**3.2 Anti-inflammatory properties**

Inflammation is an important mechanism of immune pathogenesis, which is our body's response to tissue injury, infection, and stress. Importantly, the prolonged production of inflammatory mediators by macrophage can cause damage to the host and can contribute to the pathology of many diseases including inflamm-aging, arthritis, asthma, cancer, diabetes, and atherosclerosis. Macrophage plays a key role in response to an immediate defensive mechanism of our body against attacking foreign agents, especially with a microbial lipopolysaccharide (LPS) [74]. Macrophage is activated and produces many kinds of inflammatory mediators including nitric oxide (NO), prostaglandins, and many cytokines such as interleukin 1 (IL-1), interleukin 6 (IL-6), and tumor necrosis factor (TNF)-α [75]. Many researchers have studied in vitro and in vivo models to elucidate that natural products are able to ameliorate the inflammatory response in LPS-stimulated macrophage.

During the last decade, it has been shown that anthocyanins reduce the risks of cardiovascular diseases and cancers with inflammatory, antioxidant, and chemoprotective properties [15, 76, 77]. Some reports have demonstrated that lipophilic phytochemicals contained in pigmented rice germ and bran, such as γ-oryzanol and vitamin E derivatives, exert anti-inflammatory activities [78, 79]. On the other hand, pigmented rice contains high amounts of medium polar or hydrophilic compounds such as phenolics, bioflavonoids, anthocyanin, and proanthocyanidins that have been reported for their anti-inflammatory properties, in both in vitro and in vivo models [80–82].

Pigmented rice contains a variety of bioactive compounds with antiinflammatory properties; however, there have been quite a few reports employing experimental designs that provide direct evidence to support using the extracts of pigmented rice. For the first time, our research group has demonstrated the molecular mechanisms underlying the anti-inflammatory effects. The anthocyanin-rich fraction of black rice extract significantly inhibited LPS that induced many pro-inflammatory mediators in RAW 264.7 macrophage white blood cells [31]. The pro-inflammatory mediators in this study were NO, TNF-α, and IL-6, and they effectively reduced the expression of two important inflammatory enzymes, the inducible NO synthase (iNOS) and the inducible cyclooxygenase-2 (COX-2). These results were regulated by an inhibition of the mitogen-activated protein kinase signaling pathway (MAPK pathway), leading to a decreased nuclear translocation of NF-κB and AP-1, two major transcription factors involved in the inflammation process. In testing the anti-inflammatory properties of anthocyanin and hydroxybenzoic acid, the major components were detected in the black rice extracts based on our extraction protocol, and similar results were obtained. A schematic diagram of the proposed mechanism of the anti-inflammatory properties of black rice anthocyanin is presented in **Figure 4**. In a study on cyanidin-3-glucoside and protocatechuic acid, no beneficial effects were found against inflammation induced by LPS [73]. Therefore, the anti-inflammatory properties of black rice might require the synergistic action of many phytochemicals, which are rich in anthocyanin and other phenolic compounds that play a role in this process. Interestingly, the same study has demonstrated that the cooking process did not alter the anti-inflammatory potential of black rice. In another study, other researchers reported that cyanidin-3-glucoside displays anti-inflammatory effects [8]. Our group also conducted a study on the anti-inflammatory effects of proanthocyanidin-rich red rice extract via the suppression of the MAPK, AP-1, and NF-κB pathways in RAW 264.7 macrophages that induced inflammation by LPS [7]. It was found that the red rice medium polar fraction that was enriched with polyphenols and proanthocyanidins exerted potent anti-inflammatory activities by inhibiting the production of TNF-α, IL-6, and NO in LPS-activated macrophage, whereas the red rice nonpolar fractions displayed no anti-inflammatory properties. All of the above results indicate that black rice that is rich in anthocyanins and red rice that is rich in proanthocyanidins exhibit therapeutic potential for the treatment of inflammatory diseases.

