Unraveling the nutritional and therapeutic properties of ‘Kavuni’ a traditional rice variety of...

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Journal of Plant Biochemistry andBiotechnology ISSN 0971-7811 J. Plant Biochem. Biotechnol.DOI 10.1007/s13562-014-0274-6

Unraveling the nutritional and therapeuticproperties of ‘Kavuni’ a traditional ricevariety of Tamil Nadu

R. Valarmathi, M. Raveendran, S. Robin& N. Senthil

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ORIGINAL ARTICLE

Unraveling the nutritional and therapeutic propertiesof ‘Kavuni’ a traditional rice variety of Tamil Nadu

R. Valarmathi & M. Raveendran & S. Robin & N. Senthil

Received: 22 March 2013 /Accepted: 16 December 2013# Society for Plant Biochemistry and Biotechnology 2014

Abstract ‘Kavuni’ a traditional brownish black rice variety isknown for its anti-diabetic properties and is grown under lim-ited areas of Tamil Nadu, India. The present study was aimed atprofiling the nutritional and therapeutic values of Kavuni grainsin comparison with three other popularly eaten white ricevarieties of Tamil Nadu viz., CO 50, IR64 and White Ponni.Biochemical analysis revealed the significant differences in thenutritional composition of Kavuni grains when compared toother three white rice genotypes viz., 29–35 % reduced level oftotal soluble sugars, low fat content (8–35 %), 21–52 % in-creased dietary fibre and 7–24% increased total protein content.Kavuni grains had significantly higher amount of iron (20–30 %), calcium (33–45 %), copper (9.5–14.7 %), sodium(21–38 %), potassium (7–15 %) and magnesium (8.9–26 %).GC-MS analysis showed that phenolic acids and flavonoidswere predominant in the Kavuni grains whereas sugars andfatty acids were predominant in the grains of a white rice varietyCO 50. Further, Kavuni was found to possess significantlyhigher levels of different carotenoids (β-carotene—46.2 μg/100 g; lutein—221.6 μg/100 g), total phenolics (28.8 μg/100 g) and higher anti-oxidant activity. Methanol and ethylacetate extracts of Kavuni grains were found to exhibit signif-icantly higher level of inhibitory activity against α-amylase andα-glucosidase than the extracts of CO 50 (α-amylase IC50—0.1, 1.0 μg/ml and α-glucosidase IC50 0.04, 0.05 μg/ml).

Keywords Rice . Kavuni . Biochemical analysis .

Anti-diabetic and therapeutic properties

AbbreviationsNIST National Institute of Standards and Technology,MD,

USADPPH 2,2 diphenyl-1 picrylhydrazylBHA Butylated hydroxy anisoleNBT Nitroblue tetrazoliumPMS Phenazine methosulfateFTC Ferric thiocyanate

Introduction

Rice is a major staple food for half of the world’s population,providing 50 to 80 % of daily calorie intake (Khush 2005).Carbohydrates, proteins, fatty acids and micronutrients (vita-mins and trace minerals) are the major nutrient components inrice. Traditional rice genetic resources in India are reported toharbor vast amount of genetic diversity including medicinalproperties and aroma (Das and Oudhia 2003). These tradition-al varieties are potent sources for various agronomic traits aswell as sources of many bioactive non nutrient components(Crozier et al. 2009). Due to introduction of high yielding,semi-dwarf rice varieties during first green revolution, thetraditional varieties with desirable traits were ignored andare now rare in cultivation (Rekha et al. 2011). Popularlyeaten white rice contains very low level of essentialmicronutrients, leading to the development of micronutrientdeficiency upon continuous consumption which warrants thesituation of developing micro-nutrient and vitamin rich ricethrough genetic engineering (Ye et al. 2000). Consumption ofincreased quantity of polished rice grains (high GI food) hasalso been shown to contribute for higher insulin resistance anddyslipidemia (low-high density lipoprotein) among the Asianpopulation.

Traditional coloured rice varieties are known to be rich indietary fibre, resistant starch, minerals, carotenoids, flavanoids

R. Valarmathi :M. Raveendran (*) :N. SenthilCentre for Plant Molecular Biology and Biotechnology, Tamil NaduAgricultural University, Coimbatore, Tamil Nadu, Indiae-mail: [email protected]

S. RobinCentre for Plant Breeding and Genetics, Tamil Nadu AgriculturalUniversity, Coimbatore, Tamil Nadu, India

J. Plant Biochem. Biotechnol.DOI 10.1007/s13562-014-0274-6

Author's personal copy

and polyphenols and consumption of grains of thesepigmented rice varieties help in improving human health(Hanhineva et al. 2010; Jae et al. 2010; Hudson et al. 2000;Rao et al. 2010). The bioactive phytochemicals and micro-nutrient components from these traditional rice varieties asdietary supplements are expected to play a major role inattenuating the incidence of non-communicable diseasesviz., cardiovascular diseases, diabetes, cancer and stroke(Vichapong et al. 2010). With the majority of world’s popu-lation consuming rice as staple food, developing therapeuticrice varieties with enhanced nutritive value will have greaterimpact on preventing the above life style disorders.

