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. 2024 Aug 28;34(5):1149–1159. doi: 10.1007/s10068-024-01663-4

Improvement of sleep disorders through the adenosine A receptor agonist effect of Phlomoides umbrosa Turczaninow root extract in pentobarbital-induced ICR mice

Joo-Hyun Oh 1, Yoon-Young Han 1, Eun-Bi Kim 1, Ha-Neul Jo 1, Jae-Sun Lee 1, Bo-Mi Kim 1, Ji-Min Kim 1, Young-Seob Lee 2, Dae Young Lee 3, Kwan-Woo Kim 2, Inil Lee 4, Yong-Wook Lee 1, Chan-Sung Park 1,✉, Dae-Ok Kim 4,✉
PMCID: PMC11904018  PMID: 40093550

Abstract

Sleep deprivation is a serious problem that deteriorates the health, quality of life, and social productivity of affected individuals. Research on safe and effective plant-based materials should be performed to help alleviate sleep disorders. Phlomoides umbrosa Turczaninow is a domestic plant in South Korea that has been used traditionally as a medicine to treat sleep disorders. The effects and mechanism of action of a P. umbrosa Turczaninow root extract (PUTRE) prepared using hot water extraction were investigated. ICR mice were injected with pentobarbital to induce sleep. The PUTRE-treated groups had significantly (p < 0.05) decreased sleep latency and increased sleep time compared with the controls. When PUTRE was co-administered with adenosine antagonists (caffeine and 8-cyclopentyl-1,3-dipropylxanthine), it acted specifically on the adenosine A1 receptor, because its sleep-enhancing effect was counteracted by the adenosine antagonist. This is the first study to demonstrate that PUTRE can induce sleep through adenosine A1 receptor-specific agonist activity. Therefore, PUTRE is suggested as a valuable sleep-enhancing substance.

Supplementary Information

The online version contains supplementary material available at 10.1007/s10068-024-01663-4.

Keywords: Adenosine A1 receptor, Insomnia, Pentobarbital, Phlomoides umbrosa Turczaninow root, Sleep enhancement

Introduction

Although the exact function of sleep remains uncertain, some studies have shown that sleep might be involved in energy conservation, recovery and clearance of metabolites, and brain plasticity (Porkka‐Heiskanen et al., 2013; Rasch and Born, 2013). Thus, insufficient quantity or quality of sleep can deteriorate quality of life. Sleep deprivation describes any state that affects the quality, onset, or duration of sleep and consequently affects a person’s ability to function properly while awake. Sleep disorders lead to daytime drowsiness, depression, and reduced work efficiency, including reduced concentration and learning abilities. The most common and well-known sleep disorder is insomnia, which is characterized by difficulty falling asleep, staying asleep, or both. At any given time, up to one-third of all adults suffer from insomnia, which results in poor health and poor academic or occupational performance (Buysse et al., 2008; Kyle et al., 2010; Wade, 2011).

An epidemiological study estimated that 10‒15% of adults worldwide suffer from chronic insomnia, with an additional 25‒35% suffering from intermittent insomnia (Buysse et al., 2008). Sleep disorders that cause sleep deprivation can interfere with daily life, reduce labor productivity, and cause mistakes or accidents at work, leading to increased socioeconomic costs. Additionally, chronic sleep deprivation increases the risk of high blood pressure, cardiovascular disease, diabetes, depression, and obesity. In other words, sleep deprivation not only worsens individual health, but also causes significant social and economic losses (Léger and Bayon, 2010). Therefore, it is necessary to develop appropriate treatment methods for sleep disorders. Interest in natural plant materials with proven safety and a low risk of side effects has been increasing. Domestic plants native to South Korea should be studied to develop functional materials with sleep-enhancing effects.

Phlomoides umbrosa Turczaninow (also known as Phlomis umbrosa Turczaninow and called “Han-Sok-Dan” in Korean) is a perennial plant of the Lamiaceae family that is native to northeast Asia, including South Korea, China, and Manchuria. The roots and leaves of this plant are used as a food ingredient (Chun et al., 2021). Moreover, P. umbrosa Turczaninow has traditionally been used as a medicinal plant to treat fractures, back pain, and hypogalactia (Hsu et al., 1986). An extract of P. umbrosa has also been shown to exhibit anti-inflammatory (Shang et al., 2011), osteoblast differentiation (Chun et al., 2021), anti-allergic (Shin et al., 2008), and anti-asthmatic (Pak et al., 2022) effects, among others.

