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Toxicology evaluation of a procyanidin-rich extract from grape skins and seeds
Food and Chemical Toxicology 49 (2011) 1450–1454
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Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox
Toxicology evaluation of a procyanidin-rich extract from grape skins and seeds Laura Lluís a, Mònica Muñoz a, M. Rosa Nogués a,⇑, Vanessa Sánchez-Martos a, Marta Romeu a, Montse Giralt a, Josep Valls b, Rosa Solà c a
Unit of Pharmacology, Faculty of Medicine and Health Sciences, Universitat Rovira i Virgili, Reus, Spain Shirota Functional Foods, S.L., Reus, Spain c Research Unit on Lipids and Atherosclerosis, CIBERDEM, Hospital Universitari Sant Joan, IISPV, Universitat Rovira i Virgili, Reus, Spain b
a r t i c l e
i n f o
Article history: Received 14 February 2011 Accepted 22 March 2011 Available online 2 April 2011 Keywords: Procyanidin Grape extract Genotoxicity Rat erythrocyte micronucleus Bacterial reverse mutation Chromosomal aberrations
a b s t r a c t The procyanidin-rich extract from grape seeds and skins (GSSE) has antioxidant properties which may have cardioprotective effects. Since it might be interesting to incorporate this extract into a functional food, toxicological tests need to be made to determine how safe it is. In this study we carried out a limit test to determine the acute oral toxicity and the lethal dose 50 (LD50) and some genotoxicity tests of the extract in rats. The LD50 was higher than 5000 mg/kg. Doses of up to 2000 mg/kg showed no increase in micronucleated erythrocytes 72 h after treatment. The bacterial reverse mutation test showed that the extract was weakly mutagenic to the dose of 5 mg/plate and 19.5 and 9.7 lg/ml of GSSE did not show significant differences in the frequency of aberrant metaphases in relation to negative controls. Our results indicated slight mutagenicity under the study conditions, so further studies should be conducted at lower doses to demonstrate that this extract is not toxic. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Black grapes contain a large amount of phenolic compounds in their skin, pulp and seeds. These compounds—among which are anthocyanidins, proanthocyanidins, stilbenes (resveratrol) and phenolic acids—are associated with the prevention of diseases caused by oxidative stress and they possess antioxidant, anticancer, antiinflammation, antiaging and antibacterial activities. They maintain the endothelial function, which increases the antioxidant capacity and protection against LDL oxidation, and block cellular events predisposing to atherosclerosis and coronary heart disease, so they are considered to be cardioprotective agents (Xia et al., 2010; Vitseva et al., 2005; Sugisawa et al., 2004; Kamiyama et al., 2009). Various mechanisms have been proposed to explain their cardioprotective action including scavenging of free radicals; reduc-
Abbreviations: CECT, Colección Española de Cultivos Tipo (Spanish type culture Collection); CPA, cyclophosphamide; DOX, doxorubicin; EMS, ethylmethane sulphonate; GSE, grape seed extract; GSSE, grape seed and skin extract; LDL, lowdensity lipoprotein; LD50, lethal dose 50; MEM, modified eagle’s medium; NC, negative control; PC, positive control; S9, metabolic activation system consisting of liver-derived cell extract. ⇑ Corresponding author. Tel.: +34 977 75 93 55; fax: +34 977 75 93 78. E-mail addresses: [email protected] (L. Lluís), [email protected] (M. Muñoz), [email protected] (M. Rosa Nogués), [email protected] (V. Sánchez-Martos), [email protected] (M. Romeu), [email protected] (M. Giralt), [email protected] (J. Valls), [email protected] (R. Solà). 0278-6915/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2011.03.042
tion of hydroperoxide formation; decrease in postprandial hyperlipemia, LDL and triglyceride levels; increase in cholesterol elimination with bile acids; increase in lipid-bound polyphenols, which prevents the lipids from oxidising, reduction of platelet adhesion; and aggregation and inhibition of the release of proinflammatory factors (Xia et al., 2010; Vitseva et al., 2005; Sugisawa et al., 2004; Kamiyama et al., 2009). A total of 75% of grape polyphenols are in the skin and seeds, but their concentration and composition depend on agro-geographic factors. Therefore, it is interesting to obtain extracts with high concentrations of polyphenols from grape skins and/or seeds so that they can be added to foods and beverages as a nutritional supplement. However, some reports have also shown that at high concentrations the effect of phenolic compounds on health is negative because products that are highly antioxidant are also cytotoxic (Xia et al., 2010; Ugartondo et al., 2006; Fan and Lou, 2004). Some patents on grape polyphenols use natural products (grape juice or wine) as raw materials to maximize their polyphenol contents and minimize the use of severe extraction processes with organic solvents. These natural products, however, provide high energy and fructose intake that may lead to weight gain and insulin resistance, respectively (Gollucke, 2010). In view of these nutritional applications, it is important to show that these extracts are bioactive and safe to consume. Most phenolic compounds in grapes and wine are heavily metabolized by gut flora to produce metabolites that can poten-
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tially be well absorbed into the bloodstream by passive diffusion or active transport systems. Several elements of digestion, the absorption process and the food matrix can affect the bioavailability of polyphenols, but it is generally accepted that they reach maximum plasma values between five minutes and two hours after administration, depending on the compound (Forester and Waterhouse, 2009; Gross et al., 2010). Some high molecular weight phenolics, such as oligomeric procyanidins, cannot be absorbed, but they can release monomer and dimer units on incubation with acidic gastric contents and large quantities of epicatechin that can be absorbed (Stahl et al., 2002). Several metabolites (sulfates, methylates, glucuronidates or glucosides) of these compounds have been found in rat plasma, urine, liver, lung, kidney, intestine and brain and also in human plasma and urine. The potential toxicity of some polyphenols from grapes has been investigated in various cell lines, mice, rats and human peripheral blood lymphocytes, but no results have been published on a mixed grape extract from seeds and skins. Some studies demonstrate that the mixed extracts from grape skins and seeds have better anti-inflammatory effects than individual extracts (Vitseva et al., 2005; Shanmuganayagam et al., 2002). The current study reports the results of a toxicology evaluation of a mixed extract from the seeds and skins of black grape (GSSE), with 76% of total polyphenols. The acute toxicity test, which determines the lethal dose 50 (LD50) for oral route in rats, and three tests of mutagenicity were carried out: the mammalian in vivo erythrocyte micronucleus test, the bacterial reverse mutation test and the chromosomal aberration test in cultured mammalian cells. These tests were performed according to the guidelines of the OECD (Organization for Economic Cooperation and Development) 425, 474, 471 and 473 (OEDC Guidelines for the testing of chemicals) as is required by the Spanish Agency of Food Safety and Nutrition.
