The Properties of Cranberries that make them Effective as an
Antioxidant against Cancer
An Honors Thesis (HONRS 499)
By
Kay M. Brockman
Thesis Advisor
C:;::';t~;::~
Ball State University
Muncie, Indiana
May 1,2003
May 3, 2003
Acknowledgements
Enormous thanks to Dr. James P. Rybarczyk for taking on this project with me.
Without you I never would have gotten the project off the ground.
Nature's Comer the health food store in Fort Recovery, for helping me find organic
cranberry products, especially to the owner Tricia Fisher
To my parents and fiance for always supporting me in everything I do.
2
The Properties of Cranberries that make them Effective as an
Antioxidant against Cancer
Kay M. Brockman
Department of Chemistry
Ball State University
Muncie, IN 47306
May 1, 2003
Abstract:
Several antioxidants are found in cranberries and other products containing
cranberries. The amount available within the products depends on the type and the
distributor. Organic cranberry juices contain less sugar and more metal ions such
as zinc.
Less processing of organic juices also saves some of the antioxidant
properties from being destroyed.
Polyphenols have been documented to be in
certain fruits and vegetables including high quantities in cranberries. A visual
complex forms with certain polyphenols with the addition of ion samples including
iron. Cranberries have more properties than just preventing cancer, they have also
been shown to decrease risk related to kidney stone, urinary tract infections, lower
LDL, help with ulcers, and prevent tooth decay.
Introduction:
Media and educational campaigns have begun to encourage people to eat "Five-aDay", which is five to nine servings of fruits or vegetables, to promote their health. This
results from the apparent correlation of the consumption of fruits and vegetables with
reduced risk for cancer and cardiovascular disease. Fruits and vegetables are rich in
antioxidants, but it is not known which dietary factors are responsible for the overall
3
beneficial effects. Each plant contains hundreds of phytochemicals (plant chemicals),
whose presence is dictated by hereditary factors. Only well-designed, long-term research
can determine whether any of these chemicals, taken in a pill, would be useful for
preventing specific diseases. Cranberries, with their rich deep red color, are suspected to
be satiated with antioxidants, including a large amount of polyphenols and trace metals.
The term antioxidant arose in the 1920' s to denote any substance that fought rust,
or other forms of oxidation. Reactive oxygen species (ROS) have been linked to being
formed by a variety of pathologies, including cancer and cardiovascular disease.
Reactive oxygen species are molecules such as hydrogen peroxide (H20 2), ions like the
hypochlorite ion (Ocr'), radicals like the hydroxyl radical (OH') (which is the most
reactive of them all), and the superoxide anion (02') which is both an ion and a radical. A
radical (also called a "free radical") is typically a cluster of atoms, one of which contains
an unpaired electron in its outermost shell of electrons. This is an extremely unstable
configuration, and radicals quickly react with other molecules or even other radicals to
achieve the stable configuration of 4 pairs of electrons in their outermost shell (one pair
for hydrogen). Reactive oxygen species can be formed by several different mechanisms.
One way is the interaction of ionizing radiation with a biological molecule. A common
path of formation is as an unavoidable byproduct of cellular respiration: some electrons
passing down the respiratory chain are drawn away from the main path, especially as they
pass through ubiquinone (a freely diffusible molecule) and go directly to reduce oxygen
molecules to the superoxide anion (1).
They can also be synthesized by dedicated
enzymes in phagocytic cells like neutrophils and macrophages. These enzymes include
NADPH oxidase and myeloperoxidase. (I)
4
Strong oxidants like the various ROS can damage other molecules and the cell
structures of which they are a part. Among the most important of these are the actions of
free radicals on the fatty acid side chains of lipids in the various membranes of the cell,
especially mitochondrial membranes (which are directly exposed to the superoxide
anions produced during cellular respiration).
One of the insidious things about free
radicals is that in interacting with other molecules to gain a stable configuration of
electrons, they convert that target molecule into a secondary radical. So, a chain reaction
begins that will propagate until two radicals randomly find each other, with each
contributing its unpaired electron to form a covalent bond linking the two. A common
example is the peroxyl radical, that may interact with another peroxyl radical on a nearby
side chain, crosslinking them with a covalent bond, or with another nearby carboncentered radical crosslinking them covalently. In both these cases, radical formation
comes to an end, but with the result that the fatty acid side chains of membrane lipids
may have become so deformed as to damage the membrane. The lipofuscin so
characteristic of aging cells may be formed by these mechanisms. (1)
Cells have a variety of defenses against the harmful effects of ROS. These include
two enzymes: superoxide dismutase (SOD) - which converts two superoxide anions into a
molecule of hydrogen peroxide and one of oxygen; and catalase and several small
molecules that are antioxidants, such as alpha-tocopherol (vitamin E), uric acid and
vitamin C. These enzymes can break the covalent links that ROS have formed between
fatty acid side chains in membrane lipids. Perhaps the long life span of some reptiles and
birds is attributable to their high levels of uric acid, or vitamin C in the proper, effective
5
concentration. Pharmacy shelves are filled with antioxidant preparations that people take
in the hope of warding off the damaging effects (perhaps including aging) of ROS. (2)
But it is important that the attempt to limit the production of ROS not succeed too
well because ROS actually do have important functions to perform in the cell. Some
examples include: the cells of the thyroid gland must make hydrogen peroxide in order to
attach iodine atoms to thyroglobulin in the synthesis of thyroxine; macrophages and
neutrophils must generate ROS in order to kill some types of bacteria that they engulf by
phagocytosis; and neutrophils (but not macrophages) must kill off engulfed pathogens by
using the enzyme myeloperoxidase which catalyzes the reaction of hydrogen peroxide
(made from superoxide anions) with chloride ions to produce the strongly antiseptic
hypochlorite ion. Chronic Granulomatous Disease (CGD) is a rare genetic disorder that
demonstrates the importance of ROS in protecting us from many type of bacterial
infection. It is caused by a defective gene for one of the subunits of NADPH oxidase.
However, despite some advantages, in most cases the ROS are the problem, not the
solution. (2)
By reducing ROS to harmless forms, antioxidants may prevent oxidative damage
to biomolecules including lipids, proteins, and nuclei acids. There are several
antioxidants that have been identified in living systems; they include enzymes,
endogenous proteins and non protein species, and dietary antioxidants including
tocopherols, carotenoids, and ascorbic acid. Epidemiological studies have shown that in
humans low levels of antioxidants in blood plasma are correlated with elevated risk of
disease (3) Although the levels of plasma antioxidants are related to risks, increased
dietary intake of antioxidant vitamins does not necessarily reduce risk. (3) Studies of the
6
role of dietary antioxidants in maintaining health are complicated by the fact that levels
of plasma antioxidants are a function not only of intake of nutrient antioxidants, but also
of oxidative demand of the organism. (3)
ROS can be generated by either exogenous or endogenous reactions, thus causing
an increased demand for antioxidants. Levels of plasma ascorbic acid are generally low
in smokers, perhaps because oxidative metabolism of smoke by-products consumes the
antioxidant. (3) Also the digestive tract is directly exposed to exogenous ROS when the
diet is rich in polyunsaturated fats, which are susceptible to oxidations. (3) Endogenous
ROS are produced in the digestive tract at sites of inflammation or mechanical irritation
(3) or by Fenton-type reactions involving dietary iron or other metals. (3) One thing that
could influence the demand for dietary antioxidants, such as ascorbic acids, is the level of
the ROS in the digestive tract. If nutritive antioxidants are depleted by reaction with
exogenously or endogenously formed ROS during digestion, they will be unavailable for
uptake and transport to the serum.
Phytochemical, a current buzzword in the nutrition field, is defined by the
American Cancer Society as non-nutrient plant chemicals that contain protective, diseasepreventing compounds.
A phytochemical that has been discussed quite often is
polyphenol. Polyphenols are a group of chemical compounds that are widely distributed
in nature. The basic structure of the backbone phenol can be found in Figure 1 (4). They
are responsible for the colors of many flowers, for example delphinium, fuchsia, rose,
and petunia, and of all red-berried fruit. Others are complex compounds present in bark,
roots, and leaves of plants that are used for tanning hides and skins to give leather a softer
texture. Others are simpler compounds present in most fresh fruit and vegetables, onions,
7
and tea. The term "polyphenols" was introduced some years ago to replace the term
"vegetable tannin," and using the following definition: "Water-soluble phenolic
compounds having molecular weights between 500 and 3000 and, besides giving the
usual phenolic reactions, they have special properties such as the ability to precipitate
alkaloids, gelatin and other proteins" (5). However, the subsequent use of the term has
been broadened to include lower molecular weight compounds, so that it now covers
natural products with more than one phenolic group. Polyphenols are broken down into
sub-categories, and these sub-categories are broken down even farther as their full
function has become known. Dietary phenolics may playa role in reducing the risk of
heart disease, cancer, and cataracts. Epidemiological studies generally, but not always,
indicate a reduced risk of disease with high phenolic intake.
A flow chart of
phytochemicals can be found in Figure 3.
J'
b'
•
Flgu re 1. Structure of phenol
Figure 2. Basic structure of a flavonoid.
8
PHYTOCHEMICALS
V/···
/'
Polyphenols
,
, ........... ,--..........
......
Phenolic Acids
Others, e.g.,
Stilbenes
/'
Flavonoids
C6-C3-C6 skeleton
,
-'.
.... -~"-,
...
~'... r - - - - - - - ,
Others
Anthocyanins,
Flavones,
Flavanones - e.g.,
red pepper, citrus
fru~s, cranberries,
blueberries
Flavanols
Flavonols
Monomers e.g., onions,
tomatoes
'MonomersCatechlns &
Epicatechins e.g,
tea, cocoa, apples
Oligomers Proanthocyanidins
.------~
/'
Procyanidins
e.g., cocoa,
appl es, peanut
Prodel ph ini dins
e.g., tea
Figure 3 - A flow chart of Phytochemicals (6)
Some helpful antioxidants of the polyphenols group include the group of
flavonoids, that are also important for food preservation. (3) Another flavonoid called
quercetin is found in onions, apples, tea and broccoli. This flavonoid is glycosylated in
most plants, and the position and nature of the substitution of the sugar are species
specific.
