TO EVALUATE THE EXTRACTION PROCESSES USING SOLVENT EXTRACTION TECHNIQUE FOR BIOACTIVE
3.3.3. Phytochemical properties
3.3.3.1. Total phenolic, anthocyanin, and flavonoid content
The phytochemical constituents of purple (y2) and black (y4) rice bran extract are shown in Table 3.3. The result of quantitative analysis of bran extract showed the presence of high amount phytochemical such as anthocyanin and flavonoids. The purple rice bran extract contented higher amount of TPC (901.3 mg GAE/100g), anthocyanin (20.2 mg cyanidin-3-glucoside/L) (Table 3.3) than black rice bran (18.9 mg cyanidin-3-glucoside/L) extract. Whereas, black rice bran extract contained high amount of flavonoid (17.8 mg quercetin/g) than purple rice bran (16.3 mg quercetin/g) extract as shown in Table 3.3.
3.3.3.2. Phenolic acid
The major portion of phenolic acids that existed in rice bran extract is shown in Table 3.3. In purple (y2) and black (y4) rice bran extract, seven and five numbers of phenolic acids were detected, respectively. The phenolic acids in purple and black rice bran extract was dominated by 4-hydroxybenzoic acid (284.3 and 289.6 µg/L) with lesser amounts of catechin-hydrate (4.2 and 98.1 µg/L), syringic acid (59.9 and 58 µg/L), sinapic acid (20.2 and 13.8 µg/L), and vanillic acid (87.3 and 135.2 µg/L). It was also observed that Caffeic acid (21.6 µg/L) and Chlorogenic acid (56.3 µg/L) were detected in purple rice bran extracts but were not detected in black rice bran extract. Very few studies have reported the occurrence of bound vanillic acid in cereal (Miyazawa et al., 2003). Laokuldilok et al.
(2010) reported the detection of six types of phenolic acid in the black, red and white rice bran. These observations was confirmed in the present study.
Table 3.3: Phytochemical content in rice bran extract
Phytochemical Purple rice bran
Extract
Black rice bran extract
TPC (mgGAE/100g) 901.3±8.2 852.6±4.0
Anthocyanin (mg cyanidin-3-glucoside/L) 20.2±1.3 18.9±0.7
Flavonoid (mg quercetin/g) 16.3±1.6 17.8±0.6
Phenol acid (µg/L)
1. Caffeic acid 21.6±2.0 0
2. Catechin hydrate 4.2±0.8 98.1±1.0
3. Chlorogenic acid 56.3±2.5 0
4. Syringic acid 59.9±3.2 58±0.9
5. Sinapic acid 20.2±1.7 13.8±0.7
6. Vanillic acid 87.3±3.2 135.2±2.3
7. 4-hydroxybenzoic acid 284.3±3.0 289.6±1.8
All the experiments were conducted in triplicate.
3.3.3.3. Antioxidant properties 3.3.3.3.1. DPPH scavenging activity
The DPPH scavenging activity of ethanolic extract of purple (y2) and black (y4) rice brans are shown in Table 3.4. The scavenging activity was investigated as a function of the concentration of extract. It was observed that for the increase of extract concentration, DPPH scavenging activity increase significantly (p ≤ 0.05) for both the extract. In maximum concentration, purple and black rice bran extracted showed 91.9 % and 82.2 % DPPH scavenging activity, respectively (Table 3.4). Sompong et al. (2011) investigated the scavenging activity of black rice of China, Sri Lanka. According to Sompong et al.
(2011), scavenging activity was varied from 13.0 % to 76.4 % which was quite lower than the scavenging activity of Indian purple and black rice variety. It was also illustrated from data that purple rice bran extract had a notable effect on DPPH scavenging activity than black rice bran extract, due to the presence of the high amount of TPC.
3.3.3.3.2. Reducing power
The reducing power of ethanolic purple and black rice bran extract was varied from 0.6 to 2.7 (%) and 0.5 to 2.0 (%) respectively, with the change of concentration. From Table 3.4, it was observed that as the concentrations of extract increased, the reducing power also increased significantly (p ≤ 0.05) for both the rice bran extract. It was also observed that
reducing power of purple rice bran extract was quite higher than black rice bran extract. It may be due to the presence of the high amount of TPC in purple rice bran extract than black rice bran extract, which causes the higher amount reduction of ferric (Fe3+) form to the ferrous (Fe2+) form (Ferreira et al., 2007).
Table 3.4: DPPH scavenging activity, reducing power, chelating activity, and peroxide scavenging activity of extract
Rice Bran
Concentration (%)
Radical scavenging activity (%)
Reducing power (%)
Chelating activity
(%)
Peroxide scavenging capacity (%)
Purple 20 68.7±0.8a 0.6±0.03a 20.4±2.0a 0.002
40 74.5±0.6b 0.9±0.02b 38.3±3.0b 0.004
60 77±0.8c 1.5±0.04c 54.8±2.0c 0.007
80 81.1±1.0d 2.1±0.08d 65.3±1.0d 0.011
100 91.9±2.0e 2.7±0.08e 68.6±1.0e 0.013
Black 20 45.3±2.0a 0.5±0.01a 20.2±1.0a 0.001
40 54.4±1.0b 0.7±0.01b 28.8±2.0b 0.002
60 66.3±3.0c 1.0±0.03c 44.7±3.0c 0.004
80 73.1±1.0d 1.5±0.02d 65.4±1.0d 0.008
100 82.2±4.0e 2.0±0.04e 66.3±2.0d 0.010
Superscript letters in the same column represent the significant difference in Duncan's multiple range tests at 5% level. All the experiments were conducted in triplicate.
3.3.3.3.3. Iron chelating activity
Purple and black rice bran extracts were assayed for Fe2+ chelating activity at different concentrations. The phenolic compounds in rice bran extract could interact with ferric (Fe2+) ion in the mixture and thereby changes the absorbance properties of the mixture. The purple rice bran extracts shown higher chelating activity (20.4-68.6 %) than black rice bran extract (20.2-66.3 %) for ferric (Fe2+) ion at any concentrations level (Table 3.4). From the results, it was obvious that there was a significant (p ≤ 0.05) correlation between the reduction of ferric ion and extract concentration of both the rice bran. It was illustrated from the Table 3.4 that as the concentration of the sample was increased from 20 to 100 % (v/v), the Iron chelating activity was increased from 20.2 to 66.3 and 20.4 to 68.6 (%) for black and purple rice bran extract, respectively.
3.3.3.3.4. Hydrogen peroxide scavenging capacity
The H2O2 scavenging activity is the method for the estimation of reactive oxygen scavenging ability in biological material (Paździoch-Czochra and Wideńska, 2002). It was observed from the present study (Table 3.4) that purple rice bran extract had higher potency to scavenge the free superoxide radical produced from hydrogen peroxide than black rice bran extract. Hydrogen peroxide scavenging capacity of purple and black rice bran extracts was also significantly (p ≤ 0.05) dependent upon the concentration of the extract. As the concentration of the extract in the sample was increased from 20 to 100 % (v/v), the hydrogen peroxide scavenging capacity was varied from 0.001 to 0.010 and 0.002 to 0.013
% for black and purple rice bran extract, respectively.
After conventional extraction, ultrasound was applied to efficiently extract the TPC and anthocyanin from black and purple rice bran. The conventional extraction showed that ethanol is the most effective solvent to extract the TPC. Thereby, for ultrasound-assisted extraction, only ethanol was used as extractant. Moreover, on the basis of optimizing the condition of conventional extraction the solvent to solid ratio was constant for ultrasound- assisted extraction (UAE) as 9.