2.4. Acute oral toxicity study. Limit test Acute toxicity of the extract was determined by the limit test (Test No. 425: Acute Oral Toxicity) (OECD). This method can be used to efficiently test chemicals that are likely to have low toxicity. Six Wistar female rats (200–220 g body weight) were divided into two groups: the control group and the group treated with the extract (GSSE). The GSSE was dissolved in saline solution and administered to one rat at a rate of 10 ml/kg by oral gavage at a dose of 5000 mg/kg. This rat was observed for signs of mortality and toxicity for 24 h. As it survived, two more rats were given the same dose. The three rats in the control group were treated in the same way with saline solution. Observations were continued for 14 days. At the end of the observation period, all the rats were anesthetized with an intraperitoneal (ip) injection of ketamine-xylacine (80–10 mg/kg) and sacrificed by exsanguinations from cardiac puncture. The animals were examined by necropsy. 2.5. In vivo rat erythrocyte micronucleus test The micronucleus test and scoring was carried out according to W. Schmid, 1975. The purpose of the micronucleus test is to identify substances that cause cytogenetic damage which results in the formation of micronuclei containing lagging chromosome fragments or whole chromosomes. A total of 18 female Wistar rats were divided into three groups: negative control (NC), positive control (PC) and treatment group (GSSE) (Schmid, 1975). PC rats were treated ip with two doses of 200 mg/kg of ethyl methanesulphonate (EMS) in an interval of 24 h. NC rats were given 10 ml/kg of saline solution by gavage. GSSE rats were treated with 2000 mg/kg of the extract diluted in saline solution by gavage. After 48 h of administration, peripheral blood (5 ll) was collected by piercing the ventral tail; and after 72 h, the rats were sacrificed in the same way described above and blood was collected by cardiac puncture. One drop of blood from each sample was spread on slides and air-dried. The slides were fixed in methanol, stained in Giemsa/May-Grüenwald solutions and protected by permanently mounted coverslips. To control bias, all slides were coded prior to analysis (Schmid, 1975). The micronucleus frequency (expressed as percentage of micronucleated erythrocytes) was determined on the basis of scoring at least 1000 erythrocytes per animal from an optical microscope. The data obtained were statistically analyzed with the non parametric Kruskall-Wallis test for significant differences between groups. When significant differences were found, means were compared using the Mann–Whitney test. 2.6. Bacterial reverse mutation test
2. Material and methods 2.1. Extract from grape skins and seeds A polyphenols-rich extract was obtained from the red grape marcs (variety Syrah) kindly donated in 2008 by the experimental winery Mas dels Frares (Rovira i Virgili University). 1 kg of marc was freeze-dried. Then, a solid–liquid extraction with ethanol 50% was performed, followed by vacuum evaporation and freeze drying of the liquid. A further step of Amberlite XAD7 HP (Sigma Aldrich, Germany) chromatography was applied in order to remove sugars. The retained polyphenols were later eluted with ethanol, which was again evaporated and freeze dried to obtain the final extract (30 g). The content of polyphenols was determined by well-known methodologies. The polyphenol composition of the extract was the following: 76% of total polyphenols determined by Folin method and expressed as gallic acid equivalents (Singleton and Rossi, 1965), 10% of total anthocyanins determined by the measure of the absorbance at 520 nm, using oenin-chloride (Extrasynthese) as standard (Ribéreau-Gayon and Stonestreet, 1965), and 74.8% of procyanidins as measured by the BateSmith method (Bate-Smith, 1954).
2.2. Chemicals The chemicals and reagents used in the studies were purchased from Sigma–Aldrich (Madrid, Spain).
The bacterial reverse mutation test uses amino-acid requiring strains of Salmonella typhimurium (Ta 1535, Ta 1537, TA 98, TA 100) to detect point mutations, which involve substitution, addition or deletion of one or a few DNA base pairs. The principle of this test is that it detects mutations which revert mutations present in the test strains and restore the functional capability of the bacteria to synthesize an essential amino acid. The revertant bacteria are detected by their ability to grow in the absence of the amino acid required by the parent test strain (Ames et al., 1975). This set of four bacterial strains was provided by the Spanish Type Culture Collection (CECT). The working cultures were obtained from the frozen permanent cultures after incubation overnight at 37 °C to reach a concentration of approximately 1x109 bacteria/ml in nutrient broth agar I (5 g of meat extract, 10 g of peptone, 5 g of sodium chloride and 10 ml of histidine-biotine solution [0.5/0.5 mM] recommended by CECT). The extract was examined for its mutagenic potency using the plate incorporation method (Maron and Ames, 1983), which consists of exposing the tester strains to the GSSE directly on a minimal glucose agar plate (1000 ml Vogel-Bonner minimal medium E, 1000 ml glucose solution and 3000 ml agar solution). The experiment was realized by duplicate: with and without the presence of a metabolic activator (S9 mixture) constituted by a fraction of hepatic enzymes.(Mortelmans and Riccio, 2000). Five different analyzable concentrations of the GSSE, diluted in sterile distilled water and filtered, were used for triplicate: 5, 1.58, 0.5, 0.16 and 0.05 mg/plate. Positive controls with different mutagens were included as Table 1 shows depending on the presence or absence of S9. Negative controls had sterile water. The different components (0.5 ml S9 mixture or buffer, 0.05 ml of the diluted GSSE, mutagens or sterile water and 0.05–0.10 ml overnight culture of the strains) were first added to sterile test tubes containing 2 ml molten top agar (6 g of Bacto
2.3. Animals This study was in compliance with the Ethics Committee of Animal Research, Rovira i Virgili University (Tarragona, Spain). Female Wistar rats (6 weeks old) were purchased from Charles River, for all the studies and acclimated for 1 week prior to the initiation of the tests. The animal room was maintained at a temperature of 22 ± 2 °C and 50–60% relative humidity with a 12 h light/dark photoperiod. The animals were fed a standard pellet diet from Panlab (Barcelona, Spain) and had free access to tap water and food, except for a fasting period before sacrifice. All these studies complied with the OECD (Organization for Economic Co-operation and Development) guidelines.
