**2.1 Extraction methods for bioactive compounds in pigmented rice**

Several common extraction techniques that are used in the process of rice extraction include the method of solvent extraction, which is a conventional technique used to extract bioactive compounds from pigmented rice, supercritical fluid extraction, and subcritical water extraction. With regard to the conventional technique, a number of organic solvents are commonly used such as acetone, methanol, ethanol, butanol, and water in certain proportions as the extraction solvent [28–30]. In our study, 50% ethanol was used as an extraction solvent at a proportion of 1:5 grain or bran liquid, and extraction was carried out at room temperature for 3–12 h. The extracts were then concentrated with a rotary evaporator until all ethanol residues were removed and then further partitioned against saturated butanol to obtain the medium polar bioactive compounds of the black rice extract [31] or red rice extract [7, 15, 16]. The bioactive compounds present in these fractions shall be described in a later section. In another study, 60% ethanol containing 0.1% HCL was used as an extraction solvent with a 1:10 feed to liquid proportion, and extraction was carried out for 3–12 h. The extracts were then concentrated and further partitioned against petroleum ether [8]. In another study, the rice bran was extracted with 70% ethanol for 30 min repeated three times and was then further partitioned with ethyl acetate at pH 2–3 [32]. The same method was used to extract soluble phenolic compounds in white rice, brown rice, and germinated brown rice [33].

Supercritical fluid extraction has been widely used for the extraction of functional active compounds from medicinal plants including rice and cereals. This was in common with the use of supercritical carbon dioxide as an extraction solvent in other successful experiments. Kim et al. [34] used the method of supercritical fluid extraction of rice bran oil from pigmented rice, which provided higher yields of polyunsaturated fatty acids than the conventional use of organic solvent extraction. In yet another study, supercritical carbon dioxide extraction was used, and yields of 17.5% oil were achieved from powdered rice bran, and a yield of 37% of γ-oryzanols was also obtained, which was characterized as 85% of the extraction efficiency [35].

Another extraction technique is the subcritical water extraction method that has been developed for the extraction of bioactive compounds from pigmented rice through the use of hot water at temperatures between 100 and 374°C under high pressure to maintain a liquid status. This technique is considered to be very friendly to the environment because no organic solvents are used, and this can potentially alleviate some of the problems associated with the conventional methods [36, 37].

There were differences in the extraction procedure and the varieties of the rice cultivars that were used to detect the amounts of bioactive compounds in different portions of rice such as in the whole grains, kernels, endosperm, husks, rice, and bran. More than 1000 published studies have been reviewed to make up the cited data based on this information. Some data on rice composition have been selectively recorded elsewhere [27].

#### **2.2 Various bioactive compounds present in black rice**

Phytochemical profiles of black rice are characterized by the presence of anthocyanins, which are a group of reddish to purple flavonoids that exist in black rice and other pigmented cereal grains. The main anthocyanins in black rice were found to be present in quantities more than 95% and were cyanidin 3-O-glucoside (2.8 mg/g) and peonidin-3-O-glucoside (0.5 mg/g) followed by flavones and flavonols (0.5 mg/g) and flavan-3-ols (0.3 mg/g) [38]. The concentrations of total anthocyanins in black rice cultivars significantly varied from one report to another, while much higher concentrations of anthocyanins were detected in Chinese black-purple rice that contained cyanidin 3-O-glucoside (6.3 mg/g) and peonidin 3-O-glucoside (3.6 mg/g) [39]. The variations of the anthocyanin content in the reports on black rice might be due to the use of different cultivars and the variety of differing growing conditions. The anthocyanidins or aglycons, the basic structure of anthocyanins, consist of an aromatic ring (A) that is bonded to a heterocyclic ring (C) that contains oxygen, which is bonded by a carbon–carbon bond to a third aromatic ring (B). When the anthocyanidins are bonded to a sugar moiety in the glycosidic linkage, they are known as anthocyanins. More than 500 different anthocyanins and 23 anthocyanidins have been reported. Anthocyanins exist as mono-, di-, or tri-O-linked glycosides and acyl glycosides of anthocyanidins in plants. The sugar moiety may be substituted by aliphatic, hydroxybenzoic, or hydroxycinnamic acids. The structural characteristics of anthocyanins make them highly reactive toward the reactive oxygen species (ROS) [27]. The basic structure of this is shown in **Figure 2**. Major flavone and flavonol glycosides present in black rice are taxifolin, quercetin, apigenin, and luteolin, which are comprised of monomeric and oligomeric constituents. The concentrations of the flavone and flavonol contents were

**7**

*Anthocyanins and Proanthocyanidins in Natural Pigmented Rice and Their Bioactivities*

significantly higher in black rice than in red, brown, or white rice. This was especially true with regard to taxifolin O-hexoside, quercetin 3-O-glucoside, and quercetin 3-O-rutinoside, which were detected only in black rice [38]. Abdel-Aal et al. [40] also reported that the mean anthocyanin content in black rice (3.276 mg/g) was about 35-fold higher than that of red rice (0.094 mg/g). Additionally, the contents of anthocyanin present in Northern Thai black rice cultivar obtained from Doi Saket, Chiang Mai, were 8.1 mg/g extract, which was considered very high when compared to the anthocyanin content found to be present in the Northern Thai red

