My 1

DETERMINATION OF ANTIOXIDANT CAPACITY IN CORN
GERM, WHEAT GERM AND WHEAT BRAN USING

SOLVENT AND MICROWAVE-ASSISTED SOLVENT
EXTRACTION
A Thesis
Submitted to the Graduate Faculty of the

Louisiana State University and
Agricultural and Mechanical College
in partial fulfillment of the
requirements for the degree of
Master of Science
In
The Department of Food Science
By
David Swenson Oufnac
B.S. Culinary Arts, Nicholls State University, 1999
December, 2006
DEDICATION
This thesis is dedicated to my loving parents, Murphy L. Oufnac Jr. and Marilyn
B. Oufnac, whose sacrifices and support throughout the years have inspired me to be a
better person in all aspects of life. Your selflessness and beautiful nature remind me
every day how proud I am to be your son. Thank you both for all you have done and for
what you continue to do. I love you very much.
ii
ACKNOWLEDGMENTS
I wish to convey my deepest gratitude to my major professor and advisor, Dr.
Zhimin Xu, for his guidance and patience during my three years working towards a
Master of Science degree from Louisiana State University. Working with Dr. Xu has
been a treasured experience for me while in the Department of Food Science. It has been
a pleasure and I feel much more confident in my abilities having Dr. Xu’s encouragement
and support.
I would also like to thank Dr. Ting Sun for all her help and support during my last
year of experiments. Also, a special thanks to my committee members, Dr. Witoon
Prinyawiwatkul and Dr. Christina Sabliov, for their valuable time as well as the use of
their labs. I would also like to thank my fellow students, namely Sandeep Bhale, Karen
Garcia, Amrish Chawla, Rishipal Bansode and Evi Menelaou for making my experience
at LSU so uniquely memorable. Several people who have had a colossal influence on my
life deserve thanks as well, so I would like to express my gratitude to Dr. Jerald Chesser,
Dr. Robert Harrington, Mr. And Mrs. Clarence Landry and Mitchell Rotolo.
My most sincere appreciation goes out to my parents who have disciplined and
nurtured me throughout life to give me the strength and tools to achieving any goals I
may set for myself. A special thanks goes to my loving sister and dear brother who are a
constant reminder that there are people who still inspire my world.
iii
TABLE OF CONTENTS
DEDICATION................................................................................................................... ii
ACKNOWLEDGMENTS ............................................................................................... iii
LIST OF TABLES .......................................................................................................... vii
LIST OF FIGURES ....................................................................................................... viii
ABSTRACT....................................................................................................................... x
CHAPTER 1. INTRODUCTION .................................................................................... 1
CHAPTER 2. LITERATURE REVIEW........................................................................ 4

2.1 Introduction............................................................................................................... 4
2.2 Antioxidants and Free Radicals ................................................................................ 6
2.2.1 Alpha Tocopherol (Vitamin E) .......................................................................... 9
2.2.2 Ascorbic Acid (Vitamin C).............................................................................. 10
2.2.3 Beta Carotene................................................................................................... 10
2.3 Extraction Methods................................................................................................. 12
2.4 Antioxidant Capacity Assays.................................................................................. 13
2.4.1 Trolox Equivalent Antioxidant Capacity (TEAC) Assay ................................ 14
2.4.2 DPPH (2,2-Diphenyl-1-picrylhydrazyl) Assay................................................ 16
2.4.3. Folin-Ciocalteau or Total Phenolics Assay..................................................... 17
CHAPTER 3. EXTRACTION OF ANTIOXIDANTS FROM WHEAT BRAN

USING CONVENTIONAL AND MICROWAVE-ASSISTED METHODS ............ 19
3.1 Introduction............................................................................................................. 19
3.2 Materials and Methods............................................................................................ 22
3.2.1 Chemicals and Materials.................................................................................. 22
3.2.2 Conventional Solvent Extraction ..................................................................... 22
3.2.3 Microwave-Assisted Solvent Extraction.......................................................... 23
3.2.4 Determination of Total Phenolic Compound Content ..................................... 24
3.2.5 Determination of Antioxidant Activity Using DPPH Radical Scavenging
Method ...................................................................................................................... 24
3.2.6 Statistical Analysis........................................................................................... 25
3.3 Results and Discussion ........................................................................................... 25
3.3.1 Extraction Yields of Wheat Bran Extracted by Conventional Solvent and
Microwave-assisted Solvent Extractions .................................................................. 25
3.3.2 Total Phenolic Compound of Wheat Bran Extracted by Different Solvent and
3.3.3 Free Radical Quenching Capability of Wheat Bran Extracts by Different
Solvent and Microwave-assisted Solvent Extractions .............................................. 29
3.4 Conclusions............................................................................................................. 30
iv
CHAPTER 4. EXTRACTION OF ANTIOXIDANTS FROM CORN GERM USING
CONVENTIONAL AND MICROWAVE-ASSISTED METHODS .......................... 32
4.1 Introduction............................................................................................................. 32
Method ...................................................................................................................... 36
4.3.1 Extraction Yields of Corn Germ Extracted by Conventional Solvent and
4.3.2 Total Phenolic Compound of Corn Germ Extracted by Different Solvent and
4.3.3 Free Radical Quenching Capability of Corn Germ Extracts by Different
Solvent and Microwave-assisted Solvent Extractions .............................................. 40
4.4 Conclusions............................................................................................................. 42
CHAPTER 5. EXTRACTION OF ANTIOXIDANTS FROM WHEAT GERM

USING CONVENTIONAL AND MICROWAVE-ASSISTED METHODS ............ 43
5.1 Introduction............................................................................................................. 43
Method ...................................................................................................................... 47
5.3.1 Extraction Yields of Wheat Germ Extracted by Conventional Solvent and
5.3.2 Total Phenolic Compound of Wheat Germ Extracted by Different Solvent and
Microwave-assisted Solvent Extraction.................................................................... 50
5.3.3 Free Radical Quenching Capability of Wheat Germ Extracts by Different
Solvent and Microwave-assisted Solvent Extraction................................................ 51
5.4 Conclusions............................................................................................................. 53
CHAPTER 6: SUMMARY AND CONCLUSION ...................................................... 54
REFERENCES................................................................................................................ 56
APPENDIX A. YIELD RESULTS ................................................................................ 64
APPENDIX B. PHENOLICS RESULTS ..................................................................... 65
v
APPENDIX C. DPPH RESULTS.................................................................................. 66
APPENDIX D. SAS CODE ............................................................................................ 67
VITA................................................................................................................................. 68
vi
LIST OF TABLES
Table 1. Extraction yield results (%)………………………………………………….....64
Table 2. Capabilities of scavenging DPPH free radicals (Trolox Equivalent nmol/g)…..65
Table 3. Total phenolic compound contents (Catechin Equivalent μg/g)……………….66
vii
LIST OF FIGURES
Figure 1. Free Radical Molecule…………………………………………………………. 5
Figure 2. Chemical structures of synthetic antioxidants (a) butylated hydroxyanisol

(BHA), and (b) butylated hydroxytoluene (BHT)………………………………………...6
Figure 3. Chemical structures of phenolic acids (a) Vanillic Acid, (b) Ferulic Acid, (c)
Syringic Acid, and (d) p-Coumaric Acid………………………………………………….7
Figure 4. Chemical structure of Vitamin E………………………………………………..9
Figure 5. Chemical Structure of Vitamin C...……………………………………………10
Figure 6. Chemical structures of carotenoids and tocopherols. -Carotene (I), astaxanthin

