Sleep disturbances encompass numerous disorders which have detrimental impacts on the quality of life. Sleep problems, including difficulty in falling asleep, decreased sleep duration, and inconsistent sleep/wake patterns affect 56% of people in the United States, 31% in Western Europe, 23% in Japan (1), and are a significant cause of morbidity and mortality (2). Although the importance of having a good sleep is well-established, most people suffering from low-quality sleep do not look for medication since many hypnotics and sedatives have side effects. Hypnotic drugs such as benzodiazepines are associated with memory impairment, dementia, depression, and cause dependency and withdrawal phenomena in chronic usage (3). Despite non-benzodiazepine sedatives having lesser serious side effects than benzodiazepines, they have the potential to cause anterograde amnesia, headache, dizziness, and unpleasant taste (4–7). Hence, it is required to develop new therapeutic agents with lesser side effects but as effective as hypnotics, e.g., L-theanine and GABA.

L-Theanine, a non-proteinogenic amino acid particularly found in green tea, is a well-known agent for improving sleep disturbances (8, 9). It is structurally similar to the excitatory neurotransmitter glutamate in the brain and possibly blocks glutamate receptors in the central nervous system (10). It is commonly used to treat sleep disturbance, improve non-rapid eye movement (NREM) sleep, and reduce psychological stress. Previous studies suggest that L-theanine exerts its relaxant effect by enhancing GABA levels, thereby increasing the expression of dopamine and serotonin in the brain (11–13). In animal studies, L-theanine was shown to oppose caffeine’s effect and promote sedation (11, 14–16). In addition to its relaxant potency, L-theanine has a neuroprotective role since it acts as a glutamate receptor antagonist, upregulates GABA receptors, and increases the expression of antioxidant enzymes (17–20).

Apart from L-theanine, magnesium is also a glutamate receptor antagonist and may play a critical role in regulating sleep (21). Previous studies on rodents indicated that Mg²⁺ ions serve as a critical signal for synapse formation and increased intraneuronal Mg²⁺ concentration was associated with an increase in synaptic density and plasticity in the prefrontal cortex and hippocampus in young and old rats (22–24), as well as enhancement of short/long-term memory, reduction of anxiety, reduction in depression (25–27). Besides, increased Mg²⁺ via various substances was suggested to improve memory of patients with Alzheimer’s disease by inhibiting the neuroinflammation (22, 28, 29). Moreover, molecular and animal studies have shown that Mg²⁺ pretreatment shows neuroprotective effects (30, 31). Those neuroprotective effects were attributed to Mg²⁺’s antagonistic interaction with NR2B-containing NMDA receptors, and therefore prevention of excitotoxicity.

Magnesium L-theanine is a novel compound consisting of Mg²⁺ ions and L-theanine molecules. It readily passes to the blood–brain barrier, increases the Mg²⁺ ion levels of CSF, and has a potential sleep-regulating effect similar to L-theanine (32, 33). Although magnesium and L-theanine have numerous effects on CNS and mood-related disorders separately, there has been no research concerning the effects of Mg-L-theanine compounds on sleep and brain activity. Hence, in this study, we aimed to determine the potency of different Mg-L-theanine compounds with different magnesium concentrations on sleep latency and duration, brain electrical activity, neuronal activity, and antioxidant parameters compared to L-theanine in caffeine-induced sleep disturbance and pentobarbital induced sleep models in mice.

Based on the previous research showing the improvement of L-theanine on sleep disturbance and its neuroprotective role, we examined the effects of a novel anxiolytic and cognition-enhancing agent Mg-L-theanine with different Mg²⁺ concentrations on sleep quality and brain chemical activity in comparison with L-theanine. We first analyzed the ECoG recordings to evaluate the brain waves, spike frequency, and spike amplitude. Although we did not see a general improvement in spike frequency and spike amplitude with L-theanine or Mg-T compounds on the caffeine-induced sleep disturbance model, we observed that in the Mg-T1 group, there had been a significant reduction in spike frequency after 90 min of recordings. According to PSD analyses, Mg-L-theanine with higher Mg²⁺ concentration has significantly boosted the delta waves at the 50th minute, and both Mg-L-theanine compounds have exhibited the same enhancing role at the 100th minute. Sleep is broken down into N1, N2, N3, and R stages (35), where the delta waves are high-amplitude slow brain waves, seen in N2 and N3, which correlates with sleep intensity in humans (36). Likewise, delta waves dominate NREM sleep in rodents (37) and are shown to be very important for memory consolidation by reactivating encoded neuronal memory and promoting their transference into long-term memory (38). Therefore, the enhancing effects of Mg-L-theanine compounds on delta waves may resolve insomnia, possibly intensify deep sleep stages, and regulate synaptic networks. We also performed a similar analysis for higher brain waves, in which we did not see a significant outcome. In contrast to the literature, we could not observe an improvement of L-theanine on the alpha power in our model, in which the mice were anesthetized with urethane and the recording electrodes were placed on the somatosensory cortex.

