**4.4 Quantitation**

Pigmented rice is diverse in color, mainly due to the grain's high anthocyanins content. The contents of phenolic compounds and anthocyanins are summarized in **Tables 2** and **3**, respectively, as examples of the phytochemicals that were isolated and analyzed from pigmented rice including black, red, and purple rice varieties. A range of phenolic compounds including vanillic, *p-*coumaric, protocatechuic, caffeic and ferulic acids has been detected in pigmented rice. In pigmented rice, the major phenolic acid was ferulic acid [6, 13, 25, 37]. Moreover, major flavonoids present in pigmented rice are quercetin and catechin. In addition, cyanidin-3-O-glucoside is the most predominant anthocyanins in pigmented rice. A mean of cyanidin-3-O-glucoside content in black rice was about higher than that of red rice [6, 13, 25, 37]. However, malvidin-3-O-glucoside was not observed in pigmented rice.

Our UPLC-ESI-QqQ-MS/MS analysis for phenolic acids and anthocyanins in pigmented rice showed that the retention times for the target phenolic compounds ranged between 2.9 and 4.6 min An example of a UPLC-ESI-QqQ-MS/MS chromatogram for cyanidin-3-O-glucoside for the polyphenol-rich bound fraction of black rice cv. *Hom nil* is shown in **Figure 4**. Hydroxycinnamic acids including caffeic, *p*-coumaric and ferulic acids were characterized by the loss of the carboxylic acid group (-COO; −44 *m/z*) [38]. The loss of the carboxylic acid group was observed for *p*-courmaric acid giving *m/z* at 119 [M-H-44]−, as a characteristic ion. For acids with a methoxy moiety, such as ferulic acid, the -COO loss was found by a loss of the methyl group (-CH3; −15 *m/z*, -COO; − 44 *m/z* and -CH3; −59 *m/z*). The peak with *m/z* 195 was contributed to the ferulic acid's precursor ion while the peak of product ion at *m/z* 136 was characterized to -COO and -CH3 moiety. In addition, the characterization of anthocyanin was mostly based on the loss of glucose (162 *m/z*). A molecular ion at *m/z* 449 [M + H] + corresponding with glycoside derivative of cyanidin and a major fragmentation occurring at *m/z* 287 [M + H-162] + corresponding with a cyanidin aglycone was observed in the polyphenol-rich bound fraction of black rice cv. *Hom nil* (**Figure 5(a)**)**.** The MS/MS spectrum of pelargonidin-3-glucoside in


**71**

*Phenolic Compounds and Potential Health Benefits of Pigmented Rice*

Purple rice 80%

Red rice 80%

**Extracting solvent**

Deionized water

Methanol

Methanol

Acidified methanol

Acidified methanol

Acidified methanol

Acidified methanol

Methanol

Methanol

95% Ethanol

Deionized water

Methanol

Methanol

Acidified methanol

Acidified methanol

Acidified methanol

Acidified methanol

Vanillic acid Purple Rice Methanol HPLC 0.19 ± 0.06 [34] 95% Ethanol

**Analysis method**

Reversedphase HPLC

Reversedphase HPLC

UPLC-ESI-QqQ-MS/ MS

UPLC-ESI-QqQ-MS/ MS

UPLC-ESI-QqQ-MS/ MS

UPLC-ESI-QqQ-MS/ MS

Reversedphase HPLC

Reversedphase HPLC

Reversedphase HPLC

Reversedphase HPLC

UPLC-ESI-QqQ-MS/ MS

UPLC-ESI-QqQ-MS/ MS

UPLC-ESI-QqQ-MS/ MS

UPLC-ESI-QqQ-MS/ MS

Purple Rice Methanol HPLC 0.24 ± 0.05 [34]

**Content (mg/g)**

nd

0.04 ± 0.00

0.02 ± 0.00

0.06 ± 0.00

0.01 ± 0.20

0.31 ± 0.02

nd

nd

0.02 ± 0.00

HPLC 1.02 ± 0.08

HPLC 1.26 ± 0.01

0.34 ± 0.02 [35]

0.05 ± 0.01 [13]

0.31 ± 0.02 [34]

1.60 ± 0.02 [35]

nd [13]

(Continued)

HPLC 0.77 ± 0.02

HPLC 1.15 ± 0.08

**Reference**

**rice varieties**

Hom nil

Black rice cv. Rice berry

Purple rice cv. Khoa kum

Red rice cv. Hom deang

Purple rice 80%

Red rice 80%

Purple rice 80%

Red rice 80%

Hom nil

Black rice cv. Rice berry

Purple rice cv. Khoa kum

Red rice cv. Hom deang

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

**Phenolic acids Pigmented** 

p-Coumaric acid Black rice cv.