Preliminary studies conducted by our group have resultedin the identification of a traditional dark brownish black ricevariety “Kavuni” which is known for its anti-diabetic proper-ties. Kavuni is known as a long duration, poor tillering andphotosensitive traditional rice variety and owing to these traitsit is not being cultivated widely. Morphologically, dehuskedgrains of Kavuni is dark brown to black in colour and thepolished grains are light brown (Fig. 1). No systematic scien-tific studies have been conducted to unravel the nutritional andtherapeutic properties of Kavuni. The present study reports thenutritional and therapeutic values of “Kavuni” in comparisonwith three major high yielding white rice varieties of TamilNadu viz., IR 64, CO 50 and White Ponni.

Materials and methods

Genetic material

Four rice genotypes namely, a brownish black therapeutic ricevariety “Kavuni” and three major high yielding white ricevarieties of Tamil Nadu viz., IR 64, CO 50 and White Ponniwere used in this study (Table 1). Nucleus seeds of allthe four rice genotypes were obtained and raised duringKharif’ 2010 (June to December) at Department ofRice, Centre for Plant Breeding and Genetics, TamilNadu Agricultural University, Coimbatore, Tamil Nadu,India. The well dried seeds with optimum moisturecontent (approx.13 %) were dehusked manually, powderedand stored at 4 °C until analysis.

Estimation of total soluble sugars and amylose content

Grains of all four rice genotypes were ground using a pestle andmortar till we get a fine powder. Total soluble sugars in thepowdered rice samples were estimated using phenol sulphuricacid method and starch content was estimated using anthronemethod by using glucose as the standard (Sigma, USA).The flour was defatted using 85 % methanol for 18 h,dried to equilibrate moisture content (12–15 %) andthen used for amylose estimation. Amylose fromdefatted rice flour was estimated as described bySowbhagya and Bhattacharya (1979) using potato amy-lose (Sigma, USA) as a standard.

Proximate analysis

The total nitrogen content in the grains of all the four ricegenotypes was estimated using the micro-Kjeldahl method(AOAC 2000) and the crude protein content was determinedas Total N × 5.95 (AOAC 2000). Crude lipid content wasdetermined gravimetrically after Soxhlet extraction with pe-troleum ether (60 grades) and subsequently evaporated todryness on a steam bath (AOAC 1990). Dietary fibre and totalash contents were estimated using the method described byEnglyst and Hudson (1996).

Mineral analysis

Mineral composition (Fe, Zn, Ca, Mg, Na, K, Cu etc.,) in thegrains of all four rice genotypes was determined using AtomicAbsorption Spectrophotometry (Perkin-Elmer, Analysist A700) after digestion with concentrated nitric acid (AOAC2000).

Scanning electron microscopy (SEM)

The ultrastructure of macromolecule arrangement in the grainsof Kavuni and a white rice genotype CO 50 was analyzedusing a Scanning Electron Microscope (FEI Quanta 250, Iconanalytical, FEI, USA). Dehusked grains of Kavuni and CO 50were broken along the natural fracture planes and mounted onround aluminum stubs with the aid of double side adhesive

Fig. 1 Morphological features of panicle and grains of Kavuni (a panicle; b unpolished grain; c polished grains)

J. Plant Biochem. Biotechnol.


tape. The samples were scanned and selected regions werephotographed.

Metabolite extraction and GC/MS analysis

Metabolite composition in the grains of Kavuni and a white ricevariety CO 50was studied by usingGC/MS. Finely ground riceflour (30 mg) was extracted with methanol-water and then withchloroform (Fiehn et al. 2000). Ribitol (50 μl, 0.2 mg/ml inwater) and nonadecanoic acid (50 μl, 2 mg/ml in chloroform)were added as internal standards and filtered using 0.22 μmPVDF syringe filter (Millipore, Ireland). The extracts wereconcentrated using a Speed Vac., and then derivatized byadding 80 μl of MSTFA. About 1 μl of the samples wereinjected into GC injection port (AI3000 II, Thermo FischerScientific, USA) connected to a GC/MS (TRACE™ GC Ultrawith DSQII Quadrupole mass spectrometer and capillary col-umn of 30 cm in length, 0.25 mm diameter and 0.25 μm filmthickness-Agilent, DB-5 ms Ultra Inert, 122-5532). The injec-tion port temperature was maintained at 250 °C, the oventemperature was programmed at 70 °C (5 min hold) and in-creased at the rate of 5 °C/min to 300 °C. Helium was used as acarrier gas at a flow rate of 1 ml/s. The electron ionizationsource temperature was maintained at 250 °C and the abun-dances of ions in the range of 50–600m/zwere scanned at a rateof 1.1 scans/s. Data obtained through GC/MS analysis wasanalyzed using X-Caliber software package.

