Sea buckthorn (Hippophaë rhamnoides
L.) is a thorny, deciduous shrub belonging to the Elaeagnaceae family. Six species of Hippophaë
and 12 subspecies are currently recognized, including ssp. sinensis
, ssp. mongolica
, and ssp. rhamnoides
, which are the most economically and commercially important. There are over 150 cultivars of sea buckthorn, but new thornless and easier-to-harvest varieties are still being selected. Sea buckthorn naturally grows on sea coasts and river valleys of Central and Northern Europe, Russia, China, Mongolia, Central Asia, and slopes of the Caucasus and Himalayas. The plant is cultivated mainly in the Northern Hemisphere and its largest producer is China [1
Fleshy and soft sea buckthorn fruits are yellow, orange, or red, round or oblong, and 6–15 mm in diameter [5
]. Due to the similarity of berries, the plant is commonly confused with scarlet firethorn (Pyracantha coccinea
), rock cotoneaster (Cotoneaster horizontalis
), or rowan (Sorbus aucuparia
), whose raw fruits are poisonous. The sea buckthorn aroma is compared to strawberries, peach, mango, apricot, papaya, and citrus, but mostly to pineapple, which results from a similar ester profile [6
]. In the food industry, sea buckthorn is used as a raw material enriching the pro-health value or increasing the acidity of fruit products. Berries are intended for the production of jams, juices, soft drinks, liqueurs, wine, or as an addition to beers, kefir, and cheeses. By contrast, oil obtained from seeds and pulp is used as a cosmetic and dietary supplement, and is used less frequently as a culinary product. Production residues can be a functional ingredient in meat or animal feed [1
The therapeutic properties of bark, leaves, and fruits were already known in ancient Greece as well as in Tibetan and Mongolian medicine. In the cosmetics industry, the plant is used in dermatological diseases, hair care, revitalization of wounds and skin burns, and as a form of natural protection against UV-B radiation. The results of previous in vitro and in vivo studies [2
] confirm the effectiveness of sea buckthorn extracts in the prevention of hyperglycemia, hyperinsulinemia, and hyperlipidemia, together with hepatoprotective, anti-carcinogenic, antibacterial, and antifungal effects, as well as positive functioning of the digestive system and eyesight. The properties of sea buckthorn are due to the high concentrations of flavonoids (mainly flavonols), carotenoids (principally β-cryptoxanthin and β-carotene), ascorbic acid, vitamin E (the most active form, α-tocopherol, dominates), and fatty acids (omega-3, omega-6, omega-7, and omega-9) present in seeds, skin, and flesh [6
A number of studies have been carried out on sea buckthorn in different world regions, but the knowledge about biologically active compounds and pro-health potential of cultivars grown in Poland is limited. Given the above, the aim of this study was to analyze biological activities (anti-oxidant, anti-α-amylase, anti-α-glucosidase, anti-lipase, and anti-lipoxygenase effects) relative to selected bioactive components (flavonols and phenolic acids, xanthophylls, carotenes, esterified carotenoids, tocopherols and tocotrienols, fatty acids), and the basic chemical composition (sugars, organic acid, dry matter, soluble solid, pH, titratable acidity, ash, pectins, vitamin C) of berries of six commonly grown H. rhamnoides cultivars in Poland.
α-Amylase and α-glucosidase break down polysaccharides to glucose. Therefore, their inhibition is one of the methods of postprandial hyperglycemia reduction. This effect plays a key role in treating type 2 diabetes, which, according to WHO, affects 8.5% of the