Limited cognitive benefits in Stage +2 postmenopausal women after 6 weeks of treatment with Ginkgo biloba

Original papers Limited cognitive benefits in Stage +2 postmenopausal women after 6 weeks of treatment with Ginkgo biloba Sarah Elsabagh Psychopharmacology Research Unit, Centre for Neuroscience, King s College London, London and Centre for Pharmacognosy and Phytotherapy, The School of Pharmacy, London, UK. David E. Hartley Psychopharmacology Research Unit, Centre for Neuroscience, King s College London, London, UK. Sandra E. File Psychopharmacology Research Unit, Centre for Neuroscience, King s College London, London, UK. Journal of Psychopharmacology 19(2) (2005) 173 181 2005 British Association for Psychopharmacology ISSN 0269-8811 SAGE Publications Ltd, London, Thousand Oaks, CA and New Delhi 10.1177/0269881105049038 Abstract Gingko biloba has cognitive benefits both in populations suffering from dementia and after acute treatment in healthy volunteers, with some evidence indicating that those with poorer cognitive performance show greater benefit. We have previously found that 1 week of treatment with ginkgo improved attention, memory and mental flexibility in postmenopausal women, but the evidence for any beneficial effects of longer treatment is less well-established. The present study aimed to determine whether cognitive benefits, similar to those previously found after 1 week of treatment, would persist after 6 weeks of treatment, and whether those with poorer cognitive performance would benefit more. In a placebo-controlled, double-blind study, postmenopausal women (aged 51 67 years) were randomly allocated to receive a standardized extract of ginkgo (LI 1370, Lichtwer Pharma, Marlow, UK) (one capsule/day of 120 mg, n = 45) or matching placebo (n = 42) for 6 weeks. According to an established reproductive staging system, subjects were divided into those in the early (Stage +1; mean age 55 years) and late (Stage +2: mean age 61 years) stages of menopause. At baseline and after 6 weeks of treatment, subjects completed tests of mental flexibility, planning, memory and sustained attention, and ratings of mood, sleepiness, bodily and menopausal symptoms. The only significant effects of ginkgo were in the test of mental flexibility, in which there were significant menopausal stage ginkgo interactions. This was because subjects in Stage +2 required fewer trials to complete the task and made fewer errors after ginkgo treatment, whereas those in Stage +1 showed no benefits. Subjects in Stage +2 had poorer performance at baseline compared to those in Stage +1 both in this task and the test of planning ability. The beneficial effects of ginkgo were limited to the test of mental flexibility and to those with poorer performance. Keywords attention, frontal lobe function, Ginkgo biloba, memory, mental flexibility, mood, planning, postmenopausal women Introduction Extracts from the leaves of the Ginkgo biloba tree have been used therapeutically in the Far East for millennia in the treatment of a wide variety of ailments. More recently, the beneficial effects of ginkgo in the treatment of cognitive disorders have received attention in the West, and its efficacy in the treatment of Alzheimer s disease and vascular dementia is reasonably well-established (Kanowski et al., 1996; Le Bars et al., 1997; Oken et al., 1998; Birks et al., 2002). There is also evidence that acute administration of ginkgo can improve cognition in healthy individuals. In young volunteers, acute doses (120 360 mg) improved performance on factors measuring memory and attention and in serial subtraction, which has components of working memory and attention (Kennedy et al., 2000, 2002; Scholey and Kennedy, 2002). In a slightly older healthy group (30 59 years), ginkgo (120 mg) improved shortterm memory (Rigney et al., 1999), with a greater improvement demonstrated in the oldest group (50 59 years). There is also some evidence that benefits may persist after short-term, sub-chronic, treatment. Moulton et al. (2001) demonstrated some improvement in male college students on the Sternberg memory test after 5 days of ginkgo treatment (120 mg/day), and Hartley et al. (2003) found that postmenopausal women performed better than the placebo group in tests of attention, short-term memory and mental flexibility after 7 days of ginkgo treatment (120 mg/day). Stough et al. (2001) found improved performance with ginkgo (120 mg/day) after 30 days of treatment in healthy subjects (aged 18 40 years) in digit span backwards, working memory speed, and an auditory verbal learning test. In addition, the group with lower cognitive ability improved on Trails A. However, in older groups, Corresponding author: Sarah Elsabagh, Psychopharmacology Research Unit, Centre for Neuroscience, Hodgkin Building, King s College London, Guy s Campus, London SE1 1UL, UK. Email: sarah.elsabagh@kcl.ac.uk

