Sleep-promoting Effects Research Articles

Postsynaptic competition between calcineurin and PKA regulates mammalian sleep-wake cycles.

The phosphorylation of synaptic proteins is a significant biochemical reaction that controls the sleep-wake cycle in mammals1-3. Protein phosphorylation in vivo is reversibly regulated by kinases and phosphatases. In this study, we investigate a pair of kinases and phosphatases that reciprocally regulate sleep duration. First, we perform a comprehensive screen of protein kinase A (PKA) and phosphoprotein phosphatase (PPP) family genes by generating 40 gene knockout mouse lines using prenatal and postnatal CRISPR targeting. We identify a regulatory subunit of PKA (Prkar2b), a regulatory subunit of protein phosphatase 1 (PP1; Pppr1r9b) and catalytic and regulatory subunits of calcineurin (also known as PP2B) (Ppp3ca and Ppp3r1) as sleep control genes. Using adeno-associated virus (AAV)-mediated stimulation of PKA and PP1-calcineurin activities, we show that PKA is a wake-promoting kinase, whereas PP1 and calcineurin function as sleep-promoting phosphatases. The importance of these phosphatases in sleep regulation is supported by the marked changes in sleep duration associated with their increased and decreased activities, ranging from approximately 17.3 h per day (PP1 expression) to 4.3 h per day (postnatal CRISPR targeting of calcineurin). Localization signals to the excitatory post-synapse are necessary for these phosphatases to exert their sleep-promoting effects. Furthermore, the wake-promoting effect of PKA localized to the excitatory post-synapse negated the sleep-promoting effect of PP1-calcineurin. These findings indicate that PKA and PP1-calcineurin have competing functions in sleep regulation at excitatory post-synapses.

Ginsenoside Rg1 promotes non-rapid eye movement sleep via inhibition of orexin neurons of the lateral hypothalamus and corticotropin-releasing hormone neurons of the paraventricular hypothalamic nucleus

ObjectiveThis study investigates the sleep-modulating effects of ginsenoside Rg1 (Rg1, C42H72O14), a key bioactive component of ginseng, and elucidates its underlying mechanisms. MethodsC57BL/6J mice were intraperitoneally administered doses of Rg1 ranging from 12.5 to 100 mg/kg. Sleep parameters were assessed to determine the average duration of each sleep stage by monitoring the electrical activity of the brain and muscles. Further, orexin neurons in the lateral hypothalamus (LH) and corticotropin-releasing hormone (CRH) neurons in the paraventricular hypothalamic nucleus (PVH) were ablated using viral vector surgery and electrode embedding. The excitability of LHorexin and PVHCRH neurons was evaluated through the measurement of cellular Finkel-Biskis-Jinkins murine osteosarcoma viral oncogene homolog (c-Fos) expression. ResultsRg1 (12.5–100 mg/kg) augmented the duration of non-rapid eye movement (NREM) sleep phases, while reducing the duration of wakefulness, in a dose dependent manner. The reduced latency from wakefulness to NREM sleep indicates an accelerated sleep initiation time. We found that these sleep-promoting effects were weakened in the LHorexin and PVHCRH neuron ablation groups, and disappeared in the orexin and CRH double-ablation group. Decreased c-Fos protein expression in the LH and PVH confirmed that Rg1 promoted NREM sleep by inhibiting orexin and CRH neurons. ConclusionRg1 increases the duration of NREM sleep, underscoring the essential roles of LHorexin and PVHCRH neurons in facilitating the sleep-promoting effects of Rg1.

Sleep-enhancing effect of Hongcheon-hop (Humulus lupulus L.) extract containing xanthohumol and humulone through GABAA receptor

