(Cuminum Cyminum L.) Seed Extract Under Drought

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Polyphenol Composition and Antioxidant Activity of Cumin (Cuminum Cyminum L.) Seed Extract Under Drought C: Food Chemistry

Iness Bettaieb Rebey, Nesrine Zakhama, Iness Jabri Karoui, and Brahim Marzouk

Abstract: This research evaluated the effect of drought on total and individual polyphenol contents as well as the antioxidant activities of cumin (Cuminum cyminum L.) seeds of 2 geographic origins, Tunisia (TCS) and India (ICS). Plants were treated with different levels of water deficit: control. Our results indicated that, in both varieties, moderate water deficit (MWD) improved the number of umbels per plant as well as the number of umbellets per umbel and the seed yield, in comparison to the control, but it decreased under severe water deficit (SWD). Besides, total phenolic contents were higher in the treated seeds and drought increased the level of total and individual polyphenols. This increase was appreciably more important in TCS than in ICS. Moreover, antioxidant activities of the extracts were determined by 4 different test systems, namely 2,2-diphenyl-1-picrylhydrazyl, β-carotene/linoleic acid chelating, and reducing power assays, and showed that treated seeds exhibited the highest activity, for both TCS and ICS. Keywords: antioxidant, Cuminum cyminum L., drought, geographic origin, polyphenols, seeds

Introduction Drought stress caused by soil and atmospheric water deficiency is one of the most significant environmental factors affecting plant growth and productivity worldwide. However, there are reports on the positive effect of limited water supply, as far as the biosynthesis of secondary metabolites, enzyme activities, and solute accumulation is concerned (Singh-Sangwan and others 2001). Some of these responses may be associated with the capability of the plant to survive under constraining conditions. The consumption of herbal medicines is widespread and is continuously increasing worldwide. Drought led to biochemical disorders and can change plant behavior regarding the biosynthesis of primary and secondary metabolites. Among all the secondary metabolites synthesized by plants, phenolic compounds are some of the most widespread. In plants, polyphenol biosynthesis and accumulation are generally stimulated in response to biotic/abiotic constraints (Naczk and Shahidi 2004). Consequently, one may hypothesize that optimal polyphenol yield would be obtained using stress-tolerant species (De Abreu and Mazzafera 2005). Many health-related properties, including anticancer, antiviral, antiinflammatory activities, antioxidant properties, effects on capillary fragility, and an ability to inhibit human platelet aggregation, have been ascribed more particularly to phenolics (Spignoli 2000). The development and utilization of more effective antioxidants of natural origin could, therefore, afford potential benefits for the optimization of human health (Panico and others 2005). Cumin (Cuminum cyminum L.) is a small annual and herbaceous plant belonging to the Apiaceae family. It is one of the popular

spices regularly used as a flavoring agent. It is cultivated in Arabia, India, China, and in the countries bordering the Mediterranean Sea (Thippeswamy and Naidu 2005). Cumin seeds are used as a spice for their distinctive aroma, popular in Indian, Pakistani, North African, Middle Eastern, Sri Lankan, Cuban, Northern Mexican cuisines, and the Western Chinese cuisines of Sichuan and Xinjiang (Daniel and Maria 2000). C. cyminum seeds have been used for treatment of toothache, dyspepsia, diarrhoea, epilepsy, and jaundice (Nostro and others 2005). In general, cumin is adapted to dry conditions and since its growth period coincides with winter and springs rainfall, there is no need for extra irrigation. However, where there are prolonged dry periods supplementary irrigation is effective. Excess water may reduce yield due to spread of fungal diseases (Tavosi 2000). After all the above, in order to strengthen the valorization of the Tunisian variety as a new source of antioxidant compounds, we compared it with the Indian variety by investigating their polyphenol compositions and their antioxidant activities under water deficiency conditions. The results will be important to indicate the drought effect and geographic origin on the phenolic compounds biosynthesis.

