


































Food Science and Nutrition Studies 

ISSN 2573-1661 (Print) ISSN 2573-167X (Online) 

Vol. 1, No. 1, 2017 

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43 
 

Anthocyanin, Lutein, Polyphenol Contents and Antioxidant 

Activity of Black, Red and White Pigmented Rice Varieties 

Saravanan Ponnappan1, Arun Thangavel1 & Omprakash Sahu1* 
1 Faculty of Chemical and Food Engineering, Bahir Dar Institute of Technology, Bahir Dar University, 

Ethiopia 

* Omprakash Sahu, E-mail: ops0121@gmail.com 

 

Received: April 10, 2017         Accepted: April 22, 2017       Online Published: May 16, 2017 

doi:10.22158/fsns.v1n1p43        URL: http://dx.doi.org/10.22158/fsns.v1n1p43 

 

Abstract  

Colour rice varieties are rich in antioxidants and functional based properties such as anthocyanin, 

lutein and phenolic compounds. In this experiment, two of red pigmented (TPS-1and TKM-9), one 

white (glutinous rice) and black pigmented rice varieties are cultivated from India were analysed to 

determine their antioxidants and nutrition based functional properties. Based on the result, the 

anthocyanin content was very high on black rice than other variety contents up to 244.45 mg/100 g. 

Polyphenol compound were varied significantly within the compared varieties. Highest polyphenol 

compound content (463.05 mg/100 g) was found in the black rice and also showed rich antioxidant 

properties. Obviously, black rice rich source of lutein compound was also higher than other varieties 

where under the experimental condition. DPPH (Determination of 2, 20-diphenyl-1picrylhydrazyl 

radical scavenging ability) scavenging capacity starting from 69.46% to 76.4% ranged to level of 

remain DPPH.  

Keywords 

antioxidants, anthocyanin, polyphenol compound, DPPH 

 

1. Introduction 

Rice is the essential cereal crop in developing world countries. Rice is utilizing as a staple food for one 

half of the population in developing countries (Bhattacharjee et al., 2002). But most of the developing 

countries living populations are widely eating white rice, even though they are cultivating enormous 

rice variety which have been containing various pigments such as black, red and purple color kernels. 

That kernels contains rice are rich in inevitable antioxidants such as phenolic compounds and lutein 

(Perera & Yen, 2007).  

From the report of previous study have been conducted by Lee et al. (2008), anthocyanin playing major 

role of cholesterol reduction and also inhibitory effects on in vitro allergic reaction in human body 



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(Abdel-Aal et al., 2006; Yang et al., 2008). Peonindin-3-glucoside and cyanindin-3-glucoside are all the 

major anthocyanin compounds which are present in the extracts of black rice, also these compounds are 

influencing an inhibitory effect of invasion of cells on different cancer (Chen et al., 2006). Lamberts 

and Delcour (2008) studied that carotenoids decreases the formation of cancer cells and other heart 

disease. Enormous in vitro and in vivo studies have revealed that LDL modification through the help of 

oxidation mainly plays onset of exacerbates and atherosclerosis on clinical manifestation (Siddiq, 

2004). Pigmented rice are studied as a major source of antioxidants and other vital functional properties 

especially lutein and phenolic compounds (Frei & Becker, 2005). Normally, the health benefits of 

pigmented rice varieties are very high but the production and supply are very less for the demand of 

population needs (Yawadio et al., 2007). Zeazanthin and lutein are the essential non provitamin-A 

which were present in the eye protection against both infection and disease (Rose, 1999; Tan et al., 

2005). With significant health benefits of therapeutic values in rice varieties have been known since 

from heritage scriptures and experience from the peoples (Siddiq, 2004; Johnson, 2002).  

 

2. Methods  

2.1 Sample Collection 

To study the sample experimental pigmented rice varieties (Figure 1) were selected and purchased from 

various available markets in India. The paddy of black rice (kavuni rice) was obtained from Dry Land 

Agriculture Research Station, Kanadukathan, Chettinad, India. Red rice paddy varieties TPS-1 were 

obtained from Regional Rice Research Station, Thirupathisaram and TKM-9 obtained from Rice 

Research Station, Thirurkuppam, Thiruvallur. Glutinous white rice which was sourced from Mumbai 

was purchased from Remuki Departmental stores, Madurai, India. Finally, the macerated and grounded 

ricesample flours were prepared in laboratory. Under the following methods were analysed appropriate 

compounds from different pigmented rice varieties. 