(II), canthaxanthin (III), zeaxanthin (IV), lutein (V), -cryptoxanthin (VI), lycopene
(VII), bixin (VIII), -tocopherol (IX) and -tocopherol (X). …………………………...11
Figure 7. Chemical structure of ABTS•+………………………………………………...15
Figure 8. Chemical structure of DPPH…………………………………………………..16
Figure 9. Phenolic compounds……………………………………………..…………….18
Figure 10. Diagram of a wheat kernel…………………………………………………...20
Figure 11. Extraction yield (% of fresh wheat bran) obtained by conventional solvent
extraction using different solvents and microwave-assisted solvent extraction (with
methanol) at different temperatures……………………………………………………...26
Figure 12. Total phenolic compound contents (Catechin Equivalent μg /g fresh wheat
bran) from wheat bran extracted by conventional solvent extraction using different
solvents and microwave-assisted solvent extraction (with methanol) at different
temperatures……………………………………………………………………………...28
Figure 13. Capabilities of scavenging DPPH free radicals (Trolox Equivalent nmol /g
fresh wheat bran) from wheat bran extracted by conventional solvent extraction using
different solvents and microwave-assisted solvent extraction (with methanol) at different
temperatures……………………………………………………………………………...30
Figure 14. Chemical structure of Linoleic Acid…………………………………………32
Figure 15. Extraction yield (% of fresh corn germ) obtained by conventional solvent
viii
Figure 16. Total phenolic compound contents (Catechin Equivalent μg /g fresh corn
germ) from corn germ extracted by conventional solvent extraction using different
temperatures……………………………………………………………………………...39
fresh corn germ) from corn germ extracted by conventional solvent extraction using
temperatures……………………………………………………………………………...41
Figure 18. Extraction yield (% of fresh wheat germ) obtained by conventional solvent
extractions using different solvents and microwave-assisted solvent extraction (with
germ) from wheat germ extracted by conventional solvent extraction using different
temperatures……………………………………………………………………………..51
fresh wheat germ) from wheat germ extracted by conventional solvent extractions using
temperatures……………………………………………………………………………...53
ix
ABSTRACT
Solvent extraction methods are widely used for extracting antioxidant in foods
and other sources. In this study, Traditional Solvent Extraction (TSE) and Microwave-
Assisted Solvent Extraction (MASE) were utilized to determine the content of
antioxidants in corn germ, wheat bran and wheat germ. Corn germ, wheat bran and
wheat germ all contain different levels of antioxidants; TSE and MASE were utilized to
determine the content of antioxidants in these three samples. TSE was performed at 60
ºC employing three solvents, acetone, hexane and methanol. MASE was performed using
the solvent methanol at four different temperatures, 60, 80, 100 and 120 ºC. DPPH (2,2-
Diphenyl-1-picrylhydrazyl) Assay was used to determine antioxidant capacity. For all
samples, the most antioxidant capacity extracted using the TSE was achieved using
methanol as the solvent, results ranged from 0.42-4.69 nmol Trolox/g. Using acetone and
hexane as solvents for TSE yielded much lower antioxidant capacities. The MASE
demonstrated consistent results compared with the TSE using methanol. In regard to
wheat bran and corn germ, the capacity for antioxidants and phenolics content improved
with each increase in temperature. Wheat germ yielded 3.36-3.88 nmol/g for the DPPH
assay and 1248-1575 μg catechin equivalents /g for phenolics assay establishing wheat
germ as containing the most antioxidant contents of the three samples. Corn germ and
wheat bran displayed antioxidant capacities on a much smaller scale. The sample of corn
germ’s antioxidant capacity had a range of 0.90-1.19 nmol/g and 539-730 μg catechin
equivalents /g, and wheat bran’s antioxidant capacity and phenolics ranged from 0.39-
0.72 nmol/g and 318.2-489.5 μg catechin equivalents /g. Both extraction methods were
found reliable for determining the antioxidant contents of grains when methanol was
employed.
x
CHAPTER 1. INTRODUCTION
Assessing antioxidant capacity in food products has challenged scientists for
years. According to information in the Medline database, the past ten years have seen a
340% increase in manuscripts mentioning “antioxidants”. This information was
presented in June, 2004, at the First International Congress on Antioxidant Methods
(Prior 2005). The challenge that lies before scientists is one in which the number of
methods and the variation of methods for measuring antioxidant capacity are numerous.
Traditional solvent extractions (TSE) have been widely used with debatable accuracy for
attaining total antioxidant capacities in food products. The difficulty scientists have had
is keeping the assays consistent. The variations are many; antioxidants can be extracted
using different solvents such as ethanol, acetone, methanol and hexanol, and these
solvents can each be employed with different ratios of dilution altering results.
Microwave-assisted Solvent Extraction (MASE) is a relatively new extraction method for
scientists studying antioxidant capacity in food. One prevalent application in analytical
chemistry is the use of microwave energy as auxiliary energy. However, little
documentation can be found utilizing a microwave for extracting antioxidants and
phenolics from food products.
Corn and wheat are foods rich in antioxidants, which make them excellent sources
for increased health benefits. Health advantages of diets rich in antioxidant plant
compounds include lowering the risk of cardiovascular disease, certain cancers and the
natural degeneration of the body associated with the aging process. Recent scientific
evidence is demonstrative of these advantages. The cause of the health problems listed
above is the free radical molecule. Free radicals are unstable molecules formed when the
body uses oxygen for energy. The instability of these molecules can damage tissues, alter
1
DNA and change cell structure. Ultimately, free radicals start a chain reaction resulting
in the reproduction of even more free radicals. Antioxidants interact with and stabilize
free radicals and may prevent some of the damage free radicals cause to the body. The
role antioxidants have in free radical stabilization involves the antioxidants donating one
of their own electrons to the free radical. This electron donation is done without the
antioxidant becoming unstable or damaging to the body. This remarkable action
stabilizes the free radicals as quickly as they are produced in the human body.
In this study, the traditional solvent and microwave-assisted solvent extraction
methods were investigated for extraction capabilities from corn germ, wheat germ and
wheat bran. The results can determine whether there is significance between the two
methods. Also, the traditional solvent extraction method will determine the significance
the different solvents (acetone, methanol and hexanol) have for extracting antioxidants
and phenolics. The results could provide us with valuable information the role different
solvents and/or different methods have in extracting antioxidants and phenolics. This
study may provide insight for future extraction analysis techniques for determining
antioxidant capacity in food products.
One hypothesis for this study is that TSE and MASE provide similar results
among the three samples at the same temperature. This will prove the microwave-
assisted solvent extraction method a valid method for the extraction of antioxidants. A
second hypothesis is that the antioxidant capacity extracted from the three samples will
significantly differ as the temperatures increase. Corn germ, wheat germ and wheat bran
all contain antioxidants, how these antioxidants react to heat will provide valuable
information to scientists as well as food processors.
2
The research objectives include developing two methods for extracting
antioxidants, and investigating the antioxidant capacity for corn germ, wheat germ and
wheat bran. A DPPH assay was used to determine the antioxidant activity of the samples
and Folin-Ciocalteau reagent was used in determining total phenolics content.
3
CHAPTER 2. LITERATURE REVIEW
2.1 Introduction
It is of great interest to the general public, medical and nutritional experts, and
health and food science researchers to know the antioxidant capacity and constituents in
the foods we consume. Due to the complexity of the composition of foods, separating
each antioxidant compound and studying it individually is costly and inefficient,
notwithstanding the possible synergistic interactions among the antioxidant compounds
in a food mixture. Therefore, it is most appealing to researchers to have a convenient
method for the quick quantitation of antioxidant effectiveness in preventing diseases.
A total antioxidant capacity assay using only one chemical reaction seems to be rather
unrealistic and not easy to come by, yet there are numerous published methods claiming
to measure total antioxidant capacity in vitro.
In fact, phenols are presumed to be responsible for the beneficial effects derived
from the consumption of whole grains, fruits, and vegetables. Phenolic compounds have
strong in vitro and in vivo antioxidant activities associated with their ability to scavenge
free radicals, break radical chain reactions, and chelate metals. Moreover, high phenol
consumption has been correlated with a reduced risk of cardiovascular diseases and
certain cancers (Bonoli and others 2004).
Phenolic antioxidants, a specific group of secondary metabolites, play the very
important role of protecting organisms against harmful effects of oxygen radicals and
other highly reactive oxygen species. The formation of these oxygen species in human
organisms is closely connected with the development of a wide range of degenerative and
nondegenerative diseases, mainly arteriosclerosis and other associated complications,
cancer, indispositions, and last but not least with the accelerated aging of organisms.
4
According to The Mitochondrial Free Radical Theory of Aging (1999), free radicals are a
class of molecule with a very simple definition. The nature of atomic structure and of the
covalent chemical bond (the features that give an atom its valency) are fixed by the rule
that electrons occupy orbitals of atoms, such that an orbital can contain zero, one or two
electrons, and that electrons carry less energy when they are one of a pair in an orbital
than when they are unpaired. A molecule is only a free radical if it possesses one or more
unpaired electrons (Figure 1).
Figure 1. Free Radical Molecule (www.biomatrixone.com)
Antioxidants are added in food during processing to improve food quality and
stability. The demand for natural antioxidants recently has increased because of
questions about the long-term safety and negative consumer perception of synthetic
antioxidants (figure 2) such as and butylated hydroxyanisol (BHA) and butylated
hydroxytoluene (BHT). In addition to their long term safety and capacity to improve
5
food quality and stability, these natural antioxidants may also act as nutraceuticals to
terminate free radical chain reactions in biological systems and therefore may provide
additional health benefits to consumers.
Figure 2. Chemical structures of synthetic antioxidants (a) butylated hydroxyanisol