Protocatechuic

Protocatechuic

Caffeic acid Black rice cv.

acid

acid

#### **Table 1.**

*Retention time and fragment of phenolic compounds.*


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

*Recent Advances in Rice Research*

not observed in pigmented rice.

**Qualified compound Retention time** 

*Retention time and fragment of phenolic compounds.*

**(min)**

(+)-Catechin 3.04 ± 0.01 291.10 130.01 123.03 Caffeic acid 3.29 ± 0.01 181.02 163.03 145.01 *p*-Coumaric acid 3.72 ± 0.01 162.95 118.96 92.95 Ferulic acid 3.87 ± 0.01 195.02 134.00 145.02 Quercetin 4.59 ± 0.01 300.90 178.05 150.92 Cyanidin-3-glucoside 2.87 ± 0.01 449.10 287.05 137.00 Pelargonidin-3-glucoside 3.03 ± 0.01 433.10 271.05 121.00 Peonidin-3-glucoside 3.10 ± 0.01 463.10 301.08 286.03

**m/z Precursor ion**

**m/z Quantifier**

**m/z Qualifier**

**4.4 Quantitation**

and quantification specific to each compound (**Table 1**). Positive ionization mode was selected for caffeic, ferulic acids, (+)-catechin and anthocyanins while negative ionization mode was applied for *p*-coumaric acid and quercetin, due to the chemical structures of the analyses and their ionization behavior observed in ion mode.

Pigmented rice is diverse in color, mainly due to the grain's high anthocyanins content. The contents of phenolic compounds and anthocyanins are summarized in **Tables 2** and **3**, respectively, as examples of the phytochemicals that were isolated and analyzed from pigmented rice including black, red, and purple rice varieties. A range of phenolic compounds including vanillic, *p-*coumaric, protocatechuic, caffeic and ferulic acids has been detected in pigmented rice. In pigmented rice, the major phenolic acid was ferulic acid [6, 13, 25, 37]. Moreover, major flavonoids present in pigmented rice are quercetin and catechin. In addition, cyanidin-3-O-glucoside is the most predominant anthocyanins in pigmented rice. A mean of cyanidin-3-O-glucoside content in black rice was about higher than that of red rice [6, 13, 25, 37]. However, malvidin-3-O-glucoside was

Our UPLC-ESI-QqQ-MS/MS analysis for phenolic acids and anthocyanins in pigmented rice showed that the retention times for the target phenolic compounds ranged between 2.9 and 4.6 min An example of a UPLC-ESI-QqQ-MS/MS chromatogram for cyanidin-3-O-glucoside for the polyphenol-rich bound fraction of black rice cv. *Hom nil* is shown in **Figure 4**. Hydroxycinnamic acids including caffeic, *p*-coumaric and ferulic acids were characterized by the loss of the carboxylic acid group (-COO; −44 *m/z*) [38]. The loss of the carboxylic acid group was observed for *p*-courmaric acid giving *m/z* at 119 [M-H-44]−, as a characteristic ion. For acids with a methoxy moiety, such as ferulic acid, the -COO loss was found by a loss of the methyl group (-CH3; −15 *m/z*, -COO; − 44 *m/z* and -CH3; −59 *m/z*). The peak with *m/z* 195 was contributed to the ferulic acid's precursor ion while the peak of product ion at *m/z* 136 was characterized to -COO and -CH3 moiety. In addition, the characterization of anthocyanin was mostly based on the loss of glucose (162 *m/z*). A molecular ion at *m/z* 449 [M + H] + corresponding with glycoside derivative of cyanidin and a major fragmentation occurring at *m/z* 287 [M + H-162] + corresponding with a cyanidin aglycone was observed in the polyphenol-rich bound fraction of black rice cv. *Hom nil* (**Figure 5(a)**)**.** The MS/MS spectrum of pelargonidin-3-glucoside in

**70**

**Table 1.**

*Reference: [13].*

(Continued)


