CHANGES IN ANTIOXIDANT ACTIVITY AND PHENOLIC CONTENT OF CELERY LEAVES AND ROOT DURING OSMOTIC TREATMENT

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1 Original scientific paper UDC 635.8:547.56]:65.7 CHANGES IN ANTIOXIDANT ACTIVITY AND PHENOLIC CONTENT OF CELERY LEAVES AND ROOT DURING OSMOTIC TREATMENT Milica Nićetin *, Biljana Lončar, Vladimir Filipović, Violeta Knežević, Tatjana Kuljanin, Lato Pezo, Stanislava Gorjanović Faculty of Technology, University of Novi Sad, Bulevar Cara Lazara, 000 Novi Sad, Serbia Institute of General and Physical Chemistry, University of Beograd, Studentski trg -6, 00 Beograd, Serbia * milican85@live.com Abstract Celery (Apium graveolens) is widely used as a medicinal herb or spice, with prominent antioxidant properties, due to the presence of many bioactive components, mainly phenolic compounds. An understanding of changes in phenolic content and antioxidant activity occurring during preservation of celery is important to develop appropriate drying method and optimal conditions of drying. In this study, osmotic treatment of celery leaves and root was performed in two osmotic solutions (sugar beet molasses and aqueous solution of sodium chloride and sucrose, prepared using sucrose in the quantity of 00 g/kg water, NaCl in the quantity of 350 g/kg water and distilled water), at three temperatures (0, 35 and 50 0 C), and three different immersion periods (, 3 and 5 h), under atmospheric pressure. The aim was to investigate the influence of used osmotic agent type, temperature and dehydration time on the water loss, solid gain, antioxidant activity expressed as ferric reducing antioxidant power (FRAP), while the total phenolic content was investigated by standard Folin Ciocalteau method (FC). Results have been correlated using regression analysis and statistically evaluated using analysis of variance (ANOVA). Post-hoc Tukey s HSD test at 95% confidence limit, statistically significant at p < 0.05 level, has been calculated to confirm statistically significant differences between complex samples. Accuracy of applied assays has been estimated based on coefficients of variation. Principal Component Analysis (PCA) and Cluster Analysis (CA) have been applied successfully to classify and discriminate the different samples, while Standard Score analysis (SS) was used in optimal process parameters determination. The statistically significant increase in FRAP and FC was noticed in celery leaves and root samples osmotically treated in sugar beet molasses solution, while FRAP and FC values were decreased for samples treated in sodium chloride and sucrose solution. The strong correlation between FRAP and FC assays was found. It was concluded that the optimum condition for osmotic treatment of celery leaves and root, from the aspect of antioxidant increase, were at temperature of 50 0 C and treatment time of 5 hours, in sugar beet molasses as osmotic solution. Key words: Osmotic treatment, Celery, Sugar beet molasses, Antioxidant activity, Phenolic content.. Introduction Spices and herbs have been added to foods from old times, not only as taste and flavor enhancers, but also as folk medicine and food preservatives []. There is an increasing interest both in the food industry and in the scientific research of spices and herbs, because they are rich sources of phenolic compounds [, ]. Phenolic substances in these plant materials have been shown to be the most responsible for the antioxidant activity (AOA) of plants. Also, plant phenolics could retard the process of lipid oxidation and thereby improve the quality and nutritional value of food [3]. Celery (Apium graveolens) is a medical herb and spice used as a food and also in traditional medicine, by the ancients [4]. The whole plant has a specific taste and characteristic smell, due to the presence of essential oils and aromatic substances [5, 6]. Healthful substances in celery have been claimed to contribute to its healing properties: variety of vitamins, mineral salts and many active substances [6]. The main bioactive components in celery are: flavonoids, mostly apiin and apigenin, essential oils (α-limonene and selinene, 75

