HPLC AND Mass Spectroscopic Characterization of Mango (Mangifera Indica L.) Gallotannins Following Enzymatic Hydrolysis

Abstract

Mangos contain numerous compounds that have been shown to exhibit antioxidant properties. These compounds, most of which are polyphenolics, are linked to anti-cancer and anti-inflammatory activities in the body. Mangos more specifically boast a large number of high molecular weight compounds called gallotannins, composed of gallic acid units attached to glucose via a glycosidic linkage. It is unknown if these compounds are broken down into smaller molecules through the normal course of human digestion, or if food processing operations, such as the addition of a gallotannin-active hydrolyases, could be more effective in lowering the size of these molecules to increase the absorption and potential bioactivity. This research focused on understanding the chemical changes that occur to gallotannins derived from mangos following enzymatic hydrolysis and attempted to draw inferences relating to overall human health. Polyphenolics in mangos, cv. Ataulfo were extracted using a 1:1:1 acetone:ethanol:methanol mixture and further concentrated and clarified using a reverseiv phase C18 Sep-Pak cartridge. Mango extracts were treated with 20,000 U/ml and 13,000U/ml B-glucosidase with a time course of 2, 4, 6, and 8 hours in an optimal pH 5.0 citric acid buffer, and at a constant temperature of 35 C. Changes in mango polyphenolics following enzyme hydrolysis were monitored using a Thermo Finnigan LCQ Deca XP Max MSn ion trap mass spectrometer equipped with an ESI ion source. B-glucosidase proved to be effective in the hydrolysis of some gallotannins but was incapable of hydrolyzing all gallotannins into free gallic acid. This was illustrated by the observance of an increase in penta, hexa, hepta-O- and a subsequent decrease in higher molecular weight compounds. The limitations for complete hydrolysis explains by the inability of B-glucosidase to cleave the glycosidic linkage due to steric hindrance created from having up to five gallic acid moieties attached to glucose, or from the inability of the enzyme to break m-dipside linkages between two or more galloyl groups. Incubating mango extract with both 20,000 U/mL and 13,000 U/mL resulted in an equivalent eightfold increase in free gallic acid. Enzyme concentration was not the limiting factor in the hydrolytic reaction. Additionally, reaction time did not have a significant role in the hydrolytic rate, as the amount of free gallic remained relatively constant from 2 to 8 hours. These findings indicated that it was possible to increase low molecular weight gallotannin species following enzyme hydrolysis and will aid in future studies to understand the digestion and bioavailability mango phenolics.