Composition of bioactive compounds in kernel oils recovered from sour cherry (Prunus cerasus L.) by-products: Impact of the cultivar on potential applications

Industrial Crops and Products 82 (2016) 44–50

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Composition of bioactive compounds in kernel oils recovered from sour cherry (Prunus cerasus L.) by-products: Impact of the cultivar on potential applications b ´ Paweł Górna´s a,∗ , Magdalena Rudzinska , Marianna Raczyk b , Inga Miˇsina a , c a Arianne Soliven , Dalija Seglin¸a a

Latvia State Institute of Fruit-Growing, Graudu Str. 1, Dobele, LV-3701, Latvia Institute of Food Technology of Plant Origin, Faculty of Food Science and Nutrition, Pozna´ n University of Life Sciences, Wojska Polskiego 31, 60-624 Pozna´ n, Poland c Australian Centre for Research on Separation Sciences (ACROSS), School of Science and Health, University of Western Sydney (Parramatta), Sydney, NSW, Australia b

a r t i c l e

i n f o

Article history: Received 31 August 2015 Received in revised form 25 November 2015 Accepted 7 December 2015 Available online 22 December 2015 Keywords: Prunus cerasus L. Sour cherry kernel oil Fatty acids Tocopherols Carotenoids Phytosterols

a b s t r a c t Lipophilic bioactive compounds in kernel oils recovered from fruit pits of six sour cherry (Prunus cerasus L.) cultivars were studied. Oil yields ranged between 17.5–37.1%, with an average of 31.8%. The main fatty acids were linoleic (35.50–46.06%), oleic (25.25–45.30%), ␣-eleostearic (7.43–15.76%) and palmitic (5.06–7.38%), containing 94–96% of total detected fatty acids. The range of total tocochromanols was between 118.2 and 163.6 mg/100 g of oil. Independent of the cultivar, ␥-T was the main tocochromanol and constituted between 61 and 83% of the total identified tocopherol and tocotrienol homologues in all studied samples. The total content of the carotenoids was between 0.51–1.75 mg/100 g of oil. The concentration of squalene in different sour cherry kernel oils was reached between 65.8–102.8 mg/100 g of oil. The concentration of sterols varied significantly for all of the studied cultivars and ranged between 313.6–1041.3 mg/100 g of oil. The ␤-sitosterol constituted 77–82% of the total sterol content detected in the samples studied. Two significant correlations were found between oil yield and the total content of sterols (r = −0.974, p ≤ 0.001) and carotenoids (r = −0.915, p ≤ 0.01) in sour cherry kernel oils. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Sour cherry (Prunus cerasus L.) with the production estimated to approximately 1.2 million tons per year (FAOSTAT, 2014) belongs to the group of the most common type of the fruits, which are processed in large quantities into juice, candied and frozen fruits. During the processing of sour cherries, significant amounts of fruit pits are generated without further use or with very limited utility (Yılmaz and Gökmen, 2013). However, since, the fruit pits and seeds are valuable source of oil rich in essential fatty acids, as well of minor lipophilic compounds such as: carotenoids (Cenkowski et al., 2006; Fromm et al., 2012b), squalene (Caligiani et al., 2010; Hassanien et al., 2014), phytosterols (Caligiani et al., 2010; Hassanien et al., 2014) and tocochromanols (Górna´s et al., 2014a, 2015g), they can be utilized as a potential natural source

∗ Corresponding author. Fax: +371 63781718. E-mail address: [email protected] (P. Górna´s). http://dx.doi.org/10.1016/j.indcrop.2015.12.010 0926-6690/© 2015 Elsevier B.V. All rights reserved.

of nutraceutical ingredients in the pharmaceutical industry. Sour cherry kernels were considered previously as a source of oil (Bak et al., 2010; Chandra and Nair, 1993; Matthäus and Özcan, 2009; Popa et al., 2011; Yılmaz and Gökmen, 2013), however, in most of these studies only the fatty acid profile was studied sufficiently, while the knowledge about the minor bioactive compounds is very limited. Moreover, the impact of the cultivar on the profile of minor lipophilic compounds, which have a high importance for human health—low intake of carotenoids is associated with an increased risk of skin damage, cataract, cardiovascular diseases and cancer (Aust et al., 2001); phytosterols have the ability to decrease the amount of cholesterol in blood serum (Chen et al., 2008); while tocopherols and tocotrienols are characterized by unique physicochemical activity in biological (Eitenmiller and Lee, 2004) and model (Dwiecki et al., 2007a,b; Neunert et al., 2015; Nogala-Kałucka et al., 2013) systems, has never been taken into account. It has been reported, that the concentration and composition of biologically active substances in processed by-products is significantly influenced by the cultivar of crop, for instance in apples