(BHA), and (b) butylated hydroxytoluene (BHT) (modified from chemistry.about.com)
2.2 Antioxidants and Free Radicals
Numerous antioxidants are plant-based and play a fundamental role in protecting
plants that are open to the elements such as sunlight and severe oxygen stress (Tominaga
and others 2005). It has been suggested that antioxidants may amend cellular oxidative
status and prevent biologically significant molecules such as DNA, proteins, and
membrane lipids from oxidative damage and as a result lessen the risk of several chronic
diseases including cancer and cardiovascular disease (Zhou and others 2004). The core
dietary antioxidants are vitamins A, C and E, carotenoids, terpenes, and polyphenols,
including flavonoids (Stanner and others 2004). According to Cheng and others (2006), a
previous phytochemical investigation established that phenolic acids [ferulic, syringic,
vanillic and p-coumaric acids (Figure 3)], tocopherols, and carotenoids are the natural
antioxidants present in wheat grain and its fractions.
6
Figure 3. Chemical structures of phenolic acids (a) Vanillic Acid (modified from
www.cas.astate.edu), (b) Ferulic Acid (modified from www.crscientific.com), (c) Syringic
Acid (modified from www.worldbid.com), and (d) p-Coumaric Acid (modified from
www.crscientific.com).
A sufficient ingestion of natural antioxidants in food is therefore of great
consequence for the defense of macromolecules against oxidative damage (Halliwell and
Gutteridge 1999, and Wallace 1997). According to Stratil (2006), the cells most
frequently damaged by oxidative stress are unsaturated fatty acids in lipids, cholesterol,
different functional polypeptides and proteins, and nucleic acids. Mechanisms of
antioxidants consist of free radical quenching, transition metal chelating, reducing
peroxide, and simulation of in vivo antioxidative enzyme activities (Zhou and Liangli
2004). In living systems, the antioxidants may elevate the levels of endogenous defenses.
The action of antioxidants in foods and biological systems is reliant on the systems’
composition, interfacial phenomena, and partitioning properties of the antioxidants
between lipid and aqueous phases (Diaz-Reinoso and others 2006).
7
Bulger and Helton (1998) define oxidative stress as the state in which the level of
toxic reactive oxygen intermediates (ROI) overcomes the endogenous antioxidant
defenses of its host. This results in a surplus of free radicals, which can react with
cellular lipids, proteins, and nucleic acids leading to damage and eventual organ
dysfunction. Lipids are the most exposed biomolecules to a free radical attack. Free
radicals play a vital role in a number of biological processes, some of which are
necessary for life, such as the intracellular killing of bacteria by neutrophil granulocytes.
Free radicals have also been implicated in certain cell signaling processes. The peroxyl
radical is the most common free radical in human biology, but the hydroxyl radical,
singlet oxygen, superoxide radical, and reactive nitrogen species all are present in
biological systems (Wu and others 2004). They result from molecular oxygen under
reducing conditions, because of their reactivity; these same free radicals can have a role
in unwanted side reactions causing cell damage. A variety of aging symptoms such as
atherosclerosis are credited to free radical-induced oxidation of numerous chemicals.
Since free radicals are essential for life, the body has a number of mechanisms to reduce
free radical induced damage and to restore damage that does occur, such as the enzymes,
superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase
(Campos 2003, and Pinzino 1999).
DPPH is a stable organic radical, which bears no similarity to the highly reactive
and transient peroxyl radicals involved in various oxidative reactions in vivo (Wu and
others 2004). Both alpha-tocopherol and Trolox act as antioxidants via a hydrogen atom
transfer from the phenolic group to a peroxyl radical. They are structurally alike, with
long-chain alkyl group of alpha-tocopherol replaced by a carboxyl group in Trolox
(Yousry 2000). There is considerable evidence that beta-carotene molecules act as a
8
antioxidants by forming a chemical compound with peroxyl radicals to yield the
corresponding radical cation (Liebler and McClure 1996).
2.2.1 Alpha Tocopherol (Vitamin E)
Vitamin E is an indispensable nutrient that functions as an antioxidant in the
human body. It is essential by definition, for the reason that the body cannot produce its
own vitamin E and thus it must be supplied by foods and supplements. Tocopherols are
present in oils, nuts, seeds, wheat germ and grains. Absorption is believed to be
associated with intestinal fat absorption, in which only 40% of the ingested tocopherol is
absorbed. Most tocopherols go into the blood by means of lymph where they are coupled
with chylomicrons. Vitamin E (figure 4) was shown to accumulate in adipose fat tissue.
Figure 4. Chemical structure of Vitamin E (modified from www.benbest.com)
Phospholipids of the mitochondria, endoplasmic reticulum, and plasma
membranes possess affinities for alpha tocopherol, and the vitamin tends to concentrate
in these sites. Hence, its main function is to prevent the peroxidation of membrane
phospholipids, and to avoid cell membrane damage through its antioxidant action. The
lipophilic character of tocopherol makes it possible to locate in the interior of the cell
membrane bilayers (Halliway and Gutteridge 1992, and Borg 1993).
9
2.2.2 Ascorbic Acid (Vitamin C)
Ascorbic acid is a water-soluble chain breaking antioxidant that scavenges free
radicals and reactive oxygen molecules, which are produced during metabolic pathways
of detoxification (Block and Menkes 1989). Vitamin C (figure 5) also contributes to the
regeneration of membrane-bound oxidized vitamin E. It will react with the alpha-
tocopheroxyl radical, resulting in the generation of tocopherol in this process itself being
oxidized to dehydroascorbic acid (Ward and Peters 1995).
Figure 5. Chemical structure of Vitamin C (modified from www.gma5.com)
Ascorbic acid is present in citrus fruits, potatoes, tomatoes and green leafy
vegetables. Humans must acquire ascorbic acid from dietary sources due to the absence
of the enzyme L-gulacolactone oxidase (Ensimnger and others 1991).
2.2.3 Beta Carotene
Beta-carotene’s capacity to quench singlet oxygen, scavenge free radicals and
protect the cell membrane lipids from the destructive effects of oxidative degradation is
the way it functions as an antioxidant. The quenching involves a physical reaction in
which the energy of the excited oxygen is transferred to the carotenoid, forming an
excited state molecule (Krinsky 1993). Precursors of vitamin A, carotenoids (figure 6)
are pigmented micronutrients present in fruits and vegetables.
10
Figure 6. Chemical structures of carotenoids and tocopherols. -Carotene (I), astaxanthin
(II), canthaxanthin (III), zeaxanthin (IV), lutein (V), -cryptoxanthin (VI), lycopene
(VII), bixin (VIII), -tocopherol (IX) and -tocopherol (X) (modified from Straub, 1987)
11
According to Prior and others (2005), in relation to phenolics, carotenoids are not
particularly good quenchers of peroxyl radicals and other antioxidants but are exceptional
in quenching singlet oxygen, at which most other phenolics and antioxidants are
relatively ineffective.
2.3 Extraction Methods
Literature suggests limitations on reporting antioxidants capacity in cereals based
on the various extraction methods. First, the procedure used to extract antioxidants may
be incomplete. In most of these experiments, the solvent most commonly employed is
absolute ethanol (Yu and others 2002b, and Yu and others 2002c) or ethanol:water in
different proportions (Gray 2002, Yu and others 2002a, Adom and others 2003, Liyana-
Pathirana and Shahidi 2006, and Nam and others 2006), although the extraction of
phenolic compounds could be improved by using more polar solvents such as methanol
(Zielinski 2000). Some authors use methanol:water as an extraction solvent (Handelman
and others 1999), but the solvent dilution is not regularly acidified, which has shown to
improve the extraction (Iqbal and others 2006, and Awika 2005). Second, the reported
antioxidant capacities of cereals are measured in alcoholic extracts, and the results may
differ quantitatively and qualitatively from physiological extracts from the human
gastrointestinal tract (Perez-Jimenez and Saura-Calixto 2005).
The solid-liquid extraction is a heterogeneous, multicomponent operation
involving the nonsteady transfer of solutes from a solid to a fluid. Vegetal materials
include many different solutes that can be extracted simultaneously at different rates
depending on the location (outer surface, pores, vacuoles, etc.) and their partition
coefficients (Diaz-Reinoso and others 2006).
12
Microwave-assisted extraction has begun to replace conventional extraction
techniques due to reduction in solvent consumption and increased sample throughput
(Eskilsson and Bjorklund 2000). Microwave energy is a very practical form of auxiliary
energy that has been extensively applied in analytical chemistry (Kuss 1992). Previously
microwave-assisted extraction methods have been a constant in the determination of
organic contaminants in environmental samples; recently, it has become more popular in
the fields of pharmacy (Eskilsson and others 1999) and agriculture (Croteau and others
1994). According to Pallaroni and others (2002), an important research area employing
microwave-assisted extraction within the agricultural/food sector is the determination of
mycotoxins in cereals.
According to Young (1995), microwave-assisted technology has been shown to
be a resourceful means for extracting the fungal metabolite ergosterol. Compared to
traditional solvent extraction methods, the microwave-assisted extraction procedure
provides equal or higher extraction efficiencies, and requires smaller samples and reagent
volumes. It is also quicker and is less labor intensive.
2.4 Antioxidant Capacity Assays
Most natural antioxidants are multifunctional in complex heterogeneous foods;
their activity cannot be assessed by any one method (Frankel and Meyer 2000, and
Sanchez-Moreno 2002). No single assay will accurately reflect all of the radical
foundations or all antioxidants in a mixed or complex system, and it must be appreciated
at the outset that there are no simple universal methods by which antioxidant capacity can
be measured accurately and quantitatively (Prior and others 2005); also too many
analytical methods result in inconsistent results, inappropriate application and
interpretation of assays, and improper specifications of antioxidant capacities.
13
There are two reaction mechanisms in which antioxidants can deactivate radicals.
The first of these methods is the single electron transfer assay (SET) which detects the
ability of a potential antioxidant to transfer one electron to reduce any compound,
including metals, carbonyls, and radicals (Wright and others 2001). According to Prior
(2005), SET reactions are usually slow and can require a lengthy time to reach
completion, so the antioxidant capacity calculations are based on percent decrease in
product rather than kinetics. The second method is the hydrogen atom transfer (HAT),
which measures the antioxidant’s ability to quench free radicals by hydrogen donation.
HAT reactions are solvent and pH independent and are usually quite rapid. The presence
of reducing agents, including metals, is a complication in HAT assays and can lead to
erroneously high apparent reactivity (Prior and others 2005).
There are methods utilizing both HAT and SET mechanisms. The TEAC and
DPPH assays are usually classified as SET reactions, however these two indicator
radicals in fact may be neutralized either by direct reduction via electron transfers or by
radical quenching via HAT (Jimenez and others 2004).
2.4.1 Trolox Equivalent Antioxidant Capacity (TEAC) Assay
First reported by Miller and Rice-Evans (Miller 1993), the TEAC assay is based
on the scavenging ability of antioxidants for the long-life radical anion ABTS•+ [2,2`-
azinobis (3-ethylbenzothiazoline-6-sulfonic acid)]. The ABTS•+ is oxidized by peroxyl
radicals or other oxidants to its radical cation, ABTS•+, which is intensely colored, and
antioxidant capacity is measured as the ability of test compounds to decrease the color
reacting directly with the ABTS•+ radical. Results of test compounds are expressed
relative to Trolox (Prior and others 2005).
14
There are several strengths in using the TEAC assay, the simplicity being
preeminent. This assay has been used in many laboratories for studying antioxidant
capacity. TEAC values of many compounds and food samples have been detailed in
several articles (Gil 2000, Pellegrini and others 2003, Proteggente and others 2002,
Nielsen and others 2003, Pietta and others 2000, and Cao and others 1998). ABTS•+
reacts swiftly with antioxidants, typically within 30 minutes and can be used over a wide
pH range (Lemanska and others 2001). According to Awika and others (2003), ABTS•+
(figure 7) is soluble in both aqueous and organic solvents and is not affected by ionic
strength, so can be used in multiple media to determine both hydrophilic and lipophilic
antioxidant capacities of extracts and body fluids.
Figure 7. Chemical structure of ABTS•+ (modified from www.biotium.com)
The disadvantages using the TEAC assay include the ABTS radical is not found
in mammalian biology and thus corresponds to a “nonphysiological” radical source.
Thermodynamically, a compound has the ability to reduce ABTS•+ if it has a redox
potential lower than that of ABTS (0.68 V). Many phenolic compounds have low redox
15
potentials and as a result can react with ABTS•+ (Prior 2005). Using TEAC for a
quantitative assessment of antioxidant capacity can be problematic or even impractical,
however it can be used to provide a ranking order of antioxidants (Van den Berg and
others 1999).
2.4.2 DPPH (2,2-Diphenyl-1-picrylhydrazyl) Assay
DPPH• (figure 8) is one of a few stable and commercially available organic
nitrogen radicals bearing no similarity to the highly reactive and transient peroxyl
radicals involved in various oxidative reactions in vivo (Huang 2005, and Wu 2004).
Figure 8. Chemical structure of DPPH (modified from www.chem.memphis.edu)
This assay is based on the measurement of the reducing ability of antioxidants
toward DPPH•. The ability can be evaluated by electron spin resonance or by measuring
the decrease of its absorbance. The measurement of the loss of DPPH color at 515 nm
following the reaction with test compounds is what the antioxidant assays are based on
(Prior 2005).
16
Using the DPPH assay has its advantages. It is simple, rapid and needs only a UV-
vis spectrophotometer to carry out. However, it has many disadvantages. Because DPPH
is both a radical probe and an oxidant, the assay is not a competitive reaction. DPPH
color can be lost due to radical reaction (HAT) or reduction (SET) as well as unrelated
reactions, and steric accessibility is a major factor for determining results of the reaction
(Prior 2005).
2.4.3. Folin-Ciocalteau or Total Phenolics Assay
The Folin-Ciocalteu (F-C) method has for many years been used as a means to
determine total phenolics in natural products (Prior 2005). The reaction that takes place
is an oxidation/reduction one and because this reaction is the basic mechanism, F-C can
also be considered an antioxidant capacity method. This assay has many variations.
Different reagent concentrations and timing of additions and incubation are frequently
varied (Prior 2005). Also, many studies show the recommended reference standard
(gallic acid) being replaced with tannic acid equivalents, caffeic equivalents, vanillic acid
equivalents and catechin equivalents, among others. Phenolic compounds (figure 9) can
be found in flavonoides, phenolic acids, hydroxycinnamic acid derivatives and lignans.
17
Figure 9. Phenolic compounds
18
CHAPTER 3. EXTRACTION OF ANTIOXIDANTS FROM WHEAT
BRAN USING CONVENTIONAL AND MICROWAVE-ASSISTED
METHODS
3.1 Introduction
Wheat bran is a rich source of various natural antioxidants that possess health
benefits for humans, such as preventing cardiovascular disease and certain cancers
(Halliwell 1992; Truswell 2003). Phenolics, tocopherols, and fiber are generally believed
to be primarily responsible for wheat bran’s positive effects on cardiovascular disease;
undesirable lipid oxidation reactions in the body also contribute to these disease
conditions (Alabaster and others 1997; Andreasen and others 2001; Moller and others
1988).
Cholesterol oxidation could contribute to the development of a progressive
thickening of the artery wall, due to the accumulation of cholesterol oxidation products in
low-density lipoprotein (LDL) particles after they are oxidized. Lipid oxidation reactions
in the cell membrane also result in mutation of cell duplication processes and damage to
the cell membrane that could result in various types of cancer (Jadhav and others 1996).
Recent studies have suggested that these compounds of wheat bran exhibited significant
capabilities in scavenging free radicals, chelating metal ion oxidants, and reducing lipid
oxidation at different conditions (Adom and Liu 2005; Yu and others 2002; Zhou and Yu
2004).
Similar to other cereal grains, wheat bran (figure 10) contains many different
types of phenolic antioxidant compounds, such as ferulic, vanillic, caffeic, coumaric, and
syringic acids (Li and others 2005; Kim and others 2006) and relatively high levels of
19
carotenoids, tocopherols and phytosterols (Nystrom and others 2005; Zhou and others
2005).
Figure 10. Diagram of a wheat kernel
Conventional solvent extraction at a temperature not more than the solvent boiling
point was usually used in extracting the antioxidants from wheat bran (Li and others
2005; Kim and others 2006; Zhou and Yu 2004). The polarities of those antioxidants in
wheat bran are different, which may produce different extraction yields when solvents of
different polarity are used. Small molecular weight phenolic compounds are readily
extracted from cereal by methanol (Sun and others 2006). Due to phenolic compounds’
20
polar nature, less of these compounds are extracted by the non-polar solvent, hexane.
Because of the existence of different types of antioxidants in the extracts obtained by
different extraction solvents, the extracts may have variable capabilities to prevent lipid
oxidation. Most previous studies of wheat bran antioxidant used 50% acetone (Zhou and
Yu 2004), 80% methanol (Kim and others 2006), and 100% methanol (Li and others
2005). Those solvents are efficient in extracting phenolic compounds or hydrophilic
antioxidants but may not be as efficient for lipophilic antioxidants, such as tocopherols.
In this study, the three different solvents, methanol, acetone, and hexane were used to
perform conventional solvent extraction. The concentrations of total phenolic compound
and the capabilities of scavenging free radicals of wheat bran by using those methods
were compared.
Besides the conventional solvent extraction methods, use of a relatively new
extraction technique, microwave-assisted extraction, could possibly improve the
extraction efficiency of trace compounds in foods and soils. Recent studies demonstrated
that the microwave-assisted extraction method increased the recovery of trace residues in
vegetables, fruits, coffee, tea, and beans (Diagne and others 2002; Falqui-Cao and others
2001; Ganzler and others 1986; Negeri and others 2000; Pan and others 2003; Singh and
others 2004). Also, antioxidants and antioxidant activity of asparagus were found
increased after microwave-circulated water treatment (Sun and others 2007). In this
study, the solvent having highest extraction efficiency in the conventional solvent
extraction study was used in microwave-assisted extraction media. The concentrations of
total phenolic compound and free radical scavenging capabilities of wheat bran at
different microwave-assisted extraction temperatures were evaluated as well. The
21
information of this study would be helpful in the development and utilization of wheat
bran as a food antioxidant or as an antioxidant nutritional supplement.
3.2 Materials and Methods
3.2.1 Chemicals and Materials
Wheat bran was a gift from the Bell Institute of Health and Nutrition of General
Mills Company (Minneapolis, MN). It was able to pass through a No. 40 Sieve (0.425
mm) and stored at 4°C before use. DPPH, 6-hydroxy-2,5,7,8-tetramethychroman-2-
carboxylic acid (Trolox), Folin-Ciocalteau reagent, acetic acid, α-, γ-, and δ-tocopherols
were purchased from Sigma-Aldrich (St. Louis, MO). Methanol, acetone, hexane, and
ethyl acetate were HPLC grade and purchased from Fisher Scientific (Springfield, NJ).
3.2.2 Conventional Solvent Extraction
Methanol, acetone, and hexane were the solvents used for the conventional
solvent extraction. Ten grams of wheat bran was ground fine using a coffee grinder then
weighed using an aluminum container and transferred into a test tube (25 x 150 mm).
Each solvent (40 mL) was added in a test tube and vortexed to mix with the wheat bran
sample well. Each solvent extraction (methanol, acetone, or hexane) was triplicated. The
test tubes were capped and placed in a 60oC water bath for 20 min. These test tubes were
vortexed twice during the incubation. Then, the solvent layer from each test tube was
separated by centrifugation at 2000 rpms for 15 minutes. The solvent supernatant was
transferred to clean, previously weighed and labeled test tubes. The residue was mixed
with 20 mL of the same solvent again and vortexed. The solvent supernatant was
combined with the previous one. The tube with supernatant was then placed in a vacuum
centrifuge evaporator to remove solvent. The dried extract in the test tube was weighed
22
to measure the extraction yield of the samples. All samples were placed in a freezer set at
-20oC prior to testing.
3.2.3 Microwave-Assisted Solvent Extraction
Ten grams wheat bran was weighed using a clean aluminum container and
transferred into each of three Teflon vessels of the Ethos E Microwave Extraction System
(Milestone, Inc. Monroe, CT). The Teflon vessels were covered with polymer material
that can resist high inside pressure generated when extraction temperatures are higher
than the used solvent’s boiling point. Methanol (40 mL) and a magnetic stirring rod were
added in each vessel. The three vessels were sealed and properly placed inside the
Microwave Extraction System. Then, the Microwave Extraction System was
programmed to increase to the extraction temperature with a maximum energy level of
800 Watts and held at that temperature for 20 min with an energy level of 500 Watts.
Four extraction temperatures, 60, 80, 100 and 120oC, were applied to perform the
microwave-assisted solvent extraction, respectively. After a twenty-minute cool down
period, the vessels were unsealed and transferred to each corresponding centrifuge tube.
These tubes were centrifuged at 2000 rpms for 15 min to separate the supernatant and
residue. The solvent supernatant was transferred to a clean test tube that had been
previously weighed. The residues were mixed with 20 mL of the same solvent again and
vortexed. The solvent supernatant was separated by the centrifugation and combined
with the previous one. The supernatant was then placed in a vacuum centrifuge
evaporator to remove the solvent. The dried extract in the test tube was weighed to
measure the extraction yield of the samples. The samples were stored in a -20oC freezer
prior to testing.
23
3.2.4 Determination of Total Phenolic Compound Content
The total phenolic content of wheat bran extract was determined using Folin-
Ciocalteau reagent (Velioglu and others 1998). Folin-Ciocalteau reagent was diluted 10
times with deionized water. Dried wheat bran extract (0.020 g) was re-dissolved in 1 mL
methanol and 0.1 mL of this bran extract solution was mixed with 0.75 ml diluted Folin-
Ciocalteau reagent. The reaction solution was left at room temperature for 5 min. Then
0.75 mL of sodium bicarbonate solution (60 g/L) was added. The mixture was incubated
at room temperature for 90 min and filtered through a 0.45 μm syringe filter (Pall
Corporation, Ann Arbor, MI). The absorbance of the solution was determined at 750 nm
using a UV-Visible SpectraMax Plus384 spectrophotometer (Molecular Devices,
Sunnyvale, CA). The test for each extract was triplicated. The averaged absorbance was
used in calculation. Catechin was used as a standard to prepare a standard curve. The
total phenolic compound content was expressed as μg Catechin Equivalent / gram fresh
wheat bran.

