Effects of Heating Condition and Cultivar on Phenolic Compounds and Their Radical Scavenging Activity on Sorghum

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1 Academia Journal of Biotechnology 4(10): , October 2016 DOI: /ajb ISSN Academia Publishing Research Paper Effects of Heating Condition and Cultivar on Phenolic Compounds and Their Radical Scavenging Activity on Sorghum Accepted 23 th October, 2016 Kyung ha Lee 1, Hyeonmi Ham 1, Mi Jung Kim 1, JeeYeon Ko 2, Hyun-Joo Kim 1, Sea Kwan Oh 1, Heon Sang Jeong 3 and KoanSik Woo 1 * 1 Department of Central Area Crop Science, National Institute of Crop Science, Rural Development Administration, Suwon, Gyeonggi 16429, Republic of Korea. 2 Department of Southern Area Crop Science, National Institute of Crop Science, Rural Development Administration, Miryang, Gyeongnam 50424, Republic of Korea. 3 Department of Food Science and Technology, Chungbuk National University, Cheongju, Chungbuk 28644, Republic of Korea. *Corresponding author. wooks@korea.kr. Tel: ; Fax: ABSTRACT The purpose of this study was to evaluate the effects of heating condition and cultivar on phenolic compounds and their activity in sorghum (Sorghum bicolor L. Moench). Ethanol was added to sorghum twice and heated at 120 C for 1, 2, 3, 4 and 5 h using an autoclave. After heating, total polyphenol, flavonoid contents and radical scavenging activities increased. The highest total flavonoid content was mg catechin equivalent (CE)/g ethanolic extract (EE), which was three times higher than mg CE/g EE for the untreated cultivar Nampungchal. The highest 2, 2-diphenyl-1-picrylhydrazyl (DPPH) and 2, 2 -azino-bis-3- ethylbenzthiazoline-6-sulphonic acid (ABTS) radical scavenging activities were and mg Trolox equivalent/g EE, respectively, in Nampungchal heated at 120 C for 4 h. The results of this study showed that antioxidant compounds in heated sorghum, with the addition of ethanol can be considered to have significant health benefits. Key words: Sorghum (Sorghum bicolor L. Moench), heat treatment, polyphenol, antioxidant activity. INTRODUCTION Sorghum (Sorghum bicolor L. Moench) is an important crop in the semi-arid regions of Africa and Asia as a source of energy, protein, minerals and vitamins (Gmez-Cordovs et al., 2001). Sorghum grain is an important food in developing countries of the semi-arid tropics and is also a main source of energy and protein in animal feed in both developed and developing countries (Oriaet al., 2000; Simontacchi et al., 2003). Sorghum contains phenolic compounds in the form of phenolic acids, flavonoids and condensed tannins, which have been shown to have antioxidant activity (Awikaet al., 2003; Awika et al., 2004). These phenolic compounds are concentrated in the outer layers of the sorghum grain (pericarp and testa) (Beta et al., 2000) and possess structural features that are favorable for radical scavenging and/or metal chelation, making them effective antioxidants. Recent studies have shown that thermally processed foods, especially fruits and vegetables, have higher biological activity due to changes in various compounds during heat treatment (Woo et al., 2007). The latest studies reported that heat treatment increases total polyphenolic and flavonoid contents and antioxidant activities in garlic (Kwon et al., 2006), pears (Hwang et al., 2006), tomatoes (Dewanto et al., 2002a), oranges (Peleg et al., 1991) and shiitake mushroom (Choi et al., 2006). Also, recent epidemiological studies suggested that increased consumption of whole grains, fruits and vegetables is associated with reduced risk of chronic diseases (Hu et al., 2002). This association might be attributed to natural antioxidants such as vitamin C, tocopherol, carotenoids, polyphenolics and flavonoids, which prevent free-radical damage (Choi et al., 2007). Our goal was to evaluate the

