Art13_Ercisli.indd Journal of Applied Botany and Food Quality 85, 86 - 90 (2012) 1*Department of Horticulture, Faculty of Agriculture, Ataturk University, Erzurum, Turkey 2Department of Food Science, Faculty of Agriculture, Ataturk University, Erzurum, Turkey 3Department of Food Science, Faculty of Engineering, Gumushane University, Gumushane, Turkey 4Faculty of Agriculture, Zagreb University, Zagreb, Croatia Phytochemical and antioxidant characteristics of medlar fruits (Mespilus germanica L.) S. Ercisli1*, M. Sengul2, H. Yildiz3, D. Sener2, B. Duralija4, S. Voca4, D. Dujmovic Purgar4 (Received November 19, 2011) * Corresponding author Summary Eleven medlar (Mespilus germanica L.) genotypes sampled from Turkey were analyzed for their fruit weight, fruit dimensions, fruit fi rmness, ostiole diameter, shape index, skin color, moisture (%), ash (%), reducing sugar (%), crude protein (%), pH, soluble solid content (%), vitamin C (mg/100 g), minerals (P, K, Ca, Mg, Fe, Zn, Mn), total phenolic content and total antioxidant capacity. A wide variation among genotypes on most of the searched parameters was evident. Fruit weight varied from 11.21 g to 33.24 g indicating high variability among genotypes. Determination of antioxidant activi- ties by β-carotene – linoleic acid and 2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assays resulted in average 80.8%, and 46.6 µg/ml FW DPPH, respectively. The total phenolic contents of eleven medlar genotypes varied from 114 to 293 mg gallic acid equivalent in 100 g fresh weight basis. The medlar fruits were found to be rich in terms of potassium, calcium, phosphorus, magnesium and iron. Introduction The association between a diet rich in horticultural crops and a decreased risk of cardiovascular disease and certain forms of cancer is supported by considerable epidemiological evidences (ZIEGLER, 1991; LAW and MORRIS, 1998). Guidelines of healthy nutritions have also directed the general public to eat more fresh horticultural crops, fruit and vegetables, throughout the world for prevention such kind of diseases. It is well known that horticultural crops in particular berries are the main sources of natural antioxidants (HEINONEN et al., 1998; HEGEDUS et al., 2008; TULIPANI et al., 2008). More recently underutilized fruits, small fruits, such as cornelian cherry, mountain ash, sea buckthorn, rose hip, service tree, elderberry, bilberry, mulberry, jujube are also being increasingly consumed mainly due to their pleasant fl avor and their perceived health benefi ts related to their vitamins, antioxidants and minerals (ERCISLI and ORHAN, 2007; SERTESER et al., 2008; TOMOSAKA et al., 2008). The human healthy benefi ts such as antioxidants of common con- sumed fruits have been reported (HEINONEN et al., 1998; MOHAMED et al., 2007; NETZEL et al., 2007; VOCA et al., 2008). However, chemical composition of under utilized fruits including medlar is scarce. The assessment of such properties remains can be an interesting and useful task, particularly for fi nding new sources for natural antioxidants, functional foods and nutraceuticals. In addition more recently under utilized fruit market, once restricted to local areas, has increasingly expanded to the metropolitan centers in most of the countries. Thus, information on the human healthy values of these kinds of fruits could be a great importance (ARABSHAHI- DELOUEE and UROOJ, 2007). The antioxidant activity of fruits varies considerably. These dif- ferences may be due to multiple reasons including genetic factors or cultivar differences, different environmental conditions, stage of