Effects of l-Theanine on Cognitive Function in Middle-Aged and Older Subjects: A Randomized Placebo-Controlled Study

Abstract

L-theanine (γ-glutamylethylamide), an amino acid in green tea, has been shown to affect brain functions by relieving stress disorders, improving mood, and maintaining normal sleep. However, the cognitive functions for which theanine is effective are unclear. This study aimed to clarify which cognitive functions are positively affected by the intake of L-theanine. A double-blind, randomized, placebo-controlled study was conducted. The subjects were Japanese men and women aged 50–69 years. Mini-Mental State Examination-Japanese version score was 24 or higher. Cognitrax was used as a test battery for cognitive function. Evaluations were performed before the intervention, after a single dose of L-theanine, and after 12 weeks of regular intake. The single dose of L-theanine reduced the reaction time to attention tasks (Stroop test, Part 1), and it increased the number of correct answers and decreased the number of omission errors in working memory tasks (4-Part continuous performance test, Part 4). In conclusion, our study indicated that L-theanine may contribute to improving attention, thus enhancing working memory and executive functions.

Keywords

attention; brain function; Cognitrax; executive function; green tea amino acid; working memory; Cistanche benefits.

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Introduction

L -theanine is a nonproteinogenic amino acid contained in green tea and mushrooms. Among the different tea varieties in Japan, gyokuro and matcha contain more L-theanine than sencha. L-theanine affects brain functions. Studies in mice indicate that it improves behavior under stress conditions, suppresses deterioration of learning ability under social stress, and improves memory impairment.

The epidemiologic Nakajima and Tsurugaya studies in Japan indicated that green tea may improve age-related cognitive impairment. In addition, the Hisayama study warned about a recent increase in the prevalence of Alzheimer’s disease (AD). Prevention of AD is an important problem worldwide, and it is necessary to seek a solution.

Green tea contains several substances that affect cognitive function, including caffeine, L-theanine, and catechin. Caffeine transiently improves performance after intake and has a substantial benefit on cognitive function. Our previous study indicated that the regular intake of matcha may improve attention. However, in that study, subjects did not consume matcha on the day of the test, suggesting that the improvement in attendance was not a transient effect of caffeine but involved other components of the tea. A further implication would be that the regular intake of tea is also a factor responsible for the observed improvement in attention.

L-theanine has been previously reported to have a neuroprotective effect. Because it suppresses delayed neuronal cell death in the hippocampal Cornu Ammonis area after transient ischemia, reduces excitotoxicity by suppressing the extracellular release of glutamate through inhibition of the glutamine transporter, and promotes neurogenesis, L-theanine can potentially reduce cognitive impairment. However, there are many uncertainties about the effects of L-theanine on human cognitive function.

The purpose of this study was to clarify whether the intake of L-theanine, which has a neuroprotective effect, affects the cognitive processes of attention, working memory, and executive function. In addition, to investigate whether the single response was associated with cognitive function after regular ingestion, we compared the results of single-dose and regular ingestion.

Materials and methods

The study was conducted at the Tokyo Skytree Station Medical Clinic (Tokyo, Japan) and was approved by the Research Ethics Committee of Nihonbashi Egawa Clinic (Tokyo, Japan; Approval No.: food-18071704). The study was conducted by the Declaration of Helsinki from August 8, 2018, to December 6, 2018. The study was registered with University Hospital Medical Information Network (Tokyo, Japan).

1. Test food

L-theanine (trade name: Suntheanine; Taiyo Kagaku Co., Ltd., Mie, Japan) was used as the test food. Suntheanine contains ‡98% L-theanine and was encapsulated into hard No. 1 porcine gelatin capsules and used for the test. A placebo was dispensed into the same type of capsules as L-theanine, and corn starch was used as the excipient in both the theanine and placebo capsules. Each theanine capsule contained 100.6 mg of L-theanine. The test food was manufactured at Sunsho Pharmaceutical Co., Ltd. (Shizuoka, Japan).

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2. Subjects

Sixty-nine Japanese men and women certified as healthy by a physician based on hematological and biochemical blood test results and with self-assessed declined cognitive function were initially enrolled.

Of these, subjects with a Mini-Mental State ExaminationJapanese version (MMSE-J) score of ‡24 were included, while those who had food allergies or were taking medication or undergoing treatment were excluded.

The subjects participated in the study on their initiative after receiving a full explanation of the study.

