It is now good recognized that ingestion of selected fruits and veggies is strongly linked with the reduced hazard of chronic diseases such as malignant neoplastic disease, redness, neurological and cardiovascular upsets and other degenerative diseases. [ 1 ] Antioxidant foods including vitamins, carotenoids and polyphenolics with multiple biological activities are chiefly responsible for the good wellness effects of fruits and veggies. [ 2 ] Phenolic antioxidants are deriving go oning attending due to their efficaciousness in antagonizing free groups, linked with assorted diseases. Plants phenoplasts are considered relatively more stable and are available as active phytochemicals for utilizations in different nutrient merchandises to protect them from oxidization and heightening shelf-life. On the other manus, carotenoids and vitamin C being heat sensitive are rather unstable and due to their more susceptibleness to impairment, have restricted applications in processed nutrients. [ 3 ]
Presently, there is a go oning demand of fresh or minimally processed nutrients to acquire more alimentary constituents through diet. Fruits and veggies are available in fresh signifier for really short periods, and hence, they are sooner stored at low temperatures, sometimes dried and processed into different points like juices, canned nutrients and jams, etc. under best suited conditions for retaining maximal alimentary value. It is good accepted that the conditions of storage and processing exhibit noteworthy consequence on the concentration of familial phytochemicals, particularly, carotenoids, phenolic antioxidants and vitamin C of fruits and veggies. [ 4 ] Drying/dehydration has become relatively efficient manner to minimise nutritionary losingss and microbic growing, therefore taking to heightening shelf-life of fruits.5 Effect of drying on fruit quality is non to the full understood, nevertheless, major alterations in fruits occur when they are exposed to drying at elevated temperature or at low temperature for longer periods. It is by and large considered that besides the chemical and nutritionary alterations, assorted physiological and pharmacological changes in fruit cell-wall polyoses can besides impact other quality attributes including texture, and colour of the fruits. [ 5, 6 ]
Several drying techniques are being used to dry fruits, some of which are really dearly-won and time-consuming. Fruits are by and large dried under the Sun or by utilizing an unreal drier. [ 7 ] Some literature studies show promising effects of UV irradiation on the quality of the fruits as compared to other non-thermal fruit continuing techniques. [ 8, 9 ] It was found that sun-drying may increase the antioxidant activity and entire phenolic contents of the fruits. [ 10 ] Air-drying and oven-drying are besides in pattern as an effectual and inexpensive agencies to dry fruits. [ 11 ] However, drying temperature is one of the most of import factors that may impact the quality of the fruits. For illustration, harmonizing to a recent survey, fruits dried at high temperature have higher concentrations of antioxidant compounds like phenolic acids, anthocyanins, etc. [ 12 ] , while, low drying temperature resulted in loss of fruit quality. [ 13 ]
Although a figure of scientific studies are available uncovering the possible effects of drying on the antioxidant activity of fruits, nevertheless, such effects have non yet been studied on the types of fruits selected in the present survey. Therefore, the present research work was planned to measure the effects of two common drying patterns ( ambient-drying and oven-drying ) on the antioxidant activity and phenolic acid profile of the selected fruits ( apple, plum, apricot, strawberry and mulberry ) with the chief purpose to inventing a suited method for this intents.
Material and Method
Five commercially adult fruits: apple ( Malus pumila, volt-ampere. skysuper ) , plum ( Prunus salicina, volt-ampere. Fezele manani ) , apricot ( Prunus armeniaca, volt-ampere. Nuri ) , strawberry ( Fragaria anan ( a ) Social Security Administration ) and mulberry ( Morus alba, volt-ampere. ruddy ) , were purchased from the local market of Faisalabad, Pakistan. The specimens were farther identified and authenticated by Dr. Mansoor Ahmad, Taxonomist, Department of Botany, University of Agriculture, Faisalabad, Pakistan. Three different fruit samples ( dwelling of about 1000 g ) for each fruit were taken, packed in polyethylene bags and transported to the research lab of the Department of Chemistry & A ; Biochemistry, University of Agriculture Faisalabad, Pakistan.
Chemicals and reagents
Analytic class, Merck, Sigma and Fluka trade name chemicals and reagents were used throughout the experimental work. 2, 2, -diphenyl-1-picrylhydrazyl group ( DPPH. ) ( Sigma, 90.0 % ) , linoleic acid, nutrient class man-made antioxidant butylated hydroxytoluene ( BHT ) ( 99.0 % ) , Folin-Ciocalteu reagent ( 2 N ) , and criterions of phenolic acids ( vanillic, syringic, p-coumaric, ferulic, sinapic, caffeic, and Gallic acid ) were purchased from Sigma Chemicals Co. ( St, Louis, MO, USA ) . All other chemicals ( analytical class ) i.e. ferric chloride, ammonium thiocyanate, K dihydrogen phosphate, dipotassium H phosphate, Na hydrogen carbonate, used in this survey were purchased from Merck ( Darmstadt, Germany ) .
