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Lack of toxicological effect through mutagenicity test of polyphenol extracts from peanut shells
Food Chemistry 129 (2011) 920–924
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Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Lack of toxicological effect through mutagenicity test of polyphenol extracts from peanut shells Feng Gao a,1, Haiqing Ye b,1, Yali Yu a,c,⇑, Tiehua Zhang a, Xuming Deng c,d a
Department of Food Science and Engineering, Jilin University, Changchun 130062, China Department of Food Quality and Safety, Jilin University, Changchun 130062, China c Postdoctoral Research Station of Veterinary Medicine, Jilin University, Changchun 130062, China d College of Animal Science and Veterinary Medicine, Jilin University, Changchun 130062, China b
a r t i c l e
i n f o
Article history: Received 19 February 2011 Received in revised form 1 May 2011 Accepted 8 May 2011 Available online 12 May 2011 Keywords: Peanut shells Polyphenols Toxicological effect Mutagenicity test
a b s t r a c t The toxicological effect of polyphenols extracted from peanut shells was investigated in animal models. The safety data were needed to proceed with further clinical trials. The oral LD50 of peanut shells polyphenols was determined to be higher than 15,000 mg/kg body weight. We also carried out a sperm abnormality test, a chromosomal aberration test and a micronucleus test in rats. The peanut shell polyphenols did not cause any abnormalities in the system. Furthermore, the administration of peanut shell polyphenols did not significantly alter changes in body weight or clinical signs. These results strongly indicated that peanut shell polyphenols did not induce mutagenicity. The results of this study suggested a lack of toxicological effect and supported the further use of polyphenol-rich extracts from peanut shells as a potential natural antioxidant. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Peanut shells are an abundant and inexpensive agricultural byproduct of peanut (Arachis hypogea L.). Most of this agricultural byproduct is set on fire or arbitrarily discarded, except for the small quantity that is manufactured into medium-density fibreboard, agglutinant, plastic stuffing, etc. (Akgül & Tozluog˘lu, 2008). Peanut shells have functional components, such as polyphenols, luteolin, carotene, isosaponaretin except for coarse protein, carbohydrate and ash (Han, Han, Cai, Zhao, & Tang, 2008). Grape seed proanthocyanidins (GSE) including oligomeric proanthocyanidins (Waterhouse, Ignelzi, & Shirey, 2000) have been demonstrated to exert a plethora of beneficial health effects including chemoprotective properties against oxygen free radicals and oxidative stress, as well as being antiproliferative, anti-inflammatory, and cardioprotective (Zern & Fernandez, 2005). As a result, 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 (Jimenez-Ramsey,
Rogler, Housley, Butler, & Elkin, 1994), and some of the products of hydrolytes of the higher oligomers and polymers were presumed to be absorbed through the intestinal membrane (Scalbert & Williamson, 2000), and then the absorbed procyanidins and/or hydrolytes of procyanidins might display antioxidative activity in vivo (Koga et al., 1999). Proanthocyanidin-rich extract from grape seeds was subjected to a series of toxicological tests to document its safety for use in various foods. The LD50 (median lethal dose) value of the GSE was found to be greater than 4 g/kg in male and female rats in an oral acute toxicity study. The no-observed-adverse-effect level of GSE in the sub-chronic toxicity study was 2% in the diet. And no evidence of mutagenicity in the reverse mutation test was found. These results indicated a lack of toxicity and supported use of proanthocyanidin-rich extracts from grape seeds in various foods (Yamakoshi, Saito, Kataoka, & Kikuchi, 2002). Testing must be carried out prior to the application of polyphenols extracts from peanut shells (PEPS) in food products, in order to prove that PEPS are toxicologically safe. The overall aim of this research was to investigate if PEPS have a toxicological effect through a range of tests, and to determine their possible application as new natural antioxidants to prevent lipid oxidation in foods. 2. Materials and methods
⇑ Corresponding author at: Department of Food Science and Engineering, Jilin University, Changchun 130062, China. Tel.: +86 431 87836373; fax: +86 431 87835760. E-mail address: [email protected] (Y. Yu). 1 The first two authors should be regarded as joint First Authors. 0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2011.05.046
2.1. Manufacturing process of PEPS Polyphenol-rich extract from peanut shells was prepared. A 50g sample of peanut shells was defatted with n-hexane (3400 mL)
F. Gao et al. / Food Chemistry 129 (2011) 920–924
for 10 h at room temperature, and the n-hexane remaining in peanut shells was removed under reduced pressure in a vacuum oven. The defatted peanut shells (10.0 g) were macerated for 4 min with 250 mL aqueous ethanol (75%, v/v) in a high pressure facility (DL700, Dalong High-pressure Equipment Plant of Shanghai, China) at room temperature and 300 MPa. The extract was filtered and the residue was extracted again under the same conditions. The combined filtrates were evaporated to a final volume of 10 mL under vacuum in a rotary evaporator (RE-52 Model, Anting Electronic Instruments Plant of Shanghai, China) at 35 °C. The concentrated solution was lyophilised with a freeze dryer system (FD-1C, Kangbo Experiment Instruments Ltd. of Beijing, China) to obtain the PEPS as a brown powder.
2.2. Determination of total phenolic content The total phenolic content of PEPS was determined with the Folin–Ciocalteu method (Singleton, Orthofer, & Lamuela-Raventos, 1999). Briefly, PEPS (3 mg) were dissolved in 10 mL of sterilised water. An aliquot of 100 lL of appropriate dilution of sample extract was shaken for 1 min with 500 lL of the Folin–Ciocalteu reagent freshly prepared, and 6 mL of distilled water. After the mixture was shaken, 2 mL of 15% (w/v) sodium carbonate were added and the mixture was shaken once again for 0.5 min. Finally, the solution was brought up to 10 mL by adding distilled water. After 2 h of reaction at ambient temperature, the absorbance at 750 nm was evaluated using glass cuvettes. Using gallic acid as standard, the total phenolic content of peanut shells was expressed as a gallic acid equivalent (g gallic acid/g skin).
