Food Measure (2018) 12:56–67 DOI 10.1007/s11694-017-9616-0

ORIGINAL PAPER

Extraction and in vitro antioxidant capacity evaluation of phenolic compounds from pigmented aromatic rice (Oryzae sativa L.) cultivars Farhan M. Bhat1 · Charanjit S. Riar1   

Received: 2 July 2017 / Accepted: 7 August 2017 / Published online: 28 August 2017 © Springer Science+Business Media, LLC 2017

Abstract  The aroma generating volatile components profile and in vitro antioxidant capacities of different aromatic rice cultivars was determined by GC–MS analysis and in terms of DPPH scavenging activity, lipid peroxidation inhibition, phosphomolybdenum reduction and reducing power assay. The total phenolic content including both free and bound forms in the analyzed aromatic rice cultivars, Mushki budgi (1.62 mg GAE/g), Mushki kandi (1.63 mg GAE/g) and Kamad (1.60 mg GAE/g) were found double the amount as compared to non-aromatic Koshkari (0.86 mg GAE/g) cultivar. The aromatic rice cultivars had also shown higher total flavonoid content and antioxidant activity than nonaromatic rice cultivar (Koshkari). The GC–MS results indicated 21-aromatic compounds present in sufficient quantities in aromatic cultivars and some of them were unique to these cultivars. Among the compounds identified, aldehydes were found in higher quantity followed by alkanes, ketones and esters. Among the aromatic rice cultivars, Mushki budgi and Mushki kandi were found possessing higher quantity of flavoring components such as benzaldehyde, a carcinostatic agent. The cultivars Mushki budgi and Mushki kandi indicated positive correlation of TPC, TFC and the in vitro antioxidant components largely, while the less aromatic Kamad, correlate with only two components viz DPPH and lipid peroxidation. Keywords  Antioxidant property · Aromatic rice · Aromatic compounds · GC–MS · Phenolic compounds * Charanjit S. Riar [email protected] 1



Department of Food Engineering & Technology, Sant Longowal Institute of Engineering & Technology, Longowal, Sangrur, Punjab 148106, India

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Abbreviations GAE Gallic acid equivalents RUE Rutin equivalent TFC Total flavonoid contents TPC Total phenolic contents TAC Total anthocyanin contents TBARS Thiobarbituric acid- reactive species DPPH 2,2 Diphenyl 1 picryl hydrazyl hydrate J&K Jammu & Kashmir

Introduction Rice (Oryza sativa L.) is recognized as the staple food for about half of the world’s population. Rice grain is consumed as a whole after cooking as compared to other cereal grain and is therefore considered nutritionally rich [1]. Aromatic or scented rice varieties have been in great demand in the world market for decades and are also very popular in the Indian feasts. These traditional rice cultivars along with others have occupied an important place in the valley of Kashmir since prehistoric times and are still cultivated due to their unique health and nutritional benefits. In modern days, these rice cultivars have earned high reputation and popularity throughout the valley of Jammu and Kashmir (India) due to their desirable health benefits along with their pleasant and high quality aroma [2]. These varieties constitute a small but special group of rice and are considered best in quality. Although non-aromatic Indicia rice with long and medium grains and Japonicas, short grain varieties contribute to about 79% of world trade but the aromatic basmati rice of the Indian sub-continent clinches a premium price and gets three-times higher price (US $800–$1000 BMT) than high quality non-basmati rice (US $200–$300 BMT).

Extraction and in vitro antioxidant capacity evaluation of phenolic compounds from pigmented…

The volatile profile of a rice variety not only determines the quality of whole rice or its products but also has an important role in rice breeding programmes in order to incorporate this important trait in hybrid varieties. The metabolic pathways leading to the generation of volatiles in rice are determined mainly by the variety, agronomic practices, post harvest operations and storage conditions. The main aromatic compounds identified in majority of the aromatic rice cultivars include five-member, N-heterocyclic ring compound such as 2-acetyl-1-pyrroline (2AP), having aroma threshold value as low as 0.1 ppb in water [3], however, the concentration of 2AP compounds decreases during prolonged storage at elevated temperature [4]. In the aromatic rice cultivars, in addition to 2AP, the other flavoring components of lipid oxidation such as hexanal, acetic acid, and pentanoic acids also have impact on the acceptability of aroma [5]. The volatile profile of rice has been demonstrated by several researchers using solvent extraction [6], solidphase microextraction (SPME) [7], headspace analysis, gas chromatography provided with flame ionization, or mass spectrometry [8]. More than 200 volatile compounds in aromatic rice varieties have been identified [5, 9] some of which, 2-AP, 2-acetyl-pyrrole, 2-pentylfuran and pyridine, have been identified as sweet smelling and pleasing aromatic compounds. These compounds have significant effect on the consumer acceptability of aromatic rice. Therefore, volatile profiles may have the potential to mark the identity of the variety and to interpret the quality of rice. The amino acid profiles of various aromatic rice cultivars revealed that most of these varieties possess a higher level of essential amino acids like lysine, methionine, tyrosine, phenylalanine, leucine, isoleucine, threonine and valine. This is the reason why such rice is known for their superior nutritional quality in addition to the superior taste they possess [10]. The work to explore the aromatic component profile and presence of bioactive components having high antioxidant potential has not been carried out as per the literature cited of selected aromatic rice cultivars grown in the J&K state of India. Polar solvents can be used to dissolve inorganic or ionic compounds from a substance. Different solvents having varying degree of polarity led to different dissolution of ionic compounds and water which can both donate and accept hydrogen bonds, making it an excellent solvating solutes that can donate or accept (or both) H-bonds [11]. The present research was therefore hypothesized to analyze the traditionally grown aromatic rice cultivars of Kashmir (India) for the evaluation of their aromatic volatile components profile as determined by GC–MS, free and bound phenolic compounds coupled with their antioxidant capacities evaluation in order to explore the in vitro antioxidant capacity.