Table 1 Substances used as positive control for bacterial reverse mutation test. Strain
Without S9 mix
lg/plate
With S9 mix
lg/plate
TA TA TA TA
Sodium azide ICR191 2-nitrofluorene Sodium azide
5 5 5 5
2-aminoanthracene 2-aminoanthracene 2-aminoanthracene 2-aminoanthracene
0.1 0.1 0.1 0.1
1535 1537 98 100
L. Lluís et al. / Food and Chemical Toxicology 49 (2011) 1450–1454
agar, 5 g of sodium chloride, 1000 ml purified water) supplemented with limited histidine/biotin (0.5 mM) to allow for a few cell divisions. It was important to maintain the top agar between 43 and 48 °C. Tubes were mixed and poured on glucose minimal agar plates. After the top agar had hardened, the plates were inverted and incubated at 37 °C for 48 h. The number of revertant colonies per plate was counted in the treated group, and negative and positive controls with and without S9. Three plates were used for each concentration, together with positive and negative controls. The results are expressed as the mean and standard deviation for each treatment and the data was analyzed statistically with the ANOVA test for significant differences between groups. When significant differences were found, the means were compared using the Scheffé test.
1.8 1.6 1.4 % of Micronuclei
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1.2 1 0.8
0.4 2.7. In vitro mammalian chromosomal aberration test The purpose of the in vitro chromosome aberration test is to identify agents that cause structural chromosome aberrations in cultured mammalian cells. Structural aberrations may be of two types, chromosome or chromatid. With the majority of chemical mutagens, induced aberrations are of the chromatid type, but chromosome-type aberrations also occur.The GSSE was tested in the chromosomal aberration assay using human lymphocytes. Blood samples were obtained by venous puncture from a single individual with no report of any infection or exposure to drugs. Cultures were performed with 0.4 ml of whole blood in 5 ml of Modified Eagle’s Medium (MEM), inactivated fetal bovine serum, streptomycin/penicillin, gently mixed and incubated (CO2 5%) at 37 °C for 72 h. After 48 h, the cells were exposed to the GSSE dissolved to a concentration of 19.5 and 9.7 lg/ml in MEM. The concentrations were selected on the basis of a preliminary cytotoxicity study. Positive controls included 7 lg/ml cyclophosphamide (CPA) (experiments with metabolic activation), and 110 lg/ml ethylmethane sulphonate (EMS) (experiments without metabolic activation). MEM served as the vehicle control. The test was conducted with and without S9 mixture for each experiment. After 72 h, in order to arrest the cells in metaphase, Colcemid (0.2 lg/ml) was added to each culture tube 3 h before harvesting. At the end of the experiment, cells were dropped onto clean, chilled slides, air dried, and stained with 5% Giemsa for 15 min. At least 100 well-spread metaphase cells were analyzed per slide and the chromosome aberrations were counted (Evans, 1984). The results were expressed as the percentage of metaphases with aberrations; the data was analyzed statistically with the ANOVA test for significant differences between groups.
3. Results 3.1. Acute toxicity At the limit dose of 5000 mg/kg body weight, the GSSE did not cause mortality in any of the three animals tested and there were no signs of toxicity in rats after dosing and during the observation period of 14 days thereafter. The body weight gain of treated rats was normal. No gross pathological alterations were encountered in any of the rats, as evident at terminal necropsy. On the basis of these results and under the conditions of this study, the lethal dose (LD50) of GSSE after a single oral administration in female Wistar rats was expected to be more than the limit dose level of 5000 mg/kg. 3.2. Mammalian erythrocyte in vivo micronucleus test Fig. 1 shows that there were significant statistical differences between positive controls and the other two groups (the negative controls and the treated group) at 48 and 72 h, which validates the method. There were significant statistical differences between the negative controls and the treated group at 48 h but not at 72 h of treatment. These results indicate that the extract produces a certain quantity of micronucleated cells that do not appear in the samples obtained at 72 h of treatment. 3.3. Bacterial reverse mutation test Statistical differences are significant between positive and negative controls in the specific strains TA1535 and TA1537 for the plates without metabolic activation and in the specific strains
*#
0.6
*
*
*
0.2 0 48 h
Positive Control
72 h % of Micronuclei
Negative Control
Treated
Fig. 1. Percentage of micronuclei in the different groups at different blood collection times post-treatment. The data are expressed as mean ± S.D. ⁄Significant statistical differences (p < 0.05) with the positive control. # Significant statistical differences (p < 0.05) with negative control.
