Bioactive compound profile and their biological activities of endogenous black rice from Java and East Nusa Tenggara

ABSTRACT

Indonesia has an abundance of pigmented rice cultivars with high nutritional value. The purpose of this study was to identify and characterize the phytochemical profiles, anthocyanin properties, and biological activities of endogenous Indonesian black rice (Oryza sativa L.) cultivars from Java and East Nusa Tenggara (NTT) Islands. Thirteen black rice cultivars from eastern Nusa Tenggara and West, Central, and East Java were examined. The secondary metabolites of the black rice samples included flavonoids, tannins, phenolics, quinones, glycosides, anthraquinones, leucoanthocyanidins, alkaloids, and proteins. According to the FRAP (ferric reducing antioxidant power) assay, various levels of antioxidant activity were present. The results of an alpha amylase inhibition assay indicated that the black rice samples might have antidiabetic properties. We conclude that the Java and NTT endogenous black rice cultivars have rich phytochemical compounds with high nutritional value, and that their antioxidant and antidiabetic biological activities can promote a healthy life.

KEYWORDS:

• Anthocyanin
• antidiabetic
• antioxidant
• black rice
• nutrition

Introduction

Rice (Oryza sativa L.) is a major agricultural product for human consumption in many countries and constitutes the primary diet for more than 90% of Indonesian people. There are thousands of rice cultivars in Indonesia, which are generally divided into traditional cultivars that are native to a particular area, and superior cultivars. Oryza sativa var. Javanica and var. Indica are mostly grown in Indonesia (Irianto et al., 2009). In addition to various white rice cultivars, Indonesia also features many pigmented rice cultivars, such as black, red, and brown rice, which are grown as lowland rice, upland rice, or swamp rice. In other countries, black rice is widely used in the food industry (Ito & Lacerda, 2019). In Japan, black rice is utilized in the manufacturing of alcoholic beverages with high amounts of phenolic compounds (Ito & Lacerda, 2019; Katoh et al., 2011). The freeze-dried crude extract of black rice is used as a natural antioxidant source for mayonnaise production in Thailand (Ito & Lacerda, 2019; Tananuwong & Tewaruth, 2010). Flour made from black rice is used in the production of chiffon cakes and has more bioactive compounds and significant antioxidant activity (Ito & Lacerda, 2019; Mau et al., 2017). Rice contains many secondary metabolites including flavonoids, phenolic acids, terpenoids, alkaloids, and steroids (Wang et al., 2018). The main bioactive compounds in pigmented rice are anthocyanins, for instance cyanidin-3-O-glucosides and peonidin-3-O-glucosides, cyanidin 3-rutinoside, cyanidin 3,5-diglucoside, malvidin 3-galactoside, pelargonidin 3,5-diglucoside, proanthocyanidins, flavan-3-ols, isoflavones, γ-oryzano, and ferulic acid (Deng et al., 2013; Sari et al., 2021). These compounds have broad biological functions in promoting human health as nutraceuticals to regulate cellular metabolism (Fatchiyah, Safitri, et al., 2020; Samyor et al., 2017).

Pigmented rice is currently gaining more attention. The pigments contained in rice, especially anthocyanins, have been reported to have many health benefits. Anthocyanins are natural pigments that are responsible for the red, purple, and blue colors of many fruits and flowers and have water-soluble properties (Khoo et al., 2017; Mattioli et al., 2020). The therapeutic effects of anthocyanins are mainly due to their antioxidative activities. Anthocyanin chalcones and quinoidal bases with a double bond that conjugate to the keto group are effective antioxidants since they act as free radical scavengers (Khoo et al., 2017). In addition, the glycosylated B-ring structure of anthocyanin plays a role in the antioxidant activity, which is greatly enhanced by methoxylation and orthohydroxylation (Stintzing et al., 2002). Anthocyanins have many other beneficial functions in addition to their antioxidant activities, which include antiobesity, antiapoptosis, antidiabetes, and anticancer properties (Fatchiyah, Safitri, et al., 2020; Lin et al., 2017; Sari et al., 2019, 2020)

The genotypic diversity of phytochemicals associated with pigmented rice has been presented in numerous studies (Fatchiyah, Ratih, et al., 2020; Samyor et al., 2017; Sari et al., 2021). Nevertheless, most of the research on pigmented rice focused on the relationship between anthocyanins and antioxidants, with some health-stimulating effects. In addition, the antioxidant activity of pigmented rice is commonly higher than that of non-pigmented rice. This is because the phenolic and flavonoid contents and the antioxidant activity are positively correlated. Among pigmented rice types, black rice contains the highest concentrations of anthocyanins. Black rice is therefore considered to have higher biological activity and efficacy than other pigmented rice type s (Fatchiyah, Ratih, et al., 2020).

Indonesia has many cultivars of endogenous black rice. These cultivars are widely distributed throughout the country (Anggaraini et al., 2015; Apridamayanti et al., 2017). Previous studies showed that different black rice cultivars from Java have the black coloration in different parts of the rice grain (Purwanto et al., 2018; Sholikhah et al., 2019), which suggests that environmental factors contribute to the differences in the black color morphology (Bhuvaneswari et al., 2020). In addition, our previous study revealed that anthocyanin biosynthesis and composition were significantly influenced by the genomic and proteomic profiles (Sari et al., 2021). The various metabolites present in pigmented rice have been studied in detail (Fatchiyah, Ratih, et al., 2020; J. K. Kim et al., 2013; T. J. Kim et al., 2021). However, to the best of our knowledge, no comprehensive phytochemical profiling and analysis of the biological activities of various endogenous black rice cultivars from Indonesia has yet been performed.