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Materials and methods Sample preparation For this study three different traditional rice cultivars were selected viz: Mushki budgi, Mushki kandi and Kamad. One non-aromatic rice cultivar, Koshkari was also taken as a reference for comparison purposes. Samples of these cultivars were collected from the certified agencies and the rice research centre, Khudwani of Sher-e-Kashmir Agricultural University of Science and Technology, Kashmir (India) consecutively over a period of 2 years. These research centers cultivate these rice cultivars for conservation of their valuable land races and to maintain the purity of these cultivars. The cleaned paddy grains were subjected to milling using lab rubber de-husker (Agrosa India, Pvt. Ltd.). Thereafter, brown rice was milled in a pilot scale grinding mill (Agrosa India, Pvt. Ltd.) and passed through 100 mesh sieve to get the rice flour. The resultant rice flour was stored at 4 °C in air tight containers for further analysis. All the experiments were conducted in triplicate, unless otherwise stated. Extraction of free, bound and total polyphenolic compounds The flours of brown rice cultivars (1  g, each) were extracted with 10 ml methanol for 15 min by vigorous mixing at ambient temperature in a vortex mixer (in order to achieve complete mixing or homogenizing the sample so as to achieve higher rate of leaching of phytochemicals and thus enhanced extraction of antioxidant compounds (free fractions) by means of solvent) (Fig. 1). The supernatant was separated by filtering through a vacuum pump and the extraction was repeated twice. The supernatants of each extraction step were combined together. The remaining residue was re-extracted by the addition of 10 ml of acetone/water in the ratio of 70:30 v/v basis followed by recovering of supernatant as given above. This extraction process was repeated and the supernatants were combined there off. The extracts prepared individually from both methanol and acetone/water solvents included the free fractions as reported previously by Finocchiaro et al. [12]. The final leftover residue was then used for extraction of phytochemical compounds that are covalently linked to cellular components by following the procedure of Adom and Liu [13]. For this, the residue was digested (in order to recover the bounded fraction of phytochemical compounds that were covalently linked to cellular components) with 20 ml of 2M NaOH at room temperature for 1 h. The extracts were then adjusted to pH 3 with 1N HCl. The compounds released upon hydrolysis were than extracted with 20 ml of ethyl acetate. After centrifugation at 3000 rpm for 10 min, the

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F. M. Bhat, C. S. Riar

Assay of free, bound and total flavonoid contents (TFC) The TFC were measured by following the method of Dewanto et al. [15] with slight modification. The 0.5 ml of the extracts of both free and bound fractions was mixed with 2.25 ml of distilled water, followed by addition of 0.15 ml of 5% ­NaNO2 solution. After 6 min, 0.3 ml of a 10% w/v ­(AlCl3·6H2O) solution was added and kept for 5 min before 1.0 ml of 1M NaOH was added with thorough mixing. The absorbance was instantly measured at 510 nm using spectrophotometer and the results were expressed in units of mg rutin equivalents per gram of dried sample (mg RUE/g). GC–MS analysis of volatile (odour) principles

Fig. 1  Flow sheet for the extraction of free and bound fraction of phenolic compounds form rice cultivars

supernatant was collected and the extraction was repeated once. The supernatants of ethyl estate extracts containing bound fraction were then combined and the solvent was evaporated under vacuum. The dried bound fractions were analyzed for their antioxidant activity for each cultivar by re-dissolving them in methanol as per procedures in subsequent sections below. Assay of free, bound and total phenolic contents (TPC) The TPC of the free and bound fractions of rice extracts was determined with the Folin–Ciocalteu method as described by Taga et al. [14]. To 1 ml of the prepared rice extract (both free and bound), 9 ml of distilled water and 1 ml of the Folin–Ciocalteu reagent were added. Thereafter, 10 ml of 7% (w/v) N ­ a2CO3 solution was added followed by 25 ml of distilled water with continuous stirring. The mixture was given a rest period of 90 min in dark and the absorbance was measured against the reagent blank at 750 nm using spectrophotometer. Total phenolic content was expressed as Gallic acid equivalent per gram.

