L-theanine Powder

Physiochemical properties

Theanine, like the protein-based amino acids, exists as a zwitterionic species and is a colorless crystalline solid [needles, melting point 214–216 ?C][2]. Studies on the buffering capacity of green tea extracts suggest the pKa of the theanine amino group to be[10, 12]. The pKa of the carboxyl unit was not formally quantified due to interference from other acidic species. However, comparisons with close structural analogues such as glutamine suggest the value lies in the range 2.1–2.5[13]?Theanine is stable under acidic conditions but undergoes base hydrolysis to yield glutamic acid and ethylamine[2, 14]. During infusion, theanine does not react chemically with any of the other tea components. This is in contrast to catechins, which can precipitate from solution as a result of π stacking interactions with caffeine[15, 16]or can react with proteins and enzymes such as lipoxygenase, α-amylase, pepsin, trypsin and lipase[15]. Of the tea components, theanine exhibits a higher water solubility [385 g L?1 at 0 ?C, 556 g L?1 at 100 ?C]than caffeine [21.7 g L?1]and the catechins [e.g. epigallocatechin gallate, 5 g L?1]; this permits a very effective diffusion of theanine from tea during hot-water infusions [2, 17]. The relative insolubility of theanine in organic solvents such as methanol and chloroform allows for its easy separation from caffeine and the catechins, which possess a molecular rather than a zwitterionic structure[2]. Theanine has a complex umami taste[18-20].It also exhibits a synergism with the common umami flavouring agents monosodium glutamate and the purine nucleoside inosine 5’-monophosphate, which leads to an enhancement of the umami taste experience[18].The term umami is a Japanese-derived expression and it is classified as the fifth taste after sweet, salt, bitter and sour[19, 20]. Most of the typical umami substances are divided into two groups: L-α-amino acids, usually represented by monosodium glutamate and 5’-ribonucleotides and their derivatives, usually represented by inosine 5’-monophosphate or disodium 5’-guanylate[19, 20].

Sources and synthesis

Sources The tropical and temperate regions of Asian, African, South American countries are considered the main origin of tea plant [C. sinensis or Thea sinensis]. It is a member of Theaceae family. The majority members of Theaceae family are obtained from India, Sri Lanka, China, and Japan. The physiological properties and colour e.g. black, white, green, yellow or oolong tea strongly depends upon the degree of fermentation and processing conditions[20]. L-theanine is mainly derived from a non-edible mushroom of Xerocomus badius, and C. sinensis. It is an amino acid which accumulates in the leaves of tea like C. sasanqua and C. japonica[21]. In tea, L-theanine is responsible for a strong smell [aroma]in general and in particular it is linked with tea umami taste[22]. From the compositional viewpoint, L-theanine comprises approximately 50% of the tea contents. Whereas, the dry tea contains 1–3% of L-theanine only, this higher or lower ratio of L-theanine can vary depending on several factors i.e. cultivation zone, production season, processing techniques, class of tea, time and type of harvest, etc[27]. Additionally, the harvested tea at the beginning of summer is reported to have more theanine compared with tea harvested in late summer[27]. Moreover, L-theanine concentration also depends on the type/class of tea. According to one study, a specific type of C. sinensis var. Sinensis has more L-theanine contents as compared to the C. sinensis var. Assamica[23]. Besides natural L-theanine, the synthetic L-theanine [SuntheanineTM]is also available as a racemic mixture of Dand L-forms of ethylamine and L-glutamine. It is typically obtained from ethylamine and L-glutamine with the use of glutaminase enzyme[24]. Evidently, literature evidences that L-theanine is stable in acidic conditions[25, 26]. Synthesis Theanine was first chemically synthesised in 1942 by Lichtenstein[28]with a yield of 90 g kg?1 by treating pyrrolidone-5-carboxylic acid with aqueous ethylamine for 20 days at 37 ?C. A number of other synthetic approaches have since been developed including a large-scale production method involving the reaction of γ -benzyl glutamate in the presence of trityl chloride and ethylamine [339 g kg?1][29]and a two-step approach involving initial dehydration of L-glutamic acid to L-pyrrolidone carboxylic acid followed by ring opening in the presence of ethylamine to yield theanine [374 g kg?1][30]. More recently, theanine was produced in four steps starting from commercially available N-phthaloyl-L-glutamic acid, which was dehydrated to the corresponding cyclic anhydride by reaction with acetic anhydride and then the ring was opened by reaction with ethylamine. Subsequent de-protection of the amine unit with hydrazine hydrate gave theanine with a 700 g kg?1 overall yield[31]. In the tea plant, theanine is bio-synthesised from glutamic acid and ethylamine by the enzyme theanine synthetase. However, the enzyme is very labile and cannot be used to produce the amino acid in commercial quantities[32]. Therefore, other methods for the enzymatic synthesis of theanine have been developed using bacterial enzymes such as glutaminase, glutamine synthetase and γ-glutamyl-transpeptidase. The enzyme glutamine synthetase and related enzymes, originating from Escherichia coli[33], Pseudomonas taetrolens[34].Methylovorus mays [35], and Bacillus subtilis[35], have been utilized for the synthesis of theanine from glutamic acid and ethylamine. Unlike the glutaminase biosynthetic pathway discussed previously, this glutamic acid transformation, while using a cheaper and more stable starting material, requires a continuous supply of ATP to drive the reaction[34]. Another non-ATP-dependent biosynthetic pathway, involving γ -glutamyltranspeptidase from E. coli and glutamine as the starting substrate, has also been successfully developed but the reaction requires a high concentration of ethylamine to drive the conversion of glutamine to theanine[31, 36, 37].

