(Poly)phenol toxicity in vivo following oral administration: A targeted narrative review of (poly)phenols from green tea, grape, and anthocyanin-rich extracts.

Fruit- and vegetable-derived (poly)phenols are secondary plant metabolites that may have beneficial effects on human health when consumed regularly. Recent years have seen rapid growth in both consumer demand for and research interest in (poly)phenol-rich dietary supplements, natural colorants, and functional foods. As these products continue to enter the marketplace and (poly)phenol intake patterns change from traditional food products to these sources, attention must be paid to the potential for toxicity from consuming elevated doses of (poly)phenols. To date, much remains unknown regarding the safety of high doses of (poly)phenols, especially in vivo. In this targeted narrative review, we summarize evidence from in vivo investigations of (poly)phenol toxicity after oral administration of green tea extracts, grape-derived phenolics, and anthocyanin-rich extracts. There is limited evidence of overt toxicity from oral ingestion of these (poly)phenol-rich sources, though more research on the safety of high doses-as well as defining what constitutes a "high" dose of both individual and complex mixtures of (poly)phenols-is needed before these observations can be used to create dietary guidance for consumers.

Investigation of acute, subacute and subchronic toxicities of anthocyanin derived acylation reaction products and evaluation of their antioxidant activities in vitro.

Previously, anthocyanins were successfully acylated with lauric acid using Novozym 435 lipase, and the corresponding products were confirmed to have higher stability. As novel synthetic compounds, their toxicological safety has not been evaluated. Therefore, acute, subacute and subchronic toxicities of anthocyanin-lauric acid derivatives (ALDs) were investigated while their antioxidant activities were also evaluated in vitro. The acute toxicity results showed that the 50% lethal dose (LD50) of ALDs in mice was >10 g kg-1. Subsequently, the subacute toxicity test was conducted by oral administration of ALDs at doses of 0.63, 1.25 and 2.50 g kg-1 for 28 days. No adverse effect of ALDs on body weight, food/water intake, organ coefficient and histology was observed. Though there were some fluctuations in AST and ALT, the tested biochemical parameters were maintained within the normal ranges. The subchronic toxicity test results demonstrated that less than 0.60 g of ALDs per kg BW intake did not affect mortality, body weight, food/water intake, gross pathology, histology, hematology and serum biochemistry. Furthermore, cyanidin-3-(6''-dodecanoyl)-glucoside, the main component of ALDs, had a beneficial reducing power and a strong DPPH˙, ABTS+˙, and O2-˙ scavenging activity. This study provides an imperative reference to the safety of ALDs, suggesting their application as novel colorants or antioxidants in food and therapeutics.

Smart wound dressing based on κ-carrageenan/locust bean gum/cranberry extract for monitoring bacterial infections.

A smart wound dressing based on carrageenan (κC), locust bean gum (LBG), and cranberry extract (CB) for monitoring bacterial wound infections was developed and characterized using UV-vis spectroscopy, FT-IR, and SEM. The mechanical, swelling, cytotoxic and pH sensor properties were also investigated. UV-vis spectra demonstrated that the obtained κC:LBG:CB hydrogel film exhibited a visible change of colors as it was immersed in PBS solution pH 5.0, 7.3 and 9.0. The spectra of FT-IR suggested that chemical interactions had occurred between κC and CB extract. The obtained κC:LBG:CB hydrogel film exhibited adequate mechanical properties and a swelling behavior dependent on pH. Cytotoxicity tests indicated that κC:LBG:CB hydrogel film had dose-dependent cytotoxicity against NIH 3T3 fibroblast cells. The in vitro studies using Staphylococcus aureus and Pseudomonas aeruginosa demonstrated that the color changes of the κC:LBG:CB hydrogel film could be observed by naked eyes, confirming the potential use of the obtained hydrogel film as a visual system for monitoring bacterial wound infections.

Is cytotoxicity a determinant of the different in vitro and in vivo effects of bioactives?