174 Limited cognitive benefits of treatment with Ginkgo biloba the evidence for positive effects after chronic treatment is less convincing. Cieza et al. (2003) found no improvement on tests of attention or memory after 4 weeks of treatment in healthy middleaged volunteers (50 65 years). In elderly volunteers after 6 weeks of ginkgo treatment, Mix and Crews (2000, 2002) found limited evidence of improvement, and Solomon et al. (2002) found no effect on 15 measures of memory. Persson et al. (2004) selected subjects (aged 35 80 years) who had regularly taken ginkgo supplements for 2 years and found no effects on any of the eight tests of episodic and semantic memory compared to a control group. There are several explanations for any lack of effect in the older groups after chronic treatment. One is the possibility that older people are unable to benefit from ginkgo. This would appear to be unlikely because Rigney et al. (1999) found greater improvement in the group of older subjects, Hartley et al. (2003) found improved performance in post-menopausal women and benefits have also been found in dementia patients (Kanowski et al., 1996; Oken et al., 1998). The second possibility is that the age-range and range of cognitive performance in some of the studies was too wide for potential benefits to be identified. It is possible that the benefits of ginkgo are more marked in those subjects who have lower levels of performance. The results of two studies indicate that ginkgo might have greater cognitive benefits in sub-groups with poorer cognitive performance (Rigney et al., 1999; Stough et al., 2001). Gessner et al. (1985) found that only a sub-group with a more impaired electroencephalogam (EEG) profile at baseline showed improvements in vigilance and quicker reaction times after 12 weeks of treatment with ginkgo. The contention that older subjects may benefit to a greater extent than younger subjects is supported by an animal study showing that 3 weeks of ginkgo treatment (100 mg/kg/day) improved passive avoidance learning solely in an aged group of mice (Stoll et al., 1996). The third possible explanation for a relative lack of effects after chronic treatment is that tolerance develops with longer periods of treatment. The aim of the present study was to investigate the effects of 6 weeks of treatment with ginkgo in a group of postmenopausal women of similar age-range to those previously studied. The same battery of tests was selected as was previously used by Hartley et al. (2003) to determine whether the cognitive benefits found in that study would persist after a longer period of treatment. The same dose of gingko (120 mg/day) was also selected because this is the dose at which cognitive benefits have most frequently been reported, as well as being the lower dosage-range prescribed for the chronic treatment of dementia and intermittent claudication in Germany (Upton, 2003). The benefits of ginkgo on cognition, although slight, have been previously demonstrated by 6 weeks (Mix and Crews, 2000, 2002). A secondary aim of the study was to determine whether any benefits would be more marked in those individuals with greater cognitive impairment. To investigate this, we divided our group on the basis of the time after menopause, according to a classified system of reproductive ageing (Soules et al., 2001). In Stage +1, oestrogen levels are declining, whereas they have reached a steady low level in in Stage +2. Thus, women within 5 years after menopause (Stage +1) were compared with those > 5 years after menopause (Stage +2). Materials and methods Participants Ninety-six healthy, postmenopausal women aged 51 67 years were recruited by circular e-mail at King s College London or by advertisements in the local press. King s College Ethical Committee approved the study and volunteers provided their written informed consent. Volunteers were defined as postmenopausal if they had not menstruated within the previous 12 months. Exclusion criteria included current illness, use of hormone replacement theapy (currently or within the previous 12 months), use of psychoactive or anti-coagulant medication, use of ginkgo, ginseng or isoflavone supplements or estimated verbal IQ scores < 90. They were randomly allocated to the ginkgo or placebo-treatment groups. Nine subjects (six in the placebo group and three in the ginkgo group) dropped out during the course of the study for various personal reasons, but none reported any side-effects. Nine subjects had hysterectomies (five in the ginkgo group and four in the placebo group), and one subject in the ginkgo group had a hysterectomy and oophorectomy. Five subjects were smokers: four in the ginkgo group (smoking a mean of nine cigarettes/day) and one subject in the placebo group (smoking 12 cigarettes/day). The subjects in each treatment group were stratified into those in Stage +1 (early stage) of the menopause ( 5 years after the final menstrual period; n = 25 in the placebo group, n = 18 in the ginkgo group) and those in Stage +2 (late stage, > 5 years after the final menstrual period; n = 17 in the placebo group, n = 27 in the ginkgo group) according to the guidelines of the stages of reproductive aging workshop (Soules et al., 2001). For those subjects who had hysterectomies before reaching menopause, the stage of menopause was calculated using the mean of the group age at menopause (51 years) as a guideline. One subject who had undergone a hysterectomy and oophorectomy had a natural menopause before the surgical procedure and was in Stage +2. Supplement administration The ginkgo biloba supplement used was Ginkyo One-A-Day tablets (supplied by Lichtwer Pharma UK, Marlow, UK), which contains the standardized extract LI 1370 obtained from the green leaves of the Ginkgo biloba tree. This extract comprises 25% ginkgo flavonoids and 6% terpenoids. The placebo tablets were identical in appearance (also supplied by Lichtwer). Subjects received one tablet containing 120 mg ginkgo biloba extract or the identical placebo tablet each morning for 6 weeks. Subjects were instructed to take their last tablet on the morning of the 6-week test session. Compliance was monitored by placing seven extra tablets in each bottle and asking subjects to return their tablet bottles at the end of treatment so that the remaining tablets could be counted. The administration was double-blind and neither subjects nor experimenters were aware of the treatment groups.