We next examined the levels of different inhibitory receptors, glutamate receptors, and sedative neurotransmitters, which are the key components of sleep homeostasis. GABAergic neurons are the main inhibitory neurons in sleep-inducing neural pathways, which naturally regulate wake-promoting circuitry (39). There are three types of GABA receptors, GABA_A, GABA_B, and GABA_C. GABA_A receptors are fast-acting ligand-gated ion channels (40), which are the most abundant receptors seen in GABAergic neurons. They are well-known for regulating sleep-inducing circuits and are targeted to treat sleep-related problems. In fact, most sedative-hypnotic drugs (barbiturates and benzodiazepines) act through the GABA_A receptors (41). GABA_B receptors, on the other hand, are G protein-coupled receptors that mediate slow and prolonged inhibitory action through indirectly activating K⁺ channels, inactivating Ca²⁺ channels, and decreasing cyclic adenosine monophosphate (cAMP) levels (42). They are found to be a target for sleep problems in schizophrenia since GABA_B receptor agonists induce slow-wave sleep (SWS), although they have minimal impact on (rapid-eye movement) REM sleep (43). Herein, we found that caffeine induces insomnia through inhibition of GABA_A and GABA_B receptors while L-theanine and Mg-T compounds increase their expressions. In literature, L-theanine has been shown to stimulate GABA_A receptors directly (18) and indirectly by increasing the levels of GABA and thereby opposing caffeine’s effect (44). Likewise, certain concentrations of Mg²⁺ potentiate the effect of GABA on GABA_A receptors (45). We found that Mg-T compounds are more prominent in terms of increasing the level of GABA receptors, thereby promoting sleep, compared to L-theanine. Higher GABA receptor levels with Mg-T compounds can be explained by a synergistic effect of Mg²⁺ ions and L-theanine molecules. Furthermore, we observed the inhibitory serotonin receptor 5-HT_1A levels were increased with Mg-T compounds after the caffeine-induced decline. As shown in previous studies, 5-HT_1A receptors have a complex regulatory role on sleep. Their activation promotes waking status, induces SWS, or stimulates REM sleep depending on where 5-HT_1A receptors are located (46). Besides, serotonergic system dysfunctionality – reduction in synthesis, release, and metabolism of 5-HT and reduction in pre/postsynaptic density of 5-HT_1A receptors – mainly associated with mood and anxiety disorders (47). Thus, activation of the receptor by agonists shows an anxiolytic effect as well as an increase in SWS depending on the dose and location of delivery (48, 49). This sleep-inducing effect is further attributed to the activation of inhibitory 5-HT_1A autoreceptors located in Dorsal Raphe Nuclei (49). Hence, based on the other findings in our study, Mg-T compounds possibly induce sleep and reduce anxiety through inhibitory serotonin receptors as well as GABA receptors.

Animal studies suggest that L-theanine administration increases brain serotonin and dopamine levels (50). Concordantly, we observed that Mg-T and L-theanine administrations restore serotonin, dopamine as well as melatonin levels after caffeine-induced decline while higher activity belongs to Mg-T compounds. Melatonin is a naturally produced hormone by the pineal gland, regulates the sleep-wake cycle, and improves sleep quality, onset, and duration. Therefore, with its sleep-regulatory role, melatonin is frequently used as a therapeutic for insomnia in clinics. As Mg-T compounds increase melatonin levels in the brain, they may be used for the treatment of insomnia. Serotonin, on the other hand, is the precursor of melatonin, indicating a possible sleep-inducing role by increasing melatonin levels and activating inhibitory neurons. Blocking the synthesis of serotonin has been shown to reduce SWS in rats while administration of 5-HTP or L-Tryptophan restores the sleep back (51). We also observed an increase in dopamine levels which is mediated by stimulation of NMDA receptors (33), while its effect on sleep remains unclear, although in previous research, dopamine fluctuations were shown during sleep, and a peak in dopamine release was seen to occur just after sleep onset during the light phase (52).

Conversely, glutamate receptors are the main excitatory receptors in the central nervous system (53). Recent data have shown that those receptors may have a role in sleep regulation. Miracca et al. (54) showed that the deletion of the GluN1 NMDA receptor subunits creates highly inconsistent sleep-wake patterns and dampen REM sleep, indicating insomnia. They also pointed out that the NMDA glutamate receptor signaling is important in the firing of GABAergic sleep-related neurons. Here, consistent with the prior research, we observed that caffeine-induced sleep deprivation significantly decreases the GluN1, GluN2a, and GluN2b levels. However, Mg-T compounds together with L-theanine ameliorated those subunit levels. Previous studies suggest that L-theanine improves cognitive functions by direct affinity to AMPA and NMDA (55), mediating serotonin and dopamine release (50), and indirectly relieving stress and anxiety as we showed in this study. Therefore, increased levels of NMDA and AMPA receptor subunits both after Mg-T and L-theanine administrations support GABA receptors’ sleep-inducing activity.