Material and Methods

Plant material and growth conditions Two varieties of mature green cumin (C. cyminum L.) seeds were used in this work. Tunisian cumin seeds (TCS), were collected from cultivated plants in the region of Menzel Temim (North East of Tunisia) on July 2009, while Indian cumin seeds (ICS) were purchased from a herbal market in Menzel Temime and were reported to be imported from India. The 2 provenances MS 20120100 Submitted 1/18/2012, Accepted 3/17/2012. Authors are with were cultivated under the same environmental conditions. The Laboratoire des Substances Bioactives Centre de Biotechnologie a` la Technopole de BorjC´edria (CBBC), BP 901, Hammam-Lif, Tunisia. Direct inquiries to author Rebey seeds were sown manually in 10l pots, filled with agricultural soil containing 0.22, 0.34, 0.05, and 0.08 meq (100 g)−1 of dry soil of (E-mail: [email protected]). Na+ , K+ , Ca2+ , Fe2+ , respectively.

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Journal of Food Science r Vol. 77, Nr. 6, 2012

R  C 2012 Institute of Food Technologists doi: 10.1111/j.1750-3841.2012.02731.x

Further reproduction without permission is prohibited

Composition and antioxidant activity . . .

Preparation of extracts Seed extracts were obtained by stirring 1 g of dry material powder with 10 mL of 80% acetone for 30 min. Extraction was carried out using maceration at room temperature for 24 h followed by filtration and after evaporation to dryness. Samples were stored at 4 ◦ C until analysis. Total phenolic amounts One hundred and twenty-five microliters of sample extract were dissolved in 500 μL of distilled water and 125 μL of Folin–Ciocalteu reagent (Dewanto and others 2002). The mixture was shaken, before addition of 1.25 mL of 7% Na2 CO3 , adjusting with distilled water to a final volume of 3 mL, and mixed thoroughly. After incubation in the dark for 90 min, the absorbance at 760 nm was measured versus the prepared blank. Total phenolic amounts were expressed as milligrams of gallic acid equivalents per gram of dry weight (mg GAE/g DW), through a calibration curve with gallic acid. Analysis of phenolic compounds Twenty milliliters of acetone 80% containing butylated hydroxytoluene (BHT) (1 g/L) were added to 0.5 g of a dried sample. Then 10 mL of 1 M HCl were added. The mixture was stirred carefully and sonicated for 15 min and refluxed in a water bath at 90 ◦ C for 2 h (Proestos and others 2006). The obtained mixture was injected to high-performance liquid chromatography (HPLC). The phenolic compound analysis was carried out using an Agilent Technologies (Waldbronn, Germany) 1100 series liquid chromatograph (RP–HPLC) coupled with an UV-Vis multiwavelength detector. The separation was carried out on a 250 × 4.6-mm, 4-μm Hypersil ODS C18 reversed phase column at ambient temperature. The mobile phase consisted of acetonitrile (solvent A) and water with 0.2% sulfuric acid (solvent B). The flow rate was kept at 0.5 mL/min. The gradient program was as follows: 15% A/85% B 0 to 12 min, 40% A/60% B 12 to 14 min, 60% A/40% B 14 to 18 min, 80% A/20% B 18 to 20 min, 90% A/10% B 20 to 24 min, 100% A 24 to 28 min. The injection volume was 20 μL, and peaks were monitored at 280 nm. Samples were filtered through a 0.45-μm membrane filter before injection. Peaks were identified by congruent retention times compared with standards. Analyses were performed in triplicate.

was used as positive reference while methanol was used as negative reference (Hanato and others 1998). DPPH radical-scavenging activity was expressed as the inhibition percentage (I%) and was calculated using the following formula: I% = 100 × (Ablank − Asample)/Ablank, where Ablank is the absorbance of the control at 30 min reaction and Asample is the absorbance of the sample at 30 min.