Ethnopharmacological relevanceThe bitter resins in hops (Humulus lupulus) modulate GABA receptors, leading to central nervous system suppression, which induces sedative effects and enhances sleep. Aim of the studyThis study intends to explore the sleep-enhancing properties of Hongcheon-hop extract, a hop native to Korea, by analyzing sleep structure and its mechanisms through EEG. Materials and methodsA pentobarbital-induced sleep model was used, along with EEG analysis to study sleep architecture. The receptor binding of Hongcheon-hop extract was analyzed using GABA receptor antagonists. ResultsThe extract contained 7.44 mg/g of xanthohumol, 133.04 mg/g of α-acids including 13.74 mg/g of humulone and 84.52 mg/g of β-acids. Administration of the extracts resulted in a dose-dependent enhancement of sleep duration. Both low (100 mg/kg) and high doses (200 mg/kg) of the extract increased non-rapid eye movement (NREM) sleep by enhancing δ wave sleep, which represents deep sleep. The extract exerted its sleep-promoting effects by binding to the γ-aminobutyric acid (GABA) binding site on GABAA receptor. In an insomnia model, low dose of the extract was more effective than the same dose of GABA in enhancing sleep. Administration of the extract for 3 weeks increased the expression and protein levels of 5-HT1A receptors in addition to GABAA receptor, and also increased the content of GABA in the brain. A mixture of xanthohumol (1.49 mg) and humulone (2.75 mg) within Hongcheon-hop extract (200 mg) exhibited a sleep-enhancing effect comparable to that of the extract. ConclusionThese findings suggest that Hongcheon-hop extract, which contains xanthohumol and humulone, improves sleep quality by increasing NREM sleep through the enhancement of δ-wave sleep via GABAA receptors. Hongcheon-hop shows potential as a natural therapeutic agent for treating insomnia and enhancing sleep.

Gut Bacterial Metabolites from Tryptophan and Phenylalanine Induce Melatonin Synthesis and Extend Sleep Duration in Mice.

The human gut microbiota significantly influences various physiological systems, including immune, nervous, and metabolic systems. Recent studies suggest that gut microbiota may affect sleep quality with certain bacteria and metabolites being linked to sleep patterns. However, the underlying chemical signaling pathway remains unclear. In this study, we investigated the effect of four gut bacteria-derived metabolites, tryptamine (1), indolokine A5 (2), 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE, 3), and phenethylamine (PEA, 4), on sleep characteristics in mice and melatonin biosynthesis pathways in zebrafish. Their sleep-promoting effects were evaluated in a pentobarbital-induced sleep mouse model, revealing significant increases in sleep duration and blood melatonin levels, particularly with ITE (3) and PEA (4). Further tests in zebrafish embryos showed that ITE (3) and PEA (4) increased the expression of genes for melatonin biosynthesis (Aanat1, Aanat2, Tph1a, and Hiomt) in a concentration-dependent manner, indicating their potential as therapeutic agents for sleep disorders.

Pichia pastoris secreted peptides crossing the blood-brain barrier and DSIP fusion peptide efficacy in PCPA-induced insomnia mouse models.

Pichia pastoris-secreted delta sleep inducing peptide and crossing the blood-brain barrier peptides (DSIP-CBBBP) fusion peptides holds significant promise for its potential sleep-enhancing and neurotransmitter balancing effects. This study investigates these properties using a p-chlorophenylalanine (PCPA) -induced insomnia model in mice, an approach akin to traditional methods evaluating sleep-promoting activities in fusion peptides. The research aims to elucidate the sleep-promoting mechanism of DSIP-CBBBP, exploring its impact on neurotransmitter levels and sleep regulation, and to analyze its composition and structure. Using a PCPA-induced insomnia mouse model, the study evaluates the sleep-promoting effects of DSIP-CBBBP. The peptide's influence on neurotransmitters such as 5-HT, glutamate, dopamine, and melatonin is assessed. The functions of DSIP-CBBBP are characterized using biochemical and animal insomnia-induced behavior tests and compared without CBBBP. DSIP-CBBBP demonstrates a capacity to modulate neurotransmitter levels, indicated by changes in 5-HT, glutamate, DA, and melatonin. DSIP-CBBBP shows a better restorative effect than DSIP on neurotransmitter imbalance and the potential to enhance sleep. The study underscores DSIP-CBBBP potential in correcting neurotransmitter dysregulation and promoting sleep, hinting at its utility in sleep-related therapies.

Lavender improves sleep through olfactory perception and GABAergic neurons of the central amygdala

