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  Genetic diversity in chestnuts of Kashmir valley Some date palm cultivars grown in Pakistan were biochemically characterized and the effect of fruit maturity on radical scavenging capacity (DPPH), total phenolic contents (TPC), specific activity of antioxidant enzymes, sugars profile and soluble protein contents was assessed. Higher range of differences in composition of studied phytochemicals was recorded among different cultivars. Antiradical efficiency (AE), TPC, antioxidant enzymes and soluble protein contents were recorded higher at khalal stage thereafter, declined at rutab then finally at tamar stage. The amount of glucose (11.32-32.50%) and fructose (10.95-32.41%) started accumulation from khalal stage and were in higher composition at tamar stage due to hydrolysis and inversion of sucrose (10.82-3.1%) contents. The results concluded that variation in biochemical attributes  primarily influenced by type of cultivars and different fruit developmental stages. Keywords:  Dates, fruit maturity, antioxidants, sugars, total phenolic contents INTRODUCTION Date palm is a diploid (2n=36), perennial, monocoty-ledonous and diecious plant in family Arecaceae which comprises of 183 genera and 2600 species (Dowson, 1982; Dransfield et al  ., 2008).  Phoenix  is one of the genera which consist of 14 species, all native to the tropical and subtropical regions of Southern Asia or East and North Africa (Shengji et al  ., 2010). It is leading fruit tree in many countries and is an important source of food, sugar, nutrients and antioxidants. Currently, it is cultivated in the Middle East, North Africa, parts of Central and South America, Southern Europe, India and Pakistan (Chandra et al  ., 1992; Zaid, 1999; Al-Shahib and Marshall, 2003). Pakistan is the 6 th  largest producer of dates after Egypt, Saudi Arabia, Iran, Algeria and Iraq, with a production of 557279 tons (FAO, 2011). Date palm growth is affected by different factors (Afzal et al  ., 2011; Ata et al  ., 2012). Important dates  producing provinces are Sindh (Khairpur and Sukkur), Balochistan (Makran and Panjgoor), Khyber Pakhtunkhwa (D. I. Khan) and Punjab at Jhang, Muzafargarh, Bahawalpur and D. G. Khan (Markhand et al  ., 2010). Dates fruit is berry and oblong with fleshy mesocarp and fibrous endocarp (Mansour, 2005) which constitute 85-90% of total date fruit weight (Hussein et al  ., 1998). There are five developmental stages in fruits after pollination based on change in color, texture, aroma and flavor. Internationally accepted date fruit developmental stages are in Arabic terms, viz. Hababouk (immature and pea size), kimri (green, hard, contains 80% moisture and 50% reducing sugars (glucose and fructose), khalal (colored stage, crunchy, moisture contents upto 50-60%), rutab (ripe stage, soft texture, crisp to succulent, moisture content is 35-40%) and tamar (full ripe, dry flesh, moisture content less than 20%) (Al-Shahib and Marshall, 2003; Fadel et al  ., 2006). Dates can be harvested at khalal stage depending on sugar content, i.e. sweetness, climatic conditions and market demand. Date  palm is affected by X-irradiation (Al-Enezi et al  ., 2012). Fresh dates are nutritionally superior and delicious than dried dates (Vinson et al  ., 2005; Al-Farsi et al  ., 2005). Date fruits possessed a variety of enzymatic and non- enzymatic antioxidant compounds with varying amount in different cultivars (Mansouri et al  ., 2005; Biglari et al  .  ,  2008; Awad et al  ., 2011a). The significance of antioxidants has been increasing because of their high capability in scavenging free radicals associated to variety of many damaging diseases (Silva et al  ., 2007). Chemically, dates are considered rich source of sugars (mainly reducing) and  protein contents (Amira et al  ., 2011; Rastegar et al  ., 2102) as compared to other fruits. The chemical composition of date palm varies within cultivars at different levels of maturity. It is therefore, ten date palm cultivars were selected to investigate their biochemical composition with an objective to characterize the superior cultivars with specific nutritional value at different developmental stage. MATERIALS AND METHODS Plant material  :  The fruits of ten cultivars, i.e. Zerin, Jaman, Pela Dora, Rachna, Seib, Zardo, Shado, Peli Sunder, Wahan    Pak. J. Agri. Sci., Vol. 50(4), 577-583; 2013  ISSN (Print) 0552-9034, ISSN (Online) 2076-0906 FRUIT DEVELOPMENTAL STAGES EFFECTS ON BIOCHEMICAL ATTRIBUTES IN DATE PALM M. Salman Haider 1 , Iqrar A. Khan 1 , Summar A. Naqvi 1,* , M.J. Jaskani 1 , Rashad W. Khan 1 , M. Nafees 1,2 , Maryam 1  and Imran Pasha 3   1 Institute of Horticultural Sciences, University of Agriculture, Faisalabad-38040, Pakistan; 2 University College of Agriculture & Environmental Sciences, The Islamia University of Bahawalpur, Pakistan; 3 Food Safety Lab., National Institute of Food Science and Technology, University of Agriculture, Faisalabad, Pakistan * Corresponding author:   Haider, Khan, Naqvi, Jaskani, Nafees, Maryam & Pasha 578 Wali and Champa Kali were selected at three different developmental stages (khalal, rutab and tamar) during 2012 harvesting season from the Date palm Research Station, Jhang, Pakistan. Extraction of date flesh  :  Flesh of fruits (0.5g) of each cultivar was taken at each khalal, rutab and tamar stage and grinded in mortar by pestle with 2 ml methanol solution (95% v/v) at room temperature (25 o C±4) as described by Ainsworth and Gillespie (2007) to measure antioxidant activity and total phenolic contents. The extraction of date flesh in potassium phosphate buffer (pH: 7.0) was carried out as described by Naqvi et al  . (2011) for estimation of specific activity of enzymes (catalase and peroxidase) and soluble protein contents. The extracts were filtered and centrifuged at 13,000x  g  , at 4 o C for 5 minutes. The residues were discarded and the supernatant were separated and stored at 4 o C until use. Radical scavenging assay (DPPH)  :  The antioxidant activity of the flesh of date fruits was assessed by measuring their scavenging abilities to 2, 2-diphenyl-1-picrylhydrazyl stable radicals as described by Amira et al  . (2012). The absorbance was read against a blank at 517 nm using micro-plate ELISA reader (BioTek, USA). Inhibition of free radical by DPPH in  percent (%) was calculated by following formula: I % = (A  blank   -A sample /A  blank  ) × 100   Where A  blank   is the absorbance of the control reaction mixture excluding fruit sample, and A sample  is the absorbance of the test compounds. IC 50  values, which represented the concentration of date fruit extracts that caused 50% neutralization of DPPH radicals, were calculated from the  plot of inhibition percentage against concentration. Total phenolic contents (TPC)  :  TPC was calculated by using Folin-Ciocalteu reagent method as reported by Ainsworth and Gillespie, (2007). The FC-reagent (10 mL) was dissolved in distilled water to make the solution 100 mL. In each sample (100 mL), FC-reagent (200 µL) was added and vortex thoroughly. The 700 mM Na 2 CO 3 (800 µL) was added into each sample and incubated at room temperature for 2 h. Sample (200 µL) was transferred to a clear 96-well plate and absorbance of each well was measured at 765 nm. Amount of TPC was calculated using a calibration curve for Gallic acid. The results were expressed as Gallic acid equivalent (GAE) per dry matter.  