Effect of cultivars and milling degrees on free and bound phenolic profiles and antioxidant activity of black rice

Abstract

Six black rice cultivars (Heukjinju, Sintoheugmi, Heukhyangchal 1, Bosukheukchal, Sinnongheukchal, and Josengheukchal) and varying milling degrees (step 0, 0%; step 1, 4.2%; and step 2, 10.5%, w/w) were used to evaluate the effects of cultivars and milling degrees of black rice (Oryza sativa L.) on the total phenolic contents (TPC), total flavonoid contents (TFC), antioxidant activity (2,2-diphenyl-1-picrylhydrazyl free radical assay), and phenolic composition in free and bound phenolic fractions. Unpolished (step 0) Sintoheugmi showed significantly higher TPC, TFC, antioxidant activity, phenolic acid levels, and anthocyanin levels than other unpolished cultivars (p < 0.05). As milling degree increased, TPC, TFC, antioxidant activity, phenolic acid levels, and anthocyanin levels decreased significantly (p < 0.05). TPC, TFC, and antioxidant activity were significantly higher in free phenolic fractions than bound phenolic fractions of black rice extracts, regardless of cultivars (p < 0.05). The major phenolic acid was ferulic acid, and the major anthocyanin found in free phenolic fractions in black rice samples was cyanidin-3-O-glucoside. The sum of individual phenolic acid levels (255.2 ± 0.0 μg/g) and the sum of anthocyanins levels (831.4 ± 0.3 μg/g) were significantly higher in Sintoheugmi black rice than in the other cultivars for step 0 (unpolished rice) (p < 0.05). For step 1 and step 2, Heukjinju black rice contained significantly higher sum of phenolic acid levels and sum of anthocyanin levels than the other cultivars (p < 0.05). For use as a better functional ingredient, it is, therefore, important to consider different milling degrees together with different black rice cultivars having the highest antioxidant component.

Introduction

Specialty rice, such as pigmented rice and aromatic rice, have been developed in response to increases in income levels due to social development. Black rice is produced in some parts of southern Asia, China, and Korea, and the production of this pigmented rice has greatly increased farm income. Recently, black rice species have received attention from researchers and the food industry because they are potential sources of anthocyanins. In previous studies, the total phenolic contents (TPC) and total flavonoid contents (TFC) of black rice cultivars were significantly higher than those of non-pigmented, green, and red rice cultivars [1, 2]. Wu et al. [3]. reported that the anthocyanin levels in black rice were higher than those in other available anthocyanin sources. The main anthocyanin in black rice is cyanidin-3-O-glucoside, which is followed by a minor proportion of the anthocyanin peonidin-3-O-glucoside. These anthocyanins are reported to have antioxidative, anticancer, and anti-inflammatory activities [4]. Anthocyanins may be metabolized into phenolic acids such as protocatechuic acids after consumption [5]. Anthocyanin in plants may play an antioxidant role against reactive oxygen species that result from external stresses such as ultraviolet, pathogens, and some microbes [6]. Additionally, black rice is a particularly economically important rice species that contains many beneficial compounds, such as other polyphenolics, γ-oryzanol, vitamin E, and phytic acid [7].

Black rice has a characteristically dark purple and black color from the anthocyanin pigments, which are mostly located in the aleurone and pericarp layers of the rice bran. Additionally, black rice contains phenolic acids in free and/or bound forms [8]. Free phenolic acids are absorbed in the human stomach and small intestine and can protect against oxidation of low-density lipoprotein and liposomes [9]. Insoluble, bound phenolic acids are partly digested in small intestine by enzymes, and some are digested in the colon by the colonic microflora [10]. Bound phenolics of black rice were not investigated in many studies on phenolic composition of black rice samples [11, 12].

There is little information on the varietal differences of phenolic composition in black rice. Also, different extraction solvents were used in different studies [8, 11]; thus, it is difficult to compare phenolic contents of different black rice varieties studied in previous studies. More importantly, there is little information on the changes of phenolic composition during milling process. Generally, rice is consumed in the form of rice flour and kernel after milling. The quality of raw rice before processing significantly affects the quality of rice after processing [13]. The degree of removal of rice bran during the milling process is closely related to milling yield and rice quality and is affected by the degree of milling, i.e., the ratio of the weight of removed rice bran to the weight of unpolished rice. Different milling degrees of rice are used for different purposes. Usually, textural properties of milled rice are favorable; however, consumption of milled rice may increase blood glucose levels, when measured by glycemic index, rapidly than consumption of the same amount of un-milled rice. Thus, patients with diabetes are recommended to consume un-milled rice. Also, when the rice bran and germ are completely removed, anthocyanin compounds may be also removed.

