BLBS102-c02 BLBS102-Simpson March 21, 2012 11:54 Trim: 276mm X 219mm Printer Name: Yet to Come
2 Analytical Techniques in Food Biochemistry 33Charged pigments such as the red-violet colored betalains can
be separated by electrophoresis, but reversed-phase HPLC pro-
vides more rapid resolution and quantification (Cai et al. 2005).
Betalains have been separated on reversed-phase columns using
various ion-paring or ion-suppression techniques (Schwartz and
Von Elbe 1980, Huang and Von Elbe 1985). This procedure al-
lows for more interaction of the individual betalains molecules
with the stationary phase and better separation between individ-
ual components.ANTIOXIDANTS
An increasingly important group of nonnutritive food compo-
nents are the antioxidants, a large group of biologically ac-
tive chemicals in plant and animal tissues usually in relatively
small concentrations (compared to their substrates; Halliwell
and Gutteridge 1995). As the name implies, an antioxidant miti-
gates/prevents the oxidation of other molecules that are more
easily oxidized, for example, vitamin E in oil prevents the
auto-oxidation of lipids, and antioxidants are “strong” reducing
agents. Antioxidants can accomplish this task via three possi-
ble mechanisms. The first is to prevent free-radical generating
reactions (oxidation reactions) from occurring, the second in-
volves inhibiting/scavenging for reactive species, and the third
involves the chelation/sequestering of metals that can act as cat-
alysts. Whichever pathway, antioxidants “protect” susceptible
molecules such as membrane lipids from being oxidized, thereby
maintaining their structural and functional integrity (Dilis and
Trichopoulou 2010).
These naturally occurring antioxidants consist of vitamins,
enzymes (proteins), minerals, pigments, organic acids, and
hormones (Prior et al. 2005), which include vitamins C (ascorbic
acid) and E and its isomers (α-tocopherol and tocotrienols);
selenium and manganese (minerals);α-carotene,β-carotene,
lutein, lycopene, etc. (carotenoid terpenoids); catalases, glu-
tathione (a thiol), peroxidases, reductases, and superoxide
dismutases (enzymes); flavones, flavenols, flavanones, flavanols
and their polymers, isoflavone phytoestrogens, stilbenoids, and
anthocyanins (all polyphenolic flavenoids); chlorogenic acid,
cinnamic acid (and its derivatives), gallic acid, etc. (all phenolic
acids and their esters); xanthones and eugenol (non-flavenoid
phenolics); lignin, bilirubin, citric, oxalic, phytic acids, etc.
(organic molecules); and melatonin, angiotensin, and estrogen
(hormones) (Sies 1997, Prior et al. 2005, Dilis and Trichopoulou
2010).
Chemically speaking, an oxidation reaction involves the trans-
fer of one or more electron(s) from one molecule to another (a
redox reaction), thereby creating a charged molecule/molecular
fragment called a free radical (essentially, an ion with posi-
tive or negative charge), which can further promote subsequent
reactions, leading to the generation of more free radicals. By
their very nature, free radicals are very reactive as they have an
unpaired electron in their outer shell configuration and, in an at-
tempt to reach stability, they donate/receive electrons to/from
other molecules such as lipids, proteins, and genetic mate-
rial (DNA). This interaction damages these more oxidizable
molecules, which impairs their functions. Antioxidants can ei-ther donate hydrogen atoms or donate/receive electrons to/from
free radicals. While the “intervention” of the antioxidants causes
the antioxidants to become radicals themselves, these radicals
are more stable, eventually returning to their original state.
The human body produces free radicals through normal
metabolic processes such as aerobic respiration, while inflam-
mation and disease processes, exercise, exposure to exogenous
environmental chemicals, tobacco (including tobacco smoke),
sun-exposure (radiation), and chemicals (cleaning products and
cosmetics) are also sources of free radicals (Freeman and Capro
1982, Southorn and Powis 1988). The most abundant group
of free radicals produced in the human body involve oxygen,
and they are often referred to reactive oxygen species (ROS), a
group of peroxyl radicals including superoxide anions (O 2 −), hy-
droxyl radicals (OH·), singlet oxygen radicals (^1 O 2 ·), hydrogen
peroxide (H 2 O 2 ), and ozone (O 3 ). Other free radical produc-
ers/enablers include iron and copper (transition metals acting
as catalysts), which promote the production of more aggressive
free radicals (Valko et al. 2005, Apak et al. 2007) as well as
nitric oxide. Biologically, some of the free radicals have critical
biological functions, for example, some of the ROS species are
used by the immune system to attack and kill pathogens and
nitric oxide is involved in cell (redox) signaling (Somogyi et al.
2007).
A diet high in refined sugars and grains, foods cured with ni-
trites, pesticides, and hydrogenated vegetable oils coupled with
decreased consumption of whole grains, fruits, vegetables, and
non-/minimally processed foods, and non-healthy lifestyle gen-
erates more free radicals compared to a totally opposite diet
(Bruce et al. 2000). It has been hypothesized that free-radical
generating diets can lead to oxidative stress, an unbalanced
state characterized by increased free-radical concentrations cou-
pled with decreased antioxidant concentrations. This state has
been implicated in the aging process as well as in many dis-
eases/conditions such as Alzheimer’s disease, Parkinson’s dis-
ease, cardiovascular disease (including heart attack and stroke),
various cancers, cellular DNA damage, rheumatoid arthritis, and
cataracts (Etherton et al. 2002, Stanner et al. 2004, Shenkin 2006,
Nunomura et al. 2006). Epidemiological studies have suggested
the benefits of a high antioxidant diet, but clinical trials have pro-
vided mixed results (P ́erez et al. 2009, Dilis and Trichopoulou
2010). This would seem to suggest that antioxidants may only
be partially responsible or work in synergism with other bio-
logically active molecules (Cherubini et al. 2005, Seeram et al.
2005). However, some clinical investigations have demonstrated
that antioxidants can also act as pro-oxidants in some diseases
(Bjelakovic et al. 2007). Because of their possible varied func-
tionalities in disease prevention, mitigation, or even treatment,
the food industry is very much interested in promoting the pres-
ence of either naturally occurring or added antioxidants in their
products. Pharmaceutical companies too are interested in antiox-
idants through the creation and marketing of various dietary sup-
plements or pharmaceutical drugs for the treatment of diseases.
Currently, antioxidant assays are divided into two groups, the
division being based on the underlying reaction mechanisms,
hydrogen atom transfer (HAT) and electron transfer (ET; Prior
et al. 2005), which can work in tandem in some assays. Details of