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Whole milled rice flour samples, 20 mg each were placed in 10 ml vials followed by addition of 20 µl of double distilled water containing 1 mg of 2,4,6-trimethylpyridine (TMP) as the internal standard. The vials were sealed with a Teflon lined magnetic cap and placed in an auto sampler tray maintained at room temperature until analyzed. The samples were preheated for 5 min at 80 °C prior to evaluation. Samples were desorbed for 25 s on a Varian Chrompack CP-3800 gas chromatograph with a Saturn 2000 mass spectrometer attached (GC–MS) (Varian Analytical Instruments, Walnut Creek, CA, USA). The injector temperature was held constant at 260 °C. The GC oven temperature was held at 50 °C for 1 min, then increased to 260 °C at rate of 5 °C/min, and then held at 260 °C for 4 min. A 30 mm × 0.25 mm ID with a 0.25 mm thickness of 5% diphenyl–95% dimethyl polysiloxane cross bonded liquid phase (DB-5) capillary column was used, with research grade helium (99.999%) as the carrier gas under a constant flow of 28.6 cm/s (1 ml/min), in the split less mode. The total GC cycle time consisted of a 47.0 min run and a 5 min re-stabilization time. The MS was operated in the scan mode from m/z 40 to m/z 650 with 150 count threshold. Each compound was identified by the presence of selected ions and by comparing the MS spectra to the reference spectra in the National Institute of Standards and Technology (NIST, ver. 2.0f, 2008) mass spectral database. In vitro antioxidant capacity DPPH radical scavenging activity assay Free radical scavenging activities of rice flour extracts were determined with the aid of DPPH radical as described by Sanchez-Moreno et al. [16] with slight modification. To 0.1 ml of the extracted solutions of free and bound fractions, 3.9 ml of DPPH solution prepared by dissolving 2.3 mg of DPPH radical in 100 ml methanol was added and mixed thoroughly. The solution was given a rest period of 30 min in

Extraction and in vitro antioxidant capacity evaluation of phenolic compounds from pigmented…

dark followed by measurement of the absorbance at 515 nm against reagent blank (control). The DPPH radical scavenging activity was calculated by the following equation: DPPH radical scavenging % =

(

1−

A515nm, sample A515nm, control

)

× 100

Reducing power assay The reducing power was determined by the method of Yen and Duh [17], with slight modification. To 2.5 ml of the prepared rice extracts (methanol, acetone + H 2O along with bound fractions reconstituted in methanol), 2.5 ml of phosphate buffer (2.0 M, pH 6.6) and 2.5 ml of 1% potassium ferricyanide was added. The mixtures were given a heat treatment at 50 °C for 20 min followed by addition of 2.5 ml of 10% solution of trichloro acetate and the mixture was centrifuged at 5000 rpm for 10 min. The 2.5 ml of the resulting solution was mixed with 2.5 ml distilled water and 0.5 ml of ferric chloride (0.1%) followed by measurement of absorbance at 700 nm by UV–Vis spectrophotometer. The absorbance was compared with each other for determining the strength of reducing power. Total antioxidant capacity by phosphomolybdenum reduction assay Phosphomolybdate assay system was used to determine the total antioxidant activity of the rice flour extracts according to method described by Khan et al. [18] with slight modification. The 0.3 ml of the prepared free and bound rice extracts were mixed with 3 ml of reagent solution prepared by using sulphuric acid (0.6 M), sodium phosphate (28 mM) and ammonium molybdate (4 mM). The mixtures were incubated at 95 °C in a water bath for 90 min. After cooling to room temperature, absorbance was recorded at 695 nm against reagent blank containing methanol 0.3 ml in place of extract. Total antioxidant capacity was calculated as ascorbic acid equivalence. Inhibition of lipid per oxidation in egg yolk homogenate Inhibitions of lipid per oxidation in the egg yolk was determined by using the modified thiobarbituric acid reactive species (TBARS) assay method described by Badmus et al. [19]. A 0.5 ml of egg yolk homogenate (10% in distilled water, v/v) was mixed thoroughly with 0.1 ml prepared rice extracts in a test tube and the volume was made up to 1 ml with distilled water. Finally, 0.05 ml ­FeSO4 (0.07 M) was added to the above mixture and incubated for 30 min, to induce lipid peroxidation. Thereafter, 1.5 ml of 20% acetic acid and 1.5 ml of 0.8% TBA (w/v) in 1.1% sodium dodecyl sulfate (SDS) and 0.05 ml 20% TCA were added, vortexed

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and then heated in a boiling water bath for 60 min. Then 5.0 ml of butanol was added to each tube after cooling and centrifuged at 3000 rpm for 10 min. The absorbance of the organic upper layer was measured at 532 nm and percent inhibition was calculated as. ( ) A532, sample % Inhibition = 1 − × 100 A532, control Statistical analysis All the experiments were carried out in triplicate and the results were expressed as mean ± SD. The results were analyzed by applying the Duncan’s testing procedure using SAS software version 9.1. Difference between means was declared statistically significant at (p ≤ 0.05).