This broad class of polyphenolic compounds are synthesized by plants as
defenses against stresses such as invading organism, UV light, and tissue injury.(3)
Studies have shown that quercetin inhibits the cytochrome P450-catalyzed metabolism of
9
ethoxycoumarin and of ethylresorufin by HepG2 cells at concentrations as low as 0.1
iJM. (3) Quercetin is a very potent inhibitor of Maloney mouse leukemia virus reverse
transcriptase with an IC50 value of 0.2 iJM, and inhibits HIV reverse transcriptase with Kj
= 0.52 iJM.
(3)
This Flavonoid also suppresses a-tumor necrosis factor-mediated
stimulation of interleukin-8 expression partly by inhibiting the activation of the
transcription factor, NF-kB. (3)
The activity of F1avonoids varies with each individual compound.
Several
structure-function relationships have been derived. They include:
I. Most of the free flavonols are efficient antioxidants in both the aqueous and
lipid phases, but increased hydroxyl groups on the B ring tend to increase
activity.
2. Glycosylation of flavonols decreases the antioxidant activity, but the effect is
much more marked when the substitution is on the B ring. The antioxidant
activity tends to decrease with increasing number of sugar moieties on one
position.
3. Dimerization and trimerization of (epi)-catechin increases antioxidant activity,
but the tetramer shows decreased activity.
This could be a result of the
increase in size of the molecule.
4. Galloylation of flavanols have been found to increase aqueous phase
antioxidant activity but decreased lipid phase activity. (3)
Figure 4 shows the various subclasses of flavonoids, their basic structures, and various
examples of the foods sources to which they can be found in.
10
· Exampl.. of
Flavonoid
Subcl.....
Basic Structure
Examples of Food Soure..
Fllvone
S_I red pepper, celery
Fllvonol
Onion, apple, red wine, gr..... lea, tomaloe.
o
Flavlnone
Fllvanol
-------------
,
Citrus fruits
Cocoa, chocolate, green lea, 1/'lIpe8, apples,
red wine
----t-----~+_----------------
Figure 4 - The subclasses of flavonoids, the basic structure and the foods in which they
are found,
Tannins, a fonn of polyphenols, act as a defense mechanism in plants against
pathogens, herbivores, and hostile environmental conditions,
industry. tannins are used in the processing of leather.
In the manufacturing
Tannins are extracted from
various tree barks, with the Europeans preferring sumac and oak, Animal skins are
composed of a protein called collagen (among other things), Bacteria and fungi readily
degrade this protein_ When tannins bond to the collagen, the crosslinked fibers are no
II
longer susceptible to attack. The tannin must effectively crosslink the protein, but must
also have desirable color properties and meet many other requirements.
Today, this
process still occurs, but the tannins are normally extracted from vegetables. Generally,
tannins induce a negative response when consumed by animals. These effects can be
instantaneous, like astringency or a bitter or unpleasant taste, or can have a delayed
response related to antinutritional/toxic effects. This is because polyphenols, such as
tannins, have the ability to form complexes with proteins or related biopolymers. (7)
Lignins and tannins are abundantly the most widespread of all polyphenols in
plants.
As stated above, tannins are a group of polyphenols that are used in the
commercial industry in tanning of leather and in the brewing industries. This group has
had its pharmaceutical properties investigated to help determine the structural properties
of tannins. Some interesting studies have been conducted regarding the iron complexation
of tannins for plant defense mechanisms. There are an enormous amount of Ii ving things
that require iron for other mechanisms to survive. The element Fe is involved in various
essential cellular processes. Plants are not the only things fighting for iron resources.
Pathogens and decay microorganism are also striving to obtain it. Tannins have a high
affinity for iron which helps limit the availability to other organisms. Tannins appear to
be very efficient in displacing iron from other ligands (3)
Studies dealing with bacteria and with the different strains of Erwinia
chrysanthemi, a pectinolytic bacteria, show that they can cause soft rot on a wide range of
plants. These studies showed that the binding of iron to tannins was a key property in the
protection of the plant form soft rot. When the iron concentrations were high enough to
provide both the plant and the bacteria, soft rot occurred. When there was only enough
12
iron to fulfill the tannins, no rot was observed. (3)
Iron is an essential nutrient in the human body. One of the main components of
hemoglobin, iron helps transport oxygen throughout the body and serves as a catalyst for
respiration. The iron we consume in food is generally absorbed first by the upper organs
such as the small intestines. It then travels through the liver on its way to the bone
marrow to help form hemoglobin. A certain percentage is also distributed to the body
cells, where it becomes an ingredient in nuclei chromatin and enzymes. Since it is so
important, this research project focused on correlating it with the absorption of
polyphenols.
The human diet contains several polyphenolic compounds which are obtained
from eating fruits, vegetables, spices, pulses and cereals, with high amounts in wines,
coffee, cocoa, and several varieties of teas. Some of these dietary intakes have been
noted to prevent absorption of iron in the body from simple iron forms, but this was not
noted when the iron form was more complex. There are two known low molecular
weight phenolic compounds, gallic acid and chlorogenic acid, which are shown to inhibit
absorption of iron, while catechin is shown to have no effect. (3)
13
OH
OH
R
OH
x=-CO
OH
Y1-OH
y, = - - - - -
OH
R=OH,Y.Y1, R'.X:Epigallocatechin gallate
R-Gl.Y.Y1.R'=H: Epigallocatechin
R-OH,Y-Y2.R"X: Gallocalechln gall••
R-H,Y.Y1,R'.H:
Epicaleehln
R-H. Y=Y1.R'::z X: Epicatechin gallate
R=H.Y.Y2,R''*I:
Catechin
Catechin
Figure 5 - Shows the different areas of substitutions for the Catechin compound
Tea Catechin, traditionally called tea tannin, is the special component of tea-leaf
with bitter and acerbic taste, as well as astringency. It has the effects of resisting
oxidation, resisting mutation, preventing tumor, resisting the AIDS virus, lowering blood
cholesterol, decreasing low density lipoprotein, inhibiting hyperpressure, inhibiting the
congealing of blood platelets, controlling bacteria, and preventing allergies etc.
Tea
catechins are strong active materials, formed with catechin as the main structure
combined with a gallic acid group. Those that exist in green tea are mainly (-)epigallocatechin gallate (EGCG), (-)-epigallocatechin (EGC), (-)-epicatechin gallate
(ECG), (+)-epicatechin (EC) (as seen in Figure 6), (-)-gallocatechin gallate (GCG), and ()-catechin. (8) Six different catechins can be found in Figure 5. The catechin that mainly
exists in black tea is theaflavin, which is a doubly condensed catechin substance based on
(-)- epigallocatechin-3-gallate (EGCG). The theaflvin has 4 isomers, which are
theaflavin-3-gallate, theaflavin-3'-gallate, theaflavin-3, 3'-digallate and theaflavin. The
catechin substances existing in Oolong tea are mainly catechin itself and catechin
14
oligomer. (8)
OH
H01Q(O
~I
/'
OH
Epicatechin
Figure 6 - The chemical structure of Epicatechin
Gallic acid or 3,4,5-trihydroxybenzoic acid , C6H2 (OHhC02 H, is a phenolic
antioxidant which is a colorless crystalline organic acid found in gallnuts, sumach, tea
leaves, oak bark, and many other plants, both in its free state and as part of the tannin
molecule. Since gallic acid has hydroxyl groups and a carboxylic acid group in the same
molecule, two molecules of it can react with each other to form a dimer ester, digallic
acid. Gallic acid is obtained by the hydrolysis of tannic acid with sulfuric acid. When
heated above 220°C, gallic acid loses carbon dioxide to form pyrogallol, or 1,2,3trihydroxybenzene, C6H3(OHh, which is used in the production of azo dyes and
photographic developers and in laboratories for absorbing oxygen. Its major use is in
pharmaceutical industries for manufacture of trimethoprim, an antibacterial agent, which
is usually given along with sulphonamide.
Together they have a broad spectrum of
action. The consumption coefficient of gallic acid in the manufacture of trimethoprim is
4.80. Gallic acid can increase its free radical scavenging ability when combined with
Procyanidin and Epicatechin. Gallic acid is normally found in the break down of tannins.
This polyphenol is best when used in conjunction with other antioxidants. Gallic acid is
15
also used in the enzymatic synthesis of gallic acid esters (e.g. propyl gallate), which is
used mainly as a strong antioxidant in fats, oils and beverages. Further, it also has
application in the leather industry and in pyrogallol manufacture. Gallic acid is
commonly obtained by enzimatic hydrolisis of gallotannins and further solvent
extraction. (9)
Proanthocyanidins (also called leukoanthocyanidins) are a class of natural
polyphenolic bioflavonoids that are very widespread in nature.
The structure of
proanthocyanidins can be found in Figure 7. They are found in many herbs, fruits and
vegetables, and often are the main precursors of the blue-violet and red pigments. More
than 90% of the proanthocyanidins occur as chains of two, three or more
proanthocyanidin molecules, which are called oligomeric proanthocyanidins (OPC).
These OPC's are considered most potent for their health benefits (monomers, like
catechin and epi-catechin).