β-carotene/linoleic acid bleaching assay A stock solution of β-carotene/linoleic acid mixture was prepared by dissolving 0.5 mg of β-carotene in 1 mL of chloroform and adding 40 mg of linoleic acid together with 400 mg of Tween 40. Chloroform was completely evaporated using a vacuum evaporator. Then 100 mL of oxygenated distilled water was added to the residue; 3 mL of this mixture was dispensed to test tubes and 200 μL of each extract was added. The emulsion system was incubated for 2 h at 50 ◦ C, together with 2 controls, one containing BHT as a positive control and another with the same volume of distilled water instead of the extracts. In the test tube with BHT, the yellow color is maintained during the incubation period, and the absorbance was measured at 470 nm (Tepe and others 2004). Chelating effect on ferrous ions Different concentrations of the sample were added to 0.05 mL of FeCl2 ·4H2 O solution (2 mM) and left for incubation at room temperature for 5 min. Then, the reaction was initiated by adding 0.1 mL of ferrozine (5 mM), and the mixture was adjusted to 3 mL with deionized water, shaken vigorously, and left standing at room temperature for 10 min. Absorbance of the solution was then measured spectrophotometrically at 562 nm (Zhao and others 2006). The percentage of inhibition of ferrozine-Fe2+ complex formation was calculated using the formula given below: Metal chelating effect (%) = [(A0 − A1 )/A0 ] × 100, where A0 is the absorbance of the ferrozine-Fe2+ complex and A1 is the absorbance of the test compound. Results were expressed as IC50 , efficient concentration corresponding to 50% ferrous iron chelating. Ethylene diamine tetraacetic acid (EDTA) was used as a positive control.

Reducing power One milliliter of different concentrations of organ extracts and essential oils in acetone 80% were mixed with 2.5 mL of a 0.2 M sodium phosphate buffer (pH = 6.6) and 2.5 mL of 1% potassium ferricyanide (K3 Fe (CN)6 ), and incubated in a water bath at 50 ◦ C for 20 min. Then, 2.5 mL of 10% trichloroacetic acid was added to the mixture that was centrifuged at 650 g for 10 min. The supernatant (2.5 mL) was then mixed with 2.5 mL distilled water and 0.5 mL of 0.1% ferric chloride solution. The intensity of the blue-green color was measured at 700 nm. The EC50 value (mg/mL) is the extract concentration at which the absorbance was 0.5 for the reducing power and was calculated from the graph of absorbance at 700 nm against extract concentration (Oyaizu 2,2-Diphenyl-1-picrylhydrazyl radical scavenging assay Two milliliters of the extract at different concentrations were 1986). added to 0.5 mL of a 0.2 mM 2,2-diphenyl-1-picrylhydrazyl (DPPH) methanolic solution. After shaking, the mixture was in- Statistical analysis cubated at room temperature in the dark for 30 min, and then the Data were subjected to statistical analysis using statistical program absorbance was measured at 517 nm. Butylated hydroxyanisole package STATISTICA. Percentage of each volatile compound and Vol. 77, Nr. 6, 2012 r Journal of Food Science C735

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During vegetative stage, plants were irrigated with tap water, and then divided into 3 lots subjected to different water levels: 100% (control [C]), 50% (moderate water deficit [MWD]), and 25% (severe water deficit [SWD]) of field capacity (FC). This later was determined in the pots by weight. Regular weightings (every 3 d) enabled to restore the moisture of soil at 100%, 50%, or 25% FC. The plant weight was neglected. Experiments were carried out in a greenhouse with a 14 h photoperiod (photosynthetic photon flux density, PPFD: 400 mol m−2 s−1 ) and lasted 6 mo from January 2010 to June 2010. Mean temperature and relative humidity were, respectively, 30 ± 5 ◦ C, 55 ± 5% at day and 16 ± 2 ◦ C, 90 ± 5% at night. After harvest, seed were air-dried and stored at 4 ◦ C until use for further analysis.

Composition and antioxidant activity . . .