2.2 Anthocyanins 

Potassium chloride buffer (0.03 mol/l) and sodium acetate buffer were added with 20 µl rice sample 

extracts from pigmented rice varieties. Mixed well and also allow those compounds for 15 min to 

absorb measurements on spectrophotometer at 500 nm and 700 nm (Nicoue et al., 2007). Distilled 

water used for blank purpose. The concentration of anthocyanin content (mg/l) from the extracted 

samples was calculated with standard formula and also result has expressed as cyanin-3-glucoside 

equivalents.  

Anthocyanin content = (A x MW x DF / Ɛ x 1) x1000 

Where, 

A = (Aλ700) pH 1 - (Aλ700) pH 4.5; 

MW = Mol. wt. of Cyanidin-3-glucoside; 

DF = Factor of dilution; 

Ɛ= Extinction coefficient (L x cm - 1 x mol-1) = 26,900 for Cyanidin-3-glucose;  



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where L (length) = 1.  

2.3 Polyphenols Compounds 

In test tubes, the extraction of methanol was taken and also prepare the volume up to 3 ml with 

distillation water. Then, 0.5 ml Folin-ciocalteau reagent was mixed with methanol solution. About 20 

per cent sodium carbonate was added under the volume of 2 ml after three min approximately and 

mixed well again. Absorbent measurement range on spectrophotometer wavelength range at 650 nm 

against the blank using with VIS spectrophotometer. A set of standard solutions of gallic acid prepared 

using with distilled water (10 µg-100 µg per ml) was treated in the same manner as described earlier 

and read against a blank. Polyphenol compounds was expressed in mg and also it was equivalent to 

gallic acid per 100g on FWB basis.  

2.4 Lutein Content of Selected Pigmented Rice Varieties  

Chemicals: Acetonitrile, hexane, methanol, ethanol and dichloromethane were of HPLC grade 

standard.  

Carotenoid extraction: Macerated colour rice samples flour were mixed with sodium sulfate (5 g) and 2 

mm-Rtocopherol in methanol solution. Ice-cold acetone was used to prepared colorless carotenoids 

content extracted solution (volume: 400 ml). Prepared crude extract was mixed and shaken with 100ml 

hexane. Three or four times the extraction was repeated finally make the known volume up to 250 ml. 

The mixed crude hexane was dried with anhydrous sodium sulfate (20 g) and filtered through filter 

paper (whatman No.1). An aliquot filtered known extracted volume (100 ml) solution was dried under 

the nitrogen stream and the remaining residue of the hexane solution was redissolved in 1 ml 

acetonitrile chemical. Finally, the prepared samples were analysed by HPLC.  

HPLC Analysis: SGE C-18 (ODS) column was used to separate carotenoids through HPLC instrument. 

About 0.1 per cent ammonium acetate with methanol was used as mobile phase for the carotenoids 

separation. 20 µl measured samples were injected in ODS column on HPLC equipment and also 

isocratic condition was continued at 1 ml/min flow rate. λmax values of the compound were 

determined by the time of retention and standard chromatograms assessed through the help of SPD-10 

AVD detector. In Column result where the quantified peak area were related to the standard reference. 

2.5 Anti-Oxidant Activity  

2.5.1 Diphenyl-Picryl-Hydrazyl (DPPH) Assay  

DPPH is a purple coloured stable free radical and will form yellow colour when it was reduced as 

Diphenyl-Picryl-Hydrazine complex. The sample extracts electron donation ability was calculated from 

purple colour beaching of DPPH methanol solution. Scavenging antioxidant potential indicates by 

discoloration. The DPPH assay was performed by Goupy et al. (1999). 2 ml of 60 µM 2, 2-diphenyl-1- 

picryl-hydrazyl in methanol (initial absorbance of DPPH was 0.62 ± 0.02.) was added to one ml of 

various concentrations of sample extract. Prepared 1 min vortexed mixture and kept in room 

temperature under the dark condition for 30 minutes. Decreased absorbance was measured in 

spectrophotometer under the wavelength of 517 nm with used methanol as a blank portion. Ascorbic 



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acid was applied as positive control point. Calibration curve was diagrammed indicated for using 

absorbance versus concentration of ascorbic acid and the results were noted that as a mg (vit-C) 

equivalent per 100 g of sample on FWB basis.  

