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Six varieties of pigmented and non-pigmented rice both cultivated locally and imported were investigated for their antioxidant properties. The antioxidant activity was determined by measuring the total phenolic content, total flavonoid content, anthocyanin content, ferric reducing antioxidant potential, 2,2-diphenyl-1-picrylhydrazyl free radical (DPPH) scavenging capacity and radical cation ABTS+ scavenging activity using different extracting solvent (100% methanol, 100% ethanol and water). Methanolic extract yielded the highest total phenolic content for all samples (26.27- 60.4 mg GAE/100g), for ferric reducing antioxidant potential (0.98-8.42 mM Fe (II)/g) and for anthocyanin content (0.6067-116.33 mg cyanidin-3-glucoside Eq/100g). Black rice had the highest anthocyanin content for all 3 different solvents. For 2,2-diphenyl-1-picrylhydrazyl free radical (DPPH) scavenging capacity, ethanolic yielded the highest in the range of (17.56-79.31 %) with brown rice being the highest. Radical cation ABTS+ scavenging activity had a range from 1.24-6.87 TEAC mM/100g for all rice samples with 100% ethanol yielding the highest antioxidant activity. These results indicate that pigmented and non-pigmented rice might possibly have antioxidants.
Keywords: Antioxidant, Total phenolic content, pigmented rice, extraction solvent effect
Nearly 50% of earth's population primary food is rice. There are many different kinds of rice consumed by human's, however, white rice is by far the most popular followed by brown rice, red, purple or black rice. (Nanri et al., 2010; Sompong et al., 2011). Pigmented rice is used as a constituent in many Asian cuisines as it has its own particular colour and flavour (Rhee et al., 2000). Recent studies have found an excellent source of natural antioxidants which includes many different kinds of phenolic compounds and can be found from the gramineae family of cereals (Chotimarkorn et al., 2008; Gani et al., 2012). Other compounds with antioxidants actions such as tocopherols, tocotrienols and γ-oryzanol have also been associated with rice (Chen et al., 2005). Apart from that, anthocyanins compounds can be found in the pericarp of pigmented rice, such as brown and black rice (Chaudhary et al., 2003). The inhibition of free radical damage and health benefits associated with these compounds has been long known. Pigmented rice varieties have anti-inflammatory properties and are known to prevent cancer, anti-aging, heart and nerve diseases (Kehrer et al., 1993, Tsuda et al., 2002, Manosro et al., 2012). Much interest has been generated in search for various biological activities due to the presence of various antioxidant compounds in rice. Various solvent systems has been used for extraction, however the yield of each solvent is different, as extracted antioxidant compounds are greatly influenced by the type of solvent used. Therefore the present study aims to evaluate the yield of antioxidant compounds in various pigmented and non-pigmented rice varieties using different extraction solvents.
Materials and Method
Six preferred pigmented and non pigmented rice varieties were purchased from the local Supermarket. The local non-pigmented rice varieties grown in Malaysia were the White rice (Medium grain type) and Bario rice. Pigmented rice variety used, which was grown in Malaysia was Brown rice. Black rice was the only pigmented variety used from Thailand. However, non-pigmented rice variety which was Basmati rice from Pakistan and glutinous rice from Thailand was also used in this study. Only whole rice grains without any visual defects were selected for analysis. Rice samples were ground to a fine powder (30 mesh size) and packed in air tight plastic bags. They were later stored at 4°C until further analysis.
Extraction of samples
The extraction was performed according to the method described by Atala et al., 2009. Approximately 10 g of each sample was extracted using 100 ml of distilled water, methanol and ethanol. Extracts were then placed in a shaking water bath for 90 min at room temperature before being centrifuged at 950 g for 15 min. It was later stored at −20 °C for analysis of antioxidant compounds and activities.
Determination of total phenolic content
Determination of total phenols in the extract from the rice samples was done using a modified version of the Folin-Ciocalteu assay (Singleton and Rossi, 1965). 400 μL of the sample extract was mixed with 2.0 ml of FC reagent (10 concentrations of aqueous solution of gallic acid. The absorbance was measured at 765 nm. The phenolic fold diluted with distilled water). 1.6ml of (7.5% w/v) sodium carbonate solution was added. The solutions were mixed and allowed to stand for 1h at room temperature. Gallic acid standard curve was prepared with appropriate content was expressed in mg gallic acid equivalents (GAE) per gram of sample.
