Coronary heart disease is a vital cause of morbidity and mortality worldwide, and calcium channel blockers (CCBs) are important drugs that can be used to treat cardiovascular diseases. Suxiao Jiuxin Pill (SX), a traditional Chinese medicine, is widely used as an emergency drug for coronary heart disease therapy. However, understanding its potential mechanism in intracellular calcium concentration ([Ca²⁺]_i) modulation remains a challenge. To identify the active pharmacological ingredients (APIs) and reveal a novel combination therapy for ameliorating cardiovascular diseases, the ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF MS) combined with a dual-luciferase reporter [Ca²⁺]_i assay system was applied. Ligustrazine, ferulic acid, senkyunolide I, senkyunolide A and ligustilide were identified as potential calcium antagonists in SX, and the combination of ligustrazine and senkyunolide A showed synergetic calcium antagonistic activity. Additionally, the synergetic mechanism was further investigated by live-imaging analysis with the Ca²⁺ indicator fluo-4/AM by monitoring fluorescence changes. Our results indicated that ligustrazine can block voltage-operated Ca²⁺ channels (VDCCs) effectively and senkyunolide A can exert an inhibition effect mostly on ryanodine receptors (RYRs) and partly on VDCCs. Finally, an arterial ring assay showed that the combination of ligustrazine and senkyunolide A exerted a better vasodilatation function than using any components alone. In this study, we first revealed that a pair of natural APIs in combination acting on VDCCs and RYRs was more effective on vasodilatation by regulating [Ca²⁺]_i.

冠心病是全世界发病率和死亡率的重要原因，钙通道阻滞剂（CCBs）是可用于治疗心血管疾病的重要药物。苏小酒救心丸（SX）是一种中药，被广泛用作冠心病的急诊药物。但是，了解其在细胞内钙浓度（[Ca ²⁺ ] _i）调控中的潜在机制仍然是一个挑战。为了鉴定活性药理成分（API）并揭示出改善心血管疾病的新型组合疗法，将超高效液相色谱/四极杆飞行时间质谱（UPLC / Q-TOF MS）与双荧光素酶报道分子相结合[Ca ²⁺ ] _i化验系统被应用。川gust嗪，阿魏酸，senkyunolide I，senkyunolide A和ligustilide被确定为SX的潜在钙拮抗剂，并且川gust嗪和senkyunolide A的组合具有协同的钙拮抗活性。此外，通过监测荧光变化，通过Ca ²⁺指示剂fluo-4 / AM的实时成像分析进一步研究了协同作用机理。我们的结果表明川gust嗪可以阻断电压操纵的Ca ²⁺通道（VDCCs）有效，并且senkyunolide A可以主要对ryanodine受体（RYRs）发挥抑制作用，部分对VDCCs发挥抑制作用。最后，动脉环分析显示，川gust嗪和senkyunolide A的组合比单独使用任何组分具有更好的血管舒张功能。在这项研究中，我们首先揭示了一对天然的API联合作用于VDCCs和RYRs，通过调节[Ca ²⁺ ] _i对血管舒张更为有效。