BACKGROUND: Foodstuffs of both plant and animal origin contain a wide range of bioactive compounds. Although human intervention studies are mandatory to assess the health effects of bioactives, the in vitro approach is often used to select the most promising molecules to be studied in vivo. To avoid misleading results, concentration and chemical form, exposure time, and potential cytotoxicity of the tested bioactives should be carefully set prior to any other experiments. METHODS: In this study the possible cytotoxicity of different bioactives (docosahexaenoic acid, propionate, cyanidin-3-O-glucoside, protocatechuic acid), was investigated in HepG2 cells using different methods. Bioactives were supplemented to cells at different concentrations within the physiological range in human blood, alone or in combination, considering two different exposure times. RESULTS: Reported data clearly evidence that in vitro cytotoxicity is tightly related to the exposure time, and it varies among bioactives, which could exert a cytotoxic effect even at a concentration within the in vivo physiological blood concentration range. Furthermore, co-supplementation of different bioactives can increase the cytotoxic effect. CONCLUSIONS: Our results underline the importance of in vitro cytotoxicity screening that should be considered mandatory before performing studies aimed to evaluate the effect of bioactives on other cellular parameters. Although this study is far from the demonstration of a toxic effect of the tested bioactives when administered to humans, it represents a starting point for future research aimed at verifying the existence of a potential hazard due to the wide use of high doses of multiple bioactives.

2, 2'- and 4, 4'-Cyanines are transporter-independent in vitro dopaminergic toxins with the specificity and mechanism of toxicity similar to MPP⁺.

Specific uptake through dopamine transporter followed by the inhibition of the mitochondrial complex-I have been accepted as the cause of the specific dopaminergic toxicity of 1-methyl-4-phenylpyridinium (MPP(+) ). However, MPP(+) is taken up into many cell types through other transporters, suggesting that, in addition to the efficient uptake, intrinsic vulnerability of dopaminergic cells may also contribute to their high sensitivity to MPP(+) and similar toxins. To test this possibility, two simple cyanines were employed in a comparative study based on their unique characteristics and structural similarity to MPP(+) . Here, we show that they freely accumulate in dopaminergic (MN9D and SH-SY5Y) as well as in liver (HepG2) cells, but are specifically and highly toxic to dopaminergic cells with IC50s in the range of 50-100 nM, demonstrating that they are about 1000-fold more toxic than MPP(+) under similar experimental conditions. They cause mitochondrial depolarization non-specifically, but increase the reactive oxygen species specifically in dopaminergic cells leading to the apoptotic cell death parallel to MPP(+) . These and other findings suggest that the specific dopaminergic toxicity of these cyanines is due to the inherent vulnerability of dopaminergic cells toward mitochondrial toxins that lead to the excessive production of reactive oxygen species. Therefore, the specific dopaminergic toxicity of MPP(+) must also be, at least partly, due to the specific vulnerability of dopaminergic neurons. Thus, these cyanines could be stronger in vivo dopaminergic toxins than MPP(+) and their in vivo toxicities must be evaluated. Here, we show that cationic lipophilic cyanines with structural similarity to 1-methyl-4-phenylpyridinium (MPP(+) ) freely accumulate non-specifically, but only toxic to dopaminergic cells. They are 1000-fold more toxic than MPP(+) under similar conditions. They cause mitochondrial depolarization non-specifically, but increase the ROS specifically in dopaminergic cells leading to the apoptotic cell death parallel to MPP(+) . Thus, the specific dopaminergic toxicity of MPP(+) and related toxins could be due to the intrinsic vulnerability of dopaminergic cells toward mitochondrial oxidative stress.

Anthocyanins potentiate the activity of trastuzumab in human epidermal growth factor receptor 2-positive breast cancer cells in vitro and in vivo.

Human epidermal growth factor receptor 2 (HER2) has been found to be overexpressed in ~25% of invasive breast cancer and is significantly associated with a poor prognosis in breast cancer patients. The anthocyanins cyanidin-3-glucoside (C3G) and peonidin-3-glucoside have been identified as potential drugs for the therapy of HER2­positive breast cancer. They have been used as supplements in targeted therapeutics and chemotherapeutics in Asia, however, the underlying mechanism remains to be elucidated. The aim of the present study was to investigate the synergism between C3G and trastuzumab (Trast). To address this question, the response to C3G, Trast and a combination of the two drugs, in three representative HER2­positive cell lines was evaluated. The combination treatments induced apoptosis, inhibited cell growth and affected HER2 and its downstream signaling pathway in MDA­MB­453, BT474 and HCC1569 cells, and the effects were synergistic. The combination of 3CG and Trast inhibited tumor growth in an in vivo xenograft model. The data from the present study suggested that C3G exhibits potent antitumor activity when combined with Trast under the investigated conditions.