Enzymatic antioxidant activity  :  The specific activity of antioxidant enzymes (catalase and peroxidase) were quantified using the method of Naqvi et al  . (2011). Changes in absorbance of the reaction solution of catalase (CAT) and  peroxidase (POD) were read at 240 nm and 470 nm, respectively.   Sugars profiling (HPLC)  :  Different Sugar levels were measured using high-performance liquid chromatography (HPLC) technique as described by Amira et al  . (2011). Sugars were extracted from date flesh (10g) in distilled water (20 mL) for 10 min (exciting repeatedly through magnetic stirrer in assistance dissolving the sugars). The extracts were then centrifuged at 13000x  g   for 10 min and the supernatants were separated. Each sample was filtered over 0.45 µm membrane filters and analyzed by high  performance liquid chromatography (HPLC).   Liquid chromatography conditions  :  LC separation was carried out at room temperature on a Razex RCM-Monosaccharidses Ca +2 , Phenomenex. The mobile phase was 100% double distilled water. The HPLC was connected to a refractive index detector (R  e ID) RID-10 AL (Shimadzu, Japan). The flow rate and injection volume were 1 mL/min and 20 µL, respectively. Identified sugars were quantified on the basis of peak areas of external standards consisting of glucose (1%), fructose (1%) and sucrose (1%) solutions. Total reducing sugars were obtained as a sum of glucose and fructose values. Each sample was carried out from integrated  peak areas of the sample against the corresponding standard graph. Results were expressed as percentage of dry weight.   Soluble protein contents  : The soluble proteins of the fruit extracts were determined by Bradford method (Bradford, 1976) and absorbance was taken at 595 nm. Statistical analysis  :  The experimental values were analyzed statistically using one-way analysis of variance (ANOVA) with three replications of each treatment. Means were compared using Duncan’s Multiple Range (DMR) test at 5%  probability with the help of MINITAB (15.0). RESULTS AND DISCUSSION Antiradical efficiency (DPPH)  :  There was a significant difference between antiradical efficiency (AE) of all selected date palm cultivars through the different developmental stages (Fig.1). Rachna cultivar showed the highest antiradical efficiency (3.63, 2.39 and 1.91 AE), followed by cv. Zardo cultivar (3.60, 2.56 and 1.17 AE at khalal, rutab and tamar stages, respectively. The cv. Wahan Wali showed lower (1.72, 1.33 and 1.21 AE) values at khalal, rutab and tamar stage, respectively. Selected date cultivars showed declining trend and depicted that AE was higher at khalal then gradually decreases at rutab and lower value was observed at tamar stage. The values and trend of current study was similar to those found by Awad et al  . (2011a). Some reports referred that AE of date fruits was higher at khalal stage (Allaith, 2008; Amoros et al  ., 2009) which support the obtained results. Saafi et al  . (2009) evaluated some Tunisian ripe fruits and found similar AE to those found in this study. The agro-climatic conditions may responsible for the found variation in the final values of the current study. Total phenolic concentration (TPC)  : Total phenolic contents (TPC) of studied date fruits showed significant (p < 0.05) variation in the final values within cultivars and  between stages of development as presented in Fig.2. Highest TPC was recorded in Jaman cultivar   Date palm fruit analysis 579 (569.63<281.53<183.97 mg GAE/100g, DW), followed by cv. Rachna (508.91<265.58<159.84 mg GAE/100g, DW) and lowest values (348.9<169.88<59.73 mg GAE/100g, DW) was recorded in cv. Peli Sunder at khalal < rutab < tamar stage, respectively. It is often described that the values of TPC decreases from khalal to tamar stage and showed  positive correlation with antioxidant activity of the date fruits in relation to maturity (Al-Turki et al  ., 2010; Awad