Chromatographic data processing

From the GC/MS output, deconvolution of peaks, extraction ofthe baseline corrected mass spectra and identification of theretention time of every component was carried out usingAMDIS (Automated Mass Spectral Deconvolution and Identi-fication System Program). The major mass spectral fragmentsof each peak/component across four replicates were manuallyverified for their consistency using retention time as a reference.The MSTs (Mass Spectral Tags) in each of the four replicatewere compared with the five best matches in the NIST andGolms Metabolome Database (http://csbdb.mpimp-olm.mpg.de/csbdb/gmd/gmd.html) (Kopka et al. 2005) and the bestspectral match was selected and metabolite name was assigned.The low probability matches were designated as unidentified.

Estimation of β-carotene and lutein content

Estimation of β-carotene and lutein contents in the grains ofKavuni and other three white rice genotypes was carried outaccording to Howe and Tanumihardjo (2006). Carotenoids werereleased from 0.6 g of rice grain samples by adding 3 mL ofethanol containing 0.1 % ascorbic acid (w/v) and incubated at85 °C for 5 min. Carotenoids were extracted twice with hexane(1.5mL) andwere dried using a rotoevaporator and redissolved in50:50 (v/v) dichloromethane/methanol (HPLC grade, Fisher Sci-entific, USA). The extracts were filtered through a 0.22 μmPTFE

Table 1 Details on agronomic characters of the rice genotypes used in the present study

Genotype Origin Days tomaturity (days)

Pedigree Agronomic features

Kavuni Traditional variety, Tamil Nadu 135–140 Not known Low yielding, poor tillering, long durationphotosensitive, brownish black kernel

CO 50 TNAU, Coimbatore, India 130–135 CO 43 × ADT 48 High yielding, tolerant to BLB, photo insensitiveand white kernel

IR64 IRRI, Philippines 90–120 IR 5657 × IR 2061 High yielding, fine quality, medium duration photoinsensitive and white kernel

ImprovedWhite Ponni

TNAU, Coimbatore, India 135–140 Taichung 65/2 × MayangEbos-80

High yielding, fine grain, good cooking quality,long duration, photo insensitive and white kernel

Table 2 Total soluble sugars andproximate contents in the grainsof Kavuni and three white ricevarieties of Tamil Nadu

Values are mean ± standard errorof five determinations, values aresignificantly different at p<0.05from CO 50a , IR64b and WhitePonnic

Components (g/100 g) Genotypes

Kavuni CO 50 IR64 White ponni

Total soluble sugars 56±0.002a,b,c 79±0.001 87±0.002 86±0.001

Total starch 38.7±0.004a,b,c 49.2±0.04 55.0±0.06 52.4±0.26

Total amylose in % 24.2±0.002 22.0±0.001 22.2±0.002 24.6±0.003

Crude protein (N × 5.95) 9.37±0.06a,b 7.08±0.01 7.74±0.02 8.7±0.01

Ash 0.54±0.3a,b,c 1.85±0.1 1.45±0.2 1.22±0.1

Crude lipid 1.55±0.03a,b,c 2.13±0.06 1.69±0.02 2.42±0.09

Total dietary fiber 8.56±0.4a,b,c 4.12±0.1 5.63±0.3 6.76±0.2



http://csbdb.mpimp-olm.mpg.de/csbdb/gmd/gmd.html

http://csbdb.mpimp-olm.mpg.de/csbdb/gmd/gmd.html

syringe filter (Millipore, Ireland) and used for HPLC analysis. Theanalysis was performed in a Shimadzu HPLC system with LC8Apump, PhenomenexLunaC30 column and aDAD (190–800 nm)UV detector. Separation of β-carotene and lutein was carried outusing a gradient elution with acetonitrile (60 %), equal volume ofmethanol and ethyl acetate (40 %) and detected at 450 nm.Authentic carotenoids were used as standards and individualcarotenoids were expressed as micrograms (μg) per 100 g freshweight.

Estimation of total phenolics

The total phenolic content in grains of all four rice genotypeswas determined using Folin-Ciocalteu method (Singleton et al.1999). The crude extracts obtained from 500 mg rice flourwere dissolved in 5 ml of distilled water and were oxidizedwith Folin-Ciocalteu reagent. The absorbance was measured at760 nm after 60min using Carry Varien-50 spectrophotometer.The concentrations in the test extracts were expressed as mgGallic acid (GA) equivalent/g DWof the rice powder.

Antioxidant activity

The antioxidant activity of methanol and ethyl acetate extractsin the grains of Kavuni and CO 50 were measured in terms ofDPPH radical scavenging, hydroxyl radical scavenging, su-peroxide anion scavenging, nitric oxide radical scavengingand lipid peroxidation inhibition activity as described below:

Determination of DPPH radical scavenging activityMethanol and ethyl acetate extracts of Kavuni and CO 50grains were used for estimation of their antioxidant ac-tivity in terms of hydrogen donating or radical scaveng-ing ability using DPPH method as described by Shimadaet al. (1992). About 3 ml of reaction mixture containing2.8 ml methanolic DPPH and 0.2 ml extract at variousconcentrations (2 g—0.001 μg) were mixed well andincubated for 30 min at room temperature. The degreeof reduction in absorbance wasmeasured at 517 nm usingCarry Varien-50 spectrophotometer and the percent ofinhibition was determined as

% inhibition ¼ A0−A1ð Þ=A0 � 100½ �

where A0 is the absorbance of the control and A1 is theabsorbance in the presence of sample. Gallic acid was usedas the positive control.

