Compositional Characteristics and Antioxidant Properties of Fresh and Processed Sea Cucumber ( Cucumaria frondosa )

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S: Sensory and Nutritive Qualities of Food

Compositional Characteristics and Antioxidant Components of Cherry Laurel Varieties and Pekmez

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C ESARET TIN AL ASAL VAR, MOHAMAD AL-FARSI, AND FEREIDOON SHAHIDI ESARETTIN ASALV

Introduction

C

herry laurel (Laurocerasus officinalis Roem.) is a popular fruit (dark purple or black when mature), mainly distributed in the coasts of Black Sea region of Turkey and locally called “Taflan” or “Karayemi_.” Three cultivated (Ayaz and others 1997a) and 15 wild varieties (Ustun and Tosun 2003) have been reported in the Black Sea region. Both forms could be poisonous in their early developmental stages and have a strong bitter taste but are edible when ripe. Unlike wild counterparts, the cultivated varieties are larger, sweeter, and less bitter when unripe (Ayaz and others 1997a). In Turkey, annual production of cherry laurel is not known because of its consumption as fresh fruit in local markets (less used by industry at present). Besides fresh consumption, dried, pickled, and processed (into pekmez, jam, marmalade, and fruit juice) cherry laurel products are also consumed. Apart from their use for food, both fruits and seeds of cherry laurel are well known as home medicine in Turkey and have been used for many years for the treatment of stomach ulcer, digestive system complaints, bronchitis, eczemas, hemorrhoids, and as a diuretic agent, among others (Baytop 1984). Concentrated juice of cherry laurel produced by heating (pekmez) has been in use for centuries in Turkey and is a traditional product. Fresh or dried fruits, such as grapes, are mainly used for the production of pekmez, but other sugar-rich fruits such as apples, pears, plums, mulberries, cherry laurels, watermelons, and apricots can also be used in pekmez production (Aksu and Nas 1996; Tosun and Ustun 2003). Among those fruits, pekmez from cherry laurel is becoming increasingly popular because of its potential and perceived health benefits. Most of its carbohydrate is in the form of fructose and glucose, which easily passes into blood without digesMS 20040449 Submitted 7/5/04, Revised 8/2/04, Accepted 9/21/04. Authors Alasalvar and Al-Farsi are with Faculty of Health and Life Sciences, Food Research Center, Univ. of Lincoln, Brayford Pool, Lincoln, LN6 7TS, United Kingdom. Author Shahidi is with Dept. of Biochemistry, Memorial Univ. of Newfoundland, St. John’s, Newfoundland, Canada. Direct inquiries to author Alasalvar (E-mail: [email protected]).

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tion. This is nutritionally important, especially for babies, children, athletes, and in situations demanding a rapid energy source (Batu 1993). High fruit and vegetable consumption is associated with a reduced risk of several chronic diseases such as cancer, cardiovascular disease, coronary heart disease, and atherosclerosis (Hertog and others 1993; Ness and Powles 1997; Surh 2003; Watson 2003; Shahidi and Naczk 2004). The compounds believed to be responsible for the protective effects of a fruit-rich and vegetable-rich diet include carotenoids and antioxidant vitamins. However, there is growing evidence that other phytochemicals (non-nutritive components) contribute to varying degrees to antioxidant activity of individual fruits and vegetables. In this regard, attention has been focused on the significance of phenolics such as phenolic acids, flavonoids, and in particular anthocyanins (Mazza and Miniati 1993; Hagerman and others 1998; Pearson and others 1999; Rice-Evans 2001; Surh 2003; Shahidi and Naczk 2004). Interest in the role of antioxidants in human health has prompted research in the fields of horticulture, food science, and nutrition to assess the antioxidant activity, anthocyanins, phenolics, carotenoids, and antioxidant vitamins of fruits and vegetables to determine how their content and activity can be maintained or even improved through cultivar development, degree of maturity, harvesting methods, postharvest handling procedures, processing technologies, and storage conditions (Kalt and others 2000; Connor and others 2002; Moyer and others 2002; Luximon-Ramma and others 2003; Alasalvar and others 2005). Little is known about the health-promoting components of cherry laurel (Ayaz and others 1997b; Kolayli and others 2003; Ustun and Tosun 2003) and that of its corresponding pekmez. Therefore, detailed information about the health-promoting components of cherry laurel and its concentrated products (for example, pekmez) could lead to a better understanding and an increased consumption of these, including their use as functional foods and ingredients in pharmaceuticals, nutraceuticals, and medicine. The objec-

