Research Paper. Academia Journal of Medicinal Plants 4(8): , July 2016 DOI: /ajmp ISSN: Academia Publishing

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1 Academia Journal of Medicinal Plants 4(8): , July 2016 DOI: /ajmp ISSN: Academia Publishing Research Paper Protective Effects of the Anthocyanin-Rich Fraction of Shinzami Purple Sweet Potato against Oxidative Stress and Inflammation in Mice Fed a High-Fat Diet Accepted 20 th May, 2016 ABSTRACT Song Yee Nam 1, Young Min Lee 2*, Mi Ju Kim 1, Jung Bong Kim 1, Hwan Hee Jang 1, Shin Young Park 1, Kye Won Park 3 and Sung Hyen Lee 1** 1Department of Agro-food Resources, National Institute of Agricultural Sciences, Rural Development Administration, Wanju 55365, Republic of Korea. 2Major of Food & Nutrition, Division of Applied Food System, Seoul Women s University, Seoul, 01797, Korea. 3Department of Food Science and Biotechnology, Sungkyunkwan University, Suwon 16419, Korea. *Corresponding author. ymlee@swu.ac.kr *, lshin@korea.kr ** Anthocyanins, a class of flavonoids, are natural water-soluble pigments found in vegetables and fruits. They have attractive pharmacological activities such as anti-oxidant, anti-inflammatory, and anti-obesity effects. This study evaluated the effects of the anthocyanin-rich fraction (ARF) of the purple sweet potato variety Shinzami against oxidative stress and inflammation in mice fed a high-fat diet (HD). Mice were fed a normal diet (ND), HD and HD with ARF at 10 mg/kg body weight (ARF10), or HD with ARF at 50 mg/kg body weight (ARF50) for 8 weeks. ARF supplementation had no effect on body weight, food intake and tissue weight. However, ARF50 decreased malondialdehyde and glutamicpyruvate transaminase levels compared with HD. Glutathione content decreased with HD but increased with ARF50. ARF50 also increased catalase activity. ARF50 reduced the expression of hepatic-inflammation genes IL-1β (p>0.05) and COX-2 (p<0.05). These results indicated that the ARF from Shinzami exerts anti-oxidant and anti-inflammatory activities when ingested at 50 mg/kg body weight without adversely affecting the animal s food intake, body weight, or tissue weight. Our study findings highlight the potential of using ARF as an antioxidant and anti-inflammatory agent. Key words: Hepatic enzymes, gene expression, hepatic lobular inflammation. Abbreviations: ARF: Anthocyanin-rich fraction; CAT: Catalase; C3G: Cyanidin 3- glucoside; MDA: Malondialdehyde; GOT: Glutamic oxaloacetic transaminase; GPT: Glutamic-pyruvate transaminase; GSH: Glutathione; P3G: Peonidin-3- glucoside; SOD: Superoxide dismutase. INTRODUCTION Many studies have shown that eating at least five servings of fruit and vegetables daily may reduce the risk of diseases such as heart disease, peripheral vascular disease, or stroke and that there is a strong relationship between fruit consumption and human health (Halliwell, 1999; Boyer and Liu, 2004). Anti-oxidants may protect against chronic diseases; fruits and vegetables provide a mixture of phytochemicals (Eberhardt et al., 2000). Anthocyanins and flavonoids within the polyphenol class of phytochemicals are most abundant in colored fruits, vegetables and grains and they possess a basic skeleton of 2-phenylbenzopyrylium or flavylium glycoside (Liu et al., 2014; Valenti et al., 2013). Anthocyanins have potential as therapeutic anti-oxidants (Diaconeasa et al., 2015). Their mechanisms of action in numerous experimental models in vitro and in vivo have been investigated (Halliwell, 1999; Liu et al., 2014; Diaconeasa et al., 2015). Biological systems have developed the ability to detoxify