Health effects

Green tea leaves have many beneficial health activities including anti-inflammatory, anti-carcinogenic, anti-mutagenic, antioxidative and antimicrobial and hypolipidemic effects[38, 39]. Those beneficial effects are largely originated from the biological effects from L-Theanine as below: Ingestion of theanine has been reported to facilitate the generation of alpha brain waves, which are associated with a relaxed but alert mental state[40]. In addition, theanine is reported to promote the release of the inhibitory neurotransmitterγ-aminobutyric acid [GABA], which in turn regulates dopamine and serotonin levels in the brain[41]. Thus, theanine consumption has been closely associated with relaxation and improved learning ability. A recent study found that ingestion of 50 mg of L-theanine dissolved in 100 mL of water could elicit a significant effect on the general state of mental alertness or arousal in subjects by increasing alpha-wave brain activity[42]. Another study also found a link between theanine consumption [200 mg]and the reduction of anxiety[43]. Other studies have shown that consumption of theanine in combination with caffeine could further improve concentration and learning ability. For example, the intake of a combination of 250 mg L-theanine and 150 mg caffeine was found to enhance rapid simple reaction time, fast numeric working memory reaction time and improve verification accuracy during reading tasks[44]. A separate study found that consuming a combination of 100 mg L-theanine and 50 mg caffeine improved both speed and accuracy performance during attention-switching tasks performed 60 min after ingestion and reduced susceptibility to distracting information in memory tasks at 60 and 90 min following ingestion[45]. Moreover, it is thought that theanine may provide effective prophylaxis and treatment for Alzheimer’s disease as it has been reported to exert neuro-protective effects through inhibition of the N-methyl-D-aspartate [NMDA]subtype of glutamate receptors and its related pathways in a transgenic neuronal cell model[46]. Theanine has also recently been linked to cancer prevention. Liu et al[47] found that theanine was linked to the inhibition of the in vivo and ex vivo growth of human non-small cell lung cancer and leukaemia cell lines. In another study, Friedman et al[47]found that theanine intake was associated with the induction of apoptosis in four cancer cell lines of breast, colon, hepatoma and prostate origin. In addition to enhanced antitumour activity, theanine can reduce the adverse effects of the cancer treatment drug, doxorubicin by providing protection against damage caused by doxorubicin to normal tissue[48]. It also acts as a biochemical modulator to improve the therapeutic efficacy of doxorubicin by suppressing the efflux of the drug from cancer cells, thereby increasing the effective doxorubicin concentration in the tumour[49]. Recent studies have also found that theanine was linked with regulation of blood pressure, promotion of weight loss and improvement of the immune system[51,53]. Injection of L-theanine at a dose rate of 2 g kg?1 was found to significantly reduce blood pressure in spontaneously hypertensive rats[50]. In humans, consumption of a single dose of 200 mg of theanine was also found to reduce blood pressure and, more importantly, theanine was found to antagonise the negative effect of caffeine increasing blood pressure, when the latter was consumed as a single 250 mg dose[51]. In addition, co-administration of L-theanine and L-cystine was reported to enhance antigen-specific immunoglobulin G [IgG]production, partly through augmentation of glutathione [GSH]levels and T-helper cell [Th2]-mediated responses[52]. Similarly, co-treatment of L-theanine with L-cystine was found to improve the immune response via an increase in GSH production, which significantly prevented weight loss associated with infection in aged mice[53].

