Study participants
The study was conducted with the approval of the University of Miami Institutional Review Board for human subjects research. Each subject signed informed consent and HIPAA forms prior to study entry. Potential participants (n = 144) were identified through referrals from offices at the University of Miami Miller School of Medicine and community centers in Miami-Dade, Broward, and Palm Beach counties during May 2010 to December 2011. Out of 121 eligible participants, 97 were enrolled (Ginkgo Synergy® plus Choline, n = 33; OPC Synergy® plus Catalyn®, n = 31; and placebo, n = 33). Please see Figure 1 for the CONSORT flowchart.
Study design
Participants were randomized into a three-group, parallel, placebo-controlled clinical trial to assess the efficacy of two dietary supplement formulae on cognitive and immune functioning in healthy older adults.
Inclusion and exclusion criteria
Inclusion criteria were: (a) 60+ years of age; (b) English speaking; (c) not living in a nursing facility; (d) no use of dietary supplements for cognitive functioning two weeks before enrolling and during the intervention period; and (e) a Mini-Mental State Exam (MMSE) score ≥23. Exclusion criteria were: (a) AD or related disorders; (b) schizophrenia, psychotic disorders, bipolar, major depression with psychotic features, or delirium; (c) bleeding disorders; (d) aphasia or sensory, motor, or visual disturbances that would have interfered with testing; (e) cancer, cardiovascular, pulmonary, renal, thyroid, hepatic, gastrointestinal diseases, or insulin-dependent diabetes; (f) current cigarette smoking or alcohol or substance abuse/dependence; (g) 3+ major medical or psychiatric hospitalizations in the past year; (h) a T score >70 on the Global Severity Index of the Brief Symptoms Inventory (BSI); (i) a score ≥29 on the Beck Depression Inventory-II (BDI); (j) prescription or OTC sympathomimetic amines and antihistamines within 2 days of an assessment visit; (k) cognition-enhancing drugs; or (l) Coumadin, tricyclic antidepressants, antipsychotics, or anticonvulsants.
Screening
Eligible participants completed the Short Portable Mental Status Questionnaire (SPMSQ) and the Wechsler Memory Scale III Story A (WMS-III-A). Subjects were allowed only two errors on the SPMSQ. If they scored <6 points on story A for the WMS-III-A, they were given a second opportunity and had to score a 4+ on story B. If all pre-screening criteria were achieved, subjects were scheduled for the additional screening (MMSE, BSI, and BDI) and the baseline assessment. Mental status was assessed with the MMSE, providing a rapid screen of orientation, registration, attention and calculation, recall, and language domains. As psychological distress and depression may impair cognitive function, and thus lead to poor performance on outcome measures, participants were also evaluated using the BSI and BDI.
Intervention and randomization
After screening and baseline, participants were randomly assigned to one of three conditions: (a) Ginkgo Synergy® (2 capsules/day providing 120 mg/day Ginkgo biloba leaf, 80 mg/day Ginkgo biloba whole extract, 40 mg/day grape seed extract, Gotu kola leaf (Centella asiatica), dried buckwheat leaf juice, buckwheat seed, and soybean lecithin powder) plus Choline (4 tablets/day providing 700 mg/day), (b) OPC Synergy® (2 capsules/day providing 100 mg/day grape seed extract, 50 mg/day green tea extract (60% catechins), 50 mg/day bilberry fruit (25% anthocyanins), dried buckwheat leaf and juice, green tea leaf powder, and dried carrot root) plus Catalyn® (4 tablets/day providing 312 IU/day vitamin D, 1,600 IU/day vitamin A, 5.3 mg/day vitamin C, 0.3 mg/day thiamine, 0.3 mg/day riboflavin, 1.3 mg/day vitamin B6, defatted wheat germ, carrot (root), calcium lactate, nutritional yeast, bovine adrenal, bovine liver, magnesium citrate, bovine spleen, ovine spleen, bovine kidney, dried pea (vine) juice, dried alfalfa (whole plant) juice, mushroom, oat flour, soybean lecithin, and rice bran), or (c) placebo (cellulose, lactose, and beet powder) provided by the manufacturer (Standard Process, Palmyra, WI). The table of random permutations was created by one of the authors (JEL). It was created in blocks of 10 subjects at a time, and study coordinators assigned participants to an intervention arm in sequential fashion. All subjects and investigators were blind to treatment condition and remained blinded until after data analysis; only a staff member at Standard Process knew the assignment.
Outcomes and assessments
All participants completed a basic sociodemographics and medical history questionnaire and reported their list of medications at baseline.