2 Academia Journal of Medicinal Plants; Nam et al. 024 chemically active reactive oxygen species. The anti-oxidant mechanisms include complex non-enzymatic systems such as glutathione (GSH), vitamins A, C and E and enzymes such as catalase (CAT), superoxide dismutase (SOD) and various peroxidases. Insufficient levels of anti-oxidants or inhibition of the anti-oxidant enzymes also promotes oxidative stress (Valko et al., 2007). An important component of the cellular detoxification system is the transcription factor that controls the expression of a large pool of anti-oxidant and cytoprotective genes in response to stimulation of oxidants or electrophilic molecules. An anti-inflammatory effect associated with higher intake of anthocyanins and flavonols (Cassidy et al., 2015) may be significant to reduce risk for certain chronic diseases such as heart disease, stroke, cancer and arthritis. Shinzami is a Korean sweet potato used as a source of food dye (Lee et al., 2012). Its extracts showed antioxidant effects in vitro. However, limited information is available on whether Shinzami is an anthocyanin-rich source (Lee et al., 2012; Kim et al., 2012; Kim and Kim, 2010). We hypothesized that the anthocyanin-rich fraction (ARF) of Shinzami may ameliorate the oxidative stress and inflammation that are the principal causes of metabolic disorders in vivo. We evaluated the effect of the anthocyanin-rich fraction of Shinzami against oxidative stress and inflammation in mice fed a high-fat diet. MATERIALS AND METHODS Preparation of ARF Shinzami, purchased from the Bioenergy Crop Research Center (Muan-Gun, Korea), was washed, diced, freezedried (PVTFD 10R, Ilsin Lab), and milled using a grinder (FM909T, Hanil). 50 g of the powder was added to 1.5 L of distilled water containing 0.5% trifluoroacetic acid (Merck) and mixed in a shaker (JeioTech SK-71, Lab Companion) for 3 h. The process was repeated thrice and thereafter, Whatman No.1 filter paper was used to filter the mixture. ARF was prepared serially using hexane (Sigma), ethylacetate (EtOAc, Sigma), and butanol (BuOH, Sigma) (Kim et al., 2012). The 3 g ARF sample obtained was stored at -70 C for future use. same diets were supplied to each group ad libitum during the experiment. They were then randomly assigned to four groups (n=8 per group): normal diet (ND, 10% of calories from fat); high-fat diet (HD, 60% of calories from fat); HD with ARF at 10 mg/kg body weight (ARF10); HD with ARF at 50 mg/kg body weight (ARF50). HD served as the control group. All animals and diets were purchased from Central Lab. Animal Inc. ND and HD were administered distilled water orally five times a week for eight weeks to control any effect of the administration itself. ARF groups were administered ARF orally daily for eight weeks. Body weight and food intake were measured once a week. The Institutional Animal Care and Use Committee of the National Academy of Agricultural Science (NAAS-1207) approved this experimental design. Collecting samples and analysis After the eight weeks of treatment with ARF, mice were fasted for 15 h and then euthanized with CO 2. Blood samples were obtained from the heart and serum separated by centrifugation at 3000 rpm and 4 C for 20 min and stored at -70 C until biochemical analysis. A lipid peroxidation product called malondialdehyde (MDA), a commonly used marker for oxidative stress was measured using thiobarbituric acid (Buege and Aust, 1978). The quantities of MDA, glutamic oxaloacetic transaminase (GOT), and glutamic-pyruvate transaminase (GPT) were determined by colorimetric assay using commercial kits according to the manufacturers protocols (GOT and GPT, Asan Pharm Co; MDA, Biovision Inc.). The liver, kidney, abdominal fat and retroperitoneal fat tissues of the mice were collected and weighed. Hepatic tissues were homogenized using a homogenizer (IKA) and centrifuged at 13,000 rpm for 15 min. The supernatant was used to determine antioxidant content. The hepatic GSH level in the supernatant was determined with a commercially available GSH assay kit (703002, Cayman Chemical). The activities of hepatic CAT and SOD were determined using commercially available assay kits ( and respectively, Cayman Chemical). Animals, diets, and administration of ARF Four-week-old C57BL/6J mice (18.9 to 20.0 g) were maintained at a controlled temperature (20 ± 2 C) and a 12-h light: dark cycle and housed in individual cages. They were given a one-week acclimation period and then fed ND (D12450J, Research Diets) or HD (D12492, Research Diets) for 9 weeks prior to the experiment to induce obesity; Gene expression analysis Zhang et al. (2005) in their study reported that high dietary fat intake could induce oxidative stress and inflammation. To investigate the mrna expression level of hepatic inflammation-related genes (IL-β and COX-2), hepatic RNA of 8 mice in each experimental group were isolated with the RNeasy Tissue Mini Kit (Qiagen).