3. Study design

A double-blind, randomized placebo-controlled parallel group study was conducted. The primary endpoints were the results of MMSE-J and Cognitrax, and the secondary endpoints were blood levels of amyloid b 1–40 [Ab (1–40)], Ab 1–42 [Ab (1–42)], secreted form of amyloid-b precursor protein a (sAPPa), amyloid-b precursor protein 770 (APP770), and brain-derived neurotrophic factor (BDNF). A computer-generated stratified randomized schema (Huma R&D Corp., Tokyo, Japan; Contract Research Organization) was used to assign the subjects to either the placebo or the L-theanine group, with matching based on age, sex, and MMSE-J score. The study flow diagram is shown in Figure 1.

Subjects took one capsule per day of placebo or L-theanine for 12 weeks. They took the test food after breakfast. When they did not eat breakfast, they took the test food in the morning. Subjects were required to enter into the web input system (Huma R&D Corp.), using a personal computer (PC), whether they had taken the test food. During the test period, the subjects were free to consume polyphenol-containing beverages (green tea, black tea, oolong tea, etc.), but subjects were restricted from taking any healthy foods, supplements, or medications that might affect cognitive function.

4. Evaluation items

The evaluation items are shown in Table 1. On the day of the single-dose study, the Cognitrax test was started ~50 min after capsule intake. Except for taking the capsule before undergoing the tests, the tests were performed in the same order at week 12. Hematologic tests and biochemical blood parameter measurements were conducted at baseline and 12 weeks as a safety evaluation at SRL, Inc. (Tokyo, Japan).

Mini-Mental State Examination-Japanese version. MMSE-J (Nihon Bunka Kagakusha Co., Ltd., Tokyo, Japan) is a Japanese version of MMSE, and its validity and test-retest reliability have been confirmed in Japan. The test consists of 11 items: time orientation, location orientation, memorization, attention and calculation, recall, naming, repetition, three-stage command, reading, writing, and copying, and it is evaluated with the total score. Two tests of attention and calculation were used: the backward spelling task and the serial sevens task, respectively. The score for the backward spelling task was used for the allocation of subjects into the placebo or theanine group.

Cognitrax test. Cognitrax24 is a test to evaluate cognitive function, developed by the U.S. company CNS Vital Signs (Morrisville, NC, USA). It comprises 10 test items with accompanying instructions. It measures both reaction time and several responses. Reaction time is measured in milliseconds. The test items were ordered as follows: verbal memory (VBM), visual memory (VIM), finger tapping test (FTT), symbol digit coding (SDC), Stroop test (ST), shifting attention test (SAT), continuous performance test (CPT), perception of emotions test (POET), nonverbal reasoning test (NVRT), four-part CPT (FPCPT), VBM, and VIM. These items assess various cognitive functions—VBM and VIM assess memory; ST, SAT, CPT, and FPCPT Parts 1 and 2 assess attention; POET assesses facial expression recognition; FPCPT Parts 3 and 4 assess working memory; SDC and NVRT assess visual information processing; and FTT assesses motor function.

The first VBM and VIM performed are indicative of immediate memory, while the last is indicative of delayed memory. There was ~50 min between the first-performed VBM and VIM and the last-performed VBM and VIM. Details of the test are shown in Table 2.

Biomarkers related to dementia. On the day of the test, the subjects were restricted from eating for 6 h before arriving at the hospital until the completion of the test. Serum blood collection tubes were used to estimate BDNF levels, while ethylenediamine tetra-acetate disodium tubes were used for Ab (1–40), Ab (1–42), sAPPa, and APP770 measurements. The blood was centrifuged at 3000 rpm (Kokusan Co., Ltd., Tokyo, Japan, H-19Ra). The measurement was performed using kits with the following dilutions: the blood sample for Ab (1–40) was diluted 20 times with human Ab (1–40) Full Length (FL) Assay Kit-IBL; the blood sample for Ab (1–42) was diluted 4 times with human Ab (1–42) (FL) Assay Kit-IBL; the blood sample for sAPPa was diluted four times with sAPPa (highly sensitive) Assay Kit-IBL; the blood sample for APP770 was diluted 50 times with human APP770 Assay Kit-IBL; and the blood sample for BDNF was diluted 20 times with human BDNF Enzyme-linked Immunosorbent Assay Kit (Quantikine-R&D Systems). Any measured value below the kit range was excluded due to inaccuracy. The measurement was carried out by Skylight Biotech, Inc. (Akita, Japan). The results are shown in Table 3.

5. Statistical analysis

The values are presented as mean – standard deviation. Normality was tested using the Shapiro–Wilk test. The unpaired t-test or Mann–Whitney U test was used to calculate P values, with Bonferroni correction (P < .05/3 = 0.017). This was performed at baseline, single-dose test, and 12 weeks. Data were analyzed using SAS 9.4 (SAS Institute, Inc., Cary, NC, USA).