Limited cognitive benefits of treatment with Ginkgo biloba 175 Procedure The subjects were required to attend two test sessions at the Psychopharmacology Research Unit, King s College London. The baseline test session was before the start of treatment and the second session was after 6 weeks of treatment. Subjects were tested 4 5 h after their last dose on the 6-week test session. This coincides with the time at which peak activity levels of ginkgo are reached (Upton, 2003), and at which positive effects on memory have been reported after a single dose (Kennedy et al., 2000). At the baseline test session, subjects were given a practice session to familiarize themselves with the computerized test battery. An estimate of verbal IQ was obtained using the National Adult Reading Test Revised version (NART-R) (Nelson and Willison, 1991). Thereafter the battery of tests was identical at both sessions. Measures of menopausal symptoms, anxiety and depression The Hospital Anxiety and Depression Scale (HAD) (Zigmond and Snaith, 1983) was used to determine levels of trait anxiety and depression. Menopausal symptoms were assessed by the Greene Climacteric Scale (Greene, 1998), from which four independent measures can be extracted: psychological, somatic, vasomotor symptoms and sexual dysfunction. Visual Analogue Scales Before and after cognitive testing, subjects were asked to complete Visual Analogue Scales to assess current mood state (Bond and Lader, 1974) and bodily symptoms of somatic anxiety (Tyrer, 1976). Each scale consisted of two items separated by a 100-mm vertical line. Subjects were instructed to place a perpendicular mark at the appropriate place on each line to indicate how they were feeling at that time. The current state of sleepiness was measured by the Stanford Sleepiness Scale (Hoddes et al., 1973), which was administered before and after the test session. The Epworth Sleepiness Scale (Johns, 1991, 1992) was administered after the test session and measured how sleepy the subjects usually feel. Cognitive tests Sustained attention The Paced Auditory Serial Addition Task (PASAT) was used to assess sustained attention (Spreen and Strauss, 1991). This task involves adding together successive pairs of digits read from a list of 61 numbers. There were four presentation speeds, with digits presented every 2.4, 2.0, 1.6 and 1.2 s. The first two presentation speeds were used as a practice and the two fastest presentation speeds were used to assess performance. The number of correct responses was recorded as a score out of 60 for the two fastest presentation speeds. Episodic memory There were three tests of episodic memory. In the immediate and delayed paragraph recall test (from the Weschler Memory Scale-Revised; Weschler, 1987), 25 units of information were read at the rate of 1 unit per second, and subjects were instructed to remember the paragraph as accurately as possible. They were asked for their recall immediately after hearing the story and after a delay of 30 min. The number of correctly remembered units was scored. The Delayed Matching-to-Sample (DMTS) task was from the Cambridge Neuropsychological Test Automated Battery (CANTAB CeNeS, Cambridge) and was used to assess short-term, non-verbal memory. In this test, subjects were presented with a sample pattern which they were instructed to remember. The sample pattern and three distracter patterns then appeared either simultaneously or after a delay of 0, 4 or 12 s, and the subject had to select the sample pattern that they had just been presented with. The total number of correct responses and the latency to respond were recorded. Long-term episodic memory was assessed using a picture recall task. Subjects were presented with 20 pictures of common objects for 5 s per picture, and were then asked to recall as many items as they could remember after a 25 min delay. Category generation Subjects were asked to name all the animals they could think of that belonged to a particular category (house, farm or jungle). They were given 20 s for each category. A total score was derived from all admissible answers for each category. Planning ability The Stockings of Cambridge (SoC) task is a spatial planning test from the CANTAB, based on the Tower of London task (Shallice, 1982) and is described in detail by Owen et al. (1990). Subjects were presented with an arrangement of coloured balls housed in stockings that had to be rearranged in the fewest moves possible to match a goal arrangement presented at the top of the screen. The task varied in difficulty from two to five moves. The two- and three-move problems were used for practice and the four- and five-move problems were recorded. The number of moves for each task and the subsequent thinking time were recorded Mental flexibility The Intra/Extra Dimensional Shift (IDED) task from the CANTAB involves rule learning, shifting and reversal and is a measure of mental flexibility controlled by the frontal cortex (Owen et al., 1991). In the first stage, subjects were required to learn a simple discrimination. Once the rule was correctly learned, it was then