Ethnopharmacological relevanceThe use of lavender as sleep aid or hypnotic agent can be traced back as early as ancient Romans and Greeks. Yet, objective experimental data on whether and how lavender enhances sleep duration or/and sleep quality remain lacking. Aim of the studyWe aimed to characterize the sleep-wake regulating effects of lavender in the mouse and to demonstrate the brain targets and neural circuits involved. Materials and methodsA self-made precise odor delivery system combined with chronic polysomnographic recordings was employed to assess the sleep-wake effects of inhalation with lavender essential oil (LEO, extracted from lavender) and its different constituents during the light and dark phases in free-moving C57BL/6J mice. Neuroviral labeling, in situ hybridization and pharmacogenetics were combined to identify the neural circuits and targets involved. Finally, an insomniac model of DL-4-Chlorophenylalanine (PCPA)-treated mice was established to examine the sleep-inducing potential of LEO. ResultsWe found that inhalation of LEO with a concentration at 25.0% during the light (inactive) phase significantly shortened the latency to non-rapid eye movement (NREM) sleep, increased the total amount of NREM sleep at the expense of wakefulness (W), and enhanced cortical EEG slow wave activities, notably delta power spectra density. We further identified linalool, d-limonene, 1,8-cineole, linalyl acetate and terpinene-4-ol as the major effective sleep-promoting monomer components. Importantly, we found that LEO no longer produced any of the above sleep-promoting effect following either nasal injection of zinc sulfate which interrupts the olfactory pathway, or pharmacogenetics silencing of central amygdala GABAergic neurons. Finally, LEO reestablished NREM sleep with short latency in PCPA-treated insomniac mice, effects comparable with those induced by a potent sedative diazepam. ConclusionsWe have characterized the quantitative and qualitative sleep-promoting effects of LEO and its effective components via the olfactory pathway and central amygdala GABA neuronal targets. The hypnotic property of LEO is reinforced by its ability to restore sleep in insomnia. Our study thus establishes a neurobiological basis for aromatherapy of sleep disorders using lavender.

Neurosteroids foster sedation by engaging tonic GABAA-Rs within the mesopontine tegmental anesthesia area (MPTA)

Neurosteroids are endogenous molecules with anxiolytic, anticonvulsant, sleep-promoting and sedative effects. They are biosynthesized de novo within the brain, among other tissues, and are thought to act primarily as positive allosteric modulators of high-affinity extrasynaptic GABAAδ-receptors. The location of action of neurosteroids in the brain, however, remains unknown. We have demonstrated that GABAergic anesthetics act within the brainstem mesopontine tegmental anesthesia area (MPTA) to induce and maintain anesthetic loss-of-consciousness. Here we asked whether endogenous and synthetic neurosteroids might also act in the MPTA to induce their suppressive effects. Direct exposure of the MPTA to the endogenous neurosteroids pregnenolone and progesterone, their metabolites testosterone, allopregnanolone and 3α5α-THDOC, and the synthetic neurosteroids ganaxolone and alphaxalone, was found to be pro-anesthetic. Although we cannot rule out additional sites of action, results of this study suggest that the suppressive effects of neurosteroids are due, at least in part, to actions within the MPTA, presumably by recruitment of dedicated neuronal circuitry. This undermines the usual presumption that neurosteroids, like other sedatives, endogenous somnogens and anesthetics, act by nonspecific global distribution

The role of Kupffer cells in microbiota-brain communication: Sleep and fever signaling in response to lipopolysaccharide

Microbial molecules translocated from the intestinal lumen into the host’s internal environment play a role in various physiological functions. Previously, we identified that butyrate, a short-chain fatty acid produced by intestinal bacteria, lipoteichoic acid, a cell wall component of gram-positive bacteria, and lipopolysaccharide (LPS), a cell wall component of gram-negative bacteria, induce sleep when their naturally occurring translocation is mimicked by direct delivery into the portal vein. Our findings suggested that these microbial molecules exert their sleep-promoting effects within the hepatoportal region. In the present experiments, we tested the hypothesis that resident liver macrophages, known as Kupffer cells, play a crucial role in the LPS-responsive, sleep-promoting mechanisms within the hepatoportal region. Intraportal administration of LPS induced increased sleep and fever in control rats. Remarkably, in Kupffer cell-depleted animals, both of these responses were significantly suppressed. These findings highlight the potential role of Kupffer cells in mediating the non-rapid-eye movement sleep-promoting and febrile effects of LPS translocated from the intestinal microbiota into the portal circulation. The strategic location of Kupffer cells within the hepatoportal region, coupled with their ability to rapidly take up LPS and other microbial molecules, together with their high secretory activity of multiple signaling molecules, underlie their key role in the communication between the intestinal microbiota and the brain.