P. Górna´s et al. / Industrial Crops and Products 82 (2016) 44–50

40

c

c

c

45

c

35

Oil yield, % (w/w) dw

b 30 25 a

20 15 10 5

vs ka ya Bu la tn iko

Sh ok ol ad ni ca

ai s Ze m ja s La tv i

no vs ka ya Ha rit o

Ze nt en es

Ta m

ar is

0

Fig. 1. Oil yield (%, w/w dw) recovered from the kernels of six sour cherry cultivars. Different letters indicate statistically significant differences at p ≤ 0.05.

and pears (Górna´s, 2015; Górna´s et al., 2015d,f, 2014b). Moreover, agro-climatic conditions also may affect the biochemical composition of plant material, however limited number of investigations has been done—only sour cherry samples originated from Turkey, Hungary, Greece and Iran (Bak et al., 2010; Farrohi and Mehran, 1975; Lazos, 1991; Matthäus and Özcan, 2009; Yılmaz and Gökmen, 2013) have been investigated previously. Therefore, to increase the knowledge about the possible impact of the cultivar on the oil yield and the composition of the bioactive lipophilic compounds (fatty acids, carotenoids, squalene, sterols and tocochromanols) in oils recovered from sour cherry kernels, six sour cherries cultivars, the most widely grown in the Baltic Countries and Russia were selected. The cultivar selection for specific industrial applications is crucial in order to optimize economic benefits; therefore, the present study can give a significant impact on the further utilization of the sour cherry pits.

July 2013, at the Latvia State Institute of Fruit-Growing, GPS location: N: 56◦ 36 39 E: 23◦ 17 50 . From the each of three batches of collected fresh sour cherries, the fruit pits were removed by hand equipment Delux Cherry Pitter Stoner (Homestead Harvest, Bellingham, WA, USA). Each of three fruit pit batches were crushed manually using the home hand-crusher to recover the kernels from the outer shells. Kernels were frozen (−18 ◦ C) and subsequently freeze dried at a temperature of −51 ± 1 ◦ C using a freeze-dry system (FreeZone, Labconco, Kansas City, MO, USA) under vacuum of 0.055–0.065 mBar for 48 h. Undamaged kernels (50 ± 5 g of dry weight) were selected and milled with an universal laboratory mill (KnifetecTM 1095, Foss, Höganäs, Sweden). The obtained powder was characterized by a mesh size 0.75 mm. Dry weight basis (dw) was measured gravimetrically.

2. Materials and methods

Oil was extracted according to an earlier introduced method (Górna´s et al., 2014b). In brief, ground 5 g of fruit kernels were supplemented with 25 mL of n-hexane (Sigma–Aldrich, Steinheim, Germany) in a centrifuge tube and mixed on a Vortex (REAX top, Heidolph, Schwabach, Germany) at 2500 rpm for 1 min. Samples were subjected to ultrasound treatment in the ultrasonic bath (Sonorex RK 510 H, Bandelin electronic, Berlin, Germany) for 5 min at 35 ◦ C and centrifuged (Centrifuge 5804 R, Eppendorf, Hamburg, Germany) for 5 min at 21 ◦ C and 10,000 × g . The supernatant was collected in a round bottom flask and the remaining solid residue was re-extracted (twice) as described above. The combined supernatants were evaporated in a vacuum rotary evaporator (Laborota 4000, Heidolph, Schwabach, Germany) at 40 ◦ C until constant weight. The oil content was expressed in% (w/w) dw (dry weight basis—measured gravimetrically) of seeds.