Effects of anthocyanins on the AhR-CYP1A1 signaling pathway in human hepatocytes and human cancer cell lines.

Anthocyanins are plant pigments occurring in flowers and berry fruits. Since a phenomenon of food-drug interactions is increasingly emerging, we examined the effects of 21 major anthocyanins and the extracts from 3 food supplements containing anthocyanins on the aryl hydrocarbon receptor (AhR)-cytochrome P450 CYP1A1 signaling pathway in human hepatocytes and human hepatic HepG2 and intestinal LS174T cancer cells. Pelargonidin-3-O-rutinoside (PEL-2) and cyanidin-3,5-O-diglucoside (CYA-3) dose-dependently activated AhR, as revealed by gene reporter assay. PEL-2 and CYA-3 induced CYP1A1 mRNA but not protein in HepG2 and LS174T cells. Neither compounds induced CYP1A1 mRNA and protein in four different primary human hepatocytes cultures. The effects of PEL-2 and CYA-3 on AhR occurred by ligand-dependent and ligand-independent mechanisms, respectively, as demonstrated by ligand binding assay. In a direct enzyme inhibition assay, none of the antocyanins tested inhibited the CYP1A1 marker activity to less than 50% even at 100 µM concentration. PEL-2 and CYA-3 at 100 µM inhibited CYP1A1 to 79% and 65%, respectively. In conclusion, with exception of PEL-2 and CYA-3, there were no effects of 19 major anthocyanins and 3 food supplements containing anthocyanins on AhR-CYP1A1 signaling, implying zero potential of these compounds for food-drug interactions with respect to AhR-CYP1A1 pathway.

Acute cardioprotective and cardiotoxic effects of bilberry anthocyanins in ischemia-reperfusion injury: beyond concentration-dependent antioxidant activity.

Despite being reported to reduce the risk of cardiovascular diseases, little is known about acute direct effects of bilberry anthocyanins on whole mammalian heart under ischemia-reperfusion (I-R) conditions. Bilberry anthocyanins were prepared from the ripe bilberries and analyzed using HPLC-DAD. Their antioxidant activity was evaluated by measuring the intrinsic free radical-scavenging capacity and by cellular antioxidant assay (CAA) on endothelial cells, where we quantified the intracellular capacity to inhibit the formation of peroxyl radicals. Experiments on the isolated rat hearts under I-R were carried out according to the Langendorff method. Perfusion with low concentrations of bilberry anthocyanins (0.01-1 mg/L) significantly attenuated the extent of I-R injury as evidenced by decreasing the release rate of LDH, increasing the postischemic coronary flow, and by decreasing the incidence and duration of reperfusion arrhythmias. High concentrations (5-50 mg/L) diminished cardioprotection and show cardiotoxic activity despite having their radical scavenging and intracellular antioxidant capabilities increased in a concentration-dependent manner. This study reveals the biphasic concentration-dependent bioactivity of bilberry anthocyanins under I-R, which results in strong cardioprotective activity in low concentrations and cardiotoxic activity in high concentrations.

Enhancement of HIV-1 Tat fusion protein transduction efficiency by bog blueberry anthocyanins.

Though protein transduction domains (PTDs) are well known for the delivery of exogenous therapeutic proteins into living cells, the overall low efficiency of transduction is a serious obstacle. We investigated the effect of bog blueberry anthocyanins (BBA) on protein transduction efficiency and found that BBA enhanced the transduction efficiencies of Tat-SOD fusion protein into HeLa cells and mice skin. The enzymatic activities in the cells and skin tissue in the presence of BBA were markedly increased compared to controls. Further, BBA did not demonstrate any cell toxicity at various concentrations. Although the mechanism is not fully understood, we suggest that BBA might alter the conformation of the membrane, which would indicate that BBA can be used as a protein transduction enhancer for the efficient delivery of therapeutic proteins for a variety of disorders.

