Analysis of Lipophilic and Hydrophilic Bioactive Compounds Content in Sea Buckthorn ( Hippophaë rhamnoides L.) Berries

emr00 | ACSJCA | JCA10.0.1465/W Unicode | research.3f (R3.6.i7:4236 | 2.0 alpha 39) 2014/12/19 13:33:00 | PROD-JCA1 | rq_3475208 | 4/16/2015 09:49:39 | 10 | JCA-DEFAULT Article pubs.acs.org/JAFC Analysis of Lipophilic and Hydrophilic Bioactive Compounds Content 2 in Sea Buckthorn (Hippophae ̈ rhamnoides L.) Berries 1 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 Mirosława Teleszko,† Aneta Wojdyło,*,† Magdalena Rudzińska,‡ Jan Oszmiański,† and Tomasz Golis§ † Department of Fruit and Vegetable Technology, Wrocław University of Environmental and Life Sciences, Chełmońskiego 37 Street, 51 630 Wrocław, Poland, ‡ Institute of Food Technology of Plant Origin, Poznań University of Life Sciences, Wojska Polskiego 31 Street, 60 624 Poznań, Poland § Department of Pomology, Gene Resources and Nurseries, Research Institute of Horticulture, Konstytucji 3 Maja 1/3 Street, 96-100 Skierniewice, Poland, ABSTRACT: The aim of this study was to determine selected phytochemicals in berries of eight sea buckthorn (Hippophaë rhamnoides subsp. mongolica) cultivars, including lipophilic and hydrophilic compounds. In the experiment chromatographic analyses, GC (phytosterols and fatty acids), UPLC-PDA-FL, LC-MS (polyphenols), and HPLC (L-ascorbic acid), as well spectrophotometric method (total carotenoids) were used. The lipid fraction isolated from whole fruit contained 14 phytosterols (major compounds β-sitosterol > 24-methylenecykloartanol > squalene) and 11 fatty acids in the order MUFAs > SFAs > PUFAs. Carotenoids occurred in concentrations between 6.19 and 23.91 mg/100 g fresh weight (fw) (p < 0.05). The major polyphenol group identified in berries was flavonols (mean content of 311.55 mg/100 g fw), with the structures of isorhamnetin (six compounds), quercetin (four compounds), and kaempferol (one compound) glycosides. Examined sea buckthorn cultivars were characterized also by a high content of L-ascorbic acid in a range from 52.86 to 130.97 mg/100 g fw (p < 0.05). KEYWORDS: sea buckthorn, bioactive compounds, fatty acids, phytosterols, polyphenols (58 registered cultivars in 2003), where H. rhamnoides subsp. mongolica dominates. Worldwide >150 sea buckthorn cultivars from Russia, Ukraine, Belarus, Germany, Finland, China, and Azerbaijan are known.6 In H. rhamnoides extensive variations in chemical composition have been revealed among populations, subspecies, or cultivars. For example, according to Kallio et al.7 wild berries of subsp. sinensis, native to China, contained 5−10 times more vitamin C in the juice fraction than the berries of subsp. rhamnoides from Europe and of subsp. mongolica from Russia. The fruit flesh of subsp. sinensis berries had contents of tocopherols and tocotrienols 2−3 times higher than those found in the other two subspecies. Differences in chemical composition occur also between cultivars of the same subspecies. The content of ascorbic acid among Russian cultivars (subsp. mongolica) may range from 0.5 to 3.3 g/kg,8 whereas that in berries of subsp. turkistanica ranged from 2.52 to 4.19 g/kg.9 Cultivar selection of fruit is an important problem from a food technologists’ point of view, because it allows the choice of the most valuable raw materials for different processing directions. As a consequence, the priority in cultivation is to create varieties adapted to the growing conditions of the region and suitable for mechanical harvesting, but also with preferred chemical composition and high content of biologically active substances. INTRODUCTION Sea buckthorn (Hippophaë rhamnoides L.) fruits, called also seaberry, sanddorn, or Siberian pineapple, are yellow-orange fleshy, juicy, and soft berries with 6−9 mm diameters. In Europe H. rhamnoides bushes occur along the North Sea, Baltic Sea, and the Atlantic coast of Norway. The wild form grows also in Central Asia from China through Mongolia and Siberia, eastern Afghanistan (mountain areas), and eastern Uzbekistan.1 Compared to other fruits, sea buckthorn is characterized by the unique composition of bioactive components. It is a rich source of vitamins (C, A, E, K), minerals (Fe, Mg, Na, Ca), amino acids, carotenoid pigments, and flavonoids and contains also plant sterols and fatty acids.2 The most recognizable sea buckthorn product is oil pressed from the seeds, which contains omega-3 and omega-6 fatty acids, and the pulp oil characterized by a high concentration of fatty acids from the omega-7 group.3 Phytosterols are the major constituents of the unsaponifiable fraction of sea buckthorn oils. The sterol content in different varieties ranged from 1.3 to 2%, and the major compound is sitosterol (β-sitosterol).3 The chemical composition of H. rhamnoides and high content of active compounds affect the health-promoting values. There is a lot of interesting information about the pharmacological properties of sea buckthorn and its products, such as inhibition of platelet aggregation and antioxidant, antibacterial, antiulcer, antiinflammatory, anticancer, and antihypertensive effects.4 A characteristic feature of Hippophaë is a huge biodiversity. There are 15 species and subspecies of sea buckthorn, but only 4 subspecies belonging to H. rhamnoides are being used (subsp. mongolica, subsp. sinesis, subsp. rhamnoides, and subsp. turkestanica).5 The leader in sea buckthorn breeding is Russia ■ © XXXX American Chemical Society Received: October 22, 2014 Revised: April 2, 2015 Accepted: April 10, 2015 A DOI: 10.1021/acs.jafc.5b00564 J. Agric. Food Chem. XXXX, XXX, XXX−XXX 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 Journal of Agricultural and Food Chemistry 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 Article 290 °C. The temperature in the injection system and detector was 300 °C. All samples were analyzed in triplicate. Results were expressed in micrograms of phytosterols per 100 mL of lipid extract. Determination of Fatty