reversed or shifted. The number of trials taken and the number of errors made to reach the Extra-Dimensional Shift (EDS) stage were recorded. Statistical analysis To determine whether treatment had differential effects in subjects depending on their menopausal stage, the data for the cognitive test measures were analysed using three-way analysis of variance (ANOVA). The between group factors were treatment group and menopausal stage, and the repeated measures factor was week. The three mood factors and Stanford Sleepiness Scales were analysed by four-way ANOVA, with treatment and menopausal stage as the between-group factors and repeated measures factors of time (before and after cognitive testing) and week. A four-way multivariate analysis (MANOVA) was used to analyse the factor of

176 Limited cognitive benefits of treatment with Ginkgo biloba somatic anxiety derived from the Bodily Symptoms Scale. Where this reached significance, data for the individual items were subsequently analysed by ANOVA. Where effects reached significance, both F-ratios and probability levels are quoted. Where results did not reach significance, only the F-ratios are presented and nonsignificance indicated (NS). Data were analysed using SPSS for Windows (version 10.1; SPSS Inc., Chicago, IL, USA). Results Subject characteristics The groups did not differ on predicted verbal IQ scores, body mass index, years of secondary education, typical alcohol consumption, daily caffeine consumption, or anxiety and depression ratings on the HAD Scales (treatment, menopausal stage and treatment menopausal stage interaction, F < 3.3, NS in all cases). As expected, the subjects in Stage +2 of menopause were significantly older than those in Stage +1 [F(1,83) = 71.7, p < 0.0001] (Table 1). At baseline, the subjects in Stage +1 of menopause had significantly higher ratings of vasomotor symptoms than those in Stage +2 [F(1,83) = 8.5, p < 0.005], but there were no differences in ratings of psychological symptoms or sexual dysfunction (F < 2.0, NS in all cases), or sleepiness as measured by the Epworth or Stanford Scales (Tables 2 and 3) (F < 2.0, NS in all cases). Menopausal symptoms and HAD Scales Ginkgo was without effect on menopausal symptoms or anxiety and depression as measured on the HAD Scales (week treatment and week treatment menopausal stage interactions, F < 2.5, NS in all cases). However, there was a significant effect of week for psychological symptoms [F(1,83) = 14.3, p < 0.0001], ratings of sexual dysfunction [F(1,75) = 11.5, p < 0.001] on the Greene Climacteric Scale, and anxiety ratings on the HAD A Scale [F(1,83) = 6.2, p < 0.02] because all subjects improved after 6 weeks of treatment. There was a significant week menopausal stage interaction for ratings of somatic menopausal symptoms [F(1,83) = 4.3, p < 0.05] because of improvement for the subjects in Stage +1 (Table 2). Table 1 Age, predicted verbal IQ (based on the National Adult Reading Test), years of secondary education, body mass index, daily consumption of caffeinated drinks (cups), weekly alcohol consumption (units), and scores on the Hospital Anxiety and Depression Scales (HAD A and HAD D ) of the subjects allocated to placebo and ginkgo groups in Stage +1 and Stage +2 of the menopause Stage + 1 Stage + 2 Placebo Ginkgo Placebo Ginkgo Age (years)* 55.5 ± 0.6 55.3 ± 0.6 61.4 ± 0.5 60.4 ± 0.7 IQ 116.7 ± 1.6 113.7 ± 2.1 114.2 ± 1.4 111.7 ± 1.7 Years of secondary education 5.6 ± 0.2 5.2 ± 0.3 5.5 ± 0.2 5.0 ± 0.3 Body mass index 25.4 ± 0.7 24.2 ± 0.8 26.2 ± 1.0 25.0 ± 0.5 Daily caffeinated drinks (cups) 4.4 ± 0.5 5.6 ± 0.7 5.2 ± 0.7 4.1 ± 0.7 Weekly alcohol intake (units) 5.1 ± 1.0 6.6 ± 1.8 5.6 ± 1.4 5.4 ± 1.0 HAD A 5.5 ± 0.6 6.1 ± 0.7 4.4 ± 0.6 5.8 ± 0.5 HAD D 3.0 ± 0.4 2.7 ± 0.5 2.4 ± 0.4 3.2 ± 0.4 Data are mean ± SEM. *p < 0.001 Stage +1 verses Stage +2. Table 2 Scores for Hospital Anxiety and Depression Scales (HAD A and HAD D ), psychological, somatic, vasomotor symptoms and sexual dysfunction on the Greene Climacteric Scale and sleepiness assessed by the Epworth Scale at baseline and after 6 weeks of treatment with placebo or ginkgo for subjects in Stage +1 and Stage +2 of the menopause Stage +1 Stage +2 Placebo Ginkgo Placebo Ginkgo Baseline Week 6 Baseline Week 6 Baseline Week 6 Baseline Week 6 HAD A * 5.5 ± 0.6 5.2 ± 0.7 6.2 ± 0.7 5.3 ± 0.9 4.4 ± 0.7 3.8 ± 0.6 5.8 ± 0.5 4.7 ± 0.6 HAD D 3.0 ± 0.4 2.6 ± 0.4 2.7 ± 0.5 3.0 ± 0.6 2.4 ± 0.4 2.1 ± 0.4 3.2 ± 0.4 2.4 ± 0.5 Greene Climacteric Scale Psychological symptoms*** 7.0 ± 0.8 5.5 ± 0.7 8.3 ± 1.1 7.6 ± 0.9 5.9 ± 0.9 4.4 ± 0.8 7.0 ± 0.6 5.7 ± 0.7 Somatic symptoms 2.2 ± 0.5 1.7 ± 0.3 3.6 ± 0.5 2.4 ± 0.5 2.2 ± 0.5 2.4 ± 0.6 2.8 ± 0.4 2.6 ± 0.6 Vasomotor symptoms 2.2 ± 0.4 1.8 ± 0.3 2.9 ± 0.5 2.7 ± 0.5 1.5 ± 0.4 1.5 ± 0.3 1.4 ± 0.3 1.3 ± 0.4 Sexual dysfunction*** 1.1 ± 0.2 0.8 ± 0.2 09 ± 0.2 0.9 ± 0.2 1.1 ± 0.3 0.4 ± 0.2 1.4 ± 0.2 1.2 ± 0.2 Epworth Sleepiness Scale 6.0 ± 0.6 5.5 ± 0.5 4.9 ± 0.8 5.4 ± 0.8 6.1 ± 0.6 6.1 ± 0.7 6.8 ± 0.8 6.8 ± 0.8 Data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, baseline week 6 changes in all groups. p < 0.05, Stage +1 group.

