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The effects of drying process (ambient-drying and oven-drying) on the total phenolic contents and antioxidant activity of selected fruits (strawberry, mulberry, plum, apple, and apricot) were studied. The antioxidant attributes of the fruits were evaluated following different colorimetric assays, while the composition of phenolic acids of the fresh fruits was analyzed by reverse phase high performance liquid chromatography (RP-HPLC). The amounts of total phenolics (TP) were higher in mulberry samples followed by strawberry, plum, apple, and apricot. The tested fruits exhibited appreciable, radical scavenging capacity ranging from 58.7- 82.2, and 61.8- 86.1% DPPH and inhibition of linoleic acid peroxidation, respectively. The RP-HPLC analysis on the fresh fruits revealed the presence of vanillic-, syringic-, p-coumaric-, ferulic-, sinapic-, caffeic-, and gallic- acids; p-coumaric acid being the most prominent component. The results of this study revealed that the amounts of TP and antioxidant activity of all tested fruits, except apricot, decreased after drying treatment; relatively more pronounced decline observed for the ambient-dried samples as against oven-drying. Therefore, it could be suggested that oven-drying at optimum temperature is a comparatively better means to preserve fruits retaining maximum amounts of antioxidant compounds.
Keywords: Fruits, drying, DPPH scavenging, Inhibition of per oxidation, Phenolic acids, HPLC
* Author for correspondence: Dr. Farooq Anwar, email:[email protected]
It is now well recognized that consumption of selected fruits and vegetables is strongly linked with the reduced risk of chronic diseases such as cancer, inflammation, neurological and cardiovascular disorders and other degenerative diseases. Antioxidant nutrients including vitamins, carotenoids and polyphenolics with multiple biological activities are mainly responsible for the beneficial health effects of fruits and vegetables. Phenolic antioxidants are gaining continuing attention due to their efficacy in counteracting free radicals, linked with various diseases. Plants phenolics are considered comparatively more stable and are available as active phytochemicals for uses in different food products to protect them from oxidation and enhancing shelf-life. On the other hand, carotenoids and vitamin C being heat sensitive are quite unstable and due to their more susceptibility to deterioration, have restricted applications in processed foods.
Currently, there is a continuing demand of fresh or minimally processed foods to get more nutritive components through diet. Fruits and vegetables are available in fresh form for very short periods, and therefore, they are preferably stored at low temperatures, sometimes dried and processed into different items like juices, canned foods and jams, etc. under best suitable conditions for retaining maximum nutrient value. It is well accepted that the conditions of storage and processing exhibit notable effect on the concentration of inherited phytochemicals, especially, carotenoids, phenolic antioxidants and vitamin C of fruits and vegetables. Drying/dehydration has become comparatively efficient way to minimize nutritional losses and microbial growth, thus leading to enhancing shelf-life of fruits.5 Effect of drying on fruit quality is not fully understood, however, major changes in fruits occur when they are exposed to drying at elevated temperature or at low temperature for longer periods. It is generally considered that besides the chemical and nutritional changes, various physiological and pharmacological alterations in fruit cell-wall polysaccharides can also affect other quality attributes including texture, and color of the fruits. [5, 6]
Several drying techniques are being used to dry fruits, some of which are very costly and time-consuming. Fruits are generally dried under the sun or by using an artificial dryer.  Some literature reports show promising effects of UV irradiation on the quality of the fruits as compared to other non-thermal fruit preserving techniques. [8, 9] It was found that sun-drying may increase the antioxidant activity and total phenolic contents of the fruits.  Air-drying and oven-drying are also in practice as an effective and cheap means to dry fruits.  However, drying temperature is one of the most important factors that may affect the quality of the fruits. For example, according to a recent study, fruits dried at high temperature have higher concentrations of antioxidant compounds like phenolic acids, anthocyanins, etc. , while, low drying temperature resulted in loss of fruit quality. 