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ABSTRA CT y laur el is a popular fr uit in the B lack S ea rregion egion of Tur key e cherr y laur el vvar ar ietABSTRACT CT:: Cherr Cherry laurel fruit Black Sea urkey key.. Two nativ native cherry laurel arieties az and findik, together with pekme z (made fr om concentr ated juice of the kir az vvar ar iety b y ies,, namely kir kiraz pekmez from concentrated kiraz ariety by heating) w er e examined for their pr oximate composition, antio xidant activity wer ere pro antioxidant activity,, total anthocyanins anthocyanins,, phenolics phenolics,, and car otenoids ell as phenolic acids and sugar composition. A linear corr elation existed betw een anticarotenoids otenoids,, as w well correlation between oxidant activity and total content of phenolics ((rr 2 = 0.99). The antio xidant activity and total content of phenoantioxidant lics w er e the highest in pekme z, follo w ed b y findik, and kir az. A significant ((P P < 0.01) pr opor tion of anthocyawer ere pekmez, follow by kiraz. propor oportion nins and car otenoids was lost dur ing heat pr ocessing in the pr oduction of pekme z. Ten phenolic acids (fr ee carotenoids during processing production pekmez. (free and bound) and 6 sugars w er e identified among samples esults suggest that cherr y laur el vvar ar ieties wer ere samples.. These rresults cherry laurel arieties al antio xidant, which could potentially be used in food and and pekme z ser v e as a good sour ce of natur pekmez serv source natural antioxidant, nutr aceutical supplement for mulations nutraceutical formulations mulations.. otenoids Keywor ds: cherr y laur el, pekme z, antio xidant activity anthocyanins,, car carotenoids otenoids,, phenolics phenolics,, sugars eywords: cherry laurel, pekmez, antioxidant activity,, anthocyanins

Antioxidant components of cherry laurel varieties . . . tive of this research was to compare the compositional characteristics and antioxidant components of 2 native fresh cherry laurel varieties and pekmez.

and others (2001). ORACFL values were expressed as mmol Trolox equivalents (TE) per gram of fresh weight.

Measur ement of total anthocyanins easurement

Materials and Methods Prepar ation and stor age of cherr y laur el eparation storage cherry laurel ieties and pekme z arieties pekmez var Cherry laurel (Laurocerasus officinalis Roem.) fruits of 2 native varieties, namely kiraz and findik, were harvested when fully ripe in the Giresun province of Turkey in July and August 2003, respectively. They were packed in polyethylene bags (250-g portions), frozen, and stored at –20 °C until used. Pekmez was produced from the kiraz variety by the traditional method. Basically, collected cherry laurel juice, produced by cold-pressing, was heated inside a copper container (at an open wooden fire) until reaching pekmez consistency (about 3 to 4 h). It was stirred frequently during heating. After cooling, the pekmez was poured into 250-mL bottles, sealed, and stored at 5 °C in the darkness. Cherry laurel varieties were placed inside a polystyrene box with cooling gels (pre-frozen to –20 °C) and dispatched by Turkish Airlines to the Food Research Center, Univ. of Lincoln, U.K., within the same day. Pekmez was also dispatched at the same time without cooling gel. Upon arriving to the laboratory, cherry laurel varieties and pekmez were stored at –20 °C and 5 °C, respectively, until analyzed.

Chemicals All chemicals were obtained from Sigma-Aldrich-Fluka Co. Ltd. (Dorset, U.K.) unless otherwise specified.

Proximate analysis The proximate composition of the samples were determined according to the standard AOAC procedures (1995). The moisture content was determined by vacuum oven drying (method 934.06), protein by Kjeldahl nitrogen determination (method 920.152), and ash by direct ashing analysis (method 940.26). Total lipid content was determined by the method of Bligh and Dyer (1959). The percentage of crude protein was estimated by multiplying the total nitrogen content by a factor of 6.25. Total carbohydrates were calculated by a difference method.