maturity, growth stage, soil fertilization and the part of the plant used, amongst other factors that propitiate quantitative variation in these phytochemicals (NETZEL et al., 2007; ERCISLI and ORHAN, 2008). Medlar, Mespilus germanica L. belongs to Rosaceae family and it grows mainly in frost-free areas, and on rocks and poor soils. In Turkey, they are abundant particularly in north and west-Anatolia and Marmara regions (BROWICZ, 1972). It is one of the latest maturating fruits and the ripening occurs in late October before frosts in Turkey. The fruits are used as a nutrition component by the local population and are prepared by the local people as marmalade or pickle. The fruit is consumed as a medicinal remedy for example treatment of constipation, diuretic, and to rid the kidney and bladder of stones in Turkey (BAYTOP, 1999). The increasing demand for natural antioxidants, together with the introduction of new technologies to meet the new quality standards, justifi es the search for new sources of natural antioxidants. The present study is aimed at assessing the phytochemical content of medlar fruits from Turkey, paying special attention in order to identify new sources of natural antioxidants. Materials and methods Collection and preparation of medlar fruits Approximately 3 kg fruit from each of eleven medlar genotypes were sampled from Coruh valley in Turkey. The genotypes were pre-selected according to their rising yield capacity, attractive fruit properties and absence of pest and disease indicators. Fruits were harvested at commercial maturation stage (skin brownish, pulp white, fruit hard) by hand and transferred to the laboratory for physical and phytochemical analysis. Samples were frozen immediately and then stored in about 100 g batches at -30 °C prior to analysis. Determination of fruit weight, dimensions, fi rmness and skin colors in medlar fruits Fifty fruits from each genotype were used immediately after harvest for fruit weight, dimensions, fi rmness and color determination. Fruit weight was measured by using a digital balance with a sensitivity of 0.001 g (Scaltec SPB31). Linear dimensions of fruits as length (L) and width (W) was measured by using a digital calliper gauge with a sensitivity of 0.01 mm. Fruit fi rmness was measured at 22 °C using a non-destructive fi rmness device (Aweta, NL). Skin color of medlar fruits was measured by using a CR-400 chromometer (Konica Minolta, Japan) and the color of the fruit surface was determined for the L (lightness), a (green chromaticity) and b (yellow chromaticity) values. Chroma and Hue were then calculated as described by MCGUIRE (1992): Chroma = (a2+b2)1/2 Hue angle = tan-1 (b/a) Color values for every fruit were computed as means of triplicate measures on equidistant points of each fruit. Phytochemical and antioxidant characteristics of medlar fruits 87 Determination of moisture, ash, soluble solid content (SSC), vitamin C, pH, reducing sugar and crude protein in medlar fruits For each genotype, total 50 fruits were thawed at room temperature and homogenized in a standard food blender. Homogenates were assayed for pH, reducing sugar, soluble solid content (SSC) and Vitamin C. Total soluble solid contents (TSS) were determined by a digital refractometer (Model RA-250HE, Kyoto Electronics Manufacturing Co. Ltd., Japan) at 22 °C. Moisture and ash were determined by AOAC (1984). The Kjeldahl method (BREMNER, 1996) and a Vapodest 10 Rapid Kjeldahl Distillation Unit (Gerhardt, Königswinter, Germany) were used to determine total N. Ascorbic acid (Vitamin C) and reducing sugar of samples was quantifi