Determination of total flavonoid content
The aluminium trichloride method as described by Liu et al. (2008), was used to determine the total flavonoid content. Briefly 500μL of the rice extract solution was added to a test tube with 2.5 ml of distilled water and 5% w/v, 150 μL was added to the mixture and maintained for 5 min. After which, 300 μL of aluminium trichloride (10% w/v) was added and the contents was mixed well and allowed to incubate for 6 min. 1 ml of 1 M sodium hydroxide was added. Then the mixture was diluted with 550 μL of distilled water and shaken vigorously. The absorbance was measured immediately at 510 nm and the total flavonoid content was expressed as mg quercetin equivalent (QE)/ 100g.
Determination of total anthocyanins
Total anythocyanins in the sample extract of the infloroscence was determined using the spectrophotometric pH differential method as described by Giusti et al. (2001). In brief, 0.5 ml of the extract was mixed thoroughly with 3.5 ml of 0.024 M potassium chloride buffer (at pH 1). The mixture was let to stand for 15 min before the absorbance was measured at 510 and 700 nm using UV visible spectrophotometer against a blank which contained distilled water. The extract was then added to 0.025 M sodium acetate buffer at pH 4.5 following the same procedure as previously mentioned. The absorbance was then measured again at 510 nm and 700 nm after 15 min. The total anthocyanin content was calculated using the following equation:
Total anthocyanin content (mg/l) = (A x MW x DF x 1000)/ (ε X 1)
Where A is the absorbance of the extract calculated as:
A= (A 515-A700) pH 1.0 - (A515 - A 700) pH 4.5
MW is the molecular weight for cyanidin-3-glucoside = 449.2; DF is the dilution factor and ε is the molar absorptivity of cyanidin-3-glucoside = 26900. Results were expressed as mg of cyanidin-3-glucoside equivalents for 100 g of sample.
Ferric Reducing Antioxidant Potential
Modified method described by Benzie and Strain (1996) was employed to measure the ability of the extracts to reduce ferric ions. 300 μl of sample extract was added to 4.5 ml of FRAP reagent which was prepared by mixing 300mM sodium acetate buffer at pH 3.6 (10 parts), 10 mM TPTZ solution (1 part) and 20mM FeCl3.6H2O solution (1 part). The reaction mixture was incubated in the water bath at 37°C for 4 min. The absorbance was measured at 594 nm using UV-visible spectrophotometer. Ferrous sulphate solution was used to prepare the standard calibration curve and FRAP values were expressed as mM ferrous equivalents per gram of rice.
DPPH Radical Scavenging Activity
The DPPH radical scavenging activities of extracts were measured following the method described by Blois (1958). 2 ml of the extract and 2 ml of DPPH (1,1-diphenyl 2- picrylhydrazyl) solution (0.2mM in methanol) was added and the solution was incubated in the dark for 30 min at room temperature. The control solution contained 2 ml of DPPH solution and 2 ml of distilled water, methanol and ethanol respectively. The absorbance was measured at 517 nm by using UV-visible spectrophotometer. The DPPH radical scavenging activity was calculated using the formula:
DPPH radical scavenging activity (%) = [(A0 - A1)/A0) - 100]
where A0 is the absorbance of the control reaction, and A1 is the absorbance of presence of all of the extract samples and standard.
ABTS was determined using the method described by Choi et al., (2007). 7 mM ABTSË™+ was prepared then added into 2.45 mM potassium persulfate in order to make the ABTS solution. The solution was then allowed to react overnight at room temperature and placed in a dark place. Distilled water was used to dilute the ABTS solution to get an absorbance in the range of 1.4-1.5 at 414 nm. The extracts which had different concentrations were then added to 6 ml ABTS stock solution and mix thoroughly. The absorbance was recorded after incubating the solution for 1 hour in the dark. Trolox equivalent antioxidant capacity (TEAC) was used to express ABTS radical cation scavenging activity in mM of Trolox equivalent per 100g of flour.
Colour analysis was carried out on rice flour using a colorimeter (Minolta Spectrophotometer model CM-3500d, USA). The colorimeter was calibrated first by using the zero calibration plate (CM-A120), followed by the white calibration plate (CM-A128). Twelve measurements of L*, a*, b*, Chroma were taken for each sample to obtain consistent results and mean value. L*, a*, b*, Chroma were chosen as it represents lightness (brightness), redness, yellowness and saturation (vividness).