In this study, we evaluated the effect of cultivars and milling degrees in black rice on the TFC, TPC, antioxidant activity (2,2-diphenyl-1-picrylhydrazyl free radical assay (DPPH)), and phenolic composition, including free and bound phenolic acids and anthocyanins.

Materials and methods

Chemicals and reagents

Cyanidin-3-O-glucoside and peonidin-3-O-glucoside were purchased from Extrasynthese (Lyon, France). Phenolic acid standards (i.e., gallic acid, syringic acid, vanillic acid, p-coumaric acid, and ferulic acid) and other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Rice samples and preparation

Six black rice (Oryza sativa L.) cultivars (Bosukheukchal, Heukhyangchal 1, Heukjinju, Josengheukchal, Sinnongheukchal, and Sintoheugmi) were used in this study. The Heukjinju cultivar was harvested in October 2016 at a local farm in Jindo, Jeollanam-do, Republic of Korea. All the other cultivars were harvested in October 2016 from a local farm in Hoengseong-gun, Gangwon-do, Republic of Korea. Before distribution, the grains were dried to a moisture content of about 12–13%. The rough rice samples were then placed in a sack in dark storage at room temperature (21–23 °C) for 1–2 mon before dehulling on a Satake Rice Huller (Satake, Tokyo, Japan). After dehulling, an FS-2000 rice polisher (Misul, Seoul, Korea) was used to mill the unpolished rice to three different milling degrees (step 0, 0%; step 1, 4.2%; and step 2, 10.5%, w/w). The milling degree of step 0 is typical brown rice level, and that of step 2 is typical white rice level.

Colorimetric measurements

The color of the black rice powders varied in the three different milling degrees, and the color of the samples was measured using a colorimeter of HunterLab model UltraScan PRO (Reston, VA, USA). Color characteristics were indicated by Hunter’s values: L ^* (whiteness), a ^* (redness), and b ^* (yellowness). The calibration of the colorimeter was conducted using a standard white tile (L ^* = 100.03, a ^* = 0.09, b ^* = 0.15). Samples were prepared in triplicate (n = 3). The total color difference (ΔE^*) between black rice samples of the same cultivar treated with steps 1 and 2 and the unpolished samples of step 0 were calculated using the following equation:

$$\Delta {\text{E}}^{*} = \sqrt {\Delta L^{*2} + \Delta a^{*2} + \Delta b^{*2} } .$$

Extraction of free and bound phenolics

The free and bound phenolics in the black rice samples were extracted sequentially. The first extraction used 80% methanol for free phenolic extraction, and the second extraction employed alkaline and acid hydrolysis for bound phenolic extraction [8]. The methodologies used are as follows: a sample of 100 g of black rice kernels was ground and passed through an 18-mesh sieve. The powder [0.05 g (± 1%)] was mixed with 8 mL 80% methanol, and the mixture was sonicated at 35 °C for 1 h. The supernatant was centrifuged at 4321 g for 25 min, and the extraction was performed again. The pH of combined supernatants, i.e., the free phenolic fraction, was adjusted to 4.5–5.5 using 6 M HCl.

After washing the residue with water, the residues were sonicated with 20 mL of 4 M NaOH for 2 h and the supernatant was collected. The sonication process was conducted again with the remaining residues. The combined supernatants were adjusted to 4.5–5.5 using 6 M HCl and then centrifuged at 4321 g for 25 min. The supernatants were used in the bound phenolic analysis.

The free and bound fractions were filtered through a 0.22-μm PTFE membrane filter (Merck, Darmstadt, Germany) before high-performance liquid chromatography (HPLC) analysis.

Determination of TPC and TFC

The TPC and TFC of diluted free and bound extracts of black rice samples were determined [14]. The TPC was expressed as milligrams of gallic acid equivalent per gram of the sample (mg GAE/g), and TFC was expressed as milligrams of quercetin aglycone equivalent per gram of the sample (mg QE/g).

Determination of antioxidant activity

The antioxidant activity of diluted free and bound extracts of black rice samples was determined using DPPH radical scavenging activity assays [8, 15]. The total antioxidant activity of the DPPH assays was expressed as milligrams of Trolox equivalent per gram of the sample (mg TE/g).

