Hypnotic Effects of Lactobacillus fermentum PS150TM on Pentobarbital-Induced Sleep in Mice

The bidirectional communication between the gastrointestinal tract and the central nervous system appears to be functionally linked to the intestinal microbiome, namely the microbiome–gut–brain axis (MGBA). Probiotics with health benefits on psychiatric or neurological illnesses are generally called psychobiotics, and some of them may also be able to improve sleep by targeting the MGBA. This study aimed to investigate the effects of a psychobiotic strain, Lactobacillus fermentum PS150TM (PS150TM), on sleep improvement by using a pentobarbital-induced sleep mouse model. Compared with the vehicle control group, the oral administration of PS150TM, but not the other L. fermentum strains, significantly decreased the sleep latency and increased the sleep duration of mice, suggesting strain-specific sleep-improving effects of PS150TM. Moreover, the ingestion of diphenhydramine, an antihistamine used to treat insomnia, as a drug control group, only increased the sleep duration of mice. We also found that the sleep-improving effects of PS150TM are time- and dose-dependent. Furthermore, the oral administration of PS150TM could attenuate a caffeine-induced sleep disturbance in mice, and PS150TM appeared to increase the expression of the gene encoding the adenosine 1 receptor in the hypothalamus of mice, as assessed by quantitative real-time polymerase chain reaction. Taken together, our results present a potential application of PS150TM as a dietary supplement for sleep improvement.


Introduction
Insomnia is a common disorder characterized by difficulty falling asleep and hardly maintaining sleep or waking up too early. Approximately 20-30% of adults have chronic insomnia problems (i.e., with a duration of at least 1 month), which is the most prevalent sleep disorder in the general population [1]. It is usually accompanied by psychiatric or physical diseases such as impaired attention, irritability, restlessness, anxiety, and stress or fatigue during wakefulness [2]. Insomnia also increases the risk of chronic diseases (e.g., hypertension, diabetes, stroke, and coronary artery diseases) as well as healthcare costs [3,4]. Although different drugs are used to treat insomnia, most of them are not recommended for long-term use because of their potential adverse reactions [3,5]. Thus, a search for alternative ways to treat insomnia is needed. Insomnia and gastrointestinal dysfunction have been that did not fall asleep within 15 min were excluded from the experiment. Sacrifice was performed 2 h after the end of the experiment, and the mouse brain was quickly removed and temporarily put on dry ice. After that, the brain area of interest was taken out and stored at −80 • C. The orally administrated diphenhydramine hydrochloride (DIPH, 20 mg/mL, Sigma), an antihistamine with sleep aid effects, was used in the drug control group. Caffeine (15 mg/mL, Sigma) was intraperitoneally injected 30 min before the pentobarbital-induced sleep test to induce sleep disturbance in mice [21,22].

Open Field Test
The locomotor activity of the mice was examined using the open field test as previously described [23]. In brief, each mouse was placed in an arena with Plexiglas walls (25.4 × 25.4 × 38 cm 3 ) with photobeam sensors to record locomotor activities for 10 min (Tru Scan Activity System; Coulbourn Instruments, Whitehall, PA, USA). The center area was defined as a region (12.5 × 12.5 cm 2 ) in the center of the arena. The box was cleaned with 70% ethanol after each test. Increasing time spent in the central square and the ratio of central to total locomotion are considered indications of anxiolysis [24].

Genomic DNA Extraction and PCR Analysis of L. fermentum Strains
L. fermentum strains were cultured in MRS broth overnight. Bacterial cells were collected by centrifugation at 12,000× g for 5 min. The genomic DNA was extracted as previously described [25]. The purity of genomic DNA was evaluated in terms of the A260/A280 ratio with a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). To discriminate the three L. fermentum strains, four PCR-based genomic fingerprinting methods were used, which include BOX-, (GTG) 5 -, enterobacterial repetitive intergenic consensus (ERIC)-, and random amplification of polymorphic DNA (RAPD)-PCR ( Table 1). The PCR products were analyzed by electrophoresis using 2% agarose gel with a 1× TBE (tris-borate-EDTA) buffer. The gel was stained with ethidium bromide and observed under an ultraviolet transilluminator. A 100 bp ladder (Omics Bio, New Taipei city, Taiwan) was included as a molecular weight marker.

Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) Analysis
The total RNA in the brain tissue was extracted by using an RNeasy mini kit (Qiagen, Germantown, MD, USA) [28] and converted into cDNA using a RevertAid First Strand cDNA Synthesis kit (ThermoFisher, Waltham, MA, USA). The cDNA samples in each group were diluted 20-fold with DNase-free water and were subjected to two independent repetitions of real-time PCR with specific primers (Table 1) and KAPA SYBR FAST ABI Prism (KAPA Biosystems, Woburn, MA, USA) using the StepOnePlus™ Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The cycling conditions of qRT-PCR were 95 • C for 3 min; up to 40 cycles of 95 • C for 3 s, 60 • C for 30 s; and the melt curve stage was 95 • C for 15 s, 60 • C for 30 s, and 95 • C for 15 s. The target threshold cycle (Ct) was subtracted from the Ct for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to calculate ∆Ct, and a relative quantification analysis was performed via the 2 −∆∆CT method [29].

Statistical Analysis
Data were analyzed using GraphPad Prism 5.1 (GraphPad Software) and represented as the mean ± standard error of the mean (SEM). For multiple comparisons, we used a one-way analysis of variance (ANOVA) with Tukey's post hoc test. A p-value < 0.05 was considered significant in all cases.

Strain-Specific Effects of PS150 TM on Pentobarbital-Induced Sleep in Mice
The health benefits of probiotics are generally considered to be strain-specific. To examine if PS150 TM harbors a sleep-improving effect with strain-specificity, a pentobarbital-induced sleep mouse model was used, and the activity of different L. fermentum strains was evaluated ( Figure 1a). Compared with the vehicle control (Veh) group, the oral administration of PS150 TM for 14 consecutive days significantly decreased the sleep latency (n = 10~12) and increased the sleep duration (n = 10~12) (Figure 1b,c); the recovery time (n = 4~5) was also decreased, but not significantly ( Figure 1d). However, the other two L. fermentum strains, PCC (a commercial probiotic for gastrointestinal health) and ATCC 14931 T (the taxonomic type strain of L. fermentum), did not show any sleep-improving effects. Moreover, the oral administration of a well-known antihistamine drug used as a sleep aid, diphenhydramine (DIPH), only showed increased sleep duration, but no improvement in sleep latency or recovery time was observed on day 14 of our experiment, which is consistent with previous findings [30,31].
To determine whether the L. fermentum strains under investigation have similar genetic backgrounds, three types of rep-PCR (ERIC, BOX, and (GTG) 5 ) and an RAPD-PCR were performed, and the PCR products were resolved by DNA electrophoresis. As shown in Figure 2, PS150 TM and PCC appeared to have a similar genetic background, while we observed distinguishable PCR-fingerprinting profiles between the two strains and ATCC 14931 T , especially in the ERIC-PCR profiles, suggesting two lineages of these L. fermentum strains. It is noteworthy that although PS150 TM and PCC appeared to be classified into the same lineage, only PS150 TM showed sleep-improving effects in our experiment.

PS150 TM Improved Pentobarbital-Induced Sleep in Mice in a Time-and Dose-Dependent Manner
To investigate the effects of PS150 TM supplementation with different intervention times and daily dosages on sleep improvement, mice were orally administrated 10 8 or 10 9 CFU/day of PS150 TM for 14 consecutive days, and the pentobarbital-induced sleep tests were performed on days 1, 4, 7, and 14 ( Figure 3). Compared with the vehicle control (Veh) group, the oral administration of PS150 TM of 10 9 CFU/day significantly decreased the sleep latency (n = 8~10) on days 7 and 14, increased the sleep duration (n = 8~10) on days 4, 7, and 14, and decreased the recovery time (n = 5~7) on day 14, suggesting that the intervention time was critical for the sleep-improving effects to occur. However, treatment with a lower dosage (10 8 CFU/day) did not affect the sleep pattern of mice. The oral gavage of DIPH to the drug control group 30 min before the pentobarbital-induced sleep test resulted in significantly increased sleep duration on all tested days compared with the Veh group ( Figure 3b). Nevertheless, no significant difference in sleep latency or recovery time between the DIPH group and the Veh group was found (Figure 3a,c). (Figure 1b,c); the recovery time (n = 4~5) was also decreased, but not significantly ( Figure 1d). However, the other two L. fermentum strains, PCC (a commercial probiotic for gastrointestinal health) and ATCC 14931 T (the taxonomic type strain of L. fermentum), did not show any sleep-improving effects. Moreover, the oral administration of a well-known antihistamine drug used as a sleep aid, diphenhydramine (DIPH), only showed increased sleep duration, but no improvement in sleep latency or recovery time was observed on day 14 of our experiment, which is consistent with previous findings [30,31].  To determine whether the L. fermentum strains under investigation have similar genetic backgrounds, three types of rep-PCR (ERIC, BOX, and (GTG)5) and an RAPD-PCR were performed, and the PCR products were resolved by DNA electrophoresis. As shown in Figure 2, PS150 TM and PCC appeared to have a similar genetic background, while we observed distinguishable PCRfingerprinting profiles between the two strains and ATCC 14931 T , especially in the ERIC-PCR profiles, suggesting two lineages of these L. fermentum strains. It is noteworthy that although PS150 TM and PCC appeared to be classified into the same lineage, only PS150 TM showed sleep-improving effects in our experiment.