Limited cognitive benefits of treatment with Ginkgo biloba 177 Table 3 Scores for the three mood factors extracted from the Bond and Lader (1974) mood rating scale and on the Stanford Sleepiness Scale, before (Pre) and after (Post) cognitive testing at baseline and after week 6 of treatment with placebo or ginkgo, for subjects in Stage +1 or Stage +2 of the menopause Placebo Ginkgo Baseline Week 6 Baseline Week 6 Pre Post Pre Post Pre Post Pre Post Stage +1 Anxiety*** 44.8 ± 1.9 55.9 ± 1.7 48.6 ± 2.3 52.5 ± 2.2 47.1 ± 2.2 52.5 ± 2.4 47.6 ± 2.4 54.8 ± 3.9 Alertness*** 47.7 ± 1.8 37.0 ± 1.5 44.5 ± 2.5 40.1 ± 2.4 47.2 ± 2.5 40.1 ± 2.4 47.3 ± 2.3 40.6 ± 3.5 Wellbeing*** 50.8 ± 2.6 33.2 ± 2.5 49.1 ± 3.0 41.6 ± 3.2 51.2 ± 3.5 38.3 ± 4.5 50.2 ± 3.3 37.2 ± 3.4 Stanford*** 2.1 ± 0.2 2.9 ± 0.2 2.2 ± 0.2 2.7 ± 0.3 2.2 ± 0.2 2.7 ± 0.2 2.4 ± 0.2 3.0 ± 0.4 Stage +2 Anxiety*** 43.7 ± 1.9 54.9 ± 4.1 49.1 ± 3.5 52.0 ± 2.2 42.7 ± 1.7 5.7 ± 2.4 46.5 ± 2.0 54.5 ± 2.3 Alertness*** 52.8 ± 1.6 38.6 ± 2.7 44.6 ± 2.5 41.7 ± 3.1 52.4 ± 1.6 36.1 ± 1.9 48.9 ± 1.8 42.9 ± 2.0 Wellbeing*** 54.1 ± 3.4 34.0 ± 5.7 52.9 ± 3.4 36.7 ± 4.2 54.2 ± 3.4 32.1 ± 3.3 52.0 ± 3.3 39.5 ± 3.0 Stanford *** 2.1 ± 0.2 3.0 ± 0.3 2.5 ± 0.2 2.9 ± 0.3 1.8 ± 0.2 3.1 ± 0.2 2.4 ± 0.2 2.8 ± 0.2 Data are mean ± SEM. ***p < 0.001, Pre Post cognitive testing differences in all groups. Sleepiness and Visual Analogue Self-Rating Scales Ginkgo was without effect on self-ratings of sleepiness on the Stanford and Epworth Sleepiness Scales (week treatment and week treatment menopausal stage interactions, F < 2.3, NS in all cases) (Table 3). Three independent mood factors can be extracted from the Bond and Lader Mood Rating Scale (Bond and Lader, 1974). Ginkgo was without effect on these mood factors. There were no significant week treatment or week treatment menopausal stage interactions for any of the factors alertness, anxiety or well-being (F < 1.6, NS in all cases) (Table 3). Furthermore, there was no difference between the two treatment groups in response to the stress of cognitive testing (week treatment time and week treatment menopausal stage time, F < 3.4, NS in all cases). The Bodily Symptoms Scale provides a measure of somatic anxiety. There were no significant effects of Ginkgo on this factor, and the groups did not differ in their response to the test session (MANOVA, all treatment interactions F < 1.5, NS in all cases; data not shown). Cognitive tests Sustained attention Ginkgo was without effect on this task (all treatment interactions, F < 2.5, NS in all cases) (Table 4). However, there was a significant practice effect because all subjects improved on the second test occasion [week, F(1,69) = 28.6, p < 0.0001 for 1.6 s and F(1,65) = 41.9, p < 0.0001 for 1.2 s]. There was no effect of menopausal stage in this task (F < 1.2, NS in both cases). Episodic memory There were no significant effects of ginkgo on the episodic memory tests of immediate and delayed paragraph recall, DMTS or picture recall (treatment interactions, F < 3.2, NS in all cases) and there were no effects of menopausal stage (F < 2.4, NS in all cases) (Table 4). There was a significant effect of week on all of these tasks because all subjects improved regardless of treatment group [F(1,83) = 4.3, p < 0.05 for DMTS number correct; F(1,83) = 41.7, p < 0.0001 for immediate paragraph recall; F(1,83) = 19.4, p < 0.0001 for delayed paragraph recall; F(1,83) = 60.7, p < 0.0001 for picture recall]. Category generation There were no significant effects of ginkgo on the category generation task (treatment interactions, F < 1.9, NS in both cases), and there was no effect of menopausal stage (F = 0.1, NS) (Table 4). There was a significant effect of week because all subjects improved on the task regardless of treatment [F(1,83) = 13.4, p < 0.0001]. Planning There were no effects of ginkgo on the SoC task (all treatment interactions F < 2.7, NS in all cases); however, there was a significant effect of week for the subsequent thinking time [F(1,83) = 10.3, p < 0.003 for the four-move problem and F(1,83) = 9.0, p < 0.005 for the five-move problem] because all subjects were faster on the second test occasion (Table 4). There was a significant effect of menopausal stage in this task, because subjects in Stage +2 had worse performance than subjects in Stage +1 on the number