Comparison of the Effectiveness of Plant Derived Chemicals as
Antioxidants
David E. Henderson
Chemistry Department
Abstract
Plant chemicals such as capsaicin from chilies, zingerone from ginger, and a variety of
others from oregano, basil, rosemary, thyme, and cilantro will be evaluated for their
ability to prevent oxidation of fats exposed to high temperatures and the presence of free
radicals which initiate oxidation of fats. The study will use direct measurement of the
lipid hydroperoxides formed in these reactions using separation and detection by
electrochemical and mass spectrometric means. Analysis of small molecule products of lipid
hydroperoxide decomposition will also be analyzed to complete the understanding of the
total antioxidant effectiveness.
I. Background: In the presence of oxygen from the air, all fats are subject to a
process called autooxidation in which the fats are decomposed into small molecules. This
process is responsible for fatty foods becoming rancid, and the small molecules give
rancid fat its characteristic bad smell and taste. More significantly, the same reactions
occur in our bodies as fat molecules on the lining of our arteries and our cell membranes
become oxidized. The results of this reaction in living systems are heart disease, cancer,
and ageing. Thus, oxidation of fats is one of the most significant chemical reactions for
health. Our bodies use natural antioxidants, vitamin C, vitamin E, beta carotene,
selenium, uric acid, and various enzymes to hold lipid oxidation in check. Antioxidants
are also added to foods and food packaging to prevent oxidation.
There is wide interest in finding natural product chemicals which could replace synthetic
chemical antioxidants such as BHT and BHA which are commonly used in foods, packaging for
foods, plastics, and medicines to prevent degradation by oxygen. It has recently become
clear that one of the values of many spices is that they contain natural antioxidants.
This has produced an interest in identifying and evaluating plant derived chemicals for
use as antioxidants. My own work began with a study of capsaicin, and we have demonstrated
that capsaicin is more effective in preventing oxidation of fats than vitamin E when the
oxidation is caused by the presence of free radicals, molecules with a highly reactive
unpaired electron. One of the most interesting questions raised by our present work is the
difference in antioxidant activity of Vitamin E and all of the other antioxidants we have
studied. Our data, and a recent paper by Frankel both indicate that Vitamin E actually
increases the rate of formation of lipid hydroperoxide formation but decreases the further
decomposition of the lipid hydroperoxides to small molecules. This prevents rancidity in
fats, but leads to a large buildup of lipid hydroperoxides. The reasons for this
difference and its implications for health are unclear.
During the past 7 years, I have used small molecule analysis to study the effects of
capsaicin on heated fats, and I have used direct analysis of lipid hydroperoxides to study
fats oxidized by free radicals. It is now time to complete the picture by using both types
of analysis on both types of systems. Without this, our understanding of the action of
antioxidants will remain incomplete, and it is possible to misinterpret the results. The
proposed three year study will complete this process. In addition, I will use the methods
I have developed to identify and characterize additional plant derived antioxidants.
II. Proposed Study: The oxidation of the fats will be initiated in two different
manners, by heating a 200oC to simulate cooking conditions and by the use of a free
radical initiator to model the mechanism of most lipid oxidation in living systems.
Samples with various antioxidants will be oxidized at varying oxygen and antioxidant
concentrations and the reactions will be monitored as a function of time to find out how
fast the oils are oxidized under each set of conditions. After oxidation, the two methods
for study of antioxidant effectiveness will be employed to fully characterize the nature
of the reactions. Specifically, we will analyze for lipid hydroperoxides using the LC-EC
method I developed on my 1997-98 sabbatical and faculty research leave, and the GC methods
I used in my 1991 sabbatical and subsequent work with several students. The LC method will
be expanded to take advantage of the recently acquired LCQ- Liquid Chromatograph-Mass
Spectrometer (LC-MS) system which will allow actual identification of the reaction
products when necessary. The GC method will be further refined to employ either direct
injection of solid phase micro extraction for the analysis of small volatile molecules in
the oxidized lipids. Again, we have access to two different GC-mass spectrometers on which
the reaction products can be identified.
Finally, the Chemistry Department has just acquired an additional instrument which will
facilitate this project, the Supercritical Fluid Extractor (SFE), This provides a simple
method for extracting active components from plant materials for testing as potential
antioxidants. The SFE system has been used for extracting antioxidants from spices, and is
widely used in industry for other similar purposed. It offers the advantage of selective
extraction without contaminating the extract with other chemicals. The extraction is done
with carbon dioxide or trifluoroethane which simply leaves as a gas after the extraction,
leaving a pure extract residue for use. The use of SFE will allow the study of several
plant antioxidants which are not commercially available.