C: Food Chemistry

fatty acids were the mean of 3 replicates ± SD and the differ- TCS were 1.28-fold higher compared to ICS (Table 2). TPC was ences between individual means were deemed to be significant at significantly increased when plants subjected to water deficit treatment. Thus, under MWD, TPC were about 56.82% and 40.20% P < 0.05. higher than the control one, correspondingly, for TCS and ICS. Besides, TPC were increased significantly by 1.21 and 1.13 folds Results and Discussion under SWD for TCS and ICS, respectively. On the other hand, the Effect of water deficit on yield components assessment of phenolic compounds as determined by RP-HPLC Our results indicated that MWD improved the number of um- was 12.37 and 8.18 mg/g DW in the control samples. An increase bels per plant as well as the number of umbellets per umbel. In in polyphenol content has been found under the 2 treatments as addition, both the 1000 seed weight and the seed yield per plant determined by this method. Thus, under MWD, total polyphenol increased under MWD for the 2 provenances (Table 1). In this content was about 1.48 and 1.74 times higher than that observed in context, Karam and others (2007) reported that a variety of crops control plants, respectively, for TCS and ICS. Besides, it increased have been found to benefit from deficit irrigation strategy and significantly by 24.41 and 56.84% under SWD. According to these that the application of drought at early seed formation to matu- results, the contents of phenolic compounds as assessed by RPrity induced a slight increase in seed yield of sunflower (Heliantus HPLC are too inferior to those obtained by the Folin–Ciocalteu annuus L.). Drought stress during flower development decreases method. These differences could be explained by the weak selecseed numbers and can also increase the duration from seed-set to tivity of the Folin–Ciocalteu reagent, as it reacts positively with full seed growth. Long duration of spikelet development and high different antioxidant compounds (Bettaieb and others 2010). spike weight at anthesis were positively correlated with final grain However, these 2 methods confirmed that the total phenolics yield in wheat under drought conditions (Bindraban and others content of cumin seeds was improved proportionally to the water 1998). For cereal crops, longer periods of vegetative and repro- deficit intensity and this improvement suggested the drought tolductive development are often necessary to improve reproductive erance of cumin. Depending on the water treatment, the amount potential (number of productive tillers and kernels) and also leaves of TPC of cumin seeds was highly (P < 0.05) affected by the and tillers to provide assimilate supply during the grain filling. origin of varieties. Tunisian variety was the rich one in TPC. Studies have also shown that decreased leaf area due to drought Likely, phenolic contents of sage (Salvia officinalis L.) and cumin before anthesis is correlated with reductions in the number of aerial parts content, exposed to drought were enhanced considkernels per spike (Frederick and Camberato 1995). erably (Bettaieb and others 2010; Bettaieb and others 2011). The Furthermore, yield components of the 2 ascensions are affected TPC of sprouts treated with 100 mM of NaCl was significantly by SWD (Table 1). Thus, the number of umbels and the number increased, which is similar to that of Cakile maritima and red of umbellets per umbel of Tunisian and Indian varieties decreased pepper reported by Ksouri and others (2007) and Navarro and significantly under SWD. Concomitant with the decrease in 1000 others (2006). Conversely, in the case of coriander (Coriandrum seed weight SWD, the seed yield decreased. As a consequence, sativum L.), Naffeti and others (2010) found a significant decrease this parameter pointed out variance from 3.03 to 6.62 g and 2.26 in polyphenol accumulation under different levels of salinity. to 5.14 g, respectively for TCS and ICS. This could be associated with more vegetative growth, and hence, lowers allocation of Quantitative and qualitative analysis of phenolic nutrients to the seeds. Similarly, drought caused a large decrease compounds by RP-HPLC in seed yield of sesame (Sesamum indicum L.). One cause of seed Qualitative and quantitative differences were found between the yield reduction under drought is inadequate photosynthesis owing 2 accessions (Table 3). TCS contained more phenolic acids than to stomata closure and consequently limited carbon dioxide uptake (Zhu 2001). In addition, Karam and others (2007) reported that the application of deficit irrigation at early seed formation Table 2– Water deficit effect on total phenolic contents, flavoto maturity induced a slight increase in seed yield of sunflower noids, and condensed tannins of Tunisian and Indian cumin seeds. (H. annuus L.). These results confirmed the idea that cumin is adapted to a Folin–Ciocalteu Hyrdolysis acid moderate dryness and not sensitive to drought such as caraway C TCS 18.60 ± 0.03A,c 12.37 ± 0.01A,c (Laribi and others 2009) and many other aromatic crops (Dunford ICS 14.50 ± 0.01B,c 8.18 ± 0.01B,c and Vasquez 2005; Moeini Alishah and others 2006). MWD TCS 29.17 ± 0.11A,a 18.43 ± 0.07A,a

Effect of water deficit on total phenolic content The total phenolic content (TPC) of control plants as estimated by the Folin–Ciocalteu method was of 18.60 and 14.50 mg of GAE/g DW, respectively, for TCS and ICS. TPC extracted from

SWD

20.33 ± 0.16B,a 22.51 ± 0.05A,b 16.50 ± 0.01B,b

ICS TCS ICS

14.27 ± 0.12B,a 15.39 ± 0.06A,b 12.83 ± 0.01A,B,b

The data marked with the different capital letter, for the provenance, and small letter, for the treatment, in the table, value share significant differences at P < 0.05 (Duncan test).

