Gustavo Luís Borré

HPLC-PDA method for quinovic acid glycosides assay in Cat's claw (Uncaria tomentosa) associated with UPLC/Q-TOF–MS analysis

Uncaria tomentosa (Willd.) is a medicinal plant largely used in folk medicine due to its wide range of biological activities, many of which are usually ascribed to the two main classes of secondary metabolites, namely, alkaloids and quinovic acid glycosides. In this work, a reversed phase HPLC-PDA method was developed and validated for the assay of quinovic acid glycosides in crude and dried extracts of Uncaria tomentosa (Cats claw) bark. The validation comprised tests of specificity, accuracy, linearity, intermediate precision, repeatability and limits of detection and of quantification. Alpha-hederin was used as the external standard. High coefficients of determination with lower R.S.D. were achieved for both external standard and crude extract. The structural characterization of the main quinovic acid glycosides presented in the crude extract was carried out through UPLC/Q-TOF-MS. The identities of the compounds were obtained through the comparison of their fragmentation patterns with those reported in the literature. The analytical method was successfully applied for quantifying quinovic acid glycosides in two different dried extracts from U. tomentosa and in one quinovic acid glycosides purified fraction.

COMPARISON OF METHYLXANTHINE, PHENOLICS AND SAPONIN CONTENTS IN LEAVES, BRANCHES AND UNRIPE FRUITS FROM ILEX PARAGUARIENSIS A. ST.-HIL (MATE)

The contents of the methylxanthines, saponins, and phenolics compounds were determined in different parts of Ilex paraguariensis A. St. Hil., in order to investigate the chemical differences among some well explored plant parts, leaves and branches, with an innovative raw source as the unripe fruits of the specie. Three specific LC methods were employed. In the plant raw material, the highest contents of methylxanthines (caffeine and theobromine) were found in leaves (0.86% and 0.15%), while the lowest content was observed in the fruits (0.04% and 0.04%). In the same manner the content of phenolic compounds (chlorogenic acid and rutin) was greater in leaves (0.14% and 1.09%) and lowest in unripe fruits (0.03% and not detectable). In contrast, the unripe fruits presented the highest saponin content (12.30%), followed by the leaves (4.14%), and the branches (0.94%). In a mate commercial beverage the high amounts of phenolic compounds (3.82% and 0.51%) and methylxanthines (0.79% and 0.30%) were observed.

VALIDATION OF A LC METHOD FOR POLYPHENOLS ASSAY IN CAT'S CLAW (UNCARIA TOMENTOSA)

A reversed-phase LC method was developed and validated for the separation and assay of the main polyphenols in extracts from barks of Uncaria tomentosa. The LC method consists of a RP-18 column, in gradient mode (trifluoroacetic acid-methanol) using chlorogenic acid, caffeic acid, and rutin as externals standards and UV-detection at 325 nm. The method showed a good specificity, linearity, precision, and accuracy for standards compounds and for the five major peaks from bark extract. Calibration curves were linear with determination coefficients higher than 0.99. The repeatability and intermediary precision for the five major peaks ranged from 1.09 to 5.60% and 1.25 to 6.28%, respectively. The accuracy values for chlorogenic acid, caffeic acid, and rutin in the bark extract were 97.17, 98.84, and 101.78%, respectively. The LC method was applied successfully to one commercial spray-dried and four different freeze-dried extracts produced from barks and roots of U. tomentosa.

LC-UV assay method and UPLC/Q-TOF-MS characterisation of saponins from Ilex paraguariensis A. St. Hil. (mate) unripe fruits.

INTRODUCTION Ilex paraguariensis A. St. Hil. (mate) is known in several South American countries because of the use of its leaves in stimulant herbal beverages. High saponin contents were reported in mate leaves and unripe fruits that possess a dissimilar composition. Two LC-UV methods previously reported for mate saponins assay focused on mate leaves and the quantification of the less polar saponin fraction in mate fruits. OBJECTIVE To develop and validate a LC-UV method to assay the total content of saponins in unripe mate fruits and characterise the chemical structure of triterpenic saponins by UPLC/Q-TOF-MS. METHODOLOGY From unripe fruits of mate a crude ethanolic extract was prepared (EX40) and the mate saponin fraction (MSF) purified by solid phase extraction. The LC-UV method was validated using ilexoside II as external standard. UPLC/Q-TOF-MS was adjusted from the LC-UV method to obtain the fragmentation patterns of the main saponins present in unripe fruits. RESULTS Both LC-UV and UPLC/Q-TOF-MS methods indicate a wide range of Ilex saponins polarity. The ilexoside II and total saponin content of EX40 were 8.20% (w/w) and 47.60% (w/w), respectively. The total saponin content in unripe fruits was 7.28% (w/w). The saponins present in MSF characterised by UPLC/Q-TOF-MS are derived mainly from ursolic/oleanolic, acetyl ursolic or pomolic acid. CONCLUSION The validated LC-UV method was shown to be linear, precise, accurate and to cover several saponins previously isolated from Ilex species and could be applied for the quality control of unripe fruit saponins.