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

Abstract

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 (GABA_A, adenosine A₁ and A_2A 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.

Keywords: Adenosine A1 receptor; Adenosine A2A receptor; EEG; FSD-LS; NREM; Sleep.

Fig. 1

Sleep onset (a, c) and duration (b, d) in seconds, resulting from pentobarbital-induced sleep effect (42 mg/kg) after acute and sub-chronic treatment with FSD-LS extracts (50, 100, and 150 mg/kg, p.o.) and diazepam (DZP, 1 mg/kg, i.p.) administration in ICR mice. Values are mean ± SEM. n = 9–10 (acute), 10–15 (sub-chronic) animals per group. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. DW (one-way ANOVA followed by Tukey’s post-hoc analysis)

Fig. 2

Delta and alpha wave absolute power (µV²) generated after acute (a, b) and sub-chronic (c, d) treatment with FSD-LS extract (50, 100, and 150 mg/kg, p.o.) and diazepam (DZP, 1 mg/kg, i.p.) administration in ICR mice. Values are mean ± SEM. n = 5–7 (acute), 9–16 (sub-chronic) animals per group. *P < 0.05, **P < 0.01 and ***P < 0.001 vs. DW (one- or two-way ANOVA followed by Tukey’s post-hoc analysis). ^#P < 0.05 vs. DW (t-test)

Fig. 3

Hourly average (a) and 3-h total (b) WAKE, NREM, and REM durations (min) of ICR mice after 5-day treatment of FSD-LS extract (100 mg/kg, p.o.) and diazepam (DZP, 1 mg/kg, i.p.) administration. Values are mean ± SEM. n = 10 animals per group. *P < 0.05 vs. DW (one- or two-way ANOVA followed by Tukey’s post-hoc analysis). ^#P < 0.05 vs. DW (t-test)

Fig. 4

Behavior of ICR mice in the open field test and rota-rod after acute (a, b, c, d) and sub-chronic (e, f, g, h) treatment with FSD-LS extract (50, 100, and 150 mg/kg, p.o.) and diazepam (DZP, 1 mg/kg, i.p.) administration. Values are mean ± SEM. n = 10–15 animals per group. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. DW (one-way ANOVA followed by Tukey’s post-hoc analysis)

Fig. 5

Percentage of inhibition and concentration-inhibition relationship of FSD-LS on adenosine and GABA receptors at varying concentrations (10, 30, 100, 300, and 1000 µg/mL). IC₅₀ and K_i values are expressed in µg/mL

Fig. 6

Effect of bicuculline (BIC, 1 mg/kg, i.p.) pre-treatment on sleep onset and duration (s) resulting from pentobarbital-induced sleep (42 mg/kg, i.p.) after acute (a, b) and sub-chronic (c, d) treatment of FSD-LS extract (100 mg/kg, p.o.) and diazepam (DZP, 1 mg/kg, i.p.) administration in ICR mice. Hourly average (e) and 3-h total (f) WAKE, NREM, and REM durations (min), along with delta (g) and alpha (h) wave absolute power (µV²) generated, after 5-day treatment with FSD-LS extract and DZP administration in mice pre-treated with BIC. Values are mean ± SEM. n = 10–14 animals per group. *P < 0.05, **P < 0.01, and ***P < 0.001 (one- or two-way ANOVA followed by Tukey’s post-hoc analysis or Dunnett’s post-hoc analysis). ^###P < 0.05 (t-test)

Fig. 7

Effect of caffeine (CAF, 50 mg/kg, p.o.) pre-treatment on sleep onset and duration (s) resulting from pentobarbital-induced sleep (42 mg/kg, i.p.) after acute (a, b) and sub-chronic (c, d) treatment of FSD-LS extract (100 mg/kg, p.o.) and diazepam (DZP, 1 mg/kg, i.p.) administration in ICR mice. Hourly average (e) and 3-h total (f) WAKE, NREM, and REM durations (min), along with delta (g) and alpha (h) wave absolute power (µV²) generated, after 5-day treatment with FSD-LS extract and DZP administration in mice pre-treated with CAF. Values are mean ± SEM. n = 10–16 animals per group. *P < 0.05, **P < 0.01, and ***P < 0.001 (one- or two-way ANOVA followed by Tukey’s post-hoc analysis or Dunnett’s post-hoc analysis). ^#P < 0.05 (t-test)