TA1537, TA98 and TA100 for the plates with metabolic activation. Table 2 shows the means and standard deviations of the number of revertant colonies in treated plates and the significant statistical differences between each group and the negative controls. Only two strains showed significant differences with the negative controls: TA1537 at doses of 5 and 1.58 mg with and without metabolic activation, respectively, and TA 98 at a dose of 1.58 mg with metabolic activation. 3.4. Chromosomal aberration test Positive controls with CPA and EMS induced chromosomal aberrations; the percentage of metaphases with aberrations was 73%. However, the cultures treated with GSSE at doses of 9.7 and 19.5 lg/ml in the presence and absence of S9 mix did not significantly increase the frequency of metaphases with aberrant chromosomes with respect to the negative controls. 4. Discussion In our study we have observed no acute toxic effects of the mixed extract taken via the oral route and we conclude that the LD50 is higher than 5000 mg/kg. Our results confirm the LD50 value of a grape seed extract that was estimated by Yamakoshi et al., 2002, to be higher than 4000 mg/kg.. The bibliography shows that mutagenicity studies have provided contradictory results. In our case, mutagenicity tests showed that the extract produced slight genetic damage in the models studied. In the mammalian erythrocyte in vivo micronucleus test, the mutagens in positive controls increased the percentage of micronucleated cells 48 and 72 h after treatment. We observed fewer micronucleated cells in negative controls and in the group treated with 2000 mg/kg of GSSE, and there were significant differences with the positive controls at both blood collection times. However, the samples from the treated group taken 48 h post-treatment showed a higher percentage of micronuclei than the negative controls, which might indicate a certain degree of genotoxicity that was not discerned in the samples obtained 72 h after treatment (Fig. 1). Yamakoshi et al. (2002) showed that doses up to 2 g/kg of a proanthocyanidin-rich extract from grape seeds (GSE) administered orally to male mice did not promote an increase in micronucleated peripheral reticulocytes (Yamakoshi et al., 2002). The micronu-
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L. Lluís et al. / Food and Chemical Toxicology 49 (2011) 1450–1454 Table 2 Means and standard deviations of the number of revertant colonies in each group treated with the extract. STRAIN
Without metabolic activation
With metabolic activation
TA1535 TA1537 TA98 TA100 TA1535 TA1537 TA98 TA100
DOSE 5 mg
DOSE 1.58 mg
DOSE 0.5 mg
DOSE 0.15 mg
DOSE 0.05 mg
Col./plate
p
Col./plate
p
Col./plate
p
Col./plate
p
Col./plate
p
16 ± 3 3±1 17 ± 3 35 ± 9 16 ± 7 10 ± 2 19 ± 5 66 ± 10
0.827 0.238 0.050 0.050 0.275 0.046 0.127 0.275
22 ± 6 8±1 18 ± 6 63 ± 8 12 ± 1 4±2 19 ± 6 64 ± 3
0.376 0.043 0.050 1.000 0.127 0.099 0.046 0.050
13 ± 4 3±1 11 ± 5 61 ± 4 8±5 3±1 18 ± 15 69 ± 11
0.275 0.178 0.275 0.513 0.127 0.178 0.376 0.513
12 ± 5 3±2 11 ± 6 59 ± 8 11 ± 7 6±5 12 ± 2 79 ± 15
0.275 0.487 0.500 0.513 0.127 0.099 0.513 0.513
14 ± 3 1±1 7±3 50 ± 6 13 ± 6 2±1 8±0 64 ± 7
0.513 0.487 0.513 0.050 0.127 0.814 0.487 0.127
p = statistical differences with negative controls.
cleus assay in mouse bone marrow after a GSE administration carried out by Erexson (2003) was also negative (Erexson, 2003). Other in vitro studies showed that treating lymphocytes with 2.5 lg/ml of GSE induced a significant decrease (40%) in the frequency of micronuclei (Stankovic et al., 2008). Concentrations up to 5 mg/l did not induce chromosomal damage in a human lymphoblastoid cell line and pre-treatment with GSE prevented H2O2-induced chromosomal damage (Sugisawa et al., 2004). On the other hand, Thomas et al. (2009) did not find a significant reduction in the frequency of erythrocyte micronuclei in a transgenic mouse model for Alzheimer’s disease (AD) fed with 0.07% of curcumin or 2% of a GSE. However, there was a significant reduction in micronucleated erythrocytes in mice fed with microencapsulated grape seed extract (Thomas et al., 2009). In our study, the bacterial reversal mutation test showed an increase in the number of revertant colonies at the highest dose in the TA1537 strain with significant difference in the limit with regard to the negative control. The values were 10 ± 2 and 2 ± 3 colonies/plate, respectively. TA1537 and TA 98 also increased the number of revertant colonies at doses of 1.58 mg, but not at the highest dose. So, we conclude that this extract was weakly mutagenic to the dose of 5 mg/plate, which was the highest tested by Yamakoshi in his study (Yamakoshi et al., 2002). A dose of 5 mg/ plate in TA1537 and TA1535 strains did not increase the number of revertant colonies with respect to the negative controls. A maximum dose of 1250 lg/plate was tested in TA98 and TA100 strains and no mutagenicity was detected either. Finally, the two doses of GSSE tested in the chromosomal aberration test with human lymphocytes did not show significant differences with respect to negative controls. The doses were selected for the solubility of the extract in the culture medium and they were previously subjected to the trypan-blue test to demonstrate that they were not cytotoxic. Likewise, a grape seed extract showed no toxicity under the conditions of the assay when it was subjected to the in vitro chromosomal aberration test with Chinese hamster lung cells (Yamakoshi et al., 2002). Nevertheless, several authors describe cytotoxic effects of procianidins and grape extracts in different cells to high doses, and suggest that the strongest antioxidant products were also the most cytotoxic (Ugartondo et al., 2006; Fan and Lou, 2004; Stagos et al., 2007). 5. Conclusion Our results indicated slight mutagenicity under the study conditions, so further studies should be conducted at lower doses to demonstrate that this extract is not toxic. Role of the funding source This work was supported by the project MET-DEV-FUN (1321 U07 E10 N-MET-DEV-FUN) of the Programa para la creación de