Table 1–Water deficit effect on yield components in Tunisian and Indian cumin seeds.

C MWD SWD

TCS ICS TCS ICS TCS ICS

Number of umbels/plant

Number of umbellets/umbel

1000 Seed weight (g)

Seed yield per plant (g)

3.53 ± 0.02 3.23 ± 0.01b,A 5.84 ± 0.09a,A 6.01 ± 0.08a,A 2.38 ± 0.01b,c,A 2.12 ± 0.01b,c,A

4.01 ± 0.11 3.94 ± 0.03b,A 5.23 ± 0.04a,A 5.32 ± 0.02a,A 3.26 ± 0.01b,A 3.13 ± 0.01b,A

6.62 ± 0.01 5.14 ± 0.01a,b,A 7.86 ± 0.02a,A 6.03 ± 0.01a,b,A,B 3.03 ± 0.05b,A 2.26 ± 0.03b,A

2.43 ± 0.02a,b,A 2.10 ± 0.02a,b,A 3.04 ± 0.07a,A 2.74 ± 0.04a,A 1.74 ± 0.01b,A 1.32 ± 0.01b,A

b,A

b,A

a,b,A

The data marked with the different capital letter, for the provenance, and small letter, for the treatment, in the table, value share significant differences at P < 0.05 (Duncan test).

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phenolic acids, which resulted in the enhancement of their contents by about 1.35 and 1.73 folds, respectively, for TCS and ICS. Hence, the level of p-coumaric acid increased TCS and ICS by 1.33 and 2.06 folds, consecutively, as compared to the control. Besides a significant increase was observed in the level of rosmarinic and trans-2-dihydrocinnamic acids for both varieties under moderate treatment. On the other hand, results demonstrated that also SWD enhanced significantly the biosynthesis of phenolic acids with the exception of dihydroxybenzoic acid. Analysis of the effect of drought on flavonoids contents of cumin seeds indicated that MWD improved significantly the accumulation of these metabolites by about 1.94 and 1.62 folds, respectively, for TCS and ICS. Moderate stress improved appreciably the biosynthesis of all the flavonoids as compared to the control. As well, SWD enhanced significantly the content of flavonoids by 45.13% and 59.32%. This increase was expressed mainly by the augmentation of the contents of lutelin and coumarin for the 2 varieties as compared to control. However we noticed that accumulation of flavonoids under

ICS (9.49 mg/g DW and 6.41 mg/g DW, respectively). However, TCS and ICS contained comparable flavonoids than ICS (2.88 mg/g DW and 1.77 mg/g DW, respectively). A total of 19 phenolic compounds were successfully identified in TCS and ICS. Independently of the applied treatments, p-coumaric acid was detected as the major phenolic acid for the 2 ascensions and was more present in TCS (4.83 mg/g DW) compared to ICS (2.33 mg/g DW). TCS showed higher proportions of luteolin (1.29 mg/g DW), syringic acid (0.64 mg/g DW), and even cinnamic acid (0.94 mg/g DW), compared to ICS. However, Indian seeds were richer in trans-2-dihydrocinnamic acid (1.20 mg/g DW) and flavone (0.61 mg/g DW) than TCS. Polyphenol composition and contents in plant foods can vary greatly according to many factors such as plant genetics, soil composition and growing conditions, stage of maturity, and postharvest conditions (Faller and Fialho 2009). Results indicated that, omit dihydrobenzoic acid, MWD resulted in a significant increase of the biosynthesis of the different

Table 3– Quantitative (mg/g DW) changes of phenolic compounds in Tunisian and Indian cumin seed extracts as influenced by drought.