In this study, we present a comprehensive identification of the chemical composition and biological activities of different cultivars of Indonesian black rice. We performed phytochemical analyses of the black rice cultivars Mentik, Melik, and Wajaloka from Java Island and cultivars endogenous to East Nusa Tenggara (NTT). We also determined the biological activities of these black rice cultivars based on antioxidant activity and alpha amylase inhibition assays.

Materials and methods

Rice samples and chemicals

The 13 Indonesian black rice cultivars used in this study were locally grown in East, Central, and West Java and in East Nusa Tenggara (NTT). The rice cultivars consisted of Mentik, Melik, Wajaloka, and endogenous cultivars of NTT. The eastern Java black rice cultivars were UB black rice from Batu Malang (BREJ Mentik 2), Lawang black rice (BREJ Mentik 3), Ngawi Mentik Wangi black rice (BREJ Mentik 4), Ngawi black rice (BREJ Mentik 5), Ngawi black rice 2 (BREJ Mentik 6), Jember Melik black rice (BREJ Melik), and Kepanjen Wajaloka black rice (BREJ Wajaloka). The Central Java black rice cultivars were Purworejo Mentik black rice (BRCJ Mentik 1), Klaten Mentik black rice (BRCJ Mentik 2), and Magelang Melik black rice (BRCJ Melik 2). The eastern Nusa Tenggara black rice cultivars were Blitar Endogenous black rice (BRNTT Endo 1), Sikka Bengawan Endogenous black rice (BRNTT Endo 2), and Ende Pulen Endogenous black rice (BRNTT Endo 3). Several previously studied black rice cultivars (Fatchiyah, Ratih, et al., 2020) were included in this work: Kepanjen Mentik black rice (BREJ Mentik 1), Semarang Melik black rice (BRCJ Melik 1), and Toraja-Sukabumi black rice (BRWJ Toraja).

All chemicals were purchased from Sigma – Aldrich (pro-analysis grade) and used without further purification. Alpha-amylase from Aspergillus oryzae (≥150 units/mg protein) was used for the enzyme inhibitory assay.

Extraction of black rice

The thirteen black rice cultivars were ground into fine powder, and extraction was performed using a maceration technique with 0.1% HCl (w/v) in methanol for 24 h, in a volume of 4× the dry weight. The extracts were collected through filtration using Whatman filter paper (0.45 μm) and concentrated using a rotary vacuum evaporator, with a slow speed of 700 rpm, at 45°C. The concentrated extracts were stored at 4°C for subsequent analysis. All extractions were performed in triplicate.

Phytochemical screening of black rice extracts

The phytochemical qualitative tests of the 13 black rice cultivars were conducted based on previous studies (Fatchiyah, Ratih, et al., 2020; Gul et al., 2017; Shaikh & Patil, 2020) to detect alkaloids, flavonoids, phenolic compounds, steroids, tannins, quinones, anthraquinone, leucoanthocyanidins, glycosides, and proteins. The absorbance of samples was determined using UV – Vis spectrophotometry at different wavelengths (210 nm for glycosides, 280 nm for phenolic compounds and proteins, 420 nm for quinones, 430 nm for flavonoids, 470 nm for alkaloids, 515 nm for anthraquinones, 535 nm for leucoanthocyanidins, and 700 nm for tannins) (Fatchiyah, Ratih, et al., 2020; Gul et al., 2017).

Total anthocyanin content (TAC) determination

The TAC of the rice extracts was determined with the pH differential method and expressed as cyanidin-3-glucoside (Kartini et al., 2020; Wu et al., 2015). The samples were dissolved in potassium chloride buffer (pH 1) and sodium acetate buffer (pH 4.5). The absorbance was measured at wavelengths of 520 nm and 700 nm with a UV – Vis spectrophotometer. The TAC was calculated as cyanidin-3-glucoside equivalent (% w/w), using the following formula:

$\mathrm{T}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}\mathrm{a}\mathrm{n}\mathrm{t}\mathrm{h}\mathrm{o}\mathrm{c}\mathrm{y}\mathrm{a}\mathrm{n}\mathrm{i}\mathrm{n}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{z}\left(\mathrm{m}\mathrm{g}/\mathrm{L}\right)=\frac{\mathrm{A}\times \mathrm{M}\mathrm{W}\mathrm{x}\mathrm{D}\mathrm{F}\times {10}^3}{\epsilon \times \mathrm{L}}$

where A = (A520 nm – A700 nm) at pH 1.0 – (A520 nm – A700 nm) at pH 4.5; MW = 449.2 g/mol (molecular weight of cyanidin-3-glucoside); DF = dilution factor of the solution; 10³ = conversion from g to mg; ε = molar extinction coefficient (L.mol⁻¹.cm⁻¹); and L = pathlength (1 cm).

Characterization of anthocyanins in black rice

UV – Vis spectroscopy

Black rice extracts (0.15 mg) were diluted in 3 mL of potassium chloride buffer (pH 1) and sodium acetate buffer (pH 4.5). The absorbance was measured with a Shimadzu UV – Vis 1700 spectrophotometer in a wavelength range of 200–800 nm.