Sample readying and drying process
The fruit samples were separated into two groups ; one for rating of the antioxidant activity, on the twenty-four hours of aggregation ( fresh fruit footing ) , and the other part subjected to drying under two different conditions as described below:
Fruit samples ( apple, strawberry, mulberry, plum, and apricot ) were washed with tap H2O and so dried with a paper towel. Edible parts of the fruits were cut into little pieces ( approx. 2 mm A-1 centimeter ) , utilizing a crisp steel knife and so individually subjected to ambient-drying ( room temperature drying ; mean temperature 30 oC ) up-to 7 yearss and oven-drying utilizing an electric vacuity oven ( VOC-300 SD ; EYELA, Tokyo, Japan ) at 80 oC up-to 2 yearss, until changeless weight was achieved.
Sample extraction for antioxidant activity rating
The fresh fruits ( in homogenized signifier ) and ambient-dried, and oven-dried land ( 80 mesh ) samples ( 20 g for each ) were extracted separately with 200 milliliters of aqueous methyl alcohol ( methyl alcohol: H2O, 80:20 v/v ) for 6 H at room temperature in an orbital shaker ( Gallenkamp, UK ) . The infusions were separated from the residues by filtrating through Whatman No. 1 filter paper. The residues were extracted twice with the fresh dissolver and the infusions combined. The combined infusions were concentrated and freed of dissolver under decreased force per unit area at 45 A°C, utilizing a rotary evaporator ( EYELA, SB-651, Rikakikai Co. Ltd. Tokyo, Japan ) . Infusions were weighed and stored at – 4 oC, until farther analyses.
Evaluation of antioxidant activity of fruit infusions
Determination of entire phenoplasts ( TP ) : The sum of TP was assessed utilizing the Folin-Ciocalteu reagent based spectrophotometric check. [ 14 ] Briefly, 50 milligram of petroleum infusion were assorted with 0.5 milliliters of Folin-Ciocalteu reagent and 7.5 milliliters deionized H2O. The mixture was kept at room temperature for 10 min, and added 1.5 milliliter of 20 % Na carbonate ( w/v ) to it. The mixture was incubated in a H2O bath at 40 oC for 20 min and so cooled in an ice bath ; the optical density was recorded at 755 nanometers utilizing a spectrophotometer ( U-2001, Hitachi Instruments Inc. , Tokyo, Japan ) . The sum of TP was calculated utilizing Gallic acid criterions standardization curve ( Concentration range 10-100 ppm, R2 = 0.9986 ) . The consequences were expressed as Gallic acid equivalents ( GAE ) g/100g of dry fruit. All samples were analyzed thrice and consequences averaged. The consequences are reported on dry weight footing ( DW ) .
Determination of 2, 2′-diphenyl-1-picrylhydrazyl extremist scavenging activity: The 2, 2′-diphenyl-1-picrylhydrazyl ( DPPH ) free extremist scavenging activity of the fruit extracts was assessed utilizing a antecedently described method. [ 15 ] Briefly, to 1.0 milliliter of extract incorporating 25 I?g/mL of dry infusion in methyl alcohol, 5.0 milliliter of newly prepared solution of DPPH at concentration 0.025 g/L were added. The indicant of the activity of DPPH was observed with a alteration in the colour from purple to yellow and was measured by reading the optical density at 515 nanometers utilizing a spectrophotometer ( U-2001, Hitachi Instruments Inc. , Tokyo, Japan ) . The scavenging sums of DPPH extremist ( DPPH. ) were calculated from a standardization curve. Optical density measured at 5th min was used for comparing of extremist scavenging activity of the infusions.
