Pain seriously affects human health and quality of life and has become the third major health problem after cardiovascular and cerebrovascular disease and cancer. Considering its duration, pain is typically classified as either acute or chronic. Acute pain commonly lasts for a shorter period, whereas chronic pain is conditions that persist for more than 3 months (Fillingim et al., 2016). The occurrence of chronic pain is estimated to reach as high as 38%, and the staggering figure underscores an enormous personal and economic burden on individuals and society (Tsang et al., 2008). The currently available analgesics generally come with limitations such as side effects, tolerance issues, and varying efficacy among individuals. Nonsteroidal anti-inflammatory drugs (NSAIDs) are commonly employed to mitigate chronic pain, but their long-term usage might pose gastrointestinal or renal side effects with undesirable analgesic efficacy. In some cases, opioids, despite their potency in pain management, cause adverse side effects like constipation or even the risk of dependency (Ribeiro et al., 2022). Discovering new analgesics with novel moieties enables a more tailored and effective approach, thereby providing a broader range of safer and more efficient pain management options.

Ion channels represent a prominent category of drug targets, with those distributed in peripheral nociceptors playing a crucial role in transmitting and processing nociceptive signals (Higerd-Rusli et al., 2022; Kim et al., 2023; Waxman and Zamponi, 2014). Multiple evidence collected from rodent models and human genetic studies demonstrated that modulation of ion channels is an effective strategy for treating pain (Dib-Hajj et al., 2017; Hameed, 2019; Sun et al., 2022). Voltage-gated sodium (Nav) and potassium (Kv) channels generate currents that cause the depolarizing and hyperpolarizing phases of the neuronal action potential (AP), respectively (Bean, 2007; Dib-Hajj et al., 2017). Specifically, Nav1.7 and Nav1.8 are abundantly expressed in the peripheral sensory neurons. They are critical for generating and conducting pain signals, with Nav1.7 primarily defining the threshold for AP generation and Nav1.8 forming the raising phase of an AP (Alsaloum et al., 2020; Hameed, 2019). As with Nav channels, Kv channels also fulfill a crucial role in diverse physiological processes, including regulating cellular excitability and transmission, and have been classified as delayed rectifier potassium current (IK) currents and transient outward potassium currents (IA) (Maljevic and Lerche, 2013; Oyrer et al., 2018). Notably, Kv2.1, which is enriched in nociceptive neurons, represents a major contributor to the IK currents and is crucial for regulating neuronal excitability (Fernández-Mariño et al., 2023; Misonou et al., 2005; Murakoshi H, 1999). Given the pivotal roles of these channels in nociceptive pain, the development of drugs targeting these channels is being extensively studied.

Zanthoxylum bungeanum Maxim. (Z. bungeanum), a traditional medicinal plant, finds extensive use in China, the United States, Africa and South Asia for treating toothache, arthritis and other pain conditions, and is known as the 'toothache tree' due to its excellent analgesic activity (Pereira et al., 2010; Tsunozaki et al., 2013). Over the past few decades, various structural types of analgesic monomers, including lignans (Bastos et al., 2001), alkaloids (Hu et al., 2006), flavonoids (Zhang et al., 2014), and terpenoids (Wu and Wu, 2014) have been isolated from Z. bungeanum. Among them, alkaloids were considered to be the primary active components in Z. bungeanum (Ivane et al., 2022), and topical application of the alkaloidal part or some active monomers effectively relieved the mechanical hypersensitivity in rodent inflammatory pain models (Tan et al., 2022). For example, the amide alkaloid hydroxy-α-sanshool (HAS) inhibited the excitability of Aδ mechanical injury receptors, resulting in a rapidly analgesic effect (Tsunozaki et al., 2013). As significant parts within the alkaloids, the diverse structures and promising analgesic activities of isoquinoline alkaloids have gained comprehensive attention in pharmaceutical research, and the development of new therapeutic agents based on these prototypes was thought to be a promising strategy.

Currently, numerous isoquinoline alkaloids exhibiting noteworthy analgesic properties were reported (Fig. 1). Within Corydalis yanhusuo, key analgesic compounds such as L-tetrahydropalmatine (L-THP, 1), corydaline (2), protopine (3) and dehydrocorydaline (4) effectively alleviated formalin-induced pain by inhibiting the Nav channels (Xu et al., 2021). L-stepholidine (L-SPD, 5), structurally akin to L-THP, isolated from Stephanie intermedi, also exerted analgesic effects through dopamine receptor interaction (Chu et al., 2007). The benzophenanthridine alkaloid nitidine (6), dihydrochelerythrine (7), oxyavicine (8), 8-methoxydihydrochelerythrine (9), 8-hydroxydihydrochelerythrine (10) and Rhoifoline A (RA, 11) derived from Zanthoxylum nitidum significantly relieved acetic acid-caused writhes in mice after gastric administration at 10, 40 mg/kg doses (Hu et al., 2006, 2013). Additionally, RA also exhibited notable inhibitions of formalin-induced chemical nociception and tail flicking- or hot plate-produced thermal nociception in mice, potentially mediated through the NO-cGMP signaling and L-type Ca2+ channels (Hu et al., 2013). Isoquinoline aporphine-type alkaloids crebanine (12) and stephanine (13) from Stephania yunnanenses H. S. Lo., significantly elevated the nociceptive threshold in thermal stimulus. They could also partially mitigate acetic acid torsion responses and sciatica neuralgia, potentially correlated with opioid receptors (Cui et al., 2023). Berberine (14), an isoquinoline alkaloid found in Coptis chinensis, offered relief in neuropathic pain induced by diabetes, chemotherapy and sciatic nerve injury by modulating transient receptor potential vanilloid protein 1 (TRPV1), NF-κB, μ- and δ-opioid receptors (Hashemzaei and Rezaee, 2020). Recently, we found a series of naphthylisoquinoline alkaloids from Ancistrocladus tectorius, particularly compound 2 (15), inhibiting Nav1.7 channel and effectively alleviating pain behaviors in the formalin-induced inflammatory model upon intraplantar application (Wang et al., 2023).

Nonetheless, studies on the isoquinoline alkaloids in Z. bungeanum were scarce. In this study, we first isolated and identified a novel isoquinoline alkaloid named HJ-69 from Z. bungeanum. HJ-69 (16) notably reduced the excitability of the APs in acutely isolated small-diameter dorsal root ganglion (DRG) neurons from mice. The significant inhibitory activities of HJ-69 on the endogenous Nav and Kv currents were further verified in the heterologous expression system. Remarkably, intraperitoneal application of 30 and 100 mg/kg HJ-69 notably relieved pain behaviors in the formalin-induced mouse model.