Acid Composition. Methyl esters of fatty acids (FAME) were prepared according to AOCS Method Ce 1k07.12 Diluted FAME were separated on a HP 5980 series II (HewlettPackard, Palo Alto, CA, USA) equipped with an Innowax capillary column (30 m × 0.20 mm × 0.20 μm) and flame ionization detector (FID). Hydrogen was used as the carrier gas at flow rate of 1.5 mL/ min. The column temperature was programmed from 60 to 200 °C at 12 °C/min, and the final temperature was held for 25 min. Detector and injector temperatures were set at 250 °C. Fatty acids were identified by comparison of the retention times with authentic standards, and the results were reported as weight percentages after integration and calculation using ChemStation (Agilent Technologies). All samples were analyzed in triplicate. Results were expressed in percent. Hydrophilic Compounds Analysis. Analysis of L-Ascorbic Acid Content. L-Ascorbic acid content analysis was based on the method previously described by Oszmiański and Wojdyło.13 Seedless berries (ca. 5 g) were mixed with 50 mL of 0.1 M phosphoric acid and centrifuged at 14000g for 10 min at 4 °C. The estimation of L-ascorbic acid was carried out on the Waters liquid chromatograph with a tunable absorbance detector (Waters 486) and a quaternary pump with a Waters 600 Controller apparatus (Waters Associates). A 20 μL sample was injected into a Chromolith Performance RP-18e column (100 mm × 9 mm × 4.6 mm) (Merck). The elution was carried out using 0.1 M phosphoric acid, and the flow rate was 1 mL/ min. The absorbance was monitored at 254 nm. L-Ascorbic acid was identified by comparison with the standard. The calibration curve was prepared by plotting different concentrations of the standard versus the area measurements in HPLC. All samples were analyzed in triplicate. Results were expressed in milligrams per 100 g of fw of fruits. Sample Preparation for Polyphenols Content Analysis. Extracts were prepared by mixing of 2 g of berries, 10 mL of HPLC grade methanol (30 mL/100 mL) with ascorbic acid (2 g/100 mL) and acetic acid (1 mL/100 mL), and 10 mL of hexane. Samples were sonicated for 15 min (Sonic 6D Polsonic, Warszawa, Poland), placed for 24 h at 4 °C, sonicated again for 15 min, and centrifuged (MPW380R, MPW Med. Instruments, Warszawa, Poland) for 10 min (20000g at 4 °C). The hexane layer was removed. The methanol layer was purified with Merck Samplicity Filtration System (Merck Millipore, Ireland) and collected in 10 mL PE vials. Determination of Polyphenols by UPLC Coupled to PDA and FL Detector. Conditions of quantitative polyphenols determination were previously described by Wojdyło et al.14 Analysis was carried out on a UPLC system Acquity (Waters Corp., Milford, MA, USA) with a binary solvent manager, sample manager, PDA, and fluorescence detector (FL) (model λe). For chromatographic data collection and chromatograms integration Empower 3 software was used. The UPLC analyses were performed on a BEH Shield C18 analytical column (2.1 mm × 50 mm × 1.7 μm). The flow rate was 0.42 mL/min. A partial loop injection mode with a needle overfill was set up, enabling 5 μL injection volumes when a 10 μL injection loop was used. Acetonitrile (100%) was used as a strong wash solvent and acetonitrile in water (10%. v/v) as a weak wash solvent. Two milliliters of fruit extracts was centrifuged for 10 min at 15000g at 4 °C. The analytical column was kept at 30 °C by column oven, whereas the samples were kept at 4 °C. The mobile phase was composed of solvent A (4.5% formic acid) and solvent B (acetonitrile). Elution was as follows: 0−5 min, linear gradient from 1 to 25% B; 5.0−6.5 min, linear gradient from 25 to 100%; 6.5−7.5 min, column washing; and reconditioning for 0.5 min. PDA spectra were measured over the wavelength range of 200−600 nm in steps of 2 nm. The runs were monitored at the following wavelengths: flavan-3-ols at 280 nm, hydroxycinnamates at 320 nm, and flavonol glycosides at 360 nm. Retention times (tR) and spectra were compared with those of pure standards. Calibration curves at concentrations ranging from 0.05 to 5 mg/mL (r2 = 0.9998) were made from (−)-epicatechin, (+)-catechin, Therefore, the present study has focused on the profile and content of bioactive lipophilic (carotenoids, phytosterols, fatty acids) and hydrophilic compounds (L-ascorbic acid, polyphenols) in chosen cultivated berries of Hippophaë rhamnoides subsp. mongolica. ■ MATERIALS AND METHODS Reagents and Chemicals. Quercetin glycosides (3-O-glucoside, 3-O-galactoside, 3-O-rhamnoside, 3-O- rutinoside), kaempferol glycosides (3-O-glucoside, 3-O- rutinoside), isorhamnetin glycosides (3-Oglucoside, 3-O-rutinoside), p-coumaric acid, (+)-catechin, (−)-epicatechin, and procyanidins B1 and B2 were purchased from Extrasynthese (Lyon Nord, France). Acetic acid, phloroglucinol, methanol, sterol standards, and Sylon BTZ were purchased from Sigma-Aldrich (Steinheim, Germany, and St. Louis, MO, USA). Acetonitrile for UPLC (gradient grade) and ascorbic acid were from Merck (Darmstadt, Germany). UPLC grade water, prepared by using an HLP SMART 1000s system (Hydrolab, Gdańsk, Poland), was additionally filtered through a 0.22 μm membrane filter immediately before use. Plant Material and Atmospheric Conditions in Harvesting Season. Ripe berries of eight Russian sea buckthorn (H. rhamnoides subsp. mongolica) cultivars (cv.), ‘Aromatnaja’, ‘Avgustinka’, ‘Botaniczeskaja’, ‘Botaniczeskaja Ljubitelskaja’, ‘Luczistaja’, ‘Moskwiczanka’, ‘Podarok Sadu’, and ‘Porożrachnaja’, were collected from the Institute of Horticulture in Skierniewice, Lodz province, Poland (51.59° N, 20.139° E) in September 2011. Manual harvesting fruits were picked, rinsed, and stored at −20 °C until analysis. According to data from the meteorological station in Lodz, the average temperature in 2011 was 9 °C with total annual precipitation of 483 mm, insolation of 1966 h, and 5.1 octants of average cloudiness. Lipophilic Compounds Analysis. Determination of Total Carotenoids