 

 

Figure 1. Selected Pigmented Rice Varieties for Study 

 

3. Results  

Carotenoids are essential chemical compounds of coloured rice varieties. Selected rice flour samples 

were very low levels of β-carotene content were eluted by HPLC. But the coloured rice varieties were 

good source of lutein shown in Figure 2. The lutein content found to be maximum in TPS-1 (1075 

ng/100 g) followed by black rice (280 ng/100 g), glutinous white rice (240 ng/100 g) and TKM-9 (215 

ng/100 g) respectively. The different significant ratio (P < 0.05) was noticed in terms of lutein content 

between the pigmented rice varieties and white rice. The data indicates the presence of maximum level 

of anthocyanin mainly in black rice (244.45 mg/100 g) while the other pigmented rice varieties such as 

TPS-1 and TKM-9 had anthocyanin levels of 1.39 and 1.25 mg/100 g respectively, with lowest levels 

recorded in glutinous white rice (0.32 mg/100 g). 

 

Table 1. Lutein, Anthocyanin, Polyphenols Content and Antioxidant Activity of Selected 

Pigmented Rice Varieties 

Varieties  Lutein 

(ng/100 g)  

Anthocyanin 

(mg/100 g)  

Polyphenols 

(mg/100 g)  

Antioxidant activity  

DPPH (%)  

Glutinous white Rice  240 ± 2.08  0.32 ± 0.01  88.21 ± 0.04  69.46 ± 0.05  

Black rice 280 ± 1.52  244.45 ± 0.02  463.05 ± 0.07  86.12 ± 0.05  



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TPS-1  1075 ± 4.50  1.39 ± 0.01  340.13 ± 0.08  84.42 ± 0.05  

TKM-9  215 ± 1.52  1.25 ± 0.01  208.51 ± 0.06  79.79 ± 0.03  

SEd  1.2247  0.0053  0.0156  0.0091  

CD (0.05)  2.9970**  0.0129**  0.0381**  0.0223**  

 

The polyphenol content of pigmented rice varieties was noticed to be minimum in glutinous white rice 

(88.21 mg/100 g) compared to higher levels recorded in black rice (463.05 mg/100 g), followed by red 

rice varieties TPS-1 (340.13 mg/100 g) and TKM-9 (208.51 mg/100 g).  

The data pertaining to antioxidant activity of the pigmented rice varieties is presented in Table.1. The 

stable DPPH radical is continuously used to test hydrogen donating antioxidants in enormous plant 

species. It can be inferred, that antioxidant activity of selected pigmented rice varieties was maximum 

in black rice at 15.81 per cent in terms of DPPH value followed by TPS-1 (13.42%), TKM-9 (12.68%) 

and lowest in glutinous rice (10.48%). Significant difference in terms of total antioxidant activity was 

noted between the selected varieties.  

 

 

Figure 2. Lutein Content of Selected Pigmented Rice Varieties 

 

4. Discussion 

Anthocyanin, lutein and polyphenol compounds were essential antioxidants which were majorly 

present in pigmented rice varieties. The data results shows presence the level of anthocyanin, 

polyphenol and other vital antioxidant activities were higher in black rice variety. But the lutein content 



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was maximum in TPS-1 red rice variety. Sompong et al. (2011) reported that cyanidin 3-glucoside and 

peonoidin 3-glucoside as the dominant anthocyanins in black rice varieties with contents ranging from 

19.4 to 140.8 mg/100 g DM and 11.1-12.8 mg/100 g DM respectively. Red rice varieties to have 

polyphenol compounds in the range of 79.2 and 691.4 mg FA equivalent/100 g with a mean TPC of 

364.8 mg FA equivalent/100 g. The black rice had a higher mean polyphenol compounds of 492.8 mg 

FA equivalent/100 g than the red ones. Also he was evaluated the DPPH value of three black rice 

varieties to range from 16.04 to 30.25 per cent and that of 10 red rice varieties to range from 12.99 to 

76.38 per cent. No significant difference between the black and red rice varieties were reported. 

In Conclusion, this experimental study has shown that the pigmented rice varieties were good source of 

antioxidants and other vital functional properties such as lutein, anthocyanin, and polyphenol 

compounds. The lutein content found to be maximum in TPS-1 (1075 ng/100 g) followed by black rice 

(280 ng/100 g), glutinous white rice (240 ng/100 g) and TKM-9 (215 ng/100 g) respectively. The 

different significant ratio (P < 0.05) was noticed in terms of lutein content between the pigmented rice 

varieties and white rice. Maximum level of anthocyanin mainly in black rice (244.45 mg/100 g) while 

the other pigmented rice varieties such as TPS-1 and TKM-9 had anthocyanin levels of 1.39 and 1.25 

mg/100 g respectively, with lowest levels recorded in glutinous white rice (0.32 mg/100 g). The 

polyphenol content of pigmented rice varieties was noticed to be minimum in glutinous white rice 