The anthocyanidin delphinidin mobilizes endogenous copper ions from human lymphocytes leading to oxidative degradation of cellular DNA.

Epidemiological and experimental evidence exists to suggest that pomegranate and its juice possess chemopreventive and anticancer properties. The anthocyanidin delphinidin is a major polyphenol present in pomegranates and has been shown to be responsible for these effects. Plant polyphenols are recognized as naturally occurring antioxidants but also catalyze oxidative DNA degradation of cellular DNA either alone or in the presence of transition metal ions such as copper. In this paper we show that similar to various other classes of polyphenols, delphinidin is also capable of causing oxidative degradation of cellular DNA. Lymphocytes were exposed to various concentrations of delphinidin (10, 20, 50 microM) for 1h and the DNA breakage was assessed using single cell alkaline gel electrophoresis (Comet assay). Inhibition of DNA breakage by several scavengers of reactive oxygen species (ROS) indicated that it is caused by the formation of ROS. Incubation of lymphocytes with neocuproine (a cell membrane permeable Cu(I) chelator) inhibited DNA degradation in intact lymphocytes in a dose dependent manner. Bathocuproine, which is unable to permeate through the cell membrane, did not cause such inhibition. We have further shown that delphinidin is able to degrade DNA in cell nuclei and that such DNA degradation is also inhibited by neocuproine suggesting that nuclear copper is mobilized in this reaction. These results indicate that the generation of ROS possibly occurs through mobilization of endogenous copper ions. The results are in support of our hypothesis that the prooxidant activity of plant polyphenols may be an important mechanism for their anticancer properties.

A 90-day oral toxicity study of purple corn color, a natural food colorant, in F344 rats.

A subchronic oral toxicity study of purple corn color (PCC), a natural food colorant, was performed with groups of 10 male and 10 female F344 rats fed the agent at dietary levels of 0%, 0.5%, 1.5% and 5.0% for 90 days. No mortalities occurred during the treatment period. No treatment-related changes in the body weight, food and water consumption, ophthalmology, hematology, organ weight data and histopathology were observed. Regarding general conditions and gross pathology, staining of fur and black feces were noted in rats of the 1.5% and 5.0% diet groups. Moreover, brown urine and black material in the stomach, small and large intestine were evident in rats receiving 5.0%. These changes were considered due to the anthocyanin content. On clinical chemistry analysis, total cholesterol, phospholipid and triglyceride were significantly lowered in both sexes of the 5.0% group, but these were not considered to be toxicologically significant. Thus, the No-observed-adverse-effect-level (NOAEL) was judged to be 5.0% in diet for both sexes (male: 3542 mg/kg/day, female: 3849 mg/kg/day) for PCC under the present experimental conditions.

Differential response related to genotoxicity between eggplant (Solanum melanogena) skin aqueous extract and its main purified anthocyanin (delphinidin) in vivo.

Anthocyanins are the largest group of water-soluble pigments in the plant kingdom. A number of studies have demonstrated that anthocyanins present antioxidant capacity and show inhibitory effects on the growth of some cancer cells. Thus, the goal of this study was to evaluate both the antimutagenicity/antigenotoxicity and mutagenicity/genotoxicity of aqueous extract obtained from the Solanum melanogena, a possible novel source of anthocyanin, and its main purified anthocyanin extract (delphinidin), using the single cell (comet) assay and micronucleus test. Pretreatment with higher doses of the purified anthocyanin (10 and 20mg/kg b.w.) led to a statistically significant reduction (p<0.05) in the frequency of micronuclei in polychromatic erythrocytes induced by cyclophosphamide. The pattern of reduction ranged from 48% to 57% independent of concentration. No apparent genotoxicity and mutagenicity was found for either the anthocyanin or delphinidin extracts. Taken together, these results suggest that mice pre-treated with specific compounds present in anthocyanins (delphinidin) displayed a lower incidence of mutations induced by cyclophosphamide. This finding emphasizes the potential of natural colorants to prevent mutations and also the applicability of genotoxic evaluation for improving health. Furthermore, the results presented here could be an additional argument to support the use of anthocyanins in the diet.