Measur ement of o xygen rradical adical easurement oxygen absorbance capacity assay

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An improved oxygen radical absorbance capacity (ORAC) method of Ou and others (2001), using fluorescein (FL) as the fluorescent probe, was used with slight modifications. The ORACFL assay measures the ability of antioxidative compounds in test materials to inhibit the decline in fluorescence induced by peroxyl radical AAPH (2,29-azobis [2-amidinopropane] dihydrochloride). Briefly, a 150mL sample extract was added into a 3-mL fluorescence cell, followed by 150 mL of 0.12 mM disodium FL solution, and 2.55 mL of 75 mM phosphate buffer (pH 7.4). Phosphate buffer was used as a blank, and 2.5, 5, and 10 mM Trolox (a water-soluble a-tocopherol analogue) were used as standards. The cell was incubated at 37 °C for 15 min in a water bath. The initial fluorescence (fo) was measured at the excitation of 493 nm and emission wavelength of 515 nm using a RF-540 Shimadzu spectrofluorophotometer (Shimadzu, Kyoto, Japan). After recording fo, 150 mL of 320 mM AAPH reagent as a free radical generator was added into a cell and mixed well using a glass rod. Fluorescence was measured and recorded every 5 min (f5, f10, f15…. f70) until the fluorescence of the last reading declined by >95% from the 1st reading (about 70 min). The area under curve (AUC) and relative ORACFL values were calculated according to Ou S2

Total anthocyanins were determined by the pH-differential method according to Guisti and Wrolstad (2001). All manipulations were carried out under a yellow fluorescent lighting (Thorn, U.K. [PR OVIDE MFR ’S NAME/L OCA TION] [PRO MFR’S NAME/LOCA OCATION] TION]) because anthocyanins are highly sensitive to light. Absorbance was measured in a UV-1601 Shimadzu spectrophotometer at 510 nm and at 700 nm using 2 buffer systems: potassium chloride buffer, pH 1.0 (0.025 M) and sodium acetate buffer, pH 4.5 (0.4 M). Absorbance was calculated as A = (A510nm – A700nm)pH1.0 – (A510nm – A700nm)pH4.5 with a molar extinction coefficient for cyanidin 3-glucoside of 26900. Results were expressed as mg cyanidin 3-glucoside equivalents per 100 g of fresh weight.

Measur ement of total car otenoids easurement carotenoids Total carotenoids were extracted according to the method of Talcott and Howard (1999) with slight modifications. Two grams of sample were extracted using 25 mL of acetone/ethanol (1:1, v/v) with 200 mg/L butylated hydroxytoluene (BHT) added. All manipulations were carried out under a yellow fluorescent lighting (Thorn) because carotenoids are highly sensitive to light. After extraction, sample was centrifuged at 1500 g for 15 min at 4 °C to 5 °C. The supernatant was collected and the remaining residue was re-extracted using the same method until the residue was colorless. Finally, the combined supernatants were brought to 100 mL with the extraction solvent and the absorbance at 470 nm was measured using a UV-1601 Shimadzu spectrophotometer. Total carotenoids were calculated according to Gross (1991).

Measur ement of total phenolics easurement Total soluble phenolics in the methanol extracts were determined colorimetrically using Folin-Ciocalteu reagent as described by Slinkard and Singleton (1977). Ferulic acid was used as a standard and results were expressed as mg ferulic acid equivalents per 100 g of fresh weight.

Hydr olysis and extr action of phenolic acids drolysis extraction Phenolic acids in cherry laurel varieties and pekmez were determined according to the high-performance liquid chromatographic (HPLC) method of Mattila and Kumpulainen (2002) with a slight modification. A 2-g sample was accurately weighed into a 50-mL graduated glass test tube and homogenized in 20 mL of a mixture of methanol containing 2 g/L of butylated hydroxyanisole (BHA) and 10% acetic acid (85:15, v/v) for 3 min. Then, all sample extracts were ultrasonicated for 30 min, made up to 30 mL with the extraction solvent, and mixed. After that, the samples were centrifuged at 1000 g for 15 min and 2 mL of the supernatant were finally filtered through a GELMAN Acrodisc LC13 PVDV 0.45-mm pore size syringe filter (PALL Life Sciences, Ann Arbor, Mich., U.S.A.) for the HPLC analysis of free phenolic acids. After taking the samples for analyzing free phenolic acids, 12 mL of distilled water and 5 mL of 10 M NaOH were added into the test tube, and its contents were then bubbled with nitrogen, sealed, and stirred using a magnetic stirrer at room temperature for around 16 h. The solution was then adjusted to pH 2, and liberated phenolic acids were extracted 3 times with 15 mL of a mixture of cold diethyl ether (DE) and ethyl acetate (EA, 1:1, v/v), by manually shaking and centrifuging. DE/EA layers were combined, evaporated to dryness, and dissolved in 2 mL of methanol. The HPLC run was carried out

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Antioxidant components of cherry laurel varieties . . .