Thin-Layer Chromatography (TLC)

Black rice extract (0.2 mg/mL) was spotted on F254 silica gel paper and placed in a chamber containing the mobile phase n-butanol: glacial acetic acid: water in a 3:1:1 ratio (Sari et al., 2019). After separation occurred, the silica gel paper was air dried and visualized with UV light at a wavelength of 365 nm (Sari et al., 2019) for anthocyanin detection.

Fourier transform infrared spectroscopy

The Fourier transform infrared (FTIR) spectra of the black rice extracts were recorded using an FTIR spectrophotometer (Shimadzu IRSpirit). The FTIR spectra were used to identify the functional groups of anthocyanin in the samples by measuring the infrared absorption, emission, and photoconductivity spectra of the extracts (Agustika et al., 2022). The pigmented rice extracts were ground with KBr powder and compressed to form a pellet. The IR spectra were obtained in the wavenumber range of 500–4500 cm⁻¹.

Antioxidant activity of pigmented rice using FRAP (ferric reducing antioxidant power) assay

Rice samples were diluted with 0.1% methanol containing HCl and prepared to yield concentrations of 0, 20, 40, 60, 80, and 100 μg/ml. The extracts were mixed with 2.5 mL of phosphate buffer (pH 6.6) and 2.5 mL of 1% potassium ferricyanide. The solution was incubated at 50°C for 30 min. Then, 2.5 mL of 10% TCA was added to the solution and homogenized. The upper layer of the solution was mixed with 5 mL distilled water and a freshly prepared 0.1% ferric chloride solution (1 mL). The absorbance was measured using a Shimadzu UV – Vis spectrophotometer at 700 nm. Experiments were conducted in triplicate. Antioxidant activity was calculated as follows:

$\mathrm{\%}\mathrm{A}\mathrm{n}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{x}\mathrm{i}\mathrm{d}\mathrm{a}\mathrm{n}\mathrm{t}\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{i}\mathrm{t}\mathrm{y}=\frac{Absorbancecontrol-Absorbancesample}{Absorbancecontrol}x100\mathrm{\%}$

The percentage antioxidant inhibition (Y-axis) was plotted against the sample concentration (X-axis), and the IC₅₀ values were obtained from the linear regression. Ascorbic acid was used as a positive reference.

Alpha amylase inhibitory activity of black rice

The alpha amylase inhibitory activity was determined based on the standard procedures (Agustin et al., 2021; Kazeem et al., 2013) with a minor modification. A volume of 250 μL of various concentrations of the pigmented rice extracts (10, 20, 40, 60, 80, and 100 µg/mL) was mixed with 250 µL of 50 µg/mL of alpha-amylase. The solution mixture was incubated for 20 min at 37°C. Then, 250 μL of 1% amylum starch solution was added to each sample and incubated for 10 min. Finally, 500 μL of dinitro salicylic acid reagent was added to the solution and boiled for 5 min. The absorbance was measured at 490 nm using a Shimadzu UV – Vis spectrophotometer. Experiments were performed in triplicate. The inhibition activity of the alpha amylase enzyme was calculated using the following equation:

$\mathrm{\%}\mathrm{A}\mathrm{n}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{x}\mathrm{i}\mathrm{d}\mathrm{a}\mathrm{n}\mathrm{t}\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{i}\mathrm{t}\mathrm{y}=\frac{Absorbancecontrol-Absorbancesample}{Absorbancecontrol}x100\mathrm{\%}$

The percentage of alpha amylase inhibition (Y-axis) was plotted against the sample concentration (X-axis), and the IC₅₀ values were obtained from the linear regression. Acarbose was used as a positive reference.

Data analysis

Results were expressed as the mean ± standard deviation. Statistical analyses were conducted using the GraphPad Prism5 statistical package (Graphpad Software, San Diego, CA, U.S.A) (Favaro et al., 2018). One-way analysis of variance (ANOVA) followed by Tukey’s HSD test was used to determine differences among the cultivars. Differences at p < .05 were considered statistically significant.

Results

TLC results of phytochemical compounds and anthocyanin contents of black rice extracts

All black rice cultivars were subjected to phytochemical characterization and anthocyanin content analysis. The phytochemical characterization results are presented in Figure 1. The phytochemical profiling showed that most of the black rice cultivars contained different types and amounts of secondary metabolites including flavonoids, tannins, phenolics, quinones, anthraquinones, glycosides, and leucoanthocyanidins (Figure 1a,b). In addition to the secondary metabolites, we also analyzed the level of total proteins. BREJ Mentik 2 had the lowest amounts of phytochemical compounds. BRCJ Mentik 2, BRCJ Melik 2, BRWJ Toraja, and BRNTT Endogenous 2 had higher amounts of alkaloid compounds than other cultivars (Figure 1a), and not alkaloids were detected in BREJ Mentik 2, BREJ Mentik 3, and BREJ Wajaloka. All samples contained flavonoids, tannins, phenolics, quinone, anthraquinone, leucoanthocyanidins, and glucoside compounds. Steroid compounds were detected only in BRWJ Toraja. Almost all black rice samples had high amounts of quinone compounds. BRWJ Toraja contained the highest amount of protein, and no protein was detected in BRWJ Wajaloka. These results suggest that most of the black rice cultivars are rich in phytochemical compounds, which can be beneficial for human health.