Although a number of scientific reports are available revealing the possible effects of drying on the antioxidant activity of fruits, however, such effects have not yet been studied on the types of fruits selected in the present study. Therefore, the present research work was planned to appraise the effects of two common drying practices (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 main aim to devising a suitable method for this purposes.
Material and Method
Five commercially grown fruits: apple (Malus pumila, var. skysuper), plum (Prunus salicina, var. Fezele manani), apricot (Prunus armeniaca, var. Nuri), strawberry (Fragaria anan(a)ssa) and mulberry (Morus alba, var. red), were purchased from the local market of Faisalabad, Pakistan. The specimens were further identified and authenticated by Dr. Mansoor Ahmad, Taxonomist, Department of Botany, University of Agriculture,Faisalabad, Pakistan. Three different fruit samples (consisting of about 1000 g) for each fruit were taken, packed in polythene bags and transported to the laboratory of the Department of Chemistry & Biochemistry, University of Agriculture Faisalabad, Pakistan.
Chemicals and reagents
Analytical grade, Merck, Sigma and Fluka brand chemicals and reagents were used throughout the experimental work. 2, 2,-diphenyl-1-picrylhydrazyl radical (DPPH.) (Sigma, 90.0%), linoleic acid, food grade synthetic antioxidant butylated hydroxytoluene (BHT) (99.0 %), Folin-Ciocalteu reagent (2 N), and standards 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 grade) i.e. ferrous chloride, ammonium thiocyanate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium bicarbonate, used in this study were purchased from Merck (Darmstadt, Germany).
Sample preparation and drying procedure
The fruit samples were separated into two groups; one for evaluation of the antioxidant activity, on the day of collection (fresh fruit basis), and the other portion subjected to drying under two different conditions as described below:
Fruit samples (apple, strawberry, mulberry, plum, and apricot) were washed with tap water and then dried with a paper towel. Edible parts of the fruits were cut into small pieces (approx. 2 mm Ã-1 cm), using a sharp steel knife and then separately subjected to ambient-drying (room temperature drying; average temperature 30 oC) up-to 7 days and oven-drying using an electric vacuum oven (VOC-300 SD; EYELA, Tokyo, Japan) at 80 oC up-to 2 days, until constant weight was achieved.
Sample extraction for antioxidant activity evaluation
The fresh fruits (in homogenized form) and ambient-dried, and oven-dried ground (80 mesh) samples (20 g for each) were extracted individually with 200 mL of aqueous methanol (methanol: water, 80:20 v/v) for 6 h at room temperature in an orbital shaker (Gallenkamp, UK). The extracts were separated from the residues by filtering through Whatman No. 1 filter paper. The residues were extracted twice with the fresh solvent and the extracts combined. The combined extracts were concentrated and freed of solvent under reduced pressure at 45 °C, using a rotary evaporator (EYELA, SB-651, Rikakikai Co. Ltd. Tokyo, Japan). Extracts were weighed and stored at - 4 oC, until further analyses.
Evaluation of antioxidant activity of fruit extracts
Determination of total phenolics (TP): The amount of TP was assessed using the Folin-Ciocalteu reagent based spectrophotometric assay.  Briefly, 50 mg of crude extract were mixed with 0.5 mL of Folin-Ciocalteu reagent and 7.5 mL deionized water. The mixture was kept at room temperature for 10 min, and added 1.5 mL of 20% sodium carbonate (w/v) to it. The mixture was incubated in a water bath at 40 oC for 20 min and then cooled in an ice bath; the absorbance was recorded at 755 nm using a spectrophotometer (U-2001, Hitachi Instruments Inc., Tokyo, Japan). The amount of TP was calculated using gallic acid standards calibration curve (Concentration range 10-100 ppm, R2 = 0.9986). The results were expressed as gallic acid equivalents (GAE) g/100g of dry fruit. All samples were analyzed thrice and results averaged. The results are reported on dry weight basis (DW).
