|90-Day Oral Toxicity Study of a Grape Seed Extract (IH636) in Rats
Allison F. Wren(1), Michael Cleary(2) , Christopher Frantz(3),
Shawn Melton(3), and Leslie Norris(2)
(1) The Wren Group, 900 South Meadows Parkway, Suite 2013, Reno, NV 89511
(2) Aurthor to whom correspondence should be addressed: Dry Creek Nutrition, Inc.
600 Yosemite Blvd. Modesto, CA. 95353 [telephone (209) 341-5696; fax (209) 341-4541:
(3) SRI International, Toxicology Laboratory, 333 Ravenswood Ave., Menlo Park, CA.
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.
Keywords: grape seed extract; Vitis vinifera;
flavonoid; proanthocyanidin; polyphenols; oral toxicity; rats
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.
MATERIALS AND METHODS
One hundred and sixty male and female Sprague-Dawley rats (80 rats/sex) were obtained
from Charles River Laboratories (Raleigh, NC). The animals were quarantined and
acclimatized for 7 days prior to the initiation of treatment. During this acclimatization
period, the animals were fed Purina Certified Rodent Chow 5002 (pellets). Animals were
assessed for viability, and serological evaluation of bacterial and viral infections was
performed on 3 rats/sex.
The age of the animals at study initiation was approximately 9 to 10 weeks. The weights
of the animals were 218 to 280 g and 175 to 215 g for male and female rats, respectively.
General procedures for animal care and housing were in accordance with DHHS Publication
No. (NIH) 86-23 (Revised, 1985) and the U.S. Department of Agriculture through the Animal
Welfare Act (7 USC 2131) 1985, and Animal Welfare Standards incorporated in 9
CFR Part 3, 1991. Animals were housed under conventional conditions in suspended
polycarbonate cages in groups of 2 to 3 animals/cage with Sani-Chips hardwood bedding
(P.J. Murphy Forest Products, Montville, NJ). Temperature and relative humidity of the
animal rooms were maintained at 62° to 79°F and 23 to 56%, respectively. Room
temperature varied only 3°C from the accepted temperature range of 65-79°C. This
variation occurred twice during the study and for less than 4 hours on each occasion. Four
days before study initiation, the animals were randomly assigned to treatment groups using
a computerized body weight stratification procedure.
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 (62) and Porter
(63) 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 (65). 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. (66). The oligomeric polyphenols (OPC) content of IH636 was determined
from the ethyl acetate soluble fraction by the methods of Vonk et al. (67) and Sun et al.
(68). The monomeric proanthocyanidins in IH636 were quantified from the ethyl acetate
soluble fraction according to the method of Fuleki and daSilva (69). The phytosterol
content was determined by the method of Indyk (70), 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. (71). The
polysaccharide content of IH636 was determined from the aqueous soluble fraction by the
method of Lopez-Barajas et al. (72).
Testing was performed by SRI International (Menlo Park, CA) in compliance with Good
Laboratory Practice (GLP) regulations established by the U.S. Food and Drug Administration
(FDA) under Part 58 of Title 21 of the Code of Federal Regulations (CFR), Good Laboratory
Practice for Nonclinical Laboratory Studies. The diets were provided to 4 groups of rats
(20 rats/sex/group) containing IH636 at a level of 0 (controls), 0.5, 1.0, or 2.0% for a
period of 90 days. The viability of the animals was checked twice daily Monday through
Friday, and once daily on Saturdays, Sundays, and holidays. Clinical observations were
performed once daily on Study Days 1 through 15, and weekly thereafter. One animal
underwent an additional observation after sustaining a leg injury on Day 32. Body weights
were recorded on Day 1 of the study, once weekly thereafter, and immediately before
necropsy. Animals were fed the powdered diet in specially designed feed-troughs, which
minimize spillage and contamination from feces by allowing only the head into the feeder.