Method
The wheat bran extract solution for the DPPH test was prepared by re-dissolving
0.2 g of each dried extract in 10 mL methanol. The concentration of DPPH solution was
0.025 g in 1000 mL of methanol. Two mL of the DPPH solution was mixed with 10, 20
and 40 μL of the bran extract/methanol solution and transferred to a cuvette. The
reaction solution was monitored at 515 nm, after an incubation period of 30 minutes at
room temperature, using a UV-Visible SpectraMax Plus384 spectrophotometer (Molecular
Devices, Sunnyvale, CA). The inhibition percentage of the absorbance of DPPH solution
was calculated using the following equation:
24
Inhibition%= (Abst=0 min -Abst=30 min)/ Abst=0 min ×100
Where Abst=0 min was the absorbance of DPPH at zero time and Abst=30 min was the
absorbance of DPPH after 30 min of incubation.
The inhibition percentage of the absorbance of DPPH was plotted against each
quantity of bran extract solution to obtain a regression line. Trolox (0.5 mM) was
dissolved in methanol and used as a standard to convert the inhibition capability of bran
extract solution to the Trolox equivalent. The ratio between the slopes of the regression
lines of bran extraction solution and the Trolox solution was defined as nmol Trolox
Equivalent Antioxidant Activity.
3.2.6 Statistical Analysis
The means and standard deviations of the extraction’s yield, total phenolics, and
scavenging DPPH capability were calculated and analyzed by one-way ANOVA with
multiple comparisons by Fisher's Least-Significant-Difference Test to determine
significant difference at p<0.05.
3.3 Results and Discussion
3.3.1 Extraction Yields of Wheat Bran Extracted by Conventional Solvent and

