Lang Research 2016

Project Description

The goal of this project is to understand the enzymatic oxidation of biophenols--specifically, of eugenol and methyleugenol. In order to collect data on these oxidations, we will use HPLC (High Performance Liquid Chromatography). My focus will also be on comparing new data that I have collected to previous data by Matt (Amad Pauzi), who initially worked on this project.

1. Descriptive information

Current Researcher: Sarah Lang

Faculty Research Advisor: Brad Sturgeon

Other Student Researchers/Collaborators: Ben Stillwell

Lab Notebook: (Pages)(Title)(Dates)

2. Introduction

Methyleugenol (MEU) and Eugenol (EUG), both of which are compounds found in clove oil, have been the subject of conflicting safety reports (1). According to the National Extract Manufacturer’s Association, there is no significant carcinogenic risk associated with MEU and EUG. However, a report issued by the National Toxicology Program stated that MEU was “reasonably anticipated to be a human carcinogen,” after observing increased incidences of hepatic cancers in rats after dietary administration of MEU (2). Despite these conclusions, the mechanisms by which MEU and EUG result in carcinogenesis are still largely unknown. In a study conducted by Herbert J. Sipe et al., MEU and EUG were found to undergo peroxidative metabolism to form free radicals (1). These free radicals, which are highly reactive and transient, have the potential to bind with DNA, form more stable products with each other, or contribute to other forms of cellular damage. Sipe et al. suggest that peroxidase metabolism and the resultant free radicals formed in MEU and EUG contribute to their observed carcinogenicity. However, many of the details of the metabolic processes that occur during the peroxidative metabolism of MEU and EUG still remain to be investigated. In particular, although the existence of free radicals were detected in MEU and EUG, it is unknown how these radicals behave after formation, and whether or not the products formed as a result of these free radicals also contribute to the carcinogenicity of MEU and EUG (1). In order to mimic peroxidative activity in the liver, horseradish peroxidase may be utilized in order to form free radicals in MEU and EUG. After reactions with horseradish peroxidase, the products can be analyzed through High Performance Liquid Chromatography (HPLC) in order to identify product separation as a result of peroxidative metabolism. Flash Chromatography is used in conjunction with HPLC in order to collect products associated with each peak, and these products can be further characterized using Nuclear Magnetic Resonance spectroscopy (NMR). By isolating the products formed after the metabolic oxidation of MEU and EUG and characterizing these products, it is possible to better understand how their free radicals react to form stable products and what the identities of these products are. Eventually, these products may be tested for carcinogenetic effects themselves, in order to contribute to the understanding of why MEU and EUG result in carcinogenesis.

3. Background from earlier reports

I have not completed any previous reports to discuss at this time. However, prior research on the oxidation of biophenols, with the aim of eventually studying the oxidation of methyleugenol and eugenol, was conducted by Ahmad Pauzi. The food preservative, BHA, was studied in terms of its enzymatic oxidation. BHA forms free radicals as well, which react with each other in numerous different ways. However, the free radicals are likely to tend towards the most stable products. In the case of BHA, the free radicals react to form dimers. This study’s design was then adapted for my project, where I will study the enxymatic oxidation of methyleugenol and eugenol.

4. Experimental

HPLC standards were created using 2 mM MEU/EUG in 10 mL of pH = 7.4 buffer. For the primary run, dioxane was not added to the standards. The standards were then run in the HPLC to identify standard peaks for MEU and EUG. Standards were run through the HPLC for a total of 25 minutes each in order to allow for full elution, as oils have a tendency to adhere to the HPLC column and result in slower elution times.

5. Results

Pictured are the standards for MEU and EUG, respectively. Standards were 2 mM MEU/EUG in 10 mL of pH = 7.4 buffer.

6. Discussion

The HPLC runs of MEU and EUG standards allowed me to become more comfortable with creating reaction mixtures, as well as using the instrumentation for this particular research project. The standards for MEU and EUG showed peaks that eluted ~22 minutes of the total 25 minutes of run time. This indicates that MEU and EUG, as oils, take longer to elute than other mixtures typically would. In order to address this particular problem, it is suggested that I used a 1:1 ratio of dioxane and buffer rather than 10 mL of buffer only in order to aid in the dissolution of the oils that I am working with. The standard peaks for MEU and EUG also exhibit some differences. MEU has an additional peak that is not present in EUG, which may be indicative of the differences in their structures (MEU has a dimethoxy group, and EUG only has a single methoxy group). It is also possible that this extra peak present on MEU is due to impurities in the sample, so it will be to my advantage to run the standards again using dioxane and buffer in order to compare results.

7. Conclusions

No conclusions have been reached at this time.

8. Future Directions

Following the creation of standards using 1:1 ratios of dioxane and 10 mL buffer, the next step in this project will be to carry out peroxidase reactions with MEU and EUG and analyze the samples using the HPLC. After identification of the peaks resulting from the peroxidative metabolism of MEU and EUG, these peaks will be collected using Flash Chromatography, in order to analyze the samples using NMR and characterize the products that were formed. Beyond the scope of my time with this project, but in the future as the ultimate goal for understanding the peroxidative metabolism of MEU and EUG, these products will be tested for bioactivity in order to determine whether or not they have carcinogenic effects themselves.

9. Literature references

1. Sipe, H. J., Lardinois, O. M., & Mason, R. P. (2014). Free Radical Metabolism of Methyleugenol and Related Compounds. Chemical Research in Toxicology, 27(4), 483–489.

2. Final Report on Carcinogens Background Document for Methyleugenol, Meeting of the NTP Board of Scientific Counselors, Report on Carcinogens Subcommittee, U.S. Department of Health and Human Services, Public Health Service, National Toxicology Program, Research Triangle Park, NC, (2000).

10. Signature

Sarah Lang 12/7/2016