An integrated multitarget screening strategy combining network pharmacology with cell membrane chromatography coupled to high-performance liquid chromatography for identifying candidate antidepressant compounds from Cyperus rotundus L.

ETHNOPHARMACOLOGICAL RELEVANCE: Cyperus rotundus L. (CR) is a traditional Chinese medicine (TCM) herb that has been used for centuries to soothe the liver, regulate Qi, and relieve emotional distress, with classical indications for "Yu syndrome" encompassing depressive mood, chest distension, and dysphoria. CR is incorporated into representative TCM formulas targeting depression-related conditions, such as Yueju Pill, substantiating its ethnopharmacological relevance in mood disorder management. However, its complex chemical composition presents considerable challenges for systematic bioactive compound identification, and the specific antidepressant constituents of CR remain poorly characterized, hindering the elucidation of its antidepressant material basis and the development of related therapeutics.

AIM OF THE STUDY: This study aims to establish a comprehensive multi-method strategy for the rapid screening of multi-target candidate antidepressant compounds from CR and to conduct a preliminary, in vitro evaluation of their antidepressant-relevant activity.

METHODS: Headspace gas chromatography-ion mobility spectrometry (HS-GC-IMS), headspace-solid phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS), and ultra-performance liquid chromatography-quadrupole Orbitrap mass spectrometry (UPLC-Q-Orbitrap-MS) were employed to comprehensively characterize the volatile and non-volatile chemical constituents of CR. On this basis, a multidimensional screening strategy integrating network pharmacology (NP) with a high-performance liquid chromatography-cell membrane chromatography (HPLC-CMC) online platform was developed to systematically identify candidate antidepressant compounds in CR. Molecular docking and surface plasmon resonance (SPR) were subsequently applied to evaluate the binding affinities between the identified candidate antidepressant compounds and their target receptors. Finally, with fluoxetine hydrochloride as a positive control, the neuroprotective effects of these compounds were assessed in a corticosterone-induced PC12 cell injury model.

RESULTS: HS-GC-IMS and HS-SPME-GC-MS collectively identified 115 volatile organic compounds (VOCs), with four compounds (1,8-cineole, p-cymene, terpinen-4-ol, and α-pinene) detected by both methods. UPLC-Q-Orbitrap-MS characterized 30 non-volatile organic compounds (NVOCs). A total of seven candidate antidepressant compounds were identified that showed binding affinity for 5-Hydroxytryptamine 1 A Receptor (5-HT1A), 5-Hydroxytryptamine 2 A Receptor (5-HT2A), Dopamine D1 Receptor (DRD1), and Dopamine D2 Receptor (DRD2). Molecular docking and SPR analyses confirmed direct binding between these compounds and their target receptors. In a corticosterone-induced PC12 cell injury model, most of these compounds exhibited comparable or greater in vitro neuroprotective potency than fluoxetine hydrochloride under the tested conditions. Furthermore, cyperenone and aristolone are reported here for the first time to possess antidepressant potential.

CONCLUSION: This study developed an integrated NP-HPLC-CMC screening strategy combined with HS-GC-IMS, HS-SPME-GC-MS, and UPLC-Q-Orbitrap-MS to identify seven candidate antidepressant compounds from CR targeting 5-HT1A, 5-HT2A, DRD1, and DRD2 receptors, with cyperenone and aristolone reported for the first time to possess antidepressant potential. These findings clarify the antidepressant material basis of CR and provide a reproducible framework for GPCR-targeted bioactive compound screening from TCMs, with in vivo validation of antidepressant efficacy identified as the essential next step.