Figure 1. Qualitative phytochemical screening and anthocyanin and proanthocyanidin profiles based on thin-layer chromatography. a. Phytochemical identification, b. Phytochemical distribution, c. Qualitative anthocyanin and proanthocyanidin profiles. Three samples (BREJ Mentik 1, BRCJ Melik 1, BRWJ Toraja) were obtained from Fatchiyah et al. (Stintzing et al., 2002).

The separated fraction profile of the TLC test of the black rice samples showed several different spots (Figure 1c, Table 1). Three major anthocyanin compounds, peonidin-3-O-glucoside, cyanidin-3-O-glucoside, and delphinidin-3-O-glucoside, were found in most of the black rice cultivars. All fractions from each sample had spots, which indicated the predicted peonidin-O-glucoside (Pn-3-glc) and aglycone (flavonoids). Cyanidin-O-glucoside compounds were detected in all samples except BREJ Mentik 2, BREJ Mentik 5, and BRNTT Endogenous 2. Delphinidin-O-glucoside was detected in BREJ Mentik 3, BRCJ Mentik 1, BREJ Mentik 2, BRCJ Melik 2, and BRNTT Endogenous 3. Observation of the TLC plate using UV light at 365 nm showed the presence of aglycone in all black rice cultivars. In addition, we measured the total anthocyanin content of the black rice extracts (Table 1).

Table 1. Anthocyanin prediction for black rice cultivars. Tukey’s HSD test was conducted in Graphpad Prism 9.3.1; significant differences are indicated by different letters (p < .05).

Sample	Anthocyanin Compounds				Total Anthocyanin Content (mg/L)
	Peonidin-O-glucoside (Pn-3-glc)	Cyanidin-O-glucoside (Cy-3-glc)	Delphinidin-O-glucoside (Dp-3-glc)	Aglycone (Flavonoid)
BREJ
BREJ Mentik 2	✓	-	-	✓	11.45 ± 0.44^a
BREJ Mentik 3	✓	✓	✓	✓	10.35 ± 0.08^bc
BREJ Mentik 4	✓	✓	-	✓	5.52 ± 0.002^f
BREJ Mentik 5	✓	-	-	✓	10.74 ± 0.04^b
BREJ Mentik 6	✓	✓	-	✓	3.69 ± 0.002^g
BREJ Melik	✓	✓	-	✓	3.75 ± 0.04^g
BREJ Wajaloka	✓	✓	-	✓	6.78 ± 0.01^e
BRCJ
BRCJ Mentik 1	✓	✓	✓	✓	9.99 ± 0.08^c
BRCJ Mentik 2	✓	✓	✓	✓	3.50 ± 0.02^g
BRCJ Melik 2	✓	✓	✓	✓	2.94 ± 0.002^h
BRNTT
BR NTT Endo 1	✓	✓	-	✓	5.13 ± 0.08^f
BR NTT Endo 2	✓	-	-	✓	2.13 ± 0.13^i
BR NTT Endo 3	✓	✓	✓	✓	8.83 ± 0.24^d

✓ Detected

- Not detected

Characterization of the anthocyanins in black rice samples by UV – Vis spectroscopy

UV – Vis spectroscopy was performed to analyze the anthocyanin profile in the black rice samples. The total anthocyanin content (TAC) of each sample was calculated at different pH values. Figure 2a,b shows the UV – Vis spectra (200–800 nm) at pH 1 and pH 4.5, respectively. Figure 2c shows the total anthocyanin content based on the UV – Vis spectral results. In this study, anthocyanins had a specific profile with maximum absorbance in the range of 400–600 nm.

Figure 2. Anthocyanin characterization based on UV – Vis spectroscopy. A. pH 1, B. pH 4.5, C. Total anthocyanin content of black rice extracts. Tukey’s HSD test was conducted in Graphpad Prism 9.3.1; significant differences are indicated by different letters (p < .05).

At pH 1, all black rice samples had maximum absorbance in the wavelength range of 512–528 nm (Figure 2a). BREJ Mentik 2, BREJ Mentik 5, and BRNTT Endogenous 3 showed the highest absorption of visible light (515 nm) at pH 1. At pH 4.5, when anthocyanins were degraded, the anthocyanin peak decreased (Figure 2b). BREJ Mentik 2, BREJ Mentik 3, BREJ Mentik 5, BREJ Wajaloka, and BRNTT Endogenous 3 exhibited a moderate anthocyanin peak when the absorbance was measured at pH 4.5. The decrease in absorbance indicated that the total anthocyanin content decreased due to the instability and degradation of anthocyanins at pH 4.5 (Table 2).

Table 2. Maximum wavelength and absorbance of spectral profiles at pH 1 and 4.5.

Sample	pH 1		pH 4.5
	λmax (nm)	Absorbance	λmax (nm)	Absorbance
BREJ
Mentik 2	515	2.37	542.5	0.842
Mentik 3	515	1.355	542	0.641
Mentik 4	512.5	1.875	-	-
Mentik 5	516	2.389	529	1.072
Mentik 6	515	1.178	-	-
Melik	515	1.591	-	-
Wajaloka	512.5	0.659	530	0.453
BRCJ
Mentik 1	515	1.591	-	-
Mentik 2	515	0.748	-	-
Melik 2	516	0.660	-	-
BRNTT
Endo 1	512	1.987	-	-
Endo 2	517	1.452	543	0.227
Endo 3	515	2.395	-	-

Different letters in Figure 2c symbolize a significant difference in TAC values between the samples based on Tukey’s HSD analysis; the absorbance values indicate the anthocyanin concentration in each sample. BREJ Mentik 2 had a higher TAC value than the other rice cultivars. BREJ Mentik 6 had a similar TAC value as BREJ Melik and BRCJ Mentik 2, and BRNTT Endogenous 2 had a significantly lower TAC value compared with other samples.