2 Academia Journal of Biotechnology; Lee et al. 348 phenolic compounds and radical scavenging activity of heated sorghum. MATERIALS AND METHODS Chemicals and reagents Folin-Ciocalteu reagent, sodium carbonate, gallic acid, NaNO 2, AlCl 3 6H 2O, (+)-catechin, Na 2CO 3, tannic acid, 2, 2- diphenyl-1-picrylhydrazyl (DPPH), 2, 2-azinobis(3- ethylbenothiazoline-6-sulphonic acid) diammonium salt (ABTS), potassium persulfate and Trolox were purchased from Sigma-Aldrich (St. Louis, MO, USA). HPLC-grade water was purchased from J.T. Baker (Phillipsburg, NJ, USA). Reagents were the highest grade quality. Sample preparation and extraction The sorghum (Sorghum bicolor (L.) Moench cv. Hwanggeumchal (HGC), Daepung (DP), Donganme (DAM) and Nampungchal (NPC)] cultivars were grown at the National Institute of Crop Science, Rural Development Administration, Miryang, South Korea during the 2013 growing season and stored at -20 C. Two treatments of 95% ethanol were added to sorghum, which was then placed in a sample bottle and tightly sealed. Sample bottles were heated using an autoclave (VS-1221, Vision, Bucheon, Korea). Samples were heated to 120 C for 1, 2, 3, 4, and 5 h. The heated samples were extracted five times with 95% EtOH solution. Extracts were then stored at -20 C for subsequent analysis. Determination of phenolic compounds Total phenolic contents in the sorghum bran and grain extracts were determined using the Folin-Ciocalteu method (Dewanto et al., 2002b), with some modifications and the results expressed as mg gallic acid equivalents (GAE) per gram of ethanolic extracts (EE). A standard solution of sorghum bran and grain extracts (50 μl) was mixed with 1 ml of 2% sodium carbonate solution and 50 μl of a 50% Folin-Ciocalteu reagent. After incubation for 30 min at room temperature, absorbance was measured at 750 nm. Total flavonoid contents in the sorghum bran and grain extracts were determined using colorimetric method, as described by Dewanto et al. (2002b), with some modifications and the results expressed as mg (+)-catechin equivalents (CE) per gram of EE. A standard solution of sorghum bran and grain extracts (250 μl) was mixed with 1 ml of distilled water and 75 μl of a 5% NaNO 2 solution. After 5 min, 150 μl of 10% AlCl 3 6H 2O was added. After 6 min, 500 μl of 1M NaOH was added to the mixture. The solution was well mixed and the intensity of pink color measured at 510 nm. All extracts were analyzed in triplicate. Measurement of DPPH and ABTS radical scavenging activity Scavenging activity of the samples for the DPPH radical was measured according to the methods of Choi et al. (2006) with some modifications. A 0.8-ml aliquot of 0.2 mm DPPH methanolic solution was mixed with 0.2 ml of the sample. The mixture was shaken vigorously and left to stand for 30 min under low light. The absorbance was measured at 515 nm. Scavenging activity of the extracts for the ABTS cation radical was measured according to the methods of Choi et al. (2006) with some modifications. The ABTS cation radical was generated by adding 7 mm ABTS to 2.45 mm potassium persulfate solution and storing the mixture overnight in the dark at room temperature. The ABTS cation radical solution was diluted with methanol to obtain an absorbance