Effect of Chlorogenic Acid Intake on Cognitive Function in the Elderly: A Pilot Study

Objective To evaluate the effect of chlorogenic acids (CGAs) intake on cognitive function. Methods In this pilot study, the Cogstate and CNS Vital Signs test batteries were used to evaluate cognitive function in 8 healthy elderly men and women complaining of subjective memory loss after a 6-month intake of a test beverage containing 330 mg of CGAs just before bedtime. Results After a 6-month CGA intake period, significant improvement was observed in the One Back Test of the Cogstate, the Shifting Attention Test, and Finger Tapping Test as well as in the composite memory, verbal memory, complex attention, cognitive flexibility, executive function, and motor speed domains of the CNS Vital Signs test battery. Conclusion A 6-month intake of CGAs may improve attentional, executive, and memory functions in the elderly with complaints of subjective memory loss.


Introduction
Aging is a biological process that occurs in all life forms and is accompanied by adverse effects on physical and cognitive functions in humans. The incidence of dementia, a disease strongly associated with cognitive impairment, is on the rise globally. Alzheimer's Disease International, a worldwide federation of Alzheimer's associations, estimates that the number of patients with dementia is expected to double every 20 years [1]. Early prevention of dementia is critical because no definitive therapy has been established. One study reported an association between lifestyle factors and the onset of dementia [2]. Other studies have proposed that certain foods, a key lifestyle factor, contain nutrients that prevent dementia [3,4].
High intake of fruits, vegetables, fish, nuts, and legumes and low intake of meat, high fat dairy, and sweets have been shown to delay aging-related cognitive impairment and reduce the risk of Alzheimer's disease (AD) [5]. The mechanism underlying the amelioration of cognitive impairment and dementia via fruits and vegetable intake is likely mediated by antioxidants and antioxidant vitamins [4]. In addition, the intake of n-3 polyunsaturated fatty acids (PUFAs), which are abundant in fish, has been reported to reduce the incidence of cognitive impairment and dementia [3]. Docosahexaenoic acid was found to reduce amyloid (A ) deposition and to exert neuroprotective and anti-inflammatory effects at synapses in a mouse model of AD [6]. Additionally, a crosssectional study involving dementia-free participants in the Framingham Study demonstrated that middle-aged and elderly participants who had a low red blood cell concentration of n-3 PUFAs also had small brain volumes and low cognitive function [7]. Studies have also reported an association between consumption of large amounts of green tea or coffee and reduced risk of dementia [8,9].
Studies using mice and cultured neurons have shown that CGAs protect neurons and suppress the aggregation of A through antioxidative effects [12,13]. Furthermore, caffeic acid, coumaric acid, ferulic acid, and sinapic acid, all of which are metabolites of CGAs, have been shown to improve cognitive function [13][14][15]. These findings suggest that CGA intake contributes to improved cognitive function, but the mechanism by which CGA intake affects cognitive function in humans is currently unknown.
In this study, we hypothesized that CGAs improve human cognitive function, especially in the elderly, and sought to elucidate the association between CGAs and cognitive function from the perspective of preventing dementia, the incidence of which increases with aging in this population. Therefore, in this study, we enrolled community-dwelling elderly individuals to examine the effects of a 6-month intake of CGAs on cognitive function and the association with biological data.