Functional group FTIR profiles of black rice

The functional group FTIR profiles of black rice are shown in Figures 3 and 4. The FTIR spectra of the 13 tested black rice cultivars were similar. There were eight specific regions that appeared in the FTIR spectra of all samples: the O–H stretching region, C–H aliphatic region, C=C stretching from aromatics, C=C stretching from alkanes, N=O bending from amines, CH–OH stretching from alcohols, C–O stretching from ether or ester, and the fingerprint region. However, certain wavenumbers and functional groups shifted. The FTIR image of BR Mentik and Melik data from East Java and West Java was obtained using Origin Pro 21 and is presented in Figures 3 and 4.

Figure 3. FTIR chromatogram of Mentik and Melik black rice cultivars.

Figure 4. FTIR chromatogram of Wajaloka and NTT black rice cultivars.

Based on the FTIR spectrum, Figure 3 shows the characteristics of the functional groups detected in the Mentik and Melik cultivars. These two cultivars had the same functional groups, i.e. O–H, C–H, C–O, and CH–OH (Table 3). The C–H aliphatic group was found in all samples except BREJ Mentik 3 and BREJ Melik. The aromatic groups, alkanes, ethers, and esters in the Mentik and Melik cultivars likely derived from secondary metabolites. Several anthocyanin functional groups such as O–H phenol, C–H aromatics, and carboxyl (C=O) were identified in the FTIR spectra. Protein was indicated by the amine group (N=O) in all samples of the Mentik and Melik cultivars.

Table 3. Wavenumbers and functional groups of the spectra of the Mentik and Melik black rice cultivars.

Rice	Wavenumber (cm^−1)	Functional group	Rice	Wavenumber (cm^−1)	Functional group
BREJ Mentik 2	3374.42	O–H stretching	BREJ Mentik 3	3321.65	O–H stretching
	3010.73	C–H aliphatic		2853.85–2925.16	C–H sp3 stretching
	2855.28–292659	C–H sp3 stretching		1712.88–1739.98	C=O stretching aromatic
	1718.59–1741.4	C=O stretching aromatic		1516.06–1640.14	C=C stretching alkanes, aromatic
	1518.92–1638.72	C=C stretching alkanes, aromatic		1460.44–1413.38	CH2 and CH3 (rocking C–H)
	1459.01	CH2 and CH3 (rocking C–H)		1337.79	N=O bending amines
	1337.79	N=O bending amines		1075.36–1282.16	C–O stretching Ether, ester
	1276.46	CHOH stretching alcohol		634.66–922.76	“Fingerprint region”
	1075.36	C–O stretching Ether, Ester
	634.66–777.29	“Fingerprint region”
BREJ Mentik 4	3400.09	O–H stretching	BREJ Mentik 5	3377.27	O–H stretching
	3009.31	C–H aliphatic		3010.73	C–H aliphatic
	2855.28–2916.59	C–H sp3 stretching		2855.28–2926.59	C–H sp3 stretching
	1742.83	C=O stretching aromatic		1714.31	C=O stretching aromatic
	1517.49–1630.16	C=C stretching alkanes, aromatic		1511.78–1638.72	C=C stretching alkanes, aromatic
	1460.44	CH2 and CH3 (rocking C–H)		1416.23–1459.01	CH2 and CH3 (rocking C–H)
	1370.59	N=O bending amines		1337.79	N=O bending amines
	1242.23	CHOH stretching alcohol		1206.58–1279.31	CHOH stretching alcohol
	1076.79–1169.49	C–O stretching ether, ester		1053.97–1101.04	C–O stretching ether, ester
				671.75–996.92	“Fingerprint region”
	670.32	“Fingerprint region”
BREJ Mentik 6	3367.29	O–H stretching	BREJ Melik	3378.7	O–H stretching
	3010.73	C–H aliphatic		2855.28–2926.59	C–H sp3 stretching
	2855.28–2926.59	C–H sp3 stretching		1717.16	C=O stretching aromatic
	1712.88	C=O stretching aromatic		1514.64–1637.29	C=C stretching alkanes, aromatic
	1513.21	C=C stretching alkanes, aromatic		1416.23–1457.59	CH2 and CH3 (rocking C–H)
	1417.65–1459.01	CH2 and CH3 (rocking C–H)		1339.21	N=O bending amines
	1339.21	N=O bending amines		1208–1285.02	CHOH stretching alcohol
	1208–1279.31	CHOH stretching alcohol		1052.54–1101.04	C–O stretching Ether, ester
	1052.54–1133.84	C–O stretching Ether, Ester		517.71–996.92	“Fingerprint region”
	668.89–995.5	“Fingerprint region”
BRCJ Mentik 1	3358.73	O–H stretching	BRCJ Mentik 2	3377.27	O–H stretching
	3009.31	C–H aliphatic		3009.31	C–H aliphatic
	2855.28–2926.59	C–H sp3 stretching		2855.28–2926.59	C–H sp3 stretching
	1712.88–1741.4	C=O stretching aromatic		1744.26	C=O stretching aromatic
	1517.49–1634.44	C=C stretching alkanes, aromatic		1516.06–1640.14	C=C stretching alkanes, aromatic
	1459.01	CH2 and CH3 (rocking C–H)		1460.44	CH2 and CH3 (rocking C–H)
	1339.21	N=O bending amines		1340.64–1372.02	N=O bending amines
	1239.38	CHOH stretching alcohol		1240.8	CHOH stretching alcohol
	1073.94–1162.36	C–O stretching ether, ester		1046.84–1166.64	C–O stretching ether, ester
	400–1000	“Fingerprint region”		400–1000	“Fingerprint region”
BRCJ Melik 2	3367.29	O–H stretching
	3009.31	C–H aliphatic
	2855.28–2926.59	C–H sp3 stretching
	1742.83	C=O stretching aromatic
	1514.64–1643	C=C stretching alkanes, aromatic
	1461.87	CH2 and CH3 (rocking C–H)
	1337.79–1376.29	N=O bending amines
	1203.72	CHOH stretching alcohol
	1042.56–1170.92	C–O stretching ether, ester
	648.93–668.89	“Fingerprint region”