Determination of 2, 2'-diphenyl-1-picrylhydrazyl radical scavenging activity: The 2, 2'-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity of the fruit extracts was assessed using a previously described method.  Briefly, to 1.0 mL of extract containing 25 Î¼g/mL of dry extract in methanol, 5.0 mL of freshly prepared solution of DPPH at concentration 0.025 g/L were added. The indication of the activity of DPPH was observed with a change in the color from purple to yellow and was measured by reading the absorbance at 515 nm using a spectrophotometer (U-2001, Hitachi Instruments Inc., Tokyo, Japan). The scavenging amounts of DPPH radical (DPPH.) were calculated from a calibration curve. Absorbance measured at 5th min was used for comparison of radical scavenging activity of the extracts.
Inhibition of lipid peroxidation : The antioxidant activity of the tested fruit extracts was also determined by measuring the inhibition of linoleic acid peroxidation.  Each fruit extract (5 mg) was added separately to a solution of linoleic acid (0.13 mL), 99.8% ethanol (10 mL) and 10 mL of 0.2 M sodium phosphate buffer (pH 7). The mixture was made up to 25 mL with distilled water and incubated at 40 oC up to 360 h. The extent of oxidation was measured by the peroxide value following the thiocyanate method. 16 Briefly, 10 mL of ethanol (75% v/v), 0.2 mL of aqueous solution of ammonium thiocyanate (30% w/v), 0.2 mL of sample solution and 0.2 mL of ferrous chloride (FeCl2) solution (20 mM in 3.5% HCl; v/v) were added sequentially. After 3 min of stirring, the absorption was measured at 500 nm using a spectrophotometer (U-2001, Hitachi Instruments Inc., Tokyo, Japan). A control contained all the reagents, except sample extracts. Synthetic antioxidant butylated hydroxytoluene (BHT) was used as a positive control. Percent inhibition of linoleic acid oxidation was calculated with the following equation: 100 - [(Abs. increase of sample at 360 h / Abs. increase of control at 360 h) ´ 100], to express antioxidant activity.
Sample extraction for the analysis of phenolic acids by HPLC:Extraction/hydrolysis of phenolic acids was done according to the method of Tokusoglu et al.  Briefly, 25 mL acidified methanol (1% (v/v) HCl) containing 0.5 mg mL -1 TBHQ was added to 5 g of each fruit sample, and the mixture was refluxed at 90 oC for 2 h to obtain free phenolic acids. The extract was cooled to room temperature and centrifuged at (980 Ã- g for 10 min. The upper layer was taken and sonicated for 5 min so as to remove any traces of air.
HPLC analysis of phenolic acids: Acid-hydrolyzed fresh fruit extracts were filtered through a 0.45 Î¼m (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 detector, and data acquisition class LC-10 software was used. A 20- Î¼L of the filtered sample was injected into an analytical Supelco (Supelco Inc., Supelco Park, Bellefonte, PA, USA) ODS reverse phase (C18) column (250 x 4.6 mm; 5 Î¼m particle size). The mobile phase consisted of two solvent systems A: H2O containing 0.02% Triflouroaceticacid, B: MeOH containing 0.02% triflouroacetic acid (v/v). The mobile phase was sonicated and filtered under vacuum through a 0.45 Î¼m membrane before use. The phenolic acids were separated by isocratic elution of the mobile phase (mixture of solvent 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 nm. Identification of the phenolic acids (vanillic, syringic, p-coumaric, ferulic, sinapic, caffeic, and gallic acid) was done by comparing their retention times with those of authentic standards (Sigma Chemicals Co., St Louis, MO, USA). Quantitative measurements were made using calibration curves of the related standards.
Dry matter determination: Due to varying water contents of different fruits, all calculations were made on dry mass basis. For the determination of dry matter, each sample (5-6 g of each fresh fruit) was dried in an electric vacuum drying oven (VOC- 300 SD, EYELA, Tokyo, Japan) at 70 oC, until a constant weight was achieved.
Three different samples for each fruit were assayed. Each sample was analyzed individually in triplicate and data is reported as mean (n = 3 x 3) ± SD (n = 3 x 3). Data were analyzed by analysis of variance ANOVA using the Minitab 2000 Version 13.2 statistical software (Minitab Inc. Pennsylvania, USA) at 5% significance level. A probability value of P â‰¤ 0.05 was considered to denote a statistically significant difference.
