Research Section
Safety evaluation of proanthocyanidin-rich extract from grape seeds

https://doi.org/10.1016/S0278-6915(02)00006-6Get rights and content

Abstract

Proanthocyanidins, extracted from grape seeds, are widely used mainly as nutritional supplements. However, there has not been a systematic report to investigate toxicological studies on proanthocyanidins, especially in oral administration. In our studies, proanthocyanidin-rich extract from grape seeds was subjected to a series of toxicological tests to document its safety for use in various foods. The grape seed extract (GSE) was examined for acute and subchronic oral toxicity using Fischer 344 rats and for mutagenic potential by the reverse mutation test using Salmonella typhimurium, the chromosomal aberration test using CHL cells, and the micronucleus test using ddY mice. No evidence of acute oral toxicity at dosages of 2 and 4 g/kg, and no evidence of mutagenicity in the above tests was found. Administration of GSE as a dietary admixture at levels of 0.02, 0.2 and 2% (w/w) to the rats for 90 days did not induce noticeable signs of toxicity. The no-observed-adverse-effect level (NOAEL) of GSE in the subchronic toxicity study was 2% in the diet (equal to 1410 mg/kg body weight/day in males and 1501 mg/kg body weight/day in females). The results of our studies indicate a lack of toxicity and support the use of proanthocyanidin-rich extract from grape seeds for various foods.

Introduction

Proanthocyanidins are naturally occurring compounds widely available in fruits, vegetables, nuts, seeds, flowers and bark. They are a class of phenolic compounds which take the form of oligomers or polymers of polyhydroxy flavan-3-ol units, such as (+)-catechin and (−)-epicatechin (Porter, 1986). Grape seeds are a particularly rich source of proanthocyanidins, and only the procyanidin-type of proanthocyanidins have been detected in the seeds (Fuleki, & Ricardo da Silva, 1997, Santos-Buelga et al., 1995). A few monomeric flavanols have been also detected, but other flavonoid compounds such as anthocyanins and flavonols are not contain in the seeds (Waterhouse and Walzem, 1998). Prieur et al. (1994) found that 55% of the procyanidin extracted from grape seeds consisted of more than five monomer units and determined that their mean degree of polymerization ranged from 2.3 to 15.1 (by thiolysis) and from 2.4 to 16.7 (by gel permeation chromatography). Thus, the proanthocyanidins from grape seeds contain procyanidin oligomers and polymers.

A partially purified proanthocyanidin, consisting oligomers with polymerization from 2 to 7, has been used as a pharmaceutical for their purported activity in decreasing the fragility and permeability of the peripheral vasculature in Europe (Dartenuc, Marache, & Choussat, 1980, Brasseur, 1989, Bombardelli and Morazzoni, 1995).

Recently, epidemiological data have shown that red wine may reduce the mortality rate from coronary heart disease, the so-called “French paradox” (St. Leger, Moore, & Cochrane, 1979, Renaud, bha De Lorgeril, 1992). Proanthocyanidins are the major polyphenols in red wine as well as in grape seeds, and they have potent antioxidant activity (Ariga, & Hamano, 1990, Ricardo da Silva, Fernandez, & Mitjavila, 1991), inhibit low density lipoprotein oxidation (Teissedre et al., 1996), as well as a variety of biological activities (Arii, Miki, Hosoyama, Ariga, Yamaji, & Kataoka, 1998, Dauer, Metzner, & Schimmer, 1998, Saito, Hosoyama, Ariga, Kataoka, & Yamaji, 1998, Yamakoshi, Kataoka, Koga, & Ariga, 1999, Zhao, Wang, Chen, & Agarwal, 1999). For these reasons, proanthocyanidin-rich extracts from grape seeds have appeared on the market as nutritional supplements mainly in the United States, Australia, Japan, Korea, as well as in other countries. The grape seed extract is also being used in Japan as an additive for various food applications.

However, there has not been a systematic report to investigate toxicological studies on proanthocyanidins, especially in oral administration. Bombardelli and Morazzoni (1995) reported a summary on the safety of the proanthocyanidin oligomers, described above, in which they stated that the acute oral LD50 values in rats and mice were approximately 4 g/kg, and that when were administered orally at a dose of 60 mg/kg daily for 6 months in rats and for 12 months in dogs, they were devoid of any toxic effects. The proanthocyanidins were also devoid of any mutagenic potential as well as of teratogenic effects. Although an extensive testing program had been conducted to confirm the safety of the proanthocyanidin oligomers, the data have not been published and the details are unknown.

On the other hand, mutagenicity tests of some procyanidins have been performed. Several procyanidins with different degrees of polymerization (dimers, trimers and polymers) have been found to be non-mutagenic in the Salmonella mutagenesis assay system (Yu and Swaminathan, 1987). In chromosomal aberration tests, polyploidy was induced by the procyanidin trimer and tetramer such as procyanidin C1 and procyanidin D, respectively, in human lymphocyte cultures, and it was induced by the procyanidin dimer such as procyanidin B2 in a mammalian cell line (CHL cells). However no structural aberrations were induced by these procyanidins (Popp, & Schimmer, 1991, Takahashi, Yokoo, Inoue, & Ishii, 1999).

This paper describes studies conducted to confirm the safety of proanthocyanidin-rich extract from grape seeds, especially in oral administration. The proanthocyanidin-rich extract from grape seeds for acute and subchronic oral toxicity as well as mutagenic potential were studied. Furthermore, procyanidin dimers, trimers and tetramers which were separated from the grape seed extract, for mutagenic potential by chromosomal aberration test using CHL cells were examined. The results of these studies can be used to assess the safety of proanthocyanidin-rich extracts from grape seeds when used in various foods.

Section snippets

Proanthocyanidin-rich extract from grape seeds

Proanthocyanidin-rich extract (Gravinol Super™, Kikkoman Co., Japan) was prepared from grape seeds (Vitis vinifera L.). Briefly, the grape seeds were washed with water for 2 h and then extracted with water and ethanol under reflux for 2 h. The extract was condensed to remove solvents, then the concentrate was filtrated through cellulose powder and Celite®. The filtrate was spray-dried to obtain powder of proanthocyanidin-rich extract. Total flavanols in the grape seed extract (GSE) were

Oral acute toxicity study in rats

No deaths occurred in either the control or the GSE groups. The general condition of all rats was also normal. The weight of the male and female rats in the high-dose 4 g/kg group decreased slightly on day 1 and from days 1 to 3, respectively, but increased thereafter almost the same as in the control rats. No abnormalities were found in all rats at necropsy on day 14.

Subchronic 90-day oral toxicity study in rats

GSE was administered to rats as a dietary admixture at levels of 0.02% (low-dose group), 0.2% (middle-dose group) and 2%

Discussion

Proanthocyanidin-rich extracts from grape seeds are used as nutritional supplements in the United States, Australia, Japan, Korea and other countries, and GSE is also used in Japan as a food additive.

In the past, proanthocyanidins were considered to be non-toxic because they are not absorbed. However, dimeric procyanidins have been found to be absorbed into the bloodstream (Harmand, & Blanquet, 1978, Laparra, Michaud, Lesca, Blanquet, & Masquelier, 1978, Jimenez-Ramsey, Rogler, Housley, Butler,

Acknowledgements

We wish to thank Dr Shigetaka Ishii, Dr Nobuyuki Yamaji and Dr Shoichi Tokutake, Mrs Katsuko Matsuura, Research and Development Division, Kikkoman Corporation, for their helpful discussion and suggestions.

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