Content. The content of total carotenoids in fruits was determined according to the spectrophotometric method described in Polish Standard (PN-90/A-75101/12: Fruits and vegetables preserves. Preparation of samples and physicochemical test methods. Determination of total carotenoids and β-carotene).10 All samples were analyzed in triplicate. Results were expressed in milligrams per 100 g of fresh weight (fw). Sample Preparation of Phytosterols and Fatty Acids Content Analysis. Whole fruit pulp (±20 g) was quenched with 100 mL of a chloroform and methanol mixture (2:1) with the addition of BHT (0.1%), shaken in a separating funnel for 10 min, and allowed to separate. After phase separation, the extraction mixture was three times shaken in a separatory funnel with the addition of water. The chloroform layer was collected, centrifuged for 10 min at 15000g (MPW-380R, MPW Med. Instruments, Warszawa, Poland), and filtered through anhydrous sulfate(VI). The extracts were concentrated on a rotary evaporator (Rotavapor R-215, Büchi, Flawil, Switzerland) to 50% of the original volume. Phytosterols Content Analysis. Sterol content and composition were determined according to AOCS Ch 6-91.11 Fifty milligrams of extract (the procedure described above) with 100 mg of 5αcholestanol as an internal standard were saponified with 2 mL of 1 mol of KOH in methanol, mixed, and placed for 18 h in the dark. Then 2 mL of distilled water and 5 mL of methyl tert-butyl ether (MTBE)/hexane (1:1, v/v) were added to extract the nonsaponifiable phase. The upper layer was transferred to a test tube, and the residue was washed twice by the addition of 3 and 2 mL of a mixture of MTBE/hexane. The solvent collected in the tube was evaporated to dryness under a nitrogen stream. Sterols were silylated with Sylon BTZ reagent for 4 h at 20 °C. Chromatographic separation was performed on a Hewlett-Packard 6890 equipped with flame ionization detector (FID) and a capillary column DB-35 ms, 30 mm × 0.25 mm × 0.25 μm, in 30 min. The analysis was carried out without a split, and the temperature at the time of separation was programmed. In the initial phase (5 min) the temperature was 100 °C and then increased at 25 °C/min to 250 °C, which was maintained for 1 min and then increased at 3 °C/min to B DOI: 10.1021/acs.jafc.5b00564 J. Agric. Food Chem. XXXX, XXX, XXX−XXX 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 Journal of Agricultural and Food Chemistry Article Figure 1. Total carotenoids content (mg/100 g fw) in berries of eight sea buckthorn cultivars. 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 procyanidin B1, p-coumaric acid, quercetin, isorhamnetin, and kaempferol 3-O-glucoside as standards. All samples were analyzed in triplicate. Results were presented as total flavan-3-ols, hydroxycinnamates, and flavonol glycosides content and expressed in milligrams per 100 g of fw of fruits. Analysis of Proanthocyanidins by Phloroglucinolysis. Polymeric proanthocyanidins content were determined in freeze-dried sea buckthorn berries (Alpha 1-4 LSC; Martin Christ GmbH, Osterode am Harz, Germany) according to a method described by Wojdyło et al.14 Parameters of freeze-drying process were as follows: 18 h (time), 0.960 mbar (vacuum), 26 °C (shelf temperature). A 0.5 g portion of lyophilized seedless fruits was placed into 2 mL Eppendorf vials and mixed with 0.8 mL of methanolic solution containing phloroglucinol (75 g/L) and ascorbic acid (15 g/L) and then with 0.4 mL of methanol with HCl addition in a dose of 0.3 mol/L. The vials with reaction mixture were closed and incubated for 30 min at 50 °C with all time-vortexing by thermo shaker (TS-100, Biosan, Riga, Latvia). The reaction was stopped by placing the vials in an ice bath; 0.5 mL of the reaction medium was withdrawn and diluted with 0.5 mL of 0.2 mol/L sodium acetate buffer. Next the vials were cooled in ice water and centrifuged immediately at 20000g for 10 min at 4 °C. The temperatures in the column oven and sample manager were 15 and 4 °C, respectively. The mobile phase was composed of solvent A (2.5% acetic acid) and solvent B (acetonitrile). Elution was as follows: 0−0.6 min, isocratic 2% B; 0.6−2.17 min, linear gradient from 2 to 3% B; 2.17− 3.22 min, linear gradient from 3 to 10% B; 3.22−5.00 min, linear gradient from 10 to 15% B; 5.00−6.00 min, column washing and reconditioning for 1.50 min. The fluorescence detection was recorded at an excitation wavelength of 278 nm and an emission wavelength of 360 nm. The calibration curves, which were based on peak area, were established using (+)-catechin, (−)-epicatechin, and procyanidin B1 after phloroglucinol reaction as (+)-catechin and (−)-epicatechinphloroglucinol adduct standards. The results were calculated as milligrams per 100 g of fw of fruits. Identification of Polyphenols by the Ultraperformance Liquid Chromatography−Mass Spectrometry (UPLC-MS) Method. The method was previously described by Wojdyło et al.14 Half a gram of freeze-dried powdered fruit (parameters of freezedrying process described in the previous section) was extracted twice with 15 mL of 80% acetone acidified with 1% acetic acid. The extracts were sonicated for 15 min, centrifuged at 19000g for 10 min at 4 °C, and concentrated on a vacuum rotary evaporator to a volume of ca. 3 mL. The samples were applied to the Sep-Pak C18 cartridge (Waters, Milford, MA, USA) containing 1 g of the carrier, washed with distilled water to remove the sugar and organic acid (extract = 0 °Brix), and then collected into a vacuum flask with 15 mL of 80% methanol acidified with 1% HCl. The methanol extract was evaporated to dryness. The dry residue was dissolved in 4 mL of 4.5% formic acid, centrifuged for 5 min at 15000g, and then given to the analysis. Identification of sea buckthorn polyphenols was carried out using an ACQUITY Ultra Performance LC system (UPLC) with binary solvent manager (Waters, Milford, MA, USA) and a Micromass Q-Tof Micro mass spectrometer (Waters, Manchester, UK) equipped