Date of Completion

2022

Document Type

Thesis

Degree Name

Bachelor of Science in Pharmacy

Keywords

alpha-Amylases, Quercetin

Abstract

Alpha-amylase is an enzyme that can be found in pancreatic juice and saliva. This enzyme aids in digesting carbohydrates and raises postprandial glucose levels. Natural and synthetic products that act on alpha-amylase have been used in the treatment of diabetes. There are now dietary supplements in the Philippines that contain secondary metabolites such as Quercetin that are known to inhibit alpha-amylase, among other enzymes. The USP currently recommends the use of Reversed-Phase HPLC to determine the amount of quercetin in formulation and certain plant extracts (i.e. Sophora japonica).

Weak Affinity Chromatography (WAC) is a type of HPLC that can accurately bind to quercetin and other related flavonols that are present in dietary supplements. WAC was used in this research to measure the activity of quercetin and to compare it to a widely used bioassay, the Starch-Iodine Bioassay. The Amylase Affinity column was prepared via a Schiff-based Reduction method and packed in a 2.5 mm x 50 mm stainless-steel column using the PerkinElmer HPLC system. A total of 3 nmol or 133 mgamylase/gsilica was immobilized in the affinity column. In WAC, two peaks were retained with average KD of 2227.173 and 471.515. In SIB, the average KM and KI are 2.683 and 1.5722, respectively. The results in WAC are 451 times higher than reported by Martinez-Gonzalez et al. The results of the KM and KI from SIB are 41 percent higher and 32 percent lower than the results in the study of Martinez-Gonzalez et al, respectively.

The values for the precision of WAC did not pass the requirements set by United States Pharmacopeia-National Formulary, while the precision of SIB conforms to the requirements of United States Food and Drug Administration (US FDA). However, in WAC, the use of higher sample concentration of quercetin resulted in acceptable precision as set by USP. The results show that WAC may be used to measure the activity of commercially available herbal drug supplements. WAC may be optimally suited for pharmaceutical products that contain plant extracts. Further method optimization of WAC is advised to minimize indeterminate errors in sample preparation and matrix effects (effects of other constituent/excipients on the affinity). These future studies can also consider using WAC for quantification by measuring the AUC.

First Advisor

Timothy Jay L. Bengala

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