Table 1 The gradient elution solvent A consisted of 5% methanol (v/v) and 0.3% (v/v) acetic acid in water, solvent B consisted of 80% methanol (v/v) and 0.3% (v/v) acetic acid in water. An eluent of 74% solvent A and 26% solvent B was used for the first 15 min, followed by an eluent of 60% solvent A and 40% solvent B was used for the next 15 min, an eluent of 65% solvent A and 35% solvent B was used for the next 20 min, an eluent of 20% solvent A and 80% solvent B was used for the next 10 min, an eluent of 90% solvent A and 10% solvent B was used for the next 5 min, then returned to the initial conditions to re-equilibrate for 10 min. Total analysis time per sample was 65 min. HPLC chromatograms were detected using a photo diode array UV detector. The structures for all polyphenol were confirmed by comparison of retention time, UV spectra and mass spectral analysis. Time (min)
A (%)
B (%)
0 15 30 50 60 65
74 60 65 20 90 100
26 40 35 80 10 0
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2.3. Characterisation of PEPS PEPS characterisation was carried out using an HPLC equipped with an L-6200 intelligent pump, an SIL-10Advp autosampler, and an SPD-M10A VP, UV–Vis (all Shimadzu Ltd., Kyoto, Japan) at 280 nm with a C18 column (250 4.6 mm, 5 lm) at 30 °C. Briefly, a mixture of 10 lL 0.3% (v/v) acetic acid in water and 100% methanol was used as the mobile phase with a flow rate of 1.0 mL/min. The gradient programme is shown in Table 1. 2.4. Toxicological effect studies Acute oral toxicity test and mutagenicity tests, including a sperm abnormality test, a bone marrow cell chromosomal aberration test and a rat bone marrow cell micronucleus test were performed at the Toxicology Laboratory of Jilin University. All toxicological studies complied with the Animal Welfare Act (USDA, 1985). 2.4.1. Acute oral toxicity test Twenty male and female rats, average weight 200 g, were obtained from Jilin University Laboratory Animal Centre (Changchun, China). All rats were housed in cages in a temperature-controlled animal room (23 ± 1 °C) at a relative humidity of 55 ± 5% and were fed a standard diet. In order to determine the LD50 of PEPS, its maximum tolerance dose was measured over 14 days. The PEPS (4 g) were dissolved in 12 mL of sterilised water. Each rat was exposed to 4 mL of a dilution of PEPS by oral gavage twice every 4 h. Twelve millilitres of the dilution was administered each day. Abnormal symptoms and death of rats were observed after oral gavage and the weights of all rats were recorded. Changes in the liver and kidney were observed too. 2.4.2. Sperm abnormality test Fifty male rats aged 6–8 weeks and average weight 20 g were randomly divided into 5 groups of 10. The groups included a positive control group (the rats were injected intraperitoneally with cyclophosphamide (CP) at doses of 40 mg/kg), negative control group (the rats were given solvent at the same doses), and the test groups (the PEPS was administered by oral gavage at doses of 2.5, 5 and 10 g/kg) (Erexson, 2003; Scalbert & Williamson, 2000). All the groups were exposed continuously for 5 days. The observations were continued for 35 days. On the 36th day, the rats were killed by exarticulation. Then epididymes were sliced, and abnormalities in sperm were enumerated by the methodology of Hemavathi and Rahiman using light microscope.
Fig. 1. Profiles of PEPS by HPLC: (1) pyrogallol; (2) catechol; (3) phlorglucinol; (4) quercetin; and (5) luteolin.
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Table 2 Sperm abnormality findings (means ± S.D.) after 5 days oral administration of PEPS. (a) Result of sperm abnormality test. (b) Multiple comparisons in different groups using Dunnett’s t method. Group
Animal number
Sperm number
Abnormality number
Frequency of abnormality (%)
(a) Positive control Negative control High dose Moderate dose Low dose
10 10 10 10 10
1000 10 1000 10 1000 10 1000 10 1000 10
80 13 14 13 13
8.0 ± 0.5 1.3 ± 0.1 1.4 ± 0.2 1.3 ± 0.1 1.3 ± 0.1
(I) Group
(J) Group
Mean difference (I
J)
Std. Error
Sig.
Confidence interval Lower bound
(b) Positive control
*
Upper bound
Negative control High dose Moderate dose Low dose
6.63000* 6.64000* 6.60000* 6.64000*
0.52393 0.53166 0.54148 0.52387
0.000 0.000 0.000 0.000
4.1913 4.2163 4.1880 4.2012
9.0687 9.0637 9.0120 9.0788
Negative control
Positive control High dose Moderate dose Low dose
6.63000* 0.01000 0.03000 0.01000
0.52393 0.16114 0.19105 0.13321
0.000 1.000 1.000 1.000
9.0687 0.6335 0.8171 0.5121
4.1913 0.6535 0.7571 0.5321
High dose
Positive control Negative control Moderate dose Low dose
6.64000* 0.01000 0.04000 0.00000
0.53166 0.16114 0.21134 0.16097
0.000 1.000 1.000 1.000
9.0637 0.6535 0.8771 0.6430
4.2163 0.6335 0.7971 0.6430
Moderate dose
Positive control Negative control High dose Low dose
6.60000* 0.03000 0.04000 0.04000
0.54148 0.19105 0.21134 0.19090
0.000 1.000 1.000 1.000
9.0120 -0.7571 0.7971 0.7468
4.1880 0.8171 0.8771 0.8268
Low dose
Positive control Negative control High dose Moderate dose
6.64000* 0.01000 0.00000 0.04000
0.52387 0.13321 0.16097 0.19090
0.000 1.000 1.000 1.000
9.0788 0.5321 0.6430 0.8268
4.2012 0.5121 0.6430 0.7468
The mean difference is significant at the 0.01 level.
