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DESCRIPTION:
Grape seed
proanthocyanidins refer to procyanidin mixtures extracted from grape
(Vitis vinifera) seeds. Procyanidins are derivatives of the
flavan-3-o1 class of flavonoids. This class includes (+)-catechin,
commonly referred to as catechin, and (-)-epicatechin, commonly
referred to as epicatechin. Procyanidins are dimers and oligomers of
catechin and epicatechin and their gallic acid esters. Procyanidins
are widely distributed in the plant kingdom and, in addition to
being found in grape seeds, are found in cocoa and chocolate,
apples, peanuts, almonds, cranberries, blueberries and in the bark
of pines, among other plant sources.
Grape seed
proanthocyanidins are mainly comprised of dimers, trimers and
tetramers of catechin and epicatechin and their gallates. They also
contain smaller amounts of pentamers, hexamers and heptamers of
these flavan-3-o1s and their gallates. The procyanidin dimers and
oligomers are also known as oligomeric procyanidins (OPCs) and
procyanidolic oligomers or PCOs. Grape seed proanthocyanidins
comprise approximately 60 to 70% of the polyphenol content of
grapes. The procyanidins are colorless in their pure state.
ABSTRACT:
To assess the safety of
grape seed extract with less than 5.5% catechin monomers (IH636), 4
groups of male and female Sprague-Dawley rats were provided grape
seed extract in the diet at levels of 0 (control), 0.5, 1.0, or
2.0%, for a period of 90 days. All animals survived the duration of
the study, and no significant changes in clinical signs,
hematological parameters, organ weights, ophthalmology evaluations
or histopathological findings were observed. A significant increase
in food consumption was observed in male and female rats provided
the grape seed extract diets compared to controls, especially in
male rats consuming 2.0% grape seed extract. This effect was not
accompanied by increases in body weight gains. Grape seed extract
appeared to increase the insoluble fraction of the diet. Male rats
in the high-dose group exhibited decreased serum iron levels and
decreased serum iron/total iron binding capacity ratio compared to
controls although all values were within historical limits for
Sprague-Dawley rats.
In conclusion,
administration of the grape seed extract IH636 to male and female
Sprague-Dawley rats in the feed at levels of 0.5%, 1.0%, or 2.0% for
90 days did not induce any toxicologically significant effects.
Active Component and Structure:
Proanthocyanidine is
the generic name for compounds that give rise to anthocyanidine when
decomposed with an acid. It comes in more than one structure,
depending on the type of catechins constituting it, condensing
positions, the three-dimensional arrangement of condensing
positions, and the number of polymerizations. Grape seed extract is
essentially a blend of various flavonoids.
Basic structure of
Proanthocyanidine
What is Grape Seed Extract Made of?
Molecular Structure of Grape Seed Extract:
Proanthocyanidins are
naturally occurring plant metabolites widely available in fruits,
vegetables, nuts, seeds, flowers, and bark. Other plant sources of
proanthocyanidins include wine, cranberries, and the leaves of
bilberry, birch, ginkgo, and hawthorne. Also known as procyanidins,
these substances are the main precursors of the blue-violet and red
pigments in plants.
At a symposium entitled
“Free Radicals in Biotechnology and Medicine” held in London in
1990, it was reported that esterification of (-)-epicatechin and
procyanidin B2 by gallic acid increases their free radical
scavenging ability. Information was also introduced revealing the
dimeric proanthocyanidins having the C4-C8 linkage have greater free
radical scavenging activity than the C4-C6 linkage, and that these
gallate esters are only found in the grape seed extract form.
Grape seed extract
contains OPCs made up of dimers or trimers of (+)-catechin and (-)-epicatechin.
The procyanidin dimers are comprised of procyanidins B1, B2, B3, B4,
B5, B6, B7, and B8. There are six procyanidin trimers which include
procyanidin C1 and C2. Furthermore, several gallolyl procyanidins,
which are most commonly the gallate esters of the dimeric
procyanidins, and some free gallic acid are present.
Tetramers or greater of
these flavonols would be known as polymeric proanthocyanidins and
the astringency of the molecule would increase accordingly.