with an electrospray ionization (ESI) source operating in negative mode. For instrument control data acquisition and processing MassLynxTM software (version 4.1) was used. Separations of individual polyphenols were carried out using a UPLC BEH C18 column (1.7 μm, 2.1 mm × 50 mm; Waters, Milford, MA, USA) at 30 °C. Samples (10 μL) were injected and elution completed in 12 min with a sequence of linear gradients and isocratic flow rates of 0.45 mL/min. The mobile phase was composed of solvent A (0.1 mL/100 mL formic acid, v/v) and solvent B (100 mL/100 mL of acetonitrile). The program began with isocratic elution with 99% A (0−1 min), and then a linear gradient was used for 12 min, lowering A to 0%; from 12.5 to 13.5 min, the gradient returned to the initial composition (99% A) and then was held constant to reequilibrate the column. Analysis was carried out using full scan, data-dependent MS scanning from m/z 100 to 1000. The effluent was led directly to an electrospray source with a source block temperature of 130 °C, desolvation temperature of 350 °C, capillary voltage of 2.5 kV, and cone voltage of 30 V. Nitrogen was used as a desolvation gas at flow rate of 300 L/h. Statistical Analysis. Statistical analysis (variance test and standard deviation values) was made using Statistica 10.0 (StatSoft, Kraków, Poland). Significant differences (p ≤ 0.05) between means were evaluated by one-way ANOVA and Duncan’s multiple-range test. 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 RESULTS AND DISCUSSION Analysis of Lipophilic Bioactive Compounds. Total Carotenoids Content. In raw material total carotenoids (Figure 1), phytosterols, and fatty acids content (Tables 1 and 2) were examined. The sea buckthorn fruits were characterized by a high concentration of carotenoids, 11.00 mg/100 g fw (mean value). The content of these compounds in berries was significantly correlated with cultivar and ranged from 6.19 to 23.91 mg/100 g fw (in ‘Luczistaja’ and ‘Moskwiczanka’, respectively). Similar values in nine cultivars of sea buckthorn were noted by Kruczek et al.15 (from 8.85 in ‘Botaniczeskaja Ljubitelskaja’ to 43.06 mg/ 100 g fw in ‘Botaniczeskaja’). It also should be emphasized that the content of these compounds in examined sea buckthorn fruits was comparable to that determined by Müller16 in tomatoes, young carrots, and red peppers (12.69, 9.46, and 30.37 mg/100 g of edible part, respectively). Carotenoids are located mainly in the soft parts of the fruit, giving them a characteristic orange-yellow color. In sea buckthorn berries 15−55% of all compounds of this group is β-carotene. In lower concentrations are also α-, γ-, and 289 ■ C DOI: 10.1021/acs.jafc.5b00564 J. Agric. Food Chem. XXXX, XXX, XXX−XXX 290 291 292 f1t1t2 293 t2 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 cultivarsa AR AV BOT BOT-L LUC MOS PS POR squalene kampesterol stigmasterol β-sitosterol sitostanol Δ5-avenasterol α-amyrin cycloartenol Δ7-avenasterol 28-methylobtusifoliol 24-methylenecycloartanol erythrodiol citrostadienol friedelan-3-ol 1638.27 ± 25.97 c 167.10 ± 1.25 b ndb 6145.58 ± 41.11 a 217.29 ± 1.01 b 274.08 ± 4.20 b 314.52 ± 2.82 a 441.78 ± 8.24 b 156.55 ± 0.65c 249.39 ± 0.03 a 1554.42 ± 6.26f 818.75 ± 2.61 a 663.22 ± 1.35 a 737.28 ± 3.28 a 1205.43 ± 0.60 e 139.22 ± 1.62 c 68.22 ± 2.03 a 4942.39 ± 4.91 b 172.64 ± 4.31 c 251.19 ± 4.05 c 110.48 ± 0.15 f 399.89 ± 10.50 c 152.02 ± 3.63 cd 130.10 ± 3.94 d 4048.89 ± 87.92 a 680.91 ± 7.64 b 327.09 ± 8.61 d 583.76 ± 2.16 c 2714.37 ± 7.72 a 201.32 ± 12.67 a nd 3801.64 ± 10.69 f 217.80 ± 1.57 b 377.56 ± 5.46 a 302.63 ± 13.83 a 462.48 ± 14.82 a 154.63 ± 1.62c 251.85 ± 4.48 a 2711.93 ± 36.19 c 590.29 ± 2.88 c 417.42 ± 1.56 c 606.44 ± 8.35 b 1872.42 ± 106.12 b 95.71 ± 11.57 e nd 4022.29 ± 47.14 e 110.69 ± 1.19 e 197.21 ± 1.64 d 130.68 ± 2.11 e 439.14 ± 5.54 b 138.21 ± 0.53 e 145.72 ± 1.56 c 2521.94 ± 14.64 d 367.19 ± 9.16 f 309.23 ± 4.35 e 398.64 ± 6.30 d 885.71 ± 29.16 g 82.79 ± 2.63 e nd 2049.46 ± 32.23 g 97.77 ± 0.80 f 198.22 ± 1.88 d 166.92 ± 4.82 d 299.14 ± 7.07 e 147.61 ± 2.52 d 152.03 ± 0.23 b 1994.54 ± 19.38 e 338.58 ± 2.33 g 244.82 ± 2.24 g 257.16 ± 6.94 f 1110.10 ± 37.95ef 44.37 ± 2.14 f 24.08 ± 0.06 b 2036.14 ± 59.47 g 96.50 ± 0.94 f 114.93 ± 1.27 e 112.86 ± 2.68 f 293.49 ± 3.12 e 80.80 ± 0.08 f 70.88 ± 1.40 f 1454.21 ± 10.62 g 284.02 ± 1.59 h 212.97 ± 6.52 h 232.89 ± 3.42 g 1516.27 ± 11.82 d 118.79 ± 1.39 d nd 4216.62 ± 20.01 d 254.67 ± 13.63 a 249.42 ± 2.59 c 203.24 ± 5.52 c 474.38 ± 1.18a 186.73 ± 2.39 b 89.96 ± 1.01 e 3131.95 ± 3.58 b 444.38 ± 1.55 d 444.95 ± 10.82 b 260.73 ± 1.74 f 1026.05 ± 0.92 f 127.68 ± 1.81 cd nd 4409.03 ± 14.80 c 150.00 ± 0.90 d 243.80 ± 0.31 c 228.35 ± 0.91 b 360.94 ± 1.34 d 194.97 ± 1.01 a 153.20 ± 0.72b 2661.63 ± 32.09 c 407.97 ± 0.62 e 269.61 ± 1.39 f 320.01 ± 1.77 e total 13378.22 ± 61.82 13212.23 ± 89.29 12810.36 ± 40.90 10749.06 ± 16.55 6914.75 ± 10.64 6168.24 ± 9.04 11592.07 ± 12.96 10553.23 ± 11.76 Journal of Agricultural and Food Chemistry Table 1. Phytosterols Content (Micrograms per 100 mL of Lipid Fraction) and Profile of Sea Buckthorn Berries a AR, ‘Aromatnaja’; AV, ‘Avgustinka’; BOT, ‘Botaniczeskaja’; BOT-L, ‘Botaniczeskaja Ljubitelskaja’; LUC, ‘Luczistaja’; MOS, ‘Moskwiczanka’; PS, ‘Podarok Sadu’; POR, ‘Porożrachnaja’. Entries followed by the same lower case letter constitute statistically homogeneous groups (Duncan test, p ≤ 0.05). Values are expressed as the mean ± standard deviation (n = 3). bnd, not detected. D Article DOI: 10.1021/acs.jafc.5b00564 J. Agric. Food Chem. XXXX, XXX, XXX−XXX cultivarsa AR C14:0 C16:0 C16:1 C16:2 C18:0 C18:1 C18:1 C18:2 C18:3 C20:0 C20:1 (myristic) (palmitic) n-7 (palmitoleic) n-7 (hexadecadienoic) (stearic) n-9 (oleic) n-7 (cis-vaccenic) n-6 (linoleic) n-3 (linolenic) (arachidic) n-9 (eicosenoic) ∑SFAs ∑MUFAs ∑PUFAs 0.36 37.87 33.83 0.54 0.91 7.05 6.90 11.31 0.78 0.17 0.05 ± ± ± ± ± ± ± ± ± ± ± 0.00 0.03 0.11 0.00 0.01 0.01 0.01 0.02 0.00 0.01 0.00 39.31 ± 0.12 b 47.82 ± 0.06 a 12.62 ± 0.05 c AV 