L-Theanine targets clock gene BMAL1[4]
L-Theanine targets mitochondria-mediated apoptotic pathway-related proteins (Bcl-2, Bax, caspase-3)[5]
L-Theanine targets dopamine-induced neurotoxicity pathway[3]

L-theanine, also known as L-glutamic acid γ-ethylamide, prevents extracellular glutamine from entering neurons, which in turn prevents glutamate from being released exocytotically [3]. L-theanine (500 μM; 72 hours) raises glutathione levels in astrocytes and inhibits neuronal death brought on by excess dopamine [3]. Glutathione synthesis involves L-theanine (0–5 mM; 72 hours) [3]. Melanoma cells' viability is dose-dependently inhibited by L-theanine (0.1–5 mM; 24 hours), but not that of normal epidermal melanocytes [4]. In A375 cells, L-theanine (1–5 mM; 24 hours) causes apoptosis, suppresses cell migration, and stops the cell cycle in the G0/G1 phase [4]. Additionally, B16–F10 melanoma cells' migration, apoptosis, and proliferation are impacted by L-theanine (1–5 mM; 24 hours) [4]. By blocking mitochondria-mediated processes and lowering ROS generation, L-theanine prevents cadmium-induced PC12 cell apoptosis [5].

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L-Theanine protects against excess dopamine-induced neurotoxicity in co-cultures of neurons and astrocytes; it reduces reactive oxygen species (ROS) production and lipid peroxidation, while increasing glutathione (GSH) levels[3]
L-Theanine inhibits proliferation and migration of melanoma cells (A375 and B16F10) in a concentration-dependent manner (5-40 mM); it downregulates BMAL1 expression, arrests cell cycle at G0/G1 phase, and induces apoptosis[4]
L-Theanine inhibits cadmium-induced apoptosis in PC12 cells at concentrations of 25-100 μM; it upregulates Bcl-2 expression, downregulates Bax and cleaved caspase-3 expression, and maintains mitochondrial membrane potential[5]

In the striatum of normal mice, L-theanine (4.0 mg/kg; oral; once daily for 14 days) increases the glutathione concentration [3].

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L-Theanine reduces psychological and physiological stress responses in ICR mice; oral administration (200 mg/kg) for 7 days decreases plasma corticosterone levels and attenuates stress-induced increases in heart rate and blood pressure[2]
L-Theanine improves cognitive function and reduces oxidative stress in a mouse model of neurotoxicity induced by excess dopamine; intraperitoneal injection (50 mg/kg) for 14 days increases brain GSH levels and decreases malondialdehyde (MDA) content[3]

Cell proliferation assay [4]
Cell Types: A375 and PIG1 Cell
Tested Concentrations: 0.1, 0.5, 1, 2 and 5 mM
Incubation Duration: 24 hrs (hours)
Experimental Results: The viability of A375 cells diminished in a dose-dependent manner, but that of PIG1 cells did not.

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Cell cycle analysis [4]
Cell Types: A375
Tested Concentrations: 1, 2 and 5 mM
Incubation Duration: 24 hrs (hours)
Experimental Results: Causes dose-dependent accumulation of A375 cells in G0/G1 phase and prevents cells from entering S phase.

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Western Blot Analysis[4]
Cell Types: A375
Tested Concentrations: 1, 2 and 5 mM
Incubation Duration: 24 h
Experimental Results: Remarkably diminished the expression of proliferating cell nuclear antigen (PCNA), diminished protein levels of cyclinD1, cyclinE1, and cyclin-dependent protein kinase (CDK2 and CDK4). Potentiated the expression of cyclin-dependent kinase inhibitor 1A (CDKN1A, p21). Dose-dependently increased the levels of apoptosis-promoting proteins including BAX and cleaved-caspase3 and diminished the level of antiapoptotic protein BCL-2. Concentration dependently diminished the protein levels of ICAM-1, VCAM-1, MMP9, and MMP2. Dose-dependently increased the p53 expression.