Safety and whole-body antioxidant potential of a novel anthocyanin-rich formulation of edible berries.

Edible berry extracts rich in anthocyanins possess a broad spectrum of therapeutic, pharmacologic and anti-carcinogenic properties. Six berry extracts (wild blueberry, bilberry, cranberry, elderberry, raspberry seeds and strawberry), singly and in combination, were studied in our laboratories for antioxidant efficacy, cytotoxic potential, cellular uptake and anti-angiogenic properties. Combinations of edible berry extracts were evaluated to develop a synergistic formula, OptiBerry, which exhibited high oxygen radical absorbance capacity (ORAC) value, low cytotoxicity and superior anti-angiogenic properties compared to the other combinations tested. The current study sought to determine the broad spectrum safety and antioxidant potential of OptiBerry in vivo. Acute oral LD(50) of OptiBerry was greater than 5 g/kg in rats. Acute dermal LD(50) of OptiBerry was greater than 2 g/kg. No changes in the body weight or adverse effects were observed following necropsy. Primary skin and eye irritation studies were conducted in New Zealand albino rabbits. OptiBerry was classified as slightly irritating to the skin (primary skin irritation index 0.3) and minimally irritating to the eye (maximum mean total score 6.0). The antioxidant potential of OptiBerry was investigated in rats and mice by assessing GSH redox status in tissues as well as by a unique state-of-the-art electron paramagnetic resonance (EPR) imaging of whole-body redox status. A clinically relevant hyperbaric oxygen (HBO) exposure system (2 atm, 2 h) was employed to study the antioxidant properties of OptiBerry. OptiBerry feeding (8 weeks) significantly prevented HBO-induced GSH oxidation in the lung and liver of vitamin E-deficient Sprague Dawley rats. Furthermore, OptiBerry-fed mice, when exposed to HBO, demonstrated significant protection in whole-body HBO-induced oxidation compared to the unfed controls by EPR imaging. Taken together, these results indicate that OptiBerry is reasonably safe and possess antioxidant properties.

Anthocyanidins modulate the activity of human DNA topoisomerases I and II and affect cellular DNA integrity.

In the present study, we investigated the effect of anthocyanidins on human topoisomerases I and II and its relevance for DNA integrity within human cells. Anthocyanidins bearing vicinal hydroxy groups at the B-ring (delphinidin, DEL; cyanidin, CY) were found to potently inhibit the catalytic activity of human topoisomerases I and II, without discriminating between the IIalpha and the IIbeta isoforms. However, in contrast to topoisomerase poisons, DEL and CY did not stabilize the covalent DNA-topoisomerase intermediates (cleavable complex) of topoisomerase I or II. Using recombinant topoisomerase I, the presence of CY or DEL (> or = 1 microM) effectively prohibited the stabilization of the cleavable complex by the topoisomerase I poison camptothecin. We furthermore investigated whether the potential protective effect vs topoisomerase I poisons is reflected also on the cellular level, affecting the DNA damaging properties of camptothecin. Indeed, in HT29 cells, low micromolar concentrations of DEL (1-10 microM) significantly diminished the DNA strand breaking effect of camptothecin (100 microM). However, at concentrations > or = 50 microM, all anthocyanidins tested (delphinidin, cyanidin, malvidin, pelargonidin, and paeonidin), including those not interfering with topoisomerases, were found to induce DNA strand breaks in the comet assay. All of these analogues were able to compete with ethidium bromide for the intercalation into calf thymus DNA and to replace the minor groove binder Hoechst 33258. These data indicate substantial affinity to double-stranded DNA, which might contribute at least to the DNA strand breaking effect of anthocyanidins at higher concentrations (> or = 50 microM).

Genotoxicity of phytoestrogens.