Determination of hydroxyl radical scavenging activityThe hydroxyl radical scavenging activity of rice grainextracts was measured by the deoxyribose method(Halliwell et al. 1987) and compared with that of catechin.The reaction mixture containing 0.2 ml of extracts atvarious concentrations (2 g—0.001 μg), deoxyribose(3.75 mM), H2O2 (1 mM), potassium phosphate buffer(20 mM, pH 7.4), FeCl3 (0.1 mM), EDTA (0.1 mM) andascorbic acid (0.1 mM) were incubated in water bath at

Table. 3 Mineral composition ofdehusked grains of Kavuni andother white rice varieties of TamilNadu

Values are mean ± standard errorof five determinations, values ofKavuni are significantly differentfrom CO 50a at p<0.05, IR64b atp<0.05 and White Ponnic atp<0.05

Minerals (mg/100 g) Genotypes

Kavuni CO 50 IR64 White ponni

Iron 1.86±0.006a,b,c 1.49±0.01 1.493±0.004 1.3±0.003

Calcium 13.96±0.13a,b,c 7.62±0.01 7.82±0.02 9.26±0.01

Zinc 1.18±0.01 1.21±0.04 1.13±0.04 1.22±0.02

Copper 1.36±0.02a,b,c 1.2±0.04 1.16±0.07 1.23±0.03

Manganese 2.73±0.05b 3.04±0.02 2.54±0.05 2.77±0.05

Sodium 36.7±0.05a,b,c 25.83±0.56 28.76±0.001 22.6±0.005

Potassium 282.3±0.5a,b,c 243.3±1.52 262±1 237.3±2.08

Magnesium 181.6±1.52a,b,c 155.6±3.2 165.3±0.57 133±1

Fig. 2 Scanning electronmicrographs of fractured ricegrains showing the nature ofstarch granules packed into theamyloplasts of the endospermcells; aKavuni (1,000×); bCO 50(1,000×)



37 °C for 1 h. To determine the extent of deoxyribosedegradation, TBA (1%) and TCA (2%) were added to thereaction mixtures and boiled in a water bath at 100 °C for15 min. The absorbance of resulting solution was mea-sured spectrophotometrically at 532 nm. The percentageof inhibition was calculated according to the equation.

% inhibition ¼ A0−A1ð Þ=A0 � 100½ �Determination of superoxide anion scavenging Activity

The superoxide anion-scavenging ability of the rice grainextracts was measured by the method of Nishikimi et al.(1972). One milliliter of NBT solution (150 μM NBT in100 mM phosphate buffer, pH 7.4), 1 ml nicotinamideadenine dinucleotide (NADH) solution (468 μM in100 mM phosphate buffer, pH 7.4) and 0.1 ml of extractsat various concentrations (2 g—0.001 μg) were mixed to-gether. The reaction was initiated by adding 100 μl of PMS

solution (60 μMPMS in 100 mM phosphate buffer, pH 7.4)to the mixture. The reaction mixture was incubated at 25 °Cfor 5 min, and the absorbance was measured at 560 nmagainst blank samples. The percentage of anion scavengingactivity (ASA) was calculated by the formula

% ASA ¼ 1−Asample=Ablank

� �� 100

Where Asample is the OD value of the sample or standardand Ablank is the OD value of the blank. Ascorbic acid wasused as the positive control

Assay of Nitric Oxide radical scavenging ActivityThe nitric oxide-scavenging ability of the extract was mea-sured according to the method of Marcocci et al. (1994).About 4 ml of extract at various concentrations (2 g—0.001 μg) were added to 1 ml of sodium nitroprussidesolution (25 mM) in a test tube and then incubated at

Fig. 3 GC-MS chromatogram ofmethanol extract of Kavunigrains. The identified compoundscorresponding to the peaks arelisted in Table 4

Fig. 4 GC-MS chromatogram ofmethanol extract of white ricevariety CO 50. The identifiedcompounds corresponding to thepeaks are listed in Table 5



37 °C for 1 h. The reaction mixture (0.5 ml) wasdiluted with 0.3 ml of Griess reagent (1 % sulfa-nilamide in 5 % H3PO4 and 0.1 % napthylethylene-diamine dihydrochloride). The absorbance of the chro-mophore formed during the diazotization of nitritewith sulfanilamide and subsequent coupling withnapthylethylenediamine dihydrochloride was imme-diately read at 570 nm and referred to the absor-bance of standard solutions of sodium nitrite salttreated in the same way with Griess reagent.Curcumin was used as a positive control.