of moves to complete the task for the five-move problem [F(1,83) = 4.1, p < 0.05]. Mental flexibility Only 75 subjects out of the total sample were able to reach the EDS stage of the task, and thus the results were analysed for these subjects only. Ginkgo significantly improved performance in this task, but only in the Stage +2 subjects. Thus, there were significant week treatment menopausal stage interactions for the number of trials to reach the EDS stage [F(1,71) = 4.5, p = 0.04] and the number of errors to reach the EDS stage [F(1,71) = 5.7, p = 0.02] (Fig. 1). However, the groups did not differ significantly on post-hoc tests. There were also significant main

178 Limited cognitive benefits of treatment with Ginkgo biloba Table 4 Scores on the cognitive tests at baseline and after 6 weeks of treatment with placebo or ginkgo for subjects in Stage +1 or Stage +2 of the menopause Stage + 1 Stage + 2 Placebo Ginkgo Placebo Ginkgo Baseline Week 6 Baseline Week 6 Baseline Week 6 Baseline Week 6 Picture recall (number correct)*** 9.9 ± 0.6 12.2 ± 0.6 7.6 ± 0.5 11.4 ± 0.5 9.1 ± 0.7 11.1 ± 0.6 9.2 ± 0.5 10.8 ± 0.5 Immediate paragraph recall (items recalled)*** 14.0 ± 1.1 15.4 ± 0.7 9.8 ± 0.8 12.9 ± 0.8 14.2 ± 0.8 15.9 ± 0.7 11.7 ± 0.6 14.3 ± 0.5 Delayed paragraph recall (items recalled)*** 12.7 ± 1.1 14.6 ± 0.7 8.8 ± 0.8 11.7 ± 0.9 12.8 ± 0.8 13.9 ± 1.0 11.3 ± 0.6 12.6 ± 0.7 Category generation (number of items)*** 23.4 ± 1.0 25.0 ± 1.1 23.3 ± 1.0 23.7 ± 0.7 23.5 ± 1.0 25.1 ± 0.8 22.4 ± 0.8 25.2 ± 0.9 DMTS Total number correct* 17.4 ± 0.4 17.7 ± 0.3 17.6 ± 0.4 17.7 ± 0.5 16.9 ± 0.4 17.2 ± 0.5 16.9 ± 0.4 17.6 ± 0.3 Mean correct latency 3082 ± 176 2920 ± 123 3920 ± 243 3425 ± 156 3931 ± 199 3327 ± 209 3731 ± 176 3348 ± 137 PASAT 1.6 s*** 24.5 ± 2.2 29.1 ± 2.2 24.1 ± 2.8 29.0 ± 3.3 23.2 ± 3.1 26.8 ± 3.1 23.7 ± 1.6 28.7 ± 1.8 PASAT 1.2 s*** 20.5 ± 1.5 24.6 ± 1.8 18.9 ± 2.2 23.8 ± 2.4 17.1 ± 2.7 24.7 ± 2.4 18.7 ± 1.3 21.8 ± 1.4 Stockings of Cambridge Mean subsequent thinking time(ms) Four-move problem* 1851 ± 266 1211 ± 326 2946 ± 790 1738 ± 311 2659 ± 472 1567 ± 359 2335 ± 346 1158 ± 166 Five-move problem* 632 ± 118 570 ± 128 1792 ± 377 1522 ± 398 2192 ± 541 830 ± 184 2237 ± 610 1725 ± 694 Number of moves Four-move problem 5.7 ± 0.2 5.4 ± 0.2 5.8 ± 0.2 5.6 ± 0.2 5.6 ± 0.3 5.5 ± 0.3 5.8 ± 0.1 5.5 ± 0.2 Five-move problem 6.3 ± 0.3 6.5 ± 0.2 7.2 ± 0.5 6.9 ± 0.4 8.0 ± 0.6 6.8 ± 0.4 7.5 ± 0.4 7.2 ± 0.3 Data are mean ± SEM. *p < 0.05, **p < 0.001, ***p < 0.001, baseline-week 6 change in scores for all subjects. p < 0.05, Stage +1 verses Stage +2 scores. DMTS, Delayed Matching-to-Sample; PASAT, Paced Auditory Serial Addition Task. IDED Errors Placebo Ginkgo IDED * * Trials Number of errors to criterion Change from baseline 10 5 0-5 -10 Stage +1 Stage +2 Number of trials to criterion Change from baseline 20 15 10 5 0-5 -10-15 -20 Stage +1 Stage +2 Figure 1 Mean (± SEM) change in performance from baseline to week 6, after treatment with placebo or ginkgo for subjects in Stage +1 and Stage +2 of the menopause, in the number of trials and errors to reach criterion in the Intra/Extra Dimensional Shift (IDED) task. *p < 0.05, significant week treatment menopausal stage interaction

Limited cognitive benefits of treatment with Ginkgo biloba 179 effects of menopausal stage in this task, because those in Stage +2 required more trials to reach the EDS stage [F(1,71) = 10.4, p < 0.003] and made more errors to reach this stage [F(1,71) = 10.0, p < 0.003]. Discussion This study has shown that, after 6 weeks of treatment with ginkgo, the only cognitive improvement was in mental flexibility, and this was limited to older subjects who had significantly poorer performance on this task at baseline. We utilized a fairly large group size, and positive effects of ginkgo have been detected in smaller group sizes (Hartley et al., 2003); thus, it is unlikely that a larger group size would have resulted in more significant effects, especially because there were no effects of ginkgo at marginal levels of significance. Because the test battery was identical to that used in the previous study, it is unlikely that an inappropriate selection of tests could have accounted for the results. After 1 week of treatment, we found that ginkgo improved sustained attention (PASAT) and short-term memory (DMTS), in addition to