The effectiveness of P. umbrosa Turczaninow root extract (PUTRE) and the mechanism by which it improves sleep disorders have not yet been reported. Therefore, in this work, we used in vitro and in vivo models to determine the effectiveness and mechanism of action by which PUTRE improves sleep disorders. A pentobarbital-induced mouse model was used to investigate the sleep-inducing effect of PUTRE. To identify the sleep-related mechanisms, the adenosine A1 and A2A receptors and the γ-aminobutyric acid (GABA) ion channel were examined in vitro, and sleep regulatory mechanisms were assessed in vivo. Our main objective was to verify the potential of PUTRE as a functional material for improving sleep quality.

Materials and methods

Extract preparation

Dried roots of P. umbrosa Turczaninow grown in South Korea were purchased from Chowon-Yagcho (Jecheon, South Korea). The dried P. umbrosa roots were cut into 2‒10 cm lengths and mixed with distilled water at a 1:10 (w/w) ratio. After extracting the mixture at 100 °C for 8 h, the extract was filtered through a paper filter. The filtered extract was concentrated to dryness under reduced pressure in a water bath at 50–60 °C.

Chemicals

Pentobarbital and diazepam (DZP) were purchased from Hanlim Pharm. Co., Ltd. (Seoul, South Korea) and Roche Korea Co., Ltd. (Seoul, South Korea), respectively. Caffeine and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich Co., LLC (St. Louis, MO, USA). 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX) was purchased from Tocris Biosciences (Bristol, UK).

Model animals

As model animals, 5-week-old female Institute of Cancer Research (ICR) mice (18‒20 g) supplied by Saeronbio, Inc. (Uiwang, South Korea) were used. In an animal breeding room, the mice were allowed to adapt to their surroundings in constant conditions (temperature 23 ± 2 °C, relative humidity 50% ± 5%, light/dark cycle 12 h) for 1 week. After that, eight mice were randomly assigned to each group, with a similar average body weight across all groups. During the study period, all mice were given liberal amounts of food and drinking water, and the food intake and body weights (BWs) were measured at a scheduled time once a week. The animal experiment was conducted with approval of the Institutional Animal Care and Use Committee at Kyung Hee University (KHGASP-20-253) and in compliance with the principles of laboratory animal management.

Measurements of sleep latency and sleep duration in pentobarbital-induced mice

After the adaptation period, the mice were administered the test material once a day for 2 weeks. For the sleep induction experiment, phosphate-buffered saline (PBS) was administered to the control group. The mice in the positive control group were orally administered 2 mg of DZP/kg of BW/day, and the mice in the two treatment groups (low- and high-dosage groups) were orally administered 200 and 400 mg of PUTRE/kg of BW/day, respectively. All groups were given pentobarbital (45 mg/kg of BW) intraperitoneally 45 min after the last oral administration of the test substance.

For the antagonist experiment, five groups of mice were tested: control, positive control (administration of 2 mg of DZP/kg of BW/day), PUTRE + vehicle (400 mg of PUTRE/kg of BW/day), PUTRE + caffeine (co-administration of 400 mg of PUTRE/kg of BW/day and 10 mg of caffeine/kg of BW/day), and PUTRE + DPCPX (co-administration of 400 mg of PUTRE/kg of BW/day and 5 mg of DPCPX/kg of BW/day). PUTRE, caffeine, DPCPX, and DZP were suspended in PBS for oral administration. Administration was repeated at a set time every day for 2 weeks. The mice were fasted for 24 h prior to the experiment, and all experiments were performed between 1 and 5 p.m. The experimental groups were injected intraperitoneally with a hypnotic agent (pentobarbital, 45 mg/kg of BW) 45 min after the last oral administration of the test materials. Each mouse was moved to a solitary cage and monitored as it fell asleep. The time until the righting reflex disappeared after the pentobarbital injection was measured as sleep latency. Sleep duration was measured as the time between the disappearance and reappearance of the righting reflex. The time from reappearance of the righting reflex to active movement of the mouse was measured as the recovery time. Mice whose righting reflex persisted for more than 15 min after pentobarbital injection were excluded from the analysis.

Measurement of melatonin concentration in blood

After completion of the experiment, the mice were fasted for 12 h and sacrificed through isoflurane inhalation. Blood was collected from the aorta through an abdominal incision. Serum was isolated from the collected blood by centrifugation at 1000 × g for 20 min and stored at − 80 ℃ for blood analysis. After blood collection, the liver, kidney, and spleen tissues from each mouse were harvested and weighed. To measure the blood melatonin concentration, an enzyme-linked immunosorbent assay (ELISA; MyBioSource, San Diego, CA, USA) was performed according to the provided assay protocol as follows: 50 μL each of standard and serum sample were added to each well of the melatonin-coated plate of the ELISA kit, and the same amount of detection reagent A was added to the plate and reacted at 37 ℃ for 1 h. After the plate was washed thrice, detection reagent B was added at 37 ℃ for a 45-min reaction. Subsequently, the plate was washed three times, and the substrate solution was added to each well and reacted in the dark at 37 ℃ for 20 min to confirm color development. The reaction was terminated by adding stop solution. The absorbance measured at 450 nm was used for quantification based on a standard curve.

