Antioxidant Properties of Water Extracts from Pigmented Rice Grown in Southern Thailand
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1 Antioxidant Properties of Water Extracts from Pigmented Rice Grown in Southern Thailand Asma Abru, Patcharin Pakdeechanun and Taewee T. Karrila* Department of Food science and Nutrition, Faculty of Science and Technology. Prince of Songkla University, Pattani Campus, Thailand *Corresponding author: phone: Abstract Native pigmented rice in southern Thailand are under utilization although their colour and antioxidant properties are very attractive. To promote pigmented rice its water extracts could be used as beverage. The aim of this work was to determine properties of water extracts from 8 varieties of pigmented rice from southern Thailand. The effect of extraction heating time on properties of water extracts was also studied. De-husked pigment rice grains (8 varieties both waxy and non waxy) were mixed with water (grain:water = 1:25) and heated at C for 15 min. The water extracts obtained were determined for colour parameters (L*, a* and b*), total phenol, anthocyanin and antioxidant activities (DPPH., ABTS + and SRSA). The results showed that total polyphenol and total anthocyanin contents in water extracts from all varieties were in the range of mg GAE/ml extracts and mg Cy-3-G/ml extracts, respectively. However, those from waxy rice had higher content for both polyphenol and anthocyanin and hence performed higher antioxidant activities shown by DPPH., ABTS + and SRSA radical scavenging activity (p<0.05). The water extracts from waxy rice varieties gave lower L*, a* and b* value indicated darker color. Keywords: Pigmented rice, extraction, polyphenols, anthocyanins, antioxidant activity Introduction Thailand is well known as a rice production country. It has many thousand rice varieties spread through out the country, including pigmented rice. Many different pigmented rice cultivars have been collected at Pattani Rice Research Center (PRRC), Southern Thailand. Most of them are now rarely available for consumption. Optical characteristics and antioxidant properties of 8 varieties of pigmented rice grain grown in southern Thailand have been reported by Yodmanee (2010). Those rice varieties can be grouped as dark purple color group and red brown color group. The first group has higher polyphenolic content than the later group. The phenolic content in rice grain is greatly affected by genotype and environment (Goffman and Bergman, 2004). Polyphenolic compounds are also known as natural antioxidants (Hu et al., 2003), which recognized to have protective functions against oxidative damage, and are associated with reduced risk of chronic diseases (Liu, 2007). They contain a group of reddish purple water soluble substances called anthocyanins, and that are the primary pigments in the red and black grains (Abdel-Aal et al., 2006). The major components of anthocyanin in colored rice are cyanidin-3-glucoside and peonidin-3-glucoside (Yawadio et al., 2007). Those compounds are normally extracted using methanol or inorganic solvent (Choi et al., 2007) prior to determine their content and activity, but using water is rarely found. Extract of pigmented rice has very attractive color, and could be used as food ingredients. Using water as extraction medium would be more interesting due to itsetability. The aim of this research was to expand utilization of pigmented rice grown in southern, Thailand. Therefore, the objective of this work was to determined antioxidant properties of water extracts from 8 varieties of pigmented rice available in southern Thailand. Materails and Methods 48
