HPLC/ESI-MS and NMR analysis of flavonoids and tannins in bioactive extract from leaves of Maytenus ilicifolia

Maytenus ilicifolia is an important plant in Brazilian folk medicine, used in many gastric disorder treatments. Low molecular weight components present in the leaves have been characterized as afzelechin, epiafzelechin, catechin, epicatechin, gallocatechin and epigallocatechin, as detected by HPLC and ESI-MS. Condensed tannins have also been found, consisting of di-, tri-, tetra-, and penta-, hexa, and heptamers. ESI-MS analyses were performed in positive and negative ionization modes, and in contrast with other investigations, negative ionization improved sensitivity for obtaining molecular ions. Moreover, the tandem-MS profile with negative detection provided characteristic fragments, such as those found at m/z 543 [(epi)afzelechin–(epi)afzelechin], m/z 561 [(epi)afzelechin–(epi)catechin], and m/z 577 [(epi)catechin–(epi)catechin] or [(epi)afzelechin–(epi)gallocatechin]. The analysis of the fragments also indicated the presence of additional ether linkage between C2 and C7, present in A-Type proanthocyanidins, and were identified at m/z 559 [(epi)afzelechin–(epi)catechin], m/z 575 [(epi)catechin–(epi)catechin] or [(epi)afzelechin–(epi)gallocatechin] and at m/z 591, [(epi)catechin–(epi)gallocatechin]. CID-fragmentation was used for tannins sequencing, as well as 3D NMR HMQC–TOCSY and COSY, which provided fingerprint assignments for identification of cathechin at δ 4.55/82.1 (H-2/C-2), 3.96/68.1 (H-3/C-3) and 2.82-2.50/27.7 (H-4a-H-4b/C-4), and epicathechin δ 4.78/79.1 (H-2/C-2), 4.15/66.7 (H-3/C-3) and 2.82-2.73/28.5 (H-4a-H-4b/C-4). Since HMQC–TOCSY gives a high resolution heteronuclear connectivity, it is useful for identification of other cis–trans isomers present in complex flavonoid mixtures.