Therefore, oligomeric proanthocyanidins are less astringent, bind
less strongly to proteins, and are more soluble and mobile in the
body.
What's It Made Of?
Vitamin E, flavonoids,
linoleic acid, and compounds called procyanidins (also known as
condensed tannins, pycnogenols, and oligomeric proanthocyanidins or
OPCs) are highly concentrated in grape seeds. These healthful
compounds can also be found in lower concentrations in the skin of
the grape. Procyanidins are also found in grape juice and wine, but
in lower concentrations.
Biological Properties of Grape Seed Extract:
The biological
properties of flavonoids, including proanthocyanidins, have been
extensively reviewed. In addition to their free radical scavenging
and antioxidant activity, proanthocyanidins have been reported to
have antibacterial, antiviral, anticarcinogenic, anti-inflammatory,
anti-allergic, and vasodilatory actions. Proanthocyanidins have also
been shown to inhibit lipid peroxidation, platelet aggregation,
capillary permeability and fragility, and to affect enzyme systems
including phospholipase A2, cyclooxygenase, and lipoxygenase.
The free radical
scavenging abilities of proanthocyanidins have been well documented
and command the most attention. In vivo studies have shown grape
seed proanthocyanidin extract is a better free radical scavenger and
inhibitor of oxidative tissue damage than vitamin C, vitamin E
succinate, vitamin C and vitamin E succinate combined, and beta
carotene. Moreover, in vitro experimental results have demonstrated
proanthocyanidins have specificity for the hydroxyl radicalin
addition to having the ability to non-competitively inhibit the
activity of xanthine oxidase, a major generator of free radicals,
elastase, collagenase, hyaluronidase, and beta-glucuronidase.
OPCs have also
demonstrated preferential binding to areas characterized by a high
content of glycosaminoglycans (epidermis, capillary wall,
gastrointestinal mucosa, etc.). This feature makes them useful for
decreasing vascular permeability and enhancing capillary strength,
vascular function, and peripheral circulation.
Methods of Analysis:
Grape seed extract is
a heterogeneous mixture of gallic acid, monomers, dimers, trimers,
tetramers, polymers and other oligomers. The basic building blocks
are molecules of catechin, epicatechin, epicatechin gallate, gallic
acid esters, glycosides and peptides. Due to the high degree of
heterogeneity, several analytical techniques are required to
characterize grape seed extract.
TLC (Thin Layer Chromatography)
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What is
measured:qualitative separation of phenolics, used to determine
if the extract is grape based
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Type of
method:chromatographic, based on size and polarity of phenolics
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Standard:gallic
acid, catechin, epicatechin and a commercially available grape
seed extract
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Issues:
qualitative only, can not distinguish between grape skin and
grape seed
lack of a non-partisan
"commercial" grape seed extract standard
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Grape Seed Method
Evaluation Committee has adopted this method "industry wide" to
qualitatively assess if an extract is grape based
This procedure was
proffered by ESA Laboratories and includes normal phase TLC
separation of preacetylated powder extracts
In 1998 three
independent laboratories (Alpha Chemical Laboratory, Industrial
Laboratory, and PhytoChem) validated the TLC method by testing 14
grape seed extracts, bilberry, green tea, pycnogenol and cranberry
extracts as well as 13 finished products. This is a qualitative test
designed to identify grape seed extract and differentiate it from
botanicals that may have similar components.
Physiological Functions:
Proanthocyanidine can
scavenge free oxygen radicals and inhibit lipid oxidation; in other
words, it has antioxidative properties. Grape seed extract is
considered effective in treating the diseases listed below.
INTRODUCTION:
Grape seed extract is a
natural extract from the seeds of Vitis vinifera. A multitude of
flavonoids are contained in grape seed extract. The most abundant of
these are the proanthocyanidins, which are oligomers of monomeric
flavan-3-ol units linked by carbon-carbon bonds (1-3). The major
flavan-3-ols identified in grape seed extract are (+)-catechin, (-)-epicatechin,
and (-)-epicatechin-3-O-gallate (3, 4) (Figure 1). The most basic
oligomeric proanthocyanidins are composed of flavan-3-ols units
linked together from the C4 of one unit to either the C6 or C8 of
the adjacent unit to form the B-type dimers and C-type trimers (1,
5, 6) (Figure 2). The further addition of flavan-3-ol units results
in the formation of larger proanthocyanidin oligomers and polymers.