Safety information

FDA classified L-theanine as “generally recognized as safe.” This classification means that they believe this additive to be safe when people use it as the packaging suggests.Women who are pregnant or breastfeeding should not use L-theanine.

Reference

1 Sakato Y, The chemical constituents of tea: III. A new amide theanine. Nippon Nogei Kagakukaishi 23:262–267 [1949]. 2 Wan X, Zhang Z and Li D, Chemistry and biological properties of theanine, in Tea and Tea Products, ed.byHoCT, Lin JKandShahidi F. CRC Press, Boca Raton, pp. 255–274 [2009]. 3. G. Eschenauer, B.V. Sweet, Pharmacology and therapeutic uses of theanine, Am. J. Health Syst. Pharm. 63 [1][2006]28–30. 4. D.J. White, S. de Klerk, W. Woods, S. Gondalia, C. Noonan, A.B. Scholey, Antistress, behavioural and magnetoencephalography effects of an L-theanine-based nutrient drink: a randomised, double-blind, placebo-controlled, crossover trial, Nutrients 8 [1][2016]53. 5. ?M.S. Butt, R.S. Ahmad, M.T. Sultan, M.M.N. Qayyum, A. Naz, Green tea and anticancer perspectives: updates from last decade, Crit. Rev. Food Sci. Nutr. 55 [6][2015]792–805. 6. ?V. Crespy, G. Williamson, A review of the health effects of green tea catechins in in vivo animal models, J. Nutr. 134 [12][2004]3431–3440. 7. ?R. Cooper, Green tea and theanine: health benefits, Int. J. Food Sci. Nutr. 6 [3][2012]90–97. 8. ?Juneja LR, ChuDC, Okubo T,Nagato Y and Yokogoshi H, L-Theanine – a unique amino acid of green tea and its relaxation effect in humans. Trends Food Sci Technol 10:199–204 [1999]. 9. ?Balentine DA, Harbowy ME and Graham HN, Tea: The plant and its manufacture; chemistry and consumption of the beverage, in Caffeine, ed. by Spiller GA. CRC Press, Boca Raton, pp. 35–72 [1998]. 10. Chu DC, Green tea – its cultivation, processing of the leaves for drinking materials, and kinds of green tea, in Chemistry and Applications of Green Tea, ed. by Yamamoto T, Juneja LR, Chu DC and Kim M. CRC Press, Boca Raton, pp. 1–11 [1997]. 11. Deng WW, Ogita S and Ashihara H, Biosynthesis of theanine [γ ethylamino-l-glutamic acid]in seedlings of Camellia sinensis. Phytochem Lett 1:115–119 [2008]. 12. Chu DC, Kobayashi K, Juneja LR and Yamamoto T, Theanine – its synthesis, isolation, and physiological activity, in Chemistry and Applications of Green Tea, ed. by Yamamoto T, Juneja LR, Chu DC and Kim M. CRC Press, Boca Raton, pp. 129–135 [1997]. 13. Yamano HandMiyagawa K,Buffer capacity curves of green tea extracts using a personal computer with numerically treated online software. Food Sci Technol Int Tokyo 3:69–73 [2009]. 14. Ekborg-Ott KH, Taylor A and Armstrong DW, Varietal differences in the total and enantiomeric composition of theanine in tea. J Agric Food Chem 45:353–363 [1997]. 15. Vuong VQ, Golding JB, Nguyen M and Roach PD, Extraction and isolation of catechins from tea. J Sep Sci 33:3415–3428 [2010]. 16. Ishizu T, Tsutsumi H and Sato T, Interaction between gallocatechin gallate and caffeine in crystal structure of 1 : 2 and 2 : 2 complexes. Tetrahedron Lett 50:4121–4124 [2009]. 17. Liang H, Liang Y, Dong J, Lu J, Xu H and Wang H, Decaffeination of fresh green tea leaf [Camellia sinensis]byhotwater treatment. Food Chem 101:1451–1456 [2007]. 18 Narukawa M, Morita K and Hayshi Y, L-Theanine elicits an umami taste with inosine 5’-monophosphate. Biosci Biotechnol Biochem 72:3015–3017 [2008]. 