Quantification of phenolic acids Twenty microliters of the standard and sample extracts was automatically injected into a Prodigy ODS-2 column (250 mm 3 3.2mm inner dia, Phenomenex, Cheshire, U.K.). The equipment consisted of a LaChrom L-7100 pump, LaChrom L-7455 diode array detector, LaChrom L-7200 autosampler, and LaChrom L-7300 column oven (Merck KgaA, Darmstadt, Germany). Column temperature was set at 35 °C. Gradient elution (filtered through a 0.45-mm Millipore filter before use) was used with a mobile phase consisting of 50 mM H3PO4, pH 2.5 (solution A) and acetonitrile (solution B) as follows: 0 to 5 min, isocratic elution 95% A and 5% B; 5 to 55 min, linear gradient 50% A and 50% B; 55 to 70 min, linear gradient 95% A and 5% B. Flow rate of the mobile phase was 0.7 mL/min. The wavelengths of the DAD detector were set at 254, 270, 280, and 329 nm for monitoring of phenolic acids. Tentatively identified phenolic acids were quantified on the basis of their peak areas and comparison with a calibration curve obtained with the corresponding standards. The results from free, alkaline, and acid hydrolysates were calculated to represent total phenolic acids.

Measur ement of sugars easurement Sugar levels were measured according to the HPLC method of Alasalvar and others (2003) with slight modifications. Sugars were extracted from cherry laurel (10 g) and pekmez (2.5 g) with 80 mL of acetonitrile/water (1:1, v/v) for 2 min. The extract was then kept in a water bath at 55 °C to 60 °C for 15 min (stirring frequently to aid dissolving sugars). It was subsequently centrifuged at 1500 g for 15 min at ambient temperature. The supernatant was carefully collected into a 100-mL volumetric flask and made up to a final volume of 100 mL with the extraction solvent. The HPLC column, pump, refractive index (RI) detector, and autosampler used were the same as described in a previous study (Alasalvar and others 2003). Column temperature was at ambient temperature and the mobile phase (filtered through a 0.45-mm Millipore filter and degassed before use) was a mixture of acetonitrile and HPLC-grade water at a ratio of 85:15 (v/v) at a flow rate of 0.5 mL/min. Identified sugars were quantified on the basis of peak areas and comparison with a calibration curve obtained for the corresponding standards.

Statistical analysis Results were expressed as mean value ± SD (n = 3) on a fresh weight basis. Statistical significance (t-test: 2-sample assuming equal variances) and correlation between ORACFL and phenolics were performed using the Microsoft Excel Data Analysis (Microsoft Corp., Redmond, Wash., U.S.A.). Differences at P < 0.05 were considered to be significant.

Results and Discussion Proximate analysis The proximate compositions of cherry laurel varieties and pekmez are summarized in Table 1. Significant differences (P < 0.05) URLs and E-mail addresses are active links at www.ift.org

Table 1—Proximate composition (g/100 g) of cherry laurel varieties and pekmeza,b Composition

Kiraz

Findik

Pekmezc

Protein Fat Carbohydratesd Moisture Ash

1.51 ± 0.06c 0.23 ± 0.02c 20.23 ± 0.58c 77.28 ± 0.55c 0.75 ± 0.05c

0.54 ± 0.04d 0.10 ± 0.01d 17.72 ± 0.70d 81.21 ± 0.74d 0.43 ± 0.03d

1.33 ± 0.03e 0.14 ± 0.02e 44.22 ± 0.18e 52.46 ± 0.17e 1.85 ± 0.01e

aData are expressed as mean ± SD (n = 3). bMeans ± SD followed by the same letter, within a row, are not significantly different

(P > 0.05). cPekmez was made from concentrated juice of kiraz variety by heating. dCarbohydrates were calculated by subtracting the total percent values of other components from 100.

were observed among cherry laurel varieties and pekmez. Moisture was the predominant component, followed by carbohydrates, and along with small amounts of protein and fat. These values were within the range of previously published results in the literature for cherry laurel varieties (Kolayli and others 2003; Ustun and Tosun 2003). Harvest time, maturity state, season, age of trees, geographical origin, environmental factors, storage, and handling conditions, in addition to the variety of cherry laurel, affect the composition of products (Ayaz and others 1997a; Kolayli and others 2003; Ustun and Tosun 2003).