Consorcios Estratégicos Nacionales de Investigación Técnica (CENIT), of the Ministerio de Industria, Turismo y Comercio, Spain. The Ministerio de Industria, Turismo y Comercio, Spain, and Universitat ROVIRA I VIRGILI (Reus, Spain) had no role in the design and conduct of the study; in the collection, management, analysis, and interpretation of the data; or in the preparation, review or approval of the manuscript. Conflict of Interest The authors declare that there are no conflicts of interest. Acknowledgements We are grateful to Shirota Functional Foods for the analysis of the extract composition and to the Language Service of the University for language assistance. References Ames, B.N., McCann, J., Yamasaki, E., 1975. Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test. Mutation Res. 31, 347–364. Bate-Smith, E.C., 1954. Astringency in foods. Food 23, 124–127. Erexson, G.L., 2003. Lack of in vivo clastogenic activity of grape seed and grape skin extracts in a mouse micronucleus assay. Food Chem. Toxicol. 41, 347–350. Evans, H.J., 1984. Human peripheral blood lymphocytes for the analysis of chromosome aberrations in mutagen tests. In: Kilbey, B.J., Legator, M., Nichols, W., Ramel, C. (Eds.), Handbook of Mutagenicity Test Procedures, Second ed. Elsevier Science Publishers, Amsterdam, pp. 405–427. Fan, P., Lou, H., 2004. Effects of polyphenols from grape seeds on oxidative damage to cellular DNA. Mol. Cell. Biochem. 267, 67–74. Forester, S.C., Waterhouse, A.L., 2009. Metabolites are key to understanding health effects of wine polyphenolics. J. Nutr. 139, 1824S–1831S. Gollucke, A.P., 2010. Recent applications of grape polyphenols in foods, beverages and supplements. Recent. Pat. Food Nutr. Agric. 2, 105–109. Gross, G., Jacobs, D.M., Peters, S., Possemiers, S., van Duynhoven, J., Vaughan, E.E., van de Wiele, T., 2010. In vitro bioconversion of polyphenols from black tea and red wine/grape juice by human intestinal microbiota displays strong interindividual variability. J. Agric. Food Chem. 58, 10236–10246. Kamiyama, M., Kishimoto, Y., Tani, M., Andoh, K., Utsunomiya, K., Kondo, K., 2009. Inhibition of low-density lipoprotein oxidation by Nagano purple grape (Vitis viniferax Vitis labrusca). J. Nutr. Sci. Vitaminol. (Tokyo) 55, 471–478. Maron, D.M., Ames, B.N., 1983. Revised methods for the Salmonella mutagenicity test. Mutat. Res. 113, 173–215. Mortelmans, K., Riccio, E.S., 2000. The bacterial tryptophan reverse mutation assay with Escherichia coli WP2. Mutat. Res. 455, 61–69. OECD Guidelines for the testing of chemicals. [on line]. http://www.oecd.org/ document/40/0,3343,en_2649_34377_37051368_1_1_1_1,00.html [accessed February 1, 2011]. Ribéreau-Gayon, P., Stonestreet, E., 1965. Le dosage des anthocyanes dans lev in rouge. Bull. Soc. Chim. Fr. 9, 2649–2652. Schmid, W., 1975. The micronucleus test. Mutat. Res. 31, 9–15. Shanmuganayagam, D., Beahm, M.R., Osman, H.E., Krueger, C.G., Reed, J.D., Folts, J.D., 2002. Grape seed and grape skin extracts elicit a greater antiplatelet effect when used in combination than used individually in dogs and human. J. Nutr. 132, 3592–3598. Singleton, V.L., Rossi, J.A., 1965. Colorimetry of total phenolics with phosphomdybdic-phosphotungstic acid agents. Am. J. Enol. Vitic. 16, 144–158. Stagos, D., Spanou, C., Margariti, M., Stathopoulos, C., Mamuris, Z., Kazantzoglou, G., Magiatis, P., Kouretas, D., 2007. Cytogenetic effects of grape extracts (Vitis
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Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox
Toxicology evaluation of a procyanidin-rich extract from grape skins and seeds Laura Lluís a, Mònica Muñoz a, M. Rosa Nogués a,⇑, Vanessa Sánchez-Martos a, Marta Romeu a, Montse Giralt a, Josep Valls b, Rosa Solà c a
Unit of Pharmacology, Faculty of Medicine and Health Sciences, Universitat Rovira i Virgili, Reus, Spain Shirota Functional Foods, S.L., Reus, Spain c Research Unit on Lipids and Atherosclerosis, CIBERDEM, Hospital Universitari Sant Joan, IISPV, Universitat Rovira i Virgili, Reus, Spain b
a r t i c l e
i n f o
Article history: Received 14 February 2011 Accepted 22 March 2011 Available online 2 April 2011 Keywords: Procyanidin Grape extract Genotoxicity Rat erythrocyte micronucleus Bacterial reverse mutation Chromosomal aberrations
a b s t r a c t The procyanidin-rich extract from grape seeds and skins (GSSE) has antioxidant properties which may have cardioprotective effects. Since it might be interesting to incorporate this extract into a functional food, toxicological tests need to be made to determine how safe it is. In this study we carried out a limit test to determine the acute oral toxicity and the lethal dose 50 (LD50) and some genotoxicity tests of the extract in rats. The LD50 was higher than 5000 mg/kg. Doses of up to 2000 mg/kg showed no increase in micronucleated erythrocytes 72 h after treatment. The bacterial reverse mutation test showed that the extract was weakly mutagenic to the dose of 5 mg/plate and 19.5 and 9.7 lg/ml of GSSE did not show significant differences in the frequency of aberrant metaphases in relation to negative controls. Our results indicated slight mutagenicity under the study conditions, so further studies should be conducted at lower doses to demonstrate that this extract is not toxic. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Black grapes contain a large amount of phenolic compounds in their skin, pulp and seeds. These compounds—among which are anthocyanidins, proanthocyanidins, stilbenes (resveratrol) and phenolic acids—are associated with the prevention of diseases caused by oxidative stress and they possess antioxidant, anticancer, antiinflammation, antiaging and antibacterial activities. They maintain the endothelial function, which increases the antioxidant capacity and protection against LDL oxidation, and block cellular events predisposing to atherosclerosis and coronary heart disease, so they are considered to be cardioprotective agents (Xia et al., 2010; Vitseva et al., 2005; Sugisawa et al., 2004; Kamiyama et al., 2009). Various mechanisms have been proposed to explain their cardioprotective action including scavenging of free radicals; reduc-
Abbreviations: CECT, Colección Española de Cultivos Tipo (Spanish type culture Collection); CPA, cyclophosphamide; DOX, doxorubicin; EMS, ethylmethane sulphonate; GSE, grape seed extract; GSSE, grape seed and skin extract; LDL, lowdensity lipoprotein; LD50, lethal dose 50; MEM, modified eagle’s medium; NC, negative control; PC, positive control; S9, metabolic activation system consisting of liver-derived cell extract. ⇑ Corresponding author. Tel.: +34 977 75 93 55; fax: +34 977 75 93 78. E-mail addresses: [email protected] (L. Lluís), [email protected] (M. Muñoz), [email protected] (M. Rosa Nogués), [email protected] (V. Sánchez-Martos), [email protected] (M. Romeu), [email protected] (M. Giralt), [email protected] (J. Valls), [email protected] (R. Solà). 0278-6915/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2011.03.042