19 de Araujo IET, Kringelbach ML, Rolls ET and Hobden P, Representation of umami taste in the human brain. J Neurophysiol 90:313–319 [2003]. 20 Y. Kim, K.L. Goodnera, J.D. Parkb, J. Choib, S.T. Talcott, Changes in antioxidant phytochemicals and volatile composition of Camellia sinensis by oxidation during tea fermentation, Food Chem. 129 [4][2011]1331–1342. 21 W.W. Deng, S. Ogita, H. Ashihara, Biosynthesis of theanine [γ-ethylamino-l-glutamic acid]in seedlings of Camellia sinensis, Phytochem. Lett. 1 [2][2008]115–159. 22 M. Narukawa, Y. Toda, T. Nakagita, Y. Hayashi, T. Misaka, L-Theanine elicits umami taste via the T1R1 + T1R3 umami taste receptor, Amino Acids 46 [6][2014]1583–1587. 23 D.C. Chu, Green tea – its cultivation, processing of the leaves for drinking materials, and kinds of green tea, in: T. Yamamoto, J.R. Lekh, D.C. Chu, M. Kim [Eds.], Chemistry and Applications of Green Tea, CRC Press, Boca Raton, 1997, pp. 1–11. 24 L.R. Juneja, D.C. Chu, T. Okubo, Y. Nagato, H. Yokogoshi, L-theanine—a unique amino acid of green tea and its relaxation effect in humans, Trend Food Sci. Technol. 10 [12][1999]425. 25 K. Boros, N. Jedlinszki, D. Csupor, Theanine and caffeine content of infusions prepared from commercial tea samples, Pharmacogn. Mag. 12 [45][2016]75–79. 26 J. Williams, J. Kellett, P.D. Roach, A. McKune, D. Mellor, J. Thomas, N. Naumovski, L-theanine as a functional food additive: its role in disease prevention and health promotion, Beverages 2 [13][2016], http://dx.doi.org/10. 3390/beverages2020013. 27 Q.V. Vuong, M.C. Bowyer, P.D. Roach, L-theanine: properties, synthesis and isolation from tea, J. Sci. Food Agric. 91 [11][2011]1931–1939. 28 Lichtenstein N, Preparation of c-alkylamides of glutamic acid. J Am Chem Soc 64:1021–1022 [1942]. 29 Kawagishi H and Sugiyama K, Facile and large-scale synthesis of L-theanine. Biosci Biotechnol Biochem 56:689 [1992]. 30 Yan SH, Dufour JP and Meurens M, Synthesis and characterization of highly pure theanine. J Tea Sci 23:99–104 [2003]. 31. Gu H, Jiang Y and Wang J, A practical synthesis of ethyl L-glutamine [L-theanine]. Org Prep Proced Int 36:182–185 [2004]. 32. Zhang F, Zheng QZ, Jiao QC, Liu JZ and Zhao GH, Enzymatic synthesis of theanine from glutamic acid γ -methyl ester and ethylamine by immobilized Escherichia coli cells with γ –glutamyltranspeptidase activity. Amino Acids 39:1177–1182 [2010]. 33. Miyake K and Kakita S, A novel catalytic ability of γ –glutamylcysteine synthetase of Escherichia coli and its application in theanine production. Biosci Biotechnol Biochem 73:2677–2683 [2009]. 34. Yamamoto S, Wakayama M and Tachiki T, Cloning and expression of Methylovorus mays No. 9 gene encoding γ –glutamylmethylamide synthetase: An enzyme usable in theanine formation by coupling with the alcoholic fermentation system of baker’s yeast. Biosci Biotechnol Biochem 72:101–109 [2008]. 35. Zhou X, Zhang Z, Jia X, Wu Y, Luo L and Yin Z, Mn2+ enhances theanine-forming activity of recombinant glutamine synthetase from Bacillus subtilis in Escherichia coli. World J Microb Biotechnol 24:1267–1272 [2008]. 36. Suzuki H, Izuka S, Miyakawa N and Kumagai H, Enzymatic production of theanine, an ‘umami’ component of tea, from glutamine and ethylamine with bacterial γ -glutamyltranspeptidase. Enzyme Microb Technol 31:884–889 [2002]. 