ed with the refl ectometer set of Merck Co (Merck RQfl ex). Determination of total phenolics and antioxidant activity in medlar fruits For extraction, fruit homogenates obtained with a blender were extracted with a buffer containing acetone, water, and acetic acid (70:29.5:0.5, v/v/v) for 1 h in darkness (SINGLETON and ROSSI, 1965). This extract was fi ltered and used for phytochemical analysis. Total phenolics in the methanol extracts were determined colori- metrically using Folin-Ciocalteu reagent as described by SLINKARD and SINGLETON (1977). Gallic acid was used as the standard and results were expressed as mg gallic acid equivalents per 100 g fresh weight basis. Total antioxidant capacity of samples was determined by β-carotene bleaching and 2,2-Diphenyl-1-picrylhydrazyl radical (DPPH•) assays. In the β-carotene bleaching assay, antioxidant capacity is determined by measuring the inhibition of the volatile organic compounds and the conjugated diene hydroperoxides arising from linoleic acid oxidation (KAUR and KAPOOR, 2002). Antioxidant capacities of the samples were compared with those of the synthetic antioxidant butylated hydroxyanisole (BHA) and the blank. In DPPH assay, 50 µl of various concentrations of the extracts in methanol were added to 5 ml of a 0.004% methanol solution of DPPH.. After a 30 min incubation period at room temperature, the absorbance was read against a blank at 517 nm. Inhibition of free radical DPPH in percent (I%) was calculated in following way: I% = (A= (A= ( blank-Asample/A/A/ blank) x100; where Ablank is the absorbance of the control reaction (containing all reagents except the test com- pound), and Asample is the absorbance of the test compound. Extract concentration providing 50% inhibition (IC50) was calculated from the graph plotting inhibition percentage against extract concentration. Tests were carried out in triplicate. Results were expressed as µg/ml FW (BURITS and BUCAR, 2000). Determination of mineral elements Fruit samples were oven-dried at 68 °C for 48 h and ground to pass 1 mm. Phosphorus content was determined after wet digestion using a HNO3-HClO4 acid mixture (4:1 v/v). Phosphorus in the extraction solution was measured spectrophotometrically using the indophenol-blue and ascorbic acid method with a UV/VIS Aquamat Spectrophotometer (Thermo Electron Spectroscopy LTD, Cambridge, UK). K, Ca, Mg, Fe, Mn and Zn were determined after wet digestion using a HNO3-HClO4 acid mixture (4:1 v/v) with a Perkin-Elmer 360 Atomic Absorption Spectrophotometer (Perkin- Elmer, Waltham, Massachusetts, USA). Results were expressed in mg/100g fresh mass for P, K, Ca, Mg, Fe, Mn and Zn. Statistical analysis The experiment was a completely randomized design with 5 re- plications. Data were subjected to analysis of variance (ANOVA) and means were separated by Duncan’s multiple range test at P<0.05 signifi cant level. Results and discussion Fruit weight, dimensions, fi rmness and colors of medlar fruits The fruit weight, dimensions, fi rmness, shape index, ostium diameters and colors of fruits in eleven medlar genotypes are shown in Tab. 1. Statistically signifi cant differences were recovered between the means for all the traits tested (Tab. 1). The highest fruit weight was observed in genotype M7 as 33.24 g, and followed by M3 (22.71 g) and M6 (16.42 g), respectively. Fruit dimensions are also found very variable among genotypes from 27.45 to 38.88 mm for length and 28.44 to 42.51 mm for diameter (Tab. 1). On the other hand shape index was found between 0.81 and 1.09 indicating some genotypes have pear-shaped (M2 and M8) and the