Determination of phenolic acids and anthocyanins using HPLC

The phenolic composition of free and bound extracts of black rice samples was determined using a HPLC system (Agilent 1260 Infinity II LC system, Agilent, Santa Clara, CA, USA) equipped with Agilent 1200 series VWD detector. The phenolic acid and anthocyanin profiles were determined according to Kim and Lee’s method [16]. Phenolic compounds were separated on a Zorbax Eclipse XDB-C18 (4.6 × 250 mm, 5 μm, Agilent) column after loading 20 μL of injection volume. The mobile phases consisted of A (1% TFA in water solution) and B (1% TFA in acetonitrile solution) at the flow rate of 1.0 mL/min. The solvent gradient was programmed as follows: 0–6.5 min, 10–12% B; 6.5–10.5 min, 12–13% B; 10.5–33 min, 13–17% B; 33–60 min, 17–65% B; 60–70 min, 65–95% B. The hydroxyl benzoic phenolic acids (gallic acid, syringic acid, and vanillic acid), hydroxycinnamic acids (p-coumaric acid and ferulic acid), and anthocyanins (cyanidin-3-O-glucoside and peonidin-3-O-glucoside) were detected at 280, 320, and 520 nm, respectively. The quantification linear ranges of all phenolic acids and anthocyanins were between 0.05 and 10 μg/mL, with an R ² value of over 0.9984.

Statistical analysis

Data analysis was performed with SPSS statistical software version 23 (SPSS, Inc., Chicago, IL, USA). The results were reported as mean ± standard deviation. A multivariate analysis of variance (MANOVA) at p < 0.05 was used to determine the effect of cultivar differences, milling degrees, and cultivar–milling degree interactions of black rice on all results of the analysis. The significant differences among black rice cultivars in the same milling degree and among different milling degrees in the same cultivar were determined using one-way ANOVA followed by Duncan’s multiple range test at p < 0.05. The correlation analysis among phenolic acids, anthocyanins, color parameters, TPC, TFC, and antioxidant activities was performed with Pearson’s correlation test at p < 0.05. Standardized principal component analysis (PCA) was performed using XLSTAT software (XLSTAT, ver. 2017.03, Microsoft Excel Add-in software, New York, NY, USA) to visualize clustering formation of cultivars varying in different milling degrees and the relationship between antioxidants and the differently treated black rice samples.

Results and discussion

Color of black rice samples

As milling degree increased, the L ^*, a ^*, and b ^* values significantly increased (p < 0.05), regardless of black rice cultivars (Table 1). These results concur with those of a precious study, in which the L ^* significantly increased with milling degree [17]. The L ^* value for Heukhyangchal 1 was higher than for the other cultivars for step 0 and step 2 (p < 0.05), and the L ^* value for Bosukheukchal was lower than for the other cultivars for all milling degrees (p < 0.05).

Table 1 Hunter’s color values (L ^*: whiteness, a ^*: redness, b ^*: yellowness) of black rice cultivars varying in milling degrees

For step 2, the a^* value for Sintoheugmi was higher than for other black rice cultivars and the a^* value for Heukhyangchal 1 was lower than for other black rice cultivars (p < 0.05). Because unpolished black rice (i.e., step 0) has black and dark purple bran layers, the L ^* and a ^* values were lower than for polished rice (i.e., step 1 and step 2). While bran layers are removed during milling, the purple color pigments in the aleurone and pericarp layers of the bran are removed, resulting in increased L ^* and a ^* values.

Based on ΔE^* values, there was an obvious difference between step 0 and step 2, regardless of cultivars. There were, however, smaller differences between step 0 and step 1 than between step 0 and step 2. The Heukjinju cultivar especially showed invisible difference (a ΔE^* of 0.9) between step 0 and step 2. The ΔE^* varied as milling degree increased for different cultivars. The different ΔE^* levels may have been caused by different thickness levels of the rice bran layer for the different cultivars.

Total phenolic content and total flavonoid content

The TPC and TFC of free and bound phenolic fractions of black rice samples are shown in Table 2. The MANOVA results of TPC and TFC showed significant differences in different cultivars and milling degrees (p < 0.001). There were interactions between cultivars and milling degrees (p < 0.001).