Proanthocyanidins are highly regarded for their strong
antioxidant properties, and for their functions in supporting the body's connective tissues
and capillary blood vessel system. Europeans have been studying and using various
forms of these natural proanthocyanidins for several decades for their numerous
beneficial effects. Proanthocyanidins are among the most powerful natural free radical
scavengers yet discovered. As such, proanthocyanidins have their own unique place in
the body's overall protection against harmful free radical damage. Proanthocyanidins
appear to be especially effective in neutralizing highly reactive hydroxyl and singlet
oxygen radicals. Both of these reactive oxygen species are involved in inflammatory
processes. Proanthocyanidins also support and enhance the activity of vitamin C, and are
known for their ability to help support the health of the body's capillary system and
16
connective tissues. Proanthocyanidins have been shown to bind with collagen fibers,
thereby protecting them from premature degradation. This helps maintain the natural
elasticity of collagen in skin, joints, arteries, capillaries, and other connective tissues.
(10)
Flavan-3,4-diols
~~
Anthocyanidins
Proanthocyanidins
HEAT
ACID
';0
""
-,
"~ :rJ!!
;
,IJ
Figure 7 - The structure of Proanthocyanidins and the structure of anthocyanidins
Ellagic Acid is a phenolic compound that has become a known as a potent anticarcinogenic/anti-mutagenic compound. It also has anti-bacterial and anti-viral
properties. Ellagic acid itself is not thought to be naturally present in plants. Instead,
polymers of gallic acid and hexahydroxydipenoyl (HHDP) are linked to glucose centers
to form the class of compounds known as ellagitannins. When two gallic acid groups
17
become linked side-by-side within a tannin molecule, an HHDP group is formed. Ellagic
acid is the result when the HHDP group is cleaved from the tannin molecule and
spontaneously rearranges. For example, the ellagitannins are present in red raspberries.
Some articles in which ellagitannins have been quantified refer to ellagic acid because
quantitation of ellagitannins is done by breaking them down into ellagic acid subunits and
quantifying the subunits. The Meeker red raspberry is the best source of ellagic acid,
followed by Chilliwack and Willamette. The Meeker variety is specific to the Pacific
Northwest---grown primarily for commercial use in Washington State. The Chilliwack
and Willamette varieties contain lesser variations of ellagic acid. Both of these varieties
are grown in the Pacific Northwest and may be found in lesser quantities outside the
United States. The availability to the body of ellagic acid from dietary sources has only
been confirmed with red raspberries. Other foods such as strawberries, pomegranates, and
walnuts contain far lesser amounts ellagic acid, and their bioavailability has not been
confirmed. Ellagic acid acts as a scavenger to "bind" cancer-causing chemicals, making
them inactive. It inhibits the ability of other chemicals to cause mutations in bacteria. In
addition, ellagic acid from red raspberries prevents binding of carcinogens to DNA, and
reduces the incidence of cancer in cultured human cells exposed to carcinogens. (II, 12)
Research in animal and laboratory models has found that ellagic acid inhibits the
growth of tumors caused by certain carcinogens. Studies in humans are underway to
determine the effect of long-term daily consumption of raspberries on cell activity in the
human colon. Ellagic acid has been found to cause apoptosis (cell death) in cancer cells
in the laboratory. The mechanism of how it works is not yet well understood. (13) Some
research also claims that it prevents the binding of carcinogens to DNA, and strengthens
18
connective tissue, which may keep cancer cells from spreading. Ellagic acid has also
been said to reduce heart disease, birth defects, liver fibrosis, and to promote wound
healing. Many of these claims are currently under investigation. Ellagic acid has been
demonstrated in animal models to inhibit tumor growth caused by carcinogens. A human
study is being completed at the Medical University of South Carolina Hollings Cancer
Center. Twelve participants, some of whom had undergone surgery to have cancerous
polyps removed, ate one cup of red raspberries daily for a year, with some continuing for
longer. The study was to determine if eating red raspberries could prevent colon cancer
by both inhibiting the abnormal division of cells and promoting the normal death of
healthy cells. The results of the study have not yet been published. Other studies have
also found positive effects. A recent animal study found that ellagic acid protected mice
against chromosome damage from radiation therapy. A separate study of ellagic acid
indicated that it was effective at inhibiting tumor growth from esophageal cancer cells in
mice. Animal studies may show a certain substance holds promise as a beneficial
treatment, but further studies are necessary to determine if the results apply to humans.
(11,12)
A flavonoid compound found in grapefruit, naringin, gives grapefruit its
characteristic bitter flavor. Grapefruit processors attempt to select fruits with a low
naringin content, and often blend juices obtained from different grapefruit varieties to
obtain the desired degree of bitterness. Naringin is believed to enhance human perception
of taste by stimulating the taste buds (some people consume a small amount of grapefruit
juice before a meal for this reason). Naringin may be instrumental in inhibiting cancercausing compounds and thus may have potential chemotherapeutic value. Studies have
19
also shown that naringin interferes with enzymatic activity in the intestines and, thus,
with the breakdown of certain drugs, resulting in higher blood levels of the drug. A
number of drugs that are known to be affected by the naringin in grapefruit include
calcium channel blockers, estrogen, sedatives, medications for high blood pressure,
allergies, AIDS, and cholesterol-lowering drugs. Caffeine levels and effects of caffeine
may also be extended by consuming grapefruit or grapefruit juice. While the effect of
naringin on the metabolism of a drug can increase the drug's effectiveness, it can also
result in dosages that are inadvertently too high. Therefore, it's best not to take any drugs
with grapefruit juice unless the interaction with the drug is known. In addition, the effects
of drinking grapefruit juice is cumulative, which means that if you drank a glass of
grapefruit juice daily with your medication for a week, the drug interaction would be
stronger at the end of the week than at the beginning. (14)
Vitamin C (Asorbic Acid)
Asorbic Acid (Vitamin C) is essential in the formation of collagen a fibrous
connective tissue. It is a natural antihistamine that helps in the reduction of symptoms in
allergy and asthma patients. There is little doubt that a diet high in Vitamin C reduces the
risk of cancer substantially. Its mode of action is multiple; it protects the genetic material
from free radical damage, strengthens the immune system and increases resistance to
harmful chemicals (15). Vitamin C can inhibit the conversion of a normal to a cancerous
cell after exposure to a carcinogen. Cancer cells tum back into normal-appearing cells
when you add vitamin C to the culture medium (16,17). It also blocks the initiation of
cancer by X rays (18)
Vitamin C has been linked to a decrease in stomach cancer, which at one time was
20
the most prevalent type of cancer in the United States. The decrease has been associated
with the production of frozen orange juice. Vitamin C in breakfast orange juice probably
blocks the formation of carcinogens (called nitrosamines) in the stomachs of people
eating bacon or ham and eggs (19, 20). In a randomized clinical trial in Russia, vitamin
C, along with vitamins A and E were given to prevent postoperative complications in 197
people with stomach cancer. The complication rate dropped dramatically from 30.9 to
l.9 percent. (21) Japanese generally do not drink high amounts of orange juice, and
concurrently they have a much higher rate of stomach cancer. It is believed that vitamin
C would be of value in protecting against the destructive effects of malignant
invasiveness by stabilizing the body and enhancing the formation of the cellular cement
called collagen (22) In 1991 it was reported that 294 advanced cancer patients who
received supplemental vitamin C at some stage in their illness were compared with 1,532
cancer patients not receiving supplements at the same hospital. Patients taking vitamin C
had an average survival time of 343 days compared to that of 180 days for non-vitamin C
patients (23).
When cells treated with vitamin C were examined under an electron
microscope they appeared to be disorganized, their internal structures altered, their
membranes disrupted and they were surrounded by the body's collagen cement (24, 25)
It has been concluded that the role of vitamin C in the regulation of DNA repair enzymes
and also demonstrates an antioxidant effect (26).
Studies have dealt with Vitamin C dosages and uses with various cancer
treatments. The RDA is set at 60 milligrams, but a consensus is to increase it to between
120 to 200 milligrams.
However, dosages above 400 milligrams have no evident
improvement. Vitamin C has been proven to not interfere with chemotherapy. It has
21
been documented that vitamin C can protect normal cells from the damage cause by free
radicals without interfering with the cytotoxicity of doxorubicin against tumors (27, 28).
Zinc
Zinc can playa role in the formation of powerful antioxidants; an adequate intake
is needed daily to prevent a variety of diseases, one of which is cancer. Zinc is used to
protect against damage from radiation therapy. It has been proven in experiments with
animals, that when zinc was added to the synthetic antioxidant Amifostine, the effect was
greater than with Amifostine alone (22). Experiments have documented the increased
rate of cancer in rats with lower intake of zinc. When zinc was consumed in their daily
diet and they were given a carcinogenic mineral, the cancer developed at a much lower
rate in their testes when compared to the control with no zinc intake. NCI scientist
concluded that dietary zinc deficiency appears to cause a generalized increase in the
chronic toxic effects of cadmium(22). Zinc and cadmium are both in the same chemical
family. Prostate cancer patients also have lower blood levels of zinc than patients with a
non-malignant prostate condition. (22).
An experiment was conducted in Linxian, China, which is classified as the
world's capital for esophageal and stomach cancer. From 1982 to 1991 volunteers were
given various food supplements, with dosages of zinc between two to three times higher
than the RDA. The results that were obtained demonstrated that stomach cancer among
those who received the vitamin A and zinc combination was decreased by an astonishing
62 percent, compared to those who did not receive these supplements. In addition, a
combination of beta-carotene, vitamin E, and selenium caused a 42 percent reduction in
esophageal cancer. People receiving supplements had lower overall cancer death rates as
22
well. (22) Another Chinese study monitored the effects of zinc and licorice mixture on
chemotherapy. Licorice has been used world-wide as a flavor agent and a medicine.
Glycyrrhizic acid resides naturally in the root of the licorice plant, Glycyrrhiza glabra.
Glycyrrhizic acid, which makes up from 4 to over 20% of the root, is not the only
biologically active molecule. About 300 different polyphenols, which make up 1 to 5% of
the root, are suspected antioxidants, perhaps even cancer-fighting compounds.