Measurement of GABA concentration in brain tissue

The brain tissue harvested during autopsy was washed with ice-cold PBS to remove as much blood as possible. Subsequently, 100 mg of brain tissue was added to 1 mL of PBS, and after homogenization, the mixture was ultrasonicated for 10 s. After 5 min of centrifugation at 5000 × g, a general GABA ELISA kit (MyBioSource) was used as follows. In each well of the GABA-coated plate from the kit, 50 μL each of standard and serum sample were added, and an equal amount of detection solution A was added for 1 h of reaction at 37 ℃. After the plate was washed thrice with wash buffer, detection solution B was added for a 1-h reaction at 37 ℃. Subsequently, the plate was washed five times, after which a substrate solution was added to each well for 25 min of reaction at 37 ℃ in a shaded area to check the coloration level. Then the stop solution was added to terminate the reaction. The measured optical density at 450 nm was used for quantification based on the standard curve.

Adenosine A1 receptor gene expression in brain tissue

To examine the expression pattern of the adenosine A1 receptor gene in the brain tissues of the mice, the extracted tissue was placed in TRIzol, and after homogenization, the mixture was centrifuged at 12,000 × g and 4 ℃. The acquired supernatant was used for RNA extraction using an RNeasy extraction kit (Qiagen Sciences, Germantown, MD, USA) according to the manufacturer’s protocol. For cDNA synthesis, an iScript cDNA synthesis kit (Bio-Rad Laboratories, Hercules, CA, USA) was used, and real-time polymerase chain reaction (PCR) was performed using SYBR Green (Bio-Rad Laboratories) in a real-time PCR device (Applied Biosystems, Foster City, CA, USA). As sample preparation for the real-time PCR, 100 pmol of each adenosine A1 receptor primer (forward: 5′-CAG AGC TCC ATC CTG GCT CT-3′, reverse: 5′-CGC TGA GTC ACC ACT GTC TTG-3′) and GAPDH primer (forward: 5′-CCC CAC ACA CAT GCA CTT ACC-3′, reverse: 5′-TTG CCA AGT TGC CTG TCC TT-3′) were added to 10 μL of 2× SYBR mix and 2 μL of cDNA, and then the final volume was brought to 20 μL using distilled water. During the real-time PCR, 40 cycles were run, each consisting of 8 min of hot start at 95 ℃, 15 s of denaturation at 95 ℃, 30 s of annealing at 56 ℃, and 30 s of extension at 72 ℃. At the end of the 40 cycles, a melting curve analysis to confirm primer specificity was performed using the StepOne software (version 2.3) provided by Applied Biosystems.

Adenosine A1 receptor protein production in brain tissue

The brain tissue harvested during autopsy was washed with PBS and then homogenized and lysed by adding a lysis buffer containing a protease inhibitor. The lysed tissue was placed on ice for a set time and then centrifuged at 12,000 × g and 4 ℃ to isolate the proteins. The isolated proteins were quantified using Bradford dye reagent (Bio-Rad Laboratories), separated and transferred through electrophoresis, and blocked for 1 h using 5% skim milk (Tris-buffered saline containing 0.5% Tween 20). For the primary antibody reaction, adenosine A1 receptor (Invitrogen Corp., Carlsbad, CA, USA) and beta-actin (Bethyl Laboratories, Montgomery, TX, USA) were reacted on the membrane overnight at 4 ℃. The horseradish peroxidase-conjugated secondary antibody (Cell Signaling Technology, Danvers, MA, USA) was applied to the membrane for a 60-min reaction, and after coloration through enhanced chemiluminescence, images were obtained using the chemiluminescence imaging system (AE-9300 Ez-Capture MG; ATTO, Tokyo, Japan).

In vitro functional assays for adenosine A1 and A2A receptors and GABA ion channel

Cellular and nuclear receptor functional assays for the adenosine A1 receptor, adenosine A2A receptor, and GABA ion channel were performed by Eurofins Cerep (Poitiers, France) according to the protocol of the pharmacology service provider. PUTRE was dissolved in DMSO and diluted in assay buffer to a concentration that was threefold higher than the final assay concentration. Similarly, a vehicle control and positive control were prepared to ensure that all assays were properly controlled. Samples supplied were plated in duplicate for each concentration analysis. The adenosine A1 receptor agonist effect was tested in human recombinant BA/F3 cells, and intracellular Ca2+ was measured using the fluorimetry technique. The adenosine A2A receptor agonist effect was tested in rat endogenous PC-12 cells by measuring cyclic adenosine monophosphate using the homogeneous time resolved fluorescence technique. The cellular agonist effect is expressed as a percentage of the control response to known reference agonists, N6-cyclopentyladenosine (CPA) for the adenosine A1 receptor and 5′-N-ethylcarboxamidoadenosine for adenosine A2A receptor. For GABA, a human ion channel cell-based ion flux assay was performed, and the peak internal current according to sample addition was measured and normalized to EC100 GABA.

