Study on Total Phenolic Contents and their Antioxidant Activities of Thai White, Red and Black Rice Bran Extracts

MATERIALS AND METHODS

This study was done for 12 months from February 1, 2008-March 5, 2009. All of experiment was carried out at the Department of Chemistry, Faculty of Science, Mahasarakham University, Thailand.

Materials: Thai rice cultivars used in this study were non-glutinous rice white color (Hom mali 105, Supan, Saohi, Hom chaiya and Hom jun) or red color (3256, GS 18003, 21606, 21699 and 5718) and glutinous of black rice (96041-20, 96051-37, 96004-49, 96023-35 and 96065-42). All of rice was cultured in the field of Mahasarakham University, Thailand. The rice seed were grown and then cultured in the selected area. In the culture time, the rice was supplemented with water and organic fertilizer. The environmental condition was about 30-32°C throughout the cultured periods. The age of the rice used in this study was 4 months.

Reagents and chemicals: Gallic acid, Folin-Ciocalteau reagent, 1,1-diphenyl-2-picrylhydrazyl (DPPH), linoleic acid, NaOH-phosphate buffer (pH 7), distilled water, ammonium thiocyanate, butylated hydroxyaniline, sodium carbonate and ferric chloride were purchased from Fluka (Switzeland). All other reagents and solvents used were of analytical and HPLC grade.

Preparation of crude extraction: Brans of rice samples (25 g) were extracted with methanol 3 times and each for 30 min at room temperature. The extracts were pooled and filtered through a 0.45 μm of Nylon membrane filter. The extracts were then concentrated which gradually reduced pressure on a rotary evaporator. The crude extracts were used for the determination of antioxidant activity (Osawa and Namiki, 1981).

Total phenolic content: The total phenolic contents of crude rice bran extracts were determined by spectrophotometric method using Folin-Ciocalteu’s phenol reagent (Osawa and Namiki, 1981). The crude extract (0.5 mL) was diluted to 5.0 mL with distilled water. Folin-Ciocalteu reagent (5.0 mL) was added and mixed thoroughly. After 3.0 min, 5 mL of 10% sodium carbonate solution was added and the mixture was allowed to stand for 1 h with intermittent shaking. The mixture solution was measured at 750 nm using a Thermo Spectronic 4001/4 (USA). The total phenolic content was analyzed against gallic acid calibration curve standard. This experiment was carried out in triplicate and averaged of values contents.

Thiocyanate assay: Erlenmeyer flask containing linoleic acid (0.13 mL) in 0.2 M NaOH-phosphate buffer (10 mL, pH 7) was firstly prepared then crude extract (1 mg) of different rice brans was added and adjusted volume to 25 mL with distilled water (Osawa and Namiki, 1981). The flasks were incubated at 40°C for a two weeks and the degree of oxidation was measured by thiocyanate method. Briefly, the incubation mixture (0.2 mL) was reacted with NH₄SCN (30%, 0.2 mL), 9.4 mL of 75% EtOH and 0.2 mL of FeCl₂ (2.53x10^-2 g/10 mL of 3.5 % HCl) solution. The sample was measured using spectrophotometer at 500 nm. The control solution was prepared in a similar method without some extracts, while α-tocopherol was used as standard. The experiment was carried out in triplicate and averaged of value activities.

DPPH free radical-scavenging assay: The crude extracts and BHA (5-40 mg mL^-1) were added to 1.5 mL of 0.1 mM DPPH (2,2-diphenyl-1-picrylhydrazyl) in ethanol. The mixture was shaken vigorously and left to stand for 20 min at room temperature in the dark. The absorbance was measured using spectrometer at 517 nm. The radical scavenging activity was calculated by the following equation:

where, A_c and A_s are the absorbance at 517 nm of the control and extract or standard, respectively. The experiment was carried out in triplicate and averaged for their scavenging activity.

Statistical analysis: All of data were expressed as the Mean±SD and percentage of each value.

RESULTS

Total phenolic content: With Folin-Ciocalteu reagent method using gallic acid as the standard, the average quantity of total phenolic compounds found in white, red and black rice bran extracts were in the ranged from 0.8931-0.9884, 1.0103-1.0494 and 1.0810-1.2239 mg GAE mg^-1 of extract, respectively as shown in Fig. 1. The red rice bran was remarkably the highest of total phenolic content compared to the black and white rice bran extracts. Black and white rice bran extracts showed very similar of total phenolic content and slightly lower than that of red rice. The highest value was 5718 and the next is 21606 strains for red rice. The total phenolic compounds were very similar for white rice, except Homchaiya was the lowest. For black rice, all of rice strains were similar content of total phenolic compounds. From the results, the different values of total phenolic compounds in all of rice cultivars did not dramatically different.

