Integrating spatial phytochemistry and metabolomics reveals the spatial chemical organization of Gymnadenia conopsea and its pharmacological effects in kidney-yang deficiency syndrome.

ETHNOPHARMACOLOGICAL RELEVANCE: Gymnadenia conopsea (L.) R. Br. is an important traditional medicinal plant widely used in Tibetan and Mongolian medicine for treating fatigue, weakness, and kidney-yang deficiency-related disorders. Although its traditional therapeutic value has been recognized for centuries, the tissue-specific distribution of its characteristic metabolites and the pharmacological effects underlying its kidney-tonifying application remain poorly understood. AIM OF THE STUDY: This study aimed to characterize the spatial distribution of secondary metabolites in G. conopsea and investigate its pharmacological effects and associated renal metabolic alterations in kidney-yang deficiency syndrome using an integrated spatial metabolomics strategy. MATERIALS AND METHODS: LC-MS/MS and air flow-assisted desorption electrospray ionization mass spectrometry imaging (AFADESI-MSI) were applied to characterize the chemical composition and tissue-specific distribution of metabolites in G. conopsea. A hydrocortisone-induced rat model of kidney-yang deficiency syndrome was established to evaluate the pharmacological effects of G. conopsea using biochemical analyses and histopathological examination. AFADESI-MSI-based spatial metabolomics was further performed to investigate region-specific metabolic alterations in kidney tissues and metabolic responses following G. conopsea intervention. RESULTS: A total of 93 metabolites were identified in G. conopsea, including characteristic glucosyloxybenzyl 2-isobutylbutanedioic acid derivatives, phenolic acids, stilbenoids, and phenanthrenoids. Spatial phytochemical analysis revealed distinct tissue-specific accumulation patterns of these metabolites and suggested that aerial tissues, which are traditionally discarded, may have potential phytochemical utilization value. In kidney-yang deficiency rats, G. conopsea significantly improved biochemical abnormalities and mild renal pathological alterations. Spatial metabolomics revealed extensive metabolic disturbances involving mitochondrial energy metabolism, phospholipid homeostasis, lipid mediator biosynthesis, and oxidative stress-related pathways. Treatment with G. conopsea partially reversed these metabolic alterations in a dose-dependent manner. CONCLUSION: The present study provides experimental evidence supporting the traditional use of G. conopsea in kidney-yang deficiency syndrome. The therapeutic effects of G. conopsea are associated with the regulation of renal metabolic homeostasis, particularly energy metabolism and lipid metabolic pathways. By integrating spatial phytochemistry and spatial metabolomics, this study provides new insights into the spatial chemical organization and pharmacological actions of traditional medicinal plants and supports the rational utilization of G. conopsea resources.