The IR spectra of the Wajaloka and NTT Endogenous black rice cultivars showed the characteristics of the functional groups (Figure 4). Both cultivars had the same functional groups: O–H, C–H, C=O, alkane, aromatic, N=O, and CH–OH groups (Table 4). The aliphatic C–H group was not found in BREJ Wajaloka. The Wajaloka and NTT Endogenous black rice cultivars had identical fingerprint areas in the range of 670–998 cm⁻¹.

Table 4. Wavenumber and functional groups of the spectra of the Wajaloka and NTT black rice cultivars.

Rice	Wavenumber (cm^−1)	Functional group	Rice	Wavenumber (cm^−1)	Functional group
BREJ Wajaloka	3387.25	O–H stretching	BR NTT Endo 1	3367.29	O–H stretching
	2855.28–2926.59	C–H sp3 stretching		3009.31	C–H aliphatic
	1712.88–1741.4	C=O stretching aromatic		2855.28–2926.29	C–H sp3 stretching
	1513.21–1640.14	C=C stretching alkanes, aromatic		1712.88	C=O stretching aromatic
	1416.23–1459.01	CH2 and CH3 (rocking C–H)		1516.06–1610.19	C=C stretching alkanes, aromatic
	1339.21	N=O bending amines		1457.59	CH2 and CH3 (rocking C–H)
	1209.43	CHOH stretching alcohol		1339.21–1377.72	N=O bending amines
	1055.4–1101.04	C–O stretching ether, ester		1206.58–1282.16	CHOH stretching alcohol
	670.32–998.35	“Fingerprint region”		1069.66	C–O stretching ether, ester
				670.32–929.89	“Fingerprint region”
BR NTT Endo 2	3363.01	O–H stretching	BR NTT Endo 3	3374.42	O–H stretching
	3009.31	C–H aliphatic		3009.31	C–H aliphatic
	2855.28–2926.59	C–H sp3 stretching		2855.28–2926.59	C–H sp3 stretching
	1741.4	C=O stretching aromatic		1712.88–1741.4	C=O stretching aromatic
	1611. 06–1634.44	C=C stretching alkanes, aromatic		1517.49–1640.14	C=C stretching alkanes, aromatic
	1460.44	CH2 and CH3 (rocking C–H)		1460.44	CH2 and CH3 (rocking C–H)
	1372.02	N=O bending amines		1336.36	N=O bending amines
	1202.3–1282.16	CHOH stretching alcohol		1203.72–1283.59	CHOH stretching alcohol
	1075.36	C–O stretching ether, ester		1076.79	C–O stretching ether, ester
	670.32	“Fingerprint region”		671.75	“Fingerprint region”

Biological functions of black rice

Antioxidant activity

The biological activity of black rice was determined by the antioxidant activity (Figure 5). The antioxidant activity of ascorbic acid served as a positive reference with an IC₅₀ value of 0.61 ± 0.08 µg/mL. This was followed by BRNTT Endo 3 at 0.72 ± 0.01 µg/mL, BREJ Mentik 4 at 0.90 ± 0.58 µg/mL, BRCJ Melik 2 at 1.21 ± 0.08 µg/mL, BREJ Mentik 5 at 1.39 ± 1.21 µg/mL, and BREJ Mentik 6 at 1.78 ± 1.46 µg/m. The IC₅₀ value of BRNTT Endo 2 was significantly different from that of ascorbic acid. The samples with IC₅₀ values less than 100 g/mL were categorized as having high antioxidant activity.

Figure 5. Antioxidant activities of the 13 black rice cultivars assigned as the IC₅₀ value. The designations of the samples correspond to those in the methods section. Tukey’s HSD test was conducted in Graphpad Prism 9.3.1; significant differences are indicated by different letters (p < .05).

Alpha amylase inhibition activity as a prediction of antidiabetic functions

To predict the antidiabetic biological function of the black rice extracts, we performed alpha amylase inhibition assay, with acarbose as a positive reference (Figure 6). Acarbose had the highest activity with the lowest IC₅₀ value of 1.38 µg/mL. The highest IC₅₀ value was recorded in BREJ Wajaloka, at 98.95 ± 0.57 µg/mL, indicating that this cultivar had the lowest ability to inhibit alpha amylase activity. The IC₅₀ values of BREJ Mentik 5 and BRNTT Endo 2 were not significantly different from that of acarbose. Other black rice samples had IC₅₀ values between 14.76 and 87.73 µg/mL.