2.1. Reagents Methanol, tert-butyl methyl ether, n-hexane, 2-propanol (HPLC grade), pyrogallol, sodium chloride, potassium hydroxide and 5␣-cholestane (≥97%, GC) were obtained from Sigma–Aldrich (Steinheim, Germany). Tocotrienol and tocopherol homologues (␣, ␤, ␥ and ␦) (>95%, HPLC) were received from LGC Standards (Teddington, Middlesex, UK) and Merck (Darmstadt, Germany), respectively. The fatty acid methyl ester mix and Sylon BTZ were purchased from Supelco (Steinheim, Germany) and (Bellefonte, PA, USA), respectively. 2.2. Plant material Sour cherries (P. cerasus L.) of six cultivars: ‘Bulatnikovskaya’, ‘Haritonovskaya’, ‘Latvijas Zemais’, ‘Shokoladnica’, ‘Tamaris’ and ‘Zentenes’, grown on the seedling rootstock Prunus mahaleb L., were harvested in three independent batches for each cultivar, where each of batches was harvested from at least three various trees (each batch from different trees) at the same maturity stage in

2.3. Oil extraction

2.4. Fatty acid composition GC and GC/MS The fatty acid composition of the studied oil samples were estimated using gas chromatography (GC) according to AOCS (2005). Fatty acid methyl esters were separated using a Hewlett-Packard 5890 II gas chromatography system equipped with a Supelcowax

46

P. Górna´s et al. / Industrial Crops and Products 82 (2016) 44–50

Table 1 Fatty acid composition (%) of sour cherry kernel oils. Cultivar

Fatty acid C16:0

C16:1

C18:0

C18:1

C18:2

␣-C18:3

␣-ESA C18:3

C20:0

C20:1

Tamaris

7.38 0.11d

0.33 0.03c

2.97 0.02d

25.25 0.13a

46.06 0.14d

0.48 0.02b

15.76 0.25d

1.38 0.05c

0.39 0.02a

Zentenes

5.82 0.03b

0.25 0.02b

2.41 0.07b

37.50 0.87b

43.05 0.60c

0.10 0.02b

9.09 0.42c

1.22 0.03b

0.56 0.06a,b

Haritonovskaya

6.25 0.12c

0.27 0.01b,c

2.22 0.03a

38.17 0.12b

43.47 0.22c

0.11 0.01b

8.07 0.04b

0.95 0.02a

0.48 0.01a

Latvijas Zemais

6.21 0.07c

0.31 0.04b,c

2.63 0.03c

45.30 0.05d

35.50 0.10a

0.17 0.02b

8.34 0.17b,c

1.01 0.03a

0.52 0.10a,b

Shokoladnica

6.35 0.04c

0.27 0.01b,c

2.22 0.02a

40.79 0.14c

41.00 0.24b

0.09 0.02b

7.43 0.04a

1.19 0.05b

0.67 0.06b

Bulatnikovskaya

5.06 0.05a

0.16 0.02a

3.45 0.07e

38.61 0.38b

42.55 0.46c

0.26 0.01a

8.17 0.15b

1.23 0.03b

0.50 0.08a,b

Italic values correspond to standard deviations (n = 3). Different letters in the same column indicate statistically significant differences at p ≤ 0.05. nd—not detected; ␣-ESA—␣eleostearic acid. Table 2 Sum of SFA, MUFA and PUFA (%), and fatty acids ratios of sour cherry kernel oils. Cultivar

SFA

MUFA

PUFA

Fatty acids ratio UFA/SFA

Fatty acids ratio PUFA/(SFA + MUFA)

Tamaris Zentenes Haritonovskaya Latvijas Zemais Shokoladnica Bulatnikovskaya

11.7 9.5 9.4 9.9 9.8 9.8

26.0 38.3 38.9 46.1 41.7 39.3

62.3 52.2 51.7 44.0 48.5 51.0

7.5 9.6 9.6 9.2 9.3 9.3

1.7 1.1 1.1 0.8 0.9 1.0

SFA—sum of saturated fatty acids; MUFA—sum of monounsaturated fatty acids; PUFA—sum of polyunsaturated fatty acids; UFA—sum of unsaturated fatty acids; SFA—sum of saturated fatty acids. Table 3 Content of tocochromanols and total carotenoids (mg/100 g oil) of sour cherry kernel oils. Cultivar

