Chairperson of the Institutional Review Board at Office of Research; University of West Florida; University Parkway; Pensacola, FL 32514; krasmuss@uwf.edu 41

51 Appendix B Institutional Review Board Approval Letter 42

52 43

53 44