PS150 TM Improved Pentobarbital-Induced Sleep in Mice in a Time-and Dose-Dependent Manner
To investigate the effects of PS150 TM supplementation with different intervention times and daily dosages on sleep improvement, mice were orally administrated 10 8 or 10 9 CFU/day of PS150 TM for 14 consecutive days, and the pentobarbital-induced sleep tests were performed on days 1, 4, 7, and 14 ( Figure 3). Compared with the vehicle control (Veh) group, the oral administration of PS150 TM of 10 9 CFU/day significantly decreased the sleep latency (n = 8~10) on days 7 and 14, increased the sleep duration (n = 8~10) on days 4, 7, and 14, and decreased the recovery time (n = 5~7) on day 14,  Nevertheless, no significant difference in sleep latency or recovery time between the DIPH group and the Veh group was found (Figure 3a,c).

Effects of PS150 TM on Caffeine-Induced Sleep Disruption in Mice
To further investigate the potential of PS150 TM as a sleep-improving dietary supplement, the effects of PS150 TM on caffeine-induced sleep disturbance in mice were evaluated. Mice were orally administrated PBS or PS150 TM (10 9 CFU/day) for 28 consecutive days and then intraperitoneally injected with PBS or caffeine (15 mg/kg), subjected to an open field test for 10 min, and then subjected

Effects of PS150 TM on Caffeine-Induced Sleep Disruption in Mice
To further investigate the potential of PS150 TM as a sleep-improving dietary supplement, the effects of PS150 TM on caffeine-induced sleep disturbance in mice were evaluated. Mice were orally administrated PBS or PS150 TM (10 9 CFU/day) for 28 consecutive days and then intraperitoneally injected with PBS or caffeine (15 mg/kg), subjected to an open field test for 10 min, and then subjected to a pentobarbital-induced sleep test. The oral gavage of DIPH (20 mg/kg) was used as a drug control group (Figure 4a). As shown in Figure 4b, the oral administration of PS150 TM to mice, either with or without the caffeine injection, significantly decreased the sleep latency (n = 13~15) (p < 0.05), while no significant effect of caffeine and DIPH was observed. Furthermore, compared with the Veh group, both the DIPH and the PS150 TM groups showed increased sleep duration (n = 13~15) (Figure 4c); the caffeine injection decreased the sleep duration, which could be attenuated by the supplementation of PS150 TM , but not DIPH, implying the stronger sleep-improving effects of PS150 TM . Furthermore, the supplementation of PS150 TM significantly decreased the recovery time only in mice without caffeine injection (p <0.05), and no other statistically significant differences were found (n = 4~6) (Figure 4d). These results indicate that the daily supplementation of PS150 TM for 28 days attenuated the caffeine-induced sleep disturbance in mice. Nevertheless, this sleep-improving effect of PS150 TM could not be observed on experimental day 14 (data not shown), supporting the notion that the sleep-improving effects of PS150 TM are time-dependent. to a pentobarbital-induced sleep test. The oral gavage of DIPH (20 mg/kg) was used as a drug control group ( Figure 4a). As shown in Figure 4b, the oral administration of PS150 TM to mice, either with or without the caffeine injection, significantly decreased the sleep latency (n = 13~15) (p < 0.05), while no significant effect of caffeine and DIPH was observed. Furthermore, compared with the Veh group, both the DIPH and the PS150 TM groups showed increased sleep duration (n = 13~15) ( Figure 4c); the caffeine injection decreased the sleep duration, which could be attenuated by the supplementation of PS150 TM , but not DIPH, implying the stronger sleep-improving effects of PS150 TM . Furthermore, the supplementation of PS150 TM significantly decreased the recovery time only in mice without caffeine injection (p <0.05), and no other statistically significant differences were found (n = 4~6) (Figure 4d).
These results indicate that the daily supplementation of PS150 TM for 28 days attenuated the caffeineinduced sleep disturbance in mice. Nevertheless, this sleep-improving effect of PS150 TM could not be observed on experimental day 14 (data not shown), supporting the notion that the sleep-improving effects of PS150 TM are time-dependent.