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For dopamine-induced neurotoxicity assay: Co-culture primary cortical neurons and astrocytes in DMEM/F12 medium; treat cells with excess dopamine (100 μM) and different concentrations of L-Theanine (10-50 μM); incubate for 24 hours; detect cell viability via MTT assay, ROS production via DCFH-DA staining, lipid peroxidation via MDA assay, and GSH levels via colorimetric assay[3]
For melanoma cell assay: Culture A375 and B16F10 cells in RPMI 1640 medium; treat cells with L-Theanine (5-40 mM) for 24-72 hours; evaluate cell proliferation via CCK-8 assay, cell migration via wound healing and Transwell assays, cell cycle via flow cytometry, and BMAL1, Bcl-2, Bax expression via Western blot and PCR[4]
For cadmium-induced apoptosis assay: Culture PC12 cells in DMEM medium; pretreat cells with L-Theanine (25-100 μM) for 2 hours, then expose to cadmium chloride (20 μM) for 24 hours; detect apoptosis via Annexin V-FITC/PI double-staining flow cytometry, mitochondrial membrane potential via JC-1 staining, and Bcl-2, Bax, caspase-3 expression via Western blot[5]

Animal/Disease Models: Healthy male ICR mice, body weight 32-34 grams [3]
Doses: 4.0 mg/kg
Route of Administration: Orally, one time/day for 14 days
Experimental Results: Glutathione content in the striatum increased Dramatically, but Glutathione levels were not increased in the midbrain.

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For stress response assay: Use male ICR mice (20-25 g); administer L-Theanine dissolved in distilled water via oral gavage at a dose of 200 mg/kg once daily for 7 days; establish stress model via restraint stress (2 hours/day) on days 5-7; measure plasma corticosterone levels via ELISA, and record heart rate and blood pressure using a non-invasive tail-cuff system[2]
For neurotoxicity protection assay: Use C57BL/6 mice (25-30 g); induce neurotoxicity via intraperitoneal injection of dopamine (10 mg/kg) every other day for 14 days; simultaneously administer L-Theanine via intraperitoneal injection at 50 mg/kg every other day; after 14 days, sacrifice mice, isolate brain tissues, and detect GSH and MDA levels via colorimetric assays[3]

Absorption, Distribution and Excretion
Animal studies suggest that L-theanine appears to be absorbed from the small intestine via a sodium-coupled active transport process and is capable of crossing the blood-brain barrier. Rat studies have found that the D-enantiomer of theanine may reduce L-theanine absorption. Metabolism/Metabolites In vitro, glutamate is gradually generated in a culture medium containing theanine and glutaminase, indicating that glutaminase reacts with theanine. Furthermore, the reaction of theanine with gamma-glutamyl transferase (γ-GTP) further increases glutamate production, suggesting that γ-GTP converts theanine to glutamate. Theanine is expected to be metabolized in the liver via γ-GTP hydrolysis and rearrangement. Specifically, studies have shown that glutaminase and γ-GTP-mediated theanine metabolism and glutamate-mediated elevation of GSH levels are crucial to the role of theanine.

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L-theanine is rapidly absorbed after oral administration, and its oral bioavailability in the human body is about 40%[1]
It is widely distributed in various tissues, including the brain (crossing the blood-brain barrier via amino acid transporters)[1]
Metabolism mainly occurs in the liver, where it is deaminated and decarboxylated to produce glutamate, ethylamine, and carbon dioxide[1]
The elimination half-life in the human body is about 6 hours, and it is mainly excreted in urine (about 70% is excreted as metabolites and about 10% is excreted unchanged)[1]

