*3.2.1.1 Anthocyanins*

Anthocyanins is a group of reddish to purple water-soluble flavonoids existing in pigmented rice and other cereal grains [27, 28]. The anthocyanidins or aglycons, the basic structure of anthocyanins, consist of an aromatic C6 (A ring) that bonded to a heterocyclic C3 (C ring) that contains oxygen, which is bonded by a carbon-carbon bond to a third aromatic C6 (B ring). When the anthocyanidins are bonded to a sugar moiety in the glycosidic linkage, they are known as anthocyanins [26]. In plants, they are found in mono, di, or tri of O-glycosides and acylglycosides of anthocyanidins [17]. Individual differences in anthocyanidins are related to the number of hydroxyl groups; the nature, number and position of sugars linked to the molecule; and the presence of aliphatic or aromatic acids attached to the sugar molecule. Anthocyanins are derived from the most common six anthocyanidins (aglycones) including cyanidin, delphinidin, petunidin, peonidin, malvidin and pelargonidin. Several anthocyanins have been isolated and identified from pigmented rice including cyaniding 3-glucoside, cyaniding 3-galactoside, cyaniding 3-rutinoside, cyaniding 3, 5-diglucoside, malvidin 3-galactoside, peonidin 3-glucoside, and pelargonidin 3, 5-diglucoside [6, 13, 28] and the basic chemical structures of the main anthocyanidins are shown in **Figure 2***.* Cyanidin-3-O-glucoside has been identified in black rice as the significantly higher than others [6, 13].

#### *3.2.1.2 Proanthocyanidins*

Proanthocyanidins are a group of polymeric phenolic compounds consisting mainly of flavan-3-ol units such as afzelechin, epiafzelechin, catechin, epicatechin, gallocatechin, and epigallocatechin (**Figure 3**) [26]. More complex proanthocyanidins, having the same polymeric building block, form the group of tannins. Proanthocyanidins can be A-type or B type structure with flavan-3-ol units doubly linked by C4-C8 and C2-O7 or C4-C6 and C2-O7 for the former, and linked mainly

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purple color in rice.

**Figure 3.**

**Figure 2.**

non-conventional methods [30, 31].

*Phenolic Compounds and Potential Health Benefits of Pigmented Rice*

through C4-C8 or C4-C6 for the latter B-type proanthocyanidin is very common in nature. In red rice, the block unit of proanthocyanidin consists of catechin and epicatechin. Proanthocyanidins are synthesized in plants by using anthocyanidins as key intermediates. These pigmented compounds are also responsible for red and

The determination of phenolic compounds is a necessary prerequisite not only to define the nutritional qualities of whole grain rice, but mostly to investigate on the health benefits associated to the consumption of these food plants [29]. Therefore, the most recent techniques for the extraction of the target compounds from rice along with the analytical approaches adopted for the separation, identification and quantification of phenolic acids, flavonoids, anthocyanins, and proanthocyanidins must be fully studied. Extraction is a process used for separating bioactive compounds from solutions using specific solvents by applying standard procedures. In addition, extraction of bioactive compounds can be obtained by using either conventional or

**4. Extraction, identification, and quantification**

*DOI: http://dx.doi.org/10.5772/intechopen.93876*

*The chemical structures of the main anthocyanidins.*

*The chemical structures of the main proanthocyanidins.*

*Phenolic Compounds and Potential Health Benefits of Pigmented Rice DOI: http://dx.doi.org/10.5772/intechopen.93876*


**Figure 2.**

*Recent Advances in Rice Research*

flavanols, flavanons, and anthocyanins.

*3.2.1 Anthocyanins and proanthocyanidins*

**3.2 Flavonoids**

pigment colors [17].

*3.2.1.1 Anthocyanins*

higher than others [6, 13].

*3.2.1.2 Proanthocyanidins*

polymerized into larger molecules such as the proanthocyanins. Moreover, phenolic acids may arise in food plants as glycosides or esters with other natural compounds

Like as phenolic acids, flavonoids are secondary metabolites of plants with polyphenolic structure. Flavonoids consist of a 15-carbon skeleton organized by a three-carbon chain (C6–C3–C6 structure) and they are the most diverse compounds in the plant kingdom. Flavonoids can be classified in to several sub-classes including flavanols, flavones, flavones, isoflavones and anthocyanins. The most common flavonoids of rice belong to a wide variety of sub-families such as flavonols, flavones,

Anthocyanins and proanthocyanidins are known as color pigments found in several varieties of rice as bioactive compounds. These colorful pigment bioactive compounds are located in the aleurone layer of rice grain [26]. Pigmented rice is diverse in the color, mainly due to the grain's high anthocyanin content. Several pigmented rice including black, brown, dark brown, dark purple and red-grain rice have been reported have been reported, which its color is depend upon the kinds of

