
Luteolin, a widespread flavonoid, has been known to have neuroprotective activity against various neurologic diseases such as epilepsy, and Alzheimer’s disease. However, little information is available regarding the hypnotic effect of luteolin. In this study, we evaluated the hypnotic effect of luteolin and its underlying mechanism. In pentobarbital-induced sleeping mice model, luteolin (1, and 3 mg/kg, p.o.) decreased sleep latency and increased the total sleep time. Through electroencephalogram (EEG) and electromyogram (EMG) recording, we demonstrated that luteolin increased non-rapid eye movement (NREM) sleep time and decreased wake time. To evaluate the underlying mechanism, we examined the effects of various pharmacological antagonists on the hypnotic effect of luteolin. The hypnotic effect of 3 mg/kg of luteolin was not affected by flumazenil, a GABAA receptor-benzodiazepine (GABAAR-BDZ) binding site antagonist, and bicuculine, a GABAAR-GABA binding site antagonist. On the other hand, the hypnotic effect of 3 mg/kg of luteolin was almost completely blocked by caffeine, an antagonist for both adenosine A1 and A2A receptor (A1R and A2AR), 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX), an A1R antagonist, and SCH-58261, an A2AR antagonist. From the binding affinity assay, we have found that luteolin significantly binds to not only A1R but also A2AR with IC50 of 1.19, 0.84 μg/kg, respectively. However, luteolin did not bind to either BDZ-receptor or GABAAR. From these results, it has been suggested that luteolin has hypnotic efficacy through A1R and A2AR binding.
Sleep is essential to maintain human health and well-being, and sleep disturbances can affect negatively both physical and psychological health (Krueger
To overcome the adverse effects of sleep medications, special attention has been recently focused on alternative sleep aids such as natural and herbal therapies (Cho
Among various flavonoids, apigenin and chrysin have been known to have hypnotic effects with benzodiazepine (BDZ)-binding mechanisms (Wolfman
Pentobarbital and diazepam (DZP, a reference hypnotic drug) was purchased from Hanlim Pharm. Co. Ltd. (Seoul, Korea) and Myungin Pharm. Co. Ltd. (Seoul, Korea), respectively. Luteolin, flumazenil, bicuculline and caffeine were purchased from Sigma–Aldrich Inc (St. Louis, USA). 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX), and SCH58261 were purchased from Tocris Biosciences (Avonmouth, UK). In GABAAR binding assay, the radioligand [3H] flunitrazepam (NEN Perkin-Elmer, Ontario, CA, USA) used for GABAAR-BZD agonist, and [3H] muscimol (NEN Perkin-Elmer) were used for GABAAR-GABA binding site (GABAAR-GABA) agonist. For the adenosine 1 receptor (A1R), and adenosine 2A receptor (A2AR) binding assay, the radioligand [3H] CCPA and [3H] CGS21680, respectively, were purchased from NEN Life Science Products (Boston, MA, USA).
All animals were purchased from Orient Bio Inc (Seongnam, Korea). Male Imprinting Control Region (ICR) mice of 8 weeks old (weigh 25–30 g) were used in pentobarbital-induced sleep.
Male C57BL/6 mice of 4 weeks old (weigh 20–25 g) were used for measurements of EEG architectures. Mice were housed at 24°C and 55% humidity, with food and water were ad libitum. The room maintained 12 h light/dark cycle (light on at 8:00 and off at 20:00). Animals were acclimated for over 1 week before experiments. All animals related study protocols were approved by the Committee on Animal Research at Ajou University (Permission No. 2018-0051).
Pentobarbital-induced sleep test were performed between 13:00 and 17:00. Luteolin, flumazenil, bicuculline, caffeine, and pentobarbital were suspended in saline. DPCPX and SCH58261 were suspended in 1% aqueous solution of Tween 80. Luteolin or saline was orally administered to mice 30 min before pentobarbital injection. Antagonists for GABAAR, A1R and A2AR were orally administered 15 min before luteolin administration. Control and vehicle mice were administered with saline or 1% Tween 80 at the same time for compared to the sample administration group. After pentobarbital injection (45 mg/kg i.p.), mice were placed in individual cages for the test. For sleep latency, the time was recorded from pentobarbital injection to the time when righting reflex disappeared. For sleep duration, the time was recorded until the righting reflex recovered.
EEG and EMG were recorded from 09:00 to 15:00 according the method described previously (Dela Pena
The EEG and EMG signals were routed to an 8401 conditioning/acquisition system (Pinnacle Technology, Inc.) via a tether and low-torque commutator (Part #8408, Pinnacle Technology, Inc.). Continuous sections of recorded EEG and EMG data were divided to 10 s (1 epoch). Each epoch was scored as wake, NREM or REM sleep based on which type of waveform occupied >50% of that time. The absolute EEG power recorded wakefulness, NREM and REM in the range of from 0.5 to 20 Hz. NREM, REM and wakefulness were calculated in δ (0.5–4.0 Hz), θ (5.0–9.0 Hz), α (8.0–13.0 Hz). Values measured were calculated in Microsoft Excel (WA, USA).