Microwave-assisted Solvent Extractions
The yields of acetone and hexane extraction were approximately 2.40% and not
significantly different from each other. This percentage is near to Wang and others’
(1993) result of raw wheat bran fat content (2.21%) using the petroleum-ether extraction.
However, both of them were approximately twice lower than that of the methanol
extraction, which was 4.86% of raw wheat bran. The solvent polarity from high to low is
methanol, acetone, and hexane, and it is likely that the chemical compositions of the three
extracts could be widely different (Sun and others 2006). Hexane and acetone solvent
25
may mainly extract more non- or less polar triglycerides, phytosterols, and phospholipids
of wheat bran. Wang and others (1993) reported that the content of extractable fiber and
protein were higher in the raw bran than in the whole wheat. Thus, methanol solvent
possibly extracts not only lipids and small molecule polar compounds but also some large
molecule polar compounds, such as alcohol soluble proteins and carbohydrates from
wheat bran.
WHEAT BRAN YIELD
7.00
A
Extraction Yield (% of wheat bran)
6.00 A A A A
5.00
4.00
B B
3.00
2.00
1.00
0.00
Acetone Hexane Methanol MW-60C MW-80C MW-100C MW-120C
Extraction Conditions
Figure 11. Extraction yield (% of fresh wheat bran) obtained by conventional solvent
methanol) at different temperatures. MW – Microwave assisted extraction. Bars topped
with the different letter are significantly different (P<0.05)
The extraction yields of microwave-assisted extractions with methanol as media
were in the range of 4.71% to 5.01% without any correlation to extraction temperature.
The microwave-assisted extraction did not significantly increase the extraction yield,
compared to the conventional methanol solvent extraction. The major advantage of
microwave-assisted extraction is to use microwave energy to heat the solvent and sample
26
in order to increase the mass transfer rate of solutes from the sample matrix into the
solvent (Sporring and others 2005). In this study, the particle size of wheat bran powder
was small and most probably was not a significant factor of causing the difficult
accessibility of solvent through the sample matrix under conventional extraction
conditions and consequently lowering extraction yields, or at least they could not be
observed at a mill-gram level, compared to the microwave-assisted extraction.
3.3.2 Total Phenolic Compound of Wheat Bran Extracted by Different Solvent and
The order of total phenolic compound content obtained by different conventional
solvent extractions from low to high was hexane, acetone, and methanol extraction and
59.1, 88.0, and 241.3 μg Catechin Equivalent /g of fresh wheat bran, respectively. It is
similar to the result that methanol was the best solvent in extracting phenolic compound
from oat bran (Sun and others 2006). In that study, the phenolic compound content
extracted by methanol was about 3 and 4 times higher than that extracted by acetone and
hexane, respectively. Also, microwave-assisted solvent extraction significantly increased
the phenolic compounds extracted from wheat bran when the extraction temperature was
over 80oC. The microwave-assisted extraction at 100 oC or 120oC was able to extract the
highest level of phenolic compounds from wheat bran among all studied extraction
conditions. They were over 467.5 μg Catechin Equivalent /g of fresh wheat bran and
approximately twice higher than the best conventional methanol extraction.
Higher extraction recovery of trace residue in foods, vegetables, fruits, coffee, and
beans by microwave-assisted extraction than solvent extraction was reported in several
studies (Diagne and others 2002; Falqui-Cao and others 2001; Ganzler and others 1986;
Negeri and others 2000; Pan and others 2003; Singh and others 2004).
27
WHEAT BRAN PHENOLICS
600.0
Catechin Equ. (ug/g fresh wheat bran)
A
500.0 A
B
400.0 B
300.0 B
200.0
C
100.0 C
0.0
bran) from wheat bran extracted by conventional solvent extraction using different
temperatures. MW – Microwave assisted extraction. Bars topped with the different letter
are significantly different (P<0.05)
For example, using microwave extraction, the recoveries of the three trace
pesticides from the fortified vegetable samples were increased up to 2 times, compared to
traditional solvent extraction (Singh and others 2004). Wheat bran was found to have
extractable and bound phenolic compounds (Liyana-Pathirana and Shahidi 2006; Kim
and others 2006). The total phenolic compound content was increased after wheat bran
was treated with acid or alkaline solution to hydrolyze bound phenolic compounds in
their studies. In this study, the increased extraction temperature and microwave energy
may break down or assist to hydrolyze the bonds of some bound phenolic compounds and
release or free them to be extractable phenolic compound. In a similar finding by Sun
and others (2007), the level of a glucoside called rutin found in asparagus increased after
the microwave energy treatment as well.
28
3.3.3 Free Radical Quenching Capability of Wheat Bran Extracts by Different
Solvent and Microwave-assisted Solvent Extractions
The order of the free radical quenching capabilities from high to low by different
solvent extraction was methanol, acetone, and hexane. It is in agreement with the results
of using the three solvents in extracting oat samples (Sun and others 2006). Also, the free
radical quenching capabilities was increased with increasing temperature of microwave-
assisted extraction. With the microwave-assisted extraction temperature at 120oC, the
antioxidant capability (0.72 nmol Trolox Equivalent of per gram wheat bran) was
determined to be the highest among all extractions.
The DPPH test has been used for evaluating antioxidant activity of wheat bran
extract in many studies (Adom and Liu 2005; Li and others 2005; Kim and others 2006;
Zhou and Yu 2004). The extracts from various genotypes of wheat bran provided greater
scavenging DPPH capability than its whole-wheat meal (Li and others 2005). Several
studies suggested that the total phenolics content might have positive correlation with
antioxidant activity (Emmons and others 1999; Velioglu and others 1998).
Hydrophilic phenolic antioxidants contributed more than 98% of the total
antioxidant activity of grain samples (Adom and Liu 2005). In this study, the difference
of total phenolic compound content may still contribute the difference of scavenging
DPPH capability. This suggests that important antioxidants in wheat bran scavenging the
DPPH free radical are polar phenolic compounds and likely more extractable in methanol
than either acetone or hexane, especially at increased temperature.
29
WHEAT BRAN DPPH
0.80
A
Trolox Equ. (nmol/g fresh wheat bran)
0.70 B
0.60 C
0.50 D
D
0.40
0.30 E
0.20 E
0.10
0.00
fresh wheat bran) from wheat bran extracted by conventional solvent extraction using
This would be due to the higher polarity of the methanol. The higher temperature
generated by microwave energy could also help release or hydrolyze to produce more
free polyphenolic compounds, such as phytosterols and anthocyanin (Pan and others
2003). According to Xu and others (2005) the compounds in cereals and grains were
reported to have the greater capability in scavenging free radicals.
3.4 Conclusions
In the conventional solvent extractions, methanol showed the greatest capability
in extracting phenolic antioxidants and inhibiting the free radicals produced by DPPH
among the three studied solvents. Microwave-assisted extraction with methanol as media
increased the extraction efficiencies of both hydrophilic and lipophilic antioxidants from
30
wheat bran at temperatures over 100 oC and 120oC, respectively. The significantly higher
hydrophilic antioxidants extracted by the microwave-assisted extraction resulted in
higher capability in scavenging free radicals. Therefore, microwave-assisted methanol
extraction is an efficient extraction method to extract antioxidants from wheat bran over
the conventional solvent method.
31
CHAPTER 4. EXTRACTION OF ANTIOXIDANTS FROM CORN
GERM USING CONVENTIONAL AND MICROWAVE-ASSISTED
METHODS
4.1 Introduction
It is the germ inside the corn kernel that is responsible for growing into a corn
plant. The uniqueness of any corn plant is due to the genetic information in the germ,
which is also the sole living part of the corn kernel. This genetic code is combined in the
germ with vitamins, minerals and enzymes required for the healthy growth of a plant.
One quarter of the corn germ is pure corn oil, which is the most valuable component of
the corn kernel. The remainder of the germ is used for animal feed. Corn oil is beneficial
to humans because it is high in the polyunsaturated fat, linoleic acid (figure 14). Kim and
others (2005) recognize corn as an excellent source of phytochemicals, such as
tocopherol, phytosterols, ferulic acid and carotenoids, which are all lipid soluble and
generally possess the capability of preventing oxidation.
Figure 14. Chemical structure of Linoleic Acid (modified from www.thepaleodiet.com)
Small molecular weight phenolic compounds are readily extracted from cereal by
methanol (Sun and others 2006). In general, the chemical structure of a polar compound
allows for it to be soluble in another polar compound. Likewise, non-polar compounds
32
are usually soluble in one another; therefore, polar and non-polar compounds are
insoluble in one another. Fewer compounds are extracted by hexane due to the polar
nature of the phenolic compounds. Because of the existence of different types of
antioxidants in the extracts obtained by different extraction solvents, the extracts may
have variable capabilities to prevent lipid oxidation. These solvents are efficient in
extracting phenolic compounds or hydrophilic antioxidants but may not be as efficient for
lipophilic antioxidants, such as tocopherols. In this study, the three different solvents,
methanol, acetone, and hexane were used to perform conventional solvent extraction.
The concentrations of total phenolic compound and tocopherol and the capabilities of
scavenging free radical of corn germ by using those methods were compared.
Besides the conventional solvent extraction methods, use of a relatively new
extraction technique, microwave-assisted extraction, could possibly improve the
extraction efficiency of trace compounds in foods and soils. Recent studies demonstrated
that the microwave-assisted extraction method increased the recovery of trace residues in
increased after microwave-circulated water treatment (Sun and others, 2007).
The concentrations of total phenolic compounds, and free radical scavenging
capabilities of corn germ at different microwave-assisted extraction temperatures were
evaluated as well. The information of this study would assist in understanding the
relationship between cholesterol oxidation and the role antioxidants have in corn.
Lowering the level of cholesterol oxidation products in frequently consumed foods may
reduce the possible risk of heart diseases and cancer (Xu and others 2005).
33
Corn Germ was a gift from the Bell Institute of Health and Nutrition of General
Mills Company (Minneapolis, MN). It was stored at 4°C before use. DPPH, 6-hydroxy-
2,5,7,8-tetramethychroman-2-carboxylic acid (Trolox), Folin-Ciocalteau reagent, acetic
acid, α-, γ-, and δ-tocopherols were purchased from Sigma-Aldrich (St. Louis, MO).
Methanol, acetone, hexane, and ethyl acetate were HPLC grade and purchased from
Fisher Scientific (Springfield, NJ).
solvent extraction. Ten grams of corn germ was ground fine using a coffee grinder then
Each solvent (20 mL) was added in a test tube and vortexed to mix with the wheat bran
sample well. Each solvent extraction (methanol, acetone, or hexane) was triplicated. The
test tubes were capped and placed in a 60oC water bath for 20 min. These test tubes were
34
Ten grams of ground corn germ was weighed using an aluminum container and
that can resist the high inside pressure generated when extraction temperatures are higher
800 Watts and held at that temperature for 20 min with energy level of 500 Watts. Four
extraction temperatures, 60, 80, 100 and 120oC, were applied to perform the microwave-
assisted solvent extraction, respectively. After a twenty-minute cool down period, the
vessels were unsealed and transferred to each corresponding centrifuge tube. These tubes
were centrifuged at 2000 rpms for 15 min to separate the supernatant and residue. The
solvent supernatant was transferred to a clean test tube that had been previously weighed.
The residues were mixed with 20 mL of the same solvent again and vortexed. The
solvent supernatant was separated by the centrifugation and combined with the previous
one. The supernatant was then placed in a vacuum centrifuge evaporator to remove the
solvent. The dried extract in the test tube was weighed to measure the extraction yield of
the samples. The samples were stored in a -20oC freezer prior to testing.
For this study the total phenolic content of corn germ extract was determined
using Folin-Ciocalteau reagent. Folin-Ciocalteau reagent was diluted 10 times with
35
deionized water. Dried corn germ extract (0.020 g) was re-dissolved in 1 mL methanol
and 0.1 mL of this germ extract solution was mixed with 0.75 ml diluted Folin-Ciocalteau
reagent. The reaction solution was left at room temperature for 5 min. Then 0.75 mL of
sodium bicarbonate solution (60 g/L) was added. The mixture was incubated at room
temperature for 90 min and filtered through a 0.45 μm syringe filter (Pall Corporation,
Ann Arbor, MI). The absorbance of the solution was determined at 750 nm using a UV-
Visible SpectraMax Plus384 spectrophotometer (Molecular Devices, Sunnyvale, CA). The
test for each extract was triplicated. The averaged absorbance was used in calculation.
Catechin was used as a standard to prepare a standard curve. The total phenolic
compound content was expressed as μg Catechin Equivalent / gram fresh corn germ.