Participants.
For recruitment of the participants, an explanatory meeting for this study was held at a regional community center in Yonezawa city, Yamagata, Japan, and those who voluntarily agree to participate in the study were checked for eligibility. Participants' recruitment was carried out by distribution of public relations to local residents and 42 people participated in explanatory meeting at the community center. One of the authors (K. M.) provided for participants in the explanatory meeting a 30-minute lecture on nutrition and prevention of cognitive function decline. The lecture presented previous studies which had examined the effects of CGAs intake on endothelial function [16] and body fat [17]. A beverage with the same components had been used, although the amount consumed was different from the amount in this study. Also presented was the possibility of improvement in cognitive function, as demonstrated by animal experiment [12,15]. After the lecture was over, we also explained the content of the experiment to be conducted in this study. In the explanation of the experiment content, the participants in the explanatory meeting were informed that it was uncertain whether all applicants would be able to become participants in the experiment, even if they were willing. After explaining the content of the experiment, written informed consent for participation was obtained, to apply for becoming subjects in the experiment. 28 participants agreed to participate in this study. Exclusion criteria were physical or mental disorder, visual impairment, and movement-related pain. In addition, for screening, participants completed Kihon Checklist, a self-administered questionnaire consisting of 25 questions in seven categories: instrumental activities of daily living, physical strength, nutritional status, oral function, cognitive function, and depression risk [18]. And those who checked at least one question in the cognitive function category ("Do others point out your forgetfulness or tell you 'You always ask the same thing'?," "When you want to make a call, do you usually search for the telephone number and call on your own?," or "Do you sometimes not know what the date is?") were considered eligible for enrollment. Finally, 8 participants (2 men, 6 women) were enrolled in this study and those who completed the 6-month trials were included in the analysis.

Study Design. This study was approved by the Ethics
Committee of Yonezawa University of Nutrition Sciences (Approval Number 26-3, May 28, 2014) and was conducted in accordance with the Declaration of Helsinki. In this singlearm study, participants ingested one 100 mL bottle of test beverage containing 330 mg of CGAs daily before bedtime during a 6-month period between August 2014 and May 2015. A 1month supply of test beverages was delivered to the participants' homes every month by the research staff. A diary for recording the details of test beverage consumption and daily activities was collected for evaluation every 2 months, and neurocognitive tests were performed before and after the nutritional intervention period.  [21]. All scores are age-adjusted and standardized by setting the mean score to 100 and the standard deviation (SD) to 15. High scores indicate superior neurocognitive function.

Blood Parameters.
Blood was collected early in the morning during fasting. Measurements of cortisol, dehydroepiandrosterone sulfate (DHEA-S), brain-derived neurotrophic factor (BDNF), and A 40 and A 42 levels were performed by LSI Medience Corp. (Tokyo, Japan) and Cosmo Bio Co., Ltd. (Tokyo, Japan) according to conventional methods.

Statistical
Analysis. Measurement data are expressed as mean and standard deviation (SD). Data obtained before and after the nutritional intervention were compared using the paired -test, and effect size was calculated using Cohen's ( ). In addition, parameters showing a significant difference after intervention were further analyzed to quantify the change and subjected to Pearson correlation analysis. Statistical analysis was performed using SPSS for Windows (version 22.0J; Tokyo, Japan), and significance was set at < 0.05.   Table 2 shows scores from the tests conducted before and after a 6month nutritional intervention. In OBT, the total number of errors decreased significantly from 4.0 ± 4.3 preintervention to 1.1 ± 1.9 postintervention, while the accuracy increased significantly from 89.6 ± 10.2% to 96.7 ± 5.2%, respectively.

CNS Vital Signs
(1) Neurocognitive Tests. Table 3 shows the results of another computerized neurocognitive test battery, CNS Vital Signs, conducted before and after the 6-month intervention period. The number of right taps during the FTT increased significantly from 47.6 ± 2.3 preintervention to 50.3 ± 3.7 postintervention, but the number of left taps tended to increase from 44.8 ± 5.2 to 46.9 ± 4.2, respectively. There  (2) Neurocognitive Domains.  Table 5 shows the changes in blood parameters. After the intervention, there was a significant increase in serum DHEA-S levels and a significant decrease in plasma A (1-42) and A 42/A 40 levels.

Correlation between Cognitive Function and Blood Parameters.
A correlation analysis conducted between biological data and neurocognitive test items showed a significant change due to the intervention: a significant negative correlation between verbal memory and plasma A (1-42) ( = −0.742, = 0.035) and plasma A 42/A 40 ( = −0.906, = 0.002) levels ( Figure 1).