### **3.3 Anticancer properties**

Cancer is one of the leading causes of morbidity and mortality worldwide. Notably, only 10% at the most of all cancers are due to genetic factors, while 90% are directly or indirectly correlated with an individual's lifestyle and dietary habits [83]. Many scientific reports have shown that a healthy lifestyle, including a diet rich in natural products, such as herbs, cereals, fruits, and vegetables, can help reduce the risk of cancer [84, 85]. Some of the phytochemicals found in these natural products

**13**

**Figure 4.**

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

are secondary metabolites, including phenolic compounds, bioflavonoids, terpenoids, and alkaloids. In this chapter we shall focus more on the presence of phenolic compounds and flavonoids, including anthocyanins and proanthocyanidins, as the major compounds found in pigmented rice, especially in rice germ and bran. Active components of pigmented rice bran have demonstrated anticancer properties in in vitro cancer cell models, including those involving leukemia, colon, breast, liver, and stomach cancer cells. In a study on the anticancer potential of rice bran against the proliferation of leukemic cell lines, the antioxidant activities of the active compounds found in rice bran were noted for this beneficial effect [10]. Another investigation on the tumor suppression activities of rice bran from different pigmented and nonpigmented rice varieties reported that 70% ethanolic extract of the pigmented rice bran inhibited phorbol ester-induced tumor promotion in a better manner when compared to the nonpigmented rice bran variety [11]. In yet another study, the growth inhibitory effect of rice bran polyphenols, mainly γ-oryzanol and its derivatives, has been reported in human colorectal adenocarcinoma [86]. The anticancer activity of rice bran could be varied considerably in different rice cultivars or varieties in accordance with the different chemical profiles of the active compounds. In addition, the second study had analyzed seven varieties of rice bran for their growth inhibition potential against human colorectal cancer cells and reported on variations in the degree of growth inhibition depending upon the rice bran variety [9]. Some evidences have indicated that cyanidin-3-glucoside and peonidin-3-glucoside obtained from black rice anthocyanin can be combined with doxorubicin to inhibit cancer cell growth, while both anthocyanin compounds could inhibit cancer invasion into other tissues through the downregulation of

*Schematic diagram of anti-inflammatory properties of black rice anthocyanin.*

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

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

#### **Figure 4.**

*Phytochemicals in Human Health*

in vivo models [80–82].

During the last decade, it has been shown that anthocyanins reduce the risks of cardiovascular diseases and cancers with inflammatory, antioxidant, and chemoprotective properties [15, 76, 77]. Some reports have demonstrated that lipophilic phytochemicals contained in pigmented rice germ and bran, such as γ-oryzanol and vitamin E derivatives, exert anti-inflammatory activities [78, 79]. On the other hand, pigmented rice contains high amounts of medium polar or hydrophilic compounds such as phenolics, bioflavonoids, anthocyanin, and proanthocyanidins that have been reported for their anti-inflammatory properties, in both in vitro and

Pigmented rice contains a variety of bioactive compounds with anti-

inflammatory properties; however, there have been quite a few reports employing experimental designs that provide direct evidence to support using the extracts of pigmented rice. For the first time, our research group has demonstrated the molecular mechanisms underlying the anti-inflammatory effects. The anthocyanin-rich fraction of black rice extract significantly inhibited LPS that induced many pro-inflammatory mediators in RAW 264.7 macrophage white blood cells [31]. The pro-inflammatory mediators in this study were NO, TNF-α, and IL-6, and they effectively reduced the expression of two important inflammatory enzymes, the inducible NO synthase (iNOS) and the inducible cyclooxygenase-2 (COX-2). These results were regulated by an inhibition of the mitogen-activated protein kinase signaling pathway (MAPK pathway), leading to a decreased nuclear translocation of NF-κB and AP-1, two major transcription factors involved in the inflammation process. In testing the anti-inflammatory properties of anthocyanin and hydroxybenzoic acid, the major components were detected in the black rice extracts based on our extraction protocol, and similar results were obtained. A schematic diagram of the proposed mechanism of the anti-inflammatory properties of black rice anthocyanin is presented in **Figure 4**. In a study on cyanidin-3-glucoside and protocatechuic acid, no beneficial effects were found against inflammation induced by LPS [73]. Therefore, the anti-inflammatory properties of black rice might require the synergistic action of many phytochemicals, which are rich in anthocyanin and other phenolic compounds that play a role in this process. Interestingly, the same study has demonstrated that the cooking process did not alter the anti-inflammatory potential of black rice. In another study, other researchers reported that cyanidin-3-glucoside displays anti-inflammatory effects [8]. Our group also conducted a study on the anti-inflammatory effects of proanthocyanidin-rich red rice extract via the suppression of the MAPK, AP-1, and NF-κB pathways in RAW 264.7 macrophages that induced inflammation by LPS [7]. It was found that the red rice medium polar fraction that was enriched with polyphenols and proanthocyanidins exerted potent anti-inflammatory activities by inhibiting the production of TNF-α, IL-6, and NO in LPS-activated macrophage, whereas the red rice nonpolar fractions displayed no anti-inflammatory properties. All of the above results indicate that black rice that is rich in anthocyanins and red rice that is rich in proanthocyanidins exhibit therapeutic potential for the treat-

Cancer is one of the leading causes of morbidity and mortality worldwide. Notably, only 10% at the most of all cancers are due to genetic factors, while 90% are directly or indirectly correlated with an individual's lifestyle and dietary habits [83]. Many scientific reports have shown that a healthy lifestyle, including a diet rich in natural products, such as herbs, cereals, fruits, and vegetables, can help reduce the risk of cancer [84, 85]. Some of the phytochemicals found in these natural products

**12**

ment of inflammatory diseases.