Mice
were given a combination of this mixture as well as cisplatin (a chemotherapy drug). The
mixture significantly reduced kidney, blood, and testicular toxicity. At the same time, the
mixture did not undermine chemotherapy, either in the animals or in the test tube. (2)
Scientists from India published a study in 1995, on the use of a four-supplement
pill-zinc, vitamin A, the B vitamin riboflavin, and selenium. Smoking is known to
cause breaks in DNA (genetic material). They studied the effects of this supplement on
broken DNA in heavy smokers.
Out of nearly 300 volunteers, half were given the
supplement pill and half the placebo. The end results were compiled at the end of one
year, the frequency of DNA breaks decreased by 72 to 95 percent in the supplement
group. No such effects were seen on the placebo group. There were complete remissions
in 57 percent of the people who received the supplements, while only 8 percent of those
receiving the placebo showed a similar response. (22)
The key question is whether supplementation with antioxidants (or other
phytochemicals) has been proven to do more good than harm. The answer is no, which is
why the FDA will not permit any of these substances to be labeled or marketed with
claims that they can prevent disease.
The negative publicity has not deterred
manufacturers from continuing to market antioxidants as though they have been proven
23
to be beneficial. Many have also responded by hyping new mixtures of beta-carotene and
other carotenoids, which, they suggest, may provide the same benefits as fruits and
vegetables. Many types of pills described as "concentrates" of fruits and/or vegetables
are being marketed. However, it is not possible to condense large amounts of vegetable
produce into a pill without losing fiber, nutrients, and many other phytochemicals [22].
Although some products contain significant amounts of nutrients, these nutrients are
readily obtainable at lower cost from foods. (29)
The large class of flavonoids can be found in a wide variety of foods.
Anthocyanidins such as cyaniding and delphinidin can be found in berries, grapes, fruit
skins and true fruit juices. Catechins such as catechin and epicatechin can be found in
true teas and not herbal teas.
commonly found in citrus fruits.
Flavanones including hesperetin and naringenin are
While flavones including apigenin and luteolin are
contained in grains and herbs and finally flavonols such as myricetin and quercetin are
found in fruits, onions, and botanicals
Properties in other foods and beverages
Researchers have identified and separated monomeric and oligomeric flavanols
present in cocoa and select chocolates (30). Cocoa and chocolate have been found to be
abundant in the higher oligomeric compounds, procyanidins.
Flavonoids found in
chocolate include: the flavanols, which include (-) epicatechin and (+) catechin, and a
related series of complex procyanidin oligomers built from this monomeric unit (30).
In vitro (test tube) and in vivo (in humans) studies have shown that cocoa
flavonoids and certain chocolates may decrease low-density-lipoprotein (LDL) oxidation,
may modulate platelet activation, and may positively affect the balance between certain
24
hormones, or eicosanoids. These actions can playa role in maintaining cardiovascular
health (31-38).
One study found that chocolate liquor polyphenols (CLP), an enriched
polyphenols fraction purified from chocolate liquor that is a major component of
chocolate, inhibited the production of reactive oxygen species (ROS). (Polyphenols are a
larger group to which flavonoids belong.) Decreasing ROS damage to cell membranes
and biological molecules is believed to play an important role in maintaining overall
good health throughout life (14). In other observations, in vitro studies suggest that cocoa
flavonoids may have immunoregulatory effects that go beyond their antioxidant activity
(39,40).
Blueberry, a fruit closely related to the cranberry, has been shown to reverse
certain aging characteristics. In a study found in the Iournal of Neuroscience, researchers
found that rats fed spinach and strawberries learned better than rats on a standard diet.
Then they introduced a blueberry extract into the diet. The rats that got the supplement
not only learned faster than other rats, but their motor skills improved. The rats were 19
months old (the equivalent of 60 to 65 years of age) Researchers fed them for another two
months up to 70-75 years old. The blueberry-fed rats did better on standard rat tests, like
making them swim in a water maze, or find an underwater platform in murky water. But
they also did better on tests involving a spinning rod or an inclined rod which is a good
test of coordination. Young rats six months old could stay on a rod an average of 14
seconds. Old rats fell off after six seconds. The blueberry-supplemented old rats could
stay on for 10. The blueberries did not make the rats young again, but did improve their
25
skills considerably. When the rats' brains were examined, the brain cells of the rats on
blueberries communicated better. (41)
According to some alternative clinicians, menstrual problems typical of
perimenopause can also be alleviated with large doses of flavonoids, including the
powerful anthocyanins in bilberry extract. An Italian study found that a high intake of
bilberry flavonoids (anthocyanins) may also decrease or even eliminate the soreness in
fibrocystic breasts. This may be due mainly to the anti-inflammatory action of these
phytonutients and also to their probable ability to bind to Type II estrogen receptors and
exert hormone-like effect counteracting too much estrogenic stimulation. (42)
There is extensive epidemiological evidence that populations consuming larger
quantities of polyphenols show less obesity and lower cardiovascular, cancer and
Alzheimer's disease mortality. Some examples are: Eastern Asia, for instance, with its
high intake of green tea, miso, ginger and a great variety of vegetables; and Southern
France and other Mediterranean countries, with their high consumption of red wine and
olive oil, both known to be rich in phenolic compounds. Chocolate too may have similar
benefits, due to its high content of proanthocyanidins, similar to those found in red wine.
Epidemiological studies tend to confirm a correlation between low risk for various
degenerative disorders and high consumption of polyphenols. If red wine and chocolate
do not agree with you, you can always reach for grape seed extract. (42)
We can only make an educated guess that blueberries and bilberry extract, as well
as grape seed extract, with their highly effective antioxidant power, have anti-aging
benefits and may even be able to extend life span if consumed daily in sufficient doses.
This is highly likely in view of the documented antioxidant, chelating, hypoglycemic,
26
antiatherogenic, anticarcinogenic, anti-inflammatory, immunostimulating, and enzymemodulating effects of polyphenols.
When considering the potential health benefits of flavonoids in foods and
beverages, it is important to determine not only their presence, but also their
bioavailability once ingested, since their nutritional significance will depend on their
behavior in the digestive tract. One study analyzed plasma samples from healthy men
after chocolate consumption and found that plasma concentrations of epicatechin
increased markedly, reaching maximum levels between two and three hours. Thus,
epicatechin was found to be readily absorbed from chocolate (43).
Tart cherries are another fruit that has been shown to have wonderful antioxidant
properties.
Tart cherries contain natural anti-inflammatory compounds called
anthocyanins. In laboratory tests, Michigan State University research indicates that the
tart cherry compounds are at least 10 times more active than aspirin. The advantage of
tart cherries is that they are more effective without any of the adverse side effects of
aspirin, such as stomach and kidney problems. In addition, other research has revealed
that the production of a human hormone (prostaglandin) is the cause of joint pain. The
production of this hormone is directly related to two enzymes (cyciooxgenase -1 and -2).
Twenty tart cherries appear to provide enough anthocyanin (12 to 25 milligrams) to
inhibit the enzymes that ultimately cause pain and inflammation. Tart cherries are an
excellent source of antioxidant compounds. There are 17 compounds in tart cherries with
antioxidant properties. The antioxidant activity of these tart cherry compounds, under the
MSU evaluation system, is superior when compared to vitamin E, vitamin C and some
synthetic antioxidants. Two of the antioxidant compounds (kaempferol and quercetin)
27
found in tart cherries also are found in several commercially prepared health products,
which are used to improve memory, concentration and vision. Among the antioxidant
compounds in tart cherries, there are four anthocyanins. Recent research indicates that
these cherry anthocyanins may have the potential to inhibit the growth of colon cancer
tumors. (44)
Why Cranberries?
There are thousands of naturally occurring components in fruits and vegetables
that can help promote health and reduce risks for many common diseases. With regard to
cancer prevention, the potential benefits of cranberry's rich flavonoid profile have,
understandably, received additional attention. Studies have shown that flavonoids
isolated from cranberries can inhibit bacterial adhesion, the mechanism by which
cranberry juice helps maintain urinary tract health (45), and possibly even help in the
prevention of periodontal disease (46) and the formation of certain ulcers (47).
Compounds in cranberries may also help prevent the progression of atherosceloric
plaques that lead to cardiovascular disease (48). Compared with 20 common fruits tested,
cranberries have the highest fresh-weight content of flavonoids and related phenolic
acids (49), making this berry a powerful source of potentially anti-cancer phytonutrients
and antioxidants. While findings linking cranberry's components to a decreased risk of
cancer are preliminary, in vitro studies suggest cranberries contain components that can
inhibit carcinogenesis. The polymeric proanthocyanidin fractions from lowbush
blueberry, cranberry and lingonberry exhibited the greatest anti- carcinogenic activity.
Other researchers found a proanthocyanidin-rich phenolic fraction from the American
cranberry
(Vaccinium
macrocarpon)
that
demonstrated
the
most
significant
28
anticarcinogenic activity (SO). Of the seven flavonoids in high abundance in this active
fraction, oligomeric proanthocyanidins, quercetin and myricetin were identified. Several
laboratory studies have also investigated the role of the phenolic antioxidants ellagic acid
and reversatrol, present in grapes and berries (such as cranberries) at high levels, in the
inhibition of human cancer cells. Researchers found both compounds inhibited the
growth of certain cancer cells, specifically colon and prostate cancer cells, by interfering
with the expression of a number of genes required for cell growth and activating
apoptotic-specific proteins that can trigger cell death (5 I).
Research has shown that
quercetin, a prominent cranberry flavonoid (52), inhibits the development of chemicallyinduced mammary and colon cancer (53).
