Data_Sheet_1_Humulone Modulation of GABAA Receptors and Its Role in Hops Sleep-Promoting Activity.docx

Humulus lupulus L. (hops) is a major constituent of beer. It exhibits neuroactive properties that make it useful as a sleeping aid. These effects are hypothesized to be mediated by an increase in GABAA receptor function. In the quest to uncover the constituents responsible for the sedative and hypnotic properties of hops, recent evidence revealed that humulone, a prenylated phloroglucinol derivative comprising 35–70% of hops alpha acids, may act as a positive modulator of GABAA receptors at low micromolar concentrations. This raises the question whether humulone plays a key role in hops pharmacological activity and potentially interacts with other modulators such as ethanol, bringing further enhancement in GABAA receptor-mediated effects of beer. Here we assessed electrophysiologically the positive modulatory activity of humulone on recombinant GABAA receptors expressed in HEK293 cells. We then examined humulone interactions with other active hops compounds and ethanol on GABA-induced displacement of [3H]EBOB binding to native GABAA receptors in rat brain membranes. Using BALB/c mice, we assessed humulone’s hypnotic behavior with pentobarbital- and ethanol-induced sleep as well as sedation in spontaneous locomotion with open field test. We demonstrated for the first time that humulone potentiates GABA-induced currents in α1β3γ2 receptors. In radioligand binding to native GABAA receptors, the inclusion of ethanol enhanced humulone modulation of GABA-induced displacement of [3H]EBOB binding in rat forebrain and cerebellum as it produced a leftward shift in [3H]EBOB displacement curves. Moreover, the additive modulatory effects between humulone, isoxanthohumol and 6-prenylnaringenin were evident and corresponded to the sum of [3H]EBOB displacement by each compound individually. In behavioral tests, humulone shortened sleep onset and increased the duration of sleep induced by pentobarbital and decreased the spontaneous locomotion in open field at 20 mg/kg (i.p.). Despite the absence of humulone effects on ethanol-induced sleep onset, sleep duration was increased dose-dependently down to 10 mg/kg (i.p.). Our findings confirmed humulone’s positive allosteric modulation of GABAA receptor function and displayed its sedative and hypnotic behavior. Humulone modulation can be potentially enhanced by ethanol and hops modulators suggesting a probable enhancement in the intoxicating effects of ethanol in hops-enriched beer.

啤酒花（Humulus lupulus L.）是啤酒的核心原料之一，其具备神经活性，可作为助眠制剂应用。现有假说认为，该活性通过增强γ-氨基丁酸A型受体（GABAA receptor）功能介导。为明确啤酒花中赋予其镇静催眠功效的活性成分，近期研究发现，葎草酮（humulone）——一种占啤酒花总α-酸35%~70%的异戊烯基间苯三酚衍生物——在低微摩尔浓度下可作为γ-氨基丁酸A型受体的正变构调节剂。这引发了一个关键科学问题：葎草酮是否在啤酒花的药理活性中发挥核心作用？其是否可与乙醇等其他调节剂相互作用，进一步强化啤酒中γ-氨基丁酸A型受体介导的生理效应？本研究首先采用电生理技术，评估了葎草酮对HEK293细胞中表达的重组γ-氨基丁酸A型受体的正调节活性；随后，针对大鼠脑细胞膜中的天然γ-氨基丁酸A型受体，开展[3H]EBOB放射配体结合实验，考察了葎草酮与其他啤酒花活性成分及乙醇的相互作用对γ-氨基丁酸诱导的[3H]EBOB结合置换的影响。此外，本研究使用BALB/c小鼠，通过戊巴比妥与乙醇诱导睡眠模型，以及旷场实验（open field test）检测自发活动，评估了葎草酮的催眠与镇静行为学效应。本研究首次证实，葎草酮可增强α1β3γ2型受体中γ-氨基丁酸诱导的电流反应。在天然γ-氨基丁酸A型受体的放射配体结合实验中，乙醇的加入可增强葎草酮对大鼠前脑与小脑组织中γ-氨基丁酸诱导的[3H]EBOB结合置换的调节作用，具体表现为[3H]EBOB置换曲线左移。此外，葎草酮、异黄腐酚（isoxanthohumol）与6-异戊烯基柚皮素（6-prenylnaringenin）之间存在叠加调节效应，其作用效果与各化合物单独作用时的效应之和相符。行为学实验结果显示，腹腔注射20 mg/kg剂量的葎草酮可缩短戊巴比妥诱导的睡眠潜伏期，并延长睡眠持续时间，同时降低小鼠在旷场实验中的自发活动量。尽管葎草酮对乙醇诱导的睡眠潜伏期无显著影响，但其可剂量依赖性地延长睡眠持续时间，最低有效剂量可达10 mg/kg（腹腔注射）。本研究证实，葎草酮可对γ-氨基丁酸A型受体功能产生正变构调节，并展现出镇静与催眠的行为学效应。乙醇与其他啤酒花调节剂可增强葎草酮的调节活性，这提示在添加啤酒花的啤酒中，乙醇的致醉效应可能得到进一步强化。