Nutritional Foods Division, Taiyo Kagaku Co., Ltd.,
1-3 Takaramachi, Yokkaichi, Mie 510, Japan
*Aichisyukutoku University,9, Aza-katahira, Oaza-nagakute, Nagakute-cho, Aichi-gun, Aichi 480-11
L-Theanine is an amino acid found in the leaf of green tea and its infusion, and is known to control excitement caused by caffeine. It is also known that the oral administration of L-theanine to rats results in a decrease of serotonin and an increase of catecholamine in the brain. L-Theanine has been confirmed to be safe in animal experiments.
We found that the oral intake of L-theanine caused a feeling of relaxation among the human volunteers examined. These observations led us to conduct experiments on the effects of the administration of L-theanine on brain waves. Eight female university students were selected as volunteers. Four of them were ranked as Grade I (the highest anxiety) and the remaining four as Grade V (lowest anxiety) in an investigation done by the manifest anxiety scale method. An oral administration of 200 mg of L-theanine dissolved in 100 ml of water resulted in the generation of a -electric waves in the occipital and parietal regions of the subjects brains. The emission intensity of a -brain waves (integrated as a function of investigation times and area) was significantly greater in the group of Grade I than that of Grade V.
These results indicate the possibility for L-theanine to be applied to foods and beverages as a new functional food ingredient for its relaxation effect.
Green tea has been widely consumed as a beverage since it was brought to Japan from China. Recently, a variety of the effects of green tea have been scientifically evaluated. The benefits of green tea has been noticed not only in Japan but all over the world.
The main effective components within green tea are polyphenols, which have many physiological effects including anti dental caries (1-3), deodorization (4,5), improving intestinal microflora (6), prevention of colon cancer (7), deodorant of feces (8), decrease in serum cholesterol (6), improvement of kidney function (9), and anti oxidation (10,11).
Green tea contains proteins, free amino acids, alkaloids (caffeine), polyphenols (catechin group), sugar, organic acids, lipids, pigments (chlorophyll, carotenoids), vitamins and minerals. Among the above components, the catechins and amino acids influence the taste of green tea. The content of these components changes due to variety, soil composition, weather and ripeness. In particular, the content of amino acids decreases with each harvested crop.
L-theanine is the major amino acid found in green tea, especially that of Gyokuro (highest quality green tea powder). L-theanine is bio-synthesized in the root and stored in the leaf. L-theanine, however, is converted to catechin by sun light. Therefore, Gyokuro, the highest grade green tea, is cultivated without direct sunlight to promote the accumulation of theanine.
Traditionally, L-theanine has been considered to be a flavor agent. After the chemical structure of L-theanine was reported by Sakato in 1950 (12), however, many studies were conducted on its other effects. Kimura et.al. reported that L-theanine acts antagonistically against paralysis induced by caffeine in animal models (13) and is incorporated into the brain through the blood-brain barrier (14). However, no study of L-theanine on the mental effects in humans has been reported so far.
We have studied the enzymatic synthesis of L-theanine as well as the extraction from green tea leaf for its production on an industrial scale (15,16).
In this report, the mental effect of L-theanine in humans was investigated (17,18).
Brain waves are classified as d , q , a and b waves according to the frequency of each brain wave. Each brain wave is related to an individual mental condition. The d , q , a and b waves are recorded during states of sleep, dozing, relaxation and excitement, respectively.
As people strongly look for relaxation in modern life, a variety of products that promote relaxation including perfumes (19), music and movies have appeared on the market. As these products tend to be heavily dependant on individual taste, there is a limit on its usage. Therefore, a new ingredient without influence by individual taste is needed for food applications.
Since ancient times, it has been said that the drinking of green tea induces relaxation. However, there were few studies on the exact relaxation promoting components present in green tea. We succeeded in establishing an industrial production of L-theanine by a fixed enzyme. We then investigated the efficacy of L-theanine on relaxation in order to develop its application.
Materials and Methods
Suntheanine containing more than 98% L-theanine (Taiyo Kagaku Co., Ltd.) was used in the study. Safety of Suntheanine was confirmed by acute toxicity, subacute toxicity and mutagenicity studies.
Selection of volunteers
As it was expected that the mental reactivity to theanine could vary with levels of anxiety, fifty (50) female students (18 to 22 years old) were classified into two groups, namely a high anxiety group and a low anxiety group, according to monitoring by MAS (manifest anxiety scale) (20). In this test, each subject answered 65 questions. Levels of anxiety were classified into 5 grades indicating mental and body conditions. Subjects having the strongest anxiety were classified as grade I. The clinical test was conducted with 4 high anxiety female students and 4 low anxiety students (18 to 22 years old).
The test room was divided into two rooms, a closed-environment room and a recording room. The closed-environment room was adjusted to a temperature of 25° C and lighting of 40 lux. A chair was set in the room for the brain wave measurement.
After setting the EEG electrodes on the subject in the closed-environment room and allowing to adopt to the environment, each volunteer took 100 ml of water with or without 50 mg or 200 mg Suntheanine. Brain waves were measured by the induced mono-electrode method at 12 points on the head based on an earlobe as a standard point consecutively for 60 minutes after the administration and recorded by a brain wave meter (NEC Co., Ltd., SYNAFIT 1000) and data was recorded by a Data Recorder (TEAC Co., Ltd., XR-510). After recording, each subject reported taste and odor of the test solution and their feeling during the measurement.