Novel neuropharmacological activity of citrus lime (Citrus aurantifolia): A standardized lime peel supplement enhances non-rapid eye movement sleep by activating the GABA type A receptor

Polyphenols have been well-established to exert sedative-hypnotic effects in psychopharmacology. Lime (Citrus aurantifolia) peel is rich in biologically active polyphenols; however, the effects of lime peel extract on sleep have not yet been demonstrated. A comparison was conducted in mice, between the sleep-promoting effects of a standardized lime peel supplement (SLPS) and a well-known hypnotic drug, zolpidem, and its hypnotic mechanism was investigated using in vivo and in vitro assays. The effects of SLPS on sleep were assessed using a pentobarbital-induced sleep test and sleep architecture analysis based on recording electroencephalograms and electromyograms. Additionally, a GABAA receptor binding assay, electrophysiological measurements, and in vivo animal models were used to elucidate the hypnotic mechanism. SLPS (200 and 400 mg/kg) was found to significantly decrease sleep latency and increase the amount of non-rapid eye movement sleep without altering delta activity. The hypnotic effects of SLPS were attributed to its flavonoid-rich ethyl acetate fraction. SLPS had a binding affinity to the GABA-binding site of the GABAA receptor and directly activated the GABAA receptors. The hypnotic effects and GABAA receptor activity of SLPS were completely blocked by bicuculline, a competitive antagonist of the GABAA receptor, in both in vitro and in vivo assays. To the best of our knowledge, this study is the first to demonstrate the hypnotic effects of SLPS, which acts via the GABA-binding site of the GABAA receptor. Our results suggest that lime peel, a by-product abundantly generated during juice processing, can potentially be used as a novel sedative-hypnotic.

Sedative and hypnotic effects with cortical EEG sleep-wake profiles of Millingtonia hortensis dried flower aqueous in mice

The ethnopharmacological relevance of the Millingtonia hortensis (M. hortensis) flower's aqueous extract lies in its traditional use as a herbal remedy in Southeast Asia. With a rich history in folk medicine, this aqueous has been esteemed for its purported sedative and anxiolytic properties. Our research delves into the scientific basis of these traditional claims, exploring the potential mechanisms underlying the observed effects of M. hortensis flower's aqueous extract on sleep promotion and mood regulation. This study aimed to explore the sleep-promoting effects of M. hortensis dried flower in mice, using an aqueous concentration equivalent to a human concentration of 2.7 mg/mL. Anxiolytic and antidepressant properties were evaluated using behavioural tests, while electroencephalography (EEG) analysis probed the neural mechanisms underlying sleep promotion post-consumption. The aqueous extract of M. hortensis dried flower administered to mice showed a decrease in immobility in the forced swimming test, demonstrating antidepressant-like effects. Moreover, mice treated with M. hortensis aqueous exhibited increased non-rapid eye movement (NREM) sleep duration, corroborating sleep-promoting potential. EEG analysis of mice treated with M. hortensis aqueous revealed heightened beta oscillations in the frontal and parietal cortex, while pre-treatment with M. hortensis aqueous or diazepam enhanced rapid eye movement (REM) sleep after thiopental administration. Interestingly, M. hortensis aqueous pre-treatment augmented delta frequency ranges in the frontal cortex. Overall, these findings indicate that M. hortensis dried flower's aqueous extract, at a human-equivalent dosage, exerts significant behavioural and neural effects specifically, sedative and hypnotic aspects in mice, corroborating its potential as a natural remedy to promote sleep and regulate mood.

From Garden to Pillow: Understanding the Relationship between Plant-Based Nutrition and Quality of Sleep.

The effect of diet on sleep quality has been addressed in many studies; however, whether/how plant-based diets (PBDs) impact sleep-related parameters has not been explored in detail. This review aims to give an overview of the components of PBDs and the possible mechanisms through which PBDs may improve sleep quality. Studies have indicated that diets such as PBDs, which are typically high in fruits, vegetables, nuts, seeds, whole grains, and fiber, are associated with better sleep outcomes, including less fragmented sleep and improved sleep duration. Several mechanisms may explain how PBDs impact and/or improve sleep outcomes. Firstly, PBDs are characteristically rich in certain nutrients, such as magnesium and vitamin B6, which have been associated with improved sleep patterns. Secondly, PBDs are often lower in saturated fats and higher in fiber, which may contribute to better overall health, including sleep quality. Additionally, plant bioactive compounds like phytochemicals and antioxidants in fruits, vegetables, and herbs may have sleep-promoting effects. According to available data, PBD and Mediterranean diet elements promise to enhance sleep quality; however, it is crucial to note that diets should be customized based on each person's needs.