Flavonoids and
flavan-3-ols are partially metabolized to lactones and phenolic
acids by the intestinal microflora (7, 8). These flavonoid and
flavan-3-ol metabolites are absorbed through the intestinal lumen
and are further metabolized by methylation, oxidation, or glucuronic
conjugation. Flavonoids and their metabolites are eliminated mainly
through urinary and fecal excretion and, to a certain extent, via
respired carbon dioxide (9-12).
In addition to being
present in the seeds of grapes, proanthocyanidins occur naturally in
black and green teas, chocolate, coffee, cacao, red wine, and many
fruits (7). A vast amount of literature has been published that
provides evidence that these flavonoids possess antioxidant
properties, free radical scavenging, and chelation abilities (6,
13-25). Flavonoids have been reported to exert anti-inflammatory
actions and to modulate immune function (26, 27). By reducing the
permeability and fragility of capillaries, they also have a
protective effect against vascular disorders (28). Flavonoids exert
a cholesterol-lowering effect by enhancing reverse cholesterol
transport and bile acid excretion, and by decreasing the intestinal
absorption of dietary cholesterol (29-31). The results of
epidemiological studies indicate an inverse relationship between
cancer and the consumption of flavonoid-containing foods, especially
fruits and green tea (32-35). The anti-carcinogenic properties of
flavonoids and proanthocyanidins in particular are associated with
cytotoxicity to cancer cells (36, 37) and their ability to enhance
the activity of enzymes that detoxify carcinogenic hydrocarbons by
oxidation (7, 35). Additional epidemiological studies on flavonoid
consumption indicate an inverse relationship between dietary intake
of flavonoids and coronary heart disease and stroke (38-41). By
acting as free radical scavengers proanthocyanidins inhibit lipid
peroxidation (22, 28, 42-45), a free-radical chain reaction that can
produce cytotoxicity, disrupt lipid-containing membranes, and
initiate low-density lipoprotein oxidation (46-49), a contributing
factor to the development of atherosclerosis (50, 51). Flavonoids
decrease the risk of cardiovascular disease by inhibiting platelet
aggregation and thrombosis (52-57), and by exerting a sparing effect
on other antioxidants, such as vitamins E and C (52, 58). By
reducing oxidative stress, proanthocyanidins from grape seed exert a
cardioprotective effect against ischemia reperfusion injury (59) and
also protect gastric mucosal (60) and glial cells (61) from
oxidative-stress induced injury.
Due to the increasing
interest in flavonoids as dietary supplements (taken in caplet form
with a typical daily dose being between 50 and 150mg) and a growing
understanding of their potential health benefits, the safety of
these substances must be established. The objective of the present
study was to assess the oral toxicity of a water extracted grape
seed extract with less than 5.5% catechin monomers following
administration to rats via dosed feed for a period of 90 days. At
the highest concentration of 2.0 w/w % IH636 in the chow, the rats
were consuming the extract at approximately 2g/Kg body weight/day or
10-20 times the average human intake of plant derived
proanthocyanidins.
What is grape seed extract?
Grape seed extract is a
naturally occurring plant substance that contains a concentrated
source of antioxidant nutrients known as oligomeric
proanthocyanidins. These antioxidants can help protect against the
effects of internal and external stresses. Scientists have
demonstrated that oligomeric proanthocyanidins, more commonly known
as OPCs, are more powerful antioxidants than vitamins C, E and
beta-carotene.
What is the history of grape seed extract?
Scientists first became
interested in grape seed extract in the latter part of the 20th
Century due to an outgrowth of research about the "French Paradox."
The French Paradox shows a low incidence of heart disease (nearly
half that of the United States) among the French despite a high
incidence of known dietary and other contributing factors to heart
disease. Some scientists believe that the secret of the French
Paradox is red wine, which is an integral part of the French diet.
Further research suggests that OPCs present in red wine can promote
cardiovascular health. Grape seeds have been found to be the richest
source of OPCs.