Total Antioxidant activityThe total antioxidant activitywas assayed using FTCmethod(Gülçin et al. 2007). About 500 μg of extract was dissolvedin 500 μl EtOH and added to a reaction mixture containing2.88 ml of 2.5 % linoleic acid and 9 ml of 40 mM phosphatebuffer (pH 7.0) in a vial. The vials were incubated at 40 °Cfor 96 h. At every 24 h interval 0.1 ml of sample wascollected and diluted with 9.7 ml of 75 % EtOH, 0.1 mlammonium thiocyanate (30 % v/v) and 0.1 ml FeCl2(0.002 M). The absorbance of samples was measured at500 nm using Carry Varien-50 spectrophotometer and thepercent of inhibition of linoleic acid peroxidation was deter-mined (Elmastas et al. 2007). The mixture without samplewas used as control and BHA and alpha tocopherol wereused as positive controls.

The inhibition of linoleic acid peroxidation (%) was esti-mated by the following formula:

% Inhibition ¼ 100− A1−A0ð Þ � 100ð ÞWhere A0 is the absorbance of the control and A1 is the

absorbance of the sample extracts.

Inhibitory activity against enzymes involved in carbohy-drate metabolism

Methanol and ethyl acetate extracts of Kavuni and CO 50grains were used for estimating their ability to inhibit keyenzymes linked to Type II diabetes viz., α-amylase(Apostolidis et al. 2007) and α-glucosidase (Shinde et al.

Table 4 List of metabolites in the methanol extracts of Kavuni grainsidentified through GC-MS

RT Metabolites CC RC (%)

9.12 β-resorcylic acid PA 2.08

10.622 Cinnamic acid PA 7.09

10.719 Coumarin PA 1.06

11.690 O-coumaric acid PA 28.3

12.924 Chloro phenyl cinnamic acid PA 5.65

14.028 Chlorocinnamic acid PA 8.47

16.237 Dihydrocoumarin-6-ol PA 17.1

16.498 α-Acetaminocinnamic acid PA 21.8

16.563 Ferulic acid methyl ester PA 13.4

16.599 Quercetin FLA 7.57

16.608 Flavone, 4′,5-dihydroxy-6,7-dimethoxy- FLA 7.57

16.652 3,4-Bis[(trimethylsilyl)oxy]dihydro-2(3H)-furanone

OG 21.9

16.668 p-methoxycinnamic acid PA 22.3

16.730 Kaempferide FLA 22.5

16.730 7-β-D-glucopyranoside S 7.56

18.20 N-methoxy-N-trifluoroacetyl-1-methyl-1-carbomethoxyethylamine

Amines 12.9

22.39 2-heptanone ketone 20.8

23.16 L-aspartic acid AA 22.7

23.531 β-carotene-4,4′-dione CA 2.06

23.700 Lutein CA 11.2

25.07 Pentane alkane 16.5

26.266 Coumarin PA 0.004

26.338 Riboflavin AA 0.326

27.333 Dimethyl caffeic acid PA 15.6

27.84 Pyrrolo[2,3-f]quinolin-9-ol, A 24.2

28.508 Trans-o-hydroxycinnamic acid PA 1.89

28.724 p-coumaric acid PA 15.2

28.87 β-D-Galactopyranoside S 48.2

29.314 Lycopene CA 4.24

29.84 Glycine AA 1.27

30.4 Xanthen-9-one OG 8.12

31.35 Glucopyranose S 22.5

33.09 D-glucose S 23.6

34.453 Kaempferol FLA 15.3

34.48 Hexadecanoic acid FA 0.008

35.11 Myo-inositol S 20.4

37.412 Linoleic acid FA 10.4

37.52 Oleic acid FA 11.2

37.711 Hydrocinnamic acid PA 17.9

37.75 Octadecanoic acid FA 19.9

37.807 10′-Apo- β-carotenoic acid CA 0.823

37.826 Flavone, 5-hydroxy-3,3′,4′,7-tetramethoxy- FLA 2.02

38.013 Flavone, 3,3′,4′,7-tetramethoxy- FLA 14.2

38.03 Stearic acid FA 14.2

40.15 D-xylose S 16.2

42.20 Mannose S 12.7

42.48 Naphtho[1,2-b]furan-2,8(3H,4H)-dione Ketone 8.52

Table 4 (continued)

RT Metabolites CC RC (%)

42.67 D-fructose S 12.0

43.07 Alpha glucopyranoside S 12.1

45.7 all-trans-3,3′,4,4′-Tetrahydrospirilloxanthin CA 0.653

46.39 Eicosanoic acid FA 10.8

48.84 Butanedioic acid OG 8.28

50.63 Flavone FLA 11.2

RT retention time, RC relative content (%), PA phenolic acid, S sugars andtheir derivatives, FA fatty acid, FLA flavanoids,CAcarotenoids, AAaminoacid, OG other group, A alcohol