2 butylphalide, celerin, apiol, myristicin), organic acids (chlorogenic acid, caffeic acid), bergapten, niacin, inositol, etc. [7]. Presently, there is the greatest interest in phenolic compounds derived from celery, mainly phenolic acids and flavonoids, because of their prominent antioxidant activities [8]. Phenols have been shown to be the most responsible for the antioxidant activity of celery plants, thereby for their physiological functionalities, such as: lowering cholesterol levels, anti-inflammatory, antimicrobial and anticancer activity [, 8]. Current studies have confirmed that celery can: lower blood pressure, regulate heart function, as well as the blood glucose level [7, 0]. Antioxidants in celery, also possess the potential to retard lipid oxidation, therefore celery can be used as food preservative, to improve food safety and quality [, 8]. Because of high water percentage, celery is highly perishable and susceptible to bacterial spoilage. Traditional drying processes can be used to extend its shelf life, but these treatments mainly lead to: texture degradation, color alteration and nutritional loss [, ]. Furthermore, preservation and processing might decrease antioxidant activity of celery, and consequently its health-promoting properties. An understanding of changes in phenolic content and antioxidant activity occurring during preservation of celery is important to develop appropriate drying method and optimal drying conditions [3, 4]. The application of osmotic treatment (OT) at a mild temperature is more effective in preserving valuable bioactive compounds in plant materials, as compared to the traditional drying treatments. Plants are not exposed to high temperatures, minimizing, in that way, sensory characteristic changes and retaining or even improving its initial nutritional value and functional properties [5, 6]. Additionally, OT is environmentally acceptable and energy efficient process due to the low temperature and energy requirements and low waste material [7]. OT is a water removal process, based on soaking food (mostly fruit and vegetable) in a hypertonic solution under ambient or modified environment conditions. Driving force for water removal is the concentration gradient between the surrounding hypertonic solution and the immersed plant material. The complex cellular structure of plant tissue acts as a semi-permeable membrane, which allows two main counter-current flows: water outflow from the plant tissue into the osmotic solution and the simultaneous migration of solids from solution to the tissue [8, 9]. Concentrated sucrose solution, sodium chloride solutions and their combinations are usually used as hypertonic solutions [0]. Recent research has shown that sugar beet molasses is an excellent medium for OT, primarily due to its high dry matter (80%) and specific nutrient content. The presence of complex solute compositions in molasses maintains a high transfer potential favorable to water loss and at the same time serves as nutritional enrichment of plant material []. Molasses, the thick, dark syrup obtained as a byproduct from the sugar beet processing into sucrose, consists of fermentable carbohydrates (sucrose, glucose, and fructose) and several non-sugar organic materials (betaine and other amino acids; minerals, mainly K; vitamins, especially of the B-group, etc.) []. Several studies reported that molasses is a rich source of phenolic compounds having possible roles in the prevention of several chronic diseases involving oxidative stress. Maillard browning carbohydrate-amino acid condensation products, formed during sugar processing, are also in very high concentration in molasses in a range from low organic compounds to complex aromatic polymers, and they have been reported to have antioxidant activities. It is considered that molasses has health benefits in the human diet, beyond its special taste and flavor, due to being rich in minerals and antioxidants [5, 3]. The purpose of presented work was to investigate the effects of osmotic solution type, processing time and temperature on the antioxidant activity and total phenolic content of celery leaves and roots during osmotic treatment in sugar beet molasses and aqueous ternary solution. The aim was to determine: antioxidant activity (expressed by FRAP and FC) as a function of the process variables and to find the optimum osmotic treatment conditions. Experimental results were subjected to analysis of variance (ANOVA) to show relations between applied