Table 3 Bone marrow cell chromosomal aberration findings (means ± S.D.) after 3 days oral administration of PEPS. (a) Result of bone marrow cell chromosomal aberration test. (b) Multiple Comparisons in different groups using Dunnett’s t method. Group
Animal number
Cell number
Aberration number
Frequency of aberration (%)
(a) High dose Moderate dose Low dose Positive control Negative control
10 10 10 10 10
1000 10 1000 10 1000 10 1000 10 1000 10
10 9 11 78 10
1.0 ± 0.3 0.9 ± 0.2 1.1 ± 0.3 7.8 ± 0.5 1.0 ± 0.0
(I) Group
(J) Group
Mean Difference (I-J)
Std. Error
Sig.
Confidence Interval Lower Bound
(b) Positive control
*
Upper Bound
Negative control High dose Moderate dose Low dose
5.74444* 6.44444* 6.64444* 6.44444*
0.64207 0.64207 0.64207 0.64207
0.000 0.000 0.000 0.000
3.9183 4.6183 4.8183 4.6183
7.5706 8.2706 8.4706 8.2706
Negative control
Positive control High dose Moderate dose Low dose
5.74444* 0.70000 0.90000 0.70000
0.64207 0.62494 0.62494 0.62494
0.000 0.795 0.606 0.795
7.5706 1.0774 .8774 1.0774
3.9183 2.4774 2.6774 2.4774
High dose
Positive control Negative control Moderate dose Low dose
6.44444* 0.70000 0.20000 0.00000
0.64207 0.62494 0.62494 0.62494
0.000 0.795 0.998 1.000
8.2706 2.4774 1.5774 1.7774
4.6183 1.0774 1.9774 1.7774
Moderate dose
Positive control Negative control High dose Low dose
6.64444* 0.90000 0.20000 0.20000
0.64207 0.62494 0.62494 0.62494
0.000 0.606 0.998 0.998
8.4706 2.6774 1.9774 1.9774
4.8183 0.8774 1.5774 1.5774
Low dose
Positive control Negative control High dose Moderate dose
6.44444* 0.70000 0.00000 0.20000
0.64207 0.62494 0.62494 0.62494
0.000 0.795 1.000 0.998
8.2706 2.4774 1.7774 1.5774
4.6183 1.0774 1.7774 1.9774
The mean difference is significant at the 0.05 level.
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Table 4 Bone marrow cell micronucleus aberration findings (means ± S.D.) after 3 days oral administration of PEPS. (a) Result of bone marrow cell micronucleus aberration test. (b) Multiple Comparisons in different groups using Dunnett’s t method. Group (a) Positive control Negative control High dose Moderate dose Low dose (I) Group
Sex
Animal number
PCE number
Micronucleus number
Frequency of PCEs (‰)
Female Male Female Male Female Male Female Male Female Male
5 5 5 5 5 5 5 5 5 5
1000 5 1000 5 1000 5 1000 5 1000 5 1000 5 1000 5 1000 5 1000 5 1000 5
129 131 7 6 5 7 8 9 7 6
26.0 ± 0.5
(J) Group
Mean difference (I
J)
Std. Error
1.3 ± 0.2 1.2 ± 0.6 1.7 ± 0.8 1.3 ± 0.8 Sig.
Confidence interval Lower bound
(b) Positive control
*
Upper bound
Negative control High dose Moderate dose Low dose
24.70000* 24.80000* 24.30000* 24.70000*
0.49554 0.51208 0.53852 0.53852
0.000 0.000 0.000 0.000
22.9689 23.0550 22.5158 22.9158
26.4311 26.5450 26.0842 26.4842
Negative control
Positive control High dose Moderate dose Low dose
24.70000* 0.10000 0.40000 0.00000
0.49554 0.25166 0.30185 0.30185
0.000 1.000 0.900 1.000
26.4311 0.7094 1.3937 0.9937
22.9689 0.9094 0.5937 0.9937
High dose
Positive control Negative control Moderate dose Low dose
24.80000* 0.10000 0.50000 0.10000
0.51208 0.25166 0.32830 0.32830
0.000 1.000 0.794 1.000
26.5450 0.9094 1.5555 1.1555
23.0550 0.7094 0.5555 0.9555
Moderate dose
Positive control Negative control High dose Low dose
24.30000* 0.40000 0.50000 0.40000
0.53852 0.30185 0.32830 0.36818
0.000 0.900 0.794 0.968
26.0842 0.5937 0.5555 0.7731
22.5158 1.3937 1.5555 1.5731
Low dose
Positive control Negative control High dose Moderate dose
24.70000* 0.00000 0.10000 0.40000
0.53852 0.30185 0.32830 0.36818
0.000 1.000 1.000 0.968
26.4842 0.9937 0.9555 1.5731
22.9158 0.9937 1.1555 0.7731
The mean difference is significant at the 0.05 level.