Phenolic acids Gallic acid Cafeic acid Dihydroxyphenolic acid Dihydroxybenzoic acid Chlorogenic acid Syringic acid Vanillic acid p-Coumaric acid Ferrulic acid Rosmarinic acid Trans-2-dihydrocinnamic acid Cinnamic acid Flavonoids Luteolin Catechin Coumarin Quercetin Apigenin Amentoflavone Flavone Unknown Total

TCS ICS TCS ICS TCS ICS TCS ICS TCS ICS TCS ICS TCS ICS TCS ICS TCS ICS TCS ICS TCS ICS TCS ICS TCS ICS TCS ICS TCS ICS TCS ICS TCS ICS TCS ICS TCS ICS TCS ICS TCS ICS TCS ICS TCS ICS

C

MWD

SWD

9.49 6.41 0.09 ± 0.02a,b,A 0.09 ± 0.01a,A 0.07 ± 0.00b,B 0.22 ± 0.05a,A 0.02 ± 0.00a ,B 0.20 ± 0.03a,b,A 0.39 ± 0.02a,A 0.08 ± 0.01b,B 0.22 ± 0.01a,A 0.06 ± 0.01b,B 0.64 ± 0.02a,A 0.06 ± 0.01b ,A,B 0.03 ± 0.01b ,A,B 0.35 ± 0.02b,A 4.83 ± 0.11a,b,A 2.33 ± 0.07b ,B 0.47 ± 0.03b,A 0.14 ± 0.04b,A 0.70 ± 0.04a,A 1.04 ± 0.03a,b,A 1.09 ± 0.41a,A 1.20 ± 0.03a,A 0.94 ± 0.02a,A 0.27 ± 0.00a,A 2.88 1.77 1.29 ± 0.24b,A 0.39 ± 0.11a,b,A,B 0.23 ± 0.02a,A 0.52 ± 0.03a,A 0.04 ± 0.01b,A 0.11 ± 0.05a,A 0.02 ± 0.01b,A 0.08 ± 0.01b,A 0.03 ± 0.00b,A 0.03 ± 0.01b,A 0.01 ± 0.01b,A 0.03 ± 0.00a,A 0.12 ± 0.02a,b,A,B 0.61 ± 0.07a,A 1.14 ± 0.32a,A,B 3.58 ± 0.21a,A 13.51 11.76

12.83 11.09 0.30 ± 0.01a,A 0.05 ± 0.01a,A,B 0.24 ± 0.00a,A 0.17 ± 0.03a,A 0.07 ± 0.01a,A 0.08 ± 0.01b,A 0.12 ± 0.02a,b,A 0.05 ± 0.01b,A 0.18 ± 0.04a,A 0.13 ± 0.01b ,A 0.70 ± 0.01a,A 0.48 ± 0.13a,A 0.19 ± 0.03a,A 0.52 ± 0.04b,A 6.47 ± 0.09a,A 4.80 ± 0.05a,A,B 0.80 ± 0.05a,A 0.52 ± 0.01a,b,A 1.06 ± 0.14a,A,B 2.29 ± 0.77a,A 1.50 ± 0.02a,A 1.72 ± 0.01a,A 1.20 ± 0.06a,A 0.20 ± 0.01a,A,B 5.60 3.18 3.14 ± 0.03a,A 0.93 ± 0.01a ,B 0.46 ± 0.01a,A 0.18 ± 0.01a,b,A 0.68 ± 0.04a,A 0.35 ± 0.02a,A 0.20 ± 0.03a,A 0.20 ± 0.01a,A 0.33 ± 0.01a,A 0.30 ± 0.01a,A 0.17 ± 0.01a,A 0.02 ± 0.00a,A 0.52 ± 0.05a,A 0.20 ± 0.01a,A 0.77 ± 0.03a,B 2.03 ± 0.06a,b,A 19.20 16.30