Inhibition of lipid peroxidation: The antioxidant activity of the tried fruit infusions was besides determined by mensurating the suppression of linoleic acerb peroxidation. [ 15 ] Each fruit infusion ( 5 milligram ) was added individually to a solution of linoleic acid ( 0.13 milliliter ) , 99.8 % ethyl alcohol ( 10 milliliter ) and 10 milliliter of 0.2 M Na phosphate buffer ( pH 7 ) . The mixture was made up to 25 milliliter with distilled H2O and incubated at 40 oC up to 360 h. The extent of oxidization was measured by the peroxide value following the thiocyanate method. 16 Briefly, 10 milliliter of ethyl alcohol ( 75 % v/v ) , 0.2 milliliter of aqueous solution of ammonium thiocyanate ( 30 % w/v ) , 0.2 milliliter of sample solution and 0.2 milliliter of ferric chloride ( FeCl2 ) solution ( 20 millimeter in 3.5 % HCl ; v/v ) were added consecutive. After 3 min of stirring, the soaking up was measured at 500 nanometers utilizing a spectrophotometer ( U-2001, Hitachi Instruments Inc. , Tokyo, Japan ) . A control contained all the reagents, except sample infusions. Man-made antioxidant butylated hydroxytoluene ( BHT ) was used as a positive control. Percent suppression of linoleic acerb oxidization was calculated with the undermentioned equation: 100 – [ ( Abs. addition of sample at 360 H / Abs. addition of control at 360 H ) i‚? 100 ] , to show antioxidant activity.
Sample extraction for the analysis of phenolic acids by HPLC: Extraction/hydrolysis of phenolic acids was done harmonizing to the method of Tokusoglu et Al. [ 17 ] Briefly, 25 milliliter acidified methyl alcohol ( 1 % ( v/v ) HCl ) incorporating 0.5 mg milliliter -1 TBHQ was added to 5 g of each fruit sample, and the mixture was refluxed at 90 oC for 2 Hs to obtain free phenolic acids. The infusion was cooled to room temperature and centrifuged at ( 980 A- g for 10 min. The upper bed was taken and sonicated for 5 min so as to take any hints of air.
HPLC analysis of phenolic acids: Acid-hydrolyzed fresh fruit infusions were filtered through a 0.45 I?m ( Millipore ) membrane filter, prior to analysis by RP-HPLC. An HPLC ( LC-10A, Shimadzu, Kyoto, Japan ) , equipped with binary LC-10 AS pumps, SCL-10A system control unit, Rheodyne injector, CTO-10A column oven, SPD-10A UV-Vis sensor, and informations acquisition category LC-10 package was used. A 20- I?L of the filtered sample was injected into an analytical Supelco ( Supelco Inc. , Supelco Park, Bellefonte, PA, USA ) ODS contrary stage ( C18 ) column ( 250 x 4.6 millimeters ; 5 I?m atom size ) . The nomadic stage consisted of two solvent systems A: Water incorporating 0.02 % Triflouroaceticacid, B: MeOH incorporating 0.02 % triflouroacetic acid ( v/v ) . The nomadic stage was sonicated and filtered under vacuity through a 0.45 I?m membrane before usage. The phenolic acids were separated by isocratic elution of the nomadic stage ( mixture of dissolver A and B, 50:50 v/v ) at a flow rate of 0.50 mL min-1 at 30 oC. Detection was performed at a wavelength of 280 nanometers. Designation of the phenolic acids ( vanillic, syringic, p-coumaric, ferulic, sinapic, caffeic, and Gallic acid ) was done by comparing their keeping times with those of reliable criterions ( Sigma Chemicals Co. , St Louis, MO, USA ) . Quantitative measurings were made utilizing standardization curves of the related criterions.
Dry affair finding: Due to changing H2O contents of different fruits, all computations were made on dry mass footing. For the finding of dry affair, each sample ( 5-6 g of each fresh fruit ) was dried in an electric vacuity drying oven ( VOC- 300 SD, EYELA, Tokyo, Japan ) at 70 oC, until a changeless weight was achieved.
Three different samples for each fruit were assayed. Each sample was analyzed separately in triplicate and information is reported as mean ( n = 3 x 3 ) A± SD ( n = 3 x 3 ) . Data were analyzed by analysis of discrepancy ANOVA utilizing the Minitab 2000 Version 13.2 statistical package ( Minitab Inc. Pennsylvania, USA ) at 5 % significance degree. A chance value of P a‰¤ 0.05 was considered to denote a statistically important difference.
Consequences and Discussion
In the present survey, we evaluated the effects of ambient- , and oven-drying on the entire phenolic contents and antioxidant activity of selected fruits. Changes in the antioxidant activity of the fruits were monitored by the findings of percent suppression of linoleic acerb peroxidation and DPPH extremist scavenging activity. The generated information was coupled with HPLC analysis of phenolic acids of fresh fruits.