others are apple-shaped form. Previously a wide variation on fruit weight and dimensions has been observed in medlar genotypes from 9.46 to 40.80 g for fruit weight, 23.67 to 42.51 mm for fruit length and 26.53 to 48.73 mm for fruit diameter (OZKAN et al., 1997; BOSTAN, 2002; BOSTAN and ISLAM, 2007). Our results are within the range of the values reported Tab. 1: Fruit weight, dimensions and color characteristics of samples Genotypes Fruit weight Fruit length Fruit diameter Fruit fi rmness Shape index Ostiole diameter Hue Chroma (g) (mm) (mm) (kg/cm2) (mm) (deg) (%) M1 14.32bc 32.23ab 30.75c 0.35ab 0.95ab 18.81bc 67.79bc 40.87ab M2 15.83bc 37.03ab 29.82cd 0.38ab 0.81c 15.51bc 72.85ab 33.45b M3 22.71b 34.76ab 36.62b 0.48ab 1.05ab 20.44b 61.92c 42.45ab M4 11.21c 28.73ab 28.44d 0.31b 0.99ab 16.14bc 80.54a 43.30a M5 12.94c 30.13ab 28.84cd 0.35ab 0.96ab 16.93bc 70.44b 42.21ab M6 16.42bc 32.62ab 31.68bc 0.41ab 0.97ab 18.68bc 69.63bc 39.98ab M7 33.24a 38.88a 42.51a 0.61a 1.09a 26.48a 68.85bc 33.21b M8 14.19bc 31.76ab 29.50cd 0.34ab 0.93b 13.92c 66.96bc 40.07ab M9 15.79bc 31.81ab 31.21bc 0.38ab 0.98ab 17.14bc 69.96bc 38.37ab M10 14.77bc 31.54ab 30.19c 0.35ab 0.96ab 17.30bc 69.03bc 42.45ab M11 13.74bc 27.45b 29.90cd 0.38ab 1.09a 16.52bc 68.28bc 40.67ab *Different letters indicate the statistical difference within same column among genotypes at 5% level. 88 S. Ercisli, M. Sengul, H. Yildiz, D. Sener, B. Duralija, S. Voca, D. Dujmovic Purgar in literature. Fruit fi rmness and colors (as chroma and hue) were found between 0.31 and 0.61 kg/cm2 and 33.21-43.30% for chroma and 61.92-80.54 (deg) for hue (Tab. 1). Moisture, ash, soluble solid content (SSC), vitamin C, pH, reducing sugar and crude protein in medlar fruits There were statistically signifi cant differences among genotypes in terms of above parameters except ash and reducing sugar (Tab. 2). SSC content of medlar genotypes were between 16.4-21.4% (Tab. 2). Notable the genotype M7 had relatively higher soluble solid content. Soluble solid contents of medlar genotypes previously reported between 12.5-26.0% (OZKAN et al., 1997; BOSTAN, 2002; BOSTAN and ISLAM, 2007). Among genotypes vitamin C and pH ranged from 11.5 to 15.0 mg/100 g and 3.3 to 4.2 (Tab. 2). The mean of the vitamin C contents of medlar genotypes was 12.7 mg/100 g. The genotype dependent moisture and crude protein of medlar fruits were observed between 67.4-75.6% and 3.3-4.3%, respectively (Tab. 2). As in most vegetarian diets, protein quality and quantity are major concerns. Lack of adequate proteins, either in quality or quantity contributes to low body mass, growth retardation in children, and developmental defi ciency during pregnancy. The average adult re- quires approximately 0.8 g of protein per kg of lean body mass per day to maintain normal functions, and so a person weighing 70 kg needs approximately 56 g of protein daily. To a certain extent the use of medlar genotypes in a diet may contribute to fi lling the protein gap. Vitamin C, pH, moisture and crude protein of medlar fruits was previously reported between 15.70-24.80 mg/100 g (OZKAN et al., 1997; WAZBINSKA, 2007), 2.89-6.15 (OZKAN et al., 1997; BOSTAN, 2002; BOSTAN and ISLAM, 2007); 72.2% (HACISEFEROGULLARI et al., 2005) and 3.7% (HACISEFEROGULLARI et al., 2005), respectively. The variation of SSC, vitamin C, moisture and crude proteins in medlar fruits could be due to different genotypes used, environmental conditions and the nutritional status of the plantations, as well. Total phenolics and antioxidant activity in medlar fruits The total phenolic contents of the fruits of medlar genotypes