2.4.3. Bone marrow cell chromosomal aberration test The rats and groups in this test were the same as Section 2.4.2. The groups were positive control group (the rats were injected intraperitoneally with cyclophosphamide (CP) at doses of 40 mg/ kg), negative control group (rats given solvent at the same doses), and the test groups (PEPS was administered by oral gavage at doses of 0.1, 2 and 10 g/kg) (Kavanagh et al., 2001; Scalbert et al., 2000). All the groups were exposed continuously for 3 days. On the 4th day, all rats were killed by exarticulation. One thighbone was sliced, and observed using oil immersion lens. At the each sampling time, 100 metaphases were analysed. 2.4.4. Bone marrow cell micronucleus aberration test The rats and groups in this test were the same as Section 2.4.2. The groups were positive control group (rats injected intraperitoneally with cyclophosphamide (CP) at doses of 40 mg/kg), negative control group (rats were given solvent at the same doses), and the test groups (PEPS was administered by oral gavage at doses of 0.1, 2 and 10 g/kg) (Yamakoshi et al., 2002). All the groups were exposed continuously for 3 days. On the 4th day, all the rats were killed by exarticulation. The breastbone was sliced, and observed using oil immersion lens. Genotoxicity was evaluated by measuring the frequency of polychromatic erythrocyte (PCE) cells in bone marrow. 2.5. Statistical analysis The frequency of mutagenicity tests, including a sperm abnormality test, a bone marrow cell chromosomal aberration test and
a bone marrow cell micronucleus aberration test were measured in the form of means ± S.D. Dunnett’s t method was used for multiple comparisons in different groups. The confidence interval was 99% in the sperm abnormality test and 95% in the other two mutagenicity tests.
3. Results and discussion 3.1. Characterisation of PEPS HPLC was used to characterise PEPS (Fig. 1), a complex mixture, mostly made up of luteolin, pyrogallol, catechol, phlorglucinol, and quercetin, with luteolin present at the highest amount. The total phenolic content of PEPS was 71.3 mg/g.
3.2. Acute oral toxicity test All rats treated with PEPS survived the 14-day observation period. There were no significant differences in weight between different groups and no rats died during this period. No changes were observed to the naked eye in organs studied (liver and kidney) at necropsy on the 14th day (data not shown). Therefore, the acute oral minimum lethal dose of PEPS for rats is >15,000 mg/kg body weight according to acute toxicity test (Standardization Administration of China, GB 15193.3-2003).
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The sperm abnormality test did not reveal any abnormalities with PEPS. No abnormal sperm was observed at any dose levels of PEPS (Table 2).
PEPS was added to the juice as a kind antioxidant at 1 mg/ml, about 1000 ml juice would need to be consumed. But an average juice intake 2000–3000 ml of per day. Therefore, the average total dietary intake of polyphenols for human is estimated to be considerably less than those levels that were shown to be safe in our analyses.
3.4. Bone marrow cell chromosomal aberration test
Acknowledgements
The chromosomal aberration test using bone marrow cells of rats did not reveal any abnormalities with PEPS. The frequency of aberration in the bone marrow of rat given PEPS was not significantly different from the negative control (Table 3).
We wish to thank Toxicology Laboratory of Jilin University and Jilin University Laboratory Animal Centre, for their helpful discussion and suggestions. The authors also wish to thank Ministry of Science, Technology and Innovation for the grant provided under the Intensified Research in Priority Areas Research Grant. This study was supported by Jilin Natural Science Foundation of China (20100137).
3.3. Sperm abnormality test
3.5. Bone marrow cell micronucleus aberration test In micronucleus testing in vivo, the general conditions of all test animals were observed, and body weight and dietary consumption were measured periodically. None of the rats died in any of the groups administered with PEPS. At least 5000 PCEs per animal were analysed for the frequency of micronucleus. No statistically significant increase in micronucleated PCEs was observed at any dose levels of PEPS. The frequency of PCEs in the bone marrow of rat given PEPS was not significantly different from the negative control (Table 4). 4. Conclusion Oral acute toxicity result indicated that no macroscopic abnormalities were found in any of the surviving animals. From this study, the oral LD50 of PEPS was determined to be higher than 15,000 mg/kg body weight. No abnormal symptoms or effects on body weight, food consumption, clinical chemistry or histopathological parameters were associated with a dose of 15,000 mg/kg. More recently, polyphenols intake has been assessed using dietary diaries. Total intake of polyphenols can be calculated using data on the polyphenols concentration and consumption of foods and beverages. The mean daily polyphenols intake among Finnish adults is 863 mg, with phenolic acids representing 75% of total intake, followed by proanthocyanidins (14%), anthocyanidins and other flavonoids (10%). The main polyphenols sources in this sample were coffee, cereals, fruits, mostly berries and berry products (Ovaskainen et al., 2008). The mean daily polyphenols intake from fruits and vegetables among French adults is 219 mg (males), 193 mg (females) from fruit and 78 mg (males), 67 mg (females) from vegetables (Brat et al., 2006). Scalbert and Williamson (2000) reported total dietary intake of polyphenols to be approximately 1 g/day, regardless of the methods of polyphenols concentration used such as HPLC or the Folin–Ciocalteu method. Our analysis of the total polyphenols content in peanut shell cultivated in China revealed 70.3 mg as gallic acid/1 g of peanut shells. And 1 mg/ml of PEPS can exhibit remarkably antioxidant ability. Assuming an average polyphenols intake 1 g of per day,
References Akgül, M., & Tozluog˘lu, A. (2008). Utilizing peanut husk (Arachis hypogaea L.) in the manufacture of medium-density fiberboards. Bioresource Technology, 99, 5590–5594. Brat, P., George, S., Bellamy, A., Du Chaffaut, L., Scalbert, A., Mennen, L., et al. (2006). Daily polyphenol intake in france from fruit and vegetables. Journal of Nutrition, 136, 2368–2373. Erexson, G. L. (2003). Lack of in vivo clastogenic activity of grape seed and grape skin extracts in a mouse micronucleus assay. Food and Chemical Toxicology, 41, 347–350. Han, Runping, Han, Pan, Cai, Zhaohui, Zhao, Zhenhui, & Tang, Mingsheng (2008). Kinetics and isotherms of Neutral Red adsorption on peanut husk. Journal of Environmental Sciences, 20, 1035–1041. Jimenez-Ramsey, L. M., Rogler, J. C., Housley, T. L., Butler, L. G., & Elkin, R. G. (1994). Absorption and distribution of 14C-labeled condensed tannins and related sorghum phenolics in chickens. Journal of Agricultural and Food Chemistry, 42, 963–967. Kavanagh, K. T., Hafer, L. J., Kim, D. W., Mann, K. K., Sherr, D. H., Rogers, A. E., et al. (2001). Green tea extracts decrease carcinogen-induced mammary tumor burden in rats and rate of breast cancer cell proliferation in culture. Journal of Cellular Biochemistry, 82, 387–398. Koga, T., Moro, K., Nakamori, K., Yamakoshi, J., Hosoyama, H., Kataoka, S., et al. (1999). Increase of antioxidative potential of rat plasma by oral administration of proanthocyanidin-rich extract from grape seeds. Journal of Agricultural and Food Chemistry, 47, 1892–1897. Ovaskainen, M. L., Törrönen, R., Koponen, J. M., Sinkko, H., Hellström, J., Reinivuo, H., et al. (2008). Dietary intake and major food sources of polyphenols in Finnish adults. Journal of Nutrition, 138, 562–566. Scalbert, A., & Williamson, G. (2000). Dietary intake and bioavailability of polyphenols. The Journal of Nutrition, 130, 2073S–2085S. Singleton, V. L., Orthofer, R., & Lamuela-Raventos, R. M. (1999). Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin– Ciocalteau reagent. Methods of Enzymology, 299, 152–178. Standardization Administration of The People’s Republic of China. (2004). GB 15193.3-2003. Acute toxicity test.