2008). Five hundred microliters of extracts at differentconcentrations ranging from 2 g to 0.001 μg was incubatedalong with 500 μl of 0.02 M sodium phosphate buffer (pH6.9 with 0.006 M sodium chloride) containing α-amylaseenzyme (0.5 mg/ml) at 37 °C for 10 min. After pre-incubation, 500 μl of 1 % starch solution in 0.02M sodiumphosphate buffer (pH 6.9 with 0.006 M sodium chloride)was added to each tube and incubated at 37 °C for 10 min.The reaction was stopped using 1.0 ml of dinitrosalicylicacid reagent and absorbance was measured at 540 nm usingCarry Varien-50 spectrophotometer. Gallic acid was used asa positive control. For α-glucosidase inhibition assay, α-glucosidase (20 μl, 1.5 U/ml) was premixed with 200 μl ofmethanol and ethyl acetate extracts at varying concentra-tions made up in 50 mM phosphate buffer at pH 6.8 andincubated for 5 min at 37 °C (Shinde et al. 2008). 1 mMpara-nitrophenyl-α -D glucopyranoside (200 μl) in 50 mMof phosphate buffer was added to initiate the reaction andthe mixture was further incubated at 37 °C for 20 min. Thereaction was terminated by the addition of 500 μl of 1 MNa2CO3 and the absorbance was recorded at 405 nm. Gallicacid was used as a positive control.

Statistical analysis

All the experimental data were statistically analyzed usingpaired student’s t-test and the significance between the geno-types for various traits tested are presented at p<0.05.

Results and discussion

Rice being a staple food for majority of population in Asia, itcontributes 70–75% of total body carbohydrate requirement. It isan easily digestible fine cereal but known for its high glycemicindex and hence not recommended for the patients with impairedglucose metabolism (Deepa et al. 2010). The present study wasaimed at analyzing the biochemical properties of a traditionallyknown therapeutic rice variety Kavuni possessing anti-diabeticproperties in comparison with popularly eaten white rice varietiesviz., IR 64, Improved White Ponni and CO 50. The rice geno-types used in this study exhibited significant variation for the totalsoluble sugars and starch content (Table 2). The traditional variety“Kavuni” was found to contain significantly lower level (29–35%) of total soluble sugars and starch (8–35%)when comparedto other white rice varieties. This significantly lower amount oftotal soluble sugars and starch in Kavuni may have significantinfluence on the free sugar level in the grains. The total amylosecontent among the rice varieties varied from 22 to 24.6 %. Therice varieties Kavuni (24.2 %) and White Ponni (24.6 %) hadhigher amylose content followed by IR64 and CO 50 (Table 2).Nearly all rice varieties commonly eaten by the people containonly approximately 20 % amylose (Haub et al. 2010).

Table 5 List of metabolites in the methanol extracts of a white ricevariety CO 50 identified through GC-MS analysis

RT Metabolites CC RC(%)

11.71 N,O-Bis-(trimethylsilyl)alanine AA 19.4

14.93 1-(Isopropylamino)-3-(1-naphthyloxy)-1,2-propanediol

A 11.3

15.71 Phosphoric acid ACID 12.8

16.570 Flavone, 4′,5-dihydroxy-6,7-dimethoxy- FLA 15.3

19.02 Alanine AA 15.7

20.73 9-octadecanoic acid FA 11.6

22.80 Cystathionine OG 16.4

23.08 2-Piperidinecarboxylic acid OG 26.6

25.01 Acetamide OA 16.1

27.39 Spiro[2.4]hept-5-ene OG 19.0

29.194 Hydroxystearic acid FA 43.8

29.285 Beta resorcyclic acid PA 10.1

29.482 α -d-fructopyranose S 24.1

29.648 Diphenylmethylsilyloxyheptadecane OA 24.7

29.671 1,2,3,5-tetrakis-O-(trimethylsilyl)pentofuranose

S 24.7

29.84 Pentanedioic acid OA 26.8

30.526 Palmitic acid FA 15.1


32.007 6-deoxy-1,2,3,5-tetrakis-O-(trimethylsilyl) hexofuranose

S 19.2

32.127 3H-xanthen-3-one OG 19.1

33.05 D-glucose S 21.9

33.878 17,21-dihydroxy-3,11,20-trioxopregn-4-en-9-yl thiocyanate

OG 3.07

34.383 Tri-O-methylapigenin FLA 0.094

34.402 α-tocopherol V 14.7

34.407 Kaempferol 3,7-dimethyl ether FLA 14.7

34.42 Hexadecanoic acid FA 14.5

35.073 Inositol S 20.5

35.182 α -D-glucopyranose, S 17.1

37.71 Undecanoic acid FA 20.9

37.732 Xanthen-9-one, OG 0.063

37.97 Octadecanoic acid FA 13.8

40.11 D-xylopyranose S 17.5

42.17 α -D-Mannopyranoside S 13.9


42.68 α -D-glucopyranoside S 9.01

43.01 Pamoic acid FA 0.178

44.191 Hematoporphyrin Phorphyrin 4.21

44.32 D-ribofuranose S 15.1

45.91 N-acetyl glucosamine S 17.7

46.36 Eicosanoic acid FA 10.7

47.51 Maltose S 9.76

48.80 Oleanolic acid FA 1.08

RT retention time, RC relative content (%), CC compound class, PAphenolic acid, S sugars and their derivatives, FA fatty acid, FLAflavanoids, AA amino acid, OG other group, A alcohol, OA organic acid