Entire phenolic contents ( TPC )
Phenolic resins, a good known group of works secondary metabolites, are outstanding free extremist scavengers and besides responsible for exhibiting multiple medicative and physiological maps in animate beings every bit good as in workss. [ 18 ] . Total phenolic contents ( TPC ) of the fresh and ambient-dried, and oven-dried fruits is shown in Fig. 1. The consequences indicated a important fluctuation ( P & lt ; 0.05 ) for TPC among the analyzed fruit species. Among different fruits, analyzed newly, maximal phenolic contents ( g/100 g of dry affair ) were detected in mulberry ( 3.66 ) , followed by strawberry ( 2.98 ) , plum ( 2.59 ) , apple ( 1.65 ) , and apricot ( 0.59 ) .
The influence of drying method on TPC, was non-significant ( P & lt ; 0.05 ) . However, ambient-dried fruits exhibited comparatively lower sums of TP than the fresh and oven-dried samples. Percent loss in TP contents as affected by ambient-drying ranged from 14.75 to 30.50 % . Interestingly among fruits, higher loss was noted for the apricot fruit. This loss in TP might be ascribed to greater enzymatic debasement as ambient-drying took relatively longer clip for drying taking to extra enzymatic reactions. [ 11,19, 20 ] On the other manus, TP contents of all other fruits except that of apricot fruit, , comparative to fresh samples, besides decreased ( a diminution of 10.8-33.9 % ) ; while that of apricot increased up to 18 % when fruits were oven-dried. This lessening in the phenolic contents, though of lesser extent than ambient-drying, might hold been due to chemical and enzymatic debasement, losingss by volatilization or thermic decomposition of the chemical components lending towards antioxidant effects. [ 21 ] Nonetheless, an addition in the TP contents of oven-dried apricot fruit might be ascribed to the formation of Maillard reaction merchandises taking to formation of new phenolic compounds from their precursor at high temperature. [ 22 ]
It can be presumed that bound signifier of phenoplasts with larger molecular weight, in apricort might hold been liberated into simple free signifiers by heat intervention taking to heightening over all entire phenolic contents of the samples. Several surveies reported that heat intervention is effectual towards increasing the entire phenolic content in different nutrients such as dry beans, [ 23 ] algarroba bean pulverization, [ 24 ] veggies, [ 25 ] grape seeds [ 26 ] , and peanuts. [ 27 ] Boateng et al. , [ 23 ] explained that break of the cell wall through warming or by the dislocation of indissoluble phenolic compounds as map of thermic interventions could hold led to better extractability of phenolic compounds in dry beans. Jeong et al. , [ 28 ] besides reported that simple heat intervention facilitated the transition of indissoluble phenoplasts into soluble phenoplasts but could non split covalently bounded compounds from rice hull. [ 29 ] This indicates that phenolic compounds of workss may be present in different edge signifiers and the extent of release of such compounds into simpler signifiers may differ from species to species. In add-on, the addition in entire phenoplasts of peanut meats flour in this survey may besides be attributed to the development of Maillard reaction merchandises which are reported to be formed during the roasting procedure. Yu et al. , [ 30 ] investigated that Maillard reaction merchandises might take to increase the sum of entire phenoplasts or phenolic-like composites that farther contribute to higher optical density readings measured by the Folin check.
Antioxidant Activity Evaluation of Fruits
Percent suppression of linoleic acerb peroxidation
Antioxidant activity ( AA ) of different fruit infusions was assessed by mensurating their ability to suppress oxidization of linoleic acid utilizing the thiocyanate method. [ 16 ] A important ( P & lt ; 0.05 ) fluctuation in the suppression of lipid peroxidation, exhibited by different fruit samples, was observed ( Figure 2 ) . Levels of suppression of lipid peroxidation by fresh fruit infusions, runing from 61.8 to 86.1 % , were higher than those determined in ambient- , and oven-dried fruits infusions. Mulberry exhibited the highest degree ( 86.1 % ) while the apricot sample showed the lowest extent of linoleic acid ( 61.8 % ) . Strawberry, plum, and apple besides inhibited oxidization of linoleic acid at high degrees, 80.6, 79.2 and 61.8 % , severally. As a consequence of drying under either of the conditions, suppression of linoleic acerb peroxidation of all fruit extracts, except apricot, decreased bespeaking non-significant ( P & gt ; 0.05 ) fluctuation between the methods. As expected, a larger per centum lessening in suppression was noted for ambient-dried fruits.
DPPH free extremist scavenging capacity
The effectivity of the tried fruit infusions for scavenging DPPH. extremist showed the similar tendencies as observed for suppression of linoleic acerb peoxidation. Among fresh fruits, maximal DPPH. scavenging activity was shown by mulberry ( 82.2 % ) followed by strawberry ( 80.1 % ) , plum ( 79.2 % ) , apple ( 70.3 % ) , and apricot ( 58.7 % ) . Relatively greater loss in DPPH extremist scavenging activity was observed for ambient-dried fruit samples as compared to that for oven- dried samples.
