Ashwagandha (Withania somnifera L. Dunal), an Indian medicinal plant that has been used for centuries to treat insomnia, exhibits a variety of biological activities, such as improving cognitive function, immunity and anxiety. In this study, the effect of enzyme-treated Ashwagandha root extract (EA) and on sleep was evaluated using rodent models. Starch contained in the Ashwagandha root extract was removed by amylase treatment to prepare EA. To evaluate the sleep-promoting activity of EA, a pentobarbital-induced sleep test and electroencephalogram analysis were performed. In addition, the sleep-promoting mechanism of EA was elucidated by analyzing the expression of sleep-related receptors. In the pentobarbital-induced sleep test, EA dose-dependently increased sleep duration. Additionally, electroencephalogram analysis revealed that EA significantly increased δ-wave and non-rapid eye movement sleep times, which are involved in deep sleep, thereby improving sleep quality and quantity. EA also effectively relieved caffeine-induced insomnia symptoms. Furthermore, the γ-aminobutyric acid (GABA) content in the brain and mRNA and protein expression of GABAA, GABAB1, and serotonin receptors were significantly increased by EA compared to the normal group. In particular, EA showed sleep-promoting activity by binding to various GABAA receptor sites. Collectively, EA exhibited sleep-promoting activity through the GABAergic system and may be used as a functional material to improve sleep deprivation.

Although Ziziphus jujuba Mill (jujube) is used in folk medicine for hypnotic sedative, anxiolytic, and many other purposes, to date, only a few studies have revealed its sleep-promoting effects and related mechanisms. Currently, drugs used for the treatment of sleep disorders have various side effects, so it is essential to develop safe natural materials. Therefore, we evaluated the sleep-enhancing activity and mechanism of action of an aqueous extract of jujube seeds (ZW) fermented with Lactobacillus brevis L-32 in rodent models. The starch contained in ZW was removed by enzymatic degradation and fermented with L. brevis to obtain a fermented product (ZW-FM) with a high γ-aminobutyric acid (GABA) content. To evaluate the sleep-promoting effect of ZW-FM, pentobarbital-induced sleep tests were performed on ICR mice, and electroencephalography analysis was undertaken in Sprague Dawley rats. Additionally, the awakening relief effects of ZW-FM were confirmed in a caffeine-induced insomnia model. Finally, the mechanism of sleep enhancement by ZW-FM was analyzed using GABA receptor type A (GABAA) antagonists. The ZW-FM-treated groups (100 and 150 mg/kg) showed increased sleep time, especially the δ-wave time during non-rapid eye movement (NREM) sleep. In addition, the 150 mg/kg ZW-FM treatment group showed decreased sleep latency and increased sleep time in the insomnia model. In particular, NREM sleep time was increased and REM sleep time, which was increased by caffeine treatment, was decreased by ZW-FM treatment. ZW-FM-induced sleep increase was inhibited by the GABAA receptor antagonists picrotoxin, bicuculline, and flumazenil, confirming that the increase was the result of a GABAergic mechanism. These results strongly suggest that the increased GABA in water extract from jujube seeds fermented by L. brevis acts as a sleep-promoting compound and that the sleep-promoting activity is related to GABAA receptor binding.

We aimed to analyze the increase in the sleep-promoting effects based on the mixed ratio of botanical extracts, Ziziphus jujuba seeds, Dimocarpus longan fruits, and Lactuca sativa leaves, using animal models. Behavioral analyses, including an analysis of the total sleep time of Drosophila melanogaster, were conducted to select the optimal mixed ratio of the three botanical extracts. The effects were verified in a caffeine-induced sleepless model, specific neurotransmitter receptor antagonists, and ICR mice. In D. melanogaster exposed to 2.0% of each extract, group behavior was significantly reduced, and the mixed extracts of Z. jujuba, D. longan, and L. sativa (4:1:1 and 1:4:1) significantly increased the total sleep time with individual fruit flies. In the caffeine-induced insomnia model, mixed extracts (4:1:1 and 1:4:1) led to the highest increase in total sleep time. An analysis of locomotor ability revealed a significant reduction in the mobility percentage in the mixed extract groups (0:0:1, 1:0:1, 1:1:1, 4:1:1, and 1:4:1). The administration of Z. jujuba extract and mixed extracts (4:1:1) significantly increased the expression of GABAA-R, whereas the administration of the mixed extracts (4:1:1) and (1:4:1) significantly increased the expression of GABAB-R1 and GABAB-R2, respectively. D. longan extract and the mixed ratio (1:4:1) reduced the subjective nighttime movement and increased the total sleep time in the presence of flumazenil. An analysis of ICR mice indicated that the administration of mixed extracts (4:1:1) significantly increased sleep duration in a dose-dependent manner. These results indicated that the mixed ratio of Z. jujuba, D. longan, and L. sativa extracts, particularly the mixed ratio of 4:1:1, may have sleep-enhancing effects in fruit flies and mice. The study also identified changes in gene expression related to GABA receptors, indicating the potential mechanism for the observed sleep-promoting effects.