• precise selection of the collection area with traceability,
• definition of the level of leaf maturity
• suitable drying system to preserve the assay,
• Implementation of a selection protocol according to the active ingredient content
• etc…

Abstract: Tea has been consumed for thousands of years and is an integral part of people’s daily routine, as an everyday drink and a therapeutic aid for health promotion. Consumption of tea has been linked to a sense of relaxation commonly associated with the content of the non-proteinogenic amino acid theanine, which is found within the tea leaves. The aim of this review article is to outline the current methods for synthesis, extraction and purification of theanine, as well as to examine its potential benefits related to human health. These include improvements in cognitive and immune function, cancer prevention, reduced cardiovascular risk and its potential usefulness as a functional food product.
Conclusion : L-THE is a predominant amino acid found in tea leaves that contributes to the taste sensation called “umami”. The methods of L-THE synthesis (synthetic and biosynthetic) vary in their yield and commercial viability; most are labour intensive and time consuming and have relatively low yields of L-THE. Apparently, no current technique offers an environmentally sustainable and economically viable method for commercial production of purified L-THE. As a result, there is a considerable demand for further research in devising newer extraction and purification procedures to reduce the environmental impact and manufacturing cost of producing L-THE [8]. In addition, there is immense interest in L-THE as a supplement as well as a functional food ingredient for its therapeutic health benefits, including but not limited to its effects on learning ability, immune function, cancer suppression and vascular relaxation. L-THE also promotes the generation of
-waves in the brain, inducing a state of relaxation without causing a state of drowsiness. Therefore, further research is warranted to develop L-theanine as a functional food additive and to explore its role in disease prevention and health promotion.
Keywords: theanine; green tea; alpha wave production; relaxation; functional food, bioactives
L-Theanine as a Functional Food Additive: Its Role in Disease Prevention and Health Promotion. Jackson Williams, Jane Kellett, Paul Daniel Roach, Andrew McKune, Duane Mellor, Jackson Thomas and Nenad Naumovski, Beverages
2016, 2, 13

Abstract Rationale L-Theanine (N-ethyl-L-glutamine) is an amino acid uniquely found in green tea and historically considered to be a relaxing agent. It is a glutamate derivative and has an affinity for glutamatergic receptors. However, its psychotropic effects remain unclear.
Objectives To elucidate effects of L-theanine on psychiatric disease-related behaviors in mice and its molecular basis focusing on brain-derived neurotrophic factor (BDNF) and N-methyl-D-aspartate (NMDA) receptor.
Methods We examined the effects of L-theanine on behaviors in mice by using the open-field test (OFT), forced swim test (FST), elevated plus-maze test (EPMT), and prepulse inhibition (PPI) of acoustic startle. By western blot analysis, we looked at the effect of L-theanine on the expression of BDNF and related proteins in the hippocampus and cerebral cortex. To determine whether L-theanine has agonistic action on the NMDA receptor, we performed Fluo-3 intracellular Ca2+ imaging in cultured cortical neurons.
Results Single administration of L-theanine significantly attenuated MK-801-induced deficits in PPI. Subchronic administration (3-week duration) of L-theanine significantly reduced immobility time in the FST and improved baseline PPI. Western blotting analysis showed increased expression of BDNF protein in the hippocampus after subchronic administration of L-theanine. In cultured cortical neurons, L-theanine significantly increased the intracellular Ca2+ concentration, and this increase was suppressed by competitive and noncompetitive NMDA receptor antagonists (AP-5 and MK-801, respectively).
Conclusions Our results suggest that L-theanine has antipsychotic-like and possibly antidepressant-like effects. It exerts these effects, at least in part, through induction of BDNF in the hippocampus and the agonistic action of Ltheanine on the NMDA receptor.
Keywords L-theanine .MK-801 (dizocilpine) . N-methyl Daspartate (NMDA) receptor . Schizophrenia . Prepulse inhibition . Brain-derived neurotrophic factor (BDNF) . Behavior . Glutamate . Antidepressant
Behavioral and molecular evidence for psychotropic effects in L-theanin, Chisato Wakabayashi, Tadahiro Numakawa, Midori Ninomiya, Shuichi Chiba, Hiroshi Kunugi, Psychopharmacology (2012) 219:1099–1109

