Phenolics and flavonoids content
The free, bound, and total phenolic acid contents in the bran fractions of sixteen different genotypes of pigmented rice are shown in Table 1. The free phenolic content in the bran fraction varied from 153.30 to 771.15 mg GAE/100 g DM. The bound phenolic content ranged from 102.05 to 443.55 mg GAE/100 g. The total phenolic content ranged from 269.85 to 1214.7 mg GAE/100 g DM. As shown in Table 1, black rice bran contained the highest contents of free, bound, and total phenolics (771.15, 443.55, and 1214.7 mg GAE/100 g DM, respectively), followed by red rice bran (579.46, 231.86, and 811.32 mg GAE/100 g DM, respectively) and light brown rice bran (329.65, 120.04, and 447.68 mg GAE/100 g DM, respectively).
The free flavonoid content in the bran fractions varied from 28.52 to 526.68 mg QE/100 g DM. The bound flavonoid content ranged from 11.63 to 297.20 mg QE/100 g, and the total flavonoid content ranged from 40.15 to 823.88 mg QE/100 g DM. As shown in Table 1, black rice bran possessed the highest free, bound, and total flavonoid contents (526.68, 297.20, and 823.88 mg QE/100 g DM, respectively), followed by red rice bran (324.92, 238.76, and 457.00 mg QE/100 g DM, respectively) and brown rice bran (135.18, 105.7, and 240.88 mg QE/100 g DM, respectively). In a recent study by Shen et al.  the free total flavonoid contents of white, red, and black rice were compared and it was found that the mean flavonoids content in white rice was lower than those in red and black rice.
The current results showed that the phenolic and flavonoid compounds in rice bran were mostly present in the free form, and this is an important issue for future studies. The bound forms of phenolics and flavonoids are covalently conjugated to the structures of the cell wall via ester bonds . They cannot be directly digested and can survive gastrointestinal digestion to reach the colon intact. In the colon, they are broken down by the microflora and may release the bound phenolics to exert beneficial biological actions locally . The current results are consistent with previous findings, in that phenolics and flavonoids in cereals were primarily distributed in the free form [21, 22]. Rice bran has attracted significant attention from consumers owing to its unique physiological functions and nutritional value. Some nutritional phytochemicals in rice bran primarily exist as glycosides linked to various sugar moieties or as other complexes linked to carbohydrates, lipids, organic acids, amines, and other phenols. Moreover, phytochemicals are commonly present in the bound form and as components of complex structures such as hydrolysed tannins and lignins .
Free and bound anthocyanin contents in sixteen different genotypes of pigmented rice are presented in Table 1. The total anthocyanins content in the free and bound form differed among the different genotypes of pigmented rice bran. The free and bound anthocyanin content in the bran fraction of sixteen different genotypes of pigmented rice ranged from 2.18 to 256.11 and 5.25 to 38.51 mg Cy3-GE/100 g DM, respectively. The highest anthocyanin concentration was detected in the free form. Black rice bran showed the highest content of total anthocyanins (294.62 mg Cy3-GE/100 g DM) followed by red (77.87 mg Cy3-GE/100 g DM) and brown rice (10.72 mg Cy3-GE/100 g DM). The bound form of anthocyanin was not detected in brown rice bran. It has been reported that the content of anthocyanin in rice is related to the expression levels of anthocyanin biosynthetic genes . It was found that coloured rice exhibits stronger anthocyanin and antioxidant activities than those exhibited by non-coloured rice . A recent study showed that the concentration of anthocyanin in black, blue, pink, purple, and red cereal grains was significantly dependent on the colour of the grain . Moreover, the present findings showed that the anthocyanin content in rice correlated with the colour of the grain.
Phenolics and flavonoids composition
Five phenolic compounds (protocatechuic acid, syringic acid, ferulic acid, cinnamic acid, and p-coumaric acid) and five flavonoid compounds (quercetin, apigenin, catechin, luteolin, and myrecitin) were detected in the free and bound fractions of three different pigmented rice bran (Table 2).
Protocatechuic acid only existed in free fractions, with contents ranging from 2.87 to 6.18 mg/100 g DM and the highest content (p < 0.05) was found in black rice bran. Syringic acid existed in both the free and bound fractions, with contents ranging from 11.26 to 17.5 and from 3.16 to 6.9 mg/100 g DM, respectively. Black rice bran contained the highest content of free and bound syringic acid. Ferulic acid existed in both the free and bound fractions, with contents ranging from 3.51 to 7.56 and from 14.28 to 20.58 mg/100 g DM, respectively. As shown in the data, the highest concentration of ferulic acid was detected in the bound form Black rice bran showed the highest content of free and bound ferulic acid. The contents of the free form of cinnamic acid were between 9.61 and 19.98 mg/100 g DM, and the bound form was only detected in black rice bran (5.55 mg/100 g DM). P-coumaric acid existed in both the free and bound forms, with content ranging from 4.08 to 10.41 and from 12.63 to 22.94 mg/100 g DM, respectively. Similar to that observed with ferulic acid, the highest concentration of p-coumaric acid was observed in the bound form. Black rice bran contained the highest content of free and bound p-coumaric acid. These data suggest that the contents of p-coumaric acid and ferulic acid were relatively high compared with the contents of other phenolic compounds in brown, red, and black rice bran. The highest concentration of p-coumaric acid and ferulic acid was found in the bound form.
