Nutritional Composition, Mineral Content and Antioxidant Properties of Unpolished Red Rice Cultivars of Assam, India

Tiluttama Mudoi1,*, Priyanka Das1
1Department of Biochemistry and Agricultural Chemistry, Assam Agricultural University, Jorhat-785 013, Assam, India.
Background: Assam is a rich source of different types of colored rice cultivars. Very few colored rice cultivars are studied regarding nutritional and phytochemical properties. The objective of present study was to assess the nutritional composition and antioxidant properties of traditional red rice cultivars of Assam.

Methods: Ten different red rice cultivars were collected for the study. The unpolished rice grains were analysed for proximate composition, mineral content, total phenolic, flavonoid content and DPPH free radical scavenging activity.

Result: Amylose content was found to be low in the red rice cultivars. The carbohydrate and crude protein content varied significantly among the red rice cultivars. The studied cultivars were found to be good source of Fe, Mn and Zn. Highest total phenolic (1357.22 mg /100 g) and flavonoid content (896.37 mg/100 g) were found in the Hal aus, whereas the lowest value for total phenolic (336.49 mg/100 g) and flavonoid content (127.51 mg/100 g) were observed in Basanta bahar. Antioxidant capacity of colored rice varieties ranged from 33.33 to 84.24% for DPPH radical scavenging activity. The red rice cultivars contain a significant amount of nutrients and antioxidants including phenolic and flavonoids.
Rice (Oryza sativa L.) is the staple food of Assam, North-eastern state of India. Rice plays a pivotal role in the socio-cultural life of people of Assam and also state economy. Although widely consumed as white rice, there are many special cultivars of colored rice that is characterized by its grain with red, black or dark purple color covering in different layers of the pericarp, seedcoat and aleurone. These cultivars are grown in upland, lowland and deep-water conditions of Assam (Chaudhary, 2003). Anthocynain compounds, a subclass of flavonoids are responsible for the color of grain (Abdel-Aal et al. (2006), Yawadio et al., 2007). Colored rice contains higher amounts of proteins, fiber, vitamins and other micro nutrients like iron, zinc (Itani et al., 2002, Suzuki et al., 2004). 
Phenolic compounds play an important role in decreasing the oxidative stress by scavenging free radicals (Ti et al., 2014). The phenolic compounds in rice are found in the soluble and insoluble (bound) form, with the soluble form representing 38% to 60% of the total polyphenols content in light brown rice grains and around 81% in red and black pericarp color grains (Mira et al., 2009). The type and concentration of polyphenols in the rice grain vary according to different genotypes and to the pericarp color. Colored rice varieties contains higher amount of total phenolics, flavonoids and antioxidant activity than those of light colored varieties, such as white varieties (Walter et al., 2013 and Shao et al., 2014).
Very few studies were reported on the colored rice cultivars of Assam, India (Saikia et al., 2012, (Mudoi and Das, 2018), (Mudoi and Das,2019). In this regard, different cultivars of red rice of Assam were investigated to determine the nutritional and antioxidant properties.
Collection of samples