0.24 34.47 38.01 0.51 1.04 6.67 7.92 10.32 0.52 0.14 0.04 ± ± ± ± ± ± ± ± ± ± ± 0.01 0.01 0.02 0.01 0.02 0.02 0.01 0.01 0.01 0.01 0.00 35.88 ± 0.04 b 52.63 ± 0.10 a 11.35 ± 0.40 c BOT 0.17 37.83 34.21 0.47 1.04 8.02 7.40 9.93 0.66 0.17 0.06 ± ± ± ± ± ± ± ± ± ± ± 0.00 0.02 0.08 0.01 0.02 0.04 0.01 0.04 0.00 0.00 0.01 39.21 ± 0.13 b 49.68 ± 0.14 a 11.06 ± 0.06 c BOT-L 0.36 38.25 34.54 0.81 0.97 5.25 6.66 12.20 0.69 0.17 0.05 ± ± ± ± ± ± ± ± ± ± ± 0.00 0.11 0.06 0.01 0.01 0.01 0.02 0.04 0.01 0.00 0.00 39.75 ± 0.01 b 46.50 ± 0.04 a 13.69 ± 0.03 c LUC 0.64 35.62 38.51 1.01 0.64 4.26 6.94 11.51 0.65 0.14 0.04 ± ± ± ± ± ± ± ± ± ± ± 0.01 0.17 0.11 0.01 0.01 0.02 0.01 0.06 0.01 0.01 0.00 37.03 ± 0.06 b 49.75 ± 0.05 a 13.16 ± 0.01 c MOS 0.29 37.62 36.03 0.63 0.79 6.27 7.11 10.29 0.69 0.16 0.06 ± ± ± ± ± ± ± ± ± ± ± 0.01 0.03 0.11 0.01 0.01 0.04 0.03 0.02 0.00 0.00 0.00 38.85 ± 0.03 b 49.47 ± 0.16 a 11.60 ± 0.03 c PS 0.36 33.66 31.41 0.58 0.85 7.89 9.79 14.11 0.93 0.19 0.06 ± ± ± ± ± ± ± ± ± ± ± 0.01 0.01 0.17 0.00 0.01 0.01 0.01 0.04 0.01 0.01 0.01 35.05 ± 0.04 b 49.14 ± 0.01 a 15.61 ± 0.00 c POR 0.18 39.65 32.98 0.50 1.09 7.82 7.33 9.69 0.58 0.13 0.04 ± ± ± ± ± ± ± ± ± ± ± 0.01 0.06 0.08 0.01 0.01 0.01 0.03 0.02 0.01 0.01 0.01 41.05 ± 0.05 b 48.16 ± 0.11 a 10.77 ± 0.02 c Journal of Agricultural and Food Chemistry Table 2. Fatty Acids Content (Percent) and Profile in Lipid Fraction from Sea Buckthorn Berries a AR, ‘Aromatnaja’; AV, ‘Avgustinka’; BOT, ‘Botaniczeskaja’; BOT-L, ‘Botaniczeskaja Ljubitelskaja’; LUC, ‘Luczistaja’; MOS, ‘Moskwiczanka’; PS, ‘Podarok Sadu’; POR, ‘Porożrachnaja’. SFAs, saturated fatty acids; MUFAs, monounsaturated fatty acids; PUFAs, polyunsaturated fatty acids. Entries followed by the same lower case letter constitute statistically homogeneous groups (Duncan test, p ≤ 0.05). Values are expressed as the mean ± standard deviation (n = 3). E Article DOI: 10.1021/acs.jafc.5b00564 J. Agric. Food Chem. XXXX, XXX, XXX−XXX Journal of Agricultural and Food Chemistry Article Figure 2. L-Ascorbic acid content (mg/100 g fw) in berries of eight sea buckthorn cultivars. 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 buckthorn and may exceed soybean oil by 4−20 times. βSitosterol and β-sitosterol-β-D-glucoside in sea buckthorn oils are important for the antiulcerative activity. The efficacy of the two compounds may differ depending on the cause of ulcer formation.24 Fatty Acids Composition. The fatty acid (FA) compositions of sea buckthorn pulp oil are listed in Table 2. In the examined samples 11 fatty acids were identified, including 3 PUFAs, 4 MUFAs, and 4 SFAs. The FA profile was dominated by two compounds: palmitic acid (C16:0), which constituted from 33.66 to 39.65% of the total fatty acids content (in ‘Podarok Sadu’ and ‘Porożr achnaja’, respectively), and palmitoleic acid C16:1 n-7 (from 31.41% in ‘Podarok Sadu’ to 38.51% in ‘Luczustaja’). In relatively high concentration occurred also 18-carbon unsaturated acids, that is, linoleic C18:2 n-6 (9.69−14.11% in ‘Porożrachnaja’ and ‘Podarok Sadu’, respectively), followed by cis-vaccenic C18:1 n-7 (6.66− 9.79% in ‘Botaniczeskaja Ljubitelskaja’ and ‘Podarok Sadu’) and oleic C18:1 n-9 (4.26−8.02% in ‘Luczistaja’ and ‘Botaniczeskaja’). Other compounds presented in concentrations between ∼0.5% in the case of eicosenoic acid (C20:1 n-9) and ∼1% for stearic acid (C18:0). From these results it can be concluded that MUFAs were the dominant fatty acid classes (46.50− 52.63%), followed by SFAs (35.05−41.05%) and PUFAs (10.77−15.61%). According to Dulf,25 the dominating fatty acids in sea buckthorn berry pulp/peel oils were palmitic (23−40%), oleic (20−53%), and palmitoleic (11−27%). Small or trace amounts of vaccenic, linoleic, α-linolenic, stearic, myristic, pentadecanoic, cis-7-hexadecenoic, margaric, and two long-chain fatty acids, arachidic and eicosenoic acids, were observed in all analyzed oils. As the author showed, in two from six tested cultivars (C1 and C2), the proportions of oleic acid (32.76% for C1 and 53.08% for C2) exceeded that of palmitoleic acid (19.53% for C1 and 11.05% for C2). The fatty acid profile of sea buckthorn was presented also by Khabarovet al.26 Oil samples from berry pulp and skin (species H. rhamnoides) contained the highest concentrations of palmitoleic > palmitic > and oleic acid. Our research showed that the quantified relationships between C16:1 and C16:0 acids noted by these authors occurred only in the oil fraction from ‘Avgustinka’ and ‘Luczistaja’ fruits. At the same time, a higher content of linoleic acid in all extracts was found. However, in the presented study fractions from whole berries (with skin and seeds) were examined, which could explain the observed differences. This is dihydroxy-β-carotene, lycopene, zeaxanthin, and canthaxanthin.17 The content of carotenoids in fruits is subject to extreme diversity. In the same population and species of the genus Hippophaë a 10-fold difference in the content of these components can be observed. As Yang’s18 study showed, the concentration of β-carotene ranged between 0.2 and 17 mg/ 100 g and that of total carotenoids from 1 to 120 mg/100 g fw of sea buckthorn berries. Phytosterols Content. Bioactive substances of sea buckthorn include phytosterols characterized by a documented beneficial role in the prevention of cardiovascular diseases, mainly hypercholesterolemia19,20 or cancer.21 The results of qualitative and quantitative analyses of these compounds are presented in Table 1. The total content of phytosterols in the lipid fraction from sea buckthorn pulp ranged from 6168.24 (‘Moskwiczanka’) to 13378.22 μg/100 mL (‘Aromatnaja’). The results of GC analysis showed that in the examined extracts there occurred 14 compounds belonging to three subclasses of plant sterols, that is, 4-desmethyl sterols (cholestanol derivatives, including βsitosterol, stigmasterol, campesterol, Δ5-avenasterol), 4αmonomethyl sterols (e.g., citrostadienol), and 4,4-dimethyl sterols (e.g., 