Tocochromanols ␣-T

Tamaris Zentenes Haritonovskaya Latvijas Zemais Shokoladnica Bulatnikovskaya

38.5 10.9 11.4 10.7 9.2 12.5

Total carotenoids ␤-T

± ± ± ± ± ±

1.1d 0.2b 0.4bc 0.1b 0.1a 0.2c

2.5 0.5 0.7 0.6 0.5 0.9

␥-T ± ± ± ± ± ±

0.0e 0.0a 0.0c 0.0b 0.0a 0.0d

90.8 133.3 108.5 98.7 98.3 89.1

± ± ± ± ± ±

1.9a 3.3d 2.2c 0.8b 0.5b 1.3a

␦-T

␣-T3

14.6 ± 0.2b 18.2 ± 0.7c 14.7 ± 0.3b 15.4 ± 0.1b 9.5 ± 0.1a 14.5 ± 0.2b

2.2 0.5 0.9 1.7 0.6 1.4

± ± ± ± ± ±

␥-T3 0.2d 0.1a 0.1b 0.0c 0.0a 0.0c

0.3 0.1 0.2 0.4 0.1 0.3

± ± ± ± ± ±

Total 0.0b 0.0a 0.0a 0.0b 0.0a 0.0b

148.9 163.6 136.4 127.5 118.2 118.8

± ± ± ± ± ±

3.0d 4.2e 3.0c 0.9b 0.5a 1.8a

1.75 0.61 0.51 1.04 0.54 1.18

± ± ± ± ± ±

0.06e 0.03b 0.02a 0.04c 0.02a 0.04d

Values are expressed as the mean ± standard deviation (n = 3). Different letters in the same column indicate statistically significant difference at p ≤ 0.05. T—tocopherol; T3—tocotrienol.

10 capillary column (30 m × 0.20 mm × 0.20 ␮m) and a FID detector under programmed temperature conditions: from 60 ◦ C to 200 ◦ C increased at a rate of 12 ◦ C/min, then held at 200 ◦ C for an additional 25 min. The temperature of the injection port and the detector was set at 240 ◦ C. Hydrogen was used as a carrier gas at a flow rate of 1.0 mL/min. The results were expressed as a percentage of the total peak area of all the fatty acids in the oil sample. To confirm the identification obtained by GC an Agilent Technologies 7890A GC coupled to an Agilent 7000 Triple Quad mass spectrometer (MS) with a Supelcowax-10 (30 m × 0.25 mm × 0.5 ␮m) column were used. Operating conditions for the GC/MS were as follows: helium flow 34.6 cm/s; initial oven temperature was set at 40 ◦ C (1 min) and increased to 220 ◦ C at a rate of 5 ◦ C/min and held at 220 ◦ C for an additional 30 min. Injection conditions: temperature set at 220 ◦ C, split ratio 50:1. Mass spectra were recorded in electron impact mode (70 eV) in a scan range of 33–330 m/z. The ion source temperature was 230 ◦ C, scan time 100, MS step size 0.1.

2.5. Tocopherol and tocotrienol homologues determination The oils samples were diluted in 2-propanol as was described by Górna´s (2015). Tocochromanols were determined by RP-HPLC/FLD using a PFP (pentafluorophenyl) column (ensured separation of two tocopherol and tocotrienol isomers, ␤ and ␥) on a Shimadzu HPLC system (Shimadzu Corporation, Kyoto, Japan), according to the method previously developed and validated (Górna´s et al., 2014d). The chromatographic separation was carried out on the Shimadzu HPLC system (Shimadzu, Kyoto, Japan) consisting of a pump (LC10ADvp), a degasser (DGU-14A), a low pressure gradient unit (FCV-10ALvp), a system controller (SCL-10Avp), an auto injector (SIL-10AF), a column oven (CTO-10ASvp), a fluorescence detector (RF-10AXL), and Luna PFP column (3 ␮m, 150 × 4.6 mm) with a guard column (4 × 3 mm) (Phenomenex, Torrance, CA, USA). The analysis was performed under the following conditions: isocratic mobile phase methanol:water (93:7; v/v); flow rate of 1.0 mL/min; column oven temperature set at 40 ◦ C; room temperature was 22 ± 1 ◦ C and separation run time of 13 min. The identification and quantification were performed based on previously established

P. Górna´s et al. / Industrial Crops and Products 82 (2016) 44–50

calibration curves obtained for each tocopherol and tocotrienol homologues standard using a fluorescence detector at an excitation wavelength of 295 nm and emission wavelength of 330 nm. The limits of detection (LODs) for tocopherols (Ts) and tocotrienols (T3s) were as follows: 0.051, 0.018, 0.022, 0.044, 0.061, 0.027, 0.030 and 0.019 mg/L for ␣-T, ␤-T, ␥-T, ␦-T, ␣-T3, ␤-T3, ␥-T3 and ␦-T3, respectively.