Fig. 1:	Total phenolic content of Thai rice bran extracts measured by the Folin-Ciocalteu method expressed as mg GAE/g: white rice; 1 (Mali105), 2 (Supun), 3 (Saohai), 4 (Homjun), 5 (Homchaiya), red rice; 6 (3256), 7 (GS18003), 8 (21606), 9 (21699), 10 (5718), black rice; 11 (96041-20), 12 (96051-37), 13 (96004-49), 14 (96023-35) and 15 (96065-42)

Fig. 2:	Antioxidative activity of crude extracts of Thai rice bran using thiocyanate method expressed as % inhibition of lipid peroxidation: white rice; 2 (Mali105), 3 (Supun), 4 (Saohai), 5 (Homjun), 6 (Homchaiya), red rice; 7 (3256), 8 (GS18003), 9 (21606), 10 (21699), 11 (5718), black rice; 12 (96041-20), 13 (96051-37), 14 (96004-49), 15 (96023-35) and 16 (96065-42) which compared with BHA (No. 1)

Anti-oxidative assay using thiocyanate method: With thiocyanate method, the red rice samples showed higher antioxidant activities than those of black and white rice. However, all of samples were lower antioxidant activity than BHA (Fig. 2). The bran extract of Homchaiya, however, exhibited the lowest inhibition of peroxidation of 10.15%, whereas 5718 showed the highest inhibition of peroxidation of 41.80%. The result showed that white rice was significantly lower of antioxidant activity compared to red and black rice. It is slightly surprised that black rice was similar of antioxidant activity in all of strains. Most of white strains are also composed of antioxidant activity in similar percentages, except Homchaiya. Moreover, 21606, 21699 and 5718 of red rice composed very high antioxidant.

Table 1:	Radical-scavenging activity of Thai rice bran extracts and BHA expressed by EC50 (mg mL-1)
activity. This result was positively correlated to the total phenolic content of the rice cultivars.

DPPH-free radical scavenging activity: The EC₅₀ of the rice bran extracts divided into 3 groups which were the lowest (red rice), middle (black rice) and highest (white rice) values (Table 1). In the highest group, 5718 showed the best free-radical scavenging activity (0.0057 mg mL^-1) and the next is 21699. Similar activity but lower was found in other cultivars. For middle group, 96023-35 showed the best free-radical scavenging activity (0.0218 mg mL^-1) while 96004-49 (0.0359 mg mL^-1) is the worst activity. Similar activity was also found in other cultivars. In the lowest group, Supan (0.1576 mg mL^-1) and Saohai (0.1644 mg mL^-1) appeared the highest free-radical scavenging activity whereas Mali 105 (0.2516 mg mL^-1) and Homchaiya (0.2582 mg mL^-1) gave the lowest activity in this group. The free-radical scavenging activity of all rice bran extracts were higher EC₅₀ values than BHA (0.0021 mg mL^-1).

DISCUSSION

Black and red rice are contained color pigments. These cultivars of rice have long history for people consumption, especially in Southeastern Asia (Hu et al., 2003). Rice bran extracts have been reported on a hypocholesterolemic effect as well as antioxidant activity (Sugano and Tsuji, 1997). In addition, consumption of black rice has resulted to artherosclerotic lesions (Ling et al., 2001), reduce oxidative stress and inflammatory (Xia et al., 2003) or cardiovascular protection (Xu et al., 2001). Antioxidant activity in white rice hull has been reported (Lee et al., 2003). However, pigmented rice, such as red and black rice, composed of high content of phenolic compounds (Oki et al., 2002; Clifford, 2000). They are distributed in the plant as secondary structure metabolite (Matinez-Valverde et al., 2000). Various benefits of the phenolic compounds are known to use many effects in human including oxidative damage of lipid and low density lipoproteins inhibiting platelet aggregation (Daniel et al., 1999) and reducing coronary heart disease and cancer risk (Matinez-Valverde et al., 2000; Newmark, 1996). Fruits and vegetables are major dietary sources of phenolic compounds, however, pigmented rice has also been found as an excellent source of the phenolic compounds (Tian et al., 2004).

The results from this study indicated the contents both total phenolic content and antioxidant activity were similar with many previously reported. Moreover, significantly different either total phenolic content or antioxidant activity compounds of black and red rice were higher than that of white rice. It might be suggested that both red and black rice composed of higher color pigment which were the main compounds for antioxidant activity. On the other hand, environment conditions such as temperature, light, water or soil may concern on composition of the compounds. It can not compare or imply that some substances in the rice cultured from different places. However, it is a promising that the color rice tendency compose many benefits compounds in higher than that of de-color rice. Moreover, the component of phenolic compounds in each section of the rice seed is also interesting to study. Furthermore, comparison between those of factors for rice culture should be explored.

CONCLUSION

The results showed that Thai red rice cultivars possessed relatively strong antioxidant activity. There was also a correlation between the specific antioxidation capacities and total phenolic contents of the red rice bran samples studies. The results indicated that antioxidant activities in the red rice bran extracts were largely owing to the phenolic compounds. Black rice bran extracts composed of high total phenolic content and free-radical scavenging activity as like as red rice, but in lower. On the other hand, white rice showed the lowest both total phenolic content and antioxidant activity. However, the results still appeared in high content compared to other plants. Therefore, Thai rice bran extracts should be acted as a potential source of antioxidative phytochemicals and useful ingredient for nutraceutical or functional food products.

ACKNOWLEDGMENTS

The authors thanks Dr. Perayos Khangkhun (Department of Plant Production Technology, Faculty of Technology, Mahasarakham University) for all rice samples supplement and Department of Chemistry, Faculty of Science, Mahasarakham University for a partial support of some chemicals and reagents. Thank you was also expanded to Faculty of Science and Division of Support and Development Research, Mahasarakham University and The Center of Excellence for Innovation in Chemistry (PERCH-CIC), Ministry of Education, Thailand for financial support.