A potent antioxidant:
Original OPCs are
twenty times more powerful than vitamin C and fifty times more
powerful than vitamin E in their ability to scavenge free radicals.
OPCs are able to "donate" electrons to unstable free radicals and to
neutralize them without the danger of becoming unstable themselves.
Original OPCs in Beyond
Grape Seed protects cells and tissue by neutralizing free radicals
and blocking destructive enzymes. These OPCs penetrate both water
and fat cellular membranes. OPCs even have the capacity to cross the
blood-brain barrier. They maintain the structure and integrity of
the veins, arteries and capillaries, which ensures efficient
circulation.
Beyond Grape Seed helps strengthen the entire vascular system. OPCs
prevent capillary leakage in the legs, eyes and skin reducing fluid
retention. Without OPCs, destructive enzymes are free to attack the
structure of the blood vessels. Free radicals further degenerate
these vessels, causing lesions that become traps for bad cholesterol
(LDL). Once LDL cholesterol has been trapped it attracts calcium.
Cholesterol and calcium build up to form plaque, which narrows the
arteries and encourages blood clot formation.
MECHANISM OF ACTION:
Grape seed
proanthocyanidins have been found to have a number of antioxidant
activities in the laboratory. These include scavenging of hydroxyl
and peroxyl radicals, and inhibition of the oxidation of low-density
lipoprotein (LDL). The inhibitory potential related to lipid
peroxidation appears to increase with the degree of polymerization
of the molecules. That is, grape seed proanthocyanidins with a
greater number of catechin and epicatechin units appear to have more
potent inhibitory activity than those with fewer catechin and
epicatechin units. Further, the position of linkage between inter-flavan
units also appears to influence lipid peroxidation inhibitory
activity. Procyanidin isomers with a 4-6 inter-flavan linkage appear
to show stronger inhibitory activity than those with a 4-8 linkage.
Finally, the presence of a gallate group also appears to affect the
inhibitory activity of the procyanidins with respect to lipid
peroxidation. A procyanidin dimer with a gallate group linked at the
3-hydroxy position appears to show much greater inhibition of lipid
peroxidation than a dimer without such a group.
Grape seed
proanthocyanidins have shown anti-inflammatory, anticarcinogenic and
anti-atherogenic activities, again in the laboratory. These
activities are thought to be due, in large part, to the antioxidant
activities of these molecules. These proanthocyanidins have been
found to be cytotoxic for some human cancer lines in culture.
Upregulation of apoptosis by the proanthocyanidins in these cancer
lines is another possible mechanism for their possible
anticarcinogenic activity.
Diets and Test Materials:
Animals were fed
Ralston Purina Rodent Chow pre-ground to meal form, ad libitum.
Drinking water was also provided to the animals ad libitum. Grape
seed extract with less than 5.5% catechin monomers (IH636) was
obtained from Dry Creek Nutrition, Inc. Five lots of IH636 were
blended to form a composite batch. The composite batch was blended
with the rodent chow to provide test diets containing levels of 0
(control), 0.5, 1.0, and 2.0% of IH636 (Average dose levels are
presented in Table 1). A sixth lot was used separately at the end of
the study. All lots complied with the current chemical and
microbiological specifications for the product. The standard rodent
chow without the test material was provided to the control group.
All diets were frozen
at -21° to -19°C upon receipt. The diets were refrigerated at 4° to
6°C, protected from light after opening, and were maintained at room
temperature (18° to 25°C) 24 hours prior to feeding to the animals.
The presence of Grape Seed Extract in the chow was demonstrated
using the classical Folin-Ciocalteu and Porter visible spectroscopy
methodologies for determining the presence of polyphenols and
proanthocyanidins, respectively. Samples of 0 (control), 0.5, 1.0,
and 2.0% Grape Seed Extract in chow were first extracted with
methanol then subjected to the analyses. Greater absorbance at 760
nm (Folin-Ciocalteu) and 550 nm (Porter) for the samples with added
Grape Seed Extract than the control was indicative of the addition
of Grape Seed Extract to the chow.