mental flexibility (Hartley et al., 2003). In comparing the present study with our previous study, there was no statistically significant difference in baseline scores on DMTS or PASAT; thus, it is unlikely that differences in performance between the two studies could have accounted for the lack of the effect of ginkgo on these tasks. However, the practice effects on DMTS and PASAT were greater in the present study compared to the previous study, which may have reduced the effects of ginkgo on these tasks. Alternatively, tolerance may develop to the effects of ginkgo with chronic treatment. This notion is supported by evidence that tolerance may develop to the effects of ginkgo on EEG recordings. Acute doses of ginkgo have been shown to elicit the EEG profile of a cognitive activator in healthy volunteers (Luthringer et al., 1995; Itil et al., 1996). However, after 12 weeks of treatment in subjects with ageassociated cognitive decline, there were no effects on the EEG profile, except for a subset of subjects who showed more impaired vigilance at baseline (Gessner et al., 1985). This comprises further evidence that the effects of ginkgo may depend on the target population and that, after chronic treatment, greater effects may be apparent where there is a greater degree of cognitive impairment. Importantly, mental flexibility was found to be improved in our previous study on the effects of ginkgo in postmenopausal women after 1 week of treatment (Hartley et al., 2003). In this previous study of subjects who were able to reach the ID shift stage, we found a significant effect of ginkgo in the latency to make correct responses, although further analysis of our data also showed significant effects on the number of stages completed, total trials-adjusted and total errors-adjusted (p < 0.04 in all cases), with the ginkgo group performing better. Thus, it appears that the effects of ginkgo in this task are quite robust because, after prolonged treatment, the improvement in mental flexibility is sustained. It is of particular interest that improvement was found on a test of frontal lobe function. Inhibitory functions of the frontal cortex are sensitive to the effects of ageing and this is thought to contribute to the decline in performance on other cognitive tasks involving memory and attention (Persad et al., 2002). Furthermore, Robbins et al. (1998) demonstrated that the IDED task was the most sensitive of the CANTAB tests to the effects of ageing, and found deterioration in performance of this task in the group aged 55 59 years. This supports our findings of deterioration on the task between groups with a mean age difference of 6 years. We also found evidence for poorer performance on the SoC task in the group aged over 55 years. Robbins et al. (1998) also found an age-related decline in this task, but only within the oldest age group (75 79 years). Although the SoC task is also a measure of frontal lobe function, the planning aspects of the task were found to load onto a different factor from the IDED task (Robbins et al., 1998). Thus, it is unsurprising that performance on this task was not improved by ginkgo. No effect of ginkgo was found in this task in our previous study of the effects of 1 week of treatment in postmenopausal women (Hartley et al., 2003). Although there has been limited investigation into the effects of ginkgo on frontal lobe tasks, working memory, which utilizes the frontal lobes, has been improved after acute (Rigney et al., 1999) and chronic (Stough et al., 2001) treatment with ginkgo. It is difficult to conclude whether the crucial difference between our Stage +1 and Stage +2 subjects was their age or their menopausal status. During Stage +1 of the menopause, hormonal levels continue to decline, whereas they have reached a steady low level in Stage +2 (Soules et al., 2001). A recent study has identified oestrogenic activities of ginkgo. The whole extract and the flavonoid glycoside components (quercetin, kaempferol and isorhamnetin) were found to competitively bind with the oestrogen receptors (ERs) in vitro and exert weakly oestrogenic activities shown by the induction of cell proliferation through a typical ERresponse pathway (Oh and Chung, 2004). The physiological significance of these effects has not yet been determined. However, if this is an