Contents lists available at ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Lack of toxicological effect through mutagenicity test of polyphenol extracts from peanut shells Feng Gao a,1, Haiqing Ye b,1, Yali Yu a,c,⇑, Tiehua Zhang a, Xuming Deng c,d a
Department of Food Science and Engineering, Jilin University, Changchun 130062, China Department of Food Quality and Safety, Jilin University, Changchun 130062, China c Postdoctoral Research Station of Veterinary Medicine, Jilin University, Changchun 130062, China d College of Animal Science and Veterinary Medicine, Jilin University, Changchun 130062, China b
a r t i c l e
i n f o
Article history: Received 19 February 2011 Received in revised form 1 May 2011 Accepted 8 May 2011 Available online 12 May 2011 Keywords: Peanut shells Polyphenols Toxicological effect Mutagenicity test
a b s t r a c t The toxicological effect of polyphenols extracted from peanut shells was investigated in animal models. The safety data were needed to proceed with further clinical trials. The oral LD50 of peanut shells polyphenols was determined to be higher than 15,000 mg/kg body weight. We also carried out a sperm abnormality test, a chromosomal aberration test and a micronucleus test in rats. The peanut shell polyphenols did not cause any abnormalities in the system. Furthermore, the administration of peanut shell polyphenols did not significantly alter changes in body weight or clinical signs. These results strongly indicated that peanut shell polyphenols did not induce mutagenicity. The results of this study suggested a lack of toxicological effect and supported the further use of polyphenol-rich extracts from peanut shells as a potential natural antioxidant. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Peanut shells are an abundant and inexpensive agricultural byproduct of peanut (Arachis hypogea L.). Most of this agricultural byproduct is set on fire or arbitrarily discarded, except for the small quantity that is manufactured into medium-density fibreboard, agglutinant, plastic stuffing, etc. (Akgül & Tozluog˘lu, 2008). Peanut shells have functional components, such as polyphenols, luteolin, carotene, isosaponaretin except for coarse protein, carbohydrate and ash (Han, Han, Cai, Zhao, & Tang, 2008). Grape seed proanthocyanidins (GSE) including oligomeric proanthocyanidins (Waterhouse, Ignelzi, & Shirey, 2000) have been demonstrated to exert a plethora of beneficial health effects including chemoprotective properties against oxygen free radicals and oxidative stress, as well as being antiproliferative, anti-inflammatory, and cardioprotective (Zern & Fernandez, 2005). As a result, 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 (Jimenez-Ramsey,
Rogler, Housley, Butler, & Elkin, 1994), and some of the products of hydrolytes of the higher oligomers and polymers were presumed to be absorbed through the intestinal membrane (Scalbert & Williamson, 2000), and then the absorbed procyanidins and/or hydrolytes of procyanidins might display antioxidative activity in vivo (Koga et al., 1999). Proanthocyanidin-rich extract from grape seeds was subjected to a series of toxicological tests to document its safety for use in various foods. The LD50 (median lethal dose) value of the GSE was found to be greater than 4 g/kg in male and female rats in an oral acute toxicity study. The no-observed-adverse-effect level of GSE in the sub-chronic toxicity study was 2% in the diet. And no evidence of mutagenicity in the reverse mutation test was found. These results indicated a lack of toxicity and supported use of proanthocyanidin-rich extracts from grape seeds in various foods (Yamakoshi, Saito, Kataoka, & Kikuchi, 2002). Testing must be carried out prior to the application of polyphenols extracts from peanut shells (PEPS) in food products, in order to prove that PEPS are toxicologically safe. The overall aim of this research was to investigate if PEPS have a toxicological effect through a range of tests, and to determine their possible application as new natural antioxidants to prevent lipid oxidation in foods. 2. Materials and methods
⇑ Corresponding author at: Department of Food Science and Engineering, Jilin University, Changchun 130062, China. Tel.: +86 431 87836373; fax: +86 431 87835760. E-mail address: [email protected] (Y. Yu). 1 The first two authors should be regarded as joint First Authors. 0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2011.05.046
2.1. Manufacturing process of PEPS Polyphenol-rich extract from peanut shells was prepared. A 50g sample of peanut shells was defatted with n-hexane (3400 mL)
F. Gao et al. / Food Chemistry 129 (2011) 920–924
for 10 h at room temperature, and the n-hexane remaining in peanut shells was removed under reduced pressure in a vacuum oven. The defatted peanut shells (10.0 g) were macerated for 4 min with 250 mL aqueous ethanol (75%, v/v) in a high pressure facility (DL700, Dalong High-pressure Equipment Plant of Shanghai, China) at room temperature and 300 MPa. The extract was filtered and the residue was extracted again under the same conditions. The combined filtrates were evaporated to a final volume of 10 mL under vacuum in a rotary evaporator (RE-52 Model, Anting Electronic Instruments Plant of Shanghai, China) at 35 °C. The concentrated solution was lyophilised with a freeze dryer system (FD-1C, Kangbo Experiment Instruments Ltd. of Beijing, China) to obtain the PEPS as a brown powder.