Table 2 Total polyphenol and total flavonoid contents in free and bound phenolic fractions of black rice cultivars varying in milling degrees

The TPC of free and bound fractions in unpolished rice (step 0) were 2.10–3.74 and 0.26–0.79 GAE mg/g, respectively. The TPC of the total fraction are the sum of TPC of free and bound fractions. Free phenolics were dominant in TPC for all black rice cultivars varying in milling degrees. The Sintoheugmi cultivar contained significantly higher TPC of total fraction than the other cultivars, followed by Heukjinju > Bosukheukchal > Sinnongheukchal > Heukhyangchal 1 > Josengheukchal for step 0 (p < 0.05). The TPC of free and bound fractions significantly decreased as milling degree increased in all cultivars (p < 0.05). The TPC, however, decreased at different rates for the different cultivars and milling degrees. The cultivar, Heukjinju, for example, had significantly higher TPC of total fraction (2.97 ± 0.02 GAE mg/g) than the other cultivars for step 1 (p < 0.05). The ΔE^* was also minimal in the Heukjinju cultivar when milled from step 0 to step 1. The purple and black color pigments and anthocyanin loss may, therefore, be minimal in the Heukjinju cultivar. For step 2, the cultivars Heukjinju and Sinnongheukchal showed significantly higher TPC than the other cultivars (p < 0.05).

Flavonoids have nitric oxide and oxygen scavenging activities [18]. The TFC was higher in free fraction than in bound fraction in all cultivars. For step 0, the TFC showed 0.20–0.83 and 0.09–0.22 QE mg/g in free and bound fractions, respectively. The TFC was not detected in bound fraction in step 2 for the Josengheukchal cultivar. The TFC of total fraction is the sum of TFC of free and bound fractions. For step 0, the Sintoheugmi cultivar showed a higher TFC of total fraction (1.05 ± 0.02 QE mg/g) than the other cultivars (p < 0.05), followed by Heukjinju > Bosukheukchal > Sinnongheukchal > Heukhyangchal 1 > Josengheukchal. This pattern of results is similar to that seen for the TPC. The Sintoheugmi cultivar contained significantly higher free (0.83 ± 0.14 QE) and bound (0.22 ± 0.02 QE) flavonoids than the other cultivars (p < 0.05). For step 1, the Sintoheugmi and Heukjinju cultivars contained significantly higher TFC (0.50–0.56 QE mg/g) than other black rice cultivars (0.19–0.41 QE mg/g) (p < 0.05).

DPPH radical scavenging activity

The DPPH radical scavenging activity results of black rice samples are shown in Table 3. The DPPH radical scavenging activity of free phenolic fraction was higher than that of bound phenolic fraction, as was similarly seen in the TPC and TFC results. DPPH radical scavenging activity of free and bound phenolic extracts was significantly different as milling (p < 0.05). For step 0, the DPPH radical scavenging activity of free and bound phenolic fractions was 6.31–9.36 and 1.17–1.49 TE mg/g, respectively. For step 2, the DPPH radical scavenging activity of free and bound phenolic fractions was 4.29–6.54 and 0.66–1.02 TE mg/g, respectively. The DPPH radical scavenging activity of total phenolic extracts of the Sintoheugmi cultivar was higher than the other cultivars for step 0 (p < 0.05), followed by Heukjinju > Bosukheukchal > Sinnongheukchal > Heukhyangchal 1 > Josengheukchal (p < 0.05), similar to the results of the TPC and TFC. There was, however, no significant difference in the TFC in Bosukheukchal, Heukjinju, and Sintoheugmi for step 1 (p < 0.05). For step 2, the Heukjinju cultivar showed the highest DPPH radical scavenging activity among the six black rice cultivars (p < 0.05).

Table 3 Antioxidant activity (DPPH radical scavenging activity) in free and bound phenolic fractions of black rice cultivars varying in milling degrees

Determination of individual phenolic acids and anthocyanins in free and bound phenolic fractions

A typical HPLC chromatogram for analysis of free and bound phenolics is shown in Fig. 1. The quantification of five phenolic acids (gallic acid, syringic acid, vanillic acid, p-coumaric acid, and ferulic acid) and two anthocyanins (cyanidin-3-O-glucoside and peonidin-3-O-glucoside) in black rice samples was performed using authentic standards. The free and bound phenolic acid compositions in black rice samples varied according to the three different milling degrees (step 0, 1, and 2), as shown in Table 4. The effect of cultivar, the effect of degree of milling, and the two-way interactions between cultivar and degree of milling on phenolic acid and anthocyanin levels were confirmed by the MANOVA (p < 0.001) test.