Statistical analysis

All statistical analyses in this study were performed using the Statistical Package for the Social Sciences program (version 22.0; IBM SPSS Statistical, Inc., Armonk, NY, USA), and all data are expressed as mean ± standard deviation. Analysis of variance (ANOVA) was used to determine differences between groups. When significant differences were found in the ANOVA test, Duncan’s multiple range test was used as a post-hoc test to determine statistically significant differences in the means of different groups, with the level of significance set at p < 0.05.

Results and discussion

Sleep disorder is characterized as the inability to sleep well or feeling drowsy after sleep. It leads to daytime dozing, depression, and reduced work efficiency, including reduced concentration and learning abilities. The severity of insomnia, a well-known sleep disorder, is socially underestimated compared with other diseases. Insomniac patients can suffer from severe pain that can also lead to suicidal ideations (Lim et al., 2023). Despite an increasing need for insomnia treatments to improve social productivity, such as work efficiency, no reliable solution has been proposed (Hafner et al., 2017). Drug-based treatment of insomnia currently involves the use of benzodiazepine and non-benzodiazepine drugs, benzodiazepine receptor agonists, antidepressants with sedative effects, and melatonin and antihistamine drugs; however, those drugs have side effects such as reduced memory, dizziness, haziness, and formation of resistance or dependence, so their long-term use is not suitable for students, athletes, or workers with early or intermittent anxiety or insomnia (Kallweit et al., 2023). Additionally, functional materials, such as Ecklonia cava extract (Cho et al., 2012), rice bran ethanol extract (Um et al., 2019), and milk protein hydrolysate (Kim et al., 2019), are commercially available in the health functional food market to improve sleep. Ashwagandha extract, a traditional Indian herbal medicine, has recently been certified as a functional material to improve sleep (Cheah et al., 2021). Overall, alternatives derived from natural materials that can effectively improve sleep disorders with few side effects should be developed.

Phlomoides umbrosa Turczaninow has traditionally been used as a medicinal plant, and its roots and leaves are used as food ingredients. According to Donguibogam (Heo, 2014), P. umbrosa Turczaninow can be used to treat pain and skin damage, as well as for postnatal care and muscle or bone union. It also exhibits anti-inflammatory (Shang et al., 2011), osteoblast differentiation (Chun et al., 2021), anti-allergic (Shin et al., 2008), and anti-asthmatic (Pak et al., 2022) effects. The sleep-enhancing effects of P. umbrosa Turczaninow have not been reported. Therefore, we conducted experiments using a pentobarbital-induced mouse model to assess the positive effects of PUTRE on sleep disorders and insomnia.

Changes in food efficiency rate (FER), weight gain, and organ weights

Table S1 presents the organ weights and FER, which we calculated by dividing the BW gain in each group during the experiment by the amount of food consumed. FER did not vary significantly across the groups, and no abnormal behavior affecting the food-related variables was observed. Upon analyzing the potential main indicators of toxicity in the mice, administration of the hypnotic drug and PUTRE caused no significant alteration in food intake, BW or the liver, kidney, spleen, and heart weights of the mice.

Improving effects of PUTRE on sleeping behaviors in pentobarbital-induced mice

To assess the sleep-inducing effects of PUTRE, two dosages of PUTRE, 200 and 400 mg/kg of BW/day, were repeatedly administered to ICR mice and compared with 2 mg of DZP/kg of BW/day (positive control). Forty-five minutes after the last administration of the test substances, a hypnotic agent (pentobarbital, 45 mg/kg of BW) was intraperitoneally injected to induce sleep. The measured sleep latency was 3.52 ± 0.30 and 3.07 ± 0.29 min in the groups injected with 200 and 400 mg of PUTRE/kg of BW/day, respectively. The sleep latency in the group injected with 400 mg of PUTRE/kg of BW/day was significantly lower (p < 0.05) than that in the control group (3.93 ± 0.44 min, Fig. 1A). Sleep latency also decreased significantly (p < 0.05) in the DZP-injected positive control group (2.30 ± 0.20 min, Fig. 1A). Similarly, the measured sleep duration was 68.40 ± 2.71 and 76.23 ± 3.06 min in the groups treated with 200 and 400 mg of PUTRE/kg of BW/day, respectively, a significant (p < 0.05) increase compared with the control group (50.73 ± 6.85 min, Fig. 1B). The sleep duration in the DZP-injected positive control group also increased significantly (p < 0.05) to 87.58 ± 5.93 min (Fig. 1B). Recovery time, the time between onset of the righting reflex and active movement, was 3.57 ± 0.30 and 3.54 ± 0.32 min in the groups treated with 200 and 400 mg of PUTRE/kg of BW/day, respectively, which was a significant (p < 0.05) decrease compared with the control group (4.36 ± 0.17 min, Fig. 1C). Thus, in our pentobarbital-induced mouse model, PUTRE had a remarkable effect on both sleep latency and sleep duration.