Proximate and mineral composition of rice grains

Cereals are the major sources of carbohydrates, protein and fibrefor the vegetarian population. The Total Dietary Fibre (TDF) andprotein contents were found to be significantly higher in Kavunithan the other three white rice varieties (Table 2). The TDFcontent in Kavuni was found to be 21–52 % higher than CO50, IR64 and White Ponni. Protein content in Kavuni was 7–24 % higher than the other three varieties. Ash content was lowin Kavuni (0.54 %) whereas in the other three varieties it rangedfrom 1.22 to 1.85 %. Crude lipid was found to be significantlylow in Kavuni than the other three white rice varieties andImproved White Ponni had the highest crude fat content (Ta-ble 2). Rice as a staple food is rich in carbohydrate and known forits poor mineral. Some of the brown rice genotypes are known topossess significantly higher levels of mineral ions. Kavuni wasfound to contain significantly higher amount of iron (20–30 %),calcium (33–45 %), copper (9.5–14.7 %), sodium (21–38 %),potassium (7–15 %) and magnesium (8.9–26 %) (Table 3).

Ultrastructure of rice grains

The scanning electron micrographs of Kavuni rice grainsshowed the presence of tightly packed globular starch gran-ules (Fig. 2a) whereas the amyloplasts of the white rice varietyCO 50 contained sharp irregular starch granules and were

relatively loosely packed into the endosperm (Fig. 2b). Tightlypacked starch granules in Kavuni indicate it to be a non-chalky grain.

Metabolite profiling

GC-MS analysis of grains of Kavuni and the white rice varietyCO 50 (Figs. 3 and 4) revealed the presence of about 201 and170 different compounds respectively. Using database librarysearch 82 compounds in Kavuni and 60 compounds in CO 50extracts were identified and annotated. Kavuni extracts werefound to contain 15 phenolic acids followed by sugar mole-cules, flavonoids, fatty acids and carotenoids as major com-ponents along with few minor ones belonging to alkane,amine and ketone molecules (Table 4). The GC/MS profileof CO 50 showed the presence of sugar molecules (14), fattyacids (9) and flavonoid (3) as major components along withalcohol and amino acids (Table 5). Carotenoids (lutein, β-carotene and lycopene) and phenolic acids (cinnamic acid,ferulic acid, caffeic acid, coumaric acid and their derivatives)were identified only in the Kavuni extract but not in CO 50. InKavuni, the relative amount of phenolic acids, sugars andflavonoids were higher than the other class of compounds,whereas in CO 50 the relative amount of sugars and fatty acidswere found to be higher. Foods or beverages rich in polyphe-nols have been shown to attenuate postprandial glycemic

Fig. 5 HPLC chromatogramsshowing separation of β-caroteneand lutein in unpolished grains ofdifferent rice genotypes; aAuthentic standard; bKavuni; cCO 50; d IR64 and eWhite Ponni.The peaks are indicated by arrowsand the values within parenthesisindicate the correspondingcarotenoid contents (μg/100 g).Values are mean ± standard errorof five replications; Values ofKavuni are significantly differentfrom CO 50f, IR64g and WhitePonnih at p<0.05

Table 6 IC50 values of DPPH, hydroxyl, superoxide anion and nitric oxide radical scavenging activities ofmethanol and ethyl acetate extracts of Kavuniand CO 50 grains

Extracts DPPH radical scavengingactivity IC50 (μg/ml)

Hydroxyl radical scavengingactivity IC50 (μg/ml)

Superoxide anion scavengingactivity IC50 (μg/ml)

Nitric oxide radical scavengingactivity IC50 (μg/ml)

CO50 Kavuni CO50 Kavuni CO50 Kavuni CO50 Kavuni

EA 258.6 3.4 154.2 5.8 587.5 9.3 372.1 4.7

ME 217.8 1.9 174.8 0.7 489.1 2.8 322.6 3.4

Each value is a mean of three independent assays (n=3)

MEmethanol extract, EA ethyl acetate extract



responses, fasting hyperglycemia, improved acute insulin se-cretion and insulin sensitivity (Hanhineva et al. 2010; Jae et al.2010; Hudson et al. 2000).

Estimation of carotenoids and total phenolics

Various carotenoid fractions and total phenolic contents in therice varieties were quantified using HPLC and spectrophoto-metric assays. The traditional variety Kavuni was found tocontain significantly higher quantity of β-carotene (26–65 %)and lutein (95–96 %) (Fig. 5). Total phenolics content inKavuni (28.8±0.01 μg/100 g) was higher than CO 50 (10.2±0.01 μg/100 g), IR64 (8.0±0.004 μg/100 g) and WhitePonni (12.4±0.002 μg/100 g).