3 Academia Journal of Medicinal Plants; Nam et al. 025 Table 1. Sequences of PCR primers used for IL-β and COX-2 in Real-Time PCR Target gene IL-1β Primer sequence F: 5ʹ -GCA ACT GTT CCT GAA CTC AAC T-3ʹ R: 5ʹ -ATC TTT TGG GGT CCG TCA ACT-3ʹ COX-2 F: 5ʹ -GAA GTC TTT GGT CTG GTG CCT G-3ʹ R: 5ʹ-GTC TGC TGG TTT GGA ATA GTT GC-3ʹ GAPDH F: 5ʹ -GGT GAA GGT CGG TGT GAA CG-3ʹ R: 5ʹ -CTC GCT CCT GGA AGA TGG TG-3ʹ Table 2. Scoring of hepatic lobular inflammation Assessment Number of foci per 200 field Score 0 0 Lobular inflammation > 4 3 Table 3. Effect of treatment with ARF on mouse body weight and food intake Variable ND HD ARF10 ARF50 Final body weight (g) 31.1 ± 1.7 b 44.5 ± 2.4 a 44.0 ± 4.5 a 42.0 ± 3.9 a Body weight gain (g) 8.3 ± 1.5 b 24.1 ± 2.7 a 25.1 ± 4.4 a 22.2 ± 3.7 a Food intake (g/day) 5.6 ± 0.2 a 5.4 ± 0.2 ab 4.8 ± 0.7 b 5.0 ± 0.2 b Data are expressed as Mean ± SD. Superscript letters (a, b) within a row indicate significant difference. Extracted RNA was used to synthesize cdna with a QuantiTect Reverse Transcription kit (Qiagen). Real-time PCR was performed as a two-step procedure with SYBR Green Supermix (Qiagen) and a CFX96 Real-Time PCR Detection System (Bio-Rad). Cycling conditions were as follows: 95 C for 2 min, 95 C for 15 s, 60 C for 30 s, 72 C for 30 s and by 35 cycles. By analyzing the changes in SYBR, green fluorescence relative quantification was calculated. The values obtained were the threshold cycles (Ct) at which a statistically significant increase in SYBR green emission intensity occurred. The relative expression level of each gene was determined by the 2 ΔΔCT method. Table 1 presents sequences of the PCR primers used for IL- 1β, COX-2 and GAPDH as a housekeeping gene. Histological analysis Hepatic tissue was fixed with 10% formalin and embedded in paraffin. Five sections were cut and stained with hematoxylin and eosin. Images were captured using an Olympus AX 70 Research Microscope equipped with a camera. Inflammation was scored by counting the number of foci formed by the infiltration of inflammatory cells such as lymphocytes and neutrophils per 200 field (Table 2). Statistical analysis All statistical analyses were performed with the SPSS Statistics 21. All results were analyzed with one-way ANOVAs and Duncan s multiple range test was used to detect differences among groups when ANOVA was significant at p<0.05. Duncan s multiple range test was also considered significant at p< RESULTS AND DISCUSSION Body weight and food intake For the 8 weeks of experiment, daily food intake was lower in ARF10 and ARF50 as compared with ND and final body weight and body weight gain were high in HD, ARF10 and ARF50 (Table 3). ARF50 had a slightly lower final body weight and less body weight gain than HD and ARF10, but

4 Academia Journal of Medicinal Plants; Nam et al. 026 Figure 1. Effect of ARF supplementation on serum MDA and hepatic antioxidant enzymes of mice fed a high-fat diet. (a) Serum MDA level, (b) hepatic GSH level, (c) CAT and (d) SOD activities. Data are expressed as mean ± SD. Statistical significance was determined using ANOVA followed by Duncan s multiple range test. Letters (a, b, c) within a subfigure indicate a significant difference (p< 0.05). ND: normal diet (10% of calories from fat); HD: high-fat diet (60% of calories from fat); ARF10: HD with 10 mg/kg body weight anthocyanin-rich fraction; ARF50: HD with 50 mg/kg body weight anthocyanin-rich fraction. this was not significant. Tissue weights HD, ARF10 and ARF50 had significantly higher tissue weights than ND. However, there was no significant difference among HD groups (data not shown). Serum MDA content and hepatic anti-oxidant enzymes Figure 1 shows the MDA and GSH content, CAT and SOD activities. In this study, an HD increased the serum MDA level while supplementation with ARF decreased it, indicating that ARF administration can decrease serum oxidative stress. GSH content decreased in HD compared to ND. However, GSH content increased in ARF groups in a dose-dependent manner. ARF50 showed a significantly increased CAT activity compared to those of HD and ARF10. SOD activity was higher in the ARF50 than in other groups (p> 0.05). Zhao et al. (2013) found the hepatic antioxidant effects of sweet potato anthocyanins on tetrahydroperoxide-induced oxidative stress in mice. In line with these results, anthocyanin-rich Shinzami may control the oxidative stress through anti-oxidant effects. Serum GOT and GPT levels GOT and GPT are hepatic enzymes that indicate necrosis of hepatocytes and increase the transaminase level in the blood, inducing their high serum levels (Kim, 2008; Gabriel and William, 1982). The increase in GOT level correlates with the extent and severity of cellular damage. Serum GOT and GPT activities are used as biochemical markers of early acute hepatic damage. Figure 2 shows the effects of treatment with ARF on serum GOT and GPT. ARF had no