Cyclooxygenase-2 (COX-2)-dependent mechanisms mediate sleep responses to microbial and thermal stimuli

Prostaglandins (PGs) play a crucial role in sleep regulation, yet the broader physiological context that leads to the activation of the prostaglandin-mediated sleep-promoting system remains elusive. In this study, we explored sleep-inducing mechanisms potentially involving PGs, including microbial, immune and thermal stimuli as well as homeostatic sleep responses induced by short-term sleep deprivation using cyclooxygenase-2 knockout (COX-2 KO) mice and their wild-type littermates (WT). Systemic administration of 0.4 µg lipopolysaccharide (LPS) induced increased non-rapid-eye movement sleep (NREMS) and fever in WT animals, these effects were completely absent in COX-2 KO mice. This finding underscores the essential role of COX-2-dependent prostaglandins in mediating sleep and febrile responses to LPS. In contrast, the sleep and fever responses induced by tumor necrosis factor α, a proinflammatory cytokine which activates COX-2, were preserved in COX-2 KO animals, indicating that these effects are independent of COX-2-related signaling. Additionally, we examined the impact of ambient temperature on sleep. The sleep-promoting effects of moderate warm ambient temperature were suppressed in COX-2 KO animals, resulting in significantly reduced NREMS at ambient temperatures of 30 °C and 35 °C compared to WT mice. However, rapid-eye-movement sleep responses to moderately cold or warm temperatures did not differ between the two genotypes. Furthermore, 6 h of sleep deprivation induced rebound increases in sleep with no significant differences observed between COX-2 KO and WT mice. This suggests that while COX-2-derived prostaglandins are crucial for the somnogenic effects of increased ambient temperature, the homeostatic responses to sleep loss are COX-2-independent. Overall, the results highlight the critical role of COX-2-derived prostaglandins as mediators of the sleep-wake and thermoregulatory responses to various physiological challenges, including microbial, immune, and thermal stimuli. These findings emphasize the interconnected regulation of body temperature and sleep, with peripheral mechanisms emerging as key players in these integrative processes.

Melatonin targets the paraventricular thalamus to promote non-rapid eye movement sleep in C3H/HeJ mice

Melatonin (MLT) is an important circadian signal for sleep regulation, but the neural circuitries underlying the sleep-promoting effects of MLT are poorly understood. The paraventricular thalamus (PVT) is a critical thalamic area for wakefulness control and expresses MLT receptors, raising a possibility that PVT neurons may mediate the sleep-promoting effects of MLT. Here, we found that MLT receptors were densely expressed on PVT neurons and exhibited circadian-dependent variations in C3H/HeJ mice. Application of exogenous MLT decreased the excitability of PVT neurons, resulting in hyperpolarization of membrane potential and reduction of action potential firing. MLT also inhibited the spontaneous activity of PVT neurons at both population and single-neuron levels in freely behaving mice. Furthermore, pharmacological manipulations revealed that local infusion of exogeneous MLT into the PVT promoted non-rapid eye movement (NREM) sleep and increased NREM sleep duration, whereas MLT receptor antagonists decreased NREM sleep. Moreover, we found that selectively knocking down endogenous MLT receptors in the PVT decreased NREM sleep and correspondingly increased wakefulness, with particular changes shortly after the onset of the dark or light phase. Taken together, these results demonstrate that PVT is an important target of MLT for promoting NREM sleep.

Lactobacillus brevis M2-Fermented Whey Protein Hydrolysate Increases Slow-Wave Sleep via GABAA Receptors in Rodent Models.

In this study, we investigated the effects of whey protein hydrolysate (WPH) fermented with Lactobacillus brevis on sleep behavior and GABAergic mechanisms in rodent models. Fermentation converted the glutamate in WPH to high (3.15 ± 0.21 mg/mL) levels of γ-aminobutyric acid (GABA). Fermented WPH (WP-SF) enhanced sleep duration in mice by increasing GABA content in the brain. The increase in sleep duration induced by WP-SF resulted from an increase in delta wave activity during non-rapid eye movement sleep, and its sleep-promoting effect in a caffeine-induced insomnia model was characterized by an increase in delta waves. WP-SF increased GABAergic receptors at both mRNA and protein levels. Cotreatment with GABAA receptor antagonists abolished the sleep-promoting effects of WP-SF, indicating that WP-SF shares binding sites with antagonists on GABAA receptors. Collectively, WP-SF effectively increased sleep duration by enhancing delta wave activity through GABAergic activation; thus, it is suggested as a functional food-grade ingredient for promoting sleep.