Among the five flavonoid compounds identified in the three different pigmented rice bran, quercetin, apigenin, and catechin existed in the free and bound form but luteolin and myrecitin were only detected in the free form. The contents of quercetin in the free and bound form ranged from 2.71 to 11.89 mg/100 g DM and from 0.16 to 3.66 mg/100 g DM, respectively. Apigenin existed in both the free and bound forms, with contents between 3.48 and 12.56 and 0.74 and 2.75 mg/100 g DM, respectively. Catechin also existed in both the free and bound forms, with contents between 7.27 and 15.64 and 1.69 and 6.41 mg/100 g DM, respectively. Luteolin and myrecitin only existed in the free fractions, with contents ranging from 2.35 to 10.72 mg/100 g DM and 5.68 to 12.85 mg/100 g DM, respectively.
Black rice bran contained the highest content of all the identified flavonoids in both the free and bound forms, followed by red and brown rice bran. Ferulic acid and p-coumaric acid were the most abundant phenolic compounds in brown, red, and black rice bran extracts. Further, catechin and myrecitin were the most abundant flavonoid compounds in brown and red rice bran, while apigenin and quercetin were the most abundant flavonoid compounds in black rice bran Zhou et al.  showed that brown rice contained high levels of ferulic and p-coumaric acid and low levels of gallic, vanillic, caffeic, and syringic acids, which is consistent with the findings of the present study. Arabinoxylans are present in the walls of aleurone cells, indicating that they contain high levels of ferulic acid In addition, the benefits of bound ferulic and p-coumaric acids, which are mainly present in rice bran, may be site-specific i.e. more effective in the colon. Bound forms of flavonoids and phenolic acids are covalently conjugated to the structures of the cell wall via ester bonds. The phytochemical constituents and their quality in rice grain vary considerably and this may be attributed to several factors, such as agronomic activities, environmental conditions, and genetic factors . The milling fractions obtained from different rice varieties will exhibit different chemical composition and nutritional values. The chemical composition in rice bran, polished rice, and whole brown rice grain are also different within one variety.
Nitric oxide (NO) scavenging activity
Nitric oxide scavenging activity of brown, red, and black rice bran fraction at different concentrations is shown in Fig. 1. As shown in Fig. 1, as rice bran concentration increased from 10 to 160 μg/mL, NO scavenging activity of the free and bound fractions increased significantly (p < 0.05). The NO scavenging activity of the rice bran of three different genotypes ranged from 4.0 to 89.2%. The NO scavenging activity in the free and bound fractions ranged from 13.4 to 89.2 and 4.0 to 78.0%, respectively. Significant difference (p < 0.05) in NO scavenging activity was found among the different coloured genotypes of rice. Black rice bran demonstrated the highest NO-scavenging activity followed by red and brown rice bran extracts. The NO-scavenging activity of the free fraction was higher than that of the bound fractions at all concentrations (10–160 μg/mL).
The antioxidant activities of all rice bran were lower than those of ascorbic acid and gallic acid. The IC50 values for NO scavenging activity of the free fractions of black, red, and brown rice bran were 32.0, 44.5, and 112 μg/mL, respectively. While, the IC50 values of the bound fractions of black, red, and brown rice bran were 65.7, 78.2, and 150.4 μg/mL, respectively. IC50 values of ascorbic acid and gallic acid were < 10 and 14.8 μg/mL, respectively. A lower IC50 value represents a stronger free radical inhibitor (strong free radical inhibitors are active at low concentrations). The free fractions of rice bran extracts exhibitted strong NO radical scavenging activity with low IC50 values, indicating that the antioxidant activity of free compounds in black, red, and brown rice bran was higher than that of bound compounds in these rice genotypes. It has been reported that grains with red and black pericarp demonstrated higher antioxidant activity than those demonstrated by grains with light brown pericarp . Rice bran, though in small amounts, is rich in antioxidants; hence, removal of the bran during the production of polished rice leads to lower antioxidant activity. This indicates that black rice is a good source of antioxidants when compared with brown rice, which is generally consumed in our diet. Nowadays, more rice varieties have been developed as healthy foods and have gained increasing popularity with consumers . Since nutritional imbalance in the diet can cause diseases such as obesity, diabetes, cardiovascular disease, and cancer, action is needed to promote whole rice as a “nutritious health food” and as a normal part of everyday meal consumption . In addition, safety concerns over the use of synthetic antioxidants have led to increasing interest from the food industry in identifying naturally occurring antioxidants in basic raw food materials. Rice bran, with its low cost, has great potential for applications in the food and pharmaceutical industries as a rich source of natural antioxidants .