of 1.4 to 1.5 at 735 nm (molar extinction coefficient, ε= mol -1 cm -1 ). A diluted ABTS cation radical solution (1 ml) was added to 50 ml of the extract, Trolox standard solution, or distilled water. After 30 min, the absorbance was measured at 735 nm using a spectrophotometer (model DU-650, Beckman, Fullerton, CA, USA). The DPPH radical and ABTS cation radical scavenging activity are expressed in terms of Trolox equivalent antioxidant capacity (TEAC), as milligrams of Trolox equivalents (TE) per gram of EE. Statistical analysis All data are expressed as mean ± standard deviation (SD). The significance of differences among treatment means was determined by one-way analysis of variance (ANOVA) and Duncan s multiple range test, using SAS version 9.2 (SAS Institute, Cary, NC, USA) with a significance level of Relationships between parameters were also investigated using regression analysis. RESULTS AND DISCUSSION Phenolic compounds in ethanolic extracts of heated sorghum Phenolic compounds are some of the most effective antioxidative constituents in plant foods such as fruits, vegetables and grains (Velioglu et al., 1998). Therefore, it is important to quantify polyphenolic contents and assess their contribution to anti-oxidant activity. The total polyphenol and flavonoid content in the unheated and

3 Academia Journal of Biotechnology; Lee et al. 349 Table 1: Correlations between antioxidant compounds and radical-scavenging activity of ethanolic extract of heated sorghum. Factor Polyphenol Flavonoid DPPH ABTS Polyphenol * * * Flavonoid * * DPPH * ABTS *: p< Autoclaving times time (h) (h) Figure 1: Total polyphenol content of ethanolic extracts in sorghum with heating and its variation. 1) Values with different superscripts are significantly different at p < 0.05 according to Duncan s multiple range tests heated sorghum are expressed as mg gallic acid equivalents (GAE) and mg catechin equivalents (CE), respectively, per gram of ethanolic extract (EE; Figures 1 and 2). Phenolic compounds in ethanolic extracts usually increased after being heated. The total polyphenol contents (Figure 1) for HGC, DP, DAM, and NPC of unheated sorghum were 84.29, 67.76, and mg GAE/g EE, respectively, and to , to , to and to mg GAE/g EE, respectively, for heated sorghum. The highest total polyphenol content was mg GAE/g EE after 4 h of heating; this value was four times higher than that for untreated NPC, mg CE/g EE. The total flavonoid contents (Figure 2) for HGC, DP, DAM, and NPC of unheated sorghum were 71.04, 52.62, and mg CE/g EE, respectively, and to , to , to and to mg CE/g EE, respectively, for heated sorghum. The highest total flavonoid content was mg CE/g EE, three times higher than the value for untreated NPC of mg CE/g EE. There was a significant positive correlation between levels of total polyphenol and flavonoid (R 2 = , p< 0.001; Table 1). Many anti-oxidant compounds are present in plant materials, mainly in a covalently bound form with insoluble polymers (Peleg et al., 1991). Therefore, heat treatment might disrupt cell walls and liberate antioxidant compounds from insoluble portions of sorghum, increasing the pool of bioaccessible anti-oxidant compounds. Also, the addition of ethanol in the heated treatment of sorghum was expected to facilitate separation of the bound-form components. Another reason for improved anti-oxidant activity could be the formation of novel compounds possessing anti-oxidant activity during the heat treatment or the addition of ethanol during thermal processing (Woo et al., 2007).