Results and discussion Free, bound and total phenolic contents (TPC) assay The TPC of the rice cultivars in free fractions extracted by methanol and acetone + H2O containing medium were found higher than the bound fractions. Among the rice cultivars Mushki kandi had been found containing higher amount of TPC in free fractions showing higher total TPC content of 1.63 mg GAE/g, while TPC in bound fractions were found higher in Mushki budgi (0.476  mg GAE/g) followed by Mushki kandi (0.465 mg GAE/g). In general, aromatic rice cultivars were having higher free TPC than the non-aromatic rice cultivar Koshkari containing 0.855 mg GAE/g of TPC (Table 1). The variations in the total phenolic contents of free and bound fractions as extracted by using different solvents could be attributed to the polarities of different compounds present in the cultivars. The increased amount of free phenolic contents in the aromatic rice cultivars indicated that these posses higher potential bioavailability and hence were having higher antioxidative capacity. Several researchers have shown great interest in polyphenols present in rice due to their diverse biological functionalities. These phenolics have been reported to act as reducing agents due to their hydrogen donating abilities. They also play essential role in quenching singlet oxygen species and donors of free radical hydrogen ions. It is due to these properties that the phenolics protect the cell constituents against oxidative and free radical damage. These phenolics thus have an essential role in ensuring therapeutic properties by acting as anti-carcinogenic besides preventing cardiovascular and neurological disease [20]. Mir et al. [21] reported the TPC of hybrid rice cultivars of Kashmir in the range of 0.81–1.64 mg GAE/g. The results of present research were either comparable or higher than

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Table 1  Free, bound and total phenolic contents (TPCs) and total flavonoid contents (TFCs) of the traditional pigmented aromatic and nonaromatic rice cultivars Cultivars

Total phenolic content (mg GAE/g)

Total flavonoid contents (mg RUE/g)

Methanol (free)

Acetone + H2O (free)

Bound

Total

Methanol (free)

Acetone + H2O (free)

Bound

Total

Mushki budgi

0.567 ± 0.001b

0.581 ± 0.002c

0.476 ± 0.002a

1.624 ± 0.003b

2.575 ± 0.002b

1.283 ± 0.001c

0.343 ± 0.002a

4.201 ± 0.002b

Mushki kandi

0.578 ± 0.003a

0.587 ± 0.002b

0.465 ± 0.002b

1.630 ± 0.003a

2.658 ± 0.003a

1.296 ± 0.002b

0.310 ± 0.002b

4.264 ± 0.002a

0.245 ± 0.001

c

a

c

c

1.993 ± 0.002

c

1.525 ± 0.003

a

0.273 ± 0.002

c

3.791 ± 0.003c

0.204 ± 0.002

d

1.525 ± 0.002

d

0.695 ± 0.003

d

0.207 ± 0.003

d

2.427 ± 0.003d

Kamad Koshkari

1.111 ± 0.002

d

0.468 ± 0.002

0.241 ± 0.002

d

0.183 ± 0.002

1.597 ± 0.003

d

0.855 ± 0.002

Results are mean (±SD) of three independent determinations. Values in a row with different letters superscript are significantly different (p ≤ 0.05)

the reported results. The values of bound TPC contents in the analyzed aromatic cultivars were found higher, 29.31% in Mushki budgi and 28.52% in Mushki kandi, as compared to non-aromatic Koshkari containing only 21.40% of bound phenolics. The food containing higher percentage of bound phenolics have been reported to reach the human colon without digestion in stomach and small intestines, wherein these bound phenolic may exert their beneficial effect either in colon or at other sites after absorbing in the blood stream [22]. The bound phenolics are hydrolyzed by the extracellular enzymes secreted by microorganisms, (Bifidobacterium spp. and Lactobacillus spp.) resulting in the liberation of phenolics, which promote a number of health benefits including suppression of the growth of cancer-causing microorganisms [23]. Thus consumption of these aromatic rice cultivars helps to prevent the colon, breast and prostate cancer as validated through epidemiological studies and play an important role in ensuring antioxidant property [24]. Free, bound and total flavonoid contents (TFC) assay Flavonoids represent the secondary metabolites in plants with excellent antioxidant properties and health benefits [25]. The variation in flavonoid contents among different aromatic rice cultivars could be attributed to their genotypic variation and growing environmental conditions. The TFC in free fractions as determined by methanol and acetone + H2O were found higher than the bound fractions and were a major contributor to total flavonoids as calculated by summing free and bound fractions of TFC. Among the aromatic rice cultivars Mushki kandi (4.264 mg RUE/g) was having higher TFC whereas cultivar Kamad was observed possessing lesser TFC value of 3.791 mg RUE/g. However, the TFC content in the non-aromatic cultivar Koshkari was found lowest (2.427 mg RUE/g) (Table 1). As a dietary component, flavonoids have been found to possess health-promoting properties due to their higher antioxidant properties in both in vivo and in vitro systems and are having the ability in inducing protective enzyme systems in humans [26].