Results and Discussion
In the present study, we evaluated the effects of ambient-, and oven-drying on the total phenolic contents and antioxidant activity of selected fruits. Changes in the antioxidant activity of the fruits were monitored by the determinations of percent inhibition of linoleic acid peroxidation and DPPH radical scavenging activity. The generated data was coupled with HPLC analysis of phenolic acids of fresh fruits.
Total phenolic contents (TPC)
Phenolics, a well known group of plant secondary metabolites, are prominent free radical scavengers and also responsible for exhibiting multiple medicinal and physiological functions in animals as well as in plants. . Total phenolic contents (TPC) of the fresh and ambient-dried, and oven-dried fruits is shown in Fig. 1. The results indicated a significant variation (P<0.05) for TPC among the analyzed fruit species. Among different fruits, analyzed freshly, maximum phenolic contents (g/100 g of dry matter) 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<0.05). However, ambient-dried fruits exhibited relatively lower amounts 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 degradation as ambient-drying took comparatively longer time for drying leading to additional enzymatic reactions. [11,19, 20] On the other hand, TP contents of all other fruits except that of apricot fruit,, relative to fresh samples, also decreased (a decline of 10.8-33.9%); while that of apricot increased up to 18% when fruits were oven-dried. This decrease in the phenolic contents, though of lesser extent than ambient-drying, might have been due to chemical and enzymatic degradation, losses by volatilization or thermal decomposition of the chemical constituents contributing towards antioxidant effects.  Nonetheless, an increase in the TP contents of oven-dried apricot fruit might be ascribed to the formation of Maillard reaction products leading to formation of new phenolic compounds from their precursor at high temperature. 
It can be presumed that bound form of phenolics with larger molecular weight, in apricort might have been liberated into simple free forms by heat treatment leading to enhancing over all total phenolic contents of the samples. Several studies reported that heat treatment is effective towards increasing the total phenolic content in different foods such as dry beans,  carob powder,  vegetables,  grape seeds, and peanuts.  Boateng et al.,  explained that disruption of the cell wall through heating or by the breakdown of insoluble phenolic compounds as function of thermal treatments could have led to better extractability of phenolic compounds in dry beans. Jeong et al.,  also reported that simple heat treatment facilitated the conversion of insoluble phenolics into soluble phenolics but could not cleave covalently bounded compounds from rice hull.  This indicates that phenolic compounds of plants may be present in different bound forms and the extent of liberation of such compounds into simpler forms may differ from species to species. In addition, the increase in total phenolics of peanut kernel flour in this study may also be attributed to the development of Maillard reaction products which are reported to be formed during the roasting process. Yu et al.,  investigated that Maillard reaction products might lead to increase the amount of total phenolics or phenolic-like complexes that further contribute to higher absorbance readings measured by the Folin assay.
Antioxidant Activity Evaluation of Fruits
Percent inhibition of linoleic acid peroxidation
Antioxidant activity (AA) of different fruit extracts was assessed by measuring their ability to inhibit oxidation of linoleic acid using the thiocyanate method.  A significant (P < 0.05) variation in the inhibition of lipid peroxidation, exhibited by different fruit samples, was observed (Figure 2). Levels of inhibition of lipid peroxidation by fresh fruit extracts, ranging from 61.8 to 86.1%, were higher than those determined in ambient-, and oven-dried fruits extracts. Mulberry exhibited the highest level (86.1%) while the apricot sample showed the lowest extent of linoleic acid (61.8%). Strawberry, plum, and apple also inhibited oxidation of linoleic acid at high levels, 80.6, 79.2 and 61.8%, respectively. As a result of drying under either of the conditions, inhibition of linoleic acid peroxidation of all fruit extracts, except apricot, decreased indicating non-significant (P>0.05) variation between the methods. As expected, a larger percent decrease in inhibition was noted for ambient-dried fruits.