2.6. Total carotenoids determination 0.2 g of oil sample was diluted by n-hexane in 5 mL volumetric flasks and the absorbance was measured at 450 nm with a UV-1800 spectrophotometer (Shimadzu, Kyoto, Japan). The total carotenoids concentration in oil samples were spectrophotometrically quantitated using the molar extinction coefficient for all-trans-␤-carotene (ε = 139049) and converted equations of the Beer–Lambert law and molar concentration: c=

A ×l ε

47

3. Results and discussion 3.1. Oil yield in sour cherry kernels The oil yield in kernels recovered from fruit pits of various sour cherry cultivars ranged from 17.5–37.1% (w/w) dw in cvs. ‘Tamaris’ and ‘Haritonovskaya’, respectively (Fig. 1). A wide range of oil content was recorded, due to the low value detected in cv. ‘Tamaris’. Five of six studied cultivars had an oil yield of over 30% (w/w) dw, with an average of 31.8% (w/w) dw in all investigated samples. The oil yield in sour cherry kernels of four cultivars (‘Zentenes’, ‘Haritonovskaya’, ‘Latvijas Zemais’ and ‘Shokoladnica’) was statistically insignificant (p ≤ 0.05). Farrohi and Mehran (1975) previously reported a similar range of oil yield in kernels of four sour cherry cultivars grown in Iran (20.5–33.2%), as in the present study. However, in other studies a lower (17.0%) (Yılmaz and Gökmen, 2013) and higher (50.8%) (Matthäus and Özcan, 2009) level of oil content in kernels of sour cherry were noted. Factors that may have a significant impact on the oil yield of fruit seeds may include the cultivar (Górna´s et al., 2014b), abiotic factors (Fromm et al., 2012a) and the specific method used for oil extraction (Szentmihályi et al., 2002).

m = c × MW × V where c is the concentration (mol/L); A is the absorbance; ε is the molar extinction coefficient (L/mol × cm); l represents the pathlength (cm); m is the mass of total carotenoids in study amount of the sample (g); MW is the molecular weight (g/mol); V is the volume of solution (L).

2.7. Concentration of sterols and squalene Concentration of plant sterols and squalene were determined according to AOCS (1997). In brief, the oil sample (50 mg) was saponified with 1 M KOH in methanol for 18 h at room temperature, and then unsaponifiables were extracted thrice with n-hexane/tert-butyl methyl ether (1:1, v/v). After silylation using a Sylon BTZ, sterols were separated on a HP 6890 gas chromatography system equipped with a DB-35MS capillary column (25 m × 0.20 mm × 0.33 ␮m; J&W Scientific, Folsom, CA). Samples of 0.5 ␮L were injected in split-less mode. The column temperature was held at 100 ◦ C for 5 min, then programmed to 250 ◦ C at a rate of 25 ◦ C/min, held for 1 min, then further programmed to 290 ◦ C at 3 ◦ C/min and held for an additional 20 min. The detector temperature was set at 300 ◦ C. Hydrogen was used as a carrier gas at a flow rate of 1.5 mL/min. An internal standard, 5␣-cholestane, was used for sterol quantifications. Sterols and squalene were identified by comparing the retention data of standards previously verified by mass spectrometry.

2.8. Statistical analysis The results were presented as means ± standard deviation (n = 3) from three batches of ground seeds. The p-value ≤0.05 was used to denote significant differences between mean values determined by one-way analysis of variance (ANOVA). The Bonferroni post-hoc test was used to denote statistically significant values at p ≤ 0.05. The relationship between analyzed variables was assessed by Pearson’s correlation coefficient. Its significance was evaluated by Student’s t-test. Linear regression model (y = ax + b) was calculated additionally for analysis of significant relationship between parameters. Analysis was performed with the assistance of Statistica 10.0 (StatSoft, Tulsa, OK, USA) software.