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Flavonoids have been suggested to protect the lipids against oxidative damage by various mechanisms [27]. The bound flavonoid content in the aromatic rice cultivars was found higher than the non-aromatic rice cultivar, Koshkari. Among the aromatic ones, the highly aroma generating Mushki budgi (0.343 mg RUE/g) and Mushki kandi (0.310 mg RUE/g) contained higher TFC in bound fractions. The bound flavonoids had been reported to act stronger bioactive components in suppressing of cancer, inflammation, type-2-diabetes and cardiovascular diseases [22]. The bound phenolic and flavonoid compounds are not extracted by solvent extractions and are thus obtained by alkaline hydrolysis that releases the bound phenolic compounds trapped in the cell walls, dietary fibers or proteins [28]. Rice has been regarded a potential source of bound flavonoids containing 240 μg catechin equivalents per gram of DW [29]. The bound flavonoids are transported towards the colon in which flavonoid glycosides are hydrolyzed by the residing bacteria along with the liberated flavonoid aglycones [30]. The flavonoid contents in the present aromatic rice cultivars were found higher than the aromatic rice varieties analyzed by Saikia et al. [31]. Flavonoids have been reported to play an important role in regulating several biological functions by acting as antiviral, antibacterial, anti-inflammatory, antithrombotic, anti allergic and possess free radical scavenging properties [32]. Volatile component profile of rice cultivars Typical GC–MS chromatograms of the three aromatic rice cultivars along with one non-aromatic cultivar Koshkari (as reference), indicating the location of volatile profiles are shown in Fig. 2a–d. The graphs showed a significant difference in terms of their peaks representing particular volatile aromatic component. The concentration of the different aromatic components could be validated from their peak area measurements. The aromatic cultivars were found possessing excellent desirable aromatic compounds in sufficient quantities (Table 2). Volatile compound are secondary metabolites that are determined by genetic control of

Extraction and in vitro antioxidant capacity evaluation of phenolic compounds from pigmented…

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Fig. 2  Typical GC–MS graphs of volatile components in aromatic rice cultivars of Kashmir: a Mushki budgi, b Mushki kandi, c Kamad, d Koshkari 

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Table 2  Head space GC–MS analysis of components profile of traditional pigmented aromatic and non-aromatic rice cultivars Chemical name

X X X X

X

X

X

Aldehydes  3-Methyl-butanal  Hexanal  Heptanal  Benzaldehyde  Octanal

Aroma

RT

Mushki budgi Mushki kandi Kamad Koshkari Previously reported

Malty flavor Green tomato, green, grasslike Fatty, rancid, fruity Almond like Citrus, fruity, floral, and fatty Rose–orange odor Woody and fruity

2.22 4.48

2.35 23.38

 Nonanal  Pentanal Esters  Ethyl 2-hydroxy-2-methylpropanoate  Isopropyl 2-hydroxypropanoate  Acetic acid, butoxyhydroxy-, butyl ester Ketones  2-Acetyl-1-pyrroline Popcorn, sweet, and pleasant  1-Methoxyacetone  1-Phenyl-ethanone Almond, jasmine, cherry and strawberry Carboxylic acid  Acetic acid Vinegar like, pungent and sour smell Alkanes  2-Methyl-pentane Mild gasoline odor  Hexane  Trichloro-methane Sweet-smelling ethereal odor  Dichloromethane Mild sweet odour Furans  2-Pentylfuran Floral, fruit, nutty, and bean Alcohol  1,3-Butanediol Sweet flavoring agent  2-Propanol Alcohol odour, stuffy odour

3.91 7.70

3.19 14.84

– 25.12

NRE [33, 37, 56]

8.40 2.54 11.26 1.47 13.51 2.38

– 2.28 –

– – –

– – –

[37, 55] [56] [37, 56]

18.85 3.96 2.60 5.10

3.54 3.26

3.93 –

– –

2.47

2.13

3.61

–

–

[56] [54] NRE NRE

1.57

–

–

37.96

2.32

–

–

2.74

–

NRE

2.43

4.07

6.43

–

–

NRE [33, 37, 55, 56]

1.57 15.03 16.70 1.67

22.59 5.07

– 2.53

– –

NRE NRE

6.48

12.87

7.49

NRE NRE NRE NRE NRE NRE

–

NRE

1.73 1.85 2

2.48 9.02 8.76

4.92 7.33 8.60

– – 6.35

– – 14.64

1.71

–

–

–

8.76

12.71 13.78

6.41

10.26

5.78

2.62 1.58

– –

10.70 –

– 45.70

– –

NRE NRE [37, 55, 56] NRE [56] NRE

X = compounds identified earlier in rice; NRE not reported earlier

the different cultivars and thus different rice cultivars are expected to possess different volatile aromatic compounds [33]. The main flavoring component (2-AP) reported by Buttery et al. [2], present in aromatic rice varieties had been found in the analyzed aromatic rice cultivars except Kamad and non-aromatic Koshkari. Brown rice has been reported contain higher amount of 2-AP than milled rice and was found decreases with increase in storage periods [34]. The sweet flavoring agent, 1,3-butanediol was found in the aromatic cultivar Kamad. The other compounds identified in the analyzed rice cultivars included, the 7-aldehydes, 3-esters, 3-ketones, 4-alkanes, 1-carboxylic acid and 2-alcohols.

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Among the 21 identified aromatic components aldehydes were found present in higher content followed by alkanes, ketones and esters. Aldehydes had been reported to comprise most of the volatile flavoring components in the aromatic rice varieties validated earlier by Yang et  al. [35]. Among the aromatic rice cultivars, Mushki budgi and Mushki kandi were found having higher contents of volatile flavoring components. An important flavoring compound identified in the aromatic cultivar Mushki budgi and Mushki kandi was benzaldehyde, an aldehyde that has been associated with a pleasant almond like odour and is considered as