tion of hydroperoxide formation; decrease in postprandial hyperlipemia, LDL and triglyceride levels; increase in cholesterol elimination with bile acids; increase in lipid-bound polyphenols, which prevents the lipids from oxidising, reduction of platelet adhesion; and aggregation and inhibition of the release of proinflammatory factors (Xia et al., 2010; Vitseva et al., 2005; Sugisawa et al., 2004; Kamiyama et al., 2009). A total of 75% of grape polyphenols are in the skin and seeds, but their concentration and composition depend on agro-geographic factors. Therefore, it is interesting to obtain extracts with high concentrations of polyphenols from grape skins and/or seeds so that they can be added to foods and beverages as a nutritional supplement. However, some reports have also shown that at high concentrations the effect of phenolic compounds on health is negative because products that are highly antioxidant are also cytotoxic (Xia et al., 2010; Ugartondo et al., 2006; Fan and Lou, 2004). Some patents on grape polyphenols use natural products (grape juice or wine) as raw materials to maximize their polyphenol contents and minimize the use of severe extraction processes with organic solvents. These natural products, however, provide high energy and fructose intake that may lead to weight gain and insulin resistance, respectively (Gollucke, 2010). In view of these nutritional applications, it is important to show that these extracts are bioactive and safe to consume. Most phenolic compounds in grapes and wine are heavily metabolized by gut flora to produce metabolites that can poten-
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tially be well absorbed into the bloodstream by passive diffusion or active transport systems. Several elements of digestion, the absorption process and the food matrix can affect the bioavailability of polyphenols, but it is generally accepted that they reach maximum plasma values between five minutes and two hours after administration, depending on the compound (Forester and Waterhouse, 2009; Gross et al., 2010). Some high molecular weight phenolics, such as oligomeric procyanidins, cannot be absorbed, but they can release monomer and dimer units on incubation with acidic gastric contents and large quantities of epicatechin that can be absorbed (Stahl et al., 2002). Several metabolites (sulfates, methylates, glucuronidates or glucosides) of these compounds have been found in rat plasma, urine, liver, lung, kidney, intestine and brain and also in human plasma and urine. The potential toxicity of some polyphenols from grapes has been investigated in various cell lines, mice, rats and human peripheral blood lymphocytes, but no results have been published on a mixed grape extract from seeds and skins. Some studies demonstrate that the mixed extracts from grape skins and seeds have better anti-inflammatory effects than individual extracts (Vitseva et al., 2005; Shanmuganayagam et al., 2002). The current study reports the results of a toxicology evaluation of a mixed extract from the seeds and skins of black grape (GSSE), with 76% of total polyphenols. The acute toxicity test, which determines the lethal dose 50 (LD50) for oral route in rats, and three tests of mutagenicity were carried out: the mammalian in vivo erythrocyte micronucleus test, the bacterial reverse mutation test and the chromosomal aberration test in cultured mammalian cells. These tests were performed according to the guidelines of the OECD (Organization for Economic Cooperation and Development) 425, 474, 471 and 473 (OEDC Guidelines for the testing of chemicals) as is required by the Spanish Agency of Food Safety and Nutrition.
2.4. Acute oral toxicity study. Limit test Acute toxicity of the extract was determined by the limit test (Test No. 425: Acute Oral Toxicity) (OECD). This method can be used to efficiently test chemicals that are likely to have low toxicity. Six Wistar female rats (200–220 g body weight) were divided into two groups: the control group and the group treated with the extract (GSSE). The GSSE was dissolved in saline solution and administered to one rat at a rate of 10 ml/kg by oral gavage at a dose of 5000 mg/kg. This rat was observed for signs of mortality and toxicity for 24 h. As it survived, two more rats were given the same dose. The three rats in the control group were treated in the same way with saline solution. Observations were continued for 14 days. At the end of the observation period, all the rats were anesthetized with an intraperitoneal (ip) injection of ketamine-xylacine (80–10 mg/kg) and sacrificed by exsanguinations from cardiac puncture. The animals were examined by necropsy. 2.5. In vivo rat erythrocyte micronucleus test The micronucleus test and scoring was carried out according to W. Schmid, 1975. The purpose of the micronucleus test is to identify substances that cause cytogenetic damage which results in the formation of micronuclei containing lagging chromosome fragments or whole chromosomes. A total of 18 female Wistar rats were divided into three groups: negative control (NC), positive control (PC) and treatment group (GSSE) (Schmid, 1975). PC rats were treated ip with two doses of 200 mg/kg of ethyl methanesulphonate (EMS) in an interval of 24 h. NC rats were given 10 ml/kg of saline solution by gavage. GSSE rats were treated with 2000 mg/kg of the extract diluted in saline solution by gavage. After 48 h of administration, peripheral blood (5 ll) was collected by piercing the ventral tail; and after 72 h, the rats were sacrificed in the same way described above and blood was collected by cardiac puncture. One drop of blood from each sample was spread on slides and air-dried. The slides were fixed in methanol, stained in Giemsa/May-Grüenwald solutions and protected by permanently mounted coverslips. To control bias, all slides were coded prior to analysis (Schmid, 1975). The micronucleus frequency (expressed as percentage of micronucleated erythrocytes) was determined on the basis of scoring at least 1000 erythrocytes per animal from an optical microscope. The data obtained were statistically analyzed with the non parametric Kruskall-Wallis test for significant differences between groups. When significant differences were found, means were compared using the Mann–Whitney test. 2.6. Bacterial reverse mutation test
2. Material and methods 2.1. Extract from grape skins and seeds A polyphenols-rich extract was obtained from the red grape marcs (variety Syrah) kindly donated in 2008 by the experimental winery Mas dels Frares (Rovira i Virgili University). 1 kg of marc was freeze-dried. Then, a solid–liquid extraction with ethanol 50% was performed, followed by vacuum evaporation and freeze drying of the liquid. A further step of Amberlite XAD7 HP (Sigma Aldrich, Germany) chromatography was applied in order to remove sugars. The retained polyphenols were later eluted with ethanol, which was again evaporated and freeze dried to obtain the final extract (30 g). The content of polyphenols was determined by well-known methodologies. The polyphenol composition of the extract was the following: 76% of total polyphenols determined by Folin method and expressed as gallic acid equivalents (Singleton and Rossi, 1965), 10% of total anthocyanins determined by the measure of the absorbance at 520 nm, using oenin-chloride (Extrasynthese) as standard (Ribéreau-Gayon and Stonestreet, 1965), and 74.8% of procyanidins as measured by the BateSmith method (Bate-Smith, 1954).