Ten different red pigmented rice cultivars were collected from Regional Agricultural Research station, Karimganj, Assam in the year 2015. Rata Boro, Koujapuri, Makhan Boro, Kaya Boro, Kolaboro are grouped as spring or summer rice and Basantabahar, Dadratai, Binnapali, Buriamakra, Hal aus are grouped as autumn rice.
Processing of rice grains
Rice grains were de-husked using a de-husker (Satake Corporation, Hiroshima, Japan). The unpolished rice grains were ground to flour and used for further analysis.
Amylose content
The amylose content was estimated according to Sowbhagya and Bhattacharya, 1979.
Proximate composition analysis
Moisture content, carbohydrate content, crude protein, crude fat and ash content were estimated as per AOAC method, 1995 on dry weight basis.
Calorific value
Calorific value was estimated according to Osborn and Voogt, 1978 using following equation.
Calorific value (kCal/100g) = (CP × 4) + (F × 9) + (CHO × 4)
CP means crude protein (%); F means fat (%) and CHO means carbohydrate content (%).
Mineral content
The mineral contents of rice samples were determined following the method of AOAC (1995). The ash obtained as per method was dissolved in diluted HCl (1:1), kept on a water bath at 100oC and the mixture was evaporated to dryness. 4 ml of HCl and 2 ml of glass distilled water were added, warmed and the acid soluble portion obtained after filtration was made up to 100 ml with glass distilled water. This solution was used for estimation of Fe, Zn and Mn by atomic absorption spectrometer.
Extraction of rice samples for total phenols, total flavonoid content and antioxidant activity
The rice flour (1.5 g) was extracted (1:20 w/v) at room temperature with 85% aqueous methanol for 30 min using a magnetic stirrer. The mixtures were centrifuged at 2500 g for 10 min and the supernatants were collected. The residues were re-extracted twice under the same conditions, resulting finally in 50 ml crude extract.
Determination of total phenolic content (TPC)
The TPC of extracts was determined using the Folin–Ciocalteu reagent (Singleton et al. 1999). Extract (120 µl) was added to 600 µl of freshly diluted (10-fold) Folin–Ciocalteu reagent. 7.5% Sodium carbonate solution (980 µl) was added to the mixture after 2 min reaction time. The absorbance of the resulting blue colour was measured at 760 nm against a blank after 5 min of reaction time at 50oC. TPC was expressed as mg catechol equivalents per 100 g dry sample.
Determination of total flavonoid content (TFC)
The total flavonoid content was measured by colorimetric method as described previously (Wu and Ng, 2008). Briefly, 0.5 ml of sample extract was mixed with 2 ml of distilled water, 0.15 ml of 5% sodium nitrite and 0.15 ml of 10% aluminium chloride, followed by reaction time of 6 min. Then, 4% NaOH (2 ml) was added to the mixture. After 15 min of incubation at room temperature, the absorbance of the mixture was measured at 510 nm. All values were expressed as mg quercetin equivalents per 100 g dry sample.
Determination of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity
The DPPH free radical scavenging activity was determined following the procedure of Brand-williams et al. (1995). An aliquot of 0.3 ml of diluted methanolic extract (2 times) was vigorously mixed with 1.5 ml of freshly prepared 0.004% DPPH in methanol and held in the dark for 30 min at room temperature. The absorbance was then read at 517 nm against blank (only methanol). An equal mixture of methanol and 0.004% DPPH in methanol was used as control. DPPH free radical scavenging ability was calculated by using the following formula:   