PS150 TM Did Not Alter the Spontaneous Locomotor Activity of Mice in the Open Field Test
As shown in Figure 4a, a 10-min open field test was performed to analyze the effects of PS150 TM on the locomotor activity of mice with or without the intraperitoneal injection of caffeine (n = 8~10). The drug control group of DIPH, orally administrated only on experimental day 28, was also included in the test. The open field test indicated that, compared with the (Veh + Caffeine) group, the (DIPH + Caffeine) group showed increased time spent in the central square ( Figure 5a) and increased total distance (Figure 5b), while no other significant differences were observed. PS150 TM did not alter the locomotor activity of the mice.

PS150 TM Did Not Alter the Spontaneous Locomotor Activity of Mice in the Open Field Test
As shown in Figure 4a, a 10-min open field test was performed to analyze the effects of PS150 TM on the locomotor activity of mice with or without the intraperitoneal injection of caffeine (n = 8~10). The drug control group of DIPH, orally administrated only on experimental day 28, was also included in the test. The open field test indicated that, compared with the (Veh + Caffeine) group, the (DIPH + Caffeine) group showed increased time spent in the central square ( Figure 5a) and increased total distance (Figure 5b), while no other significant differences were observed. PS150 TM did not alter the locomotor activity of the mice.

Effects of PS150 TM on the Expression of Genes Related to Adenosine Signaling in the Basal Forebrain and Hypothalamus
The inhibitory neurotransmitter adenosine is involved in sleep regulation, and the elevation of extracellular adenosine appears to be a crucial mechanism to increase sleep duration and electroencephalography (EEG) slow-wave activity [32,33]. The concentration of adenosine changes during spontaneous sleep in some sleep-related brain regions, including the basal forebrain, the preoptic area of the hypothalamus, etc. [33,34]. To explore whether PS150 TM alters sleep patterns by affecting the sleep-related pathways of mice undergoing the pentobarbital-induced sleep test, we used qRT-PCR to analyze the expression of genes encoding adenosine receptors A1 (A1R) and A2A (A2AR) as well as NT5e, the nucleotidase that synthesizes adenosine ( Figure 4a). As shown in Table  2, compared with the Veh group, the oral administration of PS150 TM for 28 consecutive days significantly increased the expression of A1R in the hypothalamus (p < 0.05) of the mice that were not subjected to the caffeine treatment. However, no other significant differences were observed in our experiment. The experimental design is shown in Figure 4a; the expression of genes was analyzed in the basal forebrain (n = 4~5) and hypothalamus (n = 6~8) of mice. Data are expressed as mean ± SEM and were analyzed by one-way ANOVA with Tukey's post hoc test. * p <0.05, compared with the Veh group.  Figure 4a; a 10-min open field test was performed after the injection of PBS or caffeine (15 mg/kg). PS150 TM was orally administrated for 28 consecutive days. DIPH (20 mg/kg), as a drug control group, was introduced on experimental day 28. Data are expressed as mean ± SEM (n = 8~10) and were analyzed by one-way ANOVA with Tukey's post hoc test. * p < 0.05, compared with the indicated groups.