Figure 6. Alpha amylase inhibition activities of the black rice extracts. The designations of the samples correspond to those in the methods section. Tukey’s HSD test was conducted in Graphpad Prism 9.3.1; significant differences are indicated by different letters (p < .05).

The IC₅₀ values of most of the black rice samples were significantly different from that of the acarbose reference: BRCJ Mentik 2 and BRNTT Endo 1 at p < .05, BREJ Mentik 3, 4, and 6 at p < .001, and BREJ Mentik 2, BREJ Melik, BREJ Wajaloka, BRCJ Mentik 1, and BRNTT Endo 3 at p < .0001. These results indicate that most of the black rice samples have high potential as alpha amylase inhibitors and can be used as natural antidiabetic agents.

Discussion

The phytochemical investigation revealed that the 13 tested black rice cultivars from Java and East Nusa Tenggara contained phenolics, flavonoids, tannins, quinones, anthraquinones, leucoanthocyanidins, and glycosides. Alkaloids were absent in BREJ Mentik 2, Mentik 3, and Wajaloka. The phytochemical tests were carried out based on changes in color after the samples reacted with the standard reagents for identification of secondary metabolites.

The FTIR spectral results supported the phytochemical profiles. FTIR analyses were performed to identify functional groups, as each functional group shows a distinct frequency in the infrared spectrum. The FTIR spectra of all black rice cultivars were similar. The bands detected at 3400–3220 cm⁻¹, 1240–1200 cm⁻¹, and 1170–1040 cm⁻¹ indicated the existence of –OH stretching regions that may have originated from carboxylic acid, ester, or carbonyl groups (Favaro et al., 2018). The C–H aliphatic group at 3010–3000 cm⁻¹ is the C–H aliphatic group from the saturated hydrocarbon group or methylene group (C–H₂). The C=C aromatic group (1740–1710 cm⁻¹) and C=C (stretch) alkanes (1640–1500 cm⁻¹) were from anthocyanins, flavonoids, or phenolic compounds (Moko & Rahardiyan, 2020). The N=O bending amines (1370–1330 cm⁻¹) may have originated from proteins or alkaloids (Favaro et al., 2018). Finally, the IR region between 670 and 640 cm⁻¹ is referred to as the fingerprinting region and is linked to the vibrations of the C–O, C–C, C–H, and C–N bonds. This region provides significant information related to organic compounds such as alcohols, organic acids, and carbohydrates that exist in black rice.

Analysis of the phytochemical composition is important to predict the biological and pharmacological activities of the plants of interest (Favaro et al., 2018; Lin et al., 2017; Sholikhah et al., 2019). All secondary metabolites detected in the phytochemical tests of the black rice samples have many biological activities. For example, phenolic and flavonoid compounds have been suggested to have high antioxidant activity (Gumul & Berski, 2021; Safitri et al., 2020; Swallah et al., 2020). Antioxidants protect the human body against oxidative stress (Rahal et al., 2014; Shahidi & Zhong, 2015).

Quinones, oxidized derivatives of aromatic compounds, are found in nature in diverse types with different properties based on the aromatic ring and chemical structure (Chien et al., 2015). In nature, quinones have aromatic ring structures, i.e. a 1-ring structure named benzoquinone, a 2-ring structure named naphtoquinone, and a 3-ring structure named anthraquinone (Chien et al., 2015). The biological functions of quinones in living organisms include the inhibition of bacterial growth (Pan et al., 2020), cancer treatment (Lu et al., 2014), and the prevention of protein misfolding and aggregation (Gong et al., 2014), as well as the inhibition of aldose reductase (Demir et al., 2019), which can be beneficial for diabetes treatment.

Interestingly, aglycone flavonoids were detected in all black rice samples from East, West, and Central Java, as well as East Nusa Tenggara. Aglycone has a potential role in health promotion and can reduce the risk of metabolic diseases (Koziara et al., 2019). Flavonoids are absorbed in the small intestine and transported to the colon and liver for further hydrolysis, and the liberated aglycones are easily digested by bacteria in the intestine (Kumar & Pandey, 2013). Glycosides are secondary metabolites that consist of a sugar portion that is linked to a non-sugar part (Xiao, 2017), and glycosidic residues improve pharmacokinetic parameters and are of great interest due to their bioactivity. Plant glycosides demonstrated antidiabetic, anti-inflammatory, antidegranulating, antistress, and antiallergic activities in many studies (Browning et al., 2010; Godinho et al., 2021; Ji et al., 2020; Xiao et al., 2016). Leucoantocyanidin, a flavan derivative, can be converted into anthocyanidin and subsequently into anthocyanin through the action of anthocyanin synthase (ANS) and glycosyl transferase (GT) (Sari et al., 2021). The leucoanthocyanidin test was positive in all black rice samples. The alkaloids and tannins detected in the black rice extracts are commonly used as antibacterial agents (Gul et al., 2017; Saha et al., 2021)