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Discussion

This study evaluated the effects of L-theanine on cognitive function using the Cognitrax test performed on a PC. We investigated whether the regular intake of theanine for 12 weeks could improve attentional function and which cognitive functions a single dose of theanine would affect.

A cup of green tea contains about 25 mg of theanine. Kuriyama et al. reported that people who drink green tea have less cognitive dysfunction. In the previous report, the amount of theanine that led to an improvement in attentional function was 50.3 mg. In this study, the dose of theanine was set at 100.6 mg to clearly show the effect of theanine alone. A single dose of L-theanine reduced reaction time in the attention task and increased correct answers and decreased the number of omission errors in the working memory task. This suggests that L-theanine may improve working memory and executive function based on the improvement in attention.

It is interesting that caffeine, which acts as a stimulant for neural activity, and L-theanine, which acts as a depressant, are both contained in green tea. Several studies have reported synergistic effects of caffeine and L-theanine on cognition and mood, as well as on tasks related to attentional function. Our results suggest that L-theanine by itself improves working memory. It remains possible that the improvement in attention, as described previously, may have contributed to this effect. In this study, after 12 weeks of regular ingestion, theanine did not affect the processes related to attentional function as previously reported, even though our subjects had ages and MMSE scores similar to those of subjects in previous studies. The difference in this effect may have been due to the combined effect of caffeine and theanine or catechin and theanine, but details of such interactions are unknown, and further investigation is required.

Studies that explored brain activity have previously reported that L-theanine intake increases a-wave activity. A study by Gomez-Ramirez et al. found that subjects who ingested 250 mg of L-theanine had increased a-wave activity for the attention task to be performed. Furthermore, a study that used a visuospatial task also showed that the intake of 250 mg of L-theanine contributed to sustained attention. Although increased a-wave activity does not simply indicate wakefulness in the brain, L-theanine may affect selective or sustained attentional function. In this study, a significant decrease in reaction time was observed for ST (Part 1), which indicates sustained attention and provides further support for the effects of L-theanine on attentional function. It will therefore be necessary to use neurophysiological techniques to study how the regular intake of theanine affects nervous system activity.

According to a study by Baddeley, working memory is composed of three factors: a phonological loop for language information processing, a visuospatial sketch pad for visuospatial information processing, an episodic buffer, and a central executive system that integrates the other three parts. The central executive system is considered to be related to attention control. The effects observed in the two back tasks (FPCPT, Part 4) are the results of L-theanine contributing to the improvement of working memory through the distribution of attention resources and movement of the focus of attention, that is, efficient shifting. Based on this observation, it is also necessary to examine brain activity during task execution using functional magnetic resonance imaging.

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Because attention and working memory are closely related, it is difficult to consider them separately. It is, therefore, necessary to examine, in more detail, attentional function and working memory, which were found to be improved by L-theanine in this study.

The limitations of this study involve the subject age and cognitive function status. This study was conducted on middle-aged and older subjects who were aware of a decline in their cognitive function. Previous studies tested elderly people, including those who had mild cognitive impairment or vascular dementia, to verify whether green tea powder could improve cognitive function. In the future, a comparative study on the preventive or restorative effects of L-theanine in younger adults, the aged, and patients with mild cognitive impairment must also be considered. We also could not explain why regular administration of L-theanine was necessary, as is accepted for epidemiological studies. Future studies are therefore required to investigate the differential effects of a single dose and daily intake of L-theanine on brain structure and cognitive function.

How does Cistanche extract improve memory for the older?

Cistanche extract, a traditional Chinese medicine, has been found to have potential benefits in improving memory for older individuals. Many studies have shown that Cistanche extract can enhance cognitive function and improve memory performance.

One possible mechanism by which Cistanche extract helps improve memory is through its ability to increase blood flow to the brain, promoting the delivery of essential nutrients and oxygen to brain cells. Additionally, Cistanche extract contains active ingredients that can reduce oxidative stress and inflammation, which are known to contribute to age-related cognitive decline.

Moreover, Cistanche extract has been demonstrated to have a positive effect on the production of nerve growth factors, proteins that support the growth and survival of nerve cells in the brain.

In conclusion, Cistanche extract shows promising potential as a natural solution for improving memory in older individuals. Further research is needed to fully understand how this traditional Chinese medicine works and to determine the most effective dosages and application methods. However, Cistanche extract represents a safe and non-invasive way to support healthy cognitive function in aging adults.

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Yoshitake Baba,1 Shun Inagaki,1 Sae Nakagawa,1 Toshiyuki Kaneko,2 Makoto Kobayashi,1 and Takanobu Takihara1

1 Central Research Institute, ITO EN, LTD., Shizuoka, Japan.

2 Tokyo Skytree Station Medical Clinic, Tokyo, Japan.