2.2. Determination of total phenolic content The total phenolic content of PEPS was determined with the Folin–Ciocalteu method (Singleton, Orthofer, & Lamuela-Raventos, 1999). Briefly, PEPS (3 mg) were dissolved in 10 mL of sterilised water. An aliquot of 100 lL of appropriate dilution of sample extract was shaken for 1 min with 500 lL of the Folin–Ciocalteu reagent freshly prepared, and 6 mL of distilled water. After the mixture was shaken, 2 mL of 15% (w/v) sodium carbonate were added and the mixture was shaken once again for 0.5 min. Finally, the solution was brought up to 10 mL by adding distilled water. After 2 h of reaction at ambient temperature, the absorbance at 750 nm was evaluated using glass cuvettes. Using gallic acid as standard, the total phenolic content of peanut shells was expressed as a gallic acid equivalent (g gallic acid/g skin).
Table 1 The gradient elution solvent A consisted of 5% methanol (v/v) and 0.3% (v/v) acetic acid in water, solvent B consisted of 80% methanol (v/v) and 0.3% (v/v) acetic acid in water. An eluent of 74% solvent A and 26% solvent B was used for the first 15 min, followed by an eluent of 60% solvent A and 40% solvent B was used for the next 15 min, an eluent of 65% solvent A and 35% solvent B was used for the next 20 min, an eluent of 20% solvent A and 80% solvent B was used for the next 10 min, an eluent of 90% solvent A and 10% solvent B was used for the next 5 min, then returned to the initial conditions to re-equilibrate for 10 min. Total analysis time per sample was 65 min. HPLC chromatograms were detected using a photo diode array UV detector. The structures for all polyphenol were confirmed by comparison of retention time, UV spectra and mass spectral analysis. Time (min)
A (%)
B (%)
0 15 30 50 60 65
74 60 65 20 90 100
26 40 35 80 10 0
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2.3. Characterisation of PEPS PEPS characterisation was carried out using an HPLC equipped with an L-6200 intelligent pump, an SIL-10Advp autosampler, and an SPD-M10A VP, UV–Vis (all Shimadzu Ltd., Kyoto, Japan) at 280 nm with a C18 column (250 4.6 mm, 5 lm) at 30 °C. Briefly, a mixture of 10 lL 0.3% (v/v) acetic acid in water and 100% methanol was used as the mobile phase with a flow rate of 1.0 mL/min. The gradient programme is shown in Table 1. 2.4. Toxicological effect studies Acute oral toxicity test and mutagenicity tests, including a sperm abnormality test, a bone marrow cell chromosomal aberration test and a rat bone marrow cell micronucleus test were performed at the Toxicology Laboratory of Jilin University. All toxicological studies complied with the Animal Welfare Act (USDA, 1985). 2.4.1. Acute oral toxicity test Twenty male and female rats, average weight 200 g, were obtained from Jilin University Laboratory Animal Centre (Changchun, China). All rats were housed in cages in a temperature-controlled animal room (23 ± 1 °C) at a relative humidity of 55 ± 5% and were fed a standard diet. In order to determine the LD50 of PEPS, its maximum tolerance dose was measured over 14 days. The PEPS (4 g) were dissolved in 12 mL of sterilised water. Each rat was exposed to 4 mL of a dilution of PEPS by oral gavage twice every 4 h. Twelve millilitres of the dilution was administered each day. Abnormal symptoms and death of rats were observed after oral gavage and the weights of all rats were recorded. Changes in the liver and kidney were observed too. 2.4.2. Sperm abnormality test Fifty male rats aged 6–8 weeks and average weight 20 g were randomly divided into 5 groups of 10. The groups included a positive control group (the rats were injected intraperitoneally with cyclophosphamide (CP) at doses of 40 mg/kg), negative control group (the rats were given solvent at the same doses), and the test groups (the PEPS was administered by oral gavage at doses of 2.5, 5 and 10 g/kg) (Erexson, 2003; Scalbert & Williamson, 2000). All the groups were exposed continuously for 5 days. The observations were continued for 35 days. On the 36th day, the rats were killed by exarticulation. Then epididymes were sliced, and abnormalities in sperm were enumerated by the methodology of Hemavathi and Rahiman using light microscope.
Fig. 1. Profiles of PEPS by HPLC: (1) pyrogallol; (2) catechol; (3) phlorglucinol; (4) quercetin; and (5) luteolin.
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Table 2 Sperm abnormality findings (means ± S.D.) after 5 days oral administration of PEPS. (a) Result of sperm abnormality test. (b) Multiple comparisons in different groups using Dunnett’s t method. Group
Animal number
Sperm number
Abnormality number
Frequency of abnormality (%)
(a) Positive control Negative control High dose Moderate dose Low dose
10 10 10 10 10
1000 10 1000 10 1000 10 1000 10 1000 10
80 13 14 13 13
8.0 ± 0.5 1.3 ± 0.1 1.4 ± 0.2 1.3 ± 0.1 1.3 ± 0.1
(I) Group
(J) Group
Mean difference (I
J)
Std. Error
Sig.