Effects of PS150 TM on the Expression of Genes Related to Adenosine Signaling in the Basal Forebrain and Hypothalamus
The inhibitory neurotransmitter adenosine is involved in sleep regulation, and the elevation of extracellular adenosine appears to be a crucial mechanism to increase sleep duration and electroencephalography (EEG) slow-wave activity [32,33]. The concentration of adenosine changes during spontaneous sleep in some sleep-related brain regions, including the basal forebrain, the preoptic area of the hypothalamus, etc. [33,34]. To explore whether PS150 TM alters sleep patterns by affecting the sleep-related pathways of mice undergoing the pentobarbital-induced sleep test, we used qRT-PCR to analyze the expression of genes encoding adenosine receptors A 1 (A 1 R) and A 2A (A 2A R) as well as NT5e, the nucleotidase that synthesizes adenosine ( Figure 4a). As shown in Table 2, compared with the Veh group, the oral administration of PS150 TM for 28 consecutive days significantly increased the expression of A 1 R in the hypothalamus (p < 0.05) of the mice that were not subjected to the caffeine treatment. However, no other significant differences were observed in our experiment. The experimental design is shown in Figure 4a; the expression of genes was analyzed in the basal forebrain (n = 4~5) and hypothalamus (n = 6~8) of mice. Data are expressed as mean ± SEM and were analyzed by one-way ANOVA with Tukey's post hoc test. * p <0.05, compared with the Veh group.