DPPH free radical scavenging capacity
The effectiveness of the tested fruit extracts for scavenging DPPH. radical showed the similar trends as observed for inhibition of linoleic acid peoxidation. Among fresh fruits, maximum DPPH. scavenging activity was shown by mulberry (82.2%) followed by strawberry (80.1%), plum (79.2%), apple (70.3%), and apricot (58.7%). Comparatively greater loss in DPPH radical scavenging activity was observed for ambient-dried fruit samples as compared to that for oven- dried samples.
The literature reveals that drying regimes, in particular, the drying temperature and drying period might affect the antioxidant activities of plant materials due to change in the chemical composition and antioxidant constituents.  In agreement to our present results, the antioxidant activity of blueberry was also noted to be decreased up to level of 52% after drying.  However, in the present investigation interestedly drying at 80 oC increased the antioxidant activity of apricot (figure 2 and figure 3).
Some previous reports have also shown an improvement in the antioxidant activity of plant materials after drying at accelerated conditions. Piga et al.,  reported an increase in the antioxidant activity of plums and prunes after drying at 85 oC for 40 h. Phenolic contents of a heat-dried plant material may increase as a result of conversion of ester-linked phenolic compounds into free phenolics that exhibit more antioxidant activity than their respective bound forms. 
Overall the results of the study indicated considerable variation in the total phenolic contents and antioxidant activity among different fruit samples with respect to processing conditions used. The antioxidant properties were superior for fresh fruits followed by those dried at 80 oC, whereas the lowest activities were recorded for ambient-dried samples.
The results of the colorimetric tests depicting the antioxidant activity of the fruits extract were compared and correlated with each other (Table 1). In the present analysis, we observed a good correlation between the results of TP and radical scavenging activity (r = 0.957). Furthermore, correlation coefficient (r) values for amounts of TP versus inhibition of oxidation and DPPH scavenging activity versus inhibition of oxidation were found to be 0.985 and 0.977, respectively predicting good positive relationships. Overall, correlation analysis data revealed that DPPH. scavenging activity and inhibition of lipid peroxidation of the fruits antioxidant extracts 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. Contents of phenolic acids varied significantly (P < 0.05) among types of fruits as well as phenolic acid components. However, drying showed non-significant (P<0.05) effects on the phenolic profile of the tested fruits. All the tested fruits mainly contained p-coumaric, ferulic and caffeic acids. Among fruits, strawberry was found to be a rich source of phenolic compounds (104.4 g/kg of dry fruit), p-coumaric acid being the principal component with contribution of 47.5 mg/ kg of dry matter, 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 also contained caffeic acid as the major phenolic compound at levels of 32.8 and 26.1 mg/kg, respectively. 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.,  i.e. 13.2 mg/kg and Suarez et al.,  i.e. 22 mg/kg of dry pomace. Such variations in of data might be attributed to differences in the varieties of the fruit used, analytical method adopted as well as due to agroclimatic factors of the harvest place.
P-coumaric acid (23.6 mg/kg) was determined to be the main phenolic acid in apricot. The amounts of ferulic, caffeic, and gallic acids determined were 13.9, 6.7, and 4.54 mg/ kg, respectively. Dragovic-Uzelac et al.,  reported somewhat lower levels of ferulic (5.09-10.81 mg/kg), caffeic (2.39-7.83 mg/kg), and gallic (2.35-3.47 mg/ kg) acids in apricot than our present results.
This study indicated that extracts of the fresh fruits possessed greater amounts of TP and thus exhibited higher antioxidant activities than the dried fruit samples. Data on the effects of drying methods showed that greater reduction in the amount of TP and also in antioxidant activity occur when samples were subjected to ambient- drying as compared with oven-drying. Therefore, on the basis of the present results it could be suggested that oven-drying at optimum temperature is a comparatively better means to preserve fruits retaining maximum antioxidant nutrients. Overall, the antioxidant potential of the tested fruits might be attributed to the presence of considerable amounts of various phenolic acids detected.