24-methylenecycloartanol). The predominant compound was β-sitosterol (26.64−42.57% of the total phytosterols in ‘Luczistaja’ and ‘Aromatnaja’, respectively). In the high concentration occurred also 24-methylenecycloartanol. This compound was determined in the range from 1454.21 to 4048.89 μg/100 mL in the lipid fraction from ‘Moskwiczanka’ and ‘Avgustinka’ berries. Sea buckthorn can be considered as a valuable source of squalene. Depending on the cultivar, the lipid fraction isolated from berry pulp contained between 885.71 and 2714.37 μg/100 mL (‘Luczistaja’ and ‘Botaniczeskaja’). Moreover, concentrations in a range from 300 to 800 μg of substances, such as erythrodiol > friedelan-3-ol > citrostadienol > or cycloartenol, were noted. In sea buckthorn trace amounts of stigmasterol were also detected. This compound was found only in two cultivars, ‘Avgustinka’ and ‘Moskwiczanka’, and its contents were 68.22 and 24.08 mg/100 mL (p < 0.05), respectively. The results of the sterols profile analysis in sea buckthorn were confirmed by studies of other authors, such as Li et al.22 or Cenkowski et al.23 According to Bal et al.,2 the major phytosterols in sea buckthorn oil are β-sitosterol and Δ5avenasterol. Other compounds are present in relatively minor quantities. The total quantity of phytosterol is quite high in sea F DOI: 10.1021/acs.jafc.5b00564 J. Agric. Food Chem. XXXX, XXX, XXX−XXX 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 Journal of Agricultural and Food Chemistry Article Figure 3. Polyphenols content (mg/100 g fw) and profile of sea buckthorn berries. 400 401 402 403 404 405 406 407 408 409 410 411 f2 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 f3 442 confirmed by George and Cenkowski.27 They proved that three major fatty acids (palmitic, palmitoleic, oleic) accounted for approximately 32.2, 26.5, and 18.7% of the total fatty acids in the sea buckthorn fruit fraction, respectively. High levels of palmitoleic acid are present in only a few plants products, such as sea buckthorn pulp or macadamia nut oils. Because this fatty acid is a major constituent of skin fat, the pulp oil is used for cosmetic and healing purposes.3 Analysis of Hydrophilic Bioactive Compounds. LAscorbic Acid Content. Except for the lipophilic active compounds, such as carotenoids and phytosterols, sea buckthorn is also rich in hydrophilic substances known for health benefits properties, including L-ascorbic acid (Figure 2). The mean content of ascorbic acid (AA) in the raw material was 80.58 mg/100 g. Likewise, as in the case of previously described compounds, the cultivar factor determined abundance of sea buckthorn in ascorbic acid (p < 0.05). The highest concentration of this compound were characterized in ‘Aromatnaja’ (130.97 mg/100 g fw) followed by ‘Avgustinka’ (88.45 mg) > ‘Podarok Sadu’ (82.61 mg) > ‘Botaniczeskaja’ (80.40 mg) > ‘Moskwiczanka’ and ‘Porożrachnaja’ (74.79 and 75.08 mg; p > 0.05) > ‘Botaniczeskaja Ljubitelskaja’ (59.48 mg) > ‘Luczistaja’ (52.86 mg). The presented results clearly showed that in terms of ascorbic acid content most of the examined sea buckthorn cultivars were definitely more valuable than many other popular fruit species including strawberries (65 mg/100 g fw), raspberries (29 mg), lemons (74.3 mg), mandarins (37.7 mg), or blackberry (21 mg).28 According to Tang,29 the content of ascorbic acid in sea buckthorn (depending on the species, variety, region) was from 28 to 2500 mg/100 g. Berries of six cultivars examined by Rop et al.30 contained between 398 and 573 mg AA/100 g fw (in ‘Buchlovicky’ and ‘Ljubitelna’, respectively). Kawecki et al.31 determined the highest content of ascorbic acid in ‘Trofimovskaja’ (197 mg/100 g) > ‘Otrodnaja’ (191 mg) > ‘Botaniczeskaja’ (178 mg) > and ‘Podarok Sadu’ (161 mg). These values are therefore higher than in our study. However, the authors paid attention not only to varietal but also seasonal variation of ascorbic acid content in H. rhamnoides berries, which explains the observed differences. Polyphenols Content and Profile. Interesting results were obtained indicating the content of polyphenols in sea buckthorn (Figure 3). Berries were characterized by a high concentration of flavonols (from 212.89 to 407.48 mg/100 g fw in ‘Botaniczeskaja Ljubitelskaja’ and ‘Botaniczeskaja’, respectively; p < 0.05). The structure of these compounds was investigated by qualitative analysis in LC-ESI/MS system performed in negative ionization mode (Table 3; Figure 4). 443 444 445 446 447 t3f4 Table 3. LC-MS Identification of Sea Buckthorn Flavonols a MSMS (m/z) no. tRa (min) λmax [M − H]− (m/z) 1 2 5.56 5.90 352 354 639 785 315 639 3 6.37 354 755 609 4 5 6.77 7.33 354 352 609 609 6 7.51 354 771 301 301, 447 625 7 8 9 7.59 8.49 8.78 355 348 353 623 593 623 315 285 315 10 11 9.03 11.28 354 356 477 869 315 723 compound isorhamnetin-3,7-diglucoside isorhamnetin-3-sophoroside7-rhamnoside quercetin-glucosiderhamnoside-7-rhamnoside quercetin-3-rutinoside quercetin-3-glucoside-7rhamnoside quercetin-3-sophoroside-7rhamnoside isorhamnetin-3-rutinoside kaempferol-3-rutinoside isorhamnetin-3rhamnosylglucoside isorhamnetin-3-glucoside isorhamnetin-3-acyloglucoside-glucoside-7rhamnoside tR, retention time. The examined sea buckthorn cultivars contained 11 flavonols, which were primarily derived of isorhamnetin (6 compounds), quercetin (4 compounds), and kaempferol (1 compound). The strongest signal in the obtained mass spectrum with a value of [MH]− at m/z 623 and a retention time of 8.78 min came from isorhamnetin-3-rhamnosylglucoside (compound 9). The source of two successive intensity signals was compound 7 with a molecular weight [M − H]− at m/z 623 and 10 ([MH]− at m/z 477). In regard to the fragmentation direction of both compounds, that is, loss of the aglycone molecule with MSMS (m/z) 315, UV−vis spectrum, and LC-MS standards analysis, as well as the literature data,32 these compounds were identified as monoglycosides: isorhamnetin-3-rutinoside (compound 7; tR 7.59 min) and isorhamnetin-3-glucoside (compound 10; tR 9.03 min). Among the quercetin derivatives, G DOI: 10.1021/acs.jafc.5b00564 J. Agric. Food Chem. XXXX, XXX, XXX−XXX 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 Journal of Agricultural and Food Chemistry Article Figure 4. LC-ESI/MS spectrum and the chromatogram (360 nm) of sea buckthorn flavonols (cv. ‘Porożrachnaja’). 