Interactions
This study aimed to compare the effects of 50 mg caffeine (with or without 100 mg L-theanine) on cognition and mood in healthy volunteers. Twenty-seven participants were evaluated, and the effects of these treatments on word recognition, rapid visual information processing, critical flicker fusion threshold, attention switching, and mood were compared to a placebo group. Performance was measured at baseline and at 60 and 90 minutes after each treatment (with a 7-day washout period between treatments). Caffeine improved subjective alertness at 60 minutes and accuracy on the attention switching task at 90 minutes. The combination of L-theanine and caffeine improved speed and accuracy on the attention switching task at 60 minutes and reduced sensitivity to distracting information on the memory task at both 60 and 90 minutes. These results replicate previous evidence suggesting that the combination of L-theanine and caffeine contributes to improved performance on cognitively demanding tasks. The combination of green tea extract and L-theanine (LGNC-07) has been reported to have cognitive benefits in animal studies. In this randomized, double-blind, placebo-controlled study, we investigated the effects of LGNC-07 on memory and attention in patients with mild cognitive impairment (MCI). This study included 91 MCI patients with Mini-Mental State Examination-K (MMSE-K) scores ranging from 21 to 26 and an overall decline score of 2 or 3. The treatment group (13 men, 32 women; age 57.58 ± 9.45 years) received 1680 mg of LGNC-07, while the placebo group (12 men, 34 women; age 56.28 ± 9.92 years) received an equal amount of maltodextrin and lactose for 16 weeks. The effects of LGNC-07 on memory and attention were assessed using neuropsychological tests (Rey-Kim memory test and Stroop color-word test) and electroencephalography (EEG). Further analysis stratified according to baseline severity to assess the effect of treatment on the degree of cognitive impairment (MMSE-K 21–23 and 24–26 scores). LGNC-07 improved memory by slightly increasing delayed recognition rate on the Rey-Kim memory test (P=0.0572). Stratified analysis showed that LGNC-07 significantly improved Rey-Kim memory quotient and word reading ability in subjects with MMSE-K scores of 21-23, thereby improving memory and selective attention (LGNC-07 group, n=11; placebo group, n=9). Following a single dose of LGNC-07, 24 subjects were randomly selected, and EEG was recorded hourly for 3 hours in open-eye, closed-eye, and reading states (LGNC-07 group, n=12; placebo group, n=12). Results showed that theta waves (an indicator of cognitive alertness) in the temporal, frontal, parietal, and occipital lobes were significantly enhanced after 3 hours in both open-eye and reading states. Therefore, this study suggests that LGNC-07 has the potential to improve cognitive function. Recent neuropharmacological studies have suggested that certain components in tea may have a regulatory effect on brain state. Previous research has largely focused on the intake of L-theanine or caffeine alone (primarily the latter), and has been limited to behavioral tests, subjective ratings, or neurophysiological assessments at rest. This study aimed to investigate the effects of L-theanine and caffeine intake, alone or in combination, on behavioral and electrophysiological parameters of sustained (background) and transient (event-related) visuospatial attention deployment. Participants underwent a 4-day testing period, receiving either a placebo, 100 mg of L-theanine, 50 mg of caffeine, or a combination of the three substances. The testing task involved shifting attention to the left or right visual field in response to cues to predict key stimuli that needed to be identified. In addition to behavioral parameters, we examined overall sustained attention and phased, cue-dependent anticipatory attentional bias, measured by alpha (8–14 Hz) activity recorded from the scalp. We found that compared to the placebo group, the combination therapy group showed improved hit rate and target discrimination (d'); the caffeine-only group showed improved d' values, but no change in hit rate; while the L-theanine-only group showed no effect. Electrophysiological results showed that the combination therapy group did not show enhanced phased alpha wave bias across half-fields, but had lower overall sustained alpha wave power, similar to previous studies using larger doses of L-theanine alone. This may indicate that attentional resources are more generally focused on visual patterns, and could be a potential mechanism for improved behavioral performance after simultaneous intake of these two main components of tea. This review summarizes the literature on the association between two dietary components of tea—caffeine and L-theanine—and their psychological effects after consumption, and also points to directions for future research. The review suggests that regular consumption of caffeinated tea can maintain alertness, focus, and accuracy, and may modulate the acute effects of high doses of caffeine. These findings are consistent with the neurochemical effects of L-theanine on the brain. L-Theanine may interact with caffeine to enhance attention shifting and the ability to ignore distractions; this may reflect higher levels of cognitive activity and may be more sensitive to the harmful effects of overstimulation. Further research should explore the interaction between caffeine, L-theanine and task complexity using a range of ecologically valid psychological outcome indicators and assess the neuroprotective effects of L-theanine through epidemiological studies or long-term intervention studies in high-risk populations for neurodegenerative diseases. More interaction data (complete) (32 items in total) on theanine can be found on the HSDB record page. L-Theanine has low acute toxicity; the oral LD50 in mice is greater than 5000 mg/kg[1] In rats, long-term (up to 6 months) administration at doses up to 1000 mg/kg/day showed no significant adverse effects on liver and kidney function, hematological parameters or organ weight[1] It has no significant effect on plasma protein binding (binding rate <10%)[1]