4 Academia Journal of Biotechnology; Lee et al. 350 Autoclaving time (h) Figure 2: Total flavonoid content of ethanolic extracts in sorghum with heating and its variation. 1) Values with different superscripts are significantly different at p < 0.05 according to Duncan s multiple range tests. Radical-scavenging activity of ethanolic extracts of heated sorghum The stable DPPH radical, which has maximum absorption at 515 nm, is widely used to evaluate the free radicalscavenging activity of hydrogen-donating anti-oxidants in many plant extracts (Wettasinghenet al., 2000). The ABTS method is widely employed for measuring the relative radical scavenging activity of hydrogen-donating and chainbreaking anti-oxidants in many plant extracts (Netzel et al., 2003). Figures 3 and 4 showed the variation in DPPH and ABTS radical-scavenging activity of unheated and heated sorghum extracts with heating time. A correlation was found between phenolic compounds and radical-scavenging activity (Table 1). The DPPH radicalscavenging activity (Figure 3) of HGC, DP, DAM, and NPC for unheated sorghum were 74.30, 49.85, and mg TE/g EE, respectively, and to 82.24, to 69.26, to and to mg TE/g EE, respectively, for heated sorghum. The highest DPPH radical-scavenging activity was mg TE/g EE, heat treated for 4 h; this value was two times higher than untreated NPC of mg TE/g EE. There was a significant positive correlation between DPPH radicalscavenging activity and levels of polyphenols (R 2 = , p< 0.001) and flavonoids (R 2 = , p< 0.001; Table 1). The ABTS radical-scavenging activity (Figure 4) of HGC, DP, DAM, and NPC for unheated sorghum were 92.62, 76.72, and mg TE/g EE, respectively, and to , to , to and to mg TE/g EE, respectively, for heated sorghum. The highest ABTS radical-scavenging activity was mg TE/g EE after 4 h heating, which was four times higher than that for untreated NPC, that is, mg TE/g EE. Significant positive correlations were found between ABTS radical-scavenging activity and levels of polyphenols (R 2 = , p< 0.001), flavonoids (R 2 = , p< 0.001) and DPPH radical-scavenging activity (R 2 = , p< 0.001; Table 1). These results confirmed that phenolic compounds might be responsible for the majority of the anti-oxidant activity of heated sorghum. Many previous studies reported a significant correlation between phenolics and antioxidant activities in fruits, barley and mushrooms (Choi et al., 2005; Woo et al., 2014). In this study, non-enzymatic browning reaction products might have been formed during prolonged heat treatment with the improvement in anti-oxidant activity. Recently, another study on tomato and coffee found that prolonged heat treatment enhanced the anti-oxidant activity of these food items (Baublis et al., 2000). Browning and antioxidant activity in tomato and coffee samples increased with heating and roasting time.

5 Academia Journal of Biotechnology; Lee et al. 351 Autoclaving times (h) Autoclaving time (h) Figure 3: The DPPH radical scavenging activity of ethanolic extracts in sorghum with heating and its variation. 1) Values with different superscripts are significantly different at p < 0.05 according to Duncan s multiple range tests. Autoclaving time (h) Figure 4: The ABTS radical scavenging activity of ethanolic extracts in sorghum with heating and its variation. 1) Values with different superscripts are significantly different at p < 0.05 according to Duncan s multiple range tests.

6 Academia Journal of Biotechnology; Lee et al. 352 In the last decade, many studies examined anti-oxidant activity after heat treatment and showed that heated products exhibit chain breaking and oxygen-scavenging activities (Jung et al., 2004). The activities of these components increase after heat treatments due to the low molecularization effects of the heating process, with the result that these active low-molecular-weight components are readily extracted. Conclusion The heated sorghum showed significantly higher antioxidant activity and contained higher polyphenolic content than did unheated sorghum in the ethanolic extracts. Our results could have a direct impact on sorghum consumption by increasing consumer awareness of the health