Confidence interval Lower bound
(b) Positive control
*
Upper bound
Negative control High dose Moderate dose Low dose
6.63000* 6.64000* 6.60000* 6.64000*
0.52393 0.53166 0.54148 0.52387
0.000 0.000 0.000 0.000
4.1913 4.2163 4.1880 4.2012
9.0687 9.0637 9.0120 9.0788
Negative control
Positive control High dose Moderate dose Low dose
6.63000* 0.01000 0.03000 0.01000
0.52393 0.16114 0.19105 0.13321
0.000 1.000 1.000 1.000
9.0687 0.6335 0.8171 0.5121
4.1913 0.6535 0.7571 0.5321
High dose
Positive control Negative control Moderate dose Low dose
6.64000* 0.01000 0.04000 0.00000
0.53166 0.16114 0.21134 0.16097
0.000 1.000 1.000 1.000
9.0637 0.6535 0.8771 0.6430
4.2163 0.6335 0.7971 0.6430
Moderate dose
Positive control Negative control High dose Low dose
6.60000* 0.03000 0.04000 0.04000
0.54148 0.19105 0.21134 0.19090
0.000 1.000 1.000 1.000
9.0120 -0.7571 0.7971 0.7468
4.1880 0.8171 0.8771 0.8268
Low dose
Positive control Negative control High dose Moderate dose
6.64000* 0.01000 0.00000 0.04000
0.52387 0.13321 0.16097 0.19090
0.000 1.000 1.000 1.000
9.0788 0.5321 0.6430 0.8268
4.2012 0.5121 0.6430 0.7468
The mean difference is significant at the 0.01 level.
Table 3 Bone marrow cell chromosomal aberration findings (means ± S.D.) after 3 days oral administration of PEPS. (a) Result of bone marrow cell chromosomal aberration test. (b) Multiple Comparisons in different groups using Dunnett’s t method. Group
Animal number
Cell number
Aberration number
Frequency of aberration (%)
(a) High dose Moderate dose Low dose Positive control Negative control
10 10 10 10 10
1000 10 1000 10 1000 10 1000 10 1000 10
10 9 11 78 10
1.0 ± 0.3 0.9 ± 0.2 1.1 ± 0.3 7.8 ± 0.5 1.0 ± 0.0
(I) Group
(J) Group
Mean Difference (I-J)
Std. Error
Sig.
Confidence Interval Lower Bound
(b) Positive control
*
Upper Bound
Negative control High dose Moderate dose Low dose
5.74444* 6.44444* 6.64444* 6.44444*
0.64207 0.64207 0.64207 0.64207
0.000 0.000 0.000 0.000
3.9183 4.6183 4.8183 4.6183
7.5706 8.2706 8.4706 8.2706
Negative control
Positive control High dose Moderate dose Low dose
5.74444* 0.70000 0.90000 0.70000
0.64207 0.62494 0.62494 0.62494
0.000 0.795 0.606 0.795
7.5706 1.0774 .8774 1.0774
3.9183 2.4774 2.6774 2.4774
High dose
Positive control Negative control Moderate dose Low dose
6.44444* 0.70000 0.20000 0.00000
0.64207 0.62494 0.62494 0.62494
0.000 0.795 0.998 1.000
8.2706 2.4774 1.5774 1.7774
4.6183 1.0774 1.9774 1.7774
Moderate dose
Positive control Negative control High dose Low dose
6.64444* 0.90000 0.20000 0.20000
0.64207 0.62494 0.62494 0.62494
0.000 0.606 0.998 0.998
8.4706 2.6774 1.9774 1.9774
4.8183 0.8774 1.5774 1.5774
Low dose
Positive control Negative control High dose Moderate dose
6.44444* 0.70000 0.00000 0.20000
0.64207 0.62494 0.62494 0.62494
0.000 0.795 1.000 0.998
8.2706 2.4774 1.7774 1.5774
4.6183 1.0774 1.7774 1.9774
The mean difference is significant at the 0.05 level.
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Table 4 Bone marrow cell micronucleus aberration findings (means ± S.D.) after 3 days oral administration of PEPS. (a) Result of bone marrow cell micronucleus aberration test. (b) Multiple Comparisons in different groups using Dunnett’s t method. Group (a) Positive control Negative control High dose Moderate dose Low dose (I) Group
Sex
Animal number
PCE number
Micronucleus number
Frequency of PCEs (‰)
Female Male Female Male Female Male Female Male Female Male
5 5 5 5 5 5 5 5 5 5
1000 5 1000 5 1000 5 1000 5 1000 5 1000 5 1000 5 1000 5 1000 5 1000 5
129 131 7 6 5 7 8 9 7 6
26.0 ± 0.5
(J) Group
Mean difference (I
J)
Std. Error
1.3 ± 0.2 1.2 ± 0.6 1.7 ± 0.8 1.3 ± 0.8 Sig.
Confidence interval Lower bound
(b) Positive control
*
Upper bound
Negative control High dose Moderate dose Low dose
24.70000* 24.80000* 24.30000* 24.70000*
0.49554 0.51208 0.53852 0.53852
0.000 0.000 0.000 0.000
22.9689 23.0550 22.5158 22.9158
26.4311 26.5450 26.0842 26.4842
Negative control
Positive control High dose Moderate dose Low dose
24.70000* 0.10000 0.40000 0.00000
0.49554 0.25166 0.30185 0.30185
0.000 1.000 0.900 1.000
26.4311 0.7094 1.3937 0.9937
22.9689 0.9094 0.5937 0.9937
High dose
Positive control Negative control Moderate dose Low dose
24.80000* 0.10000 0.50000 0.10000
0.51208 0.25166 0.32830 0.32830
0.000 1.000 0.794 1.000
26.5450 0.9094 1.5555 1.1555
23.0550 0.7094 0.5555 0.9555
Moderate dose
Positive control Negative control High dose Low dose
24.30000* 0.40000 0.50000 0.40000
0.53852 0.30185 0.32830 0.36818
0.000 0.900 0.794 0.968
26.0842 0.5937 0.5555 0.7731
22.5158 1.3937 1.5555 1.5731
Low dose
Positive control Negative control High dose Moderate dose
24.70000* 0.00000 0.10000 0.40000
0.53852 0.30185 0.32830 0.36818
0.000 1.000 1.000 0.968
26.4842 0.9937 0.9555 1.5731
22.9158 0.9937 1.1555 0.7731
The mean difference is significant at the 0.05 level.