Method
The corn germ extract solution for the DPPH test was prepared by re-dissolving
0.025 g in 1000 mL of methanol. Two mL of the DPPH solution was mixed with 10, 20
and 40 μL of the corn germ extract/methanol solution and transferred to a cuvette. The
36
quantity of corn germ extract solution to obtain a regression line. Trolox (0.5 mM) was
dissolved in methanol and used as a standard to convert the inhibition capability of corn
germ extract solution to the Trolox equivalent. The ratio between the slopes of the
regression lines of corn germ extraction solution and the Trolox solution was defined as
nmol Trolox Equivalent Antioxidant Activity.
The means and standard deviations of the extraction yields, total phenolic content,
and scavenging DPPH capability were calculated and analyzed by one-way ANOVA
with multiple comparisons by Fisher's Least-Significant-Difference Test to determine
4.3.1 Extraction Yields of Corn Germ Extracted by Conventional Solvent and

The yields from the acetone and hexane extractions were 18.36 and 14.39%
respectively and not significantly different from each other. However, both of them were
approximately eight times higher than that of the methanol extraction, which was 2.21%
of raw corn germ. The solvent polarity from high to low is methanol, acetone, and
hexane, and it is likely that the chemical compositions of the three extracts could be
widely different (Sun and others 2006). Hexane and acetone solvent may mainly extract
more non- or less polar triglycerides, phytosterols, and phospholipids of corn germ.
37
compared to the conventional methanol solvent extraction.
CORN GERM YIELD
25.00
A
Extraction Yield (% of corn germ)
20.00
B
15.00
10.00
5.00 C
C C C C
0.00
Figure 15. Extraction yield (% of fresh corn germ) obtained by conventional solvent
The major advantage of microwave-assisted extraction is to use microwave energy heat
the solvent and sample in order to increase the mass transfer rate of solutes from the
sample matrix into the solvent (Sporring et al 2005).
4.3.2 Total Phenolic Compound of Corn Germ Extracted by Different Solvent and
55, 116, and 543 μg Catechin Equivalent /g of fresh corn germ, respectively.
38
CORN GERM PHENOLICS
800
A
Catechin Equ. (ug/g fresh corn germ)
700 AB
C BC
600 C
500
400
300
200 D
100 D
0
Figure 16. Total phenolic compound contents (Catechin Equivalent μg /g fresh corn
germ) from corn germ extracted by conventional solvent extraction using different
This studies’ corn germ sample is similar to a previous experiment by Sun and
others (2006) where methanol was the best solvent in extracting phenolic compounds
from oat bran. In that study, the phenolic compound content extracted by methanol was
about 3 and 4 times higher than that extracted by acetone and hexane, respectively. Also,
microwave-assisted solvent extraction significantly increased the phenolic compounds
extracted from corn germ when the extraction temperature was over 80oC. The
microwave-assisted extraction at 100oC or 120oC was able to extract the highest level of
phenolic compounds from corn germ among all studied extraction conditions. Extracted
compounds measured over 654 μg Catechin Equivalent /g of fresh corn germ and were
well above the best conventional methanol extraction. Higher extraction recovery of
39
trace residue in foods, vegetables, fruits, coffee, and beans by microwave-assisted
extraction than solvent extraction was reported in several studies (Diagne and others
2002; Falqui-Cao and others 2001; Ganzler and others 1986; Negeri and others 2000; Pan
and others 2003; Singh and others 2004). For example, using microwave extraction, the
recoveries of the trace pesticides from fortified vegetable samples were increased up to 2
times, compared to traditional solvent extraction (Singh and others 2004). In this study,
the increased extraction temperature and microwave energy may break down or assist to
hydrolyze the bonds of some bound phenolic compounds to release or free them to be
extractable phenolic compounds.
4.3.3 Free Radical Quenching Capability of Corn Germ Extracts by Different

Solvent and Microwave-assisted Solvent Extractions
solvent extraction was methanol, acetone, and hexane. It is in agreement with the results
of using the three solvents in extracting oat samples (Sun and others 2006). Also, the free
radical quenching capabilities was increased with increasing temperature of microwave-
assisted extraction. At microwave-assisted extraction temperature at 120oC, the
capability (1.19 nmol Trolox Equivalent of per gram corn germ) was the highest among
all extractions.
The DPPH test has been used for evaluating antioxidant activity of food extracts
in many studies (Adom and Liu 2005; Li and others 2005; Kim and others 2006; Zhou
and Yu 2004). Several studies suggested that the total phenolics content might have
positive correlation with antioxidant activity (Emmons and others 1999; Velioglu and
others 1998).
40
CORN GERM DPPH
1.40
A
Trolox Equ. (nmol/g fresh corn germ)
1.20 AB
BC
1.00 C
0.80
D
0.60
0.40
E
0.20 E
0.00
fresh corn germ) from corn germ extracted by conventional solvent extraction using
Hydrophilic phenolic antioxidants contributed more than 98% of the total antioxidant
activity of grain samples (Adom and Liu 2005). In this study, the samples extracted from
methanol contained a higher quantity of antioxidants; the difference of total phenolic
compound content may still be the cause for the difference of scavenging DPPH
capability. This suggests that important antioxidants in corn germ scavenging the DPPH
free radical are polar phenolic compounds and likely more extractable in methanol than
either acetone or hexane, especially at increased temperature. The higher temperature
generated by microwave could also help release or hydrolyze to produce freer
polyphenolic compounds, such as phytosterols and anthocyanin (Pan and others 2003).
41
Those compounds in cereals and grains also were reported to have the greater capability
in scavenging free radicals (Xu and others 2005).
4.4 Conclusions
increased the extraction efficiencies of hydrophilic antioxidants from corn germ at
temperatures over 100 oC and 120oC, respectively. The significantly higher hydrophilic
antioxidants extracted by the microwave-assisted extraction resulted in higher capability
in scavenging free radicals. Therefore, microwave-assisted methanol extraction is an
efficient extraction method to extract antioxidants from corn germ over the conventional
solvent method.
42
CHAPTER 5. EXTRACTION OF ANTIOXIDANTS FROM WHEAT
GERM USING CONVENTIONAL AND MICROWAVE-ASSISTED
METHODS
5.1 Introduction
The embryo of the wheat kernel is known as the wheat germ, and is dense in
nutrients such as vitamin E, magnesium, pantothenic acid, phosphorus, thiamine and zinc.
Wheat germ is also a source of coenzyme Q10 (ubiquinine) and Para-aminobenzoic acid
(PABA). Coenzyme Q10 is an antioxidant that assists cells in oxygen, while para-
aminobenzoic acid helps to maintain the balance of intestinal flow
(http://www.healthline.com/galecontent/wheat-germ 2006). The oil from wheat germ
contains the highest tocopherol content of all vegetable oils, up to 2500 mg/kg (Shuler
1990). Sixty percent of the total phenolic content is represented by alpha-tocopherol.
Wheat germ oil is highly valuable owed to its elevated content of unsaturated fatty acids
(80%, mostly linoleic (18:2) and linolenic (18:3) (Wang and Johnson 2001). Having
immense importance in human metabolism, these two fatty acids are precursors of a
group of hormones called prostaglandins, which play an vital role in muscle contractions
as well as in proper healing of inflammatory processes (Coultate 1989). According to
Salinas (1993), linoleic acid helps to eradicate cholesterol and is a antecedent of cell
membrane phospholipids. Cholesterol oxidation could contribute to the development of a
progressive thickening of the artery wall, due to the accumulation of cholesterol
oxidation products in low-density lipoprotein (LDL) particles after they are oxidized.
Lipid oxidation reactions in the cell membrane also result in mutation of cell duplication
processes and damage to the cell membrane that could result in various types of cancer
(Jadhav and others 1996).
43
Conventional solvent extraction at a temperature not more than the solvent boiling
point was usually used in extracting the antioxidants from wheat bran (Li and others
2005; Kim and others 2006; Zhou and Yu 2004). The polarities of those antioxidants
found in wheat germ are different, which may create different extraction yields when
solvents of different polarity are used. Small molecular weight phenolic compounds are
readily extracted from cereal by methanol (Sun and others 2006). Less phenolic
compounds are extracted by hexane as a result of their being less polar. Because of the
existence of different types of antioxidants in the extracts obtained by different extraction
solvents, the extracts may have variable capabilities to prevent lipid oxidation. Acetone
and methanol are efficient in extracting phenolic compounds or hydrophilic antioxidants
but may not be as efficient for lipophilic antioxidants, such as tocopherols. In this study,
the three different solvents, methanol, acetone, and hexane were used to execute the
conventional solvent extraction. The concentrations of total phenolic compound and the
capabilities of scavenging free radical of wheat germ by using those methods were
compared.
In addition to the conventional solvent extraction methods, use of a relatively new
extraction technique, microwave-assisted extraction, could advance the extraction
efficiency of trace compounds in foods and soils. Recent studies demonstrated that the
microwave-assisted extraction method increased the recovery of trace residues in
increased after microwave-circulated water treatment (Sun and others 2007). In this
study, the solvent having highest extraction efficiency in the conventional solvent
44
extraction study was used in microwave-assisted extraction media. The concentrations of
total phenolic compound and free radical scavenging capabilities of wheat germ at
different microwave-assisted extraction temperatures were evaluated as well. The
information of this study would be helpful in the development and utilization of wheat
germ as a food antioxidant or as an antioxidant nutritional supplement.
Wheat germ was a gift from the Bell Institute of Health and Nutrition of General
Mills Company (Minneapolis, MN). The wheat germ sample was stored at 4°C before
use. DPPH, 6-hydroxy-2,5,7,8-tetramethychroman-2-carboxylic acid (Trolox), Folin-
Ciocalteau reagent, acetic acid, α-, γ-, and δ-tocopherols were purchased from Sigma-
Aldrich (St. Louis, MO). Methanol, acetone, hexane, and ethyl acetate were HPLC grade
and purchased from Fisher Scientific (Springfield, NJ).
solvent extraction. Ten grams of wheat germ was ground fine using a coffee grinder then
Each solvent (20 mL) was added in a test tube and vortexed to mix with the wheat germ
sample. Each solvent extraction (methanol, acetone, or hexane) was triplicated. The test
tubes were capped and placed in a 60oC water bath for 20 min. These test tubes were
45
Ten grams of wheat germ was weighed using an aluminum container and
that can resist high inside pressure generated when extraction temperatures are higher
800 Watts and held at that temperature for 20 min with energy level of 500 Watts. Four
extraction temperatures, 60, 80, 100 and 120oC, were applied to perform the microwave-
assisted solvent extraction, respectively. After a twenty-minute cool down period, the
vessels were unsealed and transferred to each corresponding centrifuge tube. These tubes
were centrifuged at 2000 rpms for 15 min to separate the supernatant and residue. The
solvent supernatant was transferred to a clean test tube that had been previously weighed.
The residues were mixed with 20 mL of the same solvent again and vortexed. The
solvent supernatant was separated by the centrifugation and combined with the previous
one. The supernatant was then placed in a vacuum centrifuge evaporator to remove the
solvent. The dried extract in the test tube was weighed to measure the extraction yield of
46
the samples. The samples were stored in a -20oC freezer prior to testing.
The total phenolic content of wheat germ extract was determined using Folin-
Ciocalteau reagent (Velioglu and others 1998). Folin-Ciocalteau reagent was diluted 10
times with deionized water. Dried wheat germ extract (0.020 g) was re-dissolved in 1
mL methanol and 0.1 mL of this bran extract solution was mixed with 0.75 ml diluted
Folin-Ciocalteau reagent. The reaction solution was left at room temperature for 5 min.
Then 0.75 mL of sodium bicarbonate solution (60 g/L) was added. The mixture was
incubated at room temperature for 90 min and filtered through a 0.45 μm syringe filter
(Pall Corporation, Ann Arbor, MI). The absorbance of the solution was determined at
750 nm using a UV-Visible SpectraMax Plus384 spectrophotometer (Molecular Devices,
Sunnyvale, CA). The test for each extract was duplicated. The averaged absorbance was
used in calculation. Catechin was used as a standard to prepare a standard curve. The
total phenolic compound content was expressed as μg Catechin Equivalent / gram fresh
wheat germ.