The total procyanidin content in black rice has been found to be present in high variations depending on the grain cultivar; however, it is noteworthy to mention that procyanidins are typically observed in red rice but not in black rice varieties [41–45]. Interestingly, some black cultivars have shown the presence of oligomeric procyanidins with a 2–10 degree of polymerization [38]. Furthermore, black and red rice were found to contain only one flavan-3-ol monomer, catechin. Additionally, a Canadian black rice variety also contained catechins at levels four times higher than epicatechin. Furthermore, the concentration of catechin was much higher in red rice (92 μg/g) than in black rice (20 μg/g) [46]. Other phytochemicals have been detected in black rice including all four derivatives of γ-oryzanol, such as 24-methylenecycloartenol, campesterol, cycloartenol, and β-sitosterol ferulates, along with lower levels of carotenoids. The main carotenoids detected in black rice were xanthophylls, lutein, and zeaxanthin, while lycopene and β-carotene could be detected but were found to be present as a minor component [38]. The value of the carotenoid content in black rice kernels is lower than the carotenoids found to be present in black rice bran. It was reported that values in a range of 33–41 μg/g of carotenoids were found in the bran extracts of four varieties of Thai black rice [47]. A range of phenolic compounds including vanillic acid, protocatechuic acid, chlorogenic acid, ferulic acid, and coumaric acid has been detected in black rice with the dominant phenolic acids being present in red and black rice bran [7, 31]. The contents of phenolic compounds, flavonoids, catechins, anthocyanins, and proanthocyanidins, are summarized in **Table 1** as examples of the phytochemicals that were detected in Doi Saket Thai black rice cultivar. The germ and bran extracts of the black and red rice varieties were found to have the greatest phytochemical content with decreasing amounts occurring in the rice hull and even less in the seeds or kernels. Additionally, the expected low levels of these phytochemicals were found in white rice as a consequence of the milling process.

*DOI: http://dx.doi.org/10.5772/intechopen.86962*

rice cultivar obtained from Dok Khamtai [31].

**2.3 Various bioactive compounds present in red rice**

Red rice was characterized by a high quantity of oligomeric procyanidins (0.2 mg/g) with more than 60% of total phytochemicals found in the rice seeds. Proanthocyanidins are high molecular weight polymers or complex flavan-3-ol polymers that consist mainly of catechin, epicatechin, gallocatechin, and epigallocatechin units that can also be found in rice germ and bran, particularly in pigmented rice. The degree of polymerization varied, and the reddish colored test was associated with the presence of a class of polymeric compounds of the proanthocyanidins. These could be in the sum class of the oligomer and polymer contents of the total proanthocyanidins present in the red rice bran extract fraction. The degree of polymerization and galloylation can affect their bioactivity and proanthocyanidin profiles differently depending on the food sources [27, 48]. Proanthocyanidins can be classified into several classes depending on the degree of hydroxylation of the constitutive units and the linkages between them. Our research group has reported on the type of proanthocyanidins found in the red rice that was collected from Dok Khamtai

**Figure 2.** *General structure of anthocyanins.*

#### *Anthocyanins and Proanthocyanidins in Natural Pigmented Rice and Their Bioactivities DOI: http://dx.doi.org/10.5772/intechopen.86962*

significantly higher in black rice than in red, brown, or white rice. This was especially true with regard to taxifolin O-hexoside, quercetin 3-O-glucoside, and quercetin 3-O-rutinoside, which were detected only in black rice [38]. Abdel-Aal et al. [40] also reported that the mean anthocyanin content in black rice (3.276 mg/g) was about 35-fold higher than that of red rice (0.094 mg/g). Additionally, the contents of anthocyanin present in Northern Thai black rice cultivar obtained from Doi Saket, Chiang Mai, were 8.1 mg/g extract, which was considered very high when compared to the anthocyanin content found to be present in the Northern Thai red rice cultivar obtained from Dok Khamtai [31].

The total procyanidin content in black rice has been found to be present in high variations depending on the grain cultivar; however, it is noteworthy to mention that procyanidins are typically observed in red rice but not in black rice varieties [41–45]. Interestingly, some black cultivars have shown the presence of oligomeric procyanidins with a 2–10 degree of polymerization [38]. Furthermore, black and red rice were found to contain only one flavan-3-ol monomer, catechin. Additionally, a Canadian black rice variety also contained catechins at levels four times higher than epicatechin. Furthermore, the concentration of catechin was much higher in red rice (92 μg/g) than in black rice (20 μg/g) [46]. Other phytochemicals have been detected in black rice including all four derivatives of γ-oryzanol, such as 24-methylenecycloartenol, campesterol, cycloartenol, and β-sitosterol ferulates, along with lower levels of carotenoids. The main carotenoids detected in black rice were xanthophylls, lutein, and zeaxanthin, while lycopene and β-carotene could be detected but were found to be present as a minor component [38]. The value of the carotenoid content in black rice kernels is lower than the carotenoids found to be present in black rice bran. It was reported that values in a range of 33–41 μg/g of carotenoids were found in the bran extracts of four varieties of Thai black rice [47]. A range of phenolic compounds including vanillic acid, protocatechuic acid, chlorogenic acid, ferulic acid, and coumaric acid has been detected in black rice with the dominant phenolic acids being present in red and black rice bran [7, 31]. The contents of phenolic compounds, flavonoids, catechins, anthocyanins, and proanthocyanidins, are summarized in **Table 1** as examples of the phytochemicals that were detected in Doi Saket Thai black rice cultivar. The germ and bran extracts of the black and red rice varieties were found to have the greatest phytochemical content with decreasing amounts occurring in the rice hull and even less in the seeds or kernels. Additionally, the expected low levels of these phytochemicals were found in white rice as a consequence of the milling process.