Fig. 1

HPLC chromatogram for determination of phenolic acids and anthocyanins in free (A) and bound (B) phenolic fractions of black rice (cv. Sintoheugmi)

Table 4 Total and free phenolic acid and anthocyanin contents of black rice cultivars varying in milling degrees

In all black rice cultivars, ferulic acid (59–75% of the total phenolic acids) and cyanidin-3-O-glucoside levels (93–98% of the total anthocyanins) were predominant. In all cultivars, the levels of phenolic acid and anthocyanins decreased as the milling degree increased (p < 0.05). The unpolished black rice (i.e., step 0) contained the highest total phenolic acid and anthocyanin levels for all black rice cultivars, as was noted before in a previous study [8]. The average total phenolic acid levels of all investigated cultivars were 216.3 μg/g in step 0, 152.1 μg/g in step 1, and 99.1 μg/g in step 2. The average total anthocyanin levels of all investigated cultivars were 494.4 μg/g in step 0, 385.1 μg/g in step 1, and 199.9 μg/g in step 2. The levels of ferulic acid ranged between 117.3 and 182.7 μg/g in step 0, 73.9 and 141.2 μg/g in step 1, and 37.0 and 122.2 μg/g in step 2. These values are similar to those reported by Sumczynski et al. (2016), which ranged between 162.9 and 237.0 μg/g [8]. The levels of gallic acid ranged between 3.3 and 9.8 μg/g in step 0, 0.7 and 7.1 μg/g in step 1, and 0.3 and 1.9 μg/g in step 2. The levels of syringic acid ranged between 17.5 and 29.3 μg/g in step 0, 11.6 and 23.0 μg/g in step 1, and 1.2 and 19.2 μg/g in step 2. The levels of vanillic acid ranged between 6.4 and 17.9 μg/g in step 0, trace level (detected but below LOQ) and 8.7 μg/g in step 1, and not detected and 5.4 μg/g in step 2. The levels of p-coumaric acid ranged between 23.4 and 38.1 μg/g in step 0, 19.6–26.1 μg/g in step 1, and 16.8–19.5 μg/g in step 2. In another study, the levels of gallic acid, syringic acid, vanillic acid, and p-coumaric acid in black rice ranged 12.9–23.4, 1.7–3.5, 17.3–54.4, and 3.9–50.6 μg/g, respectively [8]. The levels of cyanidin-3-O-glucoside ranged between 258.2 and 809.4 μg/g in step 0, 190.3 and 490.9 μg/g in step 1, and 72.2 and 320.0 μg/g in step 2. The levels of cyanidin-3-O-glucoside concurred with previously reported values (i.e., 515 μg/g), and anthocyanins were found only in free phenolic fraction of black rice [19]. It is, however, difficult to compare these data with other studies to show whether tested black cultivars in this study had higher levels of phenolic acids than other black rice varieties in previous studies because most prior studies did not report on milling degrees [8], and some studies used different extraction solvents [20].