Estimation of radical scavenging activity

In DPPH radical scavenging activity, methanol and ethylacetateextracts of Kavuni were found to reduce the stable pink coloredfree radical DPPH to yellow colored diphenyl picrylhydrazinesignificantly. Methanol extracts of Kavuni showed maximumDPPH radical scavenging activity than the positive standardgallic acid (IC50—2.8 μg/ml) (Table 6). Hydroxyl radical scav-enging activity of Kavuni extracts was significantly higher thancatechin (IC50—6.7 μg/ml) and CO 50 (Table 6). The

significantly higher activity of Kavuni extracts shows that theactive molecules in Kavuni might play a major role inpreventing oxidative degradation of deoxyribose in Fentonreaction (Smith et al. 1992). Superoxide anion scavengingactivities of various extracts are given in Table 6. The methanolextract of Kavuni was found to be the strongest superoxideanion scavenger and the activity was significantly higher thanthose of standard ascorbic acid (IC50—12.8μg/ml). Along withreactive oxygen species, nitric oxide radical (NO) is also asso-ciated in diabetes, inflammation, cancer and other variousdisorders (Moncada et al. 1991). Both methanol andethylacetate extracts of Kavuni showed significantly higherNO scavenging activity than the standard curcumin (IC50—15.2 μg/ml) and CO 50.

Estimation of total antioxidant activity

At 72 and 96 h of incubation, absorbance of both methanol andethyl acetate extracts of Kavuni was significantly (P<0.05)lower than the absorbance of α-tocopherol and higher than thatof BHA (Fig. 6). In contrast, CO 50 extracts had a significantlyhigher absorbance at 72 h and 96 h when compared withKavuni extracts, α-tocopherol and BHA. The percentage ofinhibition of both methanol and ethyl acetate extracts of Kavuniat 72 and 96 h were significantly higher than the inhibition ofCO 50 extracts and α-tocopherol (Fig. 4b). Higher antioxidantactivity of Kavuni extracts significantly correlated with thehigher phenolics, flavanoids and carotenoids content in Kavunigrains, suggesting the importance of these components as po-tential antioxidant biomolecules (Vichapong et al. 2010;Yodmanee et al. 2011; Krentz and Bailey 2005).

Inhibitory activity against enzymes linked to diabetes

A sudden rise in the blood glucose levels causing hypergly-cemia in type II diabetic patients happens due to hydrolysis of

Fig. 6 Antioxidant activity of methanol and ethyl acetate extracts ofKavuni and CO 50 grains along with controls; Values are mean ± SE oftriplicates. Alphabets indicate the statistical significance (P<0.05) at 72 h(lowercase) and 96 h (uppercase letters); KMEKavuni methanol extract,

KEAEKavuni ethyl acetate extract, CMECO 50 methanol extract, CEAECO 50 ethyl acetate extract, BHAbutylated hydroxy anisole and ATalphatocopherol, C control

Table 7 α-amylase and α-glucosidase inhibitory activity of methanoland ethyl acetate extracts of Kavuni and CO 50 grains

Assay Kavuni CO 50 Gallicacid

ME EAE ME EAE

α-amylase inhibition IC50 (μg/ml) 0.10 1.00 – – 0.30

α-glucosidase inhibition IC50 (μg/ml) 0.04 0.05 – – 0.15

Each experiment was repeated three times (n=3)

MEmethanol extract, EAE ethyl acetate extract



starch by pancreatic α-amylase and uptake of glucose byintestinal α-glucosidase. An effective strategy for type IIdiabetes management is the strong inhibition of α-glucosidase and mild inhibition of pancreatic α-amylase(Zhou et al. 2004). The methanol and ethyl acetate extractsof Kavuni were able to inhibit both the enzymes significantlythan that of standard compound Gallic acid; whereas CO 50did not show any inhibitiory activity against either of theenzymes (Table 7).

The present attempt on unraveling the biochemicalproperties of the traditional therapeutic rice varietyKavuni, revealed that Kavuni contain significantly lowerlevel of total soluble sugars, high levels of amylose,dietary fibre, protein, β-carotene, lutein and polyphenolsthan the popularly eaten white rice varieties. The dataalso suggests that the therapeutic variety Kavuni possessmarked health benefits in preventing oxidative stress(higher amount of antioxidants) and diabetic complica-tions by inhibiting the key enzymes (α-amylase and α-glucosidase) involved in their development. Traditionalrice varieties are rich sources of biodiversity for major-ity of the agronomic traits and as shown in the presentstudy Kavuni with rich nutritive and therapeutic proper-ties can be used as a potential donor for understandingthe genetic basis of the above traits through geneticmapping and further manipulation of these traits throughmolecular breeding. Further, efforts have been taken tounderstand the genetic basis of therapeutic properties ofKavuni and to improve the yield attributes of Kavunithrough marker assisted breeding.

Acknowledgments The authors thank Department of Biotechnology,Government of India, New Delhi for the DBT-RA fellowship to the firstauthor.

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