Plant extracts containing phytohormones are very popular as 'alternative' medicine for many kinds of diseases. They are especially favored by women who enter menopause and are concerned about the side effects of hormone replacement therapy. However, adverse health effects of phytoestrogens have often been ignored. This review examines the literature on genotoxicity and apoptotic effects of phytohormones. Genistein, coumestrol, quercetin, zearalenone, and resveratrol exerted genotoxic effects in in vitro test systems. Other phytoestrogens such as lignans, the isoflavones daidzein and glycetein, anthocyanidins, and the flavonol fisetin exhibited only weak or no effects in vitro. However, some metabolites of daidzein showed a genotoxic activity in vitro. Practically all of the phytoestrogens exhibit pro-apoptotic effects in some cell systems. Further investigations regarding dose-response-relationships and other aspects relevant for extrapolation to human exposure seem necessary. Until then, care may be advised in taking concentrated phytohormones. Nevertheless, the intake of substantial amounts of plant-food in a normal diet constitutes an important, individual contribution to cancer prevention.

Initial in vitro toxicity testing of functional foods rich in catechins and anthocyanins in human cells.

Functional foods need to be assessed for beneficial effects to support claims, but also for toxic effects. This report describes two examples of how complex food samples are initially characterized in human cells in vitro. Water extracts of green tea (GT) and black carrots (BC) were analyzed for key ingredients (catechins and anthocyanidins, respectively). Extracts, reconstituted mixtures of the major ingredients or individual compounds [(-)-epigallocatechin gallate or cyanidin, respectively] were evaluated in parallel using human colon cells (HT29 clone 19A). End points of cytotoxicity included determination of membrane integrity, proliferation inhibition, and genetic damage. Cells were pretreated with plant compounds at sub-toxic concentrations, and their resistance to toxicity of H2O2 was evaluated as a parameter of protection. The extracts reduced cell viability (BC) and cell growth (BC, GT) and caused DNA damage (BC, GT). They were more toxic than their key ingredients. Neither GT-samples nor BC protected against H2O2-induced DNA damage, whereas cyanidin did. In vitro analysis of extracts from functional foods firstly aims at defining the sub-toxic concentrations at which protective activities are then further characterized. It also allows comparing responses of complex samples and individual compounds, which is important since effects from protective food ingredients can be masked by accompanying toxic components.

Genotoxic and antigenotoxic effects of catechin and tannins from the bark of Hamamelis virginiana L. in metabolically competent, human hepatoma cells (Hep G2) using single cell gel electrophoresis.

The genotoxic and antigenotoxic activities of catechin, hamamelitannin and two proanthocyanidin fractions prepared from the bark of Hamamelis virginiana L. were investigated in a human derived, metabolically competent hepatoma cell line (Hep G2) using single cell gel electrophoresis (SCGE) for the detection of DNA-damage. DNA-migration was calculated as Olive tail moment (OTM). Catechin and a low-molecular weight proanthocyandin fraction (W(M)) caused only slight increases of OTM up to concentrations of 166 microg/ml whereas hamamelitannin and the proanthocyandin fraction with higher molecular weight (W(A)) led to a two-fold enhancement of OTM at the same concentrations. These effects were dose-independent. Treatment of the cells with the test compounds in a dose-range of 2-166 microg/ml prior to the exposure to benzo(a)pyrene (B(a)P, 10 microM, 2.5 microg/ml) led to a significant reduction of induced DNA damage which was dose-dependent for all test compounds, except for hamamelitannin. The inhibitory effects of proanthocyanidins were stronger than those of catechin and hamamelitannin; the lowest effective concentrations were about 2 microg/ml. In order to clarify the mechanisms of protection, possible effects of the test compounds on enzymes involved in toxification and detoxification of B(a)P were investigated. While B(a)P toxification by cytochrome P450 was not inhibited by the test compounds, detoxification by glutathion-S-transferase (GST) was induced by catechin and W(M). Combination experiments with the ultimate metabolite of B(a)P, (+/-)-anti-benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE; 5 microM, 1.5 microg/ml), revealed strong inhibitory effects, indicating that the observed protective effects were caused by scavenging of the ultimate mutagen by the test compounds. Exposure of Hep G2 cells to the test compounds after B(a)P treatment did not influence B(a)P induced DNA damage, demonstrating that repair mechanisms were not affected.

Lack of in vivo clastogenic activity of grape seed and grape skin extracts in a mouse micronucleus assay.