Method
The wheat germ extract solution for the DPPH test was prepared by re-dissolving
0.025 g in 1000 mL of methanol. Two mL of the DPPH solution was mixed with 2.5, 5
and 10 μL of the wheat germ extract/methanol solution and transferred to a cuvette. The
47
quantity of wheat germ extract solution to obtain a regression line. Trolox (0.5 mM) was
dissolved in methanol and used as a standard to convert the inhibition capability of wheat
germ extract solution to the Trolox equivalent. The ratio between the slopes of the
regression lines of wheat germ extraction solution and the Trolox solution was defined as
nmol Trolox Equivalent Antioxidant Activity.
The means and standard deviations of the extraction yield, total phenolic contents,
and scavenging DPPH capability were calculated and analyzed by one-way ANOVA
with multiple comparisons by Fisher's Least-Significant-Difference Test to determine
5.3.1 Extraction Yields of Wheat Germ Extracted by Conventional Solvent and

The yields of acetone and hexane extraction were approximately 7% and not
significantly different from each other. However, both of them were approximately twice
lower than that of the methanol extraction, which was 14.29% of raw wheat germ. The
solvent polarity from high to low is methanol, acetone, and hexane, and it is likely that
the chemical compositions of the three extracts could be widely different (Sun and others
48
2006). Hexane and acetone solvents, being less polar than methanol, may mainly extract
more non- or less polar triglycerides, phytosterols, and phospholipids of wheat germ.
Methanol solvent perhaps extracts not only lipids and small molecule polar compounds
but also some larger molecule polar compounds, such as alcohol soluble proteins and
carbohydrates from wheat germ.
WHEAT GERM YIELD
25.00
Extraction Yield (% of wheat germ)
A A
20.00 AB
A
15.00 BC
C
10.00
C
5.00
0.00
Figure 18. Extraction yield (% of fresh wheat germ) obtained by conventional solvent
extractions using different solvents and microwave-assisted solvent extraction (with
compared to the conventional methanol solvent extraction. The extraction yield at 100°C
is the lowest of all microwave-assisted extractions; although it is not significantly
different from the extraction yield at 120°C this low extraction yield may be the result of
49
antioxidant degradation due to the higher temperature. The major advantage of
microwave-assisted extraction is to use microwave energy to heat the solvent and sample
in order to increase the mass transfer rate of solutes from the sample matrix into the
solvent (Sporring and others 2005). In this study, the particle size of wheat germ powder
was small and may not be a significant factor of causing the complicated accessibility of
solvent through the sample matrix under conventional extraction conditions and
consequently lowering extraction yields or at least not observed at milligram level,
compared to the microwave-assisted extraction.
5.3.2 Total Phenolic Compound of Wheat Germ Extracted by Different Solvent and
Microwave-assisted Solvent Extraction
85, 205, and 1846 μg Catechin Equivalent /g of fresh wheat germ, respectively. It is
similar to the result that methanol was the best solvent in extracting phenolic compound
from oat bran (Sun and others 2006). In that study, the phenolic compound content
extracted by methanol was about 3 and 4 times higher than that extracted by acetone and
hexane, respectively.
The phenolic compounds extracted from wheat germ did not significantly differ
with higher or lower temperatures. It should be noted that the microwave-assisted
solvent extractions resulted in no predictable relationship between heat and phenolic
content. In fact, as temperatures increased from the low of 60°C, the phenolic content
slightly decreased until reaching the low of 1248 μg Catechin Equivalent /g of fresh
wheat germ at 100°C.
50
WHEAT GERM PHENOLICS
2500
A
Catechin Equ. (ug/g fresh wheat germ)
A
2000 A
A A
1500
1000
500
B
B
0
germ) from wheat germ extracted by conventional solvent extraction using different
Although a minimal increase, the phenolic content did go up to 1575 μg at 120°C,
suggesting that the phenolic compounds from wheat germ are not heat stable. These
results differ from all studies in that the conditional solvent extraction results using
methanol are higher (1846 μg) than those from the microwave-assisted solvent extraction.
5.3.3 Free Radical Quenching Capability of Wheat Germ Extracts by Different

Solvent and Microwave-assisted Solvent Extraction
solvent extraction was methanol, acetone, and hexane; results were 4.69, 0.54, and 0.36
respectively. It is in agreement with the results of using the three solvents in extracting
oat samples (Sun and others 2006). Also, the free radical quenching capabilities was
51
increased with increasing temperature of microwave-assisted extraction. At microwave-
assisted extraction temperature at 120oC, the capability (3.88 nmol Trolox Equivalent of
per gram wheat germ) was the highest among all microwave-assisted solvent extractions.
However, all microwave-assisted extraction results were not significantly different from
one another. The DPPH test has been used for evaluating antioxidant activity of wheat
extract in many studies (Adom and Liu 2005; Li and others 2005; Kim and others 2006;
Zhou and Yu 2004). Several studies suggested that the total phenolics content might
have positive correlation with antioxidant activity (Emmons and others 1999; Velioglu
and others 1998).
Hydrophilic phenolic antioxidants contributed more than 98% of the total
antioxidant activity of grain samples (Adom and Liu 2005). In this study, even though all
extracts contained a similar quantity of lipophilic tocopherol antioxidants, the difference
of total phenolic compound content may still contribute the difference of scavenging
DPPH capability. This suggests that important antioxidants in wheat germ scavenging
the DPPH free radical are polar phenolic compounds and likely more extractable in
methanol than either acetone or hexane, especially at increased temperature.
The higher temperature generated by microwave could also help release or
hydrolyze to produce freer ployphenolic compounds, such as phytosterols and
anthocyanin (Pan and others 2003). Those compounds in cereals and grains also were
reported to have the greater capability in scavenging free radicals (Xu and others 2005).
52
WHEAT GERM DPPH
6.00
A
Trolox Equ. (nmol/g fresh wheat germ)
5.00
B
B
4.00 B
B
3.00
2.00
1.00 C
C
0.00
fresh wheat germ) from wheat germ extracted by conventional solvent extractions using
5.4 Conclusions
did not increase the extraction efficiencies of either hydrophilic or lipophilic antioxidants
from wheat germ at temperatures over 100oC and 120oC, respectively. Therefore,
microwave-assisted methanol extraction is inconclusive as an efficient extraction method
to extract antioxidants from wheat germ over the conventional solvent method.
53
CHAPTER 6: SUMMARY AND CONCLUSION
Wheat germ, corn germ, and wheat bran all contain antioxidants. The study
employing these three samples in DPPH and Folin-Ciocalteau assays, were developed
and implemented based on two hypotheses: (1) TSE and MASE methods performed on
the three samples would provide similar results (at the same temperature), thereby
demonstrating the MASE method to be a valid one for the extraction of antioxidants, and
(2) The antioxidant capacity extracted from the three samples using the MASE method
would significantly differ as the temperature increased.
In both the DPPH and F-C assays the TSE method was performed using three
solvents: acetone, hexane and methanol. With each of the three samples, wheat bran,
corn germ, and wheat germ, patterns can be detected in the results. In all three, the
lowest antioxidant capacity was achieved with the solvent hexane. The solvent acetone
showed a higher, but not a significantly different result. The solvent methanol did
produce higher, significantly different results. For example, the results of the DPPH
assay on the wheat bran sample using acetone and hexane were 0.20 and 0.13 nmol
Trolox /g, respectively, while the methanol produced a much higher result (0.42). For the
wheat germ and corn germ, the same pattern was seen, with greater differences between
the results. Methanol provided results that were significantly different than those
obtained when using either acetone or hexane. The results in the phenolics assay show
the same patterns.
The results of both type assays using the MASE method showed a definite pattern
in the extractions of wheat bran and corn germ. The results using wheat germ appeared
to be less predictable.
54
In the case of corn germ, in both assays the results were not significantly different
at 60˚C (0.90 nmol Trolox /g and 539 Catechin equivalent μg/g) and at 80˚C (0.99 nmol
Trolox /g and 578 Catechin equivalent μg/g); as the temperature steadily increased so did
the antioxidant capacity and the phenolic content. The results at 100˚C (1.06 nmol/g and
654 μg/g) were not significantly different than 80˚C, and the results at 120˚C (1.19
nmol/g and 730 μg/g) were not significantly different from the results at 100˚C.
The DPPH assay results for the wheat bran sample at 60˚C (0.39 nmol/g), 80˚C
(0.53 nmol/g), 100˚C (0.64 nmol/g), and 120˚C (0.72 nmol/g) were all statistically
different than one another, the phenolic content results using the F-C assay with the
wheat bran sample at 60˚C (318 μg/g) and 80˚C (343.8 μg/g) were not statistically
different from one another, however, they were significantly different than the extractions
performed at 100˚C (467.5 μg/g) and 120˚C (489.5 μg/g) which were also found to be not
significantly different.
The results for both assays on the extractions from wheat germ were all not
significantly different. The compounds extracted from wheat bran appear to not be heat-
stable which is why these results were achieved.
These results provide us with valuable information on the capability of MASE as
a means of extracting antioxidants and phenolic compounds from food products, and may
provide insight for future extraction analysis techniques for determining antioxidants and
phenolic compounds in food products. Also, the different ways in which the three
samples’ antioxidants react to heat will provide valuable information to scientists as well
as food processors.
55
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63
APPENDIX A. YIELD RESULTS
Table 1. Extraction yield results (%)