2 Rice samples Pigmented rice samples used in this study were harvested in March 2009 and provided by Pattani Rice Research center, Pattani, Thailand. Eight varieties of pigmented rice grains, consisted of 3 non waxy rice Homkradunga, (HK), Kumyan (KN), Songyod (SY) and 5 varieties of waxy rice Red waxy rice (RWR-96060), Kramrad (KR), Black waxy rice (BWR-96044), Black waxy rice (BWR-96025) and Chormaiphai (CMP). Rough rice grains were dehusked, and brown rice obtained was stored at 4 C. Rice water extracts preparation Rice water extract of all rice varieties was prepared in the same manner as following steps. Pigmented brown rice (10g) and water (250ml) were mixed, and heated at 100 C for 15 min. Water extract was then filtered and followed by centrifuging at 1500 rpm for 10 min. Rice extracts were determined for their properties as following. Determinations of rice water extract properties Total solid and light transmission Total solid of water extract was determined using hot air oven method (AOAC, 2000). 5 ml of water extracts was weighed and evaporated on water bath and then in hot air oven until it had stable weight. Total solid content was calculated and expresses as % (w/w). Light transmission of rice water extract was measured using spectrophotometer (Libra S22, Biochrom, England) at 760 nm (Palou et al., 1999) and water was used as a blank. Color Color of water extracts was determined by HunterLab (ColorQuest XE, HunterLab, USA), color measurements were expressed as tristimulus parameters, L*, a* and b*. L* indicates lightness (100=white and 0=black) while a* and b* indicate redness-greenness and yellowness-blueness respectively (Bao et al., 2005) Total polyphenol content Total polyphenols were assayed using the Folin Ciocalteau method. Briefly, Folin-Ciocalteu reagent was diluted with water 1:9 (v/v). To 1.25 ml of this reagent, 50 µl of sample was added. After 2 min incubation at room temperature, 1 ml sodium carbonate (75 g/l) was added. The mixture was incubated for 15 min at 50 C and cooled quickly in an ice-water bath. The absorbance at 760 nm was read within 15 min. The readings were compared to a standard curve using gallic acid (Aguilar-Garcia et al., 2007) Total anthocyanin content Anthocyanins content was estimated by a ph differential method (Finocchiaro et al., 2010). Absorbance (Abs) was measured at λ max nm (500nm) and at 700 nm for elimination of interference from the background. Rice extract (400 µl) was diluted with buffer (2 ml) at ph 1.0 (0.025 mm KCl) and left at rest for 15 min before measurement, whereas the samples diluted with the buffer ph 4.5 (0.4 mm CH 3 COONa) were ready for measurement after 5 min. The corresponding pure buffer solution was used as reference sample in the spectrophotometer. The absorbance shift was calculated as: abs = (Abs 500 nm - Abs 700nm ) ph (Abs 500nm - Abs 700nm ) ph 4.5 The molar extinction coefficient of cyanidin-3-glucoside (29600 L/mol cm) was used to calculate the concentration of total anthocyanins, expressed as cyanidin glucoside equivalents in mg/ml of the extracts. 49
3 Antioxidant capacity assays Total antioxidant capacity (ABTS +, DPPH of rice extracts (0.1 mg/ml) were determined as the same method described by Choi et al. (2007). For 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) radical or ABTS + scavenging activity determination. ABTS + solution (6 ml, absorbance of at 414 nm) was added to 0.6 ml of the extracts and mixed thoroughly. The mixtures were kept at room temperature for 1 hr before an absorbance at 414 nm was read, using a spectrophotometer (Libra S22, Biochrom, England). The percentage of inhibition was expressed using the following equation: ABTS + radical scavenging activity (%) = [(Abs t=0 Abs t=60 min )/Abs t=0 ] x 100 For 1,1-diphenyl-2-picrylhydrazyl ( DPPH) radical scavenging activity determination, the extract was first added to 3 ml of 200 µm DPPH solution in methanol. After incubation at room temperature for 30 min, the absorbance at 517 nm was measured using spectrophotometer (Libra S22, Biochrom, England), and compared to that at 0 min. The percentage of inhibition was expressed using the following equation: DPPH radical scavenging activity (%) = [(Abs t=0 Abs t=30 min )/Abs t=0 ] x 100 Superoxide radical scavenging activity (SRSA) was also determined. The superoxide radical which reacts with NBT generates a colored compound with absorbance at 560 nm (Rivero-Perez et al., 2008). The reactive was made up with 50 µl NADH (77 µm), 50 µl NBT (50 µm), 5 µl of PMS (3.3 µm) and 10µL of the sample. The