UP165, standardized corn leaf extract and its active component 6-methoxybenzoxazolinone induce non-rapid eye movement sleep through melatonergic and GABAergic mechanisms

The aim of this study was to investigate the sleep-promoting effects of corn leaf extract (UP165) and its active component 6-methoxybenzoxazolinone (6-MBOA), along with the underlying mechanisms in rodent models. Sleep activity in UP165 was measured by measuring sleep duration in the pentobarbital induced sleep model, and sleep quality was evaluated through electroencephalogram (EEG) analysis. In addition, the sleep mechanism was analyzed through an in vivo sleep study co-administrated with receptor antagonists. The administrations of both UP165 and 6-MBOA effectively increased sleep duration, demonstrating a 3-fold increase sleep time at 100 mg/kg UP165 and a 2.3-fold increase at 280 μg/kg 6-MBOA compared to those of the control group. The sleep-promoting effect of UP165 was also observed in the caffeine-induced insomnia model, resulting in a 71.6% increase in sleep duration. The UP165-mediated increase in sleep time was attributed to enhanced non-rapid eye movement (NREM) sleep, specifically an increase in the δ-wave sleep measured by EEG. Mechanism analysis with co-administration of competitive antagonists in vivo revealed that UP165 and 6-MBOA enhanced sleep duration through binding to gamma-aminobutyric acid type A (GABAA) and melatonin receptors. Oral administration of UP165 for 4 weeks effectively increased both gene expression and protein levels of GABAA, melatonin 1A, and melatonin 1B receptors. High-dose administration of UP165 significantly decreased the blood level of corticosterone, while concurrently increased GABA, serotonin, and melatonin levels in brain. This study highlights the potential of UP165 and 6-MBOA as edible-grade natural ingredients for sleep promotion.

Bifidobacterium adolescentis SBT2786 Improves Sleep Quality in Japanese Adults with Relatively High Levels of Stress: A Randomized, Double-Blind, Placebo-Controlled Study.

Sleep disorders associated with lifestyle changes and unhealthy habits are major public health concerns. Our previous study showed that Bifidobacterium adolescentis SBT2786 has a potent sleep-promoting effect on fruit flies. Fruit flies share many similarities with mammals, making them suitable model organisms for studying sleep. Thus, in the present study, we conducted a randomized, double-blind, placebo-controlled clinical trial to test whether SBT2786 has sleep-enhancing effects in humans. In this study, 61 participants in the SBT2786 group and 65 participants in the placebo group were analyzed. The results showed that SBT2786 increased sleep time; however, it predominantly increased light sleep and did not improve subjective sleep quality. Interestingly, mood improvement was observed. A subgroup analysis was conducted on participants with high stress levels, and results showed that these participants experienced an increase in sleep duration and an improvement in sleepiness upon waking up and reported feeling well-rested during the day. We concluded that SBT2786 may improve sleep quality, particularly in individuals experiencing high levels of stress, and that SBT2786 can be used as a dietary supplement to improve sleep and mood.

Combination administration of alprazolam and N-Ethylmaleimide synergistically enhances sleep behaviors in mice with no potential CNS side effects.

N-Ethylmaleimide (NEM), an agonist of the potassium chloride cotransporters 2 (KCC2) receptor, has been correlated with neurosuppressive outcomes, including decreased pain perception and the prevention of epileptic seizures. Nevertheless, its relationship with sleep-inducing effects remains unreported. The present study aimed to investigate the potential enhancement of NEM on the sleep-inducing properties of alprazolam (Alp). The test of the righting reflex was used to identify the appropriate concentrations of Alp and NEM for inducing sleep-promoting effects in mice. Total sleep duration and sleep quality were evaluated through EEG/EMG analysis. The neural mechanism underlying the sleep-promoting effect was examined through c-fos immunoreactivity in the brain using immunofluorescence. Furthermore, potential CNS-side effects of the combination Alp and NEM were assessed using LABORAS automated home-cage behavioral phenotyping. Combination administration of Alp (1.84 mg/kg) and NEM (1.0 mg/kg) significantly decreased sleep latency and increased sleep duration in comparison to administering 1.84 mg/kg Alp alone. This effect was characterized by a notable increase in REM duration. The findings from c-fos immunoreactivity indicated that NEM significantly suppressed neuron activation in brain regions associated with wakefulness. Additionally, combination administration of Alp and NEM showed no effects on mouse neural behaviors during automated home cage monitoring. This study is the first to propose and demonstrate a combination therapy involving Alp and NEM that not only enhances the hypnotic effect but also mitigates potential CNS side effects, suggesting its potential application in treating insomnia.