Cognitive measures
The neuropsychological assessment targeted executive functioning, memory, and psychomotor speed and was administered at baseline and 3 and 6 months follow-up. Measures included the MMSE, the Stroop Color and Word Test (SCWT), the Trail Making Test Parts A and B (TMT-A, TMT-B), the Controlled Oral Word Association test (COWA), the Digit Symbol subtest of the Wechsler Adult Intelligence Scale, Third Edition (WAIS-III), and the Hopkins Verbal Learning Test-Revised (HVLT-R).
The SCWT assesses response inhibition [13], the effects of perceptual interference [14], concentration, and speed of processing abilities, involving three distinct tasks. TMT-A and TMT-B assess a person’s abilities to sequence and shift perceptual sets, concentration and vigilance, and visuomotor scanning and tracking speed [15], which reflect executive control functioning [13]. These instruments are exceptional measures of general brain functioning [16]. The COWA assesses verbal fluency and is sensitive to cerebral dysfunction [17]. The WAIS-III Digit Symbol subtest measures perceptual speed, during which the subject must quickly copy symbols that are linked with numbers [18]. The HVLT-R assesses verbal and visuospatial recall and recognition memory, containing two equivalent forms to allow repeat testing without correction for practice effects [19].
The administration of the battery required 60-90 minutes. Participants were provided at least one break during the testing session and were closely monitored for fatigue or emotional distress. No participants suffered from excessive fatigue to the point of needing to complete the assessment on a later date.
Immune function markers
Venous blood was obtained at baseline and 6 months. Blood samples were collected in EDTA tubes and frozen within 2 hours of collection. Samples were sent to a contract lab (Radix Biosolutions, Georgetown, TX) for analysis. A commercially-available magnetic bead-based Milliplex® MAG kit (Millipore Corporation MA) was used to analyze all samples for: interleukin (IL)-2, IL-4, IL-6, IL-8, IL-10, IL-1α, IL-1β, interferon (IFN)-γ, tumor necrosis factor (TNF)-α, monocyte chemotactic protein (MCP)-1, vascular endothelial growth factor (VEGF), and epidermal growth factor (EGF). The kit included two quality control samples, expected concentrations for each sample, and all necessary reagents. The standard curve and quality controls were prepared fresh each day. Both quality control samples were run in duplicate before and after each set of samples on each microtiter plate to produce 2 concentration values per quality control sample in each assay. Assays were set up sequentially, so all samples could be analyzed on the same day, utilizing five separate assay kits. Each sample was run neat in duplicate wells and incubated at 4°C overnight. Sample reruns were completed with the original samples and involved an additional freeze/thaw cycle. The concentration of each cytokine or growth factor in each plasma specimen was calculated from the standard curve and reported in pg/ml.
Intervention protocol
Supplements were given to each participant at the baseline and 3-month follow-up assessments to promote greater compliance. Subjects were not advised to modify eating or physical activity habits or prescription medication use. In addition, they were instructed not to consume other dietary supplements containing Ginkgo biloba, vitamin B complex nutrients, vitamin E, or any other cognitive-enhancing nutritional supplement. Subjects listed all dietary supplements taken on the health history form, and products were reviewed to ensure none of these nutrients was consumed during the course of the trial. Regardless of study arm assignment, subjects consumed 3 tablets 2 times per day with breakfast and dinner, i.e., 6 tablets per day total. Each subject was compensated $35 per assessment at baseline and 3 and 6 months follow-up.
Statistical analysis
Data were analyzed using SPSS 19 (IBM Inc., Chicago, IL) for Windows. Frequency and descriptive statistics were calculated on all variables. Analysis of variance and chi square were utilized to determine the presence of differences in background contextual variables by study arm assignment. We utilized linear mixed modeling (LMM) to assess the fixed effect of time by randomization on changes in our outcome variables from baseline to 6 months follow-up. If the type III test of the fixed effect of time by randomization was significant, then we used pairwise comparisons to determine the unique differences in effects over time by study arm between baseline and follow-up at 3 and 6 months for the cognitive assessment and between baseline and 6 months for the physiological variables. LMM with heterogeneous compound symmetry covariance allowed us to account for subject attrition, inter-correlated responses between time points, and non-constant variability. Based on the results that Mix and Crews found in response to a 6-week placebo-controlled trial of Ginkgo biloba on the SCWT color-naming task [7], we estimated an effect size of 5.25 units on the task, which gave us power of roughly 80% with 31 subjects in each group. The criterion for statistical significance was α = 0.05.