The LC-MS will be used in several ways in these studies. It allows the rapid
identification of specific chemicals in the plant extract and also allows identification
and quantitative analysis of the products of oxidation of the fats themselves. I also hope
to develop a rapid method for analysis of hexenal, in the oxidized fats using the LC-MS.
This compound is widely used as a marker for lipid hydroperoxide decomposition and will be
a simpler analysis than a full GC-MS analysis of all of the volatiles.
During the grant period, I will hire two research assistants to work during the summers,
one each of two summers, and I will supervise at least two students each year doing thesis
or independent study research. Their work will include SFE extraction of plant materials,
LC-MS analysis of the chemicals in the extracts, purification of individual chemicals from
the extracts by preparative chromatography if necessary, and the analysis of the oxidation
reactions for various fat-antioxidant-oxygen combinations. Fats to be used will include
pure fatty acids (linoleic and oleic acids) or methyl esters, and oxidation will be
carried out at high temperatures (200oC) and by the addition of a free radical initiator
(azo-bis-isobutylnitrile) in solution at 30oC. The lipid hydroperoxides will then be
analyzed using the HPLC-EC method we have developed or using the LC-MS if unidentified
components are also present. Volatile oxidation products will be analyzed using either
GC-MS or APCI LC-MS, depending on which procedure is easiest to automate and gives the
most rapid and reliable analysis.
The funds requested for research support ($8,000) will be used for supplies to support
this research. The pure lipids used for this research cost between $30 and $100 per gram
and capsaicin costs $250 per gram. Other similar plant chemicals, when available, are
similarly expensive. Thus $1500 is allocated for purchase of lipids and antioxidant
chemicals which are commercially available and fresh spices, some of which will be grown
in my garden so that they can be used in fresh form. The SFE system requires high purity
carbon dioxide, and $600 is allocated for this purpose. In addition, we will study the use
of trifluoroethane as an SFC solvent. The disadvantage of supercritical carbon dioxide is
that it is often difficult to extract polar compounds such as those found in spices.
Trifluoroethane is a much better solvent for polar compound extraction, and has not yet
been employed for extraction of spices. $400 is allocated for purchase of trifluoroethane.
The chromatography systems use various solvents, and $1500 is allocated for solvents and
chemicals required by the LC and the LC-MS. This category also provides chemicals for
purification of plant extracts when necessary. $2000 is allocated for purchase of HPLC
columns. Each column costs around $250, so this is a reasonable estimate given the fact
that at least two different systems will be in use. Other HPLC related supplies include
vials for the autosamplers, electrodes for the detector, and capillaries for the LC-MS,
etc. This will require an additional $1000. During this period, I anticipate at least two
major publications in ACS journals which require page charges. $400 is allocated to cover
these. The remaining $600 is for purchase of tools, equipment, syringes, pipetters, and
other miscellaneous lab equipment which is needed to facilitate the project.
III. Significance: Understanding the role of antioxidants in preventing lipid
oxidation is central to the prevention of a number of diseases, including cancer and heart
disease. This work will make a significant contribution to this understanding, both
through a better understanding of how antioxidants work at the molecular level and by the
identification of effective natural antioxidants for dietary and food processing use. I
anticipate several publications will result from this work. I also plan to request
external funding for this research from the USDA and from the ACS Petroleum Research Fund.
The data obtained will be central to supporting these.
Appendix 3 - Budget Summary form
(Copied from 1996 web site- actual form not provided)
Three Year Grant Program Budget
Category of Expense Year One:
6/1/99 to 5/31/00 Supplies $3000 Research Assistant $2000
Year Two:
6/1/00 to 5/31/01 Supplies $2500Research Assistant $2000
Year Three:
6/1/01 to 5/31/02 Supplies $2500
Total:
6/1/99 to 5/31/02 Supplies $8000Research Assiatants $4000
Services:
Photocopying
Other (specify):
Total Services:
Travel:
Transportation by car @ 23¢ per mile
Air / rail coach fare
Per diem expenses:
Rate ‹
Total Travel:
Supplies:
Chemicals - $1500
SFC Gases - $1000
HPLC Solvents and supplies $2500
HPLC Columns$2000
Publication Costs$400
Misc lab supplies$600
Total Other Expenses: $8000
Total Non-Personnel Expenses: (maximum = $8000) $8000
Research Assistant (s): 571.43 hours @ $7.00 per hour: (maximum = $4000) $4000
TOTAL EXPENSES: $12,000