ORA CFL ORAC Significant differences (P < 0.01) in ORACFL values were observed among cherry laurel samples and pekmez. The highest ORACFL value was measured in pekmez (19981 mmol TE/g), followed by findik (7996 mmol TE/g), and kiraz (3363 mmol TE/g). The higher ORACFL values obtained in this study compared with that of previously described ORAC method of Cao and others (1993) was because of the use of fluorescein as the fluorescent probe, which was developed by Ou and others (2001). Wang and Lin (2000) studied the antioxidant activity in fruits and leaves from different cultivars of berries, using ORAC method described by Cao and others (1993). The ORAC values ranged from 7.8 to 33.7 mmol of TE/g of fresh weight (FW) berries. Unlike other popular antioxidant activity method, the improved ORACFL assay directly measures the antioxidant activities of chain-breaking antioxidants against peroxyl radicals (Ou and others 2001, 2002). Therefore, they suggested that ORACFL values be used as a guideline for “peroxyl radical absorption capacity” of food samples. A linear correlation existed between ORACFL values and total content of phenolics (r2 = 0.99). Sample with the highest phenolic content had the highest ORAC FL, in agreement with those reported in the literature (Deighton and others 2000; Moyer and others 2002; Howard and others 2003). However, inverse relationships between ORACFL and total anthocyanins were observed, possibly because of the loss of anthocyanins as a result of heat processing in the production of pekmez (Table 2).

Total anthocyanins The average contents of total anthocyanins among kiraz, findik, and pekmez were 123.8, 174.3, and 9.3 mg of cyanidin 3-glucoside equivalents/100 g of fresh weight, respectively. A significant amount of anthocyanins was lost (92.5%) during heat processing in the production of pekmez. Raynal (1987) found that only 14.6% of initial anthocyanins remained after 1 h at 95 °C, whereas 45.6% remained at 55 °C during drying of plum. Studies have shown that anthocyanins are readily destroyed by heat during food processing (Markakis 1974; Mazza and Miniati 1993). Apart from heat, many

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after filtering through a GELMAN Acrodisc filter (PALL Life Sciences). After the above alkaline hydrolysis was completed, an acid hydrolysis was performed by adding 2.5 mL of concentrated HCl into the test tube and incubating the tube in a water bath at 85 °C for 30 min. Subsequently, the sample was allowed to cool and the pH of the medium was adjusted to 2. The DE/EA extraction performed was similar to that for alkaline hydrolysis. Evaporated extract was then dissolved into 2 mL of methanol, filtered through a GELMAN Acrodisc filter (PALL Life Sciences) and analyzed by HPLC.

Antioxidant components of cherry laurel varieties . . . Table 2—Contents of antioxidant activity (ORACFL), anthocyanins, carotenoids, and phenolics in cherry laurel varieties and pekmeza,b Sample

ORACFLc (mmol of TE/g)

Total anthocyaninsd (mg/100 g)

Total carotenoidse (mg/100 g)

Total phenolicsf (mg/100 g)

Kiraz Findik Pekmez

3363 ± 251f 7996 ± 359g 19981 ± 361h

123.8 ± 7.9f 174.3 ± 8.9g 9.3 ± 0.6h

254.0 ± 9.5f 261.3 ± 8.5f 114.0 ± 8.7g

454 ± 14f 651 ± 19g 1444 ± 20h

aData are expressed as mean ± SD (n = 3) on a fresh weight basis. bMeans ± SD followed by the same letter, within a column, are not significantly different (P > 0.05). cAntioxidant activity (ORAC : oxygen radical absorbance capacity [ORAC] using fluorescein [FL]),[CORRECT AS EDITED?] expressed as mmol of Trolox equivalents FL

(TE)/g fresh weight.

dTotal anthocyanins, expressed as mg cyanidin 3-glucoside equivalents/100 g fresh weight. eTotal carotenoids, expressed as mg/100 g fresh weight. fTotal phenolics, expressed as mg ferulic acid equivalents/100 g fresh weight.