3.2. Fatty acid composition of sour cherry kernel oils Nine fatty acids were detected in kernel oils recovered from sour cherry pits (Table 1). The composition of individual fatty acids was significantly different (p ≤ 0.05) in most of the studied sour cherry cultivars. The oleic (C18:1) and linoleic (C18:2) were the predominant fatty acids in sour cherry kernel oils (25.25–45.30 and 35.50–46.06%, respectively). Similar observations were reported previously (Bak et al., 2010; Chandra and Nair, 1993; Matthäus and Özcan, 2009; Popa et al., 2011; Yılmaz and Gökmen, 2013), where the oleic acid dominated over the linoleic acid. In the present study, oleic acid had a higher amount compared to linoleic acid only for cv. ‘Latvijas Zemais’ and nearly equal levels for both fatty acids in cv. ‘Shokoladnica’. In other cultivars, the highest content was noted for linoleic acid. The noted difference clearly shows that the composition of the two main fatty acids in kernels of sour cherry is dependent on the cultivar. A fatty acid concentration above 5% was recorded for palmitic (C16:0) (5.06–7.38%) and ␣eleostearic (␣-ESA C18:3) (7.43–15.76%) acids. The ␣-eleostearic acid was detected for the first time in sour cherry kernel oil. The GC/MS method was applied to confirm this identification. The ␣-eleostearic acid in sour cherry kernel oil was not detected in previous investigations in sour cherry samples originated from Turkey, Hungary, Greece and Iran (Bak et al., 2010; Farrohi and Mehran, 1975; Lazos, 1991; Matthäus and Özcan, 2009; Yılmaz and Gökmen, 2013). The detection of ␣-eleostearic acid in each cultivar involved in current study and the same abiotic factors for all of them propose the impact of plant material different genetic basis. However, the probability of misidentification in previous studies also cannot be excluded, since for profiling of fatty acids in the oils recovered from the sour cherry kernels was not applied combination of the GC–MS systems. The content between 1 and 5% was recorded for stearic (C18:0) (2.22–3.45%) and arachidic (C20:0) (0.95–1.38%) acids. The minor content (below 1%) was noted for palmitoleic (C16:1), ␣linolenic (␣-C18:3) and gondoic (C20:1) acids (Table 1). The range and average (%) of the different types of fatty acids in kernel oils recovered from various sour cherry cultivars were as follows: saturated (9.4–11.7, 10.0%), monounsaturated (26.0–46.1, 38.4%) and polyunsaturated (44.0–62.3, 51.6%). The ratios of UFA/SFA and PUFA/(SFA + MUFA) in sour cherry kernel oils were in the range of 7.5–9.6 and 0.8–1.7, respectively (Table 2).

102.8 2.8c 416.4 14.2c Italic values correspond to standard deviations (n = 3). Different letters in the same column indicate statistically significant differences at p ≤ 0.05. nd—not detected.

3.5 0.3a 2.9 0.3a,b 2.3 0.8a 6.3 0.7b 12.9 1.1c 24.0 1.1c 321.7 12.3b 11.8 0.4d Bulatnikovskaya

31.0 0.6c

65.8 2.0a 313.6 7.8a 2.6 0.6a 1.6 0.6a 1.2 0.4a 2.4 1.1a 13.4 0.6c 15.8 0.5a 241.0 8.8a 7.6 0.4a Shokoladnica

28.0 1.8b,c

82.4 2.4b 328.7 10.9a 2.6 1.0a 3.6 0.3b 1.6 0.3a 2.9 0.6a 8.4 0.7b 20.8 0.4b 258.3 11.5a 9.8 0.2b,c Latvijas Zemais

20.8 0.6a

75.8 4.3b 316.3 7.9a nd 2.5 0.9a,b nd 3.4 1.2a 16.2 0.4d 15.0 0.4a 250.6 5.2a 8.4 0.4a,b Haritonovskaya

20.2 1.4a

371.2 6.4b nd 1.6 0.6a 2.2 0.7a 2.5 0.9a 16.9 0.7d 16.3 0.6a 293.5 5.9b 10.9 0.6c,d Zentenes