In order to confirm our findings on receptor and neurotransmitter level alterations which were shown to be effective on sleep regulation, we performed a sleep induction test by pentobarbital administration after compounds were given with or without caffeine. Parallel to the previous studies (11), L-theanine not only reduced the sleep latency but also prolonged the sleep duration in the pentobarbital-induced sleep model. Besides, the Mg-T2 compound had the highest activity on increasing sleep duration and decreasing the time required to fall asleep. Moreover, caffeine reduces sleep duration and increases sleep latency of pentobarbital induced sleep. However, Mg-T compounds alleviate caffeine’s effect with the same efficacy. These results show that Mg-T compounds are potential molecules to increase the sleep quality of individuals who has insomnia and may diminish the side effects of pentobarbital or other hypnotic drugs.

In this study, apart from the sleep induction potencies, we also examined the effects of L-theanine and Mg-T compounds on antioxidant enzymes. Previous studies pointed out that antioxidant enzymes such as SOD, CAT, and GPx are crucial for protecting mitochondria from oxidative stress since the production of reactive species leads to mitochondrial damage, which is a key factor in the occurrence of neurodegenerative diseases (56). We found that L-theanine increases the activity of SOD, CAT but not GPx, which are similar to the findings of Zeng et al. (57). Zeng et al. indicate that L-theanine creates a significant difference in rats’ GPx levels at high doses (400 mg/kg) after brain damage. Moreover, we observed that Mg-T molecules increase the level of SOD and CAT after caffeine’s decreasing effect higher than L-theanine while they have a similar effect on GPx. In contrast to antioxidant enzyme activities, MDA, a highly active oxidative stress marker, decreases with L-theanine and Mg-T compounds while the highest effect belongs to Mg-T2 both on antioxidant enzymes and MDA levels. Thus, Mg-T molecules may exert neuroprotection by increasing the antioxidants after an oxidative stress condition such as cerebral ischemia. As shown in previous studies (58, 59), increased levels of melatonin and serotonin with Mg-T administration could further contribute antioxidant effect of novel L-theanine forms since those neurotransmitters decrease oxidative stress and play an important role in reactive oxygen species scavenging. However, a detailed study should be conducted on the protective role of Mg-T on neurodegeneration.

According to previous research, caffeine was shown to increase Ca²⁺ concentration in the vascular smooth muscle cells (VSMCs) through the cAMP pathway (60), by which it was found to increase the eNOS levels. Siamwala et al. (61) have also shown that L-theanine induces eNOS phosphorylation and increases nitric oxide (NO) production in endothelial cells, indicating possible protection against cerebrovascular diseases. Consistent with the literature, we observed an increase in eNOS levels after caffeine administration. However, contradicting the previous data, a decrease in eNOS levels was detected after L-theanine and Mg-T administrations. It is also indicated that caffeine increases the iNOS levels under basal conditions, whereas it decreases the iNOS expression after induction of inflammation by lipopolysaccharide (LPS) and IFN-γ (62, 63). Here, we observed that iNOS levels were enhanced after sole caffeine injection, similar to the previous results, while L-theanine and Mg-T injection after caffeine administration recovered that effect and reversed the increased iNOS levels. Given that hypoxic conditions such as stroke induce synthesis of NO by iNOS and this leads to exacerbation of the injury (64), Mg-L-theanine compounds may help reduction of inflammation by their anti-inflammatory role.

In conclusion, our present study shows that L-theanine reverses the effects of caffeine on sleep disturbance, sleep disturbance-related brain chemistry alterations, and the caffeine’s effects on oxidative stress, where L-theanine seems to protect against neurodegenerative disorders. Our data further indicate that Mg-L-theanine compounds with different Mg²⁺ ratios, novel L-theanine agents, show even better improvement on sleep disturbance and sleep disturbance-related neurochemical changes. Therefore, here we provided a possible neurochemical mechanism of the Mg-T compounds that are responsible for their sleep-inducing role. Furthermore, we showed the antioxidant characteristics of Mg-L-theanine molecules which can be helpful to prevent acute neurodegenerative disorders. In addition, in the present study, to visualize and analyze the effects of studied compounds on brain spike activity, we conducted ECoG recordings by placing two Ag/Ag-Cl electrodes directly on the cortex while mice were under anesthesia. The reason we have chosen to record animals under anesthesia was to ensure that data are not affected by movements and myoelectrical artifacts and the allowance of urethane for prolonged recordings. Thus, continuous EEG recording can be conducted to understand the effect of Mg-T molecules on brain waves while mice are falling asleep in more physiological conditions and under influence of circadian rhythm.

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.

The animal study was reviewed and approved by the Istanbul Medipol University, Animal Research Ethics Committee.

MD, KS, and EK designed all the experiments. MD, SE, BK, SC, and HM carried out the animal experiments and performed the statistical analysis. MB, CO, MT, and NS conducted the Western blot and ELISA experiments. SS, JK, SO, KS, and EK executed the compound synthesis. EK, KS, and SE prepared the manuscript. All authors contributed to the article and approved the submitted version.

SS, JK, and SO were employed by Nutrition21, LLC. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.