Meganatural brand grape seed extract (GSE) and grape skin extract (GSKE), containing proanthocyanidin polyphenolic compounds, are intended for use in food as functional ingredients exhibiting antioxidant activity. Proanthocyanidins, as well as the minor constituent phenolic compounds in GSE and GSKE, are present naturally in many foods such as fruits, vegetables, chocolate, tea, etc., and on average people consume 460-1000 mg/day of these combined substances. While some polyphenolic compounds, tested individually, have demonstrated antitumorigenic or antipromotional activity, at least one minor component of GSE and GSKE, quercitin, has exhibited positive activity in Salmonella and other in vitro mutagenicity assays. As part of a program to investigate the safety of GSE and GSKE, these products were tested for in vivo clastogenic activity and/or disruption of the mitotic apparatus by detecting micronuclei in polychromatic erythrocyte (PCE) cells in Crl:CD-1(ICR) BR mouse bone marrow. The appropriate test article was dissolved in 0.5% carboxymethylcellulose and dosed by oral gavage to five males/test article/dose level/harvest time point. Animals were dosed at 500, 1000 and 2000 mg/kg. Five animals dosed with either test article at 500, 1000 and 2000 mg/kg dose levels and five animals dosed with the cyclophosphamide (80 mg/kg) positive control were euthanized approximately 24 h after dosing for extraction of bone marrow. Five animals dosed with either test article at the 2000 mg/kg dose level and five animals dosed with the vehicle control article were euthanized approximately 24 and 48 h after dosing for extraction of bone marrow. At least 2000 PCEs per animal were analyzed for frequency of micronuclei. Cytotoxicity was assessed by scoring the number of PCEs and normochromatic erythrocytes (NCEs) in at least the first 500 erythrocytes for each animal. For both GSE and GSKE, no statistically significant increase in micronucleated PCEs was observed at any dose level or harvest time point. GSE produced indication of cytotoxicity (decreased PCE:NCE ratio) at the 2000 mg/kg dose level for the 48-h harvest time point, confirming that the test article reached the target bone marrow in significant amount. Meganatural GSE and Meganatural GSKE were evaluated as negative in the mouse bone marrow micronucleus assay under the conditions of this assay.

Subchronic 3-month oral toxicity study of grape seed and grape skin extracts.

Meganatural brand grape seed extract (GSE) and grape skin extract (GSKE), containing proanthocyanidin (PAC) polyphenolic compounds, are intended for use in food as functional ingredients exhibiting antioxidant activity. Proanthocyanidins, as well as the minor constituent phenolic compounds in GSE and GSKE, are present naturally in many foods such as fruits, vegetables, chocolate, tea, etc., and on average people consume 460-1000 mg/day of these combined substances. Although humans have ingested PACs for centuries without reported adverse effects, the current toxicology literature contains relatively little formal evidence regarding their safety. Accordingly, as part of a program to investigate the safety of GSE and GSKE, these products were incorporated into chow and fed to rats for at least 3 months in a GLP-compliant subchronic toxicity study. Groups of CD (Sprague-Dawley) Crl:CD IGS BR rats (20 males and 20 females per group) were fed diets containing GSE at concentrations of 0, 0.63, 1.25 or 2.5% (w/w); GSKE was fed at 2.5% (w/w) only. Clinical observations were recorded and body weight and feed consumption measured throughout the study. After 1 month, blood was obtained from 10 rats/sex/group by retrobulbar puncture for interim measurement of clinical pathology. At the end of the study the rats were subjected to a full necropsy, aortic blood samples were collected for clinical pathology, selected organs were weighed and a complete list of tissues was preserved from all animals. Histologic examination was performed on all tissues from control and high-dose GSE and GSKE groups. There were no treatment-related changes that were considered to be of toxicologic significance. Therefore, a dietary concentration of 2.5% GSE or 2.5% GSKE was considered to be a no-observed-adverse effect level (NOAEL). This was equivalent to a time-weighted average dose over the course of the study of approximately 1.78 g/kg body weight/day GSE or GSKE in male rats and 2.15 g/kg body weight/day in female rats.