___________________________

(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:
email info@drycreeknutrition.com].

(3) SRI International, Toxicology Laboratory, 333 Ravenswood Ave., Menlo Park, CA. 94025

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.

  Keywords: grape seed extract; Vitis vinifera; flavonoid; proanthocyanidin; polyphenols; oral toxicity; rats

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.

MATERIALS AND METHODS

Animals

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.

Chemical Analyses

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).

Experimental Design

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 capacity (ITC).

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 intraperitoneally. 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 Peyer’s 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 parathyroids, tongue, 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.

Statistical Analysis

Data on body weights, food consumption, clinical pathology, absolute organ weight, and organ-weight ratios were evaluated by one-way ANOVA, followed by Dunnett’s 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 microbial contamination.

Clinical Observations

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.

Weight Gains

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

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 period.

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.

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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TABLES

Table 4. 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 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

_________________________________________________________________________________________________________________

FIGURES

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