Extraction and in vitro antioxidant capacity evaluation of phenolic compounds from pigmented…

safe (GRAS) by the FDA for using as flavoring additive in a diverse range of food products [36]. Benzaldehyde has been reported to act as carcinostatic and retards or inhibits the growth of cancerous tumors. The aldehyde heptanal associated with fatty, rancid and fruity flavour was found unique for the rice cultivar Mushki budgi at retention time of 8.40  min. The furan, 2-pentyl-furan having characteristic flavor as of floral, fruit, nutty, and beany were also found present in all the aromatic cultivars including non-aromatic one with maximum peak area in aromatic cultivars Mushki budgi (13.78%) and Kamad (10.26%), while the non-aromatic Koshkari retained lesser peak area of 5.78%. Dichloromethane, an alkane found in the non aromatic Koshkari has been associated with mild sweet odour. It has been reported that milled aromatic rice varieties lose volatile components as the grains are exposed to the environmental factors and thus reduces the aroma generating capacities [6]. The characteristic aroma of raw or cooked rice has been reported due to the generated due the blend of various volatile compounds and not of a single volatile [1]. Of the three ketone compounds present, in addition to 2-AP, the 1-phenyl-ethanone and ketone were found in the present aromatic rice cultivars that are having the significant effect on generating aroma with characteristics odors of almond, jasmine, cherry and strawberry. These compounds also act as antimicrobials and thus enhance the shelf life of these rice cultivars. Alkanals, alk-2-enals, 2-pentylfuran and 2-phenylethanol are important compounds that have been found responsible for fragrance of rice cultivars and contribute to the total aromatic profile [37]. Among the three aromatic cultivars, Mushki kandi depicted higher peak area of 5.07% under the ketone. It is due to the presence of these pleasant and sweet smelling aromatic components, which ensure the increasing demand of these cultivars and motivates the consumers to fetch premium prices for these aromatic rice cultivars.

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Relationship of volatile (aromatic), free and bond phenolic components with the in vitro antioxidant capacity DPPH radical scavenging activity assay The DPPH radical scavenging activity method is considered as the better in vitro model to check the efficiency of the sample within a very short period of time. The antioxidant activity of the rice cultivars as determined by DPPH method is based on the mechanism of electron transfer and determines the reducing capacity of antioxidants in the rice kernels. The DPPH scavenging activity varied from 69.453 to 85.907% in the aromatic rice cultivars as depicted in Table 3. The DPPH activity in free fractions was found higher in aromatic rice cultivar Mushki kandi (81.857%) and lowest in the non-aromatic Koshkari (53.013%), whereas the highest DPPH activity in bound fractions was found in the aromatic rice cultivar Kamad (6.72%). The higher DPPH scavenging activity in free fractions as determined by methanolic and acetone + H2O extracts from aromatic rice cultivars could be attributed to the efficiency of these different polar extracting solvent in extracting antioxidant compounds from the respective cultivars [38]. Higher DPPH scavenging activities have been reported by Sultana et al. [39] to be linked with higher antioxidant capacity and consequently, the higher antioxidant activity in these aromatic rice cultivars could be related to their higher phenolic content [40]. The antioxidant capacity evaluated on the basis of DPPH activity is based on single electron transfer and thus determines the reducing capacity of antioxidant [41]. Considerable interest has been shown in determining the radical scavenging activity of rice varieties due to their potential health benefits by reducing the concentration of free radicals and reactive oxygen species that are having adverse effects on cells resulting in cellular damage, cancer, aging and several diseases [42]. The DPPH radical scavenging activities of the aromatic rice cultivars were found higher than the brown rice varieties of temperate areas as reported previously by Mir et al. [20].

Table 3  DPPH scavenging activity and phosphomolybdenum assay of extracted compounds (free and bound) of traditional pigmented rice cultivars Cultivars

Mushki budgi Mushki kandi Kamad Koshkari

DPPH radical scavenging activity (%)

Phosphomolybdenum reduction assay (mM AA/ml)

Methanol (free)

Acetone + H2O Bound (free)

Total

Methanol (free)

Acetone + H2O Bound (free)

Total

25.930 ± 0.02d 26.317 ± 0.01c 37.583 ± 0.02a 28.760 ± 0.02b

54.713 ± 0.01b 54.840 ± 0.02a 25.150 ± 0.02c 24.253 ± 0.01d

85.896 ± 0.05b 85.907 ± 0.01a 69.453 ± 0.06c 57.763 ± 0.03d

0.696 ± 0.02c 0.924 ± 0.01a 0.853 ± 0.03b 0.472 ± 0.02d

0.614 ± 0.01c 0.716 ± 0.02a 0.683 ± 0.01b 0.391 ± 0.03d

1.543 ± 0.02c 1.893 ± 0.03b 1.913 ± 0.03a 1.016 ± 0.03d

5.253 ± 0.01c 4.750 ± 0.02d 6.72 ± 0.003a 4.750 ± 0.01b

0.233 ± 0.01c 0.253 ± 0.03b 0.377 ± 0.01a 0.153 ± 0.01d

Results are mean (±SD) of three independent determinations. Values in a row with different letters superscript are significantly different (p ≤ 0.05)