Food consumption was measured and recorded twice weekly by cage (2 or 3 animals per cage),
by recording the difference in feed weight. An average apparent daily feed consumption was
calculated for each rat. Food spillage was not measured since this was minimal and the use
of Sani-Chip bedding (standard for GLP toxicology studies) makes the monitoring
impractical. An ophthalmologic examination was performed on all animals once prior to
study initiation and once during the final week before necropsy (Day 85).
Blood samples for clinical pathology evaluation were obtained from the animals on Days
91 to 94 via the retro-orbital sinus under CO2/O2 anesthesia.
Hematology and serum chemistry analyses were performed for all animals, and serum iron
analysis was performed for the control and high-dose groups. Blood samples were analyzed
for the following hematological parameters: red blood cell (RBC) count, hematocrit (HCT),
hemoglobin (HGB), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration
(MCC), mean corpuscular hemoglobin (MCH), white blood cell (WBC) count, WBC differential
counts [including absolute banded neutrophils (ANB), segmented neutrophils (ANS),
lymphocyte (ALY), monocyte (AMO), eosinophil (AEO), and basophil (ABA)], platelet count
(PLC), and reticulocyte count (RET). Serum samples were analyzed for the following
clinical chemistry parameters: alanine aminotransferase (ALT), albumin (ALB), alkaline
phosphatase (ALP), aspartate aminotransferase (AST), total bilirubin (TBI), blood urea
nitrogen (BUN), calcium (CAL), chloride (CHL), cholesterol (CHO), creatinine (CRE),
globulin (GLO), glucose (GLU), phosphorus (PHO), potassium (POT), total protein (TPR),
sodium (SOD), iron (IRO), total iron binding capacity (TIBC), and iron/total iron binding
At the end of the study, during Days 91 to 94, all animals were killed with
approximately 100 mg sodium phenobarbital /kg body weight administered
All animals were subjected to gross necropsy, which included an external examination of
all body orifices and surfaces, and an examination of all cranial, thoracic, and abdominal
organs. Gross pathology findings were recorded. Samples of the following tissues were
removed and were fixed in phosphate-buffered 10% formalin: adrenal glands, aorta, urinary
bladder, bone and marrow (from sternum), brain, cecum, cervix, colon, duodenum, esophagus,
epididymides, eyes, femur, gross lesions (including tissue mass and abnormal regional
lymph nodes, if identified), heart, ileum (including Peyers patches), jejunum,
kidneys, lungs and bronchi, liver, lymph nodes (mesenteric), mammary gland (to include
nipple and surrounding tissue), ovaries, pancreas, pituitary, prostate, rectum, salivary
gland, sciatic nerve, skeletal muscle, skin (abdominal; taken with mammary gland); spinal
cord (mid-thoracic), spleen, stomach, testes, thymus, thyroid and
trachea, uterus, and vagina. Organ weights were recorded for all animals for the following
(paired organs were weighed together): prostate gland and seminal vesicles, adrenal
glands, brain, heart, kidneys, liver, ovaries, spleen, testes, uterus, and thymus. No
target organs were identified by gross pathological examination in animals of the
high-dose group and histopathological examination was therefore performed on animals in
the control and high-dose groups only.
Data on body weights, food consumption, clinical pathology, absolute organ weight, and
organ-weight ratios were evaluated by one-way ANOVA, followed by Dunnetts test to
compare the mean of each dose group with the control group. P< 0.05 was the probability
level used to determine statistical significance.
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
No unscheduled deaths occurred during the study (Table 3). On Day 32, one female rat in
the 0.5% IH636 group sustained a leg injury during the closing of the cage, and 1 male rat
in the control group exhibited opacity of the left eye commencing on Day 64.
Beginning in week 5 of the study, sporadic alopecia was observed in both male and
female rats in all groups (1 to 6 rats/sex/group) (Table 2). The alopecia in some male
rats was accompanied by eschar formation (1 to 3 rats out of 20 males/group); however,
there was no significant difference between the control group and IH636-treated groups.