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 these compounds can be used as biomarkers for classification or recognition of sea buckthorn berries belonging to different species and varieties.40 Besides flavonols in sea buckthorn berries compounds belonging to three other groups of polyphenols, that is, polymeric proanthocyanidins, mono-, di-, and oligomers of flavan-3-ols, and phenolic acids, were determined. The highest contents of flavan-3-ols (88.04 mg/100 g fw) and phenolic acids (5.81 mg/100 g fw) were characterized by fruits of ‘Avgustinka’. ‘Luczistaja’ was the richest in polymeric proanthocyanidins (5.76 mg/100 g fw), but contained the least value of phenolic acids (3.11 mg/100 g fw). Similarly to the flavonols determination, also flavan-3-ols concentration was the lowest in ‘Botaniczeskaja Ljubitelskaja’ (44.94 mg/100 g fw). Compared to Hosseinian et al.42 total proanthocyanidins content in examined samples was significantly different, especially in the case of polymeric form. In the whole fruit methanol fraction the authors noted 36.56 mg of polymeric proanthocyanidins per 100 g fw basis and 98.95 mg of mono-, di-, and oligomeric compounds. In our study a similar value of simple flavan-3-ols was determined only in ‘Avgustinka’ berries. This proves, however, that the content of polyphenols in plants is a result of many factors, such as cultivar, harvest year, or geographical origin. This correlation has been previously described in the literature.43,44 The proanthocyanidins profile in sea buckthorn is varied. According to Rösch et al.45 the concentration of reaction products after acid-catalyzed cleavage (determined by HPLC) and calculated composition of sea buckthorn proanthocyanidins showed that major compounds were phloroglucinol adducts with (+)-gallocatechin, followed by (+)-catechin and (−)-epicatechin. The ratio between prodelphinidins (PD) and procyanidins (PC) was 2.1:1. On the basis of ESI-MS analysis, the authors detected compounds with different polymerization degrees, including pentameric, hexameric, heptameric, nonameric, and decameric proanthocyanidins. quercetin-3-glucoside-7-rhamnoside was dominant (compound 5; [MH]− at m/z 609, tR 7.33 min). After ion fragmentation, a strong, typical signal was registered from an ion with molecular mass MS-MS (m/z) 301. The signals from fission products of glucose ([M − H− 162]− at m/z 447) and rhamnose ([M − H− 146]− at m/z 463) were less abundant and characterized by a similar intensity. According to Yang et al.33 sea buckthorn berries of subsp. mongolica contain 80.6 mg flavonols/100 g fw, which is a significantly higher value than noted in chokeberry (23−40 mg/100 g fw),34 cranberry (20−40 mg/100 g fw),35 black currant (17−38 mg/100 g fw),36 or bilberry (10−16 mg/100 g fw).37,38 The results of our study also confirm this relationship;, however, in sea buckthorn we noted ca. a 4-fold higher content of flavonols. The most common flavonols in sea buckthorn fruits are isorhamnetin derivatives with mono-, di-, and triglycoside structures.33,39 As Pop et al.40 research showed, in fruits and leaves of H. rhamnoides L. subsp. carpatica (cv. ‘Serpenta’, ‘Serbanesti 4’, ‘Victoria’, ‘Sf. Gheorghe’, ‘Ovidiu’, and ‘Tiberiu’) 17 and 19 flavonols occurred, respectively. Isorhamnetin-3neohesperidoside, isorhamnetin-3-glucoside, isorhamnetin-3rhamnosylglucoside, isorhamnetin-3-sophoroside-7-rhamnoside, and free isorhamnetin were predominant in the case of berries. In rhamnoides subspecies, the major compounds indicated by Kallio et al.39 were isorhamnetin-3-sophoroside7-rhamnoside > quercetin-3-glucoside > quercetin-3-rutinoside > isorhamnetin-3-rutinoside > isorhamnetin-3-glucoside. Chen et al.41 isolated from the n-butanol fraction of sea buckthorn (subsp. sinensis) berries a novel acylated flavonol glycoside, isorhamnetin (3-O-[(6-O-E-sinapoyl)-β-D-glucopyranosyl-(1→ 2)]-β-D-glucopyranosyl-7-O-α-L-rhamnopyranoside), together with two known acylated flavonols glycosides, quercetin (3O-[(6-O-E-sinapoyl)-β-D-glucopyranosyl-(1→2)]-β-D-glucopyranosyl-7-O-α-L-rhamnopyranoside) and kaempferol (3-O-[(6O-E-sinapoyl)-β-D-glucopyranosyl-(1→2)]-β-D-glucopyranosyl7-O-α-L-rhamnopyranoside). Due to such significant differences of sea buckthorn berries in terms of flavonol glycosides content, H DOI: 10.1021/acs.jafc.5b00564 J. Agric. Food Chem. XXXX, XXX, XXX−XXX 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 Journal of Agricultural and Food Chemistry 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 ■ AUTHOR INFORMATION Corresponding Author *(A.W.) Phone: +48 71 320 77 06. Fax: +48 71 320 77 07. Email: aneta.wojdylo@up.wroc.pl 576 Funding 577 578 This work was supported by the European Union under Project POIG 01.01.02-00-061/09, acronym “Bioactive food”. 579 Notes 580 The authors declare no competing financial interest. 581 ACKNOWLEDGMENTS We thank Marii Bortkiewicz and Elżbiecie Buckiej for their technical assistance. 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 (5) Rongsen, L.; Ahani, H. The genetic resources of Hippophae genus and its utilization. Int. J. Scholary Res. Gate 2013, 1, 15−21. (6) Szałkiewicz, M.; Zadernowski, R. Sea buckthorn: possibilities for the production and use of fruit (in polish). Hasło Ogrodnicze. 2006, 02, http://www.ho.haslo.pl/article.php?id=2601. (7) Kallio, H.; Yang, B.; Peippo, P. Effects of different origins and harvesting time on vitamin C, tocopherols, and tocotrienols in sea buckthorn (Hippophaë rhamnoides) berries. J. Agric. Food Chem. 2002, 50, 6136−6142. (8) Kalinina, I. P.; Panteleyeva, Y. I. Breeding of sea buckthorn in the Altai. In Advances in Agricultural Science; Nauk: Moscow, Russia, 1987; pp 76−87. (9) Hussain, M.; Ali, S.; Awan, S.; Hussain, M.; Hussain, I. Analysis of minerals and vitamins in sea buckthorn (Hippophae rhamnoides) pulp collected from Ghizer and Skardu districts of Gilgit-Baltistan. Int. J. Biosci. 