27.3 0.9b

1041.3 15.2d 5.5 0.8b 6.4 0.6c 11.2 0.6b 12.5 0.7c 2.7 1.0a 30.5 0.7b,c 852.8 15.2c 41.6 0.7e Tamaris

5-Avenasterol ␤-Sitosterol Sterols Cultivar

Nine sterols (campesterol, ␤-sitosterol, 5-avenasterol, cholesterol, gramisterol, 724-methylene-cycloartanol, stigmasterol, 7-avenasterol and citrostadienol) were identified in sour cherry kernel oils. The highest levels were noted for ␤sitosterol which consisted of 77–82% of the total detected sterols. It is not surprising, since ␤-sitosterol along with stigmasterol and campesterol are the most common forms of phytosterols in the plant world (Chen et al., 2008). For instance, ␤-sitosterol represents 51–94% of total phytosterols in apple seed oils (Górna´s et al., 2014b). ␤-sitosterol in sour cherry kernel oils had similar percentages; however its content varied significantly among different cultivars (241.0–852.8 mg/100 g oil). Nevertheless, in the three cvs. ‘Haritonovskaya’, ‘Latvijas Zemais’ and ‘Shokoladnica’ statistically insignificant (p ≤ 0.05) differences were noted (250.6, 258.3 and 241.0, respectively) (Table 4). Significant concentrations were recorded also for campesterol, 5-avenasterol and 24-methylenecycloartanol (7.6–41.6, 15.0–78.2 and 20.2–31.0 mg/100 g oil, respectively). The other detected sterols had considerably lower quantities (0.0–16.9 mg/100 g oil, respectively). The total amount of sterols ranged from 313.6–1041.3 mg/100 g oil. The lowest level of sterols was recorded for kernel oil recovered from sour cherry cultivar ‘Shokoladnica’ and the highest for cv. ‘Tamaris’. The sterols concentration in sour cherry kernel oils is higher when compared to seed oils recovered from crab apple cultivars (113–314 mg/100 g oil) and similar with seed oils obtained from dessert apple cultivars

Table 4 Sterols and squalene content (mg/100 g oil) of sour cherry kernel oils.

3.4. Sterol composition in sour cherry kernel oils

78.2 1.6d

24-Methylene-cycloartanol

Cholesterol

Gramisterol

7-Stigmasterol

7-Avenasterol

Citrostadienol

Total sterols

Four tocopherol (␣, ␤, ␥ and ␦) and two tocotrienol (␣ and ␤) homologues were detected in each investigated sample. ␥-T was the main tocochromanol in sour cherry kernel oils, with levels between 89.1–133.3 mg/100 g oil in cvs. ‘Bulatnikovskaya’ and ‘Zentenes’, respectively, and constituted between 61 and 83% of the total determined tocochromanols. ␥-T was also reported to be the dominant tocopherol in other fruit kernels and seeds of plants belonging to the Rosaceae family, however the percentage range of ␥-T was wider and lower in sour cherry kernel oils, than in different cultivars of plums (Prunus domestica L. and Prunus cerasifera Ehrh.) 78–89% (Górna´s et al., 2015a), apricots (Prunus armeniaca L.) 91–95% (Górna´s et al., 2015b), sweet cherry (Prunus avium L.) 85–89% (Górna´s et al., 2015e) and pears (Pyrus communis L.) 84–88% (Górna´s et al., 2015c). Other exceptions in the Rosaceae family were reported for apple seeds and their oils (Malus domestica Borkh.) (Górna´s, 2015; Górna´s et al., 2014c) and in Japanese quince seed oil (Chaenomeles japonica (Thunb.) Lindl. ex Spach) where the ␥-T consisted of 1–24% and 2% of the total tochochromanols detected, respectively (Górna´s et al., 2014e, 2013). ␣-T and ␦-T both had significant and comparable concentrations in sour cherry kernel oils (9.2–38.5 and 9.5–18.2 mg/100 g oil, respectively). ␤-T and ␣-T3 were present in low concentrations (0.5–2.5 and 0.5–2.2 mg/100 g oil, respectively). Minor levels were detected for ␥-T3 (0.1–0.4 mg/100 g oil) (Table 3). The total tocochromanol concentration in sour cherry kernel oils was in the range 118.2–163.6 mg/100 g oil. The lowest tocochromanol concentration was recorded for sour cherry kernel oil recovered from cv. ‘Shokoladnica’ and the highest for cv. ‘Zentenes’. Presence of ␥-T (Bak et al., 2010), four tocopherol homologues (␣, ␤ + ␥ and ␦) (Yılmaz and Gökmen, 2013) and all tocopherols (␣, ␤, ␥ and ␦) and ␣-T3 (Matthäus and Özcan, 2009) in sour cherry kernel oil were previously reported. Two to five-fold lower concentrations of tocochromanols were found in those reports compared with the present study. Those differences may be associated with the cultivar, abiotic factor and sample preparation; including drying, extraction and determination.