This effect is thought to be related to fighting between the male animals. It is a common
occurrence with mid- to long-term housing of male rodents in group cages and is considered
not to be compound-related. Ophthalmologic examination revealed no compound-related
lesions in any of the animals. No other clinical observations were observed.
All groups gained weight during the study period. On Day 1 of the study, the mean body
weight of the male rats in the 1.0% IH636 group was significantly higher than in the
control group; however, this was considered to be due to a randomization artifact (Figure
3). Slightly lower mean body weights were recorded for female rats in the 1.0 and 2.0%
IH636 groups when compared to females in the control group (Figure 4). This effect was
statistically significant only in the subgroup necropsied on Day 91, and not in the groups
necropsied on Days 92 to 94. Due to the difference in body weights being only slight and
given the fact that the effect was statistically significant only in the Day 91necropsy
subgroup, this effect was considered to be of no biological significance.
Food consumption was generally higher in male rat groups fed IH636-containing diets
compared to the control group, possibly suggesting that rats preferred the flavor and
texture of the blend to conventional rodent chow (Figure 5). The estimated, increased food
consumption by male rats provided the 2.0% IH636 diets compared to males in the control
group was statistically significant as early as the Day 4 to 8 consumption measurement
period, and remained higher than the control group throughout the duration of the study.
The increase in apparent food consumption in male rats in the 0.5 and 1.0% IH636 groups
also was statistically significant; however, these increases occurred in a more random
pattern throughout the study. Sporadic, slight increases in food consumption also were
observed for IH636-treated female rats compared to the control group, and these increases
were statistically significant in only 9 out of 104 food consumption measurements (Figure
6). Increased food consumption by female rats provided the 2.0% IH636 diets compared to
the control group was statistically significant during the final 2 weeks of the study
The increases in estimated food consumption by the male and female rats
in the IH636 groups were not accompanied by increases in group body weight or in absolute
organ weights. In addition, no significant differences were observed in the serum levels
of albumin and globulin, or in total serum protein levels.
Ever since Kuhnau described plant flavonoids as
"semi-essential" components of the human diet (7), the role, if any, of these
dietary antioxidants in the maintenance of human health has been the focus of special
attention. Their metabolism appears to be highly dependent upon their degree of
polymerization (DP). Studies of chickens fed with 14 C-labelled sorghum tannins
(73) and sheep fed with 14 C-labelled Lotus pedunculatus (74) indicated
the proanthocyanidins were not absorbed. However, more recently, Déprez (75 ) compared
the in vitro absorption of radiolabelled dimer, trimer and oligomers (average degree of
polymerization was 7) and the related (+)-catechin through a cell monolayer derived from
the human intestinal cell line Caco-2. The monomer, dimer and trimer were all absorbed to
a similar extent but the polymers were not and partially adhered to the cell surface.
Further experiments by this group showed that the polymer was degraded by human colonic
micro flora grown in vitro and anaerobically. In an elegant study using 14
C-labelled proanthocyanidins from carob bean pods, Abia and Fry (76) have shown that 18h
after gavage 90-94% of the label was in the gut contents and/or feces. More than half of
this originated in condensed tannins with higher DP becoming insoluble, mainly in the form
of protein-tannin complexes.
Thus, there is a recent consensus that the monomeric, and possibly dimeric and trimeric
proanthocyanidins can be directly absorbed and that a small proportion of the higher DP
compounds are degraded by colonic bacteria prior to absorption. However the bulk of these
condensed tannins form stable complexes with both dietary and endogenous proteins and
fiber, resulting in increased fecal concentrations of these nutrients. It is this property
that has allowed these compounds to be classified as "anti-nutrients" by some
researchers. Just as there is some controversy over the metabolism of dietary
so there is evidence to support both beneficial and detrimental effects of these
carbohydrates on the nutrition of animals. This has been well reviewed by Reed (77) and is
summarised in Table 4. In short, the
proanthocyanidins interact with proteins, especially proline to form insoluble complexes,
as well as with other macromolecules and minerals in ingested food and those secreted by
the gastrointestinal tract. This adds up to the occasional finding that high levels of
procyanidins in feed results in increased consumption but no concomitant gain in body
weight gain as observed in the present study in rats. Ruiz-Roso and coworkers (78)
observed a similar phenomenon after feeding rats a diet containing 10% polyphenols
(natural carob fiber) or 10% cellulose. This has also been seen in other omnivores such as
pigs, after ingesting a diet with ~3.4% condensed tannins (79).
