Objective Temperature is a key environmental factor regulating secondary metabolism of Anoectochilus roxburghii. This study systematically investigated the effects of different cultivation temperatures on the content of secondary metabolites in A. roxburghii, providing a scientific basis for optimizing artificial cultivation conditions and improving the quality of medicinal materials.

Method Using A. roxburghii from Meihuashan as the test material, plants were treated with four temperatures (5℃, 15℃, 25℃, 35℃) for 7 days in an artificial climate chamber. Untargeted metabolomic analysis was performed using LC-MS/MS, combined with multivariate statistical methods, to systematically compare the differences in secondary metabolites, including lipids, carbohydrates and their derivatives, flavonoids and organic heterocyclic compounds, terpenoids, alkaloids and their derivatives. The contents of goodyeroside A and kinsenoside were determined by HPLC.

Result Temperature treatments significantly altered the composition of secondary metabolites in A. roxburghii. Carbohydrates and their derivatives accumulated significantly at low temperatures (5℃, 15℃), while their content dropped sharply to 1.2% of the 25℃ treatment group at high temperatures (35℃). Flavonoids and organic heterocyclic compounds significantly increased under both high and low temperatures, showing characteristics of stress-induced induction. Terpenoid substances significantly increased at low temperatures and decreased at high temperatures, but its proportion in the composition of secondary metabolites remained unchanged; while alkaloids and their derivatives, lipids, and the medicinal components kinsenoside and goodyeroside A showed an optimal temperature accumulation pattern, with peak levels at 25℃. Specifically, the contents of goodyeroside A and kinsenoside reached 103.2 mg/g and 92.43 mg/g respectively, at 25℃, and decreased by 20% to 30% under high or low temperature treatments.

Conclusion Temperature is the key environmental factor regulating the synthesis of secondary metabolites in A. roxburghii. Different secondary metabolites respond to temperature in diverse ways. A temperature of 25℃ is most conducive to the growth of A. roxburghii and the accumulation of medicinal components such as alkaloids, goodyeroside A and kinsenoside, while antioxidant components such as flavonoids and organoheterocyclic compounds can be enriched through temperature stress induction. This study provides a metabolomic basis for the precise, goal-oriented cultivation management of A. roxburghii.