Statistical analysis
All measurements were carried out in triplicate for each of the sample. All statistical analyses were carried out for the analysis of variance (ANOVA). Significance of the differences was ascribed at the 0.05 level for ANOVA.
Amylose content
The amylose content (Table 1) in unpolished red rice cultivars of Assam varied significantly from 6.31-11.93%. According to rice classification system of International Rice Research Institute (IRRI, 2009), most of the studied varieties were categorized into very low (2-9%) and low amylose (10-20%) content. Similar results were also reported by Reddy et al., 2016. The cooking quality of rice is dependent on amylose content (Yadav et al., 2007). Cooked rice with low amylose content is soft and sticky.

Table1: Biochemical composition of red rice cultivars.

Proximate composition
Fat content
Crude fat contents were found between 2.59-3.09% (Table 1). Fat content in this study were of comparatively somewhat similar and in the range values reported by Sompong et al., 2011, Gunaratne et al., 2013, Kariyawasama et al., 2016, Reddy et al., 2017. Fat content influences the taste of cooked rice as rice with high fat content tends to be tastier (Hirokadzu et al., 1979).
Protein content
Crude protein content varied significantly (3.96-13.23%) among the different red rice cultivars whereas Dadratai exhibited the highest protein content. Protein content influences the nutritional quality of rice (Sompong et al., 2011). Rice having more than 10% total crude protein is considered to be of high protein type (Resurrection et al., 1979). The protein content of cultivars Rata boro, Kaya boro, Basanta bahar, Dadratai was appreciably high (>10%). Protein content in this study was comparable to the values found by Kariyawasama et al., 2016, Reddy et al., 2017, but higher than the reports of Samyor et al. (2010).   
Ash content
Ash value ranged from 1.46-1.87% in the cultivars (Table 1). The results were similar to those reported values of Sompong et al., 2011, Kariyawasama et al., 2016, Reddy et  al. 2017 and higher than the values reported by Samyor et  al. 2015. Ash content gives an idea about the mineral contents of a food sample (Mbatchou and Dawda, 2013) and also high percentage of ash content may affect the sensory quality of the rice (Julliano, 1985).
Carbohydrate content
The carbohydrate content of all the cultivars ranged significantly from 82.33-91.38% (Table 1). All the rice cultivars exhibited higher amount of carbohydrate than the reports of Reddy et al., 2017, Sompong et al., 2011. However, the carbohydrate content of all the rice cultivars was more than 80% and thus all of them are considered as good source of carbohydrates.
Calorific value
Calorific value measures the available amount of energy obtained from food via cellular respiration (Thomas et al., 2013). Calorific values of rice cultivars obtained in the study found higher than the values of calorie contents reported by Kariyawasama et al., 2016. All the studied cultivars showed high energy values. Therefore, consumption of these nutrient rich rice cultivars may play a vital role in decreasing nutritional deficiencies.
Mineral compositions
Fe content
Red rice cultivars had Fe content in the range of 0.72-3.37 mg/100 g (Table 2). The Fe content of red rice cultivars was significantly different among all the selected rice varieties. Binnapali had the highest iron content. Fe content in all red rice cultivars is higher than the reported values of Yodmanee et al., 2011. Grain color is related to iron content and Iron content tends to be higher in colored (red and black) rice varieties than in white rice varieties (Meng et al., 2005).

Table 2: Mineral content of red rice cultivars.

Zn content
Zn content varied significantly among the selected red rice cultivars. Koujapuri had the highest Zn content of 5.85 mg/100 g and the lowest Zn content was found in Hal aus (1.84 mg/100 g). Similar range of values were also reported by Anuradha et al., 2012, Reddy et al., 2017. Zn have been found to be involved in free radicals scavenging enzyme systems in rice, as components of the superoxide dismutase (Dehury et al., 2013).
Mn content
Mn content ranged from 0.64-2.52 mg/100 g and varied significantly among the red rice cultivars. Rata boro contained the highest Mn content. Mn content of present study was comparable to the reported values of Reddy et al., 2017, Mudoi and Das, 2019.
TPC content
TPC of red rice cultivars is presented in Table 3. Significant differences in TPC were observed within the red rice cultivars. Hal aus (1357.22 mg/100g) showed the highest TPC, whereas the lowest value was noted for Basanta bahar (336.49 mg/100 g). TPC values of selected red rice cultivars were higher than the reported values of Yodmanee et al., 2011, Gunaratne et al., 2013, Samyor et al., 2015.

Table 3: Total polyphenol, flavonoid content and antioxidant activity of red rice cultivars.

TFC content
TFC in red rice cultivars ranged from 127.51 to 896.37 mg/100 g (Table 3). The highest TFC was noted in Hal aus while the lowest TFC was observed for Basanta bahar. All the cultivars exhibited higher levels of TFC than those of previous studies of red rice varieties reported by Huang and Ng, 2012 and Reddy et al., 2017.
Antioxidant properties
The antioxidant activities of red rice cultivars were analyzed by DPPH assay.  All the red rice cultivars showed significant scavenging activity towards the free radical. Dadratai exhibited the highest activity (84.24%), while the lowest was observed for Basanta bahar (33.33%). Higher antioxidant properties are due to higher phenolic compounds in the Rice bran (Zhang et al., 2010). Several authors reported that red rice cultivars have higher free radical scavenging activity than black- and white-hulled rice cultivars (Finocchiaro et al., 2010, Walter et al., 2013).
The red rice cultivars in the present study contain a significant amount of antioxidants including phenolic and flavonoids. Therefore, the present results suggest that red rice cultivars may be utilized as an effective antioxidant source due to its radical scavenging activities and phenolic compounds. The studied red rice cultivars are good source of Fe, Zn and Mn. The traditional red rice cutivars containing high proximate composition can be used for developing functional foods and also may be used as important genetic sources for breeding the excellent varieties for high quality of rice production.
The first author is grateful to Department of Biotechnology, Govt of India for offering DBT Research Associateship.

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