Table 3—Contents of free phenolic acids (mg/100 g) in cherry laurel varieties and pekmeza,b,c Phenolic acid Protocatechuicd

p-Hydroxybenzoicd Chlorogenic Vanillicd Caffeic Syringicd P-coumaric Total hydroxybenzoic acids Total hydroxycinnamic acids Total free phenolic acids

Kiraz

Findik

Pekmez

1.12 ± 0.17c 2.38 ± 0.28c 136.06 ± 6.39c 12.41 ± 0.04c nd 56.15 ± 5.09c 49.70 ± 9.42c 72.06 (27.9)f 185.76 (72.1) 257.82 ± 20.35c

nd 3.01 ± 0.46c 102.64 ± 4.27d 10.48 ± 1.73c nd 41.43 ± 0.68d 43.05 ± 4.56c 54.92 (27.4) 145.69 (72.6) 200.61 ± 4.84d

8.34 ± 0.26d nd 194.53 ± 2.16e nd 74.39 ± 1.01 nd 20.47 ± 0.39d 8.34 (2.8) 289.39 (97.2) 297.73 ± 1.12e

aData are expressed as mean ± SD ( n = 3) on a fresh weight basis. bnd = not detected. cMeans ± SD followed by the same letter, within a row, are not significantly different (P > 0.05). Numbers in parentheses indicate percent of compounds in the total

amount of free phenolic acids.

dHydroxybenzoic acid derivatives.

other factors have been reported as being responsible for the degradation of anthocyanins during processing of fruits (Mazza and Miniati 1993; Shahidi and Naczk 2004). The anthocyanin contents of a number of fruits have been reported by several researchers. However, blue, red, violet, and purple colors of most edible plant species and their fruits (berries, grapes, cherries, plums, and so forth) are due to anthocyanins (Mazza and Miniati 1993; Sellappan and others 2002; K hk nen and others 2003; Shahidi and Naczk 2004). The total anthocyanin content in the darkcolored sweet cherries ranged widely, from 82 to 298 mg/100 g and in the light-colored sweet cherries from a few milligrams to 41 mg/ 100 g of fresh weight (Gao and Mazza 1995). Cantos and others (2002) studied the varietal differences among the polyphenol profiles of 7 table grape cultivars (4 reds and 3 whites). The total anthocyanins in red grape cultivars varied from 6.9 to 15.1 mg of cyanidin 3-rutinoside equivalents/100 g of fresh weight. No anthocyanins were detected in white grape cultivars. Total content of anthocyanins in 30 genotypes of Vaccinium L. (blueberries) ranged from 34 to 515 mg of cyanidin 3-glucoside equivalents/100 g of fresh weight (on average, 230 mg/100 g) (Moyer and others 2002). Within these ranges, cherry laurel varieties were found to be good sources of anthocyanins.

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Total car otenoids carotenoids Total carotenoid contents among kiraz, findik, and pekmez were 254, 261.3, and 114 mg/100 g of fresh weight, respectively (Table 2). Similar to anthocyanins, heat processing during pekmez production caused a significant loss of carotenoids (44.9%). Al-Farsi and others (2004) observed that total carotenoids levels decreased from 2.32-4.55 mg/100 g of dry weight in fresh dates to 1.11 to 3.33 mg/100 g of dry weight in dried dates. Hart and Scott (1995) surveyed the S4

carotenoid content of vegetables and fruits commonly consumed in the U.K. The total content of carotenoids in 8 fruits ranged from 17 to 2263 mg/100 g of fresh weight, being lowest in strawberry and highest in mandarins.

Total phenolics The mean total content of phenolics among kiraz, findik, and pekmez was 454, 651, and 1444 mg/100 g of fresh weight, expressed as ferulic acid equivalents, respectively (Table 2). Moyer and others (2002) found that total content of phenolics in 30 genotypes of Vaccinium L. (blueberries) ranged from 171 to 961 mg/100 g of fresh-frozen weight expressed as gallic acid equivalents (on average, 521 mg/100 g), respectively. Subsequently, Imeh and Khokhar (2002) studied the total and free phenolics contents of 16 commonly consumed fruits (comprising 9 apples, 4 pears, and 1 each of peach, plum, and kiwi fruit cultivars). The total content of phenolics in these fruits varied from 302.3 to 535 mg/100 g of fresh weight expressed as gallic acid equivalents. Recently, Luximon-Ramma and others (2003) reported that the total phenolics of 17 Mauritian exotic fruits ranged from 11.8 to 563.8 mg/100 g of fresh weight expressed as gallic acid equivalents, being lowest in banana and highest in red Chinese guava. Among fruits reported in the literature, 1 of the genotype of Ribes L. (black currant) contained the highest amount of total phenolics (1790 mg/100 g of fresh-frozen weight) (Moyers and others 2002), which was higher than that of pekmez (1444 mg/100 g of fresh weight). Nonetheless, cherry laurel varieties and pekmez were found to serve as an excellent source of phenolics.