(88.21 mg/100 g) compared to higher levels recorded in black rice (463.05 mg/100 g), followed by red 

rice varieties TPS-1 (340.13 mg/100 g) and TKM-9 (208.51 mg/100 g). Antioxidant activity of selected 

pigmented rice varieties was maximum in black rice at 15.81 per cent in terms of DPPH value followed 

by TPS-1 (13.42%), TKM-9 (12.68%) and lowest in glutinous rice (10.48%).  

 

References 

Abdel-Aal, E. S. M., Young, J. C., & Rabalski, I. (2006). Anthocyanin composition in black, blue, pink, 

purple, and red cereal grains. Journal of Agricultural and Food Chemistry, 54, 4696-4704. 

Bhattacharjee, P., Singhal, R. S., & Kulkarni, P. R. (2002). Basmati rice: A review. International 

Journal of Food Science and Technology, 37(1), 1-12.  

Chen, P. N., Kuo, W. H., Chiang, C. L., Chiou, H. L., Hsieh, Y. S., & Chu, S. C. (2006). Black rice 

anthocyanins inhibit cancer cells invasion via repressions of MMPs and u-PA expression. 

Chemico-Biological Interactions, 163(3), 218-229.  

Frei, M., & Becker, K. (2005). Fatty acids and all-trans-beta-carotene are correlated in differently 

colored rice landraces. Journal of Science in Food and Agriculture, 85, 2380-2384. 

Johnson, J. L., Carson, K. G., & Jackson, C. L. (2002). Characteristics of intact and ruptured 

atherosclerotic plaques in brachiocephalic arteries of apolipoprotein-E knockout mice. 

Arteriosclerosis Thrombosis Vascular Biology, 22, 788-792. 

Lamberts, L., & Delcour, J. A. (2008). Carotenoids in raw and parboiled brown and milled rice. Journal 

of Agricultural Food Chemistry, 56(24), 11914-11919. 



www.scholink.org/ojs/index.php/fsns                Food Science and Nutrition Studies                     Vol. 1, No. 1, 2017 

49 
Published by SCHOLINK INC. 

Lee, J. C., Kim, J. D., Hsieh, F. H., & Eun, J.-B. (2008). Production of black rice cake using ground 

black rice and medium-grain brown rice. International Journal of Food Science and Technology, 

43(6), 1078-1082.  

Nicoue, E. E., Sylvanin, S., & Khaled, B. (2007). Anthocyanin in wild blueberries of Quebec: 

Extraction and Identification. Journal of Agriculture and Food Chemistry, 55(15), 5626-5635. 

Perera, C. O., & Yen, G. M. (2007). Functional properties of carotenoids in human health. International 

Journal of Food Properties, 10, 201-230. 

Rose, R. (1999). Atherosclerosis: An inflammatory disease. The New England Journal of Medicine, 340, 

115-126. 

Siddiq, E. A. (2004). Aromatic and medicinal rices; urgency for collection, validation and conservation 

for sustainable utilization towards value added rice farming. Compendium of papers. 

Science-Society Interface on Medicinal and Aromatic Rices. August organized by 

M.S.Swamination Research Foundation, Chennai and Kerala Agricultural University. 

Sompong, R., Siebenh, l-Ehn, S., Linsberger-Martin, G., & Berghofer, E. (2011). Physicochemical and 

antioxidative properties of red and black rice varieties from Thailand, China and Sri Lanka. Food 

Chemistry, 124, 132-140.  

Tan, J., Baisakh, N., Oliva, N., Parkhi, V., Rai, M., Torrizo, L., … Datta, S. K. (2005). The screening of 

rice germplasm, including those transgenic rice lines which accumulate beta-carotene in their 

polished seeds, for their carotenoid profile. International Journal of Food Science and Technology, 

40, 563-569. 

Yang, D. S., Lee, K. S., Jeong, O. Y., Kim, K. J., & Kays, S. J. (2008). Characterization of volatile 

aroma compounds in cooked black rice. Journal of Agricultural and Food Chemistry, 56, 235-240. 

Yawadio, R., Tanimori, S., & Morita, N. (2007). Identification of phenolic compounds isolated from 

pigmented rices and their aldose reductase inhibitory activities. Food Chemistry, 101(4), 

1616-1625.  

 

 