Fig. 1.

Fig. 1

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Effect of Phlomoides umbrosa Turczaninow root extract (PUTRE) on (A) sleep latency, (B) sleep duration, and (C) recovery time in pentobarbital-treated mice. Diazepam (DZP) and PUTRE were orally administered 45 min before an intraperitoneal injection of pentobarbital [45 mg/kg of body weight (BW)]. The control group received phosphate-buffered saline (PBS). The positive control group was orally administered 2 mg of DZP/kg of BW/day. The PUTRE-treated groups were orally administered 200 or 400 mg of PUTRE/kg of BW/day. Data are presented as the mean ± standard deviation (SD). *p < 0.05 versus control, as determined by Duncan’s multiple range test

Effect of PUTRE on serum melatonin and brain GABA concentration in pentobarbital-induced mice for sleep induction experiment

Melatonin is often called the sleep hormone because it regulates the sleep–wake cycle. In the evening, melatonin production increases to induce sleep; melatonin plays a particularly crucial role in high-quality sleep because it regulates the circadian rhythm (Zisapel, 2018). The serum melatonin concentration, a sleep-related indicator, was significantly (p < 0.05) higher in the DZP-treated positive control group (20.63 ± 3.18 pg/mL) and PUTRE-treated groups (20.63 ± 3.35 pg/mL with 200 mg of PUTRE/kg of BW/day and 22.04 ± 2.72 pg/mL with 400 mg of PUTRE/kg of BW/day) than in the control group (15.40 ± 2.58 pg/mL, Fig. 2A).

Fig. 2.

Fig. 2

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Effect of PUTRE on (A) serum melatonin and (B) brain γ-aminobutyric acid (GABA) concentration in pentobarbital-treated mice. DZP and PUTRE were orally administered 45 min before an intraperitoneal injection of pentobarbital (45 mg/kg of BW). The control group received PBS. The positive control group was orally administered 2 mg of DZP/kg of BW/day. The PUTRE-treated groups were orally administered 200 or 400 mg of PUTRE/kg of BW/day. Data are presented as the mean ± SD. *p < 0.05 versus control, as determined by Duncan’s multiple range test

GABA is a neurotransmitter in the brain that regulates several physiological mechanisms in the human body and is involved in tranquilization, stress relief, memory enhancement, and antihypertension; it alleviates depression and insomnia by improving cerebrovascular flow, increasing oxygen supply, and promoting the metabolic function of brain cells (Rashmi et al., 2018). GABA is also associated with hyperpolarization of nerve cells in the central nervous system and inhibition of neurotransmitter release. In general, suppressing GABA can cause anxiety and convulsions, and promoting GABA can have antidepressive, anticonvulsant, and sedative effects (Diana et al., 2014; Hepsomali et al., 2020; Ngo and Vo, 2019). Therefore, GABA concentration was measured in the brain tissue of mice induced with pentobarbital during the sleep induction experiment (Fig. 2B). A significant (p < 0.05) increase was seen in the DZP-treated positive control group (112.78 ± 15.04 pg/mg of protein) compared with the control group (77.44 ± 10.53 pg/mg of protein, Fig. 2B). In the treatment groups that received 200 and 400 mg of PUTRE/kg of BW/day, GABA concentrations in the brain tissue were 80.52 ± 9.03 and 88.07 ± 5.15 pg/mg of protein, respectively (Fig. 2B). The GABA concentration in the brain tissue of the PUTRE-treated groups was not significantly higher (p > 0.05) than in the control group, but it showed a tendency to increase as the PUTRE concentration increased.

Agonist effects of PUTRE on cellular and nuclear receptors

Functional assays of receptors associated with sleep were performed to identify the mechanism of the positive effect of PUTRE on sleep disorders. At concentrations above 30 μg/mL, the agonist activity of PUTRE against the adenosine A1 receptor was significantly higher (approximately 91.8%) than that of the CPA positive control (Fig. 3A). A dose–response relationship was observed for the adenosine A1 receptor, and its potential as a direct target in sleep induction was highest, with a half maximal effective concentration of 2.99 μg/mL (Fig. 3A). However, PUTRE did not exhibit any agonist activity toward the adenosine A2A receptor (Fig. 3A) or the GABA receptor (Fig. 3B).