11.21 10.01 0.17 ± 0.02a,b 0.05 ± 0.01a 0.20 ± 0.04a,A 0.15 ± 0.01a,A 0.03 ± 0.01a,A,B 0.47 ± 0.11a,A 0.06 ± 0.01c,A,B 0.33 ± 0.12a,A 0.04 ± 0.01b,A,B 0.78 ± 0.02a,A 0.62 ± 0.12a,A 0.49 ± 0.01a,A 0.15 ± 0.01a,B 1.22 ± 0.15a,A 5.03 ± 0.44a,b,A 4.51 ± 0.62a,A 1.10 ± 0.05a,A 0.80 ± 0.01a,A 1.01 ± 0.02a,A,B 2.03 ± 0.09a,A 1.72 ± 0.04a,A 1.80 ± 0.13a,A 1.08 ± 0.03a,A 0.20 ± 0.05a ,B 4.18 2.82 2.08 ± 0.11a,b,A 1.02 ± 0.02a,B 0.18 ± 0.01a,A 0.80 ± 0.01a,A 1.00 ± 0.03a,A 0.50 ± 0.02a,A,B 0.10 ± 0.01a,A 0.25 ± 0.01a,A 0.22 ± 0.02a,A 0.20 ± 0.01a,A 0.26 ± 0.04a,A 0.04 ± 0.00a,A,B 0.33 ± 0.01a 0.01 ± 0.00b,A,B 1.08 ± 0.03a,A 1.72 ± 0.08a,b,A 16.47 14.55

The data marked with the different capital letter, for the provenance, and small letter, for the treatment, in the table, value share significant differences at P < 0.05 (Duncan test).

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Composition and antioxidant activity . . .

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23.65 ± 0.87 28.12 ± 0.09a,b,A 32.75 ± 1.43b,A 77.05 ± 0.05 140.80 ± 0.04b,B 154.77 ± 0.01a,b,B

a,A

TCS

65.86 ± 0.23 42.12 ± 0.97a,A 60.23 ± 0.54b,A 15.14 ± 0.04 7.25 ± 0.01a,A,B 12.66 ± 0.01b,B

– – 43 ± 0.56

a,B

ICS

b,A

TCS

0.18 ± 0.01

C MWD SWD EDTA Ascorbic acid BHT

6.24 ± 0.64 5.03 ± 0.06a,A 5.83 ± 0.02a,b,A

c ,B

ICS

a,b,A

TCS

IC50 : the concentration of the extract generating 50% inhibition; EC50 : the effective concentration at which the absorbance was 0.5; The data marked with the different capital letter, for the provenance, and small letter, for the treatment, in the table of each IC50 or EC50 value share significant differences at P < 0.05 (Duncan test).

19.73 ± 0.83 74.52 ± 0.22b,B 102.13 ± 0.14c ,B 0.03 ± 0.01 – –

– 40 ± 0.84 –

110.34 ± 3.74 72.11 ± 1.34b,A 50.23 ± 0.77a,A

ICS ICS

a,A

TCS

c,A

Reducing power (EC50 , μg/mL) Chelating ability (IC50 , mg/mL) β-Carotene bleaching (IC50 , μg/mL) DPPH (IC50 , μg/mL)