result was expressed as % inhibition in relation to a control test (reactive mixtures without the sample) as: Superoxide radical scavenging activity (%) = (T T 1 ) x 100/ (T T 2 ) T = Abs of SRSA generation system, T 1 = Abs of control, T 2 = Abs test sample systems Statistical analysis All expressed values were mean + SD of triplicates. Data were statistically analyzed by analysis of variance (ANOVA) test procedure and significant was statistically used to test the influence of rice varieties, and Duncan s Multiple Range test was used for comparisons among rice varieties at a significant level of P<0.05. Results and Discussion Color, total solid and light transmission Color parameters, total solid and light transmission of pigmented rice water extract are presented in Table 1. The result shows that water extract from pigmented rice had lower L* value, but higher a* and b* values than those of non waxy rice varieties. This would suggest that water extracts from waxy rice had darker color. This result is agreed well with the previous study (Yodmanee, 2010). The dehusked grains of non waxy and waxy rice varieties had been determined for their colors and it was found that waxy pigmented rice varieties (BWR96025, BWR96044 and CMP) showed dark purple color while non waxy rice showed red color (SY, KR, RWR96060, HK and KN) (Yodmanee, 2010). Color of water extract from pigmented rice could be derived from anthocyanins contained in rice seed coat and is water soluble (Furukawa et al., 2007). Rice water extract, for all varieties had total solid in the range of %. This should be related to transmission of water extract samples which affected light transmission through. Water extract from BWR96044 variety showed the highest transmission and lowest total solid. Light transmission in the range of %, which were significantly different among those varieties. This should be related to total solid of water extract samples which affected light transmission. Water extract from BWR96044 variety showed the highest transmission and lowest total solid. 50
4 Total polyphenol and anthocyanin content Total polyphenol in water extract was expressed as mg gallic acid equivalents (GAE) per ml of the extracts. From Table 2 total polyphenol of all samples was in the range of mggae/ml, and ranked in descending order, as follows: BWR96025, BWR96044, CMP, RWR96060, KR, SY, KN and HK. Yodmanee (2010) determined phenolic content of rice grain from those varieties, and found that the first three highest phenolic compounds varieties were BWR96025, BWR96044 and CMP as found in this study. Many studies have reported that polyphenol contents of the extracts from pigmented cereals, including pigmented rice, are higher than colorless cereals (Finocchairo et al., 2010; Choi et al., 2007; Chotimarkorn et al., 2008; Shen et al., 2009). Table 1 Total solid, transmission and color parameters of pigmented rice extracts* Total solid Transmission Color parameters Rice varieties (%) (%) L* a* b* SY bc b f a a Non-waxy HK d b e b b rice KN bc f e c c Waxy-rice KR d a d e e RWR b d e d f CMP c c c f d BWR b e a h h BWR a f b g g *Rice grains: water ratio = 1:25, heating at 100 ο C for 15 min Mean value + standard deviation of triplicates. Mean values with different letter in the same column are significantly different (p<0.05) Total anthocyanin contents varied greatly in the range of mg Cy-3-G/ml of the extracts among rice varieties. It can be noticed that water extracts from waxy rice grain (KR, RWR96060, CMP, BWR96025 and BWR96044) had higher anthocyanin content than those of non waxy rice grain. This is related to their color parameter, especially a* and b*. The anthocyanins are composed of a group of intensely colored pigments responsible for the purple color (Escribano-Bailon et al.,2004). The two color parameters (a* and b*) are significantly correlated with phenolics, anthocyanin contents and antioxidant capacity (Shen et al., 2009; Yodmanee, 2010) Antioxidant activity In this study the antioxidant activity of pigmented rice water extracts (rice grain: water = 1: 25, heating at 100 o C for 15 