et al  ., 2011a). This decrease in TPC is due to the oxidation of TPC by polyphenol oxidase (Amiot et al  ., 1995) and decline in tannins as the dates matured to the tamar stage (Myhara et al  ., 1999). Pakistani dates showed similar values to date cultivars from America and Saudi Arabia (Al-Turki et al  ., 2010) and Tunisia (Amira et al  ., 2012). Figure 1. Antiradical efficiecy of ten date varities at three developmental stages Figure 2. Total phenolic contents of ten date varities at three developmental stages Specific activity of antioxidant enzymes  :  The specific activity of antioxidant enzymes (catalase and peroxidase) was initially higher at khalal stage then sharply decreased during rutab and finally lowest at tamar stage in all cultivars. The specific activity of catalase (CAT) was higher at khalal in cvs. Peli Sunder and Zardo, afterwards sharply decreased at rutab in cvs. Seib and Wahan Wali cultivars then absolute decrease was observed in cultivars Zerin and Seib at tamar stage (Fig.3). Similarly, specific activity of peroxidase (POD) was higher at khalal in cvs. Wahan wali and Champa Kali culivars then gradually decrease at rutab in cvs. Champa Kali and Rachna. Final decrease of Specific POD activity was recorded in cvs. Zardo and Peli Sunder at fully ripened (tamar) stage (Fig.4). Awad et al  . (2011a) studied antioxidant enzymes (CAT and POD) and depicted that their activity was higher at khalal stage and thereafter, declined at rutab and tamar stage, respectively and was in good agreement to those found in our studied cultivars. Figure 3. Specific activity of catalase of ten date varities at three developmental stages Figure 4. Specific activity of peroxidase of ten date varities at three developmental stages   Haider, Khan, Naqvi, Jaskani, Nafees, Maryam & Pasha 580 Estimation of sugars (HPLC)  :  The amount and composition of most important sugars are shown in Table 1. Analysis of variance revealed significant (p<0.05) variations between the values of sucrose and reducing sugars (glucose and fructose) among all selected date cultivars and within maturation stages. The quantity and type of sugars vary within cultivars and at stages of maturity. The amount of reducing sugars ranged (22.26-42.24 %), (30.58-55.14 %) and (38.31-64.91 %) at khalal, rutab and tamar, respectively; whereas, sucrose contents ranged (12.82-19.9 %) at khalal stage and some minor quantity were also detected at rutab stage. The cv. Seib possessed higher total sugar contents but cv. Peli Sunder showed lower values at all edible stages of date fruit development. The sucrose contents were not detected at tamar stage due to hydrolysis and later conversion into reducing sugars by the invertase enzyme (Al-Farsi and Lee, Table 1. Sugar profile of ten date palm cultivars at three different developmental stages as quantified by HPLC method Cultivars Ripening stage Sucrose (%) Glucose (%) Fructose (%) Reducing Sugars (% G/F2 Zerin Khalal 10.82±0.6j 16.65±1.1h 14.47±0.5h 31.12±0.2h 1.15 Rutab 5.76±0.2d 20.81±0.4g 20.25±1f 41.04±0.9f 1.02 Tamar nd1 25.52±0.87f 24.91±0.3f 50.43±0.2f 1.02 Jaman Khalal 15.59±1.2c 20.32±1.13b 18.32±0.8c 38.64±1.1c 1.10 Rutab 4.7±0.8h 24.57±0.3c 24.02±0.4b 48.58±0.3b 1.02 Tamar nd 30.06±0.4b 29.65±0.7b 59.7±0.7b 1.01 Pela Dora Khalal 12.82±0.5h 17.00±1.06g 15.56±1.1g 32.59±0.9g 1.09 Rutab 6.2±0.7c 20.50±0.5h 19.66±0.9g 40.16±0.7g 1.04 Tamar nd 24.91±0.9h 23.58±0.7h 48.49±0.2h 1.06 Rachna Khalal 13.79±0.3g 13.10±0.99i 11.55±0.03i 24.65±0.8i 1.13 Rutab 4.8±0.01g 16.55±1i 15.90±0.2i 32.45±0.3i 1.04 Tamar nd 21.95±0.7i 19.94±0.4i 41.89±1.01i 1.10 Seib Khalal 19.89±0.7a 21.79±0.9a 20.45±0.5a 42.24±0.9a 1.06 Rutab 5.21±0.2e 28.74±1.1a 26.4±1.3a 55.14±0.5a 1.09 Tamar nd 32.50±1.02a 32.41±1a 64.91±0.03a 1.00 Zardo Khalal 15.71±1.2b 