The literature reveals that drying governments, in peculiar, the drying temperature and drying period might impact the antioxidant activities of works stuffs due to alter in the chemical composing and antioxidant components. [ 31 ] In understanding to our present consequences, the antioxidant activity of blueberry was besides noted to be decreased up to degree of 52 % after drying. [ 32 ] However, in the present probe interestedly drying at 80 oC increased the antioxidant activity of apricot ( figure 2 and calculate 3 ) .
Some old studies have besides shown an betterment in the antioxidant activity of works stuffs after drying at accelerated conditions. Piga et al. , [ 33 ] reported an addition in the antioxidant activity of plums and prunes after drying at 85 oC for 40 h. Phenolic contents of a heat-dried works stuff may increase as a consequence of transition of ester-linked phenolic compounds into free phenoplasts that exhibit more antioxidant activity than their several edge signifiers. [ 34 ]
Overall the consequences of the survey indicated considerable fluctuation in the entire phenolic contents and antioxidant activity among different fruit samples with regard to processing conditions used. The antioxidant belongingss were superior for fresh fruits followed by those dried at 80 oC, whereas the lowest activities were recorded for ambient-dried samples.
The consequences of the colorimetric trials picturing the antioxidant activity of the fruits extract were compared and correlated with each other ( Table 1 ) . In the present analysis, we observed a good correlativity between the consequences of TP and extremist scavenging activity ( R = 0.957 ) . Furthermore, correlativity coefficient ( R ) values for sums of TP versus suppression of oxidization and DPPH scavenging activity versus suppression of oxidization were found to be 0.985 and 0.977, severally foretelling good positive relationships. Overall, correlativity analysis informations revealed that DPPH. scavenging activity and suppression of lipid peroxidation of the fruits antioxidant infusions are strongly dependent on the TP contents.
Phenolic acids profile in fruits
The concentrations of phenolic acids determined by HPLC in different fresh fruits are presented in Table 2. Contentss of phenolic acids varied significantly ( P & lt ; 0.05 ) among types of fruits every bit good as phenolic acid constituents. However, drying showed non-significant ( P & lt ; 0.05 ) effects on the phenolic profile of the tried fruits. All the tested fruits chiefly contained p-coumaric, ferulic and caffeic acids. Among fruits, strawberry was found to be a rich beginning of phenolic compounds ( 104.4 g/kg of dry fruit ) , p-coumaric acid being the chief constituent with part of 47.5 mg/ kilogram of dry affair, followed by sinapic ( 23.3 ) , ferulic ( 14.9 ) , caffeic ( 13.6 ) , and gallic ( 13.6 ) acids. Conversely, ferulic acid was the major phenolic acid of mulberry ( 32.9 mg/kg ) , followed by p-coumaric ( 22.4 ) , vanillic ( 16.9 ) , syringic ( 12.7 ) , and caffeic ( 11.9 ) acids. Plum and apple besides contained caffeic acid as the major phenolic compound at degrees of 32.8 and 26.1 mg/kg, severally. However, concentration of caffeic acid ( 26.1 ) determined in the present analysis of apple was found to be higher than that reported by Dragovic-Uzelac et al. , [ 35 ] i.e. 13.2 mg/kg and Suarez et al. , [ 36 ] i.e. 22 mg/kg of dry pomace. Such fluctuations in of informations might be attributed to differences in the assortments of the fruit used, analytical method adopted every bit good as due to agroclimatic factors of the harvest topographic point.
P-coumaric acid ( 23.6 mg/kg ) was determined to be the chief phenolic acid in apricot. The sums of ferulic, caffeic, and Gallic acids determined were 13.9, 6.7, and 4.54 mg/ kilogram, severally. Dragovic-Uzelac et al. , [ 37 ] reported slightly lower degrees of ferulic ( 5.09-10.81 mg/kg ) , caffeic ( 2.39-7.83 mg/kg ) , and Gallic ( 2.35-3.47 mg/ kilogram ) acids in apricot than our present consequences.
This survey indicated that infusions of the fresh fruits possessed greater sums of TP and therefore exhibited higher antioxidant activities than the dried fruit samples. Datas on the effects of drying methods showed that greater decrease in the sum of TP and besides in antioxidant activity occur when samples were subjected to ambient- drying as compared with oven-drying. Therefore, on the footing of the present consequences it could be suggested that oven-drying at optimal temperature is a relatively better agencies to continue fruits retaining maximal antioxidant foods. Overall, the antioxidant potency of the tried fruits might be attributed to the presence of considerable sums of assorted phenolic acids detected.