assays. Pattern recognition techniques (Principal Component Analysis - PCA and Cluster Analysis - CA) were applied to the experimental data (used as descriptors) to characterize and differentiate among the observed samples.. Materials and Methods. Osmotic treatment Celery was purchased at local market, shortly before the treatment, to be used in the fresh state. Before the OT, celery leaves were cut into small pieces (squares approximately x cm), while root was cut into cubes ( x x cm). Concentrated sugar beet molasses, with initial dry matter content of w/w, was obtained from the sugar factory Crvenka, Serbia (this solution was marked as S ). The aqueous ternary osmotic solution (S ) was made from sucrose in the quantity of,00 g/kg water, NaCl in the quantity of 350 g/kg water and distilled water. The material to solution ratio of : 0 (w/w) was used during all experiments. 76

3 The samples of celery leaves and roots were submerged in laboratory jars with these solutions at three different temperatures of 0 0 C, 35 0 C and 50 0 C, under atmospheric pressure. Samples were withdrawn from the osmotic solution at determined intervals of time (, 3, and 5 h), then lightly washed with water and gently absorbed with paper towels to remove adhering solution and excessive water from the surface. Fresh and osmotically dehydrated celery samples dried at 05 0 C in a heat chamber (Instrumentaria Sutjeska, Croatia) until constant weight, finally was grounded and extracted with boiled distilled water to obtain extracts.. Determination of total phenols Total phenol content (TPC) of celery leaves and root was determined spectrophotometrically according to a modified method of Lachman et al., with Folin-Ciocalteu s reagent [4]. Briefly, 0.5 ml of the sample extract was added into a 50 ml volumetric flask containing.5 ml of Folin-Ciocalteu s reagent, 30 ml of distilled water and 7.5 ml of 0% Na CO 3, and filled up to the mark with distilled water. After two hours the absorbance of blue coloration was measured at 765 nm against a blank sample. Gallic acid was used as the standard and the results are expressed as mg/l of gallic acid equivalents (GAE). All measurements were performed in triplicate..3 Determination of ferric reducing/ antioxidant power (FRAP assay) The ferric reducing/antioxidant power (FRAP) assay was carried out according to a standard procedure. FRAP reagent was prepared by mixing acetic buffer, TPTZ and FeCl 3 x 6 H O (0 mm water solution) at a ratio of 0 : :. Briefly, to a volume of 950 µl of FRAP reagent 50 μl of sample extract was added. After 4 min. the absorbance of blue coloration was measured against a blank sample. All measurements were performed in triplicate. Aqueous solutions of FeSO 4 7H O ( μm) were used for the calibration and the results are expressed as mmol/l Fe(II) [5]. All experiments were repeated three times, and the obtained results are presented in Table..4 Statistical analysis The experimental results were expressed by means, standard deviation (SD) for each treatment. Collected Table. Experimental results of antioxidant activity and total phenolic content during osmotic treatment of celery root and leaves in ternary solution and sugar beet molasses Ternary solution Sugar beet molasses solution Root Leaves t T FRAP FC FRAP FC 0 /.57 ± 0.00 f 0.59 ± 0.00 g.44 ± 0.07 a 0.58 ± 0.00 d 0.65 ± 0.03 e 0.40 ± 0.00 f.40 ± a 0.54 ± abcd ± 0.07 d 0.7 ± 0.00 d.44 ± a 0.56 ± 0.00 cd ± c 0.6 ± 0.00 d.47 ± 0.03 a 0.53 ± 0.00 abcd ± 0.03 de 0.33 ± e.44 ± 0.0 a 0.54 ± bcd ± 0.03 c 0.0 ± 0.00 c.374 ± 0.08 a 0.5 ± 0.00 abcd ± 0.05 b 0.5 ± 0.00 bc.39 ± 0.0 a 0.47 ± 0.00 a ± c 0.6 ± 0.00 d.383 ± 0.0 a 0.53 ± 0.00 abcd ± 0.00 b 0.4 ± 0.00 b.358 ± 0.04 a 0.50 ± 0.00 abc ± 0.03 a 0.07 ± 0.00 a.358 ± 0.03 a 0.49 ± 0.00 ab 0 /.57 ± 0.00 ab 0.59 ± 0.00 a.44 ± 0.07 a 0.58 ± 0.00 a ± a 0.6 ± ab.43 ± 0.0 a 0.6 ± 0.00 abc ± 0.09 ab 0.65 ± ab.474 ± 0.0 a 0.6 ± ab ± 0.07 b 0.6 ± 0.00 ab.475 ± 0.03 a 0.63 ± 0.00 abc ± 0.06 a 0.6 ± ab.438 ± 0.0 a 0.64 ± 0.00 abc ± 0.06 ab 0.66 ± ab.466 ± 0.05 a 0.63 ± abc ± 0.07 ab 0.66 ± 0.00 ab.467 ± 0.06 a 0.66 ± 0.00 bc ± 0.0 ab 0.67 ± 0.00 b.449 ± 0.03 a 0.65 ± abc ± 0.0 b 0.65 ± 0.00 ab.453 ± 0.07 a 0.69 ± c ± 0.00 b 0.65 ± 0.00 ab.477 ± 0.03 a 0.69 ± 0.00 c a-n Different letters written in superscript within the same column in the table show significantly different means of observed data (at p < 0.05 level), n = 3. 77

