背景:花椒.(Z.bungeanum),菊科的一员,在亚洲有丰富的传统使用历史来治疗关节炎和牙痛。作为特有的化学成分,从植物中提取了多种生物碱,并报道了其多种生物活性。然而,异喹啉生物碱的研究,一种特定类型的生物碱,在Z.bungeanum是稀缺的。
目的:本研究旨在从香根草中分离出一种新型异喹啉类生物碱,并探讨其体外药理活性和体内镇痛活性。
方法:采用色谱和光谱方法从Z.bungeanum中分离和鉴定异喹啉生物碱。全细胞膜片钳技术用于评估其对神经元兴奋性的影响,和急性分离的小鼠小直径背根神经节(DRG)神经元中的内源性电压门控钾(Kv)和钠(Nav)电流。用稳定表达Nav1.7和Nav1.8的HEK293细胞和瞬时表达Kv2.1的中国仓鼠卵巢(CHO)细胞进一步验证了其对通道的抑制作用。福尔马林炎性疼痛模型用于评估体内潜在的镇痛活性。
结果:首次从Z.bungeanum中分离并鉴定了一种新的异喹啉生物碱,命名为HJ-69(N-13-(3-甲氧基丙-1-基)卢卡地平)。HJ-69可显着抑制DRG神经元中动作电位的激发频率和振幅。始终如一,它状态依赖性地抑制DRG神经元的内源性Nav电流,失活和静息状态的半数最大抑制浓度(IC50)值为13.06±2.06μM和30.19±2.07μM,分别。HJ-69显著抑制DRG神经元的钾电流,显着抑制了延迟整流钾(IK)电流(IC50=6.95±1.29μM),并略微影响了瞬态外向钾(IA)电流(IC50=523.50±39.16μM)。更进一步,HJ-69在异源表达的Nav1.7,Nav1.8和Kv2.1通道上表现出相似的效力,它们相应地代表了神经元中的主要成分。值得注意的是,腹膜内给药30mg/kg和100mg/kgHJ-69可显着减轻福尔马林诱导的小鼠炎性疼痛模型的疼痛行为。
结论:该研究得出结论,HJ-69是一种新颖的活性异喹啉生物碱,Nav和Kv通道的抑制有助于其镇痛活性。HJ-69可能是基于异喹啉生物碱的未来镇痛药物发现的有希望的原型。
BACKGROUND: Zanthoxylum bungeanum Maxim. (Z. bungeanum), a member of the Rutaceae family, has a rich history of traditional use in Asia for treating arthritis and toothache conditions. As characteristic chemical components, numerous kinds of alkaloids have been extracted from plants and their diverse biological activities have been reported. However, research on the isoquinoline alkaloid, a specific type of alkaloids, in Z. bungeanum was scarce.
OBJECTIVE: The study aimed to isolate a novel isoquinoline alkaloid from Z. bungeanum and explore its pharmacological activity in vitro and analgesic activity in vivo.
METHODS: Isoquinoline alkaloid isolation and identification from Z. bungeanum were conducted using chromatographic and spectroscopic methods. The whole-cell patch-clamp technique was applied to assess its impact on neuronal excitability, and endogenous voltage-gated potassium (Kv) and sodium (Nav) currents in acutely isolated mouse small-diameter dorsal root ganglion (DRG) neurons. Its inhibitory impacts on channels were further validated with HEK293 cells stably expressing Nav1.7 and Nav1.8, and Chinese hamster ovary (CHO) cells transiently expressing Kv2.1. The formalin inflammatory pain model was utilized to evaluate the potential analgesic activity in vivo.
RESULTS: A novel isoquinoline alkaloid named HJ-69 (N-13-(3-methoxyprop-1-yl)rutaecarpine) was isolated and identified from Z. bungeanum for the first time. HJ-69 significantly suppressed the firing frequency and amplitudes of action potentials in DRG neurons. Consistently, it state-dependently inhibited endogenous Nav currents of DRG neurons, with half maximal inhibitory concentration (IC50) values of 13.06 ± 2.06 μM and 30.19 ± 2.07 μM for the inactivated and resting states, respectively. HJ-69 significantly suppressed potassium currents in DRG neurons, which notably inhibited the delayed rectifier potassium (IK) currents (IC50 = 6.95 ± 1.29 μM) and slightly affected the transient outward potassium (IA) currents (IC50 = 523.50 ± 39.16 μM). Furtherly, HJ-69 exhibited similar potencies on heterologously expressed Nav1.7, Nav1.8, and Kv2.1 channels, which correspondingly represent the main components in neurons. Notably, intraperitoneal administration of 30 mg/kg and 100 mg/kg HJ-69 significantly alleviated pain behaviors in the mouse inflammatory pain model induced by formalin.
CONCLUSIONS: The study concluded that HJ-69 is a novel and active isoquinoline alkaloid, and the inhibition of Nav and Kv channels contributes to its analgesic activity. HJ-69 may be a promising prototype for future analgesic drug discovery based on the isoquinoline alkaloid.