2.2. Chemicals The chemicals and reagents used in the studies were purchased from Sigma–Aldrich (Madrid, Spain).
The bacterial reverse mutation test uses amino-acid requiring strains of Salmonella typhimurium (Ta 1535, Ta 1537, TA 98, TA 100) to detect point mutations, which involve substitution, addition or deletion of one or a few DNA base pairs. The principle of this test is that it detects mutations which revert mutations present in the test strains and restore the functional capability of the bacteria to synthesize an essential amino acid. The revertant bacteria are detected by their ability to grow in the absence of the amino acid required by the parent test strain (Ames et al., 1975). This set of four bacterial strains was provided by the Spanish Type Culture Collection (CECT). The working cultures were obtained from the frozen permanent cultures after incubation overnight at 37 °C to reach a concentration of approximately 1x109 bacteria/ml in nutrient broth agar I (5 g of meat extract, 10 g of peptone, 5 g of sodium chloride and 10 ml of histidine-biotine solution [0.5/0.5 mM] recommended by CECT). The extract was examined for its mutagenic potency using the plate incorporation method (Maron and Ames, 1983), which consists of exposing the tester strains to the GSSE directly on a minimal glucose agar plate (1000 ml Vogel-Bonner minimal medium E, 1000 ml glucose solution and 3000 ml agar solution). The experiment was realized by duplicate: with and without the presence of a metabolic activator (S9 mixture) constituted by a fraction of hepatic enzymes.(Mortelmans and Riccio, 2000). Five different analyzable concentrations of the GSSE, diluted in sterile distilled water and filtered, were used for triplicate: 5, 1.58, 0.5, 0.16 and 0.05 mg/plate. Positive controls with different mutagens were included as Table 1 shows depending on the presence or absence of S9. Negative controls had sterile water. The different components (0.5 ml S9 mixture or buffer, 0.05 ml of the diluted GSSE, mutagens or sterile water and 0.05–0.10 ml overnight culture of the strains) were first added to sterile test tubes containing 2 ml molten top agar (6 g of Bacto
2.3. Animals This study was in compliance with the Ethics Committee of Animal Research, Rovira i Virgili University (Tarragona, Spain). Female Wistar rats (6 weeks old) were purchased from Charles River, for all the studies and acclimated for 1 week prior to the initiation of the tests. The animal room was maintained at a temperature of 22 ± 2 °C and 50–60% relative humidity with a 12 h light/dark photoperiod. The animals were fed a standard pellet diet from Panlab (Barcelona, Spain) and had free access to tap water and food, except for a fasting period before sacrifice. All these studies complied with the OECD (Organization for Economic Co-operation and Development) guidelines.
Table 1 Substances used as positive control for bacterial reverse mutation test. Strain
Without S9 mix
lg/plate
With S9 mix
lg/plate
TA TA TA TA
Sodium azide ICR191 2-nitrofluorene Sodium azide
5 5 5 5
2-aminoanthracene 2-aminoanthracene 2-aminoanthracene 2-aminoanthracene
0.1 0.1 0.1 0.1
1535 1537 98 100
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agar, 5 g of sodium chloride, 1000 ml purified water) supplemented with limited histidine/biotin (0.5 mM) to allow for a few cell divisions. It was important to maintain the top agar between 43 and 48 °C. Tubes were mixed and poured on glucose minimal agar plates. After the top agar had hardened, the plates were inverted and incubated at 37 °C for 48 h. The number of revertant colonies per plate was counted in the treated group, and negative and positive controls with and without S9. Three plates were used for each concentration, together with positive and negative controls. The results are expressed as the mean and standard deviation for each treatment and the data was analyzed statistically with the ANOVA test for significant differences between groups. When significant differences were found, the means were compared using the Scheffé test.
1.8 1.6 1.4 % of Micronuclei
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1.2 1 0.8
0.4 2.7. In vitro mammalian chromosomal aberration test The purpose of the in vitro chromosome aberration test is to identify agents that cause structural chromosome aberrations in cultured mammalian cells. Structural aberrations may be of two types, chromosome or chromatid. With the majority of chemical mutagens, induced aberrations are of the chromatid type, but chromosome-type aberrations also occur.The GSSE was tested in the chromosomal aberration assay using human lymphocytes. Blood samples were obtained by venous puncture from a single individual with no report of any infection or exposure to drugs. Cultures were performed with 0.4 ml of whole blood in 5 ml of Modified Eagle’s Medium (MEM), inactivated fetal bovine serum, streptomycin/penicillin, gently mixed and incubated (CO2 5%) at 37 °C for 72 h. After 48 h, the cells were exposed to the GSSE dissolved to a concentration of 19.5 and 9.7 lg/ml in MEM. The concentrations were selected on the basis of a preliminary cytotoxicity study. Positive controls included 7 lg/ml cyclophosphamide (CPA) (experiments with metabolic activation), and 110 lg/ml ethylmethane sulphonate (EMS) (experiments without metabolic activation). MEM served as the vehicle control. The test was conducted with and without S9 mixture for each experiment. After 72 h, in order to arrest the cells in metaphase, Colcemid (0.2 lg/ml) was added to each culture tube 3 h before harvesting. At the end of the experiment, cells were dropped onto clean, chilled slides, air dried, and stained with 5% Giemsa for 15 min. At least 100 well-spread metaphase cells were analyzed per slide and the chromosome aberrations were counted (Evans, 1984). The results were expressed as the percentage of metaphases with aberrations; the data was analyzed statistically with the ANOVA test for significant differences between groups.
3. Results 3.1. Acute toxicity At the limit dose of 5000 mg/kg body weight, the GSSE did not cause mortality in any of the three animals tested and there were no signs of toxicity in rats after dosing and during the observation period of 14 days thereafter. The body weight gain of treated rats was normal. No gross pathological alterations were encountered in any of the rats, as evident at terminal necropsy. On the basis of these results and under the conditions of this study, the lethal dose (LD50) of GSSE after a single oral administration in female Wistar rats was expected to be more than the limit dose level of 5000 mg/kg. 3.2. Mammalian erythrocyte in vivo micronucleus test Fig. 1 shows that there were significant statistical differences between positive controls and the other two groups (the negative controls and the treated group) at 48 and 72 h, which validates the method. There were significant statistical differences between the negative controls and the treated group at 48 h but not at 72 h of treatment. These results indicate that the extract produces a certain quantity of micronucleated cells that do not appear in the samples obtained at 72 h of treatment. 3.3. Bacterial reverse mutation test Statistical differences are significant between positive and negative controls in the specific strains TA1535 and TA1537 for the plates without metabolic activation and in the specific strains
*#
0.6
*
*
*
0.2 0 48 h
Positive Control
72 h % of Micronuclei
Negative Control
Treated
Fig. 1. Percentage of micronuclei in the different groups at different blood collection times post-treatment. The data are expressed as mean ± S.D. ⁄Significant statistical differences (p < 0.05) with the positive control. # Significant statistical differences (p < 0.05) with negative control.