4 data were subjected to ANOVA to explore the effects of process variables. Furthermore, pattern recognition techniques, including PCA and CA were applied successfully to classify and discriminate the different samples. The evaluation of RSM, ANOVA, PCA and CA of the obtained results was performed using Statistica software version (StatSoft Inc. 0, USA) [6]. The experimental data used for the analysis were derived using the Box and Behnken s fractional factorial (3 level- parameter) design, blocks, according to RSM. The RSM equations describe the effects of the test variables on the observed responses, determine test variables interrelationships and represent the combined effect of all test variables in the observed responses. The following second order polynomial (SOP) model was fitted to the experimental data. Eight models of the following form were developed to relate eight responses (Y) and two process variables (X), for each of the different osmotic treatments: first two principal components, accounting for 96.86% of the total variability for solution S and S, can be considered sufficient for data representation. Considering the map of the PCA performed on the data, FRAP leaves (which contributed 3.8% of total variance, based on correlations), FC leaves (4.4%), FRAP root (5.4%) and FC root (6.%) exhibited negative scores according to first principal component. FRAP leaves (which contributed 69.6% of total variance, based on correlations) showed the positive influence towards the second principal component, while negative impact was observed by FRAP root (0.6%). PCA graphics showed quite good discrimination between solutions S and S solutions. The influence of processing parameters can be observed in Figure, with samples processed with lower temperature located at the at the bottom side of the graphic. Samples treated in sugar beet molasses solution are located at the left side of the graphic, showing increased FRAP and FC values. = β 0 + β + β + β k k ki i kii i k i= i= Y X X X X k=-8 () where: β k 0, β ki, β kii, β k are constant regression coefficients; Y, either: FRAP or FC in root or leaves, k after OT in S or S solution, while X is time, and X is temperature. 3. Results and Discussion 3. Principal component analysis (PCA) Principal component analysis (PCA) is a mathematical procedure used as a central tool in exploratory data analysis [7, 8]. It is a multivariate technique in which the data are transformed into orthogonal components that are linear combinations of the original variables. PCA is done by Eigenvalue decomposition of a data correlation matrix [9]. This transformation is defined in such a way that the first component has the largest possible variance. This analysis is used to achieve maximum separation among clusters of parameters [7]. This approach, evidencing spatial relationship between processing parameters, enabled a differentiation between the different samples in both solutions (S and S ). The PCA, applied to the given data set, Table, has shown a differentiation between the samples according to used process parameters and is used as a tool in exploratory data analysis to characterize and differentiate neural network input parameters. As can be seen, there is a neat separation of the observed samples, according to used assays. Quality results show that the, PC : 3.78% S, 4 T=50 o C FRAPl 3 eaves S, 0. T=35 7 C 4 6 FC 0.0 leaves 9 FC root 9 FRAP 7-0. root S, S, T=0 C T=35 C S, T=0 o C PC : 93.07% 3 6 S, T=50 o C Figure. Biplot graphic of celery leaves and root osmotic treatment in sugar beet molasses solution and ternary solution 3. Cluster analysis (CA) Figure show dendrogram of CA for the osmotic treatment of celery root in sugar beet molasses solution and ternary solution. The complete linkage algorithm and City block (Manhattan) distances were used as the measure of proximity among the samples. City block distances (shown on ordinate axis) are measured as the average difference across the dimensions of the observed samples. This distance measure yields results similar to the Euclidean distance, but in this measuring technique, the effect of single large differences (outliers) is dampened (since they are not squared). The dendrogram presented in Figure is based on experimental data. The resulting dendrogram showed two main clusters; the first cluster contained samples (fresh sample) and samples - 0 (samples treated with S solution), while the second cluster contained 78

