The chemical constituents from the leaves of Ilex guayusa were investigated. Sixteen triterpenoids were isolated from the 95% ethanol extract of dried leaves of I. guayusa by silica gel, Sephadex LH-20, and ODS column chromatographies and semi-prepa-rative HPLC. Those triterpenoids were identified by NMR, HR-MS, and literature analysis: 3β-hydroxy-11α,12α-epoxy-24-nor-urs-4(23)-ene-28,13β-olide(1), 3β-hydroxy-24-nor-4(23),12-oleanadien-28-methyl ester(2), oleanolic acid(3), 3β,28-dihydroxy-12-oleanene(4), 2α,3β-dihydroxy-11α,12α-epoxy-24-\'nor-olean-4(23)-ene-28,13β-olide(5), ursolic acid(6), 3β,23-dihydroxy ursolic acid(7), 3β,28-dihydroxy-12-ursene(8), 3β-28-nor-urs-12-ene-3,17-diol(9), 3β-hydroxyurs-11-ene-28,13β-olide(10), 13β,28-epoxy-3β-hydroxy-11-ursene(11), 3β-hydroxy-28,28-dimethoxy-12-ursene(12), 3β-hydroxy-24-nor-urs-4(23),12-dien-28-oic acid(13), 3β-hydroxy-24-nor-urs-4(23),12-dien-28-methyl ester(14), 2α,3β-dihydroxy-11α,12α-epoxy-24-nor-urs-4(23)-ene-28,13β-olide(15) and 2α,3β-dihydroxy-11α,12α-epoxy-24-nor-urs-4(23),20(30)-dien-28,13β-olide(16). Compounds 1-2 were new compounds, and compounds 4-5, 7 and 9-16 were isolated from I. guayusa for the first time.

Ilex species have been consumed traditionally as medicinal teas worldwide. Though the presence of caffeine has been reported in several species, little is known about secondary-metabolite diversity within and among these taxa. Leaf samples of Ilex guayusa, Ilex paraguariensis, and Ilex vomitoria were analyzed by liquid chromatography-mass spectrometry and comparative metabolite profiling with Ilex cassine and other Ilex species to identify chemotaxonomic markers, delimit species, and provide an assessment of chemodiversity. Purine alkaloids were detected and quantified in I. guayusa, I. paraguariensis, and I. vomitoria. Reports of caffeine for I. cassine were not corroborated, suggesting that I. vomitoria was the traditional source of the Native North American tea yaupon. The tetramethyluric acid, theacrine, was detected for the first time in the genus Ilex as a low-level chemotaxonomic marker in I. vomitoria samples. Chemotaxonomy and metabolomics support a close relationship for caffeine-containing Ilex species.