Of the tested cultivars, Sintoheugmi contained the highest levels of total phenolic acids (255.2 μg/g) and anthocyanins (831.4 μg/g) in step 0, followed by Heukjinju > Bosukheukchal > Heukhyangchal 1 > Sinnongheukchal > Josengheukchal, similar to the previous antioxidant activity assay. For step 1 and step 2, Heukjinju contained the highest levels of total phenolic acids (198.4 and 161.9 μg/g in step 1 and step 2, respectively) and anthocyanins (500.6 and 326.1 μg/g in step 1 and step 2, respectively). These data are similar to the TPC, TFC, and DPPH assay results. Although unpolished Sintoheugmi black rice (step 0) contained significantly higher ferulic acid levels than other unpolished black rice cultivars (p < 0.05), polished Sintoheugmi (step 2) had significantly lower ferulic acid levels than the other cultivars (p < 0.05). In step 1, gallic acid and syringic acid levels (7.1 ± 0.8 and 23.0 ± 0.2 μg/g, respectively) in Heukjinju rice were significantly higher than those of the other cultivars (p < 0.05). In contrast, Sintoheugmi contained significantly lower levels of gallic acid and syringic acid than the other cultivars in step 1 (p < 0.05). Unpolished Sintoheugmi black rice (step 0) contained the highest cyanidin-3-O-glucoside levels (809.4 μg/g) with 1.5-fold higher levels than the second-highest cyanidin-3-O-glucoside cultivar, Heukjinju (544.9 μg/g), followed by Heukjinju > Bosukheukchal > Sinnongheukchal > Josengheukchal > Heukhyangchal 1 (p < 0.05). Partially polished black rice (step 1) of Sintoheugmi and Heukjinju cultivars, however, contained similar levels of cyanidin-3-O-glucoside (480.5 vs. 490.9 μg/g, respectively). Polished Sintoheugmi black rice (step 2) had significantly lower levels of cyanidin-3-O-glucoside (93.6 μg/g) than polished Heukjinju black rice (320.0 μg/g) (p < 0.05). The Heukjinju cultivar showed a minimal ΔE^* as a result of milling from step 0 to step 1. The anthocyanin results concurred with the trend of the ΔE^* results, shown in Table 1. These results show that the distribution of phenolics in black rice is not uniform for all cultivars and that, for example, it is likely that phenolic compounds are mainly present in the bran layer in the Sintoheugmi cultivar compared to Heukjinju cultivar, in which the phenolic compounds are evenly present in all layers. The degree of milling may, therefore, have a bigger impact on black rice cultivars with high phenolic contents in the bran layers, such as the Sintoheugmi cultivar. This observation is important because different milling degrees are used for different purposes of processing (e.g., cooking) of black rice. If black rice is used as a source of anthocyanin pigments, then unpolished Sintoheugmi or a coproduct (i.e., bran layer) of Sintoheugmi may be used as a source of the cyanidin-3-O-glucoside anthocyanin.

The predominant phenolic acid and anthocyanin (i.e., ferulic acid and cyanidin-3-O-glucoside) levels were significantly higher in Sintoheugmi (step 0) and Heukjinju (step 1–2) black rice cultivars than other black rice cultivars (p < 0.05). Some black rice cultivars, however, contained higher levels of other phenolic acids than the Sintoheugmi and Heukjinju cultivars. For example, the Josengheukchal cultivar contained significantly higher levels of vanillic acid (17.9 μg/g in step 0) than Sintoheugmi and Heukjinju cultivars (6.4–8.1 μg/g in step 0) (p < 0.05).

Gallic acid, syringic acid, vanillic acid, p-coumaric acid, and ferulic acid were found in step 0 and step 1 of all black rice cultivars, and vanillic acid was not found in Sintoheugmi, Sinnongheukchal, Bosukheukchal, and Heukhyangchal 1 cultivars after the step 2 milling degree.

In general, the sum of bound phenolic acid concentrations was higher (118.3–156.6 μg/g in the step 0, 70.1–117.2 μg/g in the step 1, and 45.4–113.1 μg/g in the step 2 black rice) than that of free phenolic acid concentrations (46.3–107.6 μg/g in the step 0, 38.2–81.2 μg/g in the step 1, and 12.6–48.7 μg/g in the step 2 black rice), as has also been shown in a previous study [21]. The major phenolic acids in free fraction were ferulic acid and syringic acid, and the major phenolic acids in bound fraction were ferulic acid and p-coumaric acid for all black rice samples, regardless of milling degrees. In black rice, phenolic acids such as ferulic acid are mainly present as a bound form esterified to macromolecules, and the linkages between the two are hydrolyzed by intestinal enzymes [10]. Ferulic acid was present in exclusively bound forms in all cultivars, concurring with the findings of a previous study [8]. Among the phenolic acids, gallic acid and syringic acid were only found in free phenolic fraction. Vanillic, p-coumaric, and ferulic acid concentrations were higher in bound fraction than in free fraction. No anthocyanins were detected in bound fractions. These results are similar to the previously reported study [21] on free and bound phenolic contents in black rice samples.