5 samples treated with S solution. The linkage distance (shown on the ordinate axis) between the main clusters was nearly 0.4. Linkage distances Complete Linkage City-block (Manhattan) distances Cases Figure. Three diagram for celery root osmotic treatment in sugar beet molasses solution and ternary solution 3.3 Response surface methodology (RSM) The ANOVA showed the significant effects of independent variables to the responses, and to show which of responses were significantly affected by the varying treatment combinations (Table ). The SOP models for all variables were found to be statistically significant and the response surfaces were fitted to these models. Linear terms of immersion time and process temperature were the most influential variables for FRAP and FC calculation. The residual variance shown in Table, where the lack of fit represents other contributions of the higher order terms. All SOP models had an insignificant lack of fit tests, which means that all the models represented the data satisfactorily. The coefficient of determination, r, is defined as the ratio of the explained variation to the total variation and is explained by its magnitude [30]. It is also the proportion of the variability in the response variable, which is accounted for by the regression analysis. A high r is indicative that the variation was accounted and that the data fitted satisfactorily to the proposed model. The r values for observed assays were very good and show the good fit of the model to experimental results. Acknowledgement These results are part of project supported by the Ministry of Science and Technological Development of the Republic of Serbia, TR-3055, Conclusions - This study was confirmed that optimization of applied drying treatment could be based on surveying of antioxidant activity, as a general indicator of potential health benefit. - Taking into account the effect of type of osmotic solution on the phenolics retention and total antioxidant activity, superiority of osmotic treatment of celery in sugar beet molasses is obvious. - OT in ternary solution has been evaluated as less convenient to be applied, due to a significant loss of total phenolics and decrease of the antioxidant activity. FRAP and FC values for celery root samples treated in ternary solution are decreased proportional to the increase of temperature and immersion time (from initially,57 to 0.90 and 0.59 to 0.07, respectively). - In the case of the leaves, decrease in FRAP and FC values is expressed to a lesser extent (from initially.44 to.358 and 0.58 to 0.49, respectively). Table. The ANOVA calculation for celery root and leaves osmotic treatment in sugar beet molasses solution and ternary solution Ternary solution Sugar beet molasses solution Root Leaves Root Leaves df FRAP FC FRAP FC FRAP FC FRAP FC T 0.08 * *.8 0-4* * * * t ** T 0.05 * * * * T t T Error r 0,98 0,989 0,659 0,700 0,894 0,87 0,96 0,937 + Significant at p < 0.0 level, * significant at p < 0.05 level, ** significant at p < 0.0 level, unmarked terms were not statistically significant. df - degrees of freedom 79

6 - On the other hand, OT in molasses provoke slight changes in FRAP and FC values or even increase them. Enhancement in FRAP values in celery root and leaves treated in molasses (from initially.57 to.54 and.44 to.477, respectively), and in FC values (from initially 0.59 to 0.67 and 0.58 to 0.69, respectively), confirms the fact that molasses is a rich source of antioxidants. These increases indicate that during OT was occurred penetration of some phenolic compounds from molasses into the tissue of celery. - According to the obtained results, it can be concluded that optimal solution for drying celery leaves and root is sugar beet molasses, optimum temperature of process is 50 0 C, while the optimum immersion time is 5 h. 5. References [] Mišan A., Dukić N. M., Sakač M., Mandić A., Sedej I., Šimurina O., and Tumbas, V. (0). Antioxidant activity of medicinal plant extracts in cookies. Journal of Food Science, 76, (9), pp [] Yao Y., Sang W., Zhou M., and Ren G. (00). 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