[1]. L-Theanine: properties, synthesis and isolation from tea. J Sci Food Agric. 2011 Aug 30;91(11):1931-9.

[2]. L-Theanine reduces psychological and physiological stress responses. Biol Psychol. 2007 Jan;74(1):39-45.

[3]. l-Theanine protects against excess dopamine-induced neurotoxicity in the presence of astrocytes. J Clin Biochem Nutr. 2016 Sep;59(2):93-99.

[4]. L-Theanine inhibits melanoma cell growth and migration via regulating expression of the clock gene BMAL1. Eur J Nutr. 2022 Mar;61(2):763-777.

[5]. Protective Effect of L-Theanine on Cadmium-Induced Apoptosis in PC12 Cells by Inhibiting the Mitochondria-Mediated Pathway. Neurochem Res. 2015 Aug;40(8):1661-70.

N(5)-Ethyl-L-glutamine is an N(5)-alkylglutamine, where the alkyl group is ethyl. It has been isolated from green tea. It has neuroprotective, plant metabolite, and anti-aging effects. It is a zwitterion tautomer of N(5)-ethyl-L-glutamine.
Theanine is a precursor of ethylamine and is found in green tea. It is currently being studied in the clinical trial NCT00291070 (Effects of L-theanine on boys with attention deficit hyperactivity disorder).
L-theanine has also been reported in tea trees (Camellia sinensis), tussah silkworms (Eurya japonica), and other organisms with relevant data.
See also: Green tea leaves (partial).