Each subject was given 100 ml water the first week, 50mg theanine solution the second week and 200mg theanine solution during the third week. Volunteers took the test solution at the fixed time on the same day, once every week. This was repeated two times, for a total of six weeks.
Analytical methods and statistics
Every 10 minutes recorded brain waves including a and q waves were analyzed by Frequent Fourier Transformation (FFT) and were mapped by EEG every 30.720 seconds. The data was analyzed for brain wave topography by the research program ATAMAP Kissei Komtech.
In the brain wave topography, when a waves were noted in the occipital and parietal regions of the brain, subjects were judged to be in a pattern of relaxation.
The intensity of the q - (4 to 7 Hz) and a - (a 1:8-10 Hz, a 2:10 13 Hz) waves observed at two positions in the parietal and occipital regions respectively, were integrated and converted into the amplitude unit (m V unit) to show the spectral data. The data was cumulated into two 30 minute segments (total 60 minutes) after starting measurement. The data was duplicated in each subject with each test solution and statistically analyzed by Fishers method.
Brain wave topography after administration with Suntheanine
Figure 4-1 showed the brain waves topography in the subjects who took water or 200 mg Suntheanine. The typical example of brain wave topography in the high anxiety group was shown in Fig. 4-2 or 4-3. In these figures, no a -brain waves were observed in the subjects with water during 60 minutes observation (Fig.4-2). However, a dose of oral administration of 200 mg of Suntheanine resulted in the generation of a -brain waves in the occipital and parietal regions of the brains of the subjects (Fig.4-3). However, a waves were not remarkably observed in the subjects with 50 mg Suntheanine.
2. Variation of intensity in emission of brain waves (integrated as function of investigation times and area).
Variation of intensity in the emission of a -brain waves for 60 minutes was shown in Figure 5. Emission intensity of a 1-, a 2- and a 1 + a 2 - brain waves for each dose during the first 30 minutes was basically unchanged. Emission intensity of a 1-, a 2- and a 1 + a 2 - brain waves for control and 50 mg Suntheanine exhibited little change during the second 30 minutes as well, but remarkably increased in subjects with 200 mg of Suntheanine. The q -brain waves were at the same level during the observation period.
Difference of intensity in the emission of brain waves in the high anxiety group.
The ratio of intensity in the emission of a -brain waves in the low and high anxiety group classified by MAS was calculated against that of the control group with water. The data is showed in Fig.6. The ratio in the low anxiety group with 50 and 200 mg of Suntheanine were 1.0 and 1.03, respectively. However, the ratio in the high anxiety group with 50 and 200 mg increased. The maximum increase was more than 2.0 times and the mean was more than 1.2 times. The ratio of emission intensity of a -brain waves in both groups with 200 mg of Suntheanine significantly increased by Fishers statistic analysis in comparison with that in the groups with water (p=0.065).
All subjects reported no change in taste and odor in the 200 mg solution and felt no difference in comparison with water and 50 mg solution. However, half of the subjects with 200 mg in the high anxiety group reported to feel warmth in their fingertips and toe-tips. It was expected that relaxation induced blood vessel expansion and consequently they felt warm.
Electodermal activity, blood flow, pulse wave, pupillary constriction, flicker test, blood pressure, heart beat, response time and tomogram of the brain are available to evaluate the effects of relaxation. In the study, however, brain wave topography was used to measure the brain waves of the eight subjects administered with water, 50 mg and 200mg Suntheanine solution.
All test solutions had no reported change in taste or odor. Consequently, a dose of oral administration of 200 mg of Suntheanine resulted in the generation of a -brain waves in the occipital and parietal regions of the brains of the subjects. The ratio of intensity in the emission of a -brain waves in the high anxiety group with 50 and 200 mg also increased. According to these observations, it was revealed that theanine induces relaxation. The q - and a - brain waves related to dozing and relaxing, respectively, were analyzed. As a result, the emission of q - brain waves remained at the same level during the observation, but a -brain waves changed. The amount of theanine administered in the study was equivalent to 10 or 40 cups of Sencha. However, as green tea contains caffeine, its theanine effect is canceled.
Theanine was absorbed in the intestinal brush-border membrane and incorporated into the brain via the leucine-preferring transport system of the blood-brain barrier (23). It has also been reported that theanine induced a decrease in Serotonin, a neurotransmitter, and an increase of catecholamine as well as a decrease of sleepiness. Therefore, theanine does not induce drowsiness. The intensity in the emission of a -brain waves was reached at the maximum level 40 minutes after administration, which shows a -brain waves were generated around 30 minutes after administration. It was supported by the report showing theanine was incorporated into the brain 30 minutes after administration in rats(14). It was reported that theanine had also been detected in the brain two hours after administration (23). Therefore, although the brain waves were observed for only one hour in this study, it was expected that relaxation is maintained at least two hours.
In this study, it was determined that a certain amount of theanine, having no taste or odor, induced the generation of a -brain waves and had a relaxation effect. In the next step, efficacy of theanine will be investigated in the application of food.
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[keywords: L-theanine, l theanine]