Fig. 3.

Fig. 3

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Agonist effect of PUTRE on the (A) adenosine A1 and A2A receptors and (B) GABA receptor. Vehicle: 0.33% DMSO as PUTRE solvent. Data are presented as the mean ± SD

Hypnotic mechanisms of PUTRE involving the adenosine A1 receptor in pentobarbital-induced mice for agonist experiment

To identify whether the adenosine receptor was involved in the sleep-enhancing effect of PUTRE, a known adenosine receptor antagonist, caffeine (adenosine pan-inhibitor; a non-selective adenosine antagonist for A1/A2A receptors) or DPCPX (adenosine A1-specific inhibitor) was administered to mice along with 400 mg of PUTRE/kg of BW/day for 2 weeks (Fig. 4). Forty-five minutes after the last administration of the test substances, the hypnotic agent pentobarbital (45 mg/kg of BW) was intraperitoneally injected to induce sleep. In the positive control group, 2 mg of DZP/kg of BW/day was used.

Fig. 4.

Fig. 4

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Effect of adenosine receptor antagonists on (A) sleep latency and (B) sleep duration in pentobarbital-treated mice co-administered with PUTRE and caffeine or 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). DZP and PUTRE were orally administered 45 min before an intraperitoneal injection of pentobarbital (45 mg/kg of BW). The control group received PBS. The positive control group was orally administered 2 mg of DZP/kg of BW/day. The PUTRE + vehicle, PUTRE + caffeine, and PUTRE + DPCPX groups were administered 400 mg of PUTRE/kg of BW/day, 400 mg of PUTRE/kg of BW/day and 10 mg of caffeine/kg of BW/day, and 400 mg of PUTRE/kg of BW/day and 5 mg of DPCPX/kg of BW/day, respectively. Data are presented as the mean ± SD. *p < 0.05 versus control; #p < 0.05 PUTRE + versus vehicle, as determined by Duncan’s multiple range test

The sleep latency was 6.24 ± 1.19 min in the control group, in which sleep was induced and no test material was administered, whereas it was 4.55 ± 0.43 min in the PUTRE-only group (PUTRE + vehicle group). A significant (p < 0.05) decrease of approximately 27.1% was observed in the latter group compared with the control group (Fig. 4A). Co-administration of PUTRE with caffeine or DPCPX resulted in sleep latency of 5.44 ± 0.56 and 5.97 ± 1.58 min, respectively, with increases of approximately 19.6% and 31.2% (p < 0.05) compared with the PUTRE + vehicle group (Fig. 4A). This suggests that the improvement of sleep onset latency caused by PUTRE was offset by the co-administered caffeine or DPCPX. The sleep latency in the DZP-administered positive control group was 4.48 ± 0.49 min, which was a significant (p < 0.05) decrease, approximately 28.2%, compared with the control group that received sleep induction alone (Fig. 4A).

In the same experiment, the sleep duration was 47.83 ± 10.11 min in the control group that received sleep induction without any test material (Fig. 4B). On the other hand, the sleep duration time in the PUTRE + vehicle group was 56.67 ± 8.29 min, a non-significant (p > 0.05) increase of approximately 18.5%, compared with the control group (Fig. 4B). The sleep duration time in the PUTRE + caffeine group was 43.50 ± 9.18 min, and that in the PUTRE + DPCPX group was 52.83 ± 12.25 min (Fig. 4B), for decreases of approximately 23.2% (p < 0.05) and 6.8%, respectively, compared with the PUTRE + vehicle group, suggesting that the improvement in sleep duration time caused by PUTRE was offset by the co-administration of caffeine or DPCPX. The sleep duration in the DZP-treated positive control group was 62.00 ± 6.54 min, a non-significant (p > 0.05) increase of approximately 29.6% compared with the control group (Fig. 4B). These results indicate that PUTRE acted as an adenosine A1 receptor agonist (and had sleep induction effect similar to the DZP used as the positive control) because its effect was reduced by both the adenosine receptor antagonists (caffeine and DPCPX). This experiment confirmed the effect of PUTRE in improving sleep latency and duration in mice via its adenosine receptor agonist activity.