2.4.3. Bone marrow cell chromosomal aberration test The rats and groups in this test were the same as Section 2.4.2. The groups were positive control group (the rats were injected intraperitoneally with cyclophosphamide (CP) at doses of 40 mg/ kg), negative control group (rats given solvent at the same doses), and the test groups (PEPS was administered by oral gavage at doses of 0.1, 2 and 10 g/kg) (Kavanagh et al., 2001; Scalbert et al., 2000). All the groups were exposed continuously for 3 days. On the 4th day, all rats were killed by exarticulation. One thighbone was sliced, and observed using oil immersion lens. At the each sampling time, 100 metaphases were analysed. 2.4.4. Bone marrow cell micronucleus aberration test The rats and groups in this test were the same as Section 2.4.2. The groups were positive control group (rats injected intraperitoneally with cyclophosphamide (CP) at doses of 40 mg/kg), negative control group (rats were given solvent at the same doses), and the test groups (PEPS was administered by oral gavage at doses of 0.1, 2 and 10 g/kg) (Yamakoshi et al., 2002). All the groups were exposed continuously for 3 days. On the 4th day, all the rats were killed by exarticulation. The breastbone was sliced, and observed using oil immersion lens. Genotoxicity was evaluated by measuring the frequency of polychromatic erythrocyte (PCE) cells in bone marrow. 2.5. Statistical analysis The frequency of mutagenicity tests, including a sperm abnormality test, a bone marrow cell chromosomal aberration test and
a bone marrow cell micronucleus aberration test were measured in the form of means ± S.D. Dunnett’s t method was used for multiple comparisons in different groups. The confidence interval was 99% in the sperm abnormality test and 95% in the other two mutagenicity tests.
3. Results and discussion 3.1. Characterisation of PEPS HPLC was used to characterise PEPS (Fig. 1), a complex mixture, mostly made up of luteolin, pyrogallol, catechol, phlorglucinol, and quercetin, with luteolin present at the highest amount. The total phenolic content of PEPS was 71.3 mg/g.
3.2. Acute oral toxicity test All rats treated with PEPS survived the 14-day observation period. There were no significant differences in weight between different groups and no rats died during this period. No changes were observed to the naked eye in organs studied (liver and kidney) at necropsy on the 14th day (data not shown). Therefore, the acute oral minimum lethal dose of PEPS for rats is >15,000 mg/kg body weight according to acute toxicity test (Standardization Administration of China, GB 15193.3-2003).
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The sperm abnormality test did not reveal any abnormalities with PEPS. No abnormal sperm was observed at any dose levels of PEPS (Table 2).
PEPS was added to the juice as a kind antioxidant at 1 mg/ml, about 1000 ml juice would need to be consumed. But an average juice intake 2000–3000 ml of per day. Therefore, the average total dietary intake of polyphenols for human is estimated to be considerably less than those levels that were shown to be safe in our analyses.
3.4. Bone marrow cell chromosomal aberration test
Acknowledgements
The chromosomal aberration test using bone marrow cells of rats did not reveal any abnormalities with PEPS. The frequency of aberration in the bone marrow of rat given PEPS was not significantly different from the negative control (Table 3).
We wish to thank Toxicology Laboratory of Jilin University and Jilin University Laboratory Animal Centre, for their helpful discussion and suggestions. The authors also wish to thank Ministry of Science, Technology and Innovation for the grant provided under the Intensified Research in Priority Areas Research Grant. This study was supported by Jilin Natural Science Foundation of China (20100137).
3.3. Sperm abnormality test
3.5. Bone marrow cell micronucleus aberration test In micronucleus testing in vivo, the general conditions of all test animals were observed, and body weight and dietary consumption were measured periodically. None of the rats died in any of the groups administered with PEPS. At least 5000 PCEs per animal were analysed for the frequency of micronucleus. No statistically significant increase in micronucleated PCEs was observed at any dose levels of PEPS. The frequency of PCEs in the bone marrow of rat given PEPS was not significantly different from the negative control (Table 4). 4. Conclusion Oral acute toxicity result indicated that no macroscopic abnormalities were found in any of the surviving animals. From this study, the oral LD50 of PEPS was determined to be higher than 15,000 mg/kg body weight. No abnormal symptoms or effects on body weight, food consumption, clinical chemistry or histopathological parameters were associated with a dose of 15,000 mg/kg. More recently, polyphenols intake has been assessed using dietary diaries. Total intake of polyphenols can be calculated using data on the polyphenols concentration and consumption of foods and beverages. The mean daily polyphenols intake among Finnish adults is 863 mg, with phenolic acids representing 75% of total intake, followed by proanthocyanidins (14%), anthocyanidins and other flavonoids (10%). The main polyphenols sources in this sample were coffee, cereals, fruits, mostly berries and berry products (Ovaskainen et al., 2008). The mean daily polyphenols intake from fruits and vegetables among French adults is 219 mg (males), 193 mg (females) from fruit and 78 mg (males), 67 mg (females) from vegetables (Brat et al., 2006). Scalbert and Williamson (2000) reported total dietary intake of polyphenols to be approximately 1 g/day, regardless of the methods of polyphenols concentration used such as HPLC or the Folin–Ciocalteu method. Our analysis of the total polyphenols content in peanut shell cultivated in China revealed 70.3 mg as gallic acid/1 g of peanut shells. And 1 mg/ml of PEPS can exhibit remarkably antioxidant ability. Assuming an average polyphenols intake 1 g of per day,
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