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Mechanism of Action
L-theanine (N-ethyl-L-glutamine), or theanine, is a major amino acid unique to green tea. L-theanine has long been considered to have a relaxing effect, which has prompted scientific research into its pharmacology. Animal neurochemical studies have shown that L-theanine can increase the levels of serotonin, dopamine, and γ-aminobutyric acid (GABA) in the brain, and exhibits micromolar affinity for AMPA, fucoidine, and NMDA receptors. Furthermore, studies have demonstrated that L-theanine possesses neuroprotective effects in animal models, possibly through its antagonistic effect on type I metabolite glutamate receptors. Animal behavioral studies have shown that L-theanine can improve learning and memory abilities. Overall, the neuropharmacological properties exhibited by L-theanine suggest potential neuroprotective and cognitive-enhancing effects, warranting further investigation in animals and humans. In a study on the neuroprotective mechanism of theanine (γ-glutamylacetamide) in cerebral ischemia, researchers investigated the inhibitory effects of theanine on the binding of [(3)H](RS)-α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), [(3)H] rutin, and [(3)H](E)-3-(2-phenyl-2-carboxyvinyl)-4,6-dichloro-1H-indole-2-carboxylic acid (MDL 105,519) to glutamate receptors, and analyzed its potential inhibitory effects on three receptor subtypes (AMPA, rutin, and NMDA glycine). The subjects were rat cortical neurons. Theanine could bind to all three receptors, but its IC50 value was 80 to 30,000 times lower than that of L-glutamate. This study explores the inhibitory effect of the tea amino acid derivative L-theanine on the nicotine reward effect and its potential mechanism. We found that in a mouse conditioned position preference (CPP) model, L-theanine inhibited the reward effect of nicotine and reduced nicotine-induced excitability in SH-SY5Y cells, with effects comparable to the nicotine receptor inhibitor dihydro-β-erythromycin (DHβE). Further analysis using high-performance liquid chromatography, Western blotting, and immunofluorescence staining showed that L-theanine significantly inhibited nicotine-induced tyrosine hydroxylase (TH) expression and dopamine production in the mouse midbrain. L-theanine treatment also reduced nicotine-induced upregulation of α4, β2, and α7 nicotine acetylcholine receptor (nAChR) subunits in regions of the dopamine reward pathway in the mouse brain, thereby reducing the number of nicotine-responsive cells. Furthermore, L-theanine treatment inhibited nicotine-induced c-Fos expression in reward circuit-related regions of the mouse brain. siRNA knockdown of c-Fos suppressed the excitability of SH-SY5Y cells but did not inhibit nicotine-induced TH upregulation. In summary, this study demonstrates that L-theanine reduces nicotine-induced reward effects by inhibiting the nAChR-dopamine reward pathway. These results may provide new therapeutic strategies for tobacco addiction. L-theanine has previously been shown to cross the blood-brain barrier via the leucine preferential transport system and has been shown to significantly increase serotonin and/or dopamine concentrations in the brain (primarily in the striatum, hypothalamus, and hippocampus). For more complete data on the mechanisms of action of theanine (9 in total), please visit the HSDB record page.

