SAHounsve Spring 2018

Chemistry/Biochemistry Research 430

Spring 2018

Selene Hounsve

Senior Biochemistry Major

Research Times

Wed: 11AM-2PM

Fri: 4-5 PM

Proposed Research Project

Project Title

Enzymatic Metabolism of Eugenol

General Information

Advisor: Dr. Bradley E. Sturgeon

Proposal

The purpose of the experiment is to report the observation of radical products of eugenol produced by horseradish peroxidase (HRP) and hydrogen peroxide by using HPLC.

Introduction

Eugenol is a phenolic derivative that can be extracted from spices and herbs like cinnamon, nutmeg, basil, cloves, and some natural oils. Eugenol is a yellow oily liquid with the characteristic fragrance of cloves. The phenol belongs to the most active natural antioxidants found in essential oils.

Figure 1: Chemical structure of eugenol

It is well known that natural antioxidants extracted from herbs and spices have vast antioxidant activity and are used in numerous food applications.¹ Most of the antioxidant potential of herbs and spices is due to the redox properties of their phenolic compounds, which permits them to act as reducing agents, hydrogen donors, and singlet oxygen quenchers.¹ Phenols are able to donate hydrogen atoms of phenol hydroxyl groups in reaction with peroxyl radicals that can produce stabilized phenoxyl radicals, thus terminating lipid peroxidation chain reactions.²

Instruments to be used

HPLC and UV-Vis

Experimental

Eugenol standard for HPLC: 15.5 ul of eugenol in 0.1 mM PBS with 10% ethanol, pH 7.4 and 1% tween. Used method "eugenol_SH_020918_b".

Oxidation reaction:

1. 1 mM Eugenol stock substrate of pH 7.4 PBS with 10% ethanol

2. 0.25 M H₂O₂ Stock

3. HRP Stock

Trial	Eugenol (1 mM)	H2O2 (0.25 M)	HRP
1	5μL	10μL	10μL
2	5μL	5μL	10μL
3	5μL	2.5μL	10μL
4	5μL	0μL	10μL

Results

Eugenol oxidized by HRP activated by hydrogen peroxide in solution is seen in Figure 2. The eugenol peak is at 13 minutes, with the top being the 1 mM eugenol standard only. The following peaks below the eugenol standard are increasing amounts of hydrogen peroxide. As the concentration of hydrogen peroxide increases, the eugenol peak decreases. This means that eugenol is being oxidized. As eugenol is being oxidized, the peak at 16 minutes could be a possible radical product because this peak increases in height as eugenol decreases. There is another peaks at 9 minutes, however we do not know what this peak is at this time.

Research pledge

I, Selene Hounsve, have read the Chem/Bioc 430 course syllabus and understand the general structure and expectations of the research program. The above material was prepared after consultation, and in conjunction with my research advisor.