Correlation among color parameters, antioxidant activity, and phenolic compounds

The correlations among the individual phenolic compounds to Hunter color parameters TPC, TFC, DPPH radical scavenging activity in free and bound phenolic fractions are shown in Table 5. All phenolic compounds in free phenolic fraction of black rice showed significantly negative correlation with L ^*, a ^*, b ^*, and ΔE (p < 0.05) and significantly positive correlation with TPC, TFC, and DPPH (p < 0.05) with exception of syringic acid and TFC (p > 0.05) (Table 5). Cyanidin-3-O-glucoside and peonidin-3-O-glucoside, the most abundant anthocyanin pigments in black rice [22], were significantly negatively correlated with ΔE^* (r = − 0.662 and r = − 0.626, respectively) (p < 0.05). Most bound vanillic, p-coumaric, and ferulic acids showed significantly negative correlation with L ^*, a ^*, and ΔE (p < 0.05). Free ferulic acid showed high positive correlation with TPC (r = 0.910) and DPPH (r = 0.825) (p < 0.001). Cyanidin-3-O-glucoside had a significantly high positive correlation with all TPC, TFC, DPPH values (r = 0.795–0.855) (p < 0.001).

Table 5 Correlation coefficients between color values, TPC, TFC, DPPH, and phenolic compounds in free and bound phenolic fractions of black rice samples

Bound vanillic, p-coumaric, and ferulic acids showed significantly negative correlation (r = − 0.646 ~ − 0.709) with ΔE^* (p < 0.05). Bound ferulic acid levels were significantly positively correlated with TFC (r = 0.563) and DPPH radical scavenging activity (r = 0.761) (p < 0.05). The DPPH radical scavenging activity showed significantly (p < 0.05) positive correlation with bound vanillic (r = 0.609), p-coumaric (r = 0.500), and ferulic acid (r = 0.761) (p < 0.05). There was no correlation between bound phenolic acids and TPC (p > 0.05). It could be suggested that individual phenolic compounds present in free and bound phenolic fractions demonstrate their antioxidant ability differently. These individual phenolic compounds may act individually, synergistically, or antagonistically. The antioxidant mechanisms of the individual phenolic compounds need to be clarified through further studies.

Principal component analysis

The PCAs, i.e., PC1 and PC2, were performed on TPC, TFC, DPPH, and individual phenolic compounds in free and bound phenolic fractions (Fig. 2). The PC1 explained approximately 54.27% of the total variance, in which step 0 black rice samples were on the right side and step 2 black rice samples were on the left side. Partially milled rice (step 1) was located between the two steps. PC2 explained approximately 21.09% of the total variance. Step 1 of Heukjinju (HJ1) was located especially close to step 0 of the black rice samples, and this may be explained by high levels of phenolic compounds in HJ1. The PCA loading plot indicated that there were high levels of antioxidant activity, TPC, TFC, and individual phenolic compounds in the step 0 samples.

Fig. 2

Principal component analysis (PCA) plots (PC1 and PC2) of free and bound phenolics and antioxidant activity of black rice cultivars varying in milling degree. The sample labels are as follows: step 0 (0), step 1 (1), step 2 (2), Bosukheukchal (BS), Heukhyangchal 1 (HH), Heukjinju (HJ), Josengheukchal (JS), Sinnongheukchal (SN), Sintoheugmi (ST), gallic acid (GA), syringic acid (SA), p-coumaric acid (pCA), ferulic acid (FA), peonidin-3-O-glucoside (P3glc), cyanidin-3-O-glucoside (C3glc) in free phenolic fraction (f) and bound phenolic fraction (b)

All antioxidant activities and individual phenolic compounds showed positive values for PC1, indicating a strong correlation with unpolished rice. Cyanidin-3-O-glucoside is located close to the TFC of free phenolics extracts, suggesting that the anthocyanin pigment, which is one type of flavonoid, is correlated with the TFC. Along PC1, antioxidant activities were on the higher side, whereas individual phenolic compounds were relatively lower in position.

In summary, the color parameters, TPC, TFC, antioxidant activity, and phenolic compositions were affected by the cultivar and milling degree of black rice. As the milling degree increased, the TPC, TFC, antioxidant activity, individual phenolic acid levels, and individual anthocyanin levels decreased significantly (p < 0.05). The decrease rate, however, was dependent on the cultivar of black rice. For example, unpolished Sintoheugmi (step 0) contained significantly higher amounts of phenolic acid and anthocyanins than other black rice cultivars (p < 0.05); however, as the milling degree increased (i.e., step 1 and 2), these levels decreased dramatically and the Heukjinju black rice cultivar was shown to have a significantly higher sum of phenolic acid and anthocyanins than other black rice cultivars (p < 0.05). It is, therefore, important to consider different milling degrees together with different black rice cultivars having the highest antioxidant component to obtain the better functional ingredient.