**3.3 Anticancer properties**

*Schematic diagram of anti-inflammatory properties of black rice anthocyanin.*

are secondary metabolites, including phenolic compounds, bioflavonoids, terpenoids, and alkaloids. In this chapter we shall focus more on the presence of phenolic compounds and flavonoids, including anthocyanins and proanthocyanidins, as the major compounds found in pigmented rice, especially in rice germ and bran.

Active components of pigmented rice bran have demonstrated anticancer properties in in vitro cancer cell models, including those involving leukemia, colon, breast, liver, and stomach cancer cells. In a study on the anticancer potential of rice bran against the proliferation of leukemic cell lines, the antioxidant activities of the active compounds found in rice bran were noted for this beneficial effect [10]. Another investigation on the tumor suppression activities of rice bran from different pigmented and nonpigmented rice varieties reported that 70% ethanolic extract of the pigmented rice bran inhibited phorbol ester-induced tumor promotion in a better manner when compared to the nonpigmented rice bran variety [11]. In yet another study, the growth inhibitory effect of rice bran polyphenols, mainly γ-oryzanol and its derivatives, has been reported in human colorectal adenocarcinoma [86]. The anticancer activity of rice bran could be varied considerably in different rice cultivars or varieties in accordance with the different chemical profiles of the active compounds. In addition, the second study had analyzed seven varieties of rice bran for their growth inhibition potential against human colorectal cancer cells and reported on variations in the degree of growth inhibition depending upon the rice bran variety [9]. Some evidences have indicated that cyanidin-3-glucoside and peonidin-3-glucoside obtained from black rice anthocyanin can be combined with doxorubicin to inhibit cancer cell growth, while both anthocyanin compounds could inhibit cancer invasion into other tissues through the downregulation of

#### *Phytochemicals in Human Health*

the degradative enzymes MMP-2 and MMP-9 [14]. Interestingly, Chen et al. [87] compared the relationship of the bioactive compounds with the growth inhibitory effects of pigmented rice bran extracts. The results revealed that the light brown bran had no effect, the purple bran exhibited a minor effect on leukemia and cervical cancer cells, and the red bran exhibited strong inhibitory effects on leukemic, cervical, and stomach cancer cells. High concentrations of protocatechuic acid and anthocyanins in purple bran and proanthocyanidins in red rice bran have been singled out for their growth inhibitory effects against human cancer cells.

Many studies on anticancer properties have been reported in Thai rice cultivars. In an important study, Kum Phayao black rice cultivar was found to be highly cytotoxic to human HepG2 cells when compared with other Northern Thai purple rice cultivars [12]. In yet another study, the alcoholic extracts of black-purple rice grain cultivar Kum Doi Saket demonstrated an antimutagenic activity against aflatoxin B1 in Ames tests [88]. The therapeutic potential of black rice anthocyanin for treating inflammatory diseases that are associated with cancer has been proposed for its mechanism via the inhibition of the MAPK signaling pathway [31]. A very recent study conducted by our research group revealed that the proanthocyanidinrich fraction isolated from the red rice germ and bran of the Kum Doi Saket cultivar grown in the northern part of Thailand significantly reduced the cell viability of HepG2 cells (IC50 value at 20 μg/ml) [13]. The proanthocyanidin-rich fraction could inhibit cell proliferation and induce cell apoptosis by increasing the apoptotic proteins, such as cleaved PARP-1, cleaved caspase 8, and cleaved caspase-3, and decreasing the anti-apoptotic protein survivin without p53 protein changes. A schematic diagram of this mechanism is presented in **Figure 5**. In addition, our previous studies have demonstrated that red rice grain extracts with high proanthocyanidin content displayed an anti-metastasis effect on invasive human breast carcinoma cells MDA-MB231 [16] and human fibrosarcoma HT1080 cell lines [15]. In addition, proanthocyanidins in other colored plants, such as grapes and blackberries, have demonstrated anticancer, anti-inflammatory, and antioxidant activities to a similar extent as the proanthocyanidins that are found in red rice germ and bran [7–9, 13].