benefits of grains. ACKNOWLEDGEMENTS This work was carried out with the support of Cooperative Research Program for Agriculture Science and Technology Development (Project No. PJ ) Rural Development Administration, Republic of Korea. REFERENCES Awika JM, Rooney LW, Waniska RD (2004). Properties of 3- deoxyanthocyanins from sorghum. J. Agric. Food Chem. 52(14): Awika JM, Rooney LW, Wu X, Prior RL, Cisneros-Zevallos L (2003). Screening methods to measure antioxidant activity of sorghum (Sorghum bicolor) and sorghum products. J. Agric. Food Chem. 51(23): Baublis AJ, Lu C, Clydesdale FM, Decker EA (2000). Potential of wheatbased breakfast cereals as a source of dietary antioxidants. J. Am. Col. Nutr. 19(3):308S-311S. Beta T, Rooney LW, Marovatsanga LT, Taylor JRN (2000). Effect of chemical treatments on polyphenols and malt quality in sorghum. J. Cereal Sci. 31(3): Choi Y, Jeong HS, Lee J (2007). Antioxidant activity of methanolic extracts from some grains consumed in Korea. Food Chem. 103(1): Choi Y, Ku JB, Chang HB, Lee J (2005). Antioxidant activities and total phenolics of ethanol extracts from several edible mushrooms produced in Korea. Food Sci. Biotechnol. 14(5): Choi Y, Lee SM, Chun J, Lee HB, Lee J (2006). Influence of heat treatment on the antioxidant activities and polyphenolic compounds of shiitake (Lentinusedodes) mushroom. Food Chem. 99(2): Dewanto V, Wu X, Adom KK, Liu RH (2002a). Thermal processing enhances the nutritional value of tomatoes by increasing total antioxidant activity. J. Agric. Food Chem. 50(10): Dewanto V, Xianzhong W, Liu RH (2002b). Processed sweet corn has higher antioxidant activity. J. Agric. Food Chem. 50(17): Gmez-Cordovs C, Bartolom B, Vieira W, Virador VM (2001). Effect of wine phenolics and sorghum tannins on tyrosinase activity and growth of melanoma cells. J. Agr. Food Chem. 49(3): Hu FB (2002). Dietary pattern analysis: a new direction in nutritional epidemiology. Curr. Opin. Lipidol. 13(1): 3-9. Hwang IG, Woo KS, Kim TM, Kim DJ, Yang MH, Jeong HS (2006). Change of physicochemical characteristics of Korean pear (PyruspyrifoliaNakai) juice with heat treatment conditions. Korean J. Food Sci. Technol. 38(3): Jung HA, Lee HJ, Kim YA, Park KE, Ahn JW, Lee BJ, Moon SG, Seo Y (2004). Antioxidant activity of Artemisia capillaris Thunberg. Food Sci. Biotechnol. 13(3): Kwon OC, Woo KS, Kim TM, Kim DJ, Hong JT, Jeong HS (2006). Physicochemical characteristics of garlic (Allium sativum L.) on the high temperature and pressure treatment. Korean J. Food Sci. Technol. 38(3): Netzel M, Strass G, Bitsch I, Könitz R, Christmann M, Bitsch R (2003). Effect of grape processing on selected antioxidant phenolics in red wine. J. Food Eng. 56(2-3): Oria MP, Hamaker BR, Axtell JD, Huang CP (2000). A highly digestible sorghum mutant cultivar exhibits a unique folded structure of endosperm protein bodies. Proc. Natl. Acad. Sci. USA. 97(10): Peleg H, Naim M, Rouseff RL, Zehavi U (1991). Distribution of bound and free polyphenolic acids in oranges (Citrus sinensis) and grapefruit (Citrus paradise). J. Sci. Food Agric. 57(3): Simontacchi M, Sadovsky L, Puntarulo S (2003). Profile of antioxidant content upon developing of Sorghum bicolor seeds. Plant Sci. 164(5): Velioglu YS, Mazza G, Gao L, Oomah BP (1998). Antioxidant activity and total polyphenolics in selected fruits, vegetables, and grain products. J. Agric. Food Chem. 46(10): Wettasinghe M, Shahidi F (2000). Scavenging of reactive-oxygen species and DPPH free radicals by extracts of borage and evening primrose meals. Food Chem. 70(1): Woo KS, Ko JY, Jeong HS (2014). Effect of milling time on antioxidant compounds and activities of methanol extracts of sorghum [Sorghum bicolor (L.) Moench]. Food Sci. Biotechnol. 23(6): Woo KS, Yoon HS, Lee YR, Lee J, Kim DJ, Hong JT, Jeong HS(2007). Characteristics and antioxidative activity of volatile compounds in heated garlic (Allium sativum). Food Sci. Biotechnol. 16(5): Cite this article as: Lee K, Ham H, Kim MJ, Ko JY, Kim HJ, Oh SK, Jeong HS, Woo K (2016). Effects of Heating Condition and Cultivar on Phenolic Compounds and Their Radical Scavenging Activity on Sorghum. Acad. J. Biotechnol. 4(10): Submit your manuscript at