According to the Alder Lake Cranberry Corporation, cranberries have several
benefits including the prevention of urinary tract infections (UTI's), anti-aging,
preventing ulcers, promoting cardiovascular health and preventing cancer. Cranberries
(Vaccinium macrocarpon) are the cousins of blueberries. Since most cranberry products
are highly sweetened, it is advisable to try unsweetened varieties to gain the full benefits
of the cranberry, especially the anti-tooth decay property. (54)
Besides being a heart-healthy source of antioxidants, cranberries were shown to
decrease total cholesterol and LDL or "bad" cholesterol levels in a recent study
conducted by the University of Wisconsin-Madison. According to Rutgers University
Cranberries contain compounds that have an anti-adhesion or anti-stick mechanism that's
been shown to be effective in maintenance of urinary tract health. Preliminary research
suggests this same anti-stick mechanism may work in the mouth and stomach, possibly
helping to prevent gum disease and ulcers.
A study conducted at the University of
29
Wisconsin-Madison found that feeding cranberry juice powder to animals with high
cholesterol decreased total cholesterol and LDL cholesterol by 22 percent. (55)
Cranberries are rich in flavonoids. These phytonutrients have been shown to
inhibit certain types of cancer. According to information obtained from the Cranberry
Institute Dr. Catherine Neto of the University of Massachusetts, Dartmouth recently
published a laboratory study in the Journal of Agricultural and Food Chemistry showing
that certain cranberry extracts inhibited the growth of a variety of tumor cells, supporting
earlier studies conducted elsewhere. While the effects in humans have not yet been
established, this work will form the basis for future research. Cranberries are a rich
source of the bioflavonoid quercetin. In a study published in the Journal Cancer
Research, quercetin, which is present in many fruits and vegetables, was shown to be
effective in inhibiting chemically induced colon and breast cancers. (56) More recently,
researchers from the University of Western Ontario demonstrated that mice injected with
human breast cancer cells had a significantly lower incidence of tumor development
when fed cranberry components. In a presentation at Experimental Biology 2000, the
annual meeting of life sciences researchers, Dr. Najla Guthrie and colleagues reported
that cranberry consumption delayed tumor development and reduced the spread of tumors
to the lungs and lymph system. (57) Guthrie concluded that the research found that mice
that received cranberry juice and cranberry products had a significantly lower number of
breast cancer tumors compared with the control group, and the development of tumors
was delayed, and the results are very preliminary, this study may suggest that cranberry
products could have cancer fighting properties in humans. (58,59,60)
30
New research suggests compounds found in Cranberries may inhibit ulcercausing bacteria from sticking to the stomach wall. From Life Extensions Magazine an
article written in 2000 suggest that cranberries might be effective against Helicobacter
pylori the bacteria implicated in stomach ulcers. In vitro research has shown that the antiadhesion property of cranberries can help combat the ulcer-causing bacteria even when
they have adhered to the lining of the stomach. Research was also presented on how this
mechanism may be effective against bacteria in the mouth in the prevention of
periodontal disease. (61, 62, 63) Apples, celery, cranberries, onions, red wine, and green
and black tea are also high in natural chemicals known as flavonoids, which appear to
inhibit H. Pylori growth and have many other health benefits. In fact cranberry juice
specifically may have properties that help prevent H. Pylori from infecting the intestinal
lining. (64)
Polyphenolic compounds found in Cranberry may help to protect against
neurodegenerative diseases, and the memory and coordination losses often associated
with aging (54).
Early findings suggest that eating plenty of high-ORAC fruits and
vegetables-such as spinach and blueberries- (see Figure 8)-may help slow the processes
associated with aging in both body and brain. Oxygen Radical Absorbance Capacity
(ORAC)-measures the ability of foods, blood plasma, and just about any substance to
subdue oxygen free radicals in the test tube. Cranberries score high on the antioxidant
scale at 1750 ORAC units per 100 g (about 3.5 oz.) of fresh fruit. 66) In studies, eating
plenty of high-ORAC foods: resulted in a raised the antioxidant power of human blood
10 to 25 percent, prevented some loss of long-term memory and learning ability in
middle-aged rats, maintained the ability of brain cells in middle-aged rats to respond to a
31
chemical stimulus-a function that nonnally decreases with age, and protected rats' tiny
blood vessels---<:apillaries-against oxygen damage. (65-70)
ORAC Values"
Cr~nb ..ri"
Strawberri.s
Spinach
R.alpberri.,
Alfalfalproutl
Broccoli
eeOCI
Orange,
Red Grop ..
Ch.rri..
o
500
1000
1500
2000
ORAC units per 100g fresh
Figure 8 - A chart containing the ORAC Values for various foods (71)
Cranberries and blueberries contain condensed tannins called Proanthocyanidins
that prevent E. coli bacteria from attaching to cells in the urinary tract. These findings
appeared in the New England Journal of Medicine. The cranberry plant transforms the
flavonoids that contribute to the fruit's bitter taste by removing part of the flavonoid
molecule and replaces it with a sugar molecule. This has the effect of sweetening the
fruit, making it more palatable as a food. This sugar molecule makes the cranberry
effective as a nutrient within the urinary tract. In the human body, different cells have
unique receptor sites. These sites can be thought of as a lock in a door requiring a unique
key to open the lock. The sugar attached to the cranberry flavonoid seeks out an
32
acceptable receptor site to attach itself. In cranberries, the sugar unlocks a receptor site on
the walls of the urinary tract. This explains some of cranberries' unique benefits.
Cranberries contain a type of flavonoid that is capable of defeating the bacteria that cause
urinary tract infections, and this flavonoid is attached to a sugar that seeks out the cells
that line the urinary tract. (72) The National Kidney Foundation recommends drinking at
least one large glass of cranberry juice a day to help maintain urinary tract health. This is
a very helpful benefit because of the development of bacterial resistance.
Another
excellent reason for decreasing the use of antibiotics is that natural remedies are not
harmless compounds. They have side effects also, such as diarrhea. The killing off of
friendl y bacteria often results in an overgrowth of harmful organisms such as Candida.
Many women experience a miserable vaginal Candida infection practically every time
they use antibiotics.
The use of potent cranberry extract has no side effects, while
producing mUltiple benefits. (42)
Cranberry juice may also be able to help reduce the risk of kidneys stones. A
preliminary study suggests drinking a glassful of blackcurrant juice may make the risk of
a certain type of kidney stone decrease. Researchers in Germany measured levels of
compounds associated with kidney stones in the urine of 12 healthy men aged 18 to 38
and determined their urinary pH levels after they consumed plum, cranberry or
blackcurrant juice on separate days. In the study, pH levels of urine increased after the
study volunteers consumed blackcurrant juice, report researchers in the October issue of
the European Journal of Clinical Nutrition. Low urinary pH, which indicates that the
urine is more acidic, is associated with uric acid kidney stones. Uric acid kidney stones
make up about 10% of kidney stones, which can also be comprised of calcium, oxalate or
33
other minerals. About 85% of stones are comprised mainly of calcium. Cranberry juice.
on the other hand, decreased the urinary pH, suggesting that it may help to treat certain
other types of kidney stones or urinary tract infections (UTIs), which affect more than
half of women in the US, according to at least one survey. UTI symptoms can be mild,
such as a frequent urge to urinate and pain on urination, or more severe, such as high
fever, pain and blood in the urine. (73)
According to current statistics in America 35% of the population is expected to
develop some form of cancer before they expire. Americans seem to be strongly keyed
into different ways to prevent various diseases including cancer. To the majority of the
population this information will be useful because almost everyone knows someone with
cancer.
Materials and Methods:
Preparation of Fresh Cranberries
Various techniques were tried and their physical alterations on cranberries and
grapes observed. The techniques used included oven drying at two different temperatures
(100 Celsius and 140 Celsius), drying in desiccators, air-drying without light exposure,
air-drying with light exposure and drying in a food dehydrator. It was concluded that the
least destructive approach to prepare the cranberries into a powder would be to dehydrate
them in the food dehydrator. Ocean SprayTM and Paradise Meadow Premium Cape Cod
Cranberries were massed and then placed on a shelf in the food dehydrator. They were
then dried using a food dehydrator (Harvest Maid - Model LD-1OOO). The dehydrator
was set at 125 degrees Fahrenheit or 50 degrees Celsius for approximately 48 hours. The
cranberries were then removed and massed and the percent water loss was calculated.
34
The cranberries were made into a fine powder by placing metal beads and the
dried cranberries into a glass jar. Then the jar was manually shaken until a fine powder
resolved. A good food processor or coffee grinder might be able to speed up the process
slightly.
Atomic Absorption
Various brands of cranberry juice were used including three cranberry juice
cocktails from Meijer, Wal-Mart and Ocean Spray, and two organic brands: Just
Cranberry and Tree of Life, a concentrate. Dilutions of each were made of .1 ml, .25 ml,
.5 ml, 1 ml, 5 mI, 10 ml, and 25 ml diluted with water to a volume of 100 milliliters for
each brand of juice. The concentrated of Tree of Life was further diluted to .1 mI and
diluted to 250 milliliters with water. They were also all tested full strength with no water
added for some of the metals.
The standards were made by considering the linear working range for Na, K, Ca,
Fe, Cu, Mg, Mn and Zn. The maximum concentration for the linear working range was
used and then 3 more samples of each were made each decreasing in value by a factor of
2. For example, if the linear working range was 2flglml then the samples were made of
2flglml, Iflg/ml, .5flglml and .25 flglml (ppm). The standard samples were all double
checked by making up another standard at each dilution and checking the concurrence of
the results. A standard curve is made for each standard by graphing the the absorption or
emission reading vs. concentration. The data that was collected from the samples are
then found on the standard curve and the concentration is extrapolated.
UV -Vis Diode Array Spectrophotometer
35
Samples of polyphenols including: Naringin, Quercetin, Flavanone, Ellagic Acid,
Catechin, Tannic Acid, 3-Hydroxyflavone, Gallic Acid, Flavone, and Epicatechin were
purchased from various laboratory supply companies. Small amounts of polyphenols
were used to find what chemical would work the best for the solvent.
Acetone,
dichloromethane and hexane were all tried alone, and then with mixtures of water. After
reviewing the results straight acetone was chosen to be the sol vent for all known
polyphenols.