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F. M. Bhat, C. S. Riar

Reducing power assay The antioxidant capacity of the experimental rice cultivars estimated by reducing power revealed that aromatic rice cultivars had greater reducing power in their free and bound fractions than the non-aromatic cultivar Koshkari. Among the aromatic rice cultivars, Mushki kandi and Mushki budgi were found to have higher total reducing potential of 1.834 and 1.858 (OD). The reducing power in bound fractions were found higher in Kamad (0.07) followed by Mushki kandi (0.06, OD) as shown in Table 4. In determination of antioxidant capacity by reducing power, the reducers that is antioxidant in the extract results in the formation of Prussian blue color due to the conversion of the ­Fe3+/ferricyanide complex into ferrous (­ Fe3+) form having maximum absorbance at 700 nm [43]. Since this method is based on the higher activity of antioxidants compounds by increasing the absorbance of reaction mixture, therefore the higher absorbance of a compound is associated with high antioxidant activity and hence their uses in diet as a natural source of antioxidant. The reducing power of these aromatic rice cultivars could be attributed to some phytochemical compounds in these rice cultivars having potent donating capabilities and hence antioxidant properties [44]. The reducing power of a rice cultivar could serve an important indicator of its antioxidant activity and are generally due to the presence of reductones that had been reported to exert antioxidant property by terminating the free radical chains by donating hydrogen atoms and quenching of oxygen radicals [45]. These reductones had also been reported to inhibit the peroxide formation by reacting with certain precursors of peroxide [46]. Phosphomolybdenum assay (PMA) The estimation of antioxidant activity by phosphomolybdenum assay shows uniqueness among various in  vitro antioxidant assays due to non induction of metal ions solution. It determine the total antioxidant capacity of rice cultivars by ensuring reduction of Mo(VI) to Mo(V) by the antioxidant compounds resulting into the formation of a

green phosphate/Mo(V) complex having maximum absorbance at 695 nm. The total antioxidant capacity (TAC) as determined by phosphomolybdenum assay measures both water and fat soluble antioxidants. PMA of the aromatic rice cultivars was observed higher than the non-aromatic cultivar Koshkari as shown in Table 3. The PMA of the methanolic extracts was found higher than acetone + H2O and bound fractions. Among the aromatic rice cultivars Mushki kandi was found having higher PMA in the free fractions depicting higher total PMA of (1.893 mM AA/ ml). The PMA in the bound fractions was found higher in Kamad (0.277 mM AA/ml) and lowest in the non-aromatic Koshkari (0.153  mM  AA/ml). PMA is the quantitative method to investigate the reduction rate between the antioxidants and the molybdenum reagent, thus indicating the strength of reducing capacity of antioxidant. Among the given rice cultivars, the aromatic cultivar Kamad showed higher reducing capacity (1.913 mM AA/ml) followed by Mushki kandi (1.893 mM AA/ml), which contributed to an increasing levels of health protection by inhibiting oxidative damage [43]. PMA have been reported to be correlated with phenolic content [47] and the antioxidant properties in rice had been reported due to phenolic compounds present in rice that act as reducing agents, hydrogen donor and singlet oxygen scavengers [48]. The aromatic compounds in rice cultivars could be considered as essential factors in determining the efficiency of antioxidants as phenolics alone cannot be sole criterion for determining antioxidant capacity [49]. Lipid peroxidation inhibition assay The inhibitions of lipid peroxidation determined in these cultivars were observed maximum in free fractions of methanol and acetone + H2O. Among the rice cultivars, the aromatic cultivar Mushki budgi was found having highest (62.237%) lipid peroxidation inhibition capacity and the non-aromatic Koshkari retained the lowest inhibition capacity of 30.447% as given in Table 4. The aromatic cultivar Kamad (0.440%) possessed higher inhibition of lipid peroxidation in bound fractions. Egg yolk lipids involved in lipid peroxidation

Table 4  Lipid peroxidation inhibitions and reducing power of extracted compounds (free and bound) of traditional pigmented rice cultivars Cultivars

Mushki budgi Mushki kandi Kamad Koshkari

Lipid peroxidation inhibition (%)

Reducing power (OD, 700 nm)

Methanol (free) Acetone + H2O (free)

Bound

Total

Methanol (free)

Acetone + H2O (free)

Bound

Total

16.530 ± 0.03b 14.730 ± 0.02c 26.457 ± 0.01a 16.570 ± 0.03b

0.350 ± 0.02b 0.253 ± 0.02d 0.440 ± 0.02a 0.327 ± 0.02c

62.237 ± 0.04a 61.546 ± 0.02b 41.630 ± 0.03c 30.447 ± 0.06d

0.970 ± 0.03a 0.953 ± 0.02b 0.874 ± 0.05c 0.523 ± 0.03d

0.834 ± 0.02a 0.815 ± 0.01b 0.713 ± 0.03c 0.463 ± 0.01d

0.05 ± 0.05c 0.06 ± 0.03b 0.07 ± 0.01a 0.04 ± 0.05d

1.858 ± 0.05a 1.834 ± 0.03b 1.660 ± 0.05c 1.028 ± 0.03d

45.357 ± 0.02b 46.563 ± 0.01a 14.733 ± 002c 13.550 ± 0.02d

Results are mean (±SD) of three independent determinations. Values in a row with different letters superscript are significantly different (p ≤ 0.05)

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Extraction and in vitro antioxidant capacity evaluation of phenolic compounds from pigmented… Fig. 3  Projections of the variables as of principal component analysis (PCA) on the factor plane for seven rice cultivars. MB Mushki budgi, MK Mushki kandi, ace acetone, RP reducing power, LP lipid peroxidation inhibition