Discussion
To our knowledge, this is the first study to show that a 6-month intake of CGAs improves cognitive function in the elderly. The CGA-induced improvement in cognitive function was reflected in the computerized neurocognitive tests OBT, SAT, and FTT. Also, a significant improvement in 6 Evidence-Based Complementary and Alternative Medicine  showed an improvement in the OBT error response and accuracy, in the SAT correct and error responses, and in the FTT tap average. Previous studies have shown an association between OBT response and working memory [22] and between SAT/FTT performance and attentional and executive functions. Furthermore, cognitive function is associated with working memory and attentional and executive functions in the prefrontal region [23]. Therefore, CGAs are presumed to improve brain function in the prefrontal region.
Previous studies have demonstrated the antioxidant effects of CGAs [24] and the improvement in cognitive function mediated by CGAs through their antioxidative and neuroprotective effects. The antioxidant effects are considered one of the most important factors to prevent the age-related neurodegenerative diseases as recent studies show that the oxidative stress links to A aggregation and following dementia due to AD [25]. In a study using mice with scopolamineinduced amnesia, administration of 5-CQA, the main component of CGAs, inhibited acetylcholinesterase activity in the prefrontal cortex [12]. Whether the results of the animal study can be applied to humans is unknown; nevertheless, the findings of the present study suggest that CGAs improve brain function in the prefrontal region in humans.
The metabolism of CGAs in the body generates gallic acid, caffeic acid, coumaric acid, ferulic acid, and sinapic acid [14], all of which have been shown to suppress the breakdown of the amyloid-precursor protein, which is pathologically related to the A (1-42) protein, lipid peroxidation, and neurite extension of hippocampal neurons [13,15]. These functions are likely involved in the improved memory following CGA intake.
We also analyzed the correlation between neurocognitive test scores and biological data and confirmed a significant decrease in plasma A 42 and A 42/A 40 levels and a significant increase in DHEA-S levels after a 6-month CGA intake period. Specifically, the change in plasma A 42 or A 42/A 40 levels was negatively correlated with an improvement in verbal memory (Figure 1), indicating that verbal memory improves as the level of A 42 or A 42/A 40 decreases. In previous cohort studies, the level of A 42 increased before the onset of AD or in the early stage of AD but decreased gradually thereafter [26][27][28]. Thus, the level of plasma A is a predictor of AD and cognitive impairment. However, no study has investigated the effect of food or dietary intervention using plasma A as an indicator. In a cross-sectional study of healthy elderly individuals aged ≥65 years with normal cognitive function, Gu et al. investigated the association between nutrient intake and plasma A levels and reported that higher dietary intake of -3 PUFAs was associated with lower plasma levels of A 42 [29]. In another study of healthy elderly individuals aged ≥65 years, exercise intervention led to an improvement in cognitive function and a significant reduction in plasma A 42/A 40 levels. However, no correlation with improvement in cognitive function and no degree of change in A 42/A 40 levels was observed [30]. These findings suggest that although it is too soon to draw conclusions about plasma A 42/A 40 levels, the correlation between the improvement in verbal memory and the change in A 42/A 40 levels in this study warrants further investigation. In this study, attentional, executive, and memory functions improved significantly after a 6-month CGA intake period in community-dwelling elderly individuals with complaints of subjective memory loss. Previous studies have shown that CGAs improve blood pressure and vascular endothelial functions, both of which are associated with the onset of dementia [17,31,32]. Because hypertension in middle age is a risk factor for dementia and cognitive impairment in old age [33], continuous consumption of CGAs may delay the onset of dementia.
This study has limitations as follows. First, this was a single-arm study with no control. Therefore, the beneficial effect observed in this study might have been due, at least in part, to placebo effects. The influences of the participants' anticipation and activities other than CGA intake cannot completely be ruled out although there seemed to be no great changes in their activities during the 6-month trial according to the diary they submitted. Second, multiple cognitive functions were carefully investigated in this study, but because of the small number of participants, further study with a larger number of participants and a control group is needed to verify the effect of CGA intake on cognitive function.

Conclusion
The findings of this study suggest that CGAs may improve cognitive function, especially attentional, executive, and memory functions in the elderly.