Effect of water deficit on antioxidant activity The DPPH radical scavenging activities of the acetonic extracts obtained from the TCS extracts showed the highest capacity to neutralize this radical. Moreover, the IC50 values obtained were 6.24 ± 0.64 μg/mL and 15.14 ± 0.15 μg/mL Tunisian and Indian varieties, respectively. The IC50 of BHT was equal to 0.18 ± 0.01 μg/mL. In this study, DPPH radical scavenging activity of test samples was in the order BHT > Tunisian variety > Indian variety. By analyzing the antioxidant activity of the different extracts under different levels of drought, it was demonstrated that they all had the capacity to scavenge DPPH free radicals (Table 4). Indeed, DPPH scavenging activity increased significantly by 19.40% and 52.11% under MWD, respectively, for TCS and ICS, as compared to the control. In the same way, the IC50 values of TCS and ICS augmented by about 6.57% and 16.83%, correspondingly, under SWD. Moreover, the DPPH radical scavenging activity was strongly (P < 0.05) affected by the cumin varieties. BenaventeGarcia and others (2000) reported that the radical scavenging activity of plant extracts depends on the amount of polyphenolic compounds in the extracts. The rate of β-carotene bleaching can be slowed down in the presence of antioxidants. TCS had the strongest ability to prevent the bleaching of β-carotene than ICS (IC50 = 65.87 μg/mL and IC50 = 77.05 μg/mL, respectively). On the other hand, TCS and ICS extracts had lower antioxidant activities than BHT with IC50 of 43 μg/mL (Table 4). Water deficit improved significantly the TCS activity by about 36.04% under MWD and still unchanged under SWD, in comparison to the control. Conversely, the aptitude to prevent the bleaching of β-carotene of ICS extracts was found to be altered significantly under MWD and SWD by about 1.82 and 2 folds, as compared to control. Thus, the β-carotenelinoleate bleaching values were highly (P < 0.05) affected by the provenance of varieties. Independent of treatment applied, both provenances presented high chelating power with IC50 of 23.65 and 19.73 mg/mL as well as a strong reducing capacity with IC50 of 110.34 and 93.87 mg/ mL, respectively, for TCS and ICS. Besides drought had no significant effect on the chelating capacity of the extracts of TCS in comparison to control. Nevertheless it was found to be damaged significantly for the ICS by 3.77 and 5.17 folds, respectively, under MWD and SWD. Finally, we should point out that TCS and ICS extracts were able to reduce Fe3+ ions in the reaction medium. Hence, the reducing power evaluation showed that both MWD and SWD increased considerably the reducing ability by 1.42 and 2.04 folds, for TCS, and 1.37 and 1.64 folds, for ICS, respectively. The reducing property is generally associated with the presence of reductones (Duh 1998), such as ascorbic acid, which have been shown to exert antioxidant action by breaking the free radical chain (Gordon 1990). On the other hand, statistical analysis revealed no effect (P < 0.05) of varieties on reducing power. TPC has been reported to be responsible for the antioxidant activities of botanical extracts. DPPH, β-carotene-linoleate

Table 4–Effect of water deficit on antioxidant activities of Tunisian and Indian cumin seed extracts.

C: Food Chemistry

SWD is less pronounced than that observed under MWD. These variations were related to the relative contents of the constituents and not to the presence of new ones or the absence of particular components. These results suggest the stimulation of isoprenoids pathway by drought, while shikimate and phenypropanoid pathways were enhanced. In general, phenolic compounds in plants are produced through the phenylpropanoid pathway, and they can be induced by environmental stresses and elicitor (Kim and others 2006; Giorgi and others 2009).

93.87 ± 2.11b,A 68.14 ± 1.19a,A 57.22 ± 1.22a,A

Composition and antioxidant activity . . .

bleaching assay, chelating ability, and reducing power have been used to measure antioxidant activity and these results should correlate with those of TPC. Do and others (2004) demonstrated that some bioactive compounds present in medicinal plant possessed high total antioxidant activity, which was due to the presence of phenolic, carotenoids, and flavonoids. In this way, drought can lead to increased production of free radicals and other oxidative species in plants, which respond by increasing their capacity to scavenge reactive oxygen species (ROS), phenolics being very significant in this field (Mittler 2002). Besides, plant resistance to various stresses is associated with antioxidant capacity and increased levels of antioxidants may prevent constraints damage (Bor and others 2003). These activities may be directly linked to the seed phenol contents and consequently to their free radical scavenging properties (Huang and others 2005), since phenolic compounds contribute directly to antioxidant activity. In comparison with our result, previous reports showed a significant correlation between the antioxidant activity and TPCs in C. sativum (Neffati and others 2010) and in Cakile maritime (Ksouri and others 2007).

Conclusion Our results confirmed the idea that cumin seeds are adapted to a moderate dryness. Thus MWD improves seed yield and yield component parameters, in comparison to control, for the 2 provenances. Additionally, drought caused a significant increase of the TPC, which was more pronounced in MWD than that observed under SWD. Finally, antioxidant activities of both TCS and ICS were positively correlated with their phenolic contents which increased under drought and which varied notably with regards to cumin varieties. Antioxidant activity of extracts from many plants are of great interest in the food industries, since their possible use as natural additives emerged from a growing tendency to replace synthetic preservatives with natural ones and this by manipulating agricultural techniques.

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Vol. 77, Nr. 6, 2012 r Journal of Food Science C739

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Composition and antioxidant activity . . .

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