37. Zhang F, Zheng QZ, Jiao QC, Liu JZ and Zhao GH, Synthesis of theanine from glutamic acid γ -methyl ester and ethylamine catalyzed by Escherichia coli having γ –glutamyltranspeptidase activity. Biotechnol Lett 32:1147–1150 [2010]. 38. G.W. Varilek, F. Yang, E.Y. Lee, W.J.S. deVilliers, J. Zhong, H.S. Oz, K.F. Westberry, C.J. McClain, Green tea polyphenol extract attenuates inflammation in interleukin-2-difeicient mice, a model of autoimmunity, J. Nutr. 131 [7][2001]2034–2039. 39. S.M. Chacko, P.T. Thambi, R. Kuttan, I. Nishigaki, Beneficial effects of green tea: a literature review, Chin. Med. 5 [2010]13. 40 Cooper R, Morr′e DJ and Morr′e DM, Medicinal benefits of green tea: Part I. Review of noncancer health benefits. J Altern Complement Med 11:521–528 [2005]. 41 Mason R, 200 mg of Zen: L-theanine boosts alpha waves, promotes alert relaxation. Altern Complement Ther 7:91–95 [2001]. 42. Nobre AC, Rao A and Owen GN, L-theanine, a natural constituent in tea, and its effect on mental state. Asia Pac J Clin Nutr 17:167–168 [2008]. 43. Lu K, Gray MA, Oliver C, Liley DT, Harrison BJ, Bartholomeusz CF, et al, The acute effects of L-theanine in comparison with alprazolam on anticipatory anxiety in humans. Hum Psychopharmacol Clin 19:457–465 [2004]. 44. Haskell CF, Kennedy DO, Milne AL, Wesnes KA and Scholey AB, The effects of L-theanine, caffeine and their combination on cognition and mood. Biol Psychol 77:113–122 [2008]. 45 Owen GN, Parnell H, Bruin EAD and Rycroft JA, The combined effects of L-theanine and caffeine on cognitive performance and mood. Nutr Neurosci 11:193–198 [2008]. 46. Di X, Yan J, Zhao Y, Zhang J, Shi Z, Chang Y, et al, L-Theanine protects the APP [Swedish mutation]transgenic SH-SY5Y cell against glutamate-induced excitotoxicity via inhibition of the NMDA receptor pathway. Neuroscience 18:778–786 [2010]. 47. Liu Q, Duan H, Luan J, Yagasaki K and Zhang G, Effects of theanine on growth of human lung cancer and leukemia cells as well as migration and invasion of human lung cancer cells. Cytotechnology 59:211–217 [2009]. 47. Friedman M, Mackey BE, Kim HJ, Lee IS, Lee KR, Lee SU, et al, Structure–activity relationships of tea compounds against human cancer cells. J Agric Food Chem 55:243–253 [2007]. 48. Sugiyama T and Sadzuka Y, Theanine, a specific glutamate derivative in green tea, reduces the adverse reactions of doxorubicin by changing the glutathione level. Cancer Lett 212:177–184 [2004]. 49. Sadzuka Y, Sugiyama T, Suzuki T and Sonobe T, Enhancement of the activity of doxorubicin by inhibition of glutamate transporter. Toxicol Lett 123:159–167 [2001]. 50. Yokogoshi H and Kobayashi M, Hypotensive effect of γ -glutamylmethylamide in spontaneously hypertensive rats. Life Sci 62:1065–1068 [1998]. 51. Rogers PJ, Smith JE, Heatherley SV and Pleydell-Pearce CW, Time for tea: Mood, blood pressure and cognitive performance effects of caffeine and theanine administered alone and together. Psychopharmacology 195:569–577 [2007]. 52. Kurihara S, Shibahara S, Arisaka H and Akiyama Y, Enhancement of antigen-specific immunoglobulin G production in mice by co-administration of L-cystine and L-theanine. J Vet Med Sci 69:1263–1270 [2007]. 53. Takagi Y, Kurihara S, Higashi N, Morikawa S, Kase T, Maeda A, et al, Combined administration of L-cystine and L-theanine enhances immune functions and protects against influenza virus infection in aged mice. J VetMed Sci 72:157–165 [2010].

