A SIMPLE METHOD FOR ASSAYING TOTAL GLUCOSINOLATE CONTENT OF PLANT MATERIALS VIA SULFATE-ION-RELEASE USING ION CHROMATOGRAPHY

Abstract

Naturally occurring phytochemicals possessing unique medicinal properties have become a matter of high interest in recent years. A good example is the enormous increase in the use of Saint John's Wort in Europe to treat depression. The anticancerous property of the vegetables of the family cruciferae, e.g., cauliflower, kale, broccoli, cabbage, mustard, and Brussels sprouts, has been attributed to a class of sulfur-containing phytochemicals known as glucosinolates. From a practical standpoint, the total glucosinolate content is generally considered the important parameter and hence that is what would generally be needed as a marker of product quality. The most frequently used method for the assay of total glucosinolates so far has been by assaying glucose which is produced stoichiometrically by the action of enzyme myrosinase. Various approaches for glucose assay have been employed and most are time-consuming and expensive. Fortunately, all glucosinolates, as a group of compounds, posses a sulfate group along with glucose which can be easily and stoichiometrically released by the action of enzymes myrosinase or sulfatase. The resulting sulfate can be easily and conveniently assayed by the use of ion chromatography. This paper reports the results of a study undertaken to develop a simple method for assaying total glucosinolates in plant materials by sulfate-ions-release and using Suppressed Ion Chromatography. Since the method is based on the sulfate ions released from the hydrolysis of glucosinolates, endogenous sulfate ions first had to be removed from the sample extracts. This was successfully accomplished via precipitation of sulfate ions by treating the extracts with 20 mM barium ions at 40 C for 60 minutes. Further, the residual (excess) barium ions were removed via precipitation by treatment of extracts with 25 mM chromate ions at 40 C for 15 minutes. Following these steps, glucosinolates were hydrolyzed by using myrosinase enzyme at a pH of 6.0 resulting in the release of sulfate moiety. Nearly 100% recovery of added sinigrin was achieved by assaying sulfate. However, the time needed for the complete hydrolysis of sinigrin by enzyme myrosinase in the presence of other salts needed for the procedure, was much longer than in their absence. Consequently, either a greater quantity of enzyme and/or a greater incubation time was necessary to achieve complete hydrolysis. Results of nine broccoli samples analyzed for total glucosinolates using the sulfate ion release method reported here were in agreement with those using glucose release method. Various parameters of the method are discussed.