2014, 4, 144−152. (10) PN-90/A-75101/12: Fruits and vegetables preserves. Preparation of samples and physico-chemical test methods. Determination of total carotenoids and β-carotene,1990. (11) AOCS Official Method Ch 6-91. Determination of the composition of the sterol fraction of animal and vegetable oils and fats by TLC and capillary GLC. (12) AOCS Official Method Ce 1k-07. Direct methylation of lipids for the determination of total fat, saturated, cis-monounsaturated, cispolyunsaturated, and trans fatty acids by chromatography. (13) Oszmiański, J.; Wojdyło, A. Effects of black currant and apple pulp blended on phenolics, antioxidant capacity and colour of juices. Czech J. Food Sci. 2009, 27, 338−351. (14) Wojdyło, A.; Teleszko, M.; Oszmiański, J. Antioxidant property and storage stability of quince juice phenolic compounds. Food Chem. 2014, 152, 261−270. (15) Kruczek, M.; Świderski, A.; Mech-Nowak, A.; Król, K. Antioxidant capacity of crude extracts containing carotenoids from the berries of various cultivars of Sea buckthorn (Hippophae rhamnoides L.). Acta Biochim. Pol. 2012, 59, 135−137. (16) Müller, H. Determination of the carotenoid content in selected vegetables and fruit by HPLC and photodiode array detection. Z. Lebensm. Unters. Forsch. A 1997, 204, 88−94. (17) Yang, B.; Kallio, H. Composition and physiological effects of sea buckthorn (Hippophaë) lipids. Trends Food Sci. Technol. 2002, 13 (5), 160−167. (18) Yang, B. Lipophilic Components in Seeds and Berries of Sea Buckthorn and Physiological Effects of Sea Buckthorn Oils. Ph.D. thesis, University of Turku, Turku, Finland, 2001. (19) Davidson, M. H.; Maki, K. C.; Umporowicz, D. M.; Ingram, K. A.; Dicklin, M. R.; Schaefer, E.; Lane, R. W.; McNamara, J. R.; RibayaMercado, J. D.; Perrone, G.; Robins, S. J.; Franke, W. C. Safety and tolerability of esterified phytosterols administered in reduced-fat spread and salad dressing to healthy adult men and women. J. Am. Coll. Nutr. 2001, 20, 307−319. (20) Neil, H. A.; Meijer, G. W.; Roe, L. S. Randomised controlled trial of use by hypercholesterolaemic patients of a vegetable oil sterolenriched fat spread. Atherosclerosis 2001, 156 (2), 329−337. (21) Woyengo, T. A.; Ramprasath, V. R.; Jones, P. J. Anticancer effects of phytosterols. Eur. J. Clin. Nutr. 2009, 63, 813−820. (22) Li, T. S. C.; Beveridge, T. H. J.; Drover, J. C. G. Phytosterol content of sea buckthorn (Hippophaë rhamnoides L.) seed oil: extraction and identification. Food Chem. 2007, 101, 1665−1671. (23) Cenkowski, S.; Yakimishen, R.; Przybylski, R.; Muir, W. E. Quality of extracted sea buckthorn seed and pulp oil. Can. Biosyst. Eng. 2006, 48, 3.9−3.16. (24) Erkkola, R.; Yang, B. Sea buckthorn oils: towards healthy mucous membranes. Agro Food Ind. Hi-Tech. 2003, May/June 2003, 43−57. (25) Dulf, F. V. Fatty acids in berry lipids of six sea buckthorn (Hippophaë rhamnoides L., subspecies carpatica) cultivars grown in Romania. Chem. Cent. J. 2012, 6, 1−12. (26) Khabarov, S. N.; Vereshchagin, A. L.; Gur’yanov, G.; Goremykina, N. V.; Bychin, N. V. Identification of the origin of sea With the phenolic acids structure taken into account, the compounds liberated from soluble esters are predominant in sea buckthorn berries (from 53.9 to 66.6%). Free phenolic acids are a minor fraction and constitute only 1.3−2.3%.46 According to Arimboor et al.,47 berry pulp contained a total of 1068 mg/ kg phenolic acids, of which 58.8% was derived from phenolic glycosides. Free phenolic acids and phenolic acid esters constituted 20.0 and 21.2%, respectively. As the authors showed, gallic acid was identified as the predominant phenolic acid in both free and bound forms (66.0% of total phenolic acids in pulp). Considerable amounts of protocatechuic (136 mg/kg), ferulic (69 mg/kg), salicylic (54 mg/kg), phydroxybenzoic (40 mg/kg), and p-coumaric acid (37 mg/ kg) were found to be present in berry pulp. The presence of vanillic (7 mg/kg), cinnamic (12 mg/kg), and caffeic acids (8 mg/kg) was also detected. Sea buckthorn berries are a valuable source of compounds with beneficial effects for human health. The high concentrations of flavonols, L-ascorbic acid, but mostly lipophilic substances, namely, carotenoids, phytosterols, and fatty acids, determine the uniqueness of its chemical composition in comparison to other fruits. Our research confirms that the content of plant bioactives in the raw material significantly affects the cultivar factor. ‘Aromatnaja’ was characterized by the highest content of L-ascorbic acid and phytosterols, whereas ‘Podarok Sadu’ berries were rich in PUFAs. In ‘Moskwiczanka’ the highest carotenoids and in ‘Botaniczeskaja’ the highest polyphenol concentrations were noted. Among the tested active compounds of sea buckthorn, the most diverse was the phytosterols profile, which consisted of 14 compounds, including 4-desmethyl sterols (e.g., β-sitosterol, stigmasterol, campesterol, Δ5-avenasterol), 4α-monomethyl sterols (e.g., citrostadienol), and 4,4-dimethyl sterols (e.g., 24methylenecycloartanol). 575 574 Article ■ ■ REFERENCES (1) Rousi, A. The genus Hippophaë L. A taxonomic study. Ann. Bot. Fenn. 1971, 8, 177−227. (2) Bal, L. M.; Meda, V.; Naik, S. N.; Satya, S. Sea buckthorn berries: a potential source of valuable nutrients for nutraceuticals and cosmoceuticals. Food Res. Int. 2011, 44, 1718−1727. (3) Fatima, T.; Snyder, C. L.; Schroeder, W. R.; Cram, D.; Datla, R.; Wishart, D.; Weselake, R. J.; Krishna, P. Fatty acid composition of developing sea buckthorn (Hippophaë rhamnoides L.) berry and the transcriptome of the mature seed. PLoS One 2012, 7, 2−18. (4) Patel, C. A.; Divakar, K.; Santani, D.; Solanki, H. K.; Thakkar, J. H. 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