17.65±1.1f 17.21±0.9d 34.86±1e 1.03 Rutab 3.1±1j 23.54±0.7d 22.61±0.7c 46.15±0.7d 1.04 Tamar nd 27.33±0.98e 26.81±1.2c 54.14±0.4d 1.01 Shado Khalal 15.16±0.4d 20.23±0.8c 19.1±0.6b 39.33±1.3b 1.05 Rutab 6.4±0.3b 24.82±0.3b 21.69±0.3d 46.51±1.2c 1.14 Tamar nd 29.30±0.2c 26.6±1.01d 55.9±0.6bc 1.10 Peli Sunder Khalal 14.55±0.02f 11.32±1.02j 10.95±1.3j 22.26±1.3j 1.03 Rutab 4.32±1.1i 15.83±0.6j 14.76±1j 30.58±0.7j 1.07 Tamar nd 20.1±0.9j 18.21±0.3i 38.31±1j 1.10 Wahan Wali Khalal 14.66±0.9e 17.89±1.03e 16.8±0.7e 34.67±0.9f 1.06 Rutab 5.31±0.3f 21.34±1e 20.48±0.7e 41.82±0.7e 1.04 Tamar nd 27.65±0.2d 25.93±0.8e 53.59±0.3e 1.06 Champa Kali Khalal 12.26±0.1i 18.74±1.2d 16.97±1f 35.7±1.1d 1.10 Rutab 7.32±0.2a 20.95±0.2f 18.35±0.5h 39.29±0.2h 1.10 Tamar nd 25.16±1.1g 23.76±0.2g 48.91±0.9g 1.06   Date palm fruit analysis 581 2008). Amira et al  . (2011) quantified the sugars from Tunisian date cultivars and reported similar findings of reducing sugars at all developmental stages. Values of reducing sugars in our results were similar to those cultivars reported by (Vayalil, 2011; Rastegar et al  ., 2012).  Quantification of soluble protein contents  :  The soluble  protein contents of date fruits are expressed in g/100 g (Fig.5). The soluble protein contents were found higher at khalal stage and substantially declined at rutab then tamar stage, respectively. The soluble protein contents ranged (5.22-5.68 g/100g), (3.67-4.56 g/100g) and (2.87-3.42 g/100g) at khalal, rutab and tamar stages respectively. The Zardo and Rachna cultivars shower higher protein contents at khalal, the cvs. Wahan Wali and Jaman at rutab and cvs. Zardo and Peli Sunder at tamar stage. This declining trend of soluble proteins may be due to its degradation by free radicals as the radical scavenging system declines during Figure 5. Soluble protein contents of ten date cultivars at three developmental stages maturity (Prochazkova et al  ., 2001) and the rising activities enzymes like proteases (Rastegar et al  ., 2012). The soluble  protein contents in this study decreases from kimri to tamar stage of date fruit maturity that is in good agreement to the date cultivars reported by Awad et al  . (2011b). Conclusion  :  This study revealed that exotic date palm germplasm available in Pakistan are the potential source of radical scavenging capability, comparable reducing sugars and protein contents compared to other famous local date  palm cultivars like Aseel and Dhakki. These cultivars can be  brought under consideration for cultivation as commercial varieties in future. Acknowledgement  :  We are thankful to the Pakistan Agriculture Research Board (PARB) and International Center for Development and Decent Work (ICDD) program, Germany-Pakistan for providing financial assistance. 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Antioxidant activity of Bahraini date  palm (  Phoenix dactylifera  L.) fruit of various cultivars. Int. J. Food Sci. & Tech. 43:1033-1040. Al-Shahib, W. and R.J. Marshall. 2003. The fruit of the date  palm: Its possible use as the best food for the future. Int. J. Food Sci. & Nut. 54:247-259. Al-Turki, S., M.A. Shahba and C. Stushnoff. 2010. Diversity of antioxidant properties and phenolic content of date  palm (  Phoenix dactylifera  L.) fruits as affected by cultivar and location. J. of Food, Agric. & Environ. 8:253-260. Amiot, J.M., M. Tacchini, S.Y. Aubert and W. Oleszek. 1995. Influence on cultivars, maturity stage and storage conditions on phenolic composition and enzymatic  browning of pear fruit. J. Agric. Food Chem. 43:1132-1137. Amira, E.A., F. Guido, S.E. Behija, I. Manel, Z. Nesrine, F. Ali, H. Mohamed, H.A. Noureddine and A. Lotfi. 2011. Chemical and aroma volatile compositions of date palm
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