Pharmacological effects and mechanisms of alkamides DDA-E and DDA-Z from Asari Radix et Rhizoma in migraine: Insights from serum pharmacochemistry, network pharmacology, and experimental validation.

ETHNOPHARMACOLOGICAL RELEVANCE: Asari Radix et Rhizoma (ARR), a traditional Chinese medicine, has been used for centuries to treat various diseases, including migraine, rheumatic pain, and toothache. Valued for its capacity to warm the meridians and promote dispersion, ARR is regarded as an essential herb for releasing the exterior, dispelling cold, and alleviating pain. Alkamides, represented by N-isobutyl-2E,4E,8Z,10E/Z-dodecatetraenamide (DDA-E/Z), were considered closely related to traditional properties of ARR and exhibited diverse biological activities. However, their underlying anti-migraine mechanisms remain unclear.

AIM OF THE STUDY: This study aimed to identify the active constituents of ARR and investigate the pharmacological effects and mechanisms of ARR-derived alkamides DDA-E and DDA-Z in migraine models through integrated network pharmacology and experimental validation.

MATERIAL AND METHODS: The chemical profile and blood entry components of ARR were analyzed by ultra-high performance liquid chromatography-quadruple time-of-flight mass spectrometry (UHPLC-Q/TOF-MS). Network pharmacology was used to identify potential targets and pathways associated with the prototypical plasma components in migraine. Analgesic and anti-inflammatory activities of the key active ingredients, DDA-E, DDA-Z and their combinations, were assessed using behavioral tests and periorbital mechanical pain thresholds. Hematoxylin and eosin, immunofluorescence analysis, enzyme-linked immunosorbent assay, Westen blot, and real-time quantitative reverse transcription PCR were conducted to explore underlying mechanisms. Functional assay and molecular docking studies investigated the ability of DDA-E and DDA-Z to activate CB1/CB2 receptors.

RESULTS: A total of 35 components were identified in ARR, with 10 of them entering the blood as prototypes. Network pharmacology revealed 209 potential targets of ARR-derived prototypical blood-entry components in migraine. DDA-E and DDA-Z showed high plasma exposure and the highest degree values in the network analysis, indicating their roles as important active ingredients of ARR for migraine. The in vitro and in vivo experiments suggested that the potential targets of DDA-E were CGRP, ERK, and AKT, which mainly acted through cAMP and PI3K-Akt pathways, while DDA-Z targeted COX-2, MAPK, and AKT through MAPK and PI3K-Akt pathways. Immunofluorescence, functional assays, and molecular docking results confirmed that DDA-E and DDA-Z tend to selectively activate CB1/CB2 receptors.

CONCLUSION: In this study, serum pharmacochemistry combined with network pharmacology identified DDA-E and DDA-Z as the key active constituents of ARR. Subsequent experimental validation elucidated their potential anti-migraine mechanisms, highlighting their selective activation of cannabinoid receptors and providing a foundation for further research and therapeutic application.