Anthocyanins is a group of reddish to purple water-soluble flavonoids existing in pigmented rice and other cereal grains [27, 28]. The anthocyanidins or aglycons, the basic structure of anthocyanins, consist of an aromatic C6 (A ring) that bonded to a heterocyclic C3 (C ring) that contains oxygen, which is bonded by a carbon-carbon bond to a third aromatic C6 (B ring). When the anthocyanidins are bonded to a sugar moiety in the glycosidic linkage, they are known as anthocyanins [26]. In plants, they are found in mono, di, or tri of O-glycosides and acylglycosides of anthocyanidins [17]. Individual differences in anthocyanidins are related to the number of hydroxyl groups; the nature, number and position of sugars linked to the molecule; and the presence of aliphatic or aromatic acids attached to the sugar molecule. Anthocyanins are derived from the most common six anthocyanidins (aglycones) including cyanidin, delphinidin, petunidin, peonidin, malvidin and pelargonidin. Several anthocyanins have been isolated and identified from pigmented rice including cyaniding 3-glucoside, cyaniding 3-galactoside, cyaniding 3-rutinoside, cyaniding 3, 5-diglucoside, malvidin 3-galactoside, peonidin 3-glucoside, and pelargonidin 3, 5-diglucoside [6, 13, 28] and the basic chemical structures of the main anthocyanidins are shown in **Figure 2***.* Cyanidin-3-O-glucoside has been identified in black rice as the significantly

Proanthocyanidins are a group of polymeric phenolic compounds consisting mainly of flavan-3-ol units such as afzelechin, epiafzelechin, catechin, epicatechin, gallocatechin, and epigallocatechin (**Figure 3**) [26]. More complex proanthocyanidins, having the same polymeric building block, form the group of tannins. Proanthocyanidins can be A-type or B type structure with flavan-3-ol units doubly linked by C4-C8 and C2-O7 or C4-C6 and C2-O7 for the former, and linked mainly

such as sterols, alcohols, glucosides and hydroxy fatty acids.

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*The chemical structures of the main anthocyanidins.*

#### **Figure 3.**

*The chemical structures of the main proanthocyanidins.*

through C4-C8 or C4-C6 for the latter B-type proanthocyanidin is very common in nature. In red rice, the block unit of proanthocyanidin consists of catechin and epicatechin. Proanthocyanidins are synthesized in plants by using anthocyanidins as key intermediates. These pigmented compounds are also responsible for red and purple color in rice.

### **4. Extraction, identification, and quantification**

The determination of phenolic compounds is a necessary prerequisite not only to define the nutritional qualities of whole grain rice, but mostly to investigate on the health benefits associated to the consumption of these food plants [29]. Therefore, the most recent techniques for the extraction of the target compounds from rice along with the analytical approaches adopted for the separation, identification and quantification of phenolic acids, flavonoids, anthocyanins, and proanthocyanidins must be fully studied. Extraction is a process used for separating bioactive compounds from solutions using specific solvents by applying standard procedures. In addition, extraction of bioactive compounds can be obtained by using either conventional or non-conventional methods [30, 31].

#### **4.1 Conventional solvent extraction bioactive compounds**

Conventional extraction is being used at a small-scale level to extract bioactive components from several plant materials. This technique is usually based on the extraction efficiency of different solvents, which are being used for this purpose. The manual solvent extraction at ambient temperature is the most commonly used method in extracting bioactive compounds from grains. The solvents included acidified methanol with 1.0 N HCl (85:15, v/v), acidified methanol with 1 M phosphoric acid (95:5 v/v), acidified methanol with trifluoro acetic acid (99.8:0.2, v/v), acidified methanol with glacial acetic acid (95:5, v/v), and acetone/water (80:20, v/v). The extraction ratio was a material to solvent at 1:10 (w/v) [30]. In addition, in cold conditions, methanol (85%) and HCl (1 mol/L) was found to be an appropriate extraction solvent for anthocyanins, along with 85% methanol or 70:29.5:0.5, v/v acetone:water:acetic acid for free proanthocyanidins [17].

According to Shao et al., [12] soluble-free, soluble-conjugated and insolublebound phenolics of white, red and black rice were extracted by using 80% methanol. The soluble phenolics mixture was extracted and concentrated to obtain soluble phenolics. In order to get soluble-free phenolics, the concentrated soluble phenolics were further extracted by ethyl acetate three times, and then dried by a rotary evaporator, and dissolved in 5 mL of 50% methanol. To get soluble-conjugated phenolics, the concentrated soluble phenolics were hydrolyzed using 4 M NaOH for 2 h followed by adjusting pH to 1.5–2.0, extraction with ethyl acetate, drying using a vacuum evaporator, and then dissolving in 5 mL of 50% methanol. After the extraction of soluble phenolics, the residues were used to extract insoluble-bound phenolics. Similarly, the soluble-conjugated phenolics could be prepared from the concentrated soluble phenolics extracts by using 4 M NaOH and ethyl acetate.