Abstract : Caffeine and L-theanine, both naturally occurring in tea, affect the ability to make rapid phasic deployments of attention to locations in space as reflected in behavioural performance and alpha-band oscillatory brain activity (8e14 Hz). However, surprisingly little is known about how these compounds affect an aspect of attention that has been more popularly associatedwith tea, namely vigilant attention: the ability to maintain focus on monotonous tasks over protracted time-periods. Twenty-seven participants performed the Sustained Attention to Response Task (SART) over a two-hour session on each of four days, on which they were administered caffeine (50 mg), theanine (100 mg), the combination, or placebo in a double-blind, randomized, cross-over fashion. Concurrently, we recorded oscillatory brain activity through high-density electroencephalography (EEG). We asked whether either compound alone, or both in combination, would affect performance of the task in terms of reduced error rates over time, and whether changes in alpha-band activitywould showa relationship to such changes in performance.When treatedwith placebo, participants showed a rise in error rates, a pattern that is commonly observed with increasing time-on-task,whereas after caffeine and theanine ingestion, error rates were significantly reduced. The combined treatment did not confer any additional benefits over either compound alone, suggesting that the individual compounds may confermaximal benefits at the dosages employed. Alpha-band oscillatory activity was significantly reduced on ingestion of caffeine, particularly in the first hour. This effect was not changed by addition of theanine in the combined treatment. Theanine alone did not affect alpha-band activity.
Conclusion : Sustained attention, or the ability to maintain vigilance over protracted periods of time, is an integral part of cognitive performance in daily life. Whereas caffeine has repeatedly been shown to improve sustained attention, theanine, another ingredient naturally present in tea has received less attention. Based on our previous studies assessing selective attention (Gomez-Ramirez et al., 2007, 2009; Kelly et al., 2008), wehypothesized that caffeine and theanine would also improve sustained attention abilities, and that the combination would confer even greater benefits. Using the wellcharacterized SART paradigm, caffeine and theanine, both alone and in combination, were indeed shown to improve vigilance as evidenced by sustained attenuation of errors that naturally occur over time. These findings concurred with reduced tonic alpha-band activity suggesting improved attentional processing. The combination treatment, however, did not confer any additional benefits over either ingredient alone, suggesting that either 50 mg of caffeine or 100 mg theanine already provides a maximal effect on sustained attention. In the context of the previous studies, it is clear that dose plays an important role, and a logical next step would be to investigate this in a doseeresponse study.
Keywords: Attention Vigilance Alpha-band Oscillations EEG Human Tea Theanine Caffeine
Assessing the effects of caffeine and theanine on the maintenance of vigilance during a sustained attention task John J. Foxe, Kristen P. Morie, Peter J. Laud, Matthew J. Rowson, Eveline A. de Bruin, Simon P. Kelly, J.J. Foxe et al. / Neuropharmacology (2012) 1-8

ABSTRACT. L-theanine (N-ethyl-L-glutamine) or theanine is a major amino acid uniquely found in green tea. L-theanine has been historically reported as a relaxing agent, prompting scientific research on its pharmacology. Animal neurochemistry studies suggest that L-theanine increases brain serotonin, dopamine, GABA levels and has micromolar affinities for AMPA, Kainate and NMDA receptors. In addition has been shown to exert neuroprotective effects in animal models possibly through its antagonistic effects on group 1 metabotrophic glutamate receptors. Behavioural studies in animals suggest improvement in learning and memory. Overall, L-theanine displays a neuropharmacology suggestive of a possible neuroprotective and cognitive enhancing agent and warrants further investigation in animals and humans.
Discussion : Pre-clinical studies suggest that L-theanine increases a number of neurotransmitters including serotonin, dopamine and GABA levels and has micromolar affinities for AMPA, kainate andNMDAreceptors. Behavioural studies in animals demonstrate improvements in learning and memory following L-theanine administration. In addition L-theanine has been shown to exert neuroprotective effects in animal models possibly through its antagonistic effects on group 1 metabotrophic glutamate receptors. While studies in humans are lacking, there is some evidence for a possible anxiolytic effect. Further studies are warranted in both animals and humans to examine L-theanine’s efficacy as a neuroprotective and cognitive enhancing agent as well as a possible anxiolytic.
KEYWORDS. Theanine, monoamines, pharmacology, neuropharmacology, cognition and anxiety, neuroprotection, cerebral infarct
The Neuropharmacology of L-Theanine(N-Ethyl-L-Glutamine): A Possible Neuroprotective and Cognitive Enhancing Agent Pradeep J. Nathan, PhD Kristy Lu, BSc (Hons) M. Gray, BSc (Hons) C. Oliver, BAgSc, Journal of Herbal Pharmacotherapy, Vol. 6(2) 2006