The stock polyphenols of Naringin and Quercetin, were diluted in acetone until
the Absorbance on the UV-Vis Diode Array Spectrophotometer was close to 1.0 AU.
The blank for this procedure was pure acetone. Serial dilutions of each sample were then
made up in order to create a Beer's Law plot. N aringin was diluted down to .1 g in 100
ml of acetone and then aliquots of this were diluted down in 100 ml volumetric flask. 20
ml, 15 ml, 10 ml and 5 ml samples were all removed and diluted down. These samples
were than run on the Spectrophotometer and the results charted. Quercetin was diluted
down by taking .05g of stock and diluting in a 100 ml volumetric flask with acetone. The
molarity from the Naringin samples used are 8.613 x 10'5, 1.722
X
10'4,2.583
X
10'4 and
3.445 x 10-4 the molarity for the Quercetin samples used are 1.476 x 10,5,2.953
4.430
X
X
10'5,
10'5, and 5.906 x 10,5. Later, 10 ml samples of each dilution were removed and
various metal ions will be added to this sample. These ions included: ferric iron, zinc to a
different sample, and both iron and zinc to a third sample. Also a sample of ferrous iron
wa~
added to a fourth sample, while a sample was saved as the control, and a blank of
acetone.
36
The samples were then analyzed to see which of the metal ions formed a complex.
For the rest of the samples of polyphenols, ferrous sulfate and ferric sulfate were used as
the ions. A.O 1 molar solution was made by taking .278g of Ferrous Sulfate and diluting
in a volumetric flask with acetone, and a .01 molar solution of Ferric Sulfate by diluting
.398 grams FeS04 in 100 mls of acetone. Approximately.5 ml or 10 drops of each
solution were added to the various dilutions of polyphenols.
Aliquots of each polyphenol sample were place in small vials in sets of four. Ion
was then added to each sample and visual changes noted. The amount of ion added was
increased in increments of .5 ml ranging from .5ml to 2.0 ml drops. After viewing the
data for the ferrous and ferric samples this portion was only used with Naringin and ferric
sulfate.
Results:
Description of berry
Turned a dark brown color and the edges looked burnt, were
Oven dried @ 140
not pliable
Turned a dark brown color and the edges looked burnt, were
Oven dried @ 100
not pliable
Dried up but took over a week, and were still pliable so there
Desiccators
was still water left to be removed
Dried up over several days
Room Air wllight
Room Air without light
Dried up over several days
Dried in about 48 hours and deep red color still present, little
Food Dehydrator
change in structure of berry
Table 1 - Observations made of the phYSIcal changes of the dned cranberrIes .
Drying Method
.
Ocean Spray #1
Ocean Spray #2
Ocean Spray #3
Paradise Meadow # 1
Paradise Meadow #2
Paradise Meadow #3
.
.
88.52%
86.91%
85.78%
84.98%
87.12%
90.06%
37
Table 2 - Percent of water in samples of cranberries (sample size 300 cranberries)
Ocean Spray Average
Paradise Meadow Average
87.07%
87.39%
Table 3- The average percent of water in both brand names of cranberries tested.
Chart 1-7 - The linear graph of the AA samples, for various ions.
ZlncAA
o
0.25
0.5
0.75
1.25
1.5
1.75
2
2.25
ppm
38
NaonAA
700
600
,' ...;;~:
'I- 580.4k~ 1.9713'
500
c
.2
400
•
.~
w
300
200
,t"
100
'.-",'
0
0
0.2
0.4
0.6
0.8
1.2
ppm
Fe on AA
0.07
0.06
0.05
0.04
'"
"" 0.03
0.02
0.01
0
0
2
4
6
8
10
12
PPM
39
MnwithAA
0.18
0.16
0.14
0.12
....'"
0.1
0.08
0.06
0.04
0.02
0
0
2
3
4
5
6
8
10
12
ppm
Ca with AA
0.45
0.'
0.35
0.3
......
0.25
0.2
0.15
0.1
0.05
2
•
ppm
40
CuonAA
0.08
'".." 0.06
.'.
"
:'.
0.04
o~----~~~~~~~~~~~~~~--~~
o
2
3
__~----~__------~
4
5
6
ppm
Potassium on AA
600~~_=
500
400
100
-1--"-:.;:.:1
o
Table 4 - Analysis of Juice on AA (all data in ppm unless labeled)
41
Berries
Na
K
Ca
Ocean Spray Man
Ocean Spray Kay
0
0.337
NA
6.5
Paradise Meadow Man
Paradise Meadow Kav
0
0.167
NA
5.7
NA
NA
257.8
Mg
Mn
Zn
Fe
Cu
NA
NA
NA
NA
NA
NA
1.41
0.27
0
0.615
0.027
0.033
NA
NA
NA
NA
NA
NA
1.11
0.24
0
0.548
0.014
0.028
NA
NA
NA
NA
55.3
1.5
0.9
0
17.1
0.35
0.13
NA
NA
NA
NA
NA
NA
NA
167.7
55.6
0.95
0
17.4
0.71
0.1
NA
NA
NA
NA
NA
NA
NA
16.7
0.8
0.1
Juice
Ocean Spray Man
Ocean Spray Kay
145.8
14
Meijer Man
Meijer Kay
145.8
16
WalmartMan
Walmart Kay
145.8
15
175.5
64.8
0.95
0.04
Just Cranberry Man
Just Cranberry Kay
73
60
480
512
135
156.4
11.413
6.25
NA
NA
NA
NA
0.13
26.3
0.81
0.32
Tree of Life Man
Tree of Life Kay
0
104
NA
0
558
.018 ppb
15
NA
NA
NA
NA
4725
0.89
87.5
12.5
2
Beer's Law for Naringin at 332nm
~ d. t"':"~,+,i';i
;"dij~,f~,:~t
i
J
06
+-~~"""""~"""4:7"""'-'-,~~"""';..,y;,~~
'·.;~<;';\"i~f\1;;;';",··
0.00005
0.0001
0.00015
0.0002
0.00025
0.0003
0.00035
0.0004
Col'IC8ntration (M)
42
Beer's Law for Quercetin at 370nm
0.00001
0.00002
0.00003
000004
0.00005
0.00006
0.00007
Concentration (M)
Charts 8-9 - Beer's Law Plots for Naringin and Quercetin
Table #1 shows the results that were obtained from the various drying methods
experimented with on grapes and cranberries. The grapes provided better observational
data because they were not previously dried like the cranberries. The dried cranberries
however still had a content of moisture remaining. Pictures of the dried cranberries along
with pictures of grapes can be found in the appendix.
Table #2 shows the percentage of water loss after drying two different brands of
cranberries in the food dehydrator. Cranberries are mainly water, and after drying, a light
air filled shell remains. They are considerably different from the texture of a grape, and
the remains have a texture similar to that of paper or dried leaves. Table #3 is an average
of both brands of cranberries dried from the data found in Table #2.
Charts 1-7 show the various atomic absorption standard curves that were made
using standard dilutions on the Atomic Absorption. Table #4 shows the final results of
43
all the data collected on the AA comparing the various brands of juices. It compares the
data from the label and the results obtained from research. The values were obtained
from the standard curves and the values obtained from the product were extrapolated to
determine the ppm or ppb of each product. The Tree of Life cranberry juice concentrate
has higher amounts compared to the other brands because the product has been
concentrated down. There are very few differences in the Meijer, Wal-mart and Ocean
Spray cranberry juice cocktails; one notable difference is that there is more potassium in
the Ocean Spray brand and also less manganese. When comparing the juices to the
whole fruit the berries have lower contents an all of the ions tested when compared to the
JUIces.
Charts 8-9 show the Beer's Law plot for both Naringin and Quercetin. After
graphing the data it was determined that Naringin has an
e value of 3306 and Quercetin's
value is 26126. Each has their own wavelength with Naringin at 332nm and Quercetin's
is set at 370nm.
In the appendix are the printouts from the data collected on the UV -Vis Diode
Array Spectrophotometer. Visible color changes were noted in the Naringin polyphenol
and Quercetin. Naringin turned a deep reddish-purple color when ferrous was added, and
no visual change could be noted after the addition of zinc the original color of naringin
was an off white color. Quercetin's yellow tinted color turned to a deep bright green
after the addition of iron and no change was noted after the addition of zinc.
The
printouts show the slight effect of ferrous sulfate on cranberry juice in comparison with
the other forms of iron. Ferrous sulfate had the most visible difference compared to the
remaining iron samples. Ferric sulfate when added to the naringin showed an analysis at
44
approximately the same position as the same naringin sample missing the iron addition.
The samples almost completely overlaid each other. While the ferrous sulfate samples
had a difference from the original naringin sample, making a notable visible difference.
Discussion:
Polyphenols are destroyed at higher temperatures, causing concern during the
manufacturing process. A food dehydrator was the least harsh process for drying the
fresh cranberries but was also very time consuming.
Since it is unclear where the
majority of polyphenols in cranberries can be found future research can entail comparing
seeds, with the rest of the berry. In grapes, for example, the skins and seeds have been
documented to contain more polyphenols than the pulp of the fruit. For cranberries the
same aspect may hold true or they might be contained throughout the berry. However
removing the seeds from the cranberry will be a very tedious process. The amount of
water in cranberries is very high for the type of berry. Cranberries are not as plush as
most berries like grapes or raspberries, their hard texture limit one to believe little water
content. Cranberries basically just have a fibrous shell left after all the water is removed,
meaning water is the main content of the berry and then the fibrous tissue gives them
their shape. For future research all the water content must be removed from the cranberry
and a method must be found to remove the polyphenol content most probably an
extraction method.
It appears that the less refining of cranberry juice provides for greater zinc content
in the juice, or this could be simply a result of the geographic growing of the cranberry.