Biological Activity

Amino acid analog of glutamine and component of green tea. Shown to bind to AMPA, Kainate, NMDA and group I mGlu receptors. Displays neuroprotective effects in vivo . Promotes self-renewal of human embryonic stem cells (hESC).

Biochem/physiol Actions

Theanine is able to bind to AMPA, kainite, and NMDA glycine receptors in rat cortical neurons but with less affinity than glutamic acid. It has been studied as a glutamate transport inhibitor, preventing glutamate uptake by M5076 ovarian sarcoma-bearing mouse cells and increasing the effect of doxorubicin on tumor growth in M5076 mice.

Chemical Structure and Natural Occurrence

Theanine exists in two isomers, L-form, and D-form, with only the L-form predominantly found in nature, particularly in tea plants. It shares a similar structure with glutamine and glutamate.

Neurotransmitter Regulation

Theanine has been recognized for its regulatory role in neurodegeneration. It binds to three glutamate receptor subtypes (NMDA, AMPA, and KA), which are excitatory neurotransmitters involved in fast synaptic transmission in the central nervous system.

Health Benefits

Theanine is associated with various health benefits, including improvements in mood, cognition, stress reduction, and anxiety relief. Supplementation of 200鈥?400 mg/day of L-theanine may assist in reducing stress and anxiety symptoms. Theanine exhibits antioxidant, anti-inflammatory, neuroprotective, anti-cancer, metabolic regulatory, cardiovascular protective, liver and kidney protective, and immune regulatory effects.

Flavor Component in Tea

L-theanine contributes to the unique taste of tea, providing a caramel flavor and alleviating the bitterness of caffeine. It is a significant index for estimating the freshness of tea and contributes to the generation of tea volatiles, imparting desirable aromas.

Applications and Distribution in Tea Plants

Due to its flavor and health benefits, L-theanine finds wide applications as a beverage ingredient or dietary supplement. It is distributed in different parts of tea plants, with higher contents in roots and leaves compared to stems.

Anticancer Properties

Studies suggest that theanine possesses anticancer effects by inhibiting various pathways involved in cancer cell proliferation, apoptosis, metastasis, migration, and invasion. Further research is needed to validate its effectiveness in cancer prevention and treatment.

Definition

ChEBI: A N5-alkylglutamine where the alkyl group is ethyl. It has been isolated from green tea.