Results and Discussion
Total Phenolic Content
The Folin-Ciocalteau method was used as it is a rapid, easy and relatively simple method to identify total phenolic content in rice samples. A close interdependence between the composition of phenolic compounds and antioxidant activity is expected as phenolic compounds are potential antioxidants and free radical scavengers (Kumar et al., 2008). Methanolic extracts generated more phenolic compound than water and ethanolic extracts in this study (Figure 1A).
The results obtained are the same as reported by (Wang et al., 2011). The highest phenolic content was obtained in black rice, followed by brown rice, white rice, bario rice, basmati and the glutinous rice. Black rice samples had the highest phenolic content in the range of 57.56 to 60.4 mg GAE/100g. However, glutinous rice had the lowest amount of phenolic content present in the range of 19.89 to 26.27 mg GAE/ 100g with ethanolic extract yielding the lowest phenolic compounds followed by basmati rice in the range of 22.56 to 23.75 mg GAE/100g. Significant difference did exist between all samples using the different extracts. The phenolic for all samples varied from 19.89 to 60.4 mg GAE/100g. Brown and black had the higher polyphenolic content compared to white rice, which propose that a large amount of polyphenolics has been lost during the milling process (Goffman and Bergman 2004). Compared to other cereals such as oats and wheat, rice had a higher total phenolic content (Dar and Sharma, 2011).
Total Flavanoid Content
Flavonoids have been linked in a previous study to reduce the risk of coronary heart disease and the moratlity caused by it (Hertog et al., 1994). Figure 1B, shows the total flavonoid content of the different rice varieties. Flavanoid content in all samples ranged from 3.27 to 16.54 mg (t) -catechin equivalent (CEQ)/g. Ethanolic extracts yielded the highest total flavonoid content followed by methanol and water for all samples except for white rice. Black rice extracts had the highest total flavonoids content in ethanolic extract, followed by methanolic extracts 13.53 and water extract 13.01 mg (t) -catechin equivalent (CEQ)/g. The lowest total flavonoid content was found in water extract for glutinous rice, 3.27 mg (t) -catechin equivalent (CEQ)/g. There is no significant difference between black and brown rice for all the different solvents. The flavonoid content in all white pigmented rice samples was generally low as the milling process removed most of it and similar findings were reported by (Shen et al., 2009). Black rice varieties have higher flavonoid content compared to other non pigmented rice samples (Zhang et al., 2010).
Based on the results in Table 1, methanolic extract gave the highest yield followed by ethanol and then water. Glutinous rice had the lowest anthocyanin content with a range of 0.30 to 0.61 mg cyanidin-3-glucoside/100g) where else the second lowest was basmati rice with a range of 1.02 to 1.46 mg cyanidin 3-glucoside/100g). Black rice had the highest anythocyanin content in this study and the results are in agreement with Sompong et al., (2011). According to Metivier et al., (1980) the most effective solvent for extracting anthocyanins are methanol and it has been proven to be 73% more effective than using water as an extracting solvent. The application of pigmented rice especially black rice as an added source of anthocyanin's in functional food formulation and as a colourant has been suggested by (Hu et al., 2003; Nontasan et al., 2012).
Ferric Reducing Antioxidant Potential (FRAP)
FRAP assay is used as it's easy to be standardized as it measures the potent of the chemical compounds present in the extract to challenge ferozine for ferrous ion (Yan et al., 2006). Once the ferric ions has been reduced to ferrous ion, the solution then changes from being yellow to bluish green in colour. The higher the FRAP value, the greater its antioxidant activity. Based on Figure 1C, methanolic extracts had the highest reducing power followed by water and then ethanolic extracts. Black rice samples had the highest reducing power in the range from 7.15-8.43 mM Fe (II)/g. No significant difference is observed between white and bario rice for both ethanolic and water extracts. Brown rice had the second highest reducing power between sample from the range of 6.32 to 7.55 mM Fe (II)/g. From the results obtained, it indicates that pigmented samples did show a significant difference on the effect of ferric reducing ability which in agreement with (Fasahat et al., 2012). It also shows methanol as a better extracting solvent when compared to the others used in the study due to its polarity and solubility of the phenolic compounds present.