77.3 1.5b

Squalene

3.3. Tocochromanols composition in sour cherry kernel oils

77.9 2.5b

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Campesterol

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(182–780 mg/100 g oil) (Górna´s et al., 2014b). Only one report has previously reported the presence of ␤-sitosterol, with the levels of 4–5% of the total components in sour cherry kernel oil (Bak et al., 2010).

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Conflict of interest None. Ethical approval

3.5. Carotenoids and squalene content in sour cherry kernel oils The sour cherry kernel oils were characterized by a substantial amount of carotenoids, 0.94 mg/100 g oil (average value). A similar concentration was reported previously (Yılmaz and Gökmen, 2013). The lowest and the highest concentration of the total carotenoids were found in cvs. ‘Haritonovskaya’(0.51 mg/100 g oil) and ‘Tamaris’ (1.75 mg/100 g oil), respectively. A comparable range of total carotenoids (0.10–1.58 mg/100 g oil) in seed oils recovered from apples was previously reported (Fromm et al., 2012b). Significant levels of squalene were noted in sour cherry kernel oils and ranged between 65.8 and 102.8 mg/100 g oil in cvs. ‘Shokoladnica’ and ‘Bulatnikovskaya’, respectively. In four oil samples (cvs. ‘Tamaris’, ‘Zentenes’, ‘Haritonovskaya’ and ‘Latvijas Zemais’) the content of squalene was very similar (77.3, 77.9, 75.8 and 82.4 mg/100 g oil, statistically insignificant, p ≤ 0.05) (Table 4). Squalene concentrations in sour cherry kernel oils are higher in comparison to levels reported in dessert apple seed oils (9.0–34.0 mg/100 g oil) which differed greatly between different cultivars. Only one previous study has noted the presence of squalene, with levels between 1 and 1.2% of the total components in sour cherry kernel oil (Bak et al., 2010). 3.6. Correlations between oil yield in sour cherry kernels and it contents of lipophilic bioactive compounds Two significant correlations between oil yield in kernels of different sour cherry cultivars and the total content of sterols (r = −0.974, p ≤ 0.001) and carotenoids (r = −0.915, p ≤ 0.01) were found. It demonstrated that higher oil yields in sour cherry kernels resulted in lower concentrations of sterols and carotenoids. These phenomenons may be applied in the future as an useful tools for preliminary estimation of the total concentration of sterols and carotenoids, based merely upon the known oil yields in sour cherry kernels. An identical finding was reported in seed oils recovered from different cultivars of dessert and crab apples, where a negative correlation between the oil yield and the total content of sterols was reported (r = −0.901) (Górna´s et al., 2014b). Nevertheless, a larger number of samples of different cultivars should be tested to confirm this phenomenon. Regarding correlations between oil yield and the total concentration of tocochromanols and squalene, such trends were not observed in our investigation. 4. Conclusion The oils recovered from kernels of different sour cherry cultivars are a valuable source of lipophilic bioactive compounds, namely, essential fatty acids, tocopherols, carotenoids, sterols, and squalene. The findings show that the content of bioactive molecules in sour cherry kernel oils is largely affected by the cultivar, as the abiotic factors were the same for all studied samples. Based on correlations between oil yield in kernels of different sour cherry cultivars and the amount of certain minor lipophilic compounds, lower concentrations of sterols and carotenoids may be expected in oils recovered from cultivars with high oil content. In this study, the presence of ␣-eleostearic acid in sour cherry kernel oils was reported for the first time (7–16% of total fatty acids). It is a significant finding, since ␣-eleostearic acid has demonstrated beneficial antitumor activity in cancer cells (Zhuo et al., 2014).

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