important mechanism underlying the cognitive effects of ginkgo, this effect may be of greater importance in Stage +2. In this study, all subjects reported improved menopausal symptoms and anxiety ratings after the 6 weeks of treatment, regardless of treatment group, which indicates a non-specific beneficial effect of participating in this trial. We found no effects of ginkgo on mood, in agreement with our results after 1 week of treatment (Hartley et al., 2003). Beneficial effects have been found in a similar age group with respect to self-rated mental health and quality of life (Cieza et al., 2003), but these are rather different from the mood scales used in the present study. However, a study by Cockle et al. (2000) indicated a significant improvement in ratings of activities of daily living and self-mood ratings, which assessed anxiety, depression, sedation and sleep behaviour after 1 4 months of treatment with ginkgo (120 mg/day) in an elderly group of subjects (mean age 69 years). Although the reported benefits were more pronounced for the group aged under 60 years, a limitation was that the study was not placebo-controlled, and the importance of this is highlighted by the significant placebo-effect noted in the present study. A variety of mechanisms have been proposed for the effects of ginkgo and it has been suggested that the polyvalent activity of the total extract is responsible for the overall beneficial cognitive effects (DeFeudis and Drieu, 2000). However, it is likely that after

180 Limited cognitive benefits of treatment with Ginkgo biloba acute/short-term treatment, only some mechanisms are able to take effect. For example, it is possible that, in healthy subjects, ginkgo may act immediately to increase cerebral blood flow and blood glucose levels (Krieglstein et al., 1986), which are related to cognitive performance (Gur et al., 1987; Benton and Owens, 1993) but, after repeated administration, tolerance may develop to these effects. However, there is evidence from a forearm blood flow study that increased flow persists for at least 6 weeks of treatment in healthy subjects (Mehlsen et al., 2002). Animal studies have shown ginkgo to increase the release of neurotransmitters such as acetylcholine (DeFeudis, 1991) and noradrenaline (Brunello et al., 1985) in the rat brain after acute or short-term treatment, although there is a down-regulation of the associated receptors after longer periods of 21 27 days. This may represent an adaptive response of the brain underlying the development of tolerance to cognitive benefits after longer periods of treatment with ginkgo in healthy populations. Ginkgo has been shown to have cognitive benefits over treatment periods of up to 52 weeks in patients suffering from Alzheimer s disease, vascular dementia and cerebral insufficiency syndrome (Taillandier et al., 1986; Le Bars et al., 1997), but it is possible that there are different mechanisms underlying ginkgo s effects in patient versus healthy populations. For example, the neuroprotective effects of ginkgo, such as prevention of oxidative mitochondrial damage (Sastre et al., 1998; Spinnewyn et al., 1995) and attenuation of Aß-mediated toxicity (Bastianetto et al., 2000), are more pertinent to patient populations where oxidative damage is a major contributor to the clinical symptoms of cognitive decline. Such mechanisms may be of limited use in non-clinical populations. Our finding that improvement in mental flexibility was only sustained in the older and more cognitively impaired agegroup may indicate that there is a threshold of cognitive ability at which ginkgo is effective after chronic treatment periods. In summary, the range of benefits from ginkgo appears to be reduced after 6 weeks of treatment, although the improved mental flexibility is maintained in an older group of subjects. If a benefit is retained after longer periods of treatment, this could prove to be of very real value, particularly if the effect is more marked in older and/or more cognitively impaired individuals. Acknowledgements This work was funded by a grant from the Dunhill Medical Trust. We would like to thank Dr R. Middleton of Lichtwer Pharma UK for the generous gift of Ginkyo and matched placebo tablets and for help in recruiting volunteers. 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