Anthocyanins are the most important flavonoid pigments in plants. The six main anthocyanin glycoside compounds are pelargonidin, cyanidin, delphinidin, peonidin, petunidin, and malvidin. Three of these were discovered in samples in this study: peonidin-3-O-glucoside, cyanidin-3-O-glucoside, and delphinidin-3-O-glucoside. Similar to our study of black rice anthocyanins using UHPLC analysis (Sari et al., 2021), we detected peonidin-3-O-glucosides and cyanidin-3-O-glucoside in the black rice extracts from Java and Nusa Tenggara. In our previous study (Fatchiyah, Ratih, et al., 2020), those anthocyanins were also present in BREJ Mentik 1, BRCJ Melik, and BRWJ Toraja. The UV – Vis spectra of the black rice extracts provided anthocyanin profiles at two pH values and the anthocyanin concentrations in the samples. Anthocyanin peaks were identified in the visible light region (510–530 nm). In agreement with previous published studies (Ahliha et al., 2018; Saha et al., 2021; Wu et al., 2015), anthocyanin, as an unstable colorant, may undergo gradual degradation through changes in the environment, such as pH, light, and oxygen, and these changes can be followed to calculate the anthocyanin concentrations. The pH distinction method is a fast and simple spectrophotometric method based on the pH-dependent structural conversion of anthocyanin (oxonium form at pH 1.0 and hemiketal form at pH 4.5) (Favaro et al., 2018). At pH 1, anthocyanins are more stable than at higher pH values, which cause faster degradation rates (Saha et al., 2021). At pH 4.5, the anthocyanin peaks decreased, and there was a complete loss of anthocyanin peaks in some samples. The TAC value ranged from 2.13–11.45 mg/L; these values are lower than those in our previous study (Fatchiyah, Ratih, et al., 2020).

The biological functions of black rice presented here are based on antioxidant and alpha amylase inhibition assays. The FRAP assay was used to determine the antioxidant activity. The advantages of the FRAP assay are that this test is simple, rapid, affordable, and requires no special instruments. Antioxidant activity was calculated based on the reduction of the ferric ion (Fe³⁺)–ligand complex to the intensely blue-colored ferrous (Fe²⁺) complex by antioxidants in acidic media (Prastiwi et al., 2020). The results showed that even though black rice samples may have various levels of antioxidant activities, the ones used in this study had high antioxidant activities with IC₅₀ values lower than 100 μg/mL. Several black rice cultivars used in this study had antioxidant activity comparable to that of ascorbic acid (BREJ Mentik 2, 4, 5, and 6, BRCJ Mentik 1, Melik 2, and BRNTT Endo 1 and 3). The highest TAC level was measured in BREJ Mentik 2, while the highest antioxidant activity was observed in BRNTT Endo 3. These findings suggest that the phytochemical compounds responsible for the antioxidant activity of black rice were not only anthocyanins but also other phytochemicals such as phenolics, flavonoids, or quinones. This is in accordance with a previous study, which showed that phenolic and flavonoid compounds were predominantly responsible for the antioxidant activity of pigmented rice (Ghasemzadeh et al., 2018).

The final in vitro biochemical assay was the alpha amylase inhibition assay. BREJ Mentik 2 had the highest TAC content, and BREJ Mentik 5 and BRNTT Endo 2 had the highest potential as inhibitors of alpha amylase. These results support the evidence that phytochemical compounds in black rice, other than anthocyanins, contribute to the biological functions of black rice. The inhibition of the enzyme involved in hydrolyzing carbohydrates (alpha amylase) is an important approach for reducing hyperglycemia (Meidinna & Fatchiyah, 2019; Oyedemi et al., 2017). Thus, alpha amylase inhibition activity can be used to determine potential antidiabetic activity. Acarbose, which was used as a positive control, is a known competitive inhibitor of alpha amylase and is used as a drug to treat diabetes mellitus (Dinicolantonio et al., 2015; Hedrington & Davis, 2019). Investigation of the biological activity of black rice extracts, especially their ability to inhibit alpha amylase, may reveal black rice as a potential functional food for patients with diabetes mellitus.

Several in silico studies of alpha amylase inhibitory activity have been performed and have shown that the polyphenolic compounds in black rice (flavonoids and anthocyanins) are tightly bound to alpha amylase by hydrogen bonds (Jhong & Chia, 2015; Meidinna & Fatchiyah, 2019; Safitri et al., 2020). Another proposed hypothesis is that polyphenolic compounds bind covalently to alpha amylase and change its activity due to the formation of quinones or lactones that react with nucleophilic groups on the enzyme molecule (Bhutkar & Bhise, 2012; Oyedemi et al., 2017). Nevertheless, further in vitro or in vivo studies are needed to elucidate the biological functions of the phytochemical compounds of Indonesian endogenous black rice.

Conclusion

This study presents a complete phytochemical profiling of Indonesian endogenous black rice cultivars as well as their in vitro biological activities, in particular as antioxidants and inhibitors of alpha amylase. The phytochemical compounds discovered in the black rice samples include flavonoids, tannins, phenolics, quinones, anthraquinones, and leucoanthocyanidins. Anthocyanins, which contribute to the pigment of black rice, were also detected in high concentrations. The black rice samples have high potential as antioxidants and also as alpha amylase inhibitors, as their IC₅₀ values with regard to their biological activities were lower than 100 μg/mL. These findings provide an understanding of the relationship between black rice phytochemical compounds and their biological activities. This study also supports the application of Indonesian black rice as a functional food for healthy individuals and patients.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

The work was supported by the RISPRO-PRN-LPDP National Research Grant from Ministry of Research & technology BRIN and Ministry of Finance, Republic of Indonesia [4/E1/PRN.2020] and The Professor Grants by Brawijaya University.