Phenolic acids The content of free and bound phenolic acids in cherry laurel

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Antioxidant components of cherry laurel varieties . . . Table 4—Contents of bound phenolic acids (mg/100 g) in cheery laurel varieties and pekmeza,b,c Alkaline hydrolysis

Acid hydrolysis

Phenolic acid

Kiraz

Findik

Pekmez

Kiraz

Findik

Pekmez

p-Hydroxybenzoicd Vanillicd Caffeic Syringicd P-coumaric Ferulic o-Coumaric Total hydroxybenzoic acids Total hydroxycinnamic acids Total bound phenolic acids

nd nd 4.96 ± 0.80c 74.83 ± 2.44c nd 12.89 ± 1.90c nd nd 4.96 (5.4)f 87.72 (94.6) 92.68 ± 5.12c

nd nd 6.33 ± 0.86c 64.64 ± 1.40d nd 11.49 ± 0.26c 6.18 ± 0.34 nd 6.33 (7.1) 82.31 (92.9) 88.64 ± 1.20c

53.74 ± 3.42 11.40 ± 1.31 5.46 ± 0.37c 191.43 ± 3.05e nd 28.09 ± 3.40d nd nd 70.6 (24.3) 219.52 (75.7) 290.12 ± 0.90d

37.57 ± 2.15c 2.32 ± 0.08c nd nd nd nd nd nd 38.89 (100)f 0.00 (0.0) 39.89 ± 2.16c

16.54 ± 2.01d 2.77 ± 0.49c 1.47 ± 0.19 1.37 ± 0.31c 0.98 ± 0.14c nd nd 2.24 ± 0.09 21.76 (85.8) 3.61 (14.2) 25.37 ± 1.45d

222.34 ± 12.04e 16.60 ± 0.91d

Gallicd

6.50 ± 0.45d 1.98 ± 0.21d nd nd nd 240.92 (97.4) 6.5 (2.6) 247.42 ± 13.17e

a Data are expressed as mean ± SD ( n = 3) on a fresh weight basis. b nd = not detected. cMeans ± SD followed by the same letter, within a row, are not significantly different (P > 0.05). Numbers in parentheses indicate percent of compounds in the total

amount of bound phenolic acids. d Hydroxybenzoic acid derivatives.

Table 5—Sugar composition (g/100 g) of cherry laurel varieties and pekmeza,b,c Sample

Xylose

Arabinose

Fructose

Glucose

Sorbitol

Sucrose

Total

Kiraz Findik Pekmez

0.19 ± 0.02c 0.23 ± 0.01c 0.48 ± 0.05d

0.08 ± 0.01c 0.07 ± 0.02c 0.21 ± 0.01d

5.16 ± 0.51c 4.84 ± 0.19c 13.76 ± 0.04d

5.88 ± 0.42c 5.43 ± 0.21c 16.39 ± 0.12d

4.80 ± 0.45c 1.51 ± 0.08d 7.43 ± 0.05e

tr tr 0.05 ± 0.00

16.11 ± 1.40c 12.08 ± 0.47d 38.3 ± 0.21e

a Data are expressed as mean ± SD (n = 3) on a fresh weight basis. b tr = trace. cMeans ± SD followed by the same letter, within a column, are not significantly different (P > 0.05).

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were present in the highest content, respectively. The cherry laurel varieties used in this study were different from varieties used by Ayaz (2001). Many factors have been reported to influence the concentration and variability of phenolic compounds within the same fruit type (Sellappan and others 2002; Gonçalves and others 2004).