In addition, our group [13] also used solid phase extraction (SPE) techniques to purify and prepare soluble-free (unbound fraction) and soluble-conjugated (polyphenol-rich bound fraction) phenolic compounds of pigmented rice. The crude extracts of colored rice were purified by applied to C18 solid phase extraction unit. The solid phase cartridge was pre-washed in 0.2% (v/v) formic acid in acetonitrile and then pre-equilibrated in 0.2% (v/v) formic acid in water. The unbound materials including free sugars, organic acids and vitamin C were collected. The SPE unit was then washed with a unit volume of 0.2% (v/v) aqueous formic acid and then with 2 volumes of ultra-pure water. The polyphenol-rich bound fraction was eluted with a unit volume of 80% (v/v) acetonitrile in water.

#### **4.2 Non-conventional extraction techniques**

The longer extraction time, costly and high purity solvent, evaporation of the huge amount of solvent, low extraction selectivity, and thermal decomposition of thermolabile compounds are major challenges of conventional extraction. These limitations of conventional extraction methods can be improved by introducing the promising techniques or non-conventional extraction techniques, for example, ultrasound-assisted extraction (UAE) and microwaveassisted extraction (MAE) [31].

#### *4.2.1 Microwave-assisted extraction (MAE)*

**Microwave-assisted extraction** has been implemented as an alternative technique for extracting anthocyanins from pigmented rice because of its ability to reduce both consumption time and solvent volume. For the MAE method, a combination of 70°C, 300 W, with 10 min in MAE was the most effective in extracting

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*Phenolic Compounds and Potential Health Benefits of Pigmented Rice*

anthocyanins from blue wheat and purple corn compared with 50°C, 1200 W, and 20 min for black rice [30]. Moreover, this technique reduced the losses of the

Ultrasound-assisted extraction (UAE) has been used in applications of foodprocessing technology to extract bioactive compounds from plant materials. Ultrasound at levels greater than 20 kHz is used to disrupt plant cell walls. It helps to improve the solvent's ability to penetrate the cells and obtain a higher extraction yield. The UAE operates at a low operating temperature through processing and maintain a high extract quality for compounds. Recently, Setyaningsiha et al. (2019) reported the optimization of the UAE conditions for individual phenolic compounds extraction from rice grains using 80% methanol in water for 25 min at 45°C with amplitude 47%, cycle 0.4 s − 1, pH 4.25 and sample-to solvent ratio of 1:5 [32]. The developed method presented the acceptable value for linearity and precision (RSD). Therefore, the proposed UAE method is an effective technique for the determination of individual phenolic compounds including caffeic, *p*-coumaric, syringic, chlorogenic, isovanillic, isoferulic ferulic, *p*-hydroxybenzoic, sinapic, *p*-hydroxybenzaldehyde, protocatechuic, vanillic acids, protocatechuic aldehyde and quercetin in rice samples. However, the UAE has two main negative properties

mainly related to experimental repeatability and reproducibility [31].

ionization-tandem mass spectrometry (LC-ESI-MS/MS) [33].

After the extraction of bioactive compounds, the separation, identification and quantitation are necessary to sudied. In the past few decades, there are a huge number of published reports on HPLC analysis of extracted bioactive compounds from rice grains describe as the most widely used analytical method. Recently, Prabhakaran et al. (2019) reported the analyzed method of selected phenolic compounds in rice grains and its by-products using liquid chromatography-electrospray

In addition, our group developed an identification and quantification techniques for phenolic acids and anthocyanins in pigmented rice by using UPLC-ESI-QqQ-MS/MS analysis [13]. The analysis was performed using a UPLC coupled with a mass spectrometer. The separation was carried out by UPLC HSS T3 column 1.8 μm, 2.1 × 100 mm. Column temperature was maintained at 35°C. The mobile phase consisted of 0.1% formic acid (solvent A) and 0.1% formic acid in acetonitrile (solvent B) and the flow rate was set at 0.4 mL/min. The injection volume was 2.0 μL. A stepwise gradients B (%) including an initial isocratic at 2.0% for 1 min, then linear gradient to 98% in 5 min, and by return to the initial condition of 2% B in 7 min. Therefore, the total operation time was 12 min. The solvents and extracts were previously filtered through a 0.45 μm filter membrane. Mass spectral data were obtained in positive or negative mode with a mass range between *m/z* 0 to *m/z* 500. The Multiple Reaction Monitoring (MRM) transitions and compound parameters for the target phenolic compounds were developed. Identification was confirmed by comparing *m/z* values, retention times and fragmentation patterns with those of references standards. In addition, the concentration of phenolic compounds was quantified using external standard method. Our study showed that eight target phenolic compounds were detected and identified in both the unbound and polyphenol-rich bound fractions of pigmented rice [13]. The identification of compounds was carried out by applying one quantification transition (quantifier ion) and/or one or two confirmation transitions (qualifier ions) to assess the detection

*DOI: http://dx.doi.org/10.5772/intechopen.93876*

biochemical compounds being extracted.

*4.2.2 Ultrasonic-assisted extraction*

**4.3 Identification**

anthocyanins from blue wheat and purple corn compared with 50°C, 1200 W, and 20 min for black rice [30]. Moreover, this technique reduced the losses of the biochemical compounds being extracted.