TA1537, TA98 and TA100 for the plates with metabolic activation. Table 2 shows the means and standard deviations of the number of revertant colonies in treated plates and the significant statistical differences between each group and the negative controls. Only two strains showed significant differences with the negative controls: TA1537 at doses of 5 and 1.58 mg with and without metabolic activation, respectively, and TA 98 at a dose of 1.58 mg with metabolic activation. 3.4. Chromosomal aberration test Positive controls with CPA and EMS induced chromosomal aberrations; the percentage of metaphases with aberrations was 73%. However, the cultures treated with GSSE at doses of 9.7 and 19.5 lg/ml in the presence and absence of S9 mix did not significantly increase the frequency of metaphases with aberrant chromosomes with respect to the negative controls. 4. Discussion In our study we have observed no acute toxic effects of the mixed extract taken via the oral route and we conclude that the LD50 is higher than 5000 mg/kg. Our results confirm the LD50 value of a grape seed extract that was estimated by Yamakoshi et al., 2002, to be higher than 4000 mg/kg.. The bibliography shows that mutagenicity studies have provided contradictory results. In our case, mutagenicity tests showed that the extract produced slight genetic damage in the models studied. In the mammalian erythrocyte in vivo micronucleus test, the mutagens in positive controls increased the percentage of micronucleated cells 48 and 72 h after treatment. We observed fewer micronucleated cells in negative controls and in the group treated with 2000 mg/kg of GSSE, and there were significant differences with the positive controls at both blood collection times. However, the samples from the treated group taken 48 h post-treatment showed a higher percentage of micronuclei than the negative controls, which might indicate a certain degree of genotoxicity that was not discerned in the samples obtained 72 h after treatment (Fig. 1). Yamakoshi et al. (2002) showed that doses up to 2 g/kg of a proanthocyanidin-rich extract from grape seeds (GSE) administered orally to male mice did not promote an increase in micronucleated peripheral reticulocytes (Yamakoshi et al., 2002). The micronu-
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L. Lluís et al. / Food and Chemical Toxicology 49 (2011) 1450–1454 Table 2 Means and standard deviations of the number of revertant colonies in each group treated with the extract. STRAIN
Without metabolic activation
With metabolic activation
TA1535 TA1537 TA98 TA100 TA1535 TA1537 TA98 TA100
DOSE 5 mg
DOSE 1.58 mg
DOSE 0.5 mg
DOSE 0.15 mg
DOSE 0.05 mg
Col./plate
p
Col./plate
p
Col./plate
p
Col./plate
p
Col./plate
p
16 ± 3 3±1 17 ± 3 35 ± 9 16 ± 7 10 ± 2 19 ± 5 66 ± 10
0.827 0.238 0.050 0.050 0.275 0.046 0.127 0.275
22 ± 6 8±1 18 ± 6 63 ± 8 12 ± 1 4±2 19 ± 6 64 ± 3
0.376 0.043 0.050 1.000 0.127 0.099 0.046 0.050
13 ± 4 3±1 11 ± 5 61 ± 4 8±5 3±1 18 ± 15 69 ± 11
0.275 0.178 0.275 0.513 0.127 0.178 0.376 0.513
12 ± 5 3±2 11 ± 6 59 ± 8 11 ± 7 6±5 12 ± 2 79 ± 15
0.275 0.487 0.500 0.513 0.127 0.099 0.513 0.513
14 ± 3 1±1 7±3 50 ± 6 13 ± 6 2±1 8±0 64 ± 7
0.513 0.487 0.513 0.050 0.127 0.814 0.487 0.127
p = statistical differences with negative controls.
cleus assay in mouse bone marrow after a GSE administration carried out by Erexson (2003) was also negative (Erexson, 2003). Other in vitro studies showed that treating lymphocytes with 2.5 lg/ml of GSE induced a significant decrease (40%) in the frequency of micronuclei (Stankovic et al., 2008). Concentrations up to 5 mg/l did not induce chromosomal damage in a human lymphoblastoid cell line and pre-treatment with GSE prevented H2O2-induced chromosomal damage (Sugisawa et al., 2004). On the other hand, Thomas et al. (2009) did not find a significant reduction in the frequency of erythrocyte micronuclei in a transgenic mouse model for Alzheimer’s disease (AD) fed with 0.07% of curcumin or 2% of a GSE. However, there was a significant reduction in micronucleated erythrocytes in mice fed with microencapsulated grape seed extract (Thomas et al., 2009). In our study, the bacterial reversal mutation test showed an increase in the number of revertant colonies at the highest dose in the TA1537 strain with significant difference in the limit with regard to the negative control. The values were 10 ± 2 and 2 ± 3 colonies/plate, respectively. TA1537 and TA 98 also increased the number of revertant colonies at doses of 1.58 mg, but not at the highest dose. So, we conclude that this extract was weakly mutagenic to the dose of 5 mg/plate, which was the highest tested by Yamakoshi in his study (Yamakoshi et al., 2002). A dose of 5 mg/ plate in TA1537 and TA1535 strains did not increase the number of revertant colonies with respect to the negative controls. A maximum dose of 1250 lg/plate was tested in TA98 and TA100 strains and no mutagenicity was detected either. Finally, the two doses of GSSE tested in the chromosomal aberration test with human lymphocytes did not show significant differences with respect to negative controls. The doses were selected for the solubility of the extract in the culture medium and they were previously subjected to the trypan-blue test to demonstrate that they were not cytotoxic. Likewise, a grape seed extract showed no toxicity under the conditions of the assay when it was subjected to the in vitro chromosomal aberration test with Chinese hamster lung cells (Yamakoshi et al., 2002). Nevertheless, several authors describe cytotoxic effects of procianidins and grape extracts in different cells to high doses, and suggest that the strongest antioxidant products were also the most cytotoxic (Ugartondo et al., 2006; Fan and Lou, 2004; Stagos et al., 2007). 5. Conclusion Our results indicated slight mutagenicity under the study conditions, so further studies should be conducted at lower doses to demonstrate that this extract is not toxic. Role of the funding source This work was supported by the project MET-DEV-FUN (1321 U07 E10 N-MET-DEV-FUN) of the Programa para la creación de
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