Interestingly, a similar finding occurs when rats are fed a cellulose-enhanced diet.
Freeman (80) reported increased consumption without weight gain during a 90-day study of
microcrystalline cellulose (Avicel®) administered at either 2.5% or 5.0% w/w in the diet.
The author noted that the NOEL exceeded 50,000mg/Kg diet since there was no evidence of
toxicity at the highest dose. In another study of a fibrous cellulose, Cellulon,
given at 5% and 10% of the diet for 13 weeks, increased food consumption occurred in all
the groups fed the fiber but there were no differences in body weight between fiber-fed
and control groups. This was attributed to the altered nutritional value of the diet.
Again, there was no evidence of treatment related effects (81).
An alternative explanation for the apparent toxicity of a high-tannin diet may lie in
the inhibition of post-digestive metabolism, or a systemic effect. In a study with rats,
Mole et al. (82) found that the toxic effects of dietary condensed tannins were due
to the impaired efficiency with which digested and absorbed nutrients were converted to
new body substance and did not involve inhibition of food consumption or digestion. Butler
and Rogler (83) also suggested possible systemic effects to include direct inhibition of a
key metabolic pathway and/or the diversion of metabolism into detoxification of
polyphenols or their degradation products.
Gross necropsy findings did not demonstrate any adverse effects in any organ. Organ
weights were examined as a % of body weight and as a % of brain weight. No statistically
significant differences in organ weights were present in any of the male and female rats
receiving the IH636 diets when compared to the control group. Histopathological evaluation
of the tissues from rats in the high-dose and control groups revealed a number of lesions,
the incidence of which was similar between the 2 groups (Tables 5 and 6). These types of
lesions are commonly associated with Sprague-Dawley rats of this age and are considered to
be related to spontaneous or iatrogenic causes. They were similar in severity in all
groups and were graded as either minimal or mild and are therefore not considered
Few significant differences in hematological and clinical chemistry values were
observed between GSE-fed groups and the control group (Tables 7 and 8). Male rats in the
1.0 and 2.0% IH636 groups exhibited slight, statistically significant increases in serum
sodium levels. Compared to the control group, slight, statistically significant decreases
in blood urea nitrogen and creatine levels of females provided the diet containing 2.0%
IH636 were observed. These serum chemistry changes were not accompanied by
histopathological findings and are considered not to be of toxicological significance.
Serum iron analysis was performed for the control and high-dose groups only (Table 9).
The levels of serum iron (IRO) were significantly decreased (14 to 17% lower) in male rats
in the 2.0% IH636 group compared to the control group. Consequently, the serum iron/total
iron binding capacity (ITC) ratio was significantly reduced. No significant changes were
observed for total iron binding capacity (TIBC). There were no significant differences in
clinical chemistry values for serum iron, total iron binding capacity, and iron/total iron
binding capacity between the females in the 2.0% IH636 group and females in the control
In the present rat study, the serum iron levels of male rats in the control and 2.0%
IH636 groups were 175 and 151 µg/dL, respectively. Historically, the levels of serum iron
in male Sprague-Dawley rats average 202 ± 49 µg/dL in rats 6 to 8 weeks of age and 152
± 70 µg/dL in rats 19 to 21 weeks of age (94). While the mean serum iron levels
of the rats in the control and 2.0% IH636 groups varied statistically, the levels of both
groups are within the range of these historical limits. No histological evidence
supporting any physiological changes from a pronounced decrease in serum iron was
observed. In addition, the results of the clinical hematology analyses showed no changes
in male rats in the IH636-fed groups in hematological parameters that would be expected to
be altered by a decrease in circulating iron, such as red blood cells, hemoglobin, and
hematocrit (Table 10).