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2

Kamad Kamad Kamad

1.5

TPCAce+H2O LP methanol PMAbound DPPHmethanol RPbound LPbound TFCace+H2O

F2 (38.94 %)

1

PMAace+H2O PMAmethanol

DPPHbound

RPmethanol RPace+H2O

0.5

TFCbound 0

TFCmethanol MB MB MB MK MK MK

-0.5

TPCbound TPC methanol DPPHace+H2O LPace+H2O

Koshkari Koshkari Koshkari

-1 -2

-1.5

-1

-0.5

0

0.5

1

1.5

2

F1 (56.16 %)

inhibition test undergoes non-enzymatic peroxidation upon mixing with ferrous sulphate instantly that in turn produces malonodialdehyde along with other aldehydes leading to the formation of pinkish red chromogen with maximum absorbance at 532 nm [50]. The products of lipid peroxidation formed due to oxidation of polyunsaturated lipids by free radicals such as malondialdeyde, 4-hydroxy 2-nonenal had been reported to have harmful effects on the functioning and biochemistry of cells, as these cause permeability of cell membrane, destruction of cellular constituents, alter ions transportation across membranes and inhibits metabolic processes causing carcinogenic effects [51, 52]. The higher values of lipid peroxidation inhibition in aromatic rice cultivars depicts, that these rice cultivars play an important role to inhibit the free radical induced damage on the cellular bi-layer membrane thus ensure the proper functioning of cells. The inhibition of lipid peroxidation exhibited by these rice cultivars could be attributed to the phytochemical content present in these cultivars. The higher lipid peroxidation inhibition in aromatic rice cultivars could be attributed to their higher stability against undesirable changes like lipid auto-oxidation that leads to the deterioration of quality of rice grains by producing rancid and stale flavours [53]. The consumption of rice cultivars possessing higher lipid peroxidation inhibition could be linked with prevention of various pathological diseases [54]. Multivariate analysis

the viability of correlation among the various variables assessed in the research and validates the accuracy of the results recorded by experimentations. From the PCA analysis of the relationship between experimental factors and variable (Fig. 3), the Factor-1 (F1) revealed a variability of 56.16%, having positively correlatiion with TPC, TFC and reducing power in all the fractions along with DPPH activity and lipid peroxidation in acetone + H2O fractions which justified the higher antioxidant capacity of highly aroma producing cultivars in the present study. The second component (­ F2 = 38.94%) showed a positive correlation with DPPH scavenging activity and lipid peroxidation inhibition in methanol and bound fractions. These variables evaluated by PCA graph provided an important criterion in differentiating these cultivars from each other. The highly aromatic cultivars Mushki budgi and Mushki kandi positioned on the left side of component ­F2 were linked to TPC, TFC and antioxidant components largely, while the less aromatic Kamad positioned on the right side of F1, were found to correlate with only two variables of antioxidant components viz. DPPH activity and lipid peroxidation. This validated results of the given research wherein the less aromatic cultivar showed lesser antioxidant property as compared to highly aromatic cultivars. The negative correlation associated with the non-aromatic cultivar (Koshkari) can be attributed to its lesser antioxidant capacity as depicted by the results obtained in the present research.

The principal components analysis (PCA) was done in order to differentiate the rice cultivars on the basis of their parameters estimated in this research. PCA also indicates

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Conclusion Present research revealed the sufficient in vitro antioxidant potential of aromatic rice cultivars which was higher than the non-aromatic cultivar used as reference. The TPC, TAC and antioxidant activity were found higher in free fractions as compared to bound fractions. The antioxidant capacity of the experimental rice cultivars revealed that aromatic rice cultivars possessed greater antioxidant activity in their free and bound fractions than the non-aromatic cultivar Koshkari. The 21-volatile compounds detected by GC–MS analysis included, 7-aldehydes, 3-esters, 3-ketones, 4-alkanes, 1-carboxylic acid and 2-alcohols. The non-aromatic cultivar, Koshkari accounted for the least number of volatile aromatic components while the aromatic cultivars Mushki budgi had the largest number of volatile aromatic compounds. The main aromatic compound 2-AP was found absent in nonaromatic cultivar Koshkari and in aromatic cultivar Kamad, that justified the lesser aroma producing characteristics of Kamad despite having some of the important aromatic volatile compounds like nonanal, 1-phenyl-ethanone, trichloromethane, 2-pentylfuran and 1,3-butanediol. PCA revealed that the highly aromatic cultivars Mushki budgi and Mushki kandi correlated to TPC, TFC and in vitro antioxidant capacity largely, while the less aromatic Kamad correlate with two variables of antioxidant components viz. DPPH and lipid peroxidation. Acknowledgements  The authors are highly thank full for the rice breeding station Khudwani, (SKAUST) for providing traditional aromatic paddy cultivars in order to reveal the desirable bioactive components from these cultivars and their excellent antioxidative properties. Authors are also thankful to SLIET, Longowal, for providing TEQIP-II fellowship for research help. Funding  Funding was provided by MHRD, India. Compliance with ethical standards  Conflict of interest  The authors declare that they have no conflict of interest.

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