2,2-diphenyl-1-picrylhydrazyl free radical (DPPH) scavenging capacity
DPPH radical scavenging method is widely used as it is fast and convenient method for determining radical scavenging activity of many samples without being dependant on sample polarity (Koleva et al., 2001). The electron-/hydrogen donating ability of antioxidants are what affect's DPPH radical scavenging activity. Its characteristic absorption is at 517 nm and exists as a stable, organic free radical which gives a deep purple colour in methanolic solution. As the reaction between radicals and antioxidants progress, a decrease is observed in absorption of DPPH radical (Biswas et al., 2010). Rice varieties extracted with 100 % ethanol exhibited the highest antioxidant activity in terms of the different solvents used (Figure 1D).
Black rice had the highest DPPH in the range from 62.41 to 79.31% and glutinous rice had the lowest, in the range of 14.25 to 17.56%. The results obtained from this study are similar to the results obtained by (Maisuthisakul et al., 2009), which stated that pigmented rice has higher DPPH radical scavenging activity. Significant differences did exist between all the rice samples, hence confirming that different percentage DPPH free radical scavenging activity of different rice varieties is affected by the extracting solvent.
Radical cation ABTS+ scavenging activity
Evaluating the corresponding radical scavenging activity of hydrogen contributors and chain disintegrating antioxidants in many plant extracts is usually assessed by using the ABTS method (Netzel et al., 2003). All samples showed a higher value for ABTS when extracted with 100% ethanol, followed by water and then 100% methanol (Table 2).
Brown rice extracted with 100% ethanol showed much higher radical scavenging activity followed by water and methanolic extract. Black rice extracted with 100% ethanol recorded a 6.34 TEAC mM/100g followed by water 5.87 TEAC mM/100g and then 100% methanol (4.9 TEAC mM/100g). It has been reported that reddish brown rice has high ABTS activity compared to other pigmented rice's (Yodmandee et al., 2011; Jun et al., 2012).
Different solvents for extraction
For this study, the extraction and determination of antioxidants present in rice was determined by using three different solvent systems which are ethanol, methanol and water. Previous studies have reported that different solvents used for extraction does play an important role in the antioxidant yield (Mohammedi et al., 2011 ; Marinova et al., 1997). Insoluble, bound phenolics, esters both free and soluble can be found in various cereals such as wheat, rice and many others (Shahidi, 1997) Ethanol and methanol has been used widely as an extraction solvent for antioxidant and is generally recognized as safe (GRAS). Water on the other hand is commonly used for cooking rice. Compared to methanol and ethanol, the OH group present in water has a much higher polarity. Phenolic compounds are more soluble with a rise in solvent polarity. Apart from polarity, another factor affecting yield is the chemical composition of extractable components made available due to their difference in nature (Shaflan et al., 2009). Phenolic compounds are not present and distributed uniformly in tissue and can be found free, conjugated or form complexes with plant components, proteins and carbohydrates (Luthria et al., 2006). According to Alothman et al., (2009) phenolic compounds solubility in the extraction solvent has an effect on the recovery of polyphenols from rice.
For phenolic content, pure methanol had the highest yield, followed by water and pure ethanol. Even though, phenol compounds can dissolve better in ethanol, the results showed otherwise. Water in contrast to methanol used as a solvent for extraction might be a better medium when toxicity and price are thought about. Therefore, choosing the appropriate solvent is one of the most important factors in obtaining extracts with a high content of bioactive compounds.
Grain colour is another quality indicator and it does have an impact on the rice price sold in the market (Guzman et al., 2008). The values obtained for a* which corresponds for redness and b* which corresponds for yellowness, were in the range of 0.21-8.19 and 4.79-17.05 respectively (table 3). Significant differences within the tested rice varieties were found, of which glutinous rice had the highest and black rice had the lowest L* which express the brightness. Black rice gave the lowest reading for b* and brown rice gave the highest value for a*. Chroma, a measure of vividness of colour was very high in all rice samples.
The research showed that different rice varieties does show potential in being a source for phenolic, flavonoids, anthocyanins and many others antioxidant compounds. The rice varieties extracted with methanol showed the highest total extract yield for phenolic content followed by water and then ethanol. However for flavonoid and DPPH, ethanolic yielded the highest antioxidant activity. Pigmented rice, black and brown rice had the highest antioxidant activity for all tests except for DPPH radical scavenging activity. White rice recorded the highest DPPH activity. From the results obtained, it shows that rice grains especially pigmented rice has many health benefits and consumer consciousness regarding this fact should be spread. It can be used in many other industrial applications.