Various mechanisms, such as free radical-scavenging, reducing capacity, metal ion-chelation, and inhibition of lipid peroxidation, have been studied to explain how rice bran extracts could be used as effective antioxidants [13, 29]. DPPH radical-scavenging assays are based on the transfer of electrons from a donor molecule to the corresponding radical. This method is the simplest method to measure the ability of antioxidants to intercept free radicals.
The DPPH radical-scavenging effects of all rice bran extracts (free and bound) increased with increasing concentration (Fig. 2). DPPH activity was significantly influenced (p < 0.05) by the colour of the rice bran. Black rice bran extract demonstrated the highest DPPH activity followed by red and brown rice extracts. The DPPH activity of the rice bran of three different pigmented rice bran ranged from 10.7 to 87.9%. The DPPH activity in the free and bound fractions ranged from 22.6 to 87.9 and 10.7 to 76.1%, respectively. Black rice bran exhibited the highest DPPH activity, followed by red and brown rice bran extracts. DPPH activity in free fractions was higher than that of bound fractions at all concentrations (10–160 μg/mL). The DPPH activity of all rice bran extracts was lower than those of the positive controls (ascorbic acid and gallic acid). The IC50 values of the free fractions of black, red, and brown rice bran against DPPH activity were 25, 32, and 51 μg/mL, respectively. IC50 values for DPPH radical scavenging activity of the bound fractions in black, red, and brown rice bran extract were 39.1, 64.7, and 87.1 μg/mL, respectively. The lowest IC50 value was obtained in the free form, indicating that free compounds exhibit potent antioxidant property compared to that of bound compounds. The IC50 values of ascorbic acid and gallic acid were 12.4 and 19.2 μg/mL, respectively.
The highest DPPH activity and the lowest IC50 value of the rice bran extracts was observed with black rice, which contained the highest content of phenolic and flavonoid compounds. The higher levels of secondary metabolites (flavonoids, phenolic acids, and anthocyanins) in black rice compared to those of red and brown rice might be responsible for the high antioxidant activity. Previous studies have reported that the concentration of total phenolics and flavonoids in rice grains positively correlated with the antioxidant activity [18, 30, 31]. Oki et al.  reported that in red pericarp grains, a strong correlation between antioxidant activity and the content of proanthocyanidins was observed; however, in the case of black pericarp grains, the correlation was dependent on the content of anthocyanins. These results suggest that phenolic compounds were primarily responsible for the antioxidant activity of rice grains .
The free fractions of black, red, and brown rice bran extracts were subjected to antiproliferative assays since it demonstrated the highest antioxidant activity and phytochemical content. The antiproliferative activity of the bran extracts of black, red, and brown rice (25–400 μg/mL) was evaluated against the breast cancer cell lines MCF-7 and MDA-MB-231 (Fig. 3). The MTT assay indicated that black, red, and brown rice bran reduced the viability of MCF-7 and MDA-MB-231 cells in a dose-dependent manner. The IC50 values differed significantly (p < 0.05) among the different pigmented rice bran. The black rice bran extracts exhibited potent antiproliferative activity, with IC50 values of 148.6 and 119.2 mg/mL against MCF-7 and MDA-MB-231 cells, compared to those of red rice bran extracts (175 and 151 mg/mL, respectively) and brown rice bran (382.3 and 346.1 mg/mL, respectively) (Fig. 3). The biological properties and reactions to certain agents differ between different breast cancer cell lines. MDA-MB-231 cells were the most sensitive to treatment with different pigmented rice bran followed by the MCF-7 cell line; therefore, their pro-apoptotic responses to different pigmented rice bran were analysed. The IC50 values of all the extracts were higher that of than tamoxifen (MDA-MB-231 = 40.2 mg/mL; MCF-7 = 22.8 mg/mL), which is a breast cancer drug. Previous studies have reported the anticancer activity of brown rice bran on breast cancer cell lines such as MDA-MB-468 [34, 35], MCF-7  and MDA-MB-231 . It has been reported that the cytotoxic activity of rice bran against breast cancer cell lines is influenced by the variety of rice, growing conditions, cultivation process, and the type of cancer cell lines [35, 37].
Extracts of black, red, and brown rice bran at concentrations between 25 and 400 μg/mL did not exert toxic effect against normal cells (MCF-10A), with viability between 77 and 98% (Fig. 4). In food supplements, one ingredient may provide the desired therapeutic benefits while others may exert toxic effects. The American National Cancer Institute recommends that crude herbal extracts that do not reduce the viability of normal cells below 76% is safe for human consumption . In the current study, black rice bran contained the highest content of secondary metabolites, including anthocyanins, phenolics, and flavonoids, and exhibited the highest antioxidant and antiproliferative activity. Therefore, in general, it appears that the antioxidant and antiproliferative activities of black rice bran are attributed to the high level of phytochemicals. However, further research is needed to understand the relationship between these phytochemicals and the antiproliferative activity in black rice bran extracts. The current results corroborate the findings of a great deal of previous work in this field.