**73**

*Phenolic Compounds and Potential Health Benefits of Pigmented Rice*

**Extracting solvent**

Acidified methanol

Acidified methanol

Deionized water

methanol

Acidified methanol

Acidified methanol

Acidified methanol

Acidified methanol

Acidified methanol

Acidified methanol

Deionized water

Thai black rice Acidified

**Analysis method HPLC**

Purple Rice Methanol HPLC 21.09 ± 1.09 [34] Ethanol HPLC 8.40 ± 0.00

> UPLC-ESI-QqQ-MS/ MS

> UPLC-ESI-QqQ-MS/ MS

> UPLC-ESI-QqQ-MS/ MS

> UPLC-ESI-QqQ-MS/ MS

> UPLC-ESI-QqQ-MS/ MS

> UPLC-ESI-QqQ-MS/ MS

> UPLC-ESI-QqQ-MS/ MS

> UPLC-ESI-QqQ-MS/ MS

Purple Rice Methanol HPLC n.d [34] Ethanol HPLC n.d

HPLC n.d

**Content (mg/g) Reference**

HPLC 137.41 ± 16.66 [6]

HPLC 142 ± 1.5 [36]

n.d

47.80 ± 0.44

11.75 ± 0.32

0.06 ± 0.00

0.07 ± 0.00

0.03 ± 0.00

n.d

(Continued)

0.23 ± 0.01 [13]

132.96 ± 393 [13]

HPLC 19.39 ± 0.09

HPLC n.d

**rice varieties**

Black rice cv. Niaw Dam Pleuak Khao

Black rice cv. Niaw Dam Pleuak Dam

Thai Jasmine red rice

Black rice cv. Hom nil

Black rice cv. Rice berry

Purple rice cv. Khoa kum

Red rice cv. Hom deang

Black rice cv. Hom nil

Black rice cv. Rice berry

Purple rice cv. Khoa kum

Red rice cv. Hom deang

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

**Anthocyanins Pigmented** 

Cyanidin 3-glucoside

Pelargonidin 3-glucoside

Malvidin 3-glucoside

**Table 2.** *Phenolic compound contents in pigmented rice.*


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

*Recent Advances in Rice Research*

**Phenolic acids Pigmented** 

Ferulic acid Black rice cv.

**rice varieties**

Hom nil

Black rice cv. Rice berry

Purple rice cv. Khoa kum

Red rice cv. Hom deang

Black rice cv. Hom nil

Black rice cv. Rice berry

Purple rice cv. Khoa kum

Red rice cv. Hom deang

Black rice cv. Hom nil

Black rice cv. Rice berry

Purple rice cv. Khoa kum

*Phenolic compound contents in pigmented rice.*

Quercetin Purple rice 80%

Purple rice 80%

Red rice 80%

Purple rice 80%

Red rice 80%

**Extracting solvent**

Methanol

Methanol

Acidified methanol

Acidified methanol

Acidified methanol

Acidified methanol

Methanol

Methanol

Deionized water

Acidified methanol

Acidified methanol

Acidified methanol

Acidified methanol

Methanol

Methanol

Acidified methanol

Acidified methanol

Acidified methanol

Red rice 80%

Catechin Purple Rice Methanol HPLC 0.09 ± 0.01 [34] 95% Ethanol

**Analysis method**

Reversedphase HPLC

Reversedphase HPLC

UPLC-ESI-QqQ-MS/ MS

UPLC-ESI-QqQ-MS/ MS

UPLC-ESI-QqQ-MS/ MS

UPLC-ESI-QqQ-MS/ MS

Reversedphase HPLC

Reversedphase HPLC

UPLC-ESI-QqQ-MS/ MS

UPLC-ESI-QqQ-MS/ MS

UPLC-ESI-QqQ-MS/ MS

UPLC-ESI-QqQ-MS/ MS

Reversedphase HPLC

UPLC-ESI-QqQ-MS/ MS

UPLC-ESI-QqQ-MS/ MS

UPLC-ESI-QqQ-MS/ MS

**Content (mg/g)**

nd

0.40 ± 0.03

0.24 ± 0.02

0.57 ± 0.02

0.34 ± 0.05

0.10 ± 0.01

0.01 ± 0.00

0.91 ± 0.08

n.d

0.08 ± 0.00

0.10 ± 0.01

HPLC 1.73 ± 0.05

HPLC 0.37 ± 0.09

0.98 ± 0.32 [35]

0.25 ± 0.01 [13]

1.03 ± 0.02 [12]

0.12 ± 0.01 [13]

0.29 ± 0.22 [35]

0.07 ± 0.03 [13]

**Reference**

**72**

**Table 2.**

*n.d: not detectable.*

(Continued)