Abstract Sleep deprivation is associated with an elevated risk of various diseases and leads to a poor quality of life and negative socioeconomic consequences. Sleep inducers such as drugs and herbal medicines may often lead to dependence and other side effects. L-Theanine (γ-glutamyl ethylamide), an amino acid naturally found abundant in tea leaves, has anxiolytic effects via the induction of α brain waves without additive and other side effects associated with conventional sleep inducers. Anxiolysis is required for the initiation of high-quality sleep. In this study, we review the mechanism(s), safety, and efficacy of L-theanine. Collectively, sleep studies based on an actigraph, the obstructive sleep apnea (OSA) sleep inventory questionnaire, wakeup after sleep onset (WASO) and automatic nervous system (ANS) assessment, sympathetic and parasympathetic nerve activities, and a pediatric sleep questionnaire (PSQ) suggest that the administration of 200 mg of L-theanine before bed may support improved sleep quality not by sedation but through anxiolysis. Because L-theanine does not induce daytime drowsiness, it may be useful at any time of the day. The no observable adverse effect level (NOAEL) for the oral administration of L-theanine was determined to be above 2000 mg/kg bw/day. KEY TEACHING POINTS: Sleep deprivation-associated morbidity is an increasing public health concern posing a substantial socioeconomic burden. Chronic sleep disorders may seriously affect quality of life and may be etiological factors in a number of chronic diseases such as depression, obesity, diabetes, and cardiovascular diseases. Most sleep inducers are sedatives and are often associated with addiction and other side effects. L-Theanine promotes relaxation without drowsiness. Unlike conventional sleep inducers, L-theanine is not a sedative but promotes good quality of sleep through anxiolysis. This review suggests that L-theanine is a safe natural sleep aid.
Keywords: OSA questionnaire; actigraph; automatic nervous system (ANS); l-theanine; sleep; wake up after sleep onset (WASO).
In Search of a Safe Natural Sleep Aid, Theertham P Rao, Motoko Ozeki, Lekh R Juneja , J Am Coll Nutr . 2015;34(5):436-47

Abstract: As the population ages, neurodegenerative diseases such as Parkinson’s disease (PD) and Alzheimer’s disease (AD) impose a heavy burden on society and families. The pathogeneses of PD and AD are complex. There are no radical cures for the diseases, and existing therapeutic agents for PD and AD have diverse side effects. Tea contains many bioactive components such as polyphenols, theanine, caffeine, and theaflavins. Some investigations of epidemiology have demonstrated that drinking tea can decrease the risk of PD and AD. Tea polyphenols can lower the morbidity of PD and AD by reducing oxidative stress and regulating signaling pathways and metal chelation. Theanine can inhibit the glutamate receptors and regulate the extracellular concentration of glutamine, presenting neuroprotective effects. Additionally, the neuroprotective mechanisms of caffeine and theaflavins may contribute to the ability to antagonize the adenosine receptor A2AR and the antioxidant properties, respectively. Thus, tea bioactive components might be useful for neuronal degeneration treatment in the future. In the present paper, the neuro protection and the mechanisms of tea and its bioactive components are reviewed. Moreover, the potential challenges and future work are also discussed.
Discussion (partial) : Theanine can inhibit the glutamate receptors and regulate the extracellular concentration of glutamine, presenting neuroprotective effects. Theanine is a unique amino acid of tea, accounting for 1–2% (dry weight) in tea fresh leaves. It has the positive health effects of improving learning and memory abilities, sedation, relaxation, anticancer properties, etc.. Theanine can cross the blood brain barrier via the leucinepreferring transport system. In recent years, numerous studies have shown that theanine has a neuroprotective effect (...) Dodd et al. and Haskell et al. have found that caffeine combined with theanine (amounting to 1–2 cups of tea) is beneficial for focusing attention and improving reaction speed and accuracy.
Keywords: neuronal degeneration; Parkinson’s disease; Alzheimer’s disease; polyphenols; theanine; caffeine; theaflavins
Neuroprotective Effects and Mechanisms of Tea Bioactive Components in Neurodegenerative Diseases Shu‐Qing Chen, Ze‐Shi Wang, Yi‐Xiao Ma, Wei Zhang, Jian‐Liang Lu, Yue‐Rong Liang and Xin‐Qiang Zheng, Molecules 2018, 23, 512

