Abstract:Using 6 species of Calanthe s. l. plants and 2 kinds of Epiendroidea plants as materials, we determined indican, isatin, indigo and indirubin contents in flowers and leaves before and after freezing treatment with liquid chromatographytandem triple quadrupole mass spectrometer (LCMSQQQ). Analyzed the content of indoxyl derivatives of Calanthe s. l. plants and the differences between species. The results showed that: (1) these 4 indoxyl derivatives were detected in all the 6 Calanthe s. l. plants, but not in the 2 Epidendroidea plants Doritis pulcherrima and Eria coronaria. (2) In the flowers and leaves of 6 Calanthe s. l. plants, the contents of indigo, indirubin and isatin increased significantly after freezing treatment, while the content of indican decreased significantly. At the same time, the content of indoxyl derivatives in flowers was higher than that in leaves. (3) The total content of indoxyl derivatives in the flowers and leaves of the 6 Calanthe s. l. plants were the highest in Cephalantheropsis gracilis flowers and the lowest in Calanthe triplicata leaves. This work has shown that the massive conversion of indican to indigo caused by freezing treatment is the main reason for the blue color in the tissues of the Calanthe s. l. after freezing. It is speculated that indoxyl derivatives may also be a class of compounds related to plant defense and play an important role in plant resistance to adversity.