#### *n.d: not detectable.*

#### **Table 3.**

*Anthocyanins contents in pigmented rice.*

**75**

**Figure 5.**

*Phenolic Compounds and Potential Health Benefits of Pigmented Rice*

the positive ionization mode (**Figure 5(b)**) of the polyphenol rich bound fraction of black rice cv. *Hom nil* showed protonated molecular ions at *m/z* 433 [M + H]+. The product ion of aglycone at *m/z* 271 [M + H-162] + corresponds to the loss of a pelargonidin moiety. The positive ion mass spectrum of the polyphenol-rich bound fraction of black rice cv. *Hom nil* (**Figure 5(c)**) showed its molecular ion at *m/z* 463 [M + H] + and a major fragment at *m/z* 301 [M + H-162]+. These data indicated the

*(a) MS/MS spectrum of cyanidin 3-O-glucoside. (b) MS/MS spectrum of pelargonidin-3-O-glucoside.* 

Proanthocyanidins are high molecular weight of flavan-3-ol polymers that consist of catechin, epicatechin, gallocatechin, and epigallocatechin units that can also be found in rice germ and bran, particularly in pigmented rice. Proanthocyanidins were only detected in red and black rice, and their contents were dependent on the genotypes which differed from 15.57 mg/kg to 1417.12 mg/kg, respectively. In red rice, the majority of proanthocyanidins are oligomers of 5–8 mers (40%), whereas the

presence of a peonidin aglycone and a hexose moiety.

*(c) MS/MS spectrum of peonidin 3-O-glucoside [13].*

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

**Figure 4.** *Chromatogram of cyanidin 3-O-glucoside [13].*

#### **Figure 5.**

*Recent Advances in Rice Research*

Peonidin-3 glucoside

*n.d: not detectable.*

*Anthocyanins contents in pigmented rice.*

*Chromatogram of cyanidin 3-O-glucoside [13].*

**Table 3.**

**Anthocyanins Pigmented** 

**rice varieties**

Black rice cv. Niaw Dam Pleuak Khao

Black rice cv. Niaw Dam Pleuak Dam

Black rice cv. Hom nil

Black rice cv. Rice berry

Purple rice cv. Khoa kum

Red rice cv. Hom deang

Thai Jasmine red rice

Thai black rice Acidified

**Extracting solvent**

Acidified methanol

Acidified methanol

Acidified methanol

Acidified methanol

Acidified methanol

Acidified methanol

methanol

Acidified methanol

**Analysis method HPLC**

UPLC-ESI-QqQ-MS/ MS

UPLC-ESI-QqQ-MS/ MS

UPLC-ESI-QqQ-MS/ MS

UPLC-ESI-QqQ-MS/ MS

**Content (mg/g) Reference**

19.56 ± 0.39 [13]

HPLC 11.07 ± 0.97 [6]

6.94 ± 0.26

5.29 ± 0.10

0.01 ± 0.0

HPLC 98 ± 0.5 [36]

n.d

HPLC n.d

HPLC 12.75 ± 0.51

**74**

**Figure 4.**

*(a) MS/MS spectrum of cyanidin 3-O-glucoside. (b) MS/MS spectrum of pelargonidin-3-O-glucoside. (c) MS/MS spectrum of peonidin 3-O-glucoside [13].*

the positive ionization mode (**Figure 5(b)**) of the polyphenol rich bound fraction of black rice cv. *Hom nil* showed protonated molecular ions at *m/z* 433 [M + H]+. The product ion of aglycone at *m/z* 271 [M + H-162] + corresponds to the loss of a pelargonidin moiety. The positive ion mass spectrum of the polyphenol-rich bound fraction of black rice cv. *Hom nil* (**Figure 5(c)**) showed its molecular ion at *m/z* 463 [M + H] + and a major fragment at *m/z* 301 [M + H-162]+. These data indicated the presence of a peonidin aglycone and a hexose moiety.

Proanthocyanidins are high molecular weight of flavan-3-ol polymers that consist of catechin, epicatechin, gallocatechin, and epigallocatechin units that can also be found in rice germ and bran, particularly in pigmented rice. Proanthocyanidins were only detected in red and black rice, and their contents were dependent on the genotypes which differed from 15.57 mg/kg to 1417.12 mg/kg, respectively. In red rice, the majority of proanthocyanidins are oligomers of 5–8 mers (40%), whereas the

polymers (DP > 10) accounted for 29% [12]. Moreover, total proanthocyanidins of the traditional Sri Lankan red-grained rice varieties ranged from 11.95 to 24.70 mg/g in bran samples and from 1.07 to 2.27 mg/g in brown rice samples [39].