Dietary iron absorption may be affected by the formation of insoluble iron complexes
within the intestinal lumen (95) or by altered intestinal permeability (96).
Various studies have been published that aim to determine the effects of flavonoids on
iron absorption in laboratory animals and humans (97-99). Red wine, but not white
wine, was reported to decrease non-heme iron absorption in humans, and, in the presence of
food (a bread roll, high in phytate), the inhibitory effect on iron absorption was doubled
(97). In human volunteers, tea consumption decreased iron absorption from solutions
of ferric chloride and ferrous sulphate, from bread, rice, and from uncooked, but not
cooked hemoglobin (98,99). These inhibitory effects were overcome by other nutrient
factors in the diet, such as ascorbic acid (100) and meat (96). In rats,
although iron absorption was decreased with the administration of iron in a tea solution,
substitution of drinking water with tea for a period of 3 days did not affect iron
Other published investigations have focused on the actions of the monomeric
constituents of flavonoids, such as (+)-catechin, (-)-epicatechin, (+)-gallocatechin, and
(-)-epigallocatechin. In a study designed to measure the inhibitory effect of different
polyphenol structures on iron absorption in humans, researchers at the University of
Goteburg provided a bread meal with a variety of phenolic acids, catechin, or tannic acid
with glucose (101). Tannic acid, gallic acid, and chlorogenic acid caused decreases
in iron absorption of 88, 50, and 30%, respectively, while catechin had no effect,
indicating that the content of iron-binding galloyl groups may determine the inhibitory
effect of flavonoids on dietary iron absorption. The weight % value for gallation of IH636
was identified using thiolysis followed by HPLC (results not shown). Thiolysis degrades
the oligomers to their respective monomer flavan-3-ols as well as providing the proportion
of galloylated units and the ratio of trihydroxylated to dihydroxylated units).
Approximately 10% w/w of IH636 is gallated; therefore, according to the hypothesis put
forth by Brune and coworkers (101), if iron complexation is dependent upon the
presence of gallated flavonoids, only 10% of ingested IH636 could contribute to inhibition
of dietary iron absorption.
While several studies provide some evidence for an inhibition of dietary iron
absorption by flavonoids through complexation in the intestinal lumen, no general
consensus has been reached as to whether this effect has physiological consequences. In
addition, the various nutrient factors in the diet that enhance or inhibit the absorption
of iron are confounding factors in determining the effect on iron absorption of one
particular dietary component. There is no evidence of iron deficiency in vegetarians,
whose diet usually contains exaggerated quantities of fruit. They are able to obtain on
average >90% of their daily iron intake (primarily as non-heme iron) from foods other
than meat. In a study designed to evaluate the responsiveness of serum and fecal ferritin
to differences in iron absorption, 21 women ate a non-vegetarian or an ovolactovegetarian
diet for 2 separate 8-week periods (102). While non-heme iron absorption in women
consuming the ovolactovegetarian was less than 50% of the absorption in women consuming
the meat diet, no effects were observed in hemoglobin, transferrin saturation, serum
ferritin, or erythrocyte protoporphyrin, indicating a possible physiological adaptation to
lowered iron absorption or intestinal responsiveness to iron bioavailability (103).