Certain soils contain differences in the trace metals that are available to the plants that
grow there. Also it was interesting to find out that Ocean Spray, Meijer and Wal-Mart
labeled cranberry juice cocktail all have 10 times less sodium than what their label has
45
listed. After trying to contact the companies and gaining no information. it is assumed
that this may be a mis-labeling, or there was another reason behind this perhaps. Sodium
is a concern to patients of high blood pressure, making manufactures want to limit the
amount they add to their products. Maybe they actually have lowered the amount of
sodium they add to their product, while not changing the label.
From the analysis of 5 juice brands with metal ion content, organic cranberry
juice is the route to take. Both organic juices had a higher amount of zinc, which is
shown to help in the fight against cancer, and less sodium. Sodium is additive which be
closely monitored by people suffering from hypertension, so less is better.
Organic
brands also contain fewer preservatives and more natural, less refined products. In future
research the amount of polyphenols in each brand needs to be examined both in quantity
and quality.
Preliminary studies of the pure juice showed no positive, definite
identification of any polyphenol.
Ferrous sulfate, which was the ion used to form complexes with the polyphenols,
is the same form as iron supplements taken by anemic patients. This form of iron is more
readily absorbed by the body, making it more useful to humans. Ferric sulfate was also
looked at with the flavanol naringin. Future research will include at looking at more
metal ions and their effects. Some metals have different ion charges, so correlating the
charge of the ion related to the reaction of the polyphenols content will be critical. It will
be key to understand how it is absorbed into the body and which metal ions make it more
readably absorbed. It will also be key to determine if there is a mole ratio for each
polyphenol and ions. Preliminary research suggested a high mole ratio with ferrous
sulfate and naringin possibly around 34 moles of iron per mole of naringin, since this was
46
preliminary data future research will be utilized to determine if the calculated number is
correct.
Beer's law needs to be completed on more polyphenols to see the remaining
epsilon values.
Naringin has an E value of 3306 and Quercetin's value is 26126.
Preliminary work was begun to find a molar ratio between the metal ions as they complex
using the Diode Array Spectrophotometer. This method can also be utilized in future
research.
Dramatic visible changes occurred when adding certain metal ions to the
polyphenols samples of Naringin and Quercetin.
When viewing this on the
spectrophotometer it was difficult to determine where the dramatic difference occurred.
Future research can include determining what happens in the UV -Vis spectrum during the
complexing of the polyphenols with various metal ions, including if they all have the
same reaction per charge or not. While trying to find analyze the Quercetin sample on
the Spectrophotometer the color began to fade away at a very rapid rate, it needs to be
determined in future research if time or light degraded the complex. Light is believed to
be the main reason because while in the Spectrophotometer box, shielded from the light,
the color of the sample continued to fade at a rate visible to the box.
In the future the main goal needs to be to quantitatively and qualitatively
determining which polyphenols are indeed in cranberries and their products.
A
correlation between the manufacturing process and finished polyphenols content will be
crucial for the consumer. When buying a product, one wants the best product for the
purpose he or she is buying it.
So if they are purchasing solely for the antioxidant
benefits, fresh cranberries are better than processed juices.
47
Since there is little documentation on cranberries and their polyphenols, it will be
interesting to see what the future holds for this research area. With every experiment
another door was opened and ideas on how to try new things evolved. Hopefully in the
near future, it will be clearly understood the full value of a cranberry and just how good
polyphenols are for us as antioxidants.
48
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53
Appendix
Raw data from the Atomic Absorption Analysis
ABS
Zn
0
0.25
0.5
1
2
5
Na
Emission
0
0.05
0.1
0.25
0.5
0
0.5
1
2
5
10
0
1
2
5
10
15
0
0.003
0.006
0.013
0.031
0.065
0
0.05
0.095
0.207
0.389
0.54
ABS
Cu
0
33
52
138
288
580
ABS
Fe
ABS
Ca
0
0.036
0.064
0.128
0.249
0.54
0
0.25
0.5
2
5
K
0
0.007
0.014
0.026
0.046
0.114
Emission
0
0.05
0.1
0.25
1
0
33
61
130
511
ABS
Mn
0
0.25
0.5
1
2
5
0
0.0096
0.0206
0.0376
0.0686
0.169
54
Diode Array Spectrophotometer Printouts - On the following pages
55
Cranberry Control.
0_8
~
~
0_6
0
I~
I~
004
0_2 -
--------------
0
·~~~I-r-,-r-r_,--'-.-r~~-~TI~-~~'I
~_ _~~
200 _ _~220~
1----------C;:nb';"ri
+ Zinc
~~
I
I~
~
o
0_8
0_6
C
<0
-eo
w
:1
OA
Cranberry + Zinc, Iron
N
0_8
0.6
OA
~260~_ _~260~_ _~3D~~__~~~_ _~~~_ _~300
------------.--
1-
.......,...---;'-.-~·~~~I~-~~'I-~
Wa,leienqth {l1lll
~~--~~~~~-
CranberrY- -Control
--_._-
0.8
0.6
0.4 .
0.2'
~~~~.~~.-~f~~/_!
o
t
200
3X)
~'Tt--~--.
4.X)
i
)
!5CX)
i i i
ffX)
i
t
700
i
--'-,
---1
Wavel~nl1th .I..iltlli
1.
5
::sw
0
c
0.80.6
m
-e0
~
.0
0.4
<[
0.2
o·
200
Cranberry + Zinc, Iron
1.2
I
15:'S
0.8
i~
l;ij
0.6
'l5
n'"
0.4
I
0.2
<{
L1__
I
~30 ~'t".-'_ -_T_-_-_T_-_-_T._-_-_-_,-_-",4O :t~ ~ ~ ~ ~_-_-_"_'-_-"-SXl:t:~~-_~-_____r_-_-_'~-_'o o-, 't:~:.~ :.~_-·_·~-_~ - "70 , ,~, - _- -,!w- ~-~e-,I-"'-l~-'h-i{-nl]
-_T_-._--_'_ _
0.4
0.2
L_---'2OO""-___ -"'22O<"-_ _ _2£4O"'--_ _-'26J"""_ _---'''''._ _--''''''-_ _ _32O~'___
340 ___ .__. 36J
Wavelength (nm
==~~-------------------------------------------I
Ferrous Chloride
0.6
0.5
0.4
0.3
0.2
0.1
200
1--
----Terrou~
-------1
Sulfa=lc - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - e
osl
I
oJ\
.~
0.3
I
'~~
~
0.2
0.1
--.------.--...----T'----.---.--.-----j-,
L -_ _ _
"-..
~----. - - - - - - - _ . _ -
--------__Il
~~~I~~-~'I--~ r-..,.....,-~_,j------.--------_______r--T -,~--'----l---.-------.---
~2OO~___~220~~___~240~_ _ _~26J~~____2BO~~_ _~300~____~320~~.
I
i
_~340~_ _ _~36J~~_ _V~'\~!a~ve~i£~f~"~fl~Il~(I~"""ill
1
I
'-~~-~~--'I-~~'-~~··~I-~~
5CXl
Ferrous Chloride
0.8·
0.7
i
0.6
IS
0.5~
:'0
Ij
0
~
"'
"
0.40.3
0.2
0.1
LO
200
- Ferrous Sulfate
600
I
700
'-~~--.-,
VVGlio.=lcn ill 'riiTl
Cranberry + Iron
N
1.2
s~
w
0
c
ro
ti
""'
O.B
0.6
~
0.4
0.2
-----C=ra=n'"ber=-ry + FerrOUs Chloride
0.9
O.B
0.7
0.6
0.5.
0.4
0.3
_I
0.2
0.1 .
~--------
i-~~.~,------~-,__--~~-,,~----~,,----~~-,,~------,,------~-,____~~-.'~'--'-~"------~'
-.,~~~
I________~2OO~______~220~~____~2~40~______~260~____~260~~____~300~______~320~~____~340~~____~360~____~V~VEa~v"~t"~r~'g~tI~;~t'~"rill'l
Cran~rry + Ferrous Sulfate
1.2
O.B
0.6
\
0.4
0.2
-----------------------------
O~-C-.,---__~-, -.-.~_,'---T-__" __---·~~·---'~ ~~ _I---~·
200
____
____
____
____
_____
____
__
.~220~
~2~40~
~260~
~260~
~300~
,320~
~340~
. =-1
VVavdcngtll (nm/
Cranberry "+ Iron
1.2
0.8
0.6
0.4
0.2
r-----Cran~rry + Ferrous Chloride
0.8
~
~
0.6
~
c
,u
~
0.4
"'
-<
.0
0.2
~~~----~---~----~~~~~-~~~
o
L
Cranberry + Ferrous Sulfate
1.2
I
I
«Xl
500
rk"
~ wavt!I~;19th
(;]
Grape Control
1-
0.8
:::>
~
w
0.6
0
c
ro
-e
0
w
0.4
.0
«
N
co
ro
0.2
OJ")
'"
m
0220
200
~2
,
1
:5
'"
w
0
,c
ru
i§
240
260
260
340
360
WaIJehmgth (nm)
.
l
Grape + ZinC
N\
0
08
0.6
w
.0
<i
I
0.4
0.2
l_~_:2OO
~
I
I~
o
c
.f!
~
.0
'"
....CO
<D
U)
220
240
260
280
N
:lOO
'=,
320
360
I
I
Wavd8ngth
(nmil
Wavelength
!nrn~
Grape + Zinc, Iron
N
N
1
0.8
0.6
0.4
0.2
....co
ro
OJ")
o J,
200
(~
'gj
"220
240
260
280
:lOO
320
340
360
-'-r-- ...............
0.8
s-
:::.
~
0.6
0
iii
-e0
0.4
if>
.0
""
0.2
0
r
I
I
I
I
·~~-r·-~--
200
3:XJ
-«Xl
5CXl
5CXl
700
Wa"'t;;l~n tn nm
Grape + Zinc
1.2
s-
:::.