varied from 114 mg GAE/100 g FW in M11 genotype to 293 mg GAE/100 g in M5 genotype (Tab. 3). The average total phenolic content of genotypes was 194 mg GAE/100 g FW. It can be said that medlar germplasm from Coruh valley is rich in total phenolics. This phenomenon could be due to an induction of synthesis of antioxidant enzymes and an increase in polyphenolic concentration due to the greater exposure of the unsheltered medlar plants to extremes of temperature, and infecting/damaging organisms in the valley. Phenolic compound biosynthesis is a typical stress-defense reaction. Total antioxidant capacity of medlar genotypes is shown in Tab. 3. The genotype seemed to infl uence the extent of antioxidant activity in medlar fruits. Determination of antioxidant activities by β-carotene – linoleic acid and 2-diphenyl-1-picryhydrazyl (DPPH) free radical scavenging assays resulted in average 80.8 % and 46.6 µg/ml FW DPPH, respectively. In β-carotene linoleic acid assay, antioxidant capacity was in order of 92.85% (M9) > 89.01% (M11) > 87.05% (M5) > 85.42% (M7) > 84.75% (M10) > 83.25% (M8) > 82.07% (M4) > 81.60% (M6) > 69.86% (M1) > 68.90% (M2) > 64.63% (M3) (Tab. 3). In DPPH assay, the antioxidant activity was between 22.3-57.7 µg/ml FW DPPH. The genotype M10 had the highest antioxidant capacity with 22.3 µg/ml FW DPPH, whereas the genotype M3 had the lowest one (57.7 µg/ml FW DPPH). Tab. 2: Moisture, ash, reducing sugar, soluble solid content, vitamin C and crude protein of samples Genotypes Moisture Ash Reducing sugar SSC Vitamin C Crude protein (%) (%) (%) (%) (mg/100 g FW) (%) M1 69.3ab 1.9NS 3.3NS 19.4ab 12.7ab 4.1ab M2 71.4ab 2.1 2.9 18.0bc 11.3b 3.6ab M3 68.7ab 1.8 3.2 20.2ab 13.8ab 4.0ab M4 73.4ab 2.3 2.6 17.4bc 11.9ab 3.4ab M5 70.6ab 2.0 2.7 18.6b 14.4ab 3.7ab M6 72.3ab 2.2 2.6 17.6bc 13.3ab 3.5ab M7 67.4b 1.8 3.3 21.4a 15.0a 4.3a M8 74.9ab 2.3 2.4 16.8c 11.9ab 3.5ab M9 75.6a 2.4 2.4 16.4c 12.0ab 3.3b M10 73.1ab 2.4 2.7 17.3bc 12.2ab 3.3b M11 70.4ab 2.0 2.9 18.5b 11.5b 3.6ab *Different letters indicate the statistical difference within same column among genotypes at 5% level. Tab. 3: Total phenolic content (TPC), antioxidant activity (β-carotene) and free radical scavenging capacity (DPPH) of samples Genotypes TPC DPPH β-carotene (mg GAE/100 g FW) (µg/ml FW) bleaching assay (%) M1 152d 54.0ab 69.7c M2 199c 43.3bc 68.9cd M3 119e 57.7a 64.6d M4 238bc 44.0bc 82.1b M5 293a 32.3c 87.1ab M6 232bc 53.7ab 81.6bc M7 244b 53.3ab 85.4ab M8 176cd 56.0ab 83.3ab M9 218bc 45.6b 92.9a M10 147d 22.3d 84.8ab M11 114e 50.0ab 89.0ab Average 194 46.6 80.8 BHA 21.24 94.33 *Different letters indicate the statistical difference within same column among genotypes at 5% level. Phytochemical and antioxidant characteristics of medlar fruits 89 These results indicate that medlar fruits can function as important natural antioxidant sources. These results agree with those previously reported for medlars in which a good antioxidant capacity had been described (CAMPANELLA et al., 2003; SERTESER et al., 2008). It was previously reported that the genotype effects antioxidant capacity in different fruit species such as strawberries (TULIPANI et al., 2008), mulberries (ERCISLI and ORHAN, 2007) and currants (HEGEDUS et al., 2008). Many under utilized fruits possess high concentrations of phenolic acids, some fl avonols, and other phenolic classes, which have antioxidant activity in vitro (TOMOSAKA et al., 2008; IKRAM et al., 2009). The results of our study show large variations on physico-chemical properties of medlar genotypes. A wide diversity among genotypes in Turkey, presumably the one of