Overall, the ingestion of IH636 at dietary levels of up to 2% was well tolerated by
male and female Sprague-Dawley rats. Results from this study do not provide any evidence
of toxicity at 2% IH636 in the diet as demonstrated by the findings in clinical
observations, body weight and food consumption measurements, ophthalmoscopic examinations,
hematology, serum chemistry, organ weights, or histopathology. Although slight increases
in serum sodium levels in male rats in the 1.0 and 2.0% IH636 groups, and decreased blood
urea nitrogen and creatine levels in females provided 2.0% IH636 were observed, the serum
chemistry changes, in the absence of histopathological findings, were not considered to be
of toxicological significance. Similarly, although decreased levels of serum iron and the
serum iron/total iron binding capacity (14 to 17% lower) were observed in male rats in the
2.0% IH636, the serum iron levels were within range of the historical levels reported by
Loeb and Quimby (1989). In conclusion, at 2.0% in the diet, IH636 produced no significant
compound-related toxicity inSprague-Dawley rats.
Of greater importance to the consuming public is the effect that
ingesting normal levels of proanthocyanidins might have on the nutrient value of the diet.
In man, there are no toxicological studies per se but in a recent paper Dubnick and
Omaye (104) have reviewed more than 150 reports of the effect of wine, grape and tea
antioxidant polyphenols on atherosclerosis and ischemic heart disease. In the majority of
these studies, the doses of proanthocyanidins ingested were between 75mg and 300mg or
those present in 1-3 glasses of wine or a few cups of tea; in other words a small increase
above normal consumption. Not only did the authors conclude that the available data
indicate that wine and tea polyphenols possess biological activity that may modify risk
factors associated with cardiovascular disease, but also that the rare side effects have
only been observed after the ingestion of pharmacological doses. For example, some stomach
discomfort was noted in subjects taking ~1g/day epigallocatechin gallate supplements. This
is approximately the dose in >10 cups of green tea/day (105).
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Table 1. Experimental Dose Groups
Table 2. Mortality and Clinical Observations for Male and Female
Fed Diets Containing up to 2.0% IH636 for 90 Days
Table 3. Effects of
proanthocyanidins (condensed tannins) on the nutritive value of ingested food
antioxidant cytoprotective effects on the GI tract
||Halliwell et. al.
||McAllister et. al.
|Low to moderate
concentrations increase N retention by complexing protein at the pH of the lumen,
releasing it in lower stomachs of ruminants
||McNabb et. al. ( 86)
digestibility of amino acids (esp. glycine, proline and histidine), and proteins and
increases fecal N excretion
Cousins et. al (79)
Sheep: McNabb et. al. (87)
Humans: Bressani et.al. (88)
|Prevents bloat in
enzymes trypsin anda -amylase in rats by formation of
||Griffiths, D. W. (89)
of urea recycled to the rumen
||Waghorn et. al. (90)
||Reduces food intake
by lowering palatability due to astringency
and Duncan (91)
|Better performance in
high-producing dairy cows if moderate tannins in diet
||Marten and Ehle (92)
absorption in rat ileum
||Silverstein et. al.
Mean Terminal Body weights and Absolute Organ Weight
(g) of Male and Female
Sprague-Dawley Rats Fed Diets Containing up to 2.0% IH636 for 90 Days
Table 5. Terminal Histopathologic Observations for Male Sprague-Dawley Rats Fed
Containing 0 or 2.0% IH636 for 90 Days
Table 6. Terminal Histopathologic Observations
for Female Sprague-Dawley Rats Fed
Diets Containing 0 or 2.0% IH636 for 90 Days
Table 7. Terminal
Hematological Values for Sprague-Dawley Rats Fed Diets Containing
up to 2.0% IH636 for 90 Days
Table 8. Clinical Chemistry Values for
Sprague-Dawley Rats Fed Diets Containing up to 2.0% IH636 for 90 Days
Table 9. Terminal Serum Iron Chemistry Values for
Male and Female Sprague-Dawley
Rats Fed Diets Containing 0 or 2.0% IH636 for 90 Days
Table 10. Summary of the Results of the Serum Iron
and Clinical Hematology Analyses of Male
Sprague-Dawley Rats Fed Diets Containing up to 2.0% IH636 for 90 Days
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
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
Figure 6. Cumulative food consumption (g) for female