Heukharang (Lactuca sativa L.) extracts enhanced the sleep behavior of mice: potential involvement of adenosine A1 and A2A receptors.

A significant proportion of the world's population suffers from insomnia, a disorder characterized by complications in initiating and maintaining sleep. Many medications used to treat insomnia target the γ-aminobutyric acid (GABA) neurotransmitter system. However, these substances, such as benzodiazepines, induce significant adverse consequences, including dependence and memory impairment, after prolonged use. Thus, current studies are aimed at developing therapeutic hypnotics derived from natural sources that may cause less severe side effects. Heukharang is a variety of lettuce from Korea that was discovered to contain sleep-promoting compounds. Therefore, we investigated the potential effects of sub-chronic administration of Heukharang extract (FSD-LS) on sleep behavior (pentobarbital-induced sleeping test), brain wave activity and sleep architecture (electroencephalography), and physiological behavior (open-field test and rota-rod) in mice, along with radioligand binding assays (GABAA, adenosine A1 and A2A receptors). We found that FSD-LS prolonged the total sleep duration and reduced the onset time of sleep, and enhanced delta wave power and non-rapid eye movement (NREM) sleep duration, all indicating persistent sleep-enhancing effects. FSD-LS lacked adverse effects on the spontaneous locomotor activity and motor coordination of mice, unlike diazepam. Pharmacological blocking using caffeine and bicuculline supported the possible involvement of adenosine receptors in the sleep-promoting effects of FSD-LS, with partial contribution from GABA receptor activity. Overall, our study recommends FSD-LS as a potential source for the development of sleep-aiding therapeutics.

Effects of chronic administration and withdrawal of phlorotannin supplement on sleep-wake profiles in mice: A comparative study with the hypnotic drug diazepam

The aim of this study was to investigate the effects of chronic administration (21 days) of phlorotannin supplement (PS) on sleep and compare them with the effects of diazepam (DZP), a benzodiazepine hypnotic drug. DZP significantly decreased sleep latency and increased non-rapid eye movement sleep amount on day 1 of administration compared with those at baseline; however, its effects diminished over the administration period. On the contrary, PS showed significant sleep-promoting effects throughout the study period, with no significant difference in efficacy between days 1 and 21. Notably, PS did not cause any changes in the slow wave activity (SWA) throughout the treatment period; whereas, DZP decreased the SWA on day 1 of administration. These results suggest that chronic administration of PS does not induce tolerance, implying its consistent sleep-maintaining effects.

The Antimelanogenic Activity of the Extract of Heukharang Lettuce (Lactuca sativa L.) Leaf

Heukharang (Lactuca sativa L.), the artificially developed lettuce cultivar for enhancement of lactucin content, exhibits antioxidant activities and sleep-promoting effects. However, potential of Heukharang as a raw material for the skin-whitening agent has not been investigated yet. This study evaluated the effects of Heukharang extract (HHE) on α-melanocyte-stimulating hormone (α-MSH)-induced melanogenesis in vitro and in vivo models. Our findings revealed that HHE (25–100 μg/mL) effectively inhibited α-MSH-induced melanin synthesis in both 2D and 3D cell culture environments without compromising the viability of the B16F10 murine melanoma cell line. Furthermore, HHE suppressed intracellular tyrosinase activity, along with both gene and protein expression of tyrosinase (TYR) and tyrosinase-related protein (TRP)-1 and TRP-2. HHE downregulated the mRNA level of microphthalmia-associated transcription factor (MITF) and the phosphorylation of cAMP response element-binding (CREB) protein. The antimelanogenic activity of HHE (50 and 100 μg/mL) was also confirmed using a zebrafish embryo model, highlighting its efficacy in inhibiting α-MSH-induced melanogenesis through the downregulating CREB/MITF/TYR signaling pathways. Moreover, our study demonstrated that a corresponding amount of lactucin (5–20 μM) to the HHE inhibited and regulated melanogenesis. These results collectively suggest that HHE, enriched with lactucin, holds promise as a potential skin-whitening agent for nutricosmetic industries.