Yield Results – Mean Scores a
Conventional Solvent Extraction Methods
Method Wheat Bran Corn Germ Wheat Germ
a a
Acetone 2.42 18.36 6.97 a
± 0.63 (b) ± 3.38 (a) ± 2.25 (c)
a a
Hexane 2.45 14.39 7.01 a
± 0.15 (b) ± 0.39 (b) ± 1.02 (c)
Methanol 4.86 a 2.21 a 14.29 a
± 0.21 (a) ± 0.08 (c) ± 2.97 (a)
Microwave Assisted Solvent Extraction Methods
o a a
60 C 4.76 2.49 14.63 a
± 0.79 (a) ± 0.01 (c) ± 4.88 (a)
o a a
80 C 4.71 2.72 14.39 a
± 0.79 (a) ± 0.01 (c) ± 4.80 (a)
o a a
100 C 5.01 2.94 10.11 a
± 0.83 (a) ± 0.01 (c) ± 3.37 (b,c)
120oC 4.84 a 3.51 a 13.68 a
± 0.81 (a) ± 0.01 (c) ± 4.56 (a,b)
a
Data is represented as mean ± standard deviation.
Mean values in the same column not followed by the same letter are significantly
different at p≤ 0.05.
64
APPENDIX B. PHENOLICS RESULTS
Table 3. Total phenolic compound contents (Catechin Equivalent μg/g)

Phenolic Content Results – Mean Scores a
a a
Acetone 88.0 116 205 a
± 16.1 (c) ± 34 (d) ± 8 (b)
Hexane 59.1 a 55 a 85 a
± 15.8 (c) ± 18 (d) ± 37 (b)
Methanol 241.3 a 543 a 1846 a
± 20.7 (b) ± 61 (c) ± 374 (a)
o a a
60 C 318.2 539 1389 a
± 112.2 (b) ± 19 (c) ± 492 (a)
o a a
80 C 343.8 578 1353 a
± 43.2 (b) ± 32 (b,c) ± 279 (a)
100oC 467.5 a 654 a 1248 a
± 18.3 (a) ± 13 (a,b) ± 325 (a)
120oC 489.5 a 730 a 1575 a
± 24.7 (a) ± 22 (a) ± 556 (a)
a
65
APPENDIX C. DPPH RESULTS
Table 2. Capabilities of scavenging DPPH free radicals (Trolox Equivalent nmol/g)

DPPH Results – Mean Scores a
a a
Acetone 0.20 0.19 0.54 a
± 0.05 (e) ± 0.03 (e) ± 0.17 (c)
a a
Hexane 0.13 0.11 0.36 a
± 0.02 (e) ± 0.02 (e) ± 0.07 (c)
Methanol 0.42 a 0.58 a 4.69 a
± 0.04 (d) ± 0.01 (d) ± 0.66 (a)
o a a
60 C 0.39 0.90 3.71 a
± 0.02 (d) ± 0.05 (c) ± 0.13 (b)
o a a
80 C 0.53 0.99 3.43 a
± 0.02 (c) ± 0.12 (b,c) ± 0.06 (b)
o a a
100 C 0.64 1.06 3.36 a
± 0.02 (b) ± 0.04 (a,b) ± 0.19 (b)
120oC 0.72 a 1.19 a 3.88 a
± 0.02 (a) ± 0.05 (a) ± 0.05 (b)
a
66
APPENDIX D. SAS CODE
ANOVA
dm 'log;clear;output;clear';
data one;
input Grain $ sample $ Yield;
datalines;
;
proc means mean std n maxdec=2;by sample;
var Yield;
proc anova;
class sample;
model Yield = sample;
means sample/tukey lines;
run;
67
VITA
David Swenson Oufnac was born April 22, 1974, in Baton Rouge, Louisiana. Since the
age of fifteen, David has worked in the hospitality industry eventually working his way
up to the position of Executive Chef. He became the first graduate from the Chef John
Folse Culinary Institute at Nicholls State University where in December of 1999 he
earned his Bachelor of Science in Culinary Arts. The next five years, he occupied
positions of Executive Chef, Restaurant General Manager and Restaurant Owner. David
is now a candidate for a Master of Science from the Department of Food Science at
Louisiana State University and Agricultural and Mechanical College in December 2006.
68

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DETERMINATION OF ANTIOXIDANT CAPACITY IN CORN

GERM, WHEAT GERM AND WHEAT BRAN USING

Submitted to the Graduate Faculty of the

The Department of Food Science

what you continue to do. I love you very much.

I wish to convey my deepest gratitude to my major professor and advisor, Dr.

year of experiments. Also, a special thanks to my committee members, Dr. Witoon

ACKNOWLEDGMENTS ............................................................................................... iii

LIST OF TABLES .......................................................................................................... vii

LIST OF FIGURES ....................................................................................................... viii

CHAPTER 1. INTRODUCTION .................................................................................... 1

CHAPTER 2. LITERATURE REVIEW........................................................................ 4

CHAPTER 3. EXTRACTION OF ANTIOXIDANTS FROM WHEAT BRAN

CHAPTER 5. EXTRACTION OF ANTIOXIDANTS FROM WHEAT GERM

CHAPTER 6: SUMMARY AND CONCLUSION ...................................................... 54

APPENDIX A. YIELD RESULTS ................................................................................ 64

APPENDIX B. PHENOLICS RESULTS ..................................................................... 65

APPENDIX D. SAS CODE ............................................................................................ 67

Table 1. Extraction yield results (%)………………………………………………….....64

Table 2. Capabilities of scavenging DPPH free radicals (Trolox Equivalent nmol/g)…..65

Table 3. Total phenolic compound contents (Catechin Equivalent μg/g)……………….66

Figure 1. Free Radical Molecule…………………………………………………………. 5

Figure 2. Chemical structures of synthetic antioxidants (a) butylated hydroxyanisol

Figure 4. Chemical structure of Vitamin E………………………………………………..9

Figure 5. Chemical Structure of Vitamin C...……………………………………………10

Figure 6. Chemical structures of carotenoids and tocopherols. -Carotene (I), astaxanthin

Figure 7. Chemical structure of ABTS•+………………………………………………...15

Figure 8. Chemical structure of DPPH…………………………………………………..16

Figure 9. Phenolic compounds……………………………………………..…………….18

Figure 10. Diagram of a wheat kernel…………………………………………………...20

Figure 14. Chemical structure of Linoleic Acid…………………………………………32

Assisted Solvent Extraction (MASE) were utilized to determine the content of

Diphenyl-1-picrylhydrazyl) Assay was used to determine antioxidant capacity. For all

Assessing antioxidant capacity in food products has challenged scientists for

340% increase in manuscripts mentioning “antioxidants”. This information was

presented in June, 2004, at the First International Congress on Antioxidant Methods

Microwave-assisted Solvent Extraction (MASE) is a relatively new extraction method for

scientists studying antioxidant capacity in food. One prevalent application in analytical

chemistry is the use of microwave energy as auxiliary energy. However, little

documentation can be found utilizing a microwave for extracting antioxidants and

phenolics from food products.

antioxidant becoming unstable or damaging to the body. This remarkable action

In this study, the traditional solvent and microwave-assisted solvent extraction

antioxidant capacity in food products.

information to scientists as well as food processors.

and Folin-Ciocalteau reagent was used in determining total phenolics content.

each antioxidant compound and studying it individually is costly and inefficient,

notwithstanding the possible synergistic interactions among the antioxidant compounds

in a food mixture. Therefore, it is most appealing to researchers to have a convenient

method for the quick quantitation of antioxidant effectiveness in preventing diseases.

to measure total antioxidant capacity in vitro.

certain cancers (Bonoli and others 2004).

Phenolic antioxidants, a specific group of secondary metabolites, play the very

nondegenerative diseases, mainly arteriosclerosis and other associated complications,

unpaired electrons (Figure 1).

Figure 1. Free Radical Molecule (www.biomatrixone.com)

antioxidants (figure 2) such as and butylated hydroxyanisol (BHA) and butylated

additional health benefits to consumers.

Figure 2. Chemical structures of synthetic antioxidants (a) butylated hydroxyanisol

2.2 Antioxidants and Free Radicals

Numerous antioxidants are plant-based and play a fundamental role in protecting

dietary antioxidants are vitamins A, C and E, carotenoids, terpenes, and polyphenols,

previous phytochemical investigation established that phenolic acids [ferulic, syringic,