Discussion
Recent studies have correlated the gut microbiome with immune function, nutrient metabolism, circadian rhythms, and mood disorders [35,36]. Through the MGBA, the gut microbiome influences not only the digestive, immune, and metabolic functions but also the sleep and mental states of the host [37]. To manipulate the gut microbiome for improving host health, probiotics are generally utilized, mostly for digestive and immune functions [38,39]. There is considerable evidence showing that probiotics may be effective in reducing stress and anxiety and alleviating low moods [40]. However, specific probiotic strains that can be used for sleep improvement are rare. Dietary supplementation of heat-killed Lactobacillus brevis SBC8803 can modulate circadian locomotion and sleep rhythms, as assessed by an EEG analysis [41]. The oral administration of Japanese sake yeast promotes non-rapid eye movement (NREM) sleep in mice via the activation of adenosine A 2A but not A 1 receptors [42]. Moreover, the daily consumption of Lactobacillus casei Shirota or heat-inactivated Lactobacillus gasseri CP2305 improved stress-related symptoms and sleep quality in exploratory clinical trials [43,44].
In this study, we used a pentobarbital-induced sleep mouse model to identify potential probiotics with sleep-improving effects, which is the most commonly used method for screening of sedative-hypnotic agents [45,46]. Numerous strains belonging to L. fermentum and other Lactobacillus spp. were tested, but only PS150 TM showed sleep-improving effects in our experiment. Pentobarbital is a barbituric acid that activates GABA type A receptors, leads to cellular hyperpolarization within the CNS, and produces dose-dependent sedation and hypnosis [47]. Sleep latency and sleep duration obtained in the pentobarbital-induced sleep test are commonly used as indicators for assessing the sedative and hypnotic effects of dietary supplements (most of them are plant extracts) and drugs including DIPH, diazepam (a longer-acting benzodiazepine), and 5-ydroxytryptophan (a clinically effective serotonin precursor) [21,46,48,49]. The administration of caffeine has also been shown to increase sleep latency and decrease sleep duration in a dose-dependent manner assessed by the pentobarbital-induced sleep test [50]. Moreover, the hypnotic effect and the interaction of caffeine with pentobarbital have been studied in 42 medical and surgical patients [51]. To our knowledge, this is the first study that used the pentobarbital-induced sleep mouse model to evaluate the potential sleep-improving effects of probiotics; moreover, the caffeine-induced sleep disturbance appeared to be ameliorated by the supplementation of PS150 TM . However, these results are preliminary and need to be confirmed by further animal and clinical studies.
The dietary supplementation of PS150 TM for more than 14 consecutive days appeared to decrease the sleep latency and increase the sleep duration and recovery time of mice with pentobarbital-induced sleep. Sleep recovery, measured from the recovery of righting reflex to the beginning of a movement, reflects the subsequent effects of hypnotics on the CNS [21]. An ideal hypnotic should not affect awakening after sleep, and PS150 TM seemed to have this feature. As shown in Figure 3, on experimental day 1, the oral administration of PS150 TM did not affect the sleep pattern of mice. However, PS150 TM increased the sleep duration from day 4, decreased the sleep latency from day 7, and decreased the recovery time on day 14. We speculated that PS150 TM may need to colonize the gut mucosa and interact with the host microbiota to gradually regulate the sleep pattern via the MGBA. It is known that probiotics can alter the gut microbiota by competing for nutrients, producing antimicrobial compounds, or modulating host immunity [52]. The possibility that PS150 TM modulates the host gut microbiota remains to be investigated. On the other hand, the oral gavage of DIPH, which exerts its hypnotic effects via the antihistamine pathway [30], only prolonged sleep duration but did not affect sleep latency or recovery time. Based on these findings, we suggest that PS150 TM regulates sleep patterns differently from DIPH, although the exact mechanism is not yet clear.
Various neurotransmitters, including gamma-aminobutyric acid (GABA), serotonin or 5-hydroxytryptamine (5-HT), adenosine, histamine, and orexin, affect different brain nuclei to regulate the switch between wakefulness and sleep [2,53]. Although PS150 TM has been demonstrated to prevent the stress-mediated reduction of 5-HT and neurodegeneration in the rat brain, in this study, the oral administration of PS150 TM did not affect the levels of 5-HT or 5-hydroxyindole acetic acid (5-HIAA) in specific mouse brain areas (striatum, prefrontal cortex, hippocampus, and hypothalamus) assessed by high-performance liquid chromatography-electrochemical detection (data not shown). Specific strains of Lactobacillus spp. can produce GABA, the chief inhibitory neurotransmitter in the mammalian CNS. The ability to synthesize GABA is considered to be an important feature of psychobiotics [54]. However, PS150 TM did not seem to produce GABA in vitro from its precursor monosodium glutamate, as assessed by a thin-layer chromatography analysis of its culture supernatant (data not shown). However, PS150 TM appeared to increase the expression of adenosine A 1 R in the hypothalamus (Table 2). Adenosine is an inhibitory neurotransmitter that has been proposed to decrease the activity of orexinergic and histaminergic neurons via A 1 R in different areas of the hypothalamus to promote sleep [55][56][57]. Moreover, the histaminergic output from the hypothalamus plays an important role in mediating forebrain arousal [58]. Whether or not the sleep-improving effects of PS150 TM are involved in these adenosine A 1 R-mediated regulations in the hypothalamus awaits further investigation.
Although its pathogenesis is not fully understood, important features of insomnia include difficulty initiating or maintaining sleep, and waking up earlier than desired [59]. Many studies have shown that insomnia is often accompanied by neurophysiological abnormalities and poor health [5]. The use of medications is a dominant approach to treat insomnia, which involves four fundamental pharmacodynamic categories with key actions related to receptors of GABA, melatonin, histamine, or orexin/hypocretin [59]. Nevertheless, side effects associated with hypnotics are common [59]. Thus, the development of alternative treatment strategies with higher safety, including dietary supplements, is needed. Probiotics, which include strains of Lactobacillus, Bifidobacterium, and Saccharomyces, have a long history of safe and effective use as dietary supplements. Moreover, emerging evidence has shown the potential of psychobiotics to improve CNS-related illnesses, particularly in stress-related, anxiety, and depressive disorders [60]. In this study, we have shown the potential sleep-improving effects of PS150 TM , which has also been demonstrated as a psychobiotic capable of alleviating abnormal behaviors induced by mild chronic stress in rats [16]. The use of PS150 TM as a dietary supplement is generally considered as safe, since PS150 TM is classified as the species L. fermentum, with the qualified presumption of safety (QPS) status suggested by the European Food Safety Authority [61]. One major limitation of this study is the non-use of polysomnography; moreover, possible interactions between pentobarbital and probiotics cannot be excluded. To further understand the sleep-improving effects of PS150 TM , we are now performing EEG and electromyogram analyses to investigate if PS150 TM affects sleep architecture in mice.

Conclusions
The present data demonstrate that the psychobiotic strain L. fermentum PS150 TM can potentially bring sleep-improving effects to both normal and caffeine-treated (mimicking short-term insomnia) mice without affecting their locomotor activities. The potential hypotonic effects of PS150 TM are strain-specific, showing some time-and dose-dependency, and may be involved in the regulation of the histaminergic system via adenosine A 1 receptor. This suggests a new direction for the future development of novel dietary supplements as sleep aids.