Sugar composition Figure 1 illustrates a typical chromatographic separation of sugars extracted from pekmez. Six sugars were positively identified in cherry laurel varieties and pekmez; these included monosaccharides (xylose, arabinose, fructose, and glucose), a sugar alcohol (sorbitol), and trace amounts of the disaccharide sucrose. Among these, xylose and arabinose were identified for the 1st time in cherry laurel varieties and pekmez. Monosaccharides, which represented 80.5% of the total sugar in pekmez, can easily pass into the blood stream without digestion. The highest content of average total sugar was

Figure 1—High-performance liquid chromatographic (HPLC) sugar profile of Pekmez

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varieties and pekmez are listed in Table 3 and 4, respectively. The samples studied contained hydroxylated derivatives of benzoic acid (gallic acid, protocatechuic acid, P-hydroxybenzoic acid, vanillic acid, and syringic acid) and cinnamic acid (chlorogenic acid, caffeic acid, P-coumaric acid, ferulic acid, and o-coumaric acid). In total, 8, 9, and 8 phenolic acids (free and bound) were tentatively identified and quantified in kiraz, findik, and pekmez, respectively. Free and bound phenolic acids, using 3 different hydrolysis procedures, in the same tube was carried out. Chlorogenic acid was the main hydroxycinnamic acid derivative that represented 52.8%, 51.2%, and 65.3% of the total free phenolic acids in kiraz, findik, and pekmez, respectively. Under alkaline conditions, chlorogenic acid was hydrolyzed to caffeic acid (Antolovich and others 2000), which was the major compound that represented 80.7%, 72.9%, and 66% of the total bound phenolic acids in kiraz, findik, and pekmez, respectively. Following alkaline hydrolysis, acid hydrolysis was performed to liberate the rest of the bound phenolic acids. Gallic acid predominated in this fraction and represented 94.2%, 65.2%, and 89.9% of the total bound phenolic acids in kiraz, findik, and pekmez, respectively. Gallic acid is unstable under alkaline conditions (Mattila and Kumpulainen 2002). The hydroxycinnamic acid derivatives predominated in free and alkaline hydrolysis, whereas hydroxybenzoic acid derivatives were the major compounds upon acid hydrolysis. Ayaz and others (1997b) found 5 phenolic acids (protocatechuic acid, P-hydroxybenzoic acid, vanillic acid, caffeic acid, and P-coumaric acid) in 3 cultivated and 1 wild cherry laurel varieties, among which vanillic acid was the major compound present in both forms. Subsequently, Ayaz (2001) studied the changes in phenolic acids of cultivated cherry laurel during maturation. He identified and quantified 7 phenolic acids (benzoic acid, 4-hydroxybenzoic acid, Pcoumaric acid, vanillic acid, 3,4-dihydroxybenzoic acid, caffeic acid, and ferulic acid), among which benzoic, caffeic, and vanillic acids

Antioxidant components of cherry laurel varieties . . . found in pekmez (38.32 g/100 g), followed by kiraz (16.11 g/100 g) and findik (12.08 g/100 g) (Table 5). Among sugars, glucose was the predominant component in all samples (5.43 to 16.39 g/100 g), followed by fructose (4.84 to 13.76 g/100 g), and sorbitol (1.51 to 7.43 g/100 g). The remaining 3 sugars (xylose, arabinose, and sucrose) were present only in small amounts. Ayaz and others (1997a, 1998) identified 4 sugars (fructose, glucose, sorbitol, and trace amount of sucrose) in 3 cultivated and 1 wild cherry laurel varieties and found varietal differences among them. The average total sugar content in cherry laurel varieties was in good agreement with those in the literature. Sugars are responsible for the sweetness of foods. Individual sugars possess different relative sweetness scores; fructose has been reported to be the sweetest (1.1 to 1.8), followed by sucrose (1.0) and glucose (0.5 to 0.8) (Alexander 1998).

Conclusions

T

he results presented in this work suggest that cherry laurel varieties and pekmez serve as a good source of natural antioxidant that could potentially be used in food and nutraceutical supplement formulations. Pekmez, which contains a high amount of total soluble sugar (38.32 g/100 g), can also be used as a sport drink for rapid energy supply. Cherry laurel varieties had different patterns of phenolics. Most of the phenolic acids in cherry laurel varieties were in the free form, whereas in pekmez, they[[ EDIT OK?] existed in the bound form. A significant (P < 0.01) amount of anthocyanins and carotenoids was lost during heat processing in the production of pekmez.

Acknowledgments We thank the Giresun Intl. Society of Education for their financial support. The authors are also grateful to Salih Ala•alvar and Mehmet KŸlekci for providing cherry laurel samples and pekmez, respectively.

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