The neurotransmitter adenosine is one of several sleep-related compounds found in the human body. Adenosine has various physiological functions and is characteristically engaged in the sleep–wakefulness mechanism (Fredholm, 2014). Adenosine is an endogenic mediator of sleep deprivation, and its increased release accompanies the accumulated sleep demand, suggesting its involvement in homeostatic feedback control of sleep onset (Benington et al., 1995). Previous experiments in a mouse model showed that the adenosine level in the hippocampus was highest a few hours before the mice exhibited sleep-like behavior (Huston et al., 1996). Along with its role in sleep induction, adenosine affects biological rhythms (Jagannath et al., 2021). The effects of adenosine are mostly mediated through the activation of its receptors. Among the four adenosine receptor subtypes (A1, A2A, A2B, and A3), the adenosine A1 receptor contributes to the induction of sleep in specific body parts (Thakkar et al., 2003). Thus, the A1 receptor regulates the sleep–wakefulness cycle, whereas the A2A receptor induces sleep (Huang et al., 2014).

Caffeine is a widely used stimulant that acts by blocking the activity of adenosine receptors in the basal forebrain (Lazarus et al., 2011). Whereas caffeine is a nonspecific antagonist that can inhibit all four adenosine receptor subtypes (A1, A2A, A2B, and A3), DPCPX is a powerful and selective antagonist of the adenosine A1 receptor, so it can stimulate the brain and behaviors, similar to the role of caffeine (Van Dort et al., 2009).

PUTRE had a remarkable effect on sleep induction (Fig. 1). The specific association of its sleep-enhancing effect with the adenosine A1 receptor was verified via in vitro functional assays (Fig. 3). To verify the association with the adenosine A1 receptor in vivo, mice were treated with PUTRE in combination with adenosine receptor antagonists (caffeine and DPCPX), and those results showed that the sleep-enhancing effect of PUTRE was offset by both adenosine antagonists (Fig. 4).

Effects of PUTRE and adenosine antagonists on GABA concentration in brain tissue in pentobarbital-induced mice

To determine how co-administering PUTRE with caffeine or DPCPX affected the GABA concentration in the brain tissues of pentobarbital-induced mice, brain tissues extracted during autopsy were homogenized, and an ELISA was performed. Those results are shown in Fig. 5. The GABA concentration in the control group and positive control group treated with DZP (GABA receptor agonist) was 84.88 ± 6.01 and 105.33 ± 8.50 pg/mg of protein, respectively, with a significant (p < 0.05) increase in GABA concentration of approximately 24.1% in the latter group compared with the former group. The GABA concentration (99.17 ± 11.36 pg/mg of protein) increased significantly (p < 0.05), by approximately 16.8%, in the group treated with only PUTRE (PUTRE + vehicle group) compared with the control group. The GABA concentrations in the PUTRE + caffeine and PUTRE + DPCPX groups were 78.22 ± 8.76 and 75.93 ± 8.27 pg/mg of protein, respectively, significantly (p < 0.05) lower, by approximately 21.1% and 23.4%, respectively, than in the PUTRE + vehicle group. GABA is a sleep hormone, so the increased GABA levels in brain tissue from mice treated with PUTRE were counteracted by co-treatment with an adenosine receptor antagonist (caffeine or DPCPX). Melatonin has previously been reported to increase brain GABA concentrations (Rosenstein and Cardinali, 1986; Xu et al., 1995). However, in this study, PUTRE had no direct effect on the GABA ion channel (Fig. 3B), suggesting that the increase in GABA concentration in the brain associated with PUTRE could be due to indirect factors such as melatonin.

Fig. 5.

Fig. 5

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Effect of PUTRE and adenosine receptor antagonists on GABA concentration in brain tissue from pentobarbital-treated mice co-administered with PUTRE and caffeine or DPCPX. DZP and PUTRE were orally administered 45 min before an intraperitoneal injection of pentobarbital (45 mg/kg of BW). The control group received PBS. The positive control group was orally administered 2 mg of DZP/kg of BW/day. The PUTRE + vehicle, PUTRE + caffeine, and PUTRE + DPCPX groups were administered 400 mg of PUTRE/kg of BW/day, 400 mg of PUTRE/kg of BW/day and 10 mg of caffeine/kg of BW/day, and 400 mg of PUTRE/kg of BW/day and 5 mg of DPCPX/kg of BW/day, respectively. Data are presented as the mean ± SD. *p < 0.05 versus control; #p < 0.05 versus PUTRE + vehicle, as determined by Duncan’s multiple range test

Effects of PUTRE and adenosine antagonists on adenosine A1 receptor expression in pentobarbital-induced mice

To determine the effects of PUTRE administration on the expression of the adenosine A1 receptor gene in brain tissue from pentobarbital-induced mice, reverse transcription PCR was performed. The expression of the adenosine A1 receptor gene in mouse brain tissue increased significantly (p < 0.05), by approximately 35.3%, in the group treated with DZP, a known agonist, compared with the control group (Fig. 6A), and it increased by approximately 22.8% in the PUTRE + vehicle group compared with the control group (Fig. 6A). The PUTRE + caffeine group had a non-significant (p > 0.05) decrease in the expression of the adenosine A1 receptor gene, by approximately 9.2%, compared with the PUTRE + vehicle group, whereas the PUTRE + DPCPX group had a significant (p < 0.05) decrease in adenosine A1 receptor gene expression, by approximately 14.6%, compared with PUTRE + vehicle group (Fig. 6A).