0.8
~
0
c
-e0
0.6
«
0.4-
ru
if>
D
02
0
I
3:XJ
200
I
I
I
I
_="-______
-"""
5CXl______-''''''''-_______
5CXl
700'''''-- __Wavt:!len
-«Xl
th nm
Grape + Zinc, Iron
1.2
Is-
::s
0.8
~
0
c
ro
0.6
-e0
w
.0
«
0.4
0.2
0
200
-«Xl
5CXl
700
VVa ... ",Jen th nm
Grape + Iron
o
N
0.8
5
:0w
0.6
0
C
<U
-f'
0
"'
n
0.4-
""
0.2
C>
~I
0200
z!o
240
I~
~I
I
280
340
Wavelenatn (nm
Grape + Ferrous Chloride
1.2
::J
~
~
c
-eg
0.8
0.6
.0
""
0.4·
0.2
200
I
I
I
i
i
I
I
I
220
240
280
280
200
320
340
36J
i
Waveieflgth (nml
Grape ... Ferrous Sulfate
0.8
::J
:0-
0.6
,
w
c
0
"
-f'
I~
~
0.4-
I
I,
I
L
0.2
0
200
~~I
I
I
i
I
I
I
I
I
220
240
280
280
200
320
340
36J
i
'--1
Wavelengtt, {nm)1
--
Grape + Iron
---
0.6
0.6
0.4
I
Grape + Ferrous Chloride
1.2
I
0.2
L °200'1-'~-~'~-~"r-~~-~~-,,-:==:==:==:==~,==:==:==:::~::::~,::=:~'~'~~~
3lJ
4XJ
5(X)
€(X)
700
WaveJenmh fnmj
I
I1GraPe-+FerrouS6u'fate
I
I
0.8
s-
0.6
11
0.4
"-
I~
\~~~~~~~-~
0.2
I,
I
I
\
0
-,-----.,------.------r---
2(0.
XO
~
4:XJ
..
::::::::='
;:=:=::=::=:;:="
&Xl
700
'~-:-"'--.j
Wavelt'!f1qth (nmj
1.4
1.2
S
::;
ID
0
0.8
c
m
"§
'"
0.6
.0
"
,
1'1
0.4
0.2
o
ro
0
200
:lOCI
400
WaveJen h nm
Sample/Result Table
------------------Abs (AU)
Peaks (run)
Abs (AU) Valleys (nm)
# Name
-------------------------------------------------------------------1 Quercetin Iml/lO
370.0
0.36836
566.0 -5.6458E-4
1
708.0
7.4768E-3
642.0 -4.5776E-5
774.0
4.5776E-5
1
794.0
7.2632E-3
3.0212E-3
2 Quercetin 2ml/l0
372.0
0.76578
642.0
794.0
1.4816E-2
586.0
3.2196E-3
2
2
4.2114E-3
758.0
1.4526E-2
632.0
3 Quercetin 3ml/l0
370.0
1.14010
642.0
1.6953E-2
3
712.0
3.2074E-2
566.0
1.9058E-2
3
792.0
3.1326E-2
***
***
370.0
1.0712E-2
4 Quercetin 4ml/l0
1. 52960
566.0
1.3702E-2
4
796.0
4.0359E-2
590.0
1.9775E-2
4
582.0
2.6688E-2
780.0
----------------
0.8
0_6
0.4
0.2
o
-.----~-~,~~
70J Wavelen th nm
Sample/Result Table
Name
Naringin 5ml/100 1
1
1
Naringin 10ml/l0 2
2
2
Naringin 15ml/10 3
3
3
Naringin 20ml/10 4
4
4
#
Name
Naringin 5ml/100
Naringin 10ml/10
Naringin 15ml/10
Naringin 20ml/10
Peaks(nrn)
328.0
712.0
792.0
330.0
708.0
696.0
330.0
708.0
794.0
332.0
708.0
758.0
Abs (AU)
0.30014
1.7395E-2
1.5106E-2
0.58672
2.7298E-2
2.5406E-2
0.88664
3.7003E-2
3.6972E-2
1.14900
3.7354E-2
3.5568E-2
-~-·---i
!
0.8
0.6
0.4
0.2
o
I
Ferric Chloride
1.4
0
1
I
1.2
OJ
~
w
u
c
ro
-"
is
w
-"
.
OB
0.6
0.4
0.2
co
<t
l&
0
200
Ferric
1.4
....
EDl
N~rate +
700
Wavelen th nm
CranbelTjl
1.2
OJ
~
w
c
0.8
u
.l'l
isw
.
0.6
-"
0.4
0.2
CO CO
CO CO
~I
0
200
!DJ
<0
N
I
I
EDl
700
<t
1'-.
';1 "'I
J
Wavelen h nm
dl.lerCetin 1m~100mf~>.a;g
---
Sample + Ferne Sulfate
o
I
~I.
I
~~
N
CD
--&
:g °1 .:&
I
~--='F'~- _ _~I
~-'--~~~-'I~---'----'··"'---~~~"--~~T'~'---,-~"~~---,,'-----'1 --~f"'
300
38)
4'X)
420
440
-
4W
iii
l
Quercetin 2m~100m1->.a;g
Sample + Ferric Sulfate
0.8
~..
0.7
0.6
S-
0.5
1;;-,
,
0.4
2
0
•
<t
0.3
.0
0.2
<t
"--1
Wavelength (nm 1
I
I
/
V
0.1
0
N'
-0.1
3X)
320
34)
380
300
Wavelen h nm
r~----n.;;=~=nn;;;,----::n~------------~--------
Quercetin 3m1/100m1->.a;g
Sample + Ferric Sulfate
..,o
s-
0.8
:'S
~
0
c
0.6
ro
-e0
•
.0
<t
--I
0.4
o:r
3X)
F
I
320
I
34)
I
300
I
380
,~,
--
I
I:]~"'''''~~''
l
. Sample + Ferric Sulfafe
1.6
1.4 -
1_
::l
II
6w
«
"'
.~
I
I
1.2
1
I
0.8
I
0.6
0.4
0.2
/J
0
300
340
320
380
440
Wavelen th nm
1.6
1.4
1.2
I
e"
0.8
I
I
"'«
0.6
I,
S
~
w
c
0
w
0.4
\
I
"
N
COo
CO
01
~11S!~1it
0.2
0
300
325
375
400
425
450
Wavelen th nm'
1--
Quercetin 1 mVl 00 ml->.CEg
Sample + Ferrous Sulfate
I
0.5
s
0.4
I
~
ID
0
c
ro
.c
I~
0.3
I
01
0.2
o
IlL
0.1
0
:nl
0.8
0.6
S
~
ID
0
c
ro
-ea
1'1
0.4
«
375
Wavelen h nm
425
Queicetin 3rnV100m1->.CEg
Sample + Ferrous Sulfate
1.2
S
~
ID
0
O.B
C
ro
.c
<;
0.6
~
.c
«
0.4
0.2
0
:nl
320
340
360
3BO
420
440
Wavele
h nm
1- -
NaringinsmIi100niJ~.1g·-­
Sample + Ferrous Sulfate
0.5
I~
0.4
0.3
II
0.2
I
0.1
I~
0
300
320
340
360
380
Waveren th nm
Naringin 1OmV1CDnl ~.1g
Sample + Ferrous Sulfate
~
0.8
0.7
S
0.6
~
0.5
u
c
ro
0.4
ru
€
..'"
0
.c
0.3
0.2
0.1 -
~
.
~'~i
i
0
300
320
Wavelenath InmJ
Naringin 15mV100m1-?1g
Sample + Ferrous Sulfate
I~
=>
0.8
~
ru
u
c
0.6
«'"
0.4
.ea
.c
CO
2~
~~ "'.
N
0.2
I
0
300
320
340
WavE!ten th nm
-
r
0.3
0.25~
0.2
v
ro
Ij
I
I
Sample + Feme Sulfate
0.35
u
c
.n
Narirlgin 5mUl00mI->.1 g~­
-
0.15·
'\,
0.1
C>
~L
0.05
LOm
l
Naringin IOmU10Jm1->.lg
Sample + Ferric Sulfate
0.6
0.5
S-
~
v
u
c
ro
"§
OA
I
;
0.3
~
.0
<{
0.2
C>
0.1
0
m
3BO
320
«Xl
420
440
460
Wavelen th nm
Naringin 15mUl(J()n1....>.1 9
Sample + Ferrjc Sulfate
0.9-
;:!;
0.8
0.7
0.6
0.5
0.4
0.3
0.2
~f= ~~ I~ ~.
o~~~~-~32O'1-~~~34J'"~~-~3BO-'1~~~:3BO~1=;:::::=~«Xl~':::::;~=:=42O~':::::;=::::::440::;=:=;::::::-;=~~:;:::·
~alvelenOth (nmJ
0.1
~4
I ;2
Sample ... Ferrous Sulfate
I
0.8
1lc
.e
0.6
1<
:;
~
t=1~-~ 320T'~ ~ 340- -"~ ~-30 "-~ ~-= 30 ~'= =:=4CXl~'
~
:>
------[
Naringin 2OmV100m1->.lg
=~ a~!,=el;:~= : : h=n;:m=
:::::::=:=;:420::;::'::::;:::::::=:=440::;::=::::;:::::::=::;=1::::::=Ii;:.
Naringin 2Om1l100m1-> 19
Sample + Ferric Sulfate
(\
0.8
,
"'~
0
c
ro
I
0.6-
-e0
~
.c
<t
0.4
0.2
I/f
0
;D)
,~ co
~~
320
340
I
440
Wavelenath Inm
1no"
Cranberries on the left and Grapes on the right, both baked in an oven at 100 degrees.
Cranberries on the left and Grapes on the right, both baked at 140 degrees.
Room Temperature with
Sunlight
Cranberries on the left and Grapes on the right, at room temperature in the presence of
sunlight.
Room Temperature
Cranberries on the left and Grapes on the right room lighting and temperature.