min) was determined by DPPH, ABTS + and SRSA assays. The results are presented in Figure 1. The water extract from waxy rice had DPPH radical inhibition activity in the range of %, and were higher than those of non-waxy rice ( %). The first three highest varieties were BWR96025, BWR96044 and CMP ABTS + method is employed for measuring the relative radical inhibition activity of hydrogen donating and chain breaking antioxidants (Netzel et al., 2003). From this result, water extract of waxy rice varieties had higher ABTS + radical inhibition activity than those of non-waxy rice. The first three higher values of rice extracts were from BWR96025, BWR96044 and CMP varieties, which were 84.02, and 76.20% respectively. 51
5 Table 2 Total polyphenol and total anthocyanin contents of pigmented rice extracts* Non-waxy rice Waxy-rice Rice varieties Total polyphenol (mg GAE/ml extracts) Total anthocyanin** (mg Cy-3-G/ml extracts) SY b b HK a a KN b b KR c c RWR d c CMP e d BWR g e BWR f e *Rice grains: water ratio = 1:25, heating at 100 o C for 15 min ** λ max of anthocyanin was 500 nm. Mean value + standard deviation of triplicates. Mean values with different letter in the same column are significantly 80 (a) different. DPPH radical scavenging activity (%) A B C D E F G H ABTS radical scavenging activity (%) (b) A B C D E F G H 52
6 SRSA radical scavenging activity (%) (c) A B C D E F G H Figure 1 Radical inhibition (%) of water extract (0.1 mg/ml) from pigmented rice. (a), DPPH, (b) ABTS + and (c) and SRSA A = SY; B,= HK; C= KN; D = KR; E= RWR96060; F= CMP; G= BWR96025; H = BWR96044 The analysis of SRSA inhibition activity of determined oxygen free radicals could be considered as a first approach of the potential healthy preventive effect of the moderate consumption of the extracts (Zhang et al., 2006). The SRSA radical inhibition activity of the water extracts from waxy rice and non waxy rice varieties were in the ranged of % and %, respectively. Similar to DPPH and ABTS+ results, the three higher SRSA radical scavenging activity were waxy rice varieties, BWR96025, BWR96044 and CMP. This result is agreed well with the previous study. Yodmanee (2010) has reported for methanolic extracts of these 8 varieties pigmented rice on DPPH and ABTS + scavenging activity.the result obtained also shows that BWR96044, BWR96025 and CMP varieties are higher than other varieties. Choi et al. (2007) reported that, the DPPH radical scavenging activity of the extracts from red sorghum and black rice showed relatively higher radical scavenging activity than non pigmented samples. Conclusions Eight varieties of pigmented rice grown in southern Thailand were determined for their water extracts properties particularly color and antioxidant properties. Water extract of three varieties of waxy pigmented rice BWR96025, BWR96044 and CMP had higher antioxidant, both content and activity, as well as darker color than those of others. The results obtained provide very useful information for selecting rice variety to expand pigmented rice utilization, especially for values added and healthy products. References Abdel-Aal, E.M., Young, J.C, and Rabalski, I Anthocyanin composition in black, blue, pink, purple and red cereal grains. Journal of Agricultural and Food Chemistry. 54, Aguilar-Garcai, C., Gavino, G., Baragoño-Mosqueda, M., Hevia, P. and Gavino, V.C Correlation of tocopheral, tocotrienol, γ-oryzanol and total polyphenol content in rice bran with different antioxidant capacity assays. Food Chemistry. 120, AOAC Official Method of Analysis. 16 th ed. Verginia: The Associate Analysis Chemists. Bao, J.S., Cai, Y., Sun, M., Wang, G.Y, and Corke, H Anthocyanins, flavonols, and free radical scavenging activity of Chinese bayberry (Myrica rubra) extracts and their color properties and stability. Journal of Agricultural and Food Chemistry. 53, Cevallos-Casals, B.A. and Cisneros-Zevallos, L Stability of anthocyanin-based aqueous extracts of Andean purple corn and red-fleshed sweet potato compared to synthetic and natural colorants. Food Chemistry. 86,
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