Attainment of the
target concentrations and content uniformity of the Grape Seed
Extract in the different test diets were determined by absorbance
spectroscopy at 280 nm of methanol extracts of the chow. Standard
regression analyses were performed. Target concentration attainment
and content uniformity were defined as sample concentrations being
within +/- two methodology standard deviations of the mean
concentration at the three levels of IH636 in the different test
diets.
The stability of IH636
in the rodent chow was determined by absorbance spectroscopy at 280
nm and comparison to a "fingerprint" HPLC chromatogram. Standard
regression analyses were performed on the UV spectroscopy. Stability
was defined as sample concentrations being within +/- two
methodology standard deviations of the mean concentration at the
three levels of IH636 in the rodent chow and no new unidentified
chromatographic peaks at greater than 0.01 area percent appearing in
the "fingerprint" chromatogram. For the "fingerprint" HPLC analysis,
methanol extracts of the chow samples were dried. The dried samples
were reconstituted with 30% ethanol/70% water to make 1000 ppm
solutions. 25m l of the 1000 ppm solution were injected onto a
Zorbax SB-C18 4.6 x 150 mm, 5µ column maintained at 30 ºC. The
injected material was eluted at 0.5 ml/min with a mobile phase
gradient from 2.5% acetic acid/97.5% water to 2.5% acetic acid/17.5%
water/80% acetonitrile in 85 minutes. The eluting material was
monitored at 280 nm.
Grape Seed Extraction:
Grape Seed isolates
were prepared by batch extraction with 100% water from dried grape
seeds at up to 82 ºC for up to 40 minutes. These isolates were
purified by ultrafiltration and chromatography according to the
process of Nafisi-Movaghar et al. (64) to produce IH636.
Loss on drying (LOD)
and ash were determined on neat IH636 by AOAC methods 925.09 and
923.03, respectively. IH636 is a complicated mixture of chemical
classes. It was separated into an ethyl acetate and aqueous soluble
fractions to facilitate chemical analysis by the method of
Oszmianski and Sapis. The amino acid content of IH636 was then
determined by hydrolysis of an aliquot of the aqueous soluble
fraction followed by the methodology of Battaglia et al. The
oligomeric polyphenols (OPC) content of IH636 was determined from
the ethyl acetate soluble fraction by the methods of Vonk et al. and
Sun et al. The monomeric proanthocyanidins in IH636 were quantified
from the ethyl acetate soluble fraction according to the method of
Fuleki and daSilva. The phytosterol content was determined by the
method of Indyk, after a saponification and extraction of an aliquot
of the ethyl acetate soluble fraction. The fatty acid content was
quantified from the ethyl acetate soluble fraction by the method of
Mehta et al. The polysaccharide content of IH636 was determined from
the aqueous soluble fraction by the method of Lopez-Barajas et al..
RESULTS AND DISCUSSION
Grape
Seed Extract Characterization and Stability:
The chemical
composition of IH636 is shown in Table 2. It was possible to
complete a mass balance of IH636 with 100% accountability. The non-polyphenolic
components in IH636 are typical compounds found in plant material.
The target concentrations, content uniformity, and stability of the
Grape Seed Extract in the various chows were demonstrated to a 95%
confidence level. No losses of IH636 were incurred due to
instability under the storage conditions of –6 to –20 ºC for 94
days. No new unidentified compounds were detected in the IH636 in
rodent chow during the course of the stability study. All six
batches of IH636 used in the study complied with the Final Product
Specifications with respect to the overall proanthocyanidin and
monomer contents, heavy metal analysis and microbial contamination.
Figure 1.
Structures of the major flavan-3-ols identified in grape seed
extract.
Proanthocyanidin B-1 Dimer
Proanthocyanidin C-1 Trimmer
Figure 2.
Structures of proanthocyanidin oligomers. The oligomeric
proanthocyanidins are composed of flavan-3-ols units linked together
from the C4 of one unit to either the C6 or C8 of the adjacent unit.
Figure 3.
Body weights (g) for male Sprague-Dawley Rats
Figure 4.
Body weights (g) for female Sprague-Dawley Rats
Body weights (g) for female Sprague-Dawley Rats
Figure 5.
Cumulative food consumption (g) for male Sprague-Dawley Rats
Figure 6.
Cumulative food consumption (g) for female Sprague-Dawley Rats
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