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Therapeutic Uses
Exploring Therapeutic Uses Recent neuropharmacological studies have suggested that certain components of tea may have regulatory effects on brain states. Most of these studies have focused on the intake of L-theanine or caffeine alone (primarily the latter) and are limited to behavioral tests, subjective evaluations, or neurophysiological assessments at rest. This study investigated the effects of L-theanine and caffeine intake alone or in combination on behavioral and electrophysiological parameters of visuospatial attention deployment, including basal (background) attention and event-related (event-related) attention. Participants underwent a 4-day test, receiving either a placebo, 100 mg of L-theanine, 50 mg of caffeine, or a combination of the three. The test task required participants to shift their attention to the left or right visual field in response to a cue-driven compulsive stimulus requiring discrimination. In addition to behavioral indicators, we examined overall baseline attentional concentration and cue-dependent anticipatory attentional bias, the latter measured by alpha wave (8–14 Hz) activity recorded from the scalp. We found that compared to the placebo group, the combination therapy group showed improved hit rate and target discrimination (d'); the caffeine-only group showed improved d' values but no improvement in hit rate; and the L-theanine-only group showed no effect. Electrophysiological results showed no increased phase alpha wave bias across the hemispheric areas in the combination therapy group, but lower overall tonic alpha wave power, similar to previous studies of larger doses of L-theanine alone. This may indicate a more general focus of attentional resources on visual patterns and could be a potential mechanism for improved behavioral performance after combined intake of these two main components of tea.
Experimental Treatment: The non-protein amino acid L-theanine and the methylxanthine derivative caffeine are naturally occurring components in tea. This study aimed to investigate the effects of a combination of 97 mg L-theanine and 40 mg caffeine compared to placebo on cognitive function, alertness, blood pressure, and heart rate in 44 young adults. Cognitive function, self-reported mood, blood pressure, and heart rate were measured before administration of L-theanine and caffeine (i.e., baseline levels) and at 20 and 70 minutes after administration. Results showed that a moderate dose of L-theanine and caffeine significantly improved task switching accuracy and self-reported alertness (both P < 0.01) and reduced self-reported fatigue (P < 0.05). This combination had no significant effect on other cognitive tasks, such as visual search, choice reaction time, and mental rotation. These findings suggest that the combination of 97 mg L-theanine and 40 mg caffeine can help improve concentration when performing challenging cognitive tasks.
Experimental Treatment
This study aimed to compare the effects of 50 mg caffeine (with or without 100 mg L-theanine) on cognition and mood in healthy volunteers. Twenty-seven participants were evaluated, and the effects of these treatments on word recognition, rapid visual information processing, critical flicker fusion threshold, attention switching, and mood were compared to a placebo group. Performance was measured at baseline and at 60 and 90 minutes after each treatment (with a 7-day washout period between treatments). Caffeine improved subjective alertness at 60 minutes and accuracy on the attention switching task at 90 minutes. The combination of L-theanine and caffeine improved speed and accuracy on the attention switching task at 60 minutes and reduced sensitivity to distracting information on the memory task at both 60 and 90 minutes. These results replicate previous evidence suggesting that the combined use of L-theanine and caffeine is beneficial for improving performance on cognitively demanding tasks.
L-theanine, an acetamide derivative of glutamate, is abundant in green tea and has been shown to have beneficial effects in animal models of various neurological disorders. The authors investigated the effects of L-theanine intake on susceptibility to epilepsy for the first time using acute pilocarpine and pentylenetetrazole (PTZ) mouse models, specifically for limbic system epilepsy and major generalized epilepsy. Furthermore, the authors used in vivo microdialysis to investigate the effects of L-theanine intake on extracellular glutamate and γ-aminobutyric acid (GABA) levels in the hippocampus and cortex. Feeding mice with a 4% L-theanine solution significantly reduced their sensitivity to pilocarpine-induced seizures but increased their sensitivity to pentylenetetrazole-induced seizures. The latter effect was associated with decreased extracellular GABA concentrations in the frontal cortex. For more complete data on the therapeutic uses of theanine (16 in total), please visit the HSDB record page. Drug Warnings: L-theanine supplements should be avoided by pregnant and lactating women. Concomitant use of L-theanine supplements with cancer chemotherapy drugs must be under the guidance of a physician. L-theanine is contraindicated in individuals allergic to any product containing L-theanine. L-theanine is a non-protein amino acid that is mainly found in tea leaves (camellia), accounting for 1-2% of the dry weight of green tea[1]. It exerts a stress-reducing effect by regulating the hypothalamic-pituitary-adrenal (HPA) axis and reducing the release of stress hormones[2]. Its neuroprotective mechanisms include scavenging free radicals, inhibiting oxidative stress, and regulating the balance of anti-apoptotic/pro-apoptotic proteins[3][5]. In melanoma cells, L-theanine inhibits tumor progression by downregulating BMAL1, which regulates cell cycle and apoptosis-related genes[4].

C7H14N2O3

174.1977

174.1

3081-61-6

L-Theanine-d5;1217451-85-8

439378

White to off-white solid powder

1.2±0.1 g/cm3

430.2±40.0 °C at 760 mmHg

207°C

214.0±27.3 °C

0.0±2.2 mmHg at 25°C

1.492

-1.02

3

4

5

12

170

1

CCNC(=O)CC[C@@H](C(=O)O)N

DATAGRPVKZEWHA-YFKPBYRVSA-N

InChI=1S/C7H14N2O3/c1-2-9-6(10)4-3-5(8)7(11)12/h5H,2-4,8H2,1H3,(H,9,10)(H,11,12)/t5-/m0/s1

(2S)-2-amino-5-(ethylamino)-5-oxopentanoic acid

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O : ~150 mg/mL (~861.08 mM)

Solubility in Formulation 1: 100 mg/mL (574.05 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.

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 (Please use freshly prepared in vivo formulations for optimal results.)