the centre of origin and diversity of Mespilus germanica, offers scope for selecting the better ones. The results also imply that dietary polyphenolic phytochemicals from medlar may supply substantial antioxidants, which, in turn, may provide health-promoting effects to consumers. Mineral element contents of medlar fruits The mineral contents of medlar genotypes are shown in Tab. 4. The statistical differences between the genotypes were observed based on P, K, Ca, Mg and Fe contents (Tab. 4). The average P, K, Ca, Mg and Fe values of medlar genotypes were 39, 792, 73, 55 and 7.2 mg/ 100 g (Tab. 4), respectively. Data obtained from medlar genotypes show that they have very high nutritional potential, particularly Ca, Fe, P, K, Mg and their nutritional value is greater than that of some cultivated fruits presented in Tab. 5 (ANON., 2007). GLEW et al. (2003) reported that medlar fruits are richer in Ca than in P and Mg. Macro and trace elements play an important role in many metabolic processes and functions throughout the life cycle. Studies on humans as well as on animals revealed that optimal intakes of elements such as potassium, magnesium, calcium, sodium, manganese, copper and zinc could reduce individual risk factors, including those related to cardiovascular disease (MERTZ, 1982). With respect to their Ca and Fe content, the medlar genotypes considered by this study may offer a better nutritional potential. Due to the high content of K, P and Mg, the medlar genotypes have the potential to meet the daily K, P and Mg requirements of an adult. References ANONYMOUS, 2007: www.healthalternatives2000.com/minerals-nutrition- chart.html. AOAC., 1984: Offi cials methods of analysis (14th ed.). VA, USA: Association of Offi cial Analytical Chemist, Arlington. Tab. 4: Mineral content of medlar genotypes Genotypes mg/100g K Ca Mg P Fe Mn Zn M1 828ab 73ab 51bc 42bc 7.6ab 0.6NS 0.7NS M2 788ab 72ab 60ab 38cd 7.0ab 0.3 0.5 M3 830ab 76ab 57ab 44b 8.1a 0.5 0.7 M4 754ab 68ab 54b 34d 6.8ab 0.4 0.6 M5 793ab 73ab 61ab 35cd 7.4ab 0.3 0.3 M6 774ab 70ab 56ab 39c 6.2b 0.5 0.3 M7 841a 80a 62a 48a 7.5ab 0.4 0.6 M8 762ab 69ab 50bc 36cd 6.2b 0.5 0.5 M9 740b 67b 49c 30de 7.0ab 0.4 0.6 M10 768ab 72ab 51bc 32de 7.4ab 0.6 0.3 M11 834ab 77ab 50bc 45ab 6.2b 0.5 0.5 Average 792 73 55 39 7.2 0.5 0.5 *Different letters indicate the statistical difference within same column among genotypes at 5% level. Tab. 5: Mineral content of some selected fruits compared to medlar fruits K Ca Mg P Fe Mn Zn Fruits mg/100g Medlar 792 73 55 39 7.2 0.5 0.5 Apple 158 9.5 7 9.5 - - -* Avacado 1204 22 78.4 82.4 2 - - Banana 467 7 43 27 0.4 - - Blackberries 282 46 28 30 0.8 1.9 0.4 Grapes 176 13 4.6 9 0.4 - - Kiwi 588 46 53 71 0.7 - 0.3 Mango 323 20.7 18.6 22.8 0.3 - - Orange 237 52 13 18 - - - Peach 193 5.0 69 12 - - - * : Trace amount 90 S. Ercisli, M. Sengul, H. Yildiz, D. Sener, B. Duralija, S. Voca, D. Dujmovic Purgar ARABSHAHI-DELOUEE, S., UROOJ, A., 2007:Antioxidant properties of various solvent extracts of mulberry (Morus indica L.) leaves. Food Chem. 102,1233-1240. BAYTOP, T., 1999: Therapy with medicinal plants in Turkey (past and present). Capa-Istanbul, Nobel Tıp Press (2nd ed.). BOSTAN, S.Z., 2002: Interrelationships among pomological traits and selection of medlar (Mespilus germanica L.) types in Turkey. J. Amer. Pomolog Soc. 56, 215-218. 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ZIEGLER, R.G., 1991: Vegetables, fruits, and carotenoids and the risk of cancer. Am. J. Clin. Nutr. 53, 251-259. Address of the corresponding author: S. Ercisli, Tel.: +90 442 2312599, E-mail: sercisli@hotmail.com