Fig. 6.

Fig. 6

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Effects of PUTRE and adenosine A1 receptor antagonist on (A) mRNA expression, (B) protein expression, and (C) the adenosine A1 receptor/β-actin ratio in pentobarbital-treated mice co-administered with PUTRE and caffeine or DPCPX. DZP and PUTRE were orally administered 45 min before an intraperitoneal injection of pentobarbital (45 mg/kg of BW). The control group received PBS. The positive control group was orally administered 2 mg of DZP/kg of BW/day. The PUTRE + vehicle, PUTRE + caffeine, and PUTRE + DPCPX groups were administered 400 mg of PUTRE/kg of BW/day, 400 mg of PUTRE/kg of BW/day and 10 mg of caffeine/kg of BW/day, and 400 mg of PUTRE/kg of BW/day and 5 mg of DPCPX/kg of BW/day, respectively. Data are presented as the mean ± SD. *p < 0.05 versus control; #p < 0.05 versus PUTRE + vehicle, as determined by Duncan’s multiple range test

To determine the effect of PUTRE administration on protein expression of the adenosine A1 receptor in brain tissue from pentobarbital-induced mice, western blot analyses were performed (Fig. 6B). Adenosine A1 receptor protein production in brain tissue from the DZP-administered positive control group was significantly (p < 0.05) higher, by approximately 535.3%, than in the control group (Fig. 6C). Adenosine A1 receptor protein production also increased significantly (p < 0.05), by approximately 280.1%, in the PUTRE + vehicle group compared with the control group. On the other hand, the PUTRE + caffeine and PUTRE + DPCPX groups had significant (p < 0.05) decreases in adenosine A1 receptor protein production, by approximately 42.1% and 42.8%, respectively, compared with the PUTRE + vehicle group (Fig. 6C). These results confirm that PUTRE directly increased the production of the adenosine A1 receptor, and the adenosine receptor antagonists caffeine and DPCPX offset the positive responses induced by PUTRE. Thus, the sleep-enhancing activity of PUTRE is due to its adenosine receptor agonist activity.

The gene and protein expressions of the adenosine A1 receptor in brain tissue increased significantly (p < 0.05) with PUTRE administration, and co-administration of PUTRE with caffeine or DPCPX suppressed the increase in adenosine A1 receptor levels. Therefore, PUTRE exerts its agonistic activity on the expression of the adenosine A1 receptor at the gene level.

We identified four specific marker components in PUTRE using a high-performance liquid chromatography–photodiode array system: sesamoside, shanzhiside methylester, umbroside, and verbascoside (data not shown). Verbascoside is a caffeoyl phenylethanoid glycoside found in PUTRE. It has various pharmacological health benefits, such as antimicrobial, anti-inflammatory, and antioxidant effects (Pardo et al., 1993; Speranza et al., 2009). Also, it has previously been reported that verbascoside significantly decreased sleep latency and significantly increased sleeping time in a pentobarbital-induced mouse model (Razavi et al., 2017).

In conclusion, this study is the first to demonstrate that PUTRE could serve as an agonist for sleep induction, similar to the DZP used as the positive control. Compared with the control group, the mice treated with PUTRE showed reduced sleep latency and increased sleep duration. The recovery time between the onset of wakefulness and active movements was also reduced, implying that PUTRE has a positive effect on not only sleep duration but also sleep quality. Our analysis of PUTRE’s mechanism of action revealed that the sleep-enhancing effect was due to adenosine A1 receptor-specific agonist activity. Future studies should elucidate the comprehensive mechanisms responsible for the regulatory effect of PUTRE and its active compounds, such as verbascoside, on the adenosine A1 receptor and downstream signaling. Additionally, a clinical study should be conducted to determine whether the sleep-enhancing effect of PUTRE confirmed in our in vivo study remains valid in human.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 18 KB) (18KB, docx)

Acknowledgements

This work was supported by the National Institute of Horticultural and Herbal Science, Rural Development Administration (Project No. PJ014195-04), Republic of Korea.

Declarations

Conflict of interest

On behalf of all authors, the corresponding author states that there is no conflict of interest.

Footnotes

Publisher's Note

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Contributor Information

Chan-Sung Park, Email: cspark@naturalendo.co.kr.

Dae-Ok Kim, Email: DOKIM05@khu.ac.kr.

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