The radioligand GABAAR binding assay was performed with Eurofins (St. Charles, IL, USA) Pharmacology Services. The cell membrane pellet from brain tissues of mice was obtained by the method described previously (Risa
The radioligand binding assay for A1R and A2AR was performed with Eurofins Pharmacology Services as described by Rivkees
All data are expressed as mean ± SEM. Significance was evaluated by analysis of variance (ANOVA). Differences between two-group with
To evaluate hypnotic activity of luteolin, we examined its effect on pentobarbital-induced sleep in mouse. After injection of a hypnotic dose of pentobarbital (45 mg/kg, i.p.), sleep latency and duration of the control group were 248.0 ± 5.8 sec and 49.6 ± 2.7 min, respectively (Fig. 1). As expected, a wellknown GABAAR-BDZ receptor agonist, DZP (1 mg/kg, p.o.) significantly promoted sleep latency and duration (
After oral administration of luteolin (3 mg/kg, p.o.) to the mice, the sleep architectures (NREM sleep, REM sleep, wake) were recorded. As shown in Fig. 2, compared to control group (Vehicle), luteolin significantly increased NREM sleep (approximately 16.7%). In addition, wake time was significantly decreased by luteolin approximately 40.9%. Luteolin (3 mg/kg) also changed EEG power density so that in NREM sleep, the power density of δ-wave significantly increased about 9.7% (Fig. 3).
In order to investigate whether GABAAR is involved in the hypnotic mechanisms of luteolin, we used pharmacological antagonists for GABAAR-BDZ (flumazenil), and GABAAR-GABA (bicuculline). As shown in Fig. 4, the GABAAR-BDZ agonist, DZP (1 mg/kg), significantly decreased sleep latency and increased sleep duration–an effect that was blocked by administration of flumazenil. Unlike DZP, the hypnotic effect of luteolin (3 mg/kg) was not affected by either flumazenil or bicuculline. We also evaluated the binding affinity of luteolin for GABAAR-BDZ or GABAAR-GABA. As expected, luteolin was not displace both [3H] flunitrazepam, and [3H] muscimol binding (Table 1). These results suggested that, hypnotic effect of luteolin were not related with GABAAR-BDZ, and GABAAR-GABA.
To test whether the hypnotic effects of luteolin were related with AR, we used caffeine (an A1R, A2AR antagonist), DPCPX (an A1R antagonist), and SCH58261 (an A2AR antagonist). As shown in Fig. 5, the hypnotic effect of luteolin (3 mg/kg) was antagonized by caffeine, DPCPX and SCH58261. We also evaluated the binding affinity of luteolin for A1R, and A2AR. Luteolin was found to displace over 90% of both [3H] CCPA, and [3H] CGS21680 binding at 10 μg/ml. Its IC50 values for A1R and A2AR were 1.19 and 0.84 μg/ml, respectively (Table 1). These results suggest that hypnotic effect of luteolin involves both A1R and A2AR.
The major finding of this study is that luteolin improves quantity and quality of sleep, especially NREM, and its underlying mechanisms may involve A1R and A2AR binding, but neither GABAAR-BDZ nor GABAAR-GABA binding.
Sleep disorders can be caused by lack of sleep, excessive amounts of sleep, or abnormal movements during sleep. Among various sleep disorders, insomnia is that the patient reports not only dissatisfaction with sleep (sleep-onset insomnia or sleep-maintenance insomnia) but also other daytime symptoms (sleepiness, attention disorders, mood disorders) for at least 3 nights per week and last for more than 3 months (K. Pavlova and Latreille, 2019). Most epidemiological studies show that about one third (30%–36%) of adults have at least one symptom of insomnia, such as difficulty in starting or maintaining sleep (Ohayon, 2002). Insomnia increases the public health burden and health care costs, and decreased work ability in many people (Atkin
Luteolin, one of the important bioactive flavonoids, has been suggested to be a useful therapeutic constituent for neurodegenerative diseases based on not only its anti-inflammatory effects which has a crucial role in the neuroprotection, but also its lipophilic property indicating the possibility to cross blood brain barrier (Orhan
Adenosine is known as an endogenous sleep-promoting substance (Zhang
In this study, we found that luteolin directly bound to both A1R and A2AR and with IC50 value of 1.19 μg/ml and 0.84 μg/ml, respectively. We also found that the sleep-improving effect of luteolin was significantly inhibited by DPCPX and SCH58261, suggesting that the sleep-promoting effect of luteolin may be mediated via activation of not only A1R but A2AR. Further study remains to clarify whether the level of GABA or adenosine in brain tissue can be changed by luteolin. In conclusion, this study for the first time demonstrate that luteolin improves quantity and quality of sleep, especially for NREM in mice. As a mechanism for the effect of luteolin, the involvement of both A1R and A2AR activation, but neither GABAAR-BDZ nor GABAAR-GABA binding seems to be very convincing. From these results, it is suggested that luteolin may have a potential for sleep-promoting agent.
This research was supported by the Commercialization Promotion Agency for R&D Outcomes (COMPA) funded by the Ministry of Science and ICT (MSIT) (2018K000277), the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (HI18C0920), and the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2018R1D1A1B07048729), Republic of Korea.
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