L-theanine (gamma-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 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.
Effects of l-Theanine on Cognitive Function in Middle-Aged and Older Subjects: A Randomized Placebo-Controlled Study. Baba Y, Inagaki S, Nakagawa S, Kaneko T, Kobayashi M, Takihara T, march 2021 J Med Food. 2021

Context: gamma-Aminobutyric acid (GABA) is the main inhibitory neurotransmitter and it is well established that activation of GABAa receptors favours sleep. L-Theanine, a naturally occurring amino acid first discovered in green tea, is a well-known anti-anxiety supplement with proven relaxation benefits. Objective: This study investigated the potential synergistic sleep enhancement effect of GABA/L-theanine mixture.
Materials and methods: Pentobarbital-induced sleep test was applied to find proper concentration for sleep-promoting effect in ICR mice. Electroencephalogram (EEG) analysis was performed to investigate total sleeping time and sleep quality in normal SD rats and caffeine-induced awareness model. Real-time polymerase chain reaction (RT-PCR) was applied to investigate whether the sleep-promoting mechanism of GABA/L-theanine mixture involved transcriptional processes.
Results: GABA/L-theanine mixture (100/20mg/kg) showed a decrease in sleep latency (20.7 and 14.9%) and an increase in sleep duration (87.3 and 26.8%) compared to GABA or theanine alone. GABA/L-theanine mixture led to a significant increase in rapid eye movement (REM) (99.6%) and non-REM (NREM) (20.6%) compared to controls. The use of GABA/L-theanine mixture rather than GABA or L-theanine alone restored to normal levels sleep time and quality in the arousal animal model. The administration of GABA/L-theanine led to increased expression of GABA and the glutamate GluN1 receptor subunit.
Conclusions: GABA/L-theanine mixture has a positive synergistic effect on sleep quality and duration as compared to the GABA or L-theanine alone. The increase in GABA receptor and GluN1 expression is attributed to the potential neuromodulatory properties of GABA/L-theanine combination, which seems to affect sleep behaviour.
Discussion : In conclusion, our results demonstrate that the combined use of GABA and L-theanine increase sleep activity to more than a single administration of either amino acid or these synergistic sleep-promoting effects are likely mediated via changes in GABA and/or glutamate receptor expression in the brain. In summary, this result suggests that GABA/L-theanine mixture could be used for treatment for insomnia and sleep disorders as a concept of nonpharmacological management of sleep.
GABA and L-theanine mixture decreases sleep latency and improves NREM sleep Suhyeon Kim, Kyungae Jo, Ki-Bae Hong, Sung Hee Han and Hyung Joo Suh, Pharmaceutical Biology, 2019, Vol. 57, No. 1, 65–73

Abstract: Epidemiological studies in Japan, including the Nakajima study and the Tsurugaya study, have indicated that green tea consumption may improve cognitive impairment. Catechins, which are typical polyphenols contained in green tea, have been reported to have antioxidative, anti-inflammatory, and neuroprotective effects. However, their impact on human cognitive function remains unclear. Therefore, we performed a double-blind, randomized, controlled study to investigate the effect of 336.4 mg of decaffeinated green tea catechins (GTC) on cognitive function after a single dose and after 12 weeks of daily intake. This study included Japanese adults between the ages of 50 and 69 years with a Mini-Mental State Examination Japanese version score of min 24 and self-assessed cognitive decline. The Cognitrax testing battery was used to evaluate cognitive function. The incorrect response rate on the Continuous Performance Test significantly decreased after a single dose of GTC. After 12 weeks of daily GTC intake, the response time for Part 4 of the 4-part Continuous Performance Test, which is a two-back test, was shortened. These results suggest that daily intake of GTC might have beneficial effects on working memory.
Keywords: green tea; catechins; cognitive function; Cognitrax; middle-aged; randomized placebo-controlled trial
Effect of Daily Intake of Green Tea Catechins on Cognitive Function in Middle-Aged and Older Subjects: A Randomized, Placebo-Controlled Study Yoshitake Baba, Shun Inagaki, Sae Nakagawa, Toshiyuki Kaneko, Makoto Kobayashi and Takanobu Takihara