5 Academia Journal of Medicinal Plants; Nam et al. 027 Figure 2. Effect of ARF on serum (a) GOT and (b) GPT on mice fed a high-fat diet. Data are expressed as mean ± SD. Statistical significance was determined using ANOVA followed by Duncan s multiple range test. Letters (a, b, c) within a subfigure indicate a significant difference (p< 0.05). ND: normal diet (10% of calories from fat); HD: high-fat diet (60% of calories from fat); ARF10: HD with 10 mg/kg body weight anthocyanin-rich fraction; ARF50: HD with 50 mg/kg body weight anthocyanin-rich fraction. Figure 3. Effect of ARF on hepatic inflammation of mice fed a high-fat diet. Data are expressed as mean ± SD. Statistical significance was determined using ANOVA followed by Duncan s multiple range test. Indicate a significant difference (p< 0.05). ND: normal diet (10% of calories from fat); HD: high-fat diet (60% of calories from fat); ARF10: HD with 10 mg/kg body weight anthocyanin-rich fraction; ARF50: HD with 50 mg/kg body weight anthocyanin-rich fraction. effect on serum GOT levels in ARF groups (p>0.05). GPT level was significantly higher in HD as compared with ND, but ARF supplementation decreased GPT levels in ARF50. In a previous study, administration of a purple sweet potato powder decreased the serum GPT level as with this experiment (Cho et al., 2003). These results showed that ARF could suppress HD-induced lipotoxic hepatic injury at 50 mg/kg body weight. Hepatic histopathology Figure 3 shows the lobular inflammation scores in liver tissues. Hepatic inflammation grade scores increased

6 Academia Journal of Medicinal Plants; Nam et al. 028 Figure 4. Effect of ARF supplementation on mrna levels of hepatic inflammation-related genes (a) IL-1β and (b) COX-2 of mice fed a high-fat diet. Data are expressed as Mean ± SD. Statistical significance was determined using ANOVA followed by Duncan's multiple range test. Within a sub-figure indicate a significant difference (p< 0.05). ND: normal diet (10% of calories from fat); HD: high fat diet (HD, 60% of calories from fat); ARF50: HD with 50 mg/kg body weight anthocyanin-rich fraction. sevenfold in HD over ND. ARF50 showed a significantly decreased hepatic inflammation score compared with HD. Hepatic mrna expression of inflammation-related genes As shown in Figure 4, the expression of IL-1β and COX-2 were significantly higher in the HD group than the ND group. However, COX-2 mrna expression levels decreased in the ARF50 group. Therefore, administration of ARF at 50 mg/kg body weight may have a protective effect on inflammatory diseases. Thus, the anthocyanin-rich fraction from the purple sweet potato variety Shinzami can significantly control oxidative stress and inflammation without affecting food intake, body weight, or tissue weights. To explain the mechanism of this action further studies are required. Conclusions These results indicate that the ARF from Shinzami exerts anti-oxidant and anti-inflammatory activities when ingested at 50 mg/kg body weight, without adversely affecting the animal s food intake, body weight, or tissue weight. Our study findings highlight the potential of using ARF as an anti-oxidant and anti-inflammatory agent. ACKNOWLEDGMENTS This work was carried out with the support of Cooperative Research Program for Agriculture Science and Technology Development (PJ ), Rural Development Administration, Republic of Korea. CONFLICT OF INTEREST The authors declare that they have no competing interests. AUTHOR CONTRIBUTIONS Sung Hyen Lee and Young Min Lee conducted the experiments, wrote, and revised the manuscript. Song Yee Nam and Mi Ju Kim conceived the study and were actively involved in manuscript preparation. Hwan Hee Jang, Jung Bong Kim, Shin Young Park, and Kye Won Park conceived the study and designed the experiments. All authors reviewed and approved the final manuscript. REFERENCES Boyer J, Liu RH (2004). Apple phytochemicals and their health benefits. Nutr. J. 3: 5. Buege JA, Aust SD (1978). Microsomal lipid peroxidation. Methods Enzymol. 52:

7 Academia Journal of Medicinal Plants; Nam et al. 029 Cassidy A, Rogers G, Peterson JJ, Dwyer JT, Lin H, Jacques PF (2015). Higher dietary anthocyanin and flavonol intakes are associated with anti-inflammatory effects in a population of US adults. Am. J. Clin. Nutr. 102: Cho YJ, Kim HA, Bang MA, Oh YB, Jeong BC, Moon YH, Jeong WJ (2003). Protective effect of purple sweet potato (Ipomoea batatas) on hepatotoxicity rats induced by carbon tetrachlolide. Korean J Food Culture. 18: Diaconeasa Z, Leopold L, Rugina D, Ayvaz H, Socaciu C (2015). Antiproliferative and antioxidant properties of anthocyanin rich extracts from blueberry and blackcurrant juice. Int. J. Mol. Sci. 6: Eberhardt MV, Lee CY, Liu RH (2000). Nutrition: antioxidant activity of fresh apples. Nature 405: Gabriel LP, William RH (eds) (1982). Principles and methods of toxicology, Raben Press, New York. pp Halliwell B (1999). Establishing the significance and optimal intake of dietary antioxidants: the biomarker concept. Nutr. Rev. 57: Kim HW, Kim JB, Cho SM, Chung MN, Lee YM, Chu SM, Che JH, Kim SN, et al (2012). Anthocyanin changes in the Korean purple-fleshed sweet potato, Shinzami, as affected by steaming and baking. Food Chem. 130: Kim SJ, Kim JS (2010). Antioxidative effects of purple sweet potato extracts. Agricultural Research Bull Kyungpook National University. 28: Kim YJ (2008). Interpretation of liver function tests. Korean J. Gastroenterol. 51: Lee YM, Bae JH, Kim JB, Kim SY, Chung MN, Park MY, Ko JS, Song J, Kim JH (2012) Changes in the physiological activities of four sweet potato varieties by cooking condition. Korean J. Nutr. 45: Liu Y, Li D, Zhang Y, Sun R, Xia M (2014). Anthocyanin increases adiponectin secretion and protects against diabetes-related endothelial dysfunction. Am. J. Physiol. Endocrinol. Metab. 306: E Valenti L, Riso P, Mazzocchi A, Porrini M, Fargion S, Agostoni C (2013). Dietary anthocyanins as nutritional therapy for nonalcoholic fatty liver disease. Oxid. Med. Cell. Longev. 2013: Valko M, Leibfritz D, Moncol J, Cronin MT, Mazur M, Telse J (2007). Free radicals and antioxidants in normal physiological functions and human disease. Int. J. Biochem. Cell Biol. 39: Zhang X, Dong F, Ren J, Driscoll MJ, Culver B (2005). High dietary fat induces NADPH oxidase-associated oxidative stress and inflammation in rat cerebral cortex. Exp. Neurol. 191: Zhao JG, Yan QQ, Lu LZ, Zhag YQ (2013). In vivo antioxidant, hypoglycemic, and anti-tumor activities of anthocyanin extracts from purple sweet potato. Nutr. Res. Pract. 7: Cite this article as: Nam SY, Lee YM, Kim MJ, Kim JB, Jang HH, Park SY, Park KW, Lee SH (2016). Protective Effects of the Anthocyanin-Rich Fraction of Shinzami Purple Sweet Potato against Oxidative Stress and Inflammation in Mice Fed a High-Fat Diet. Acad. J. Med. Plant. 4(8): Submit your manuscript at

Sunghyen Lee 1, Youngmin Lee 2, Sungjoon Lee 3, Haejeung Lee 4, Hyun Lillehoj 5

Dec 8, 2015 Sunghyen Lee 1, Youngmin Lee 2, Sungjoon Lee 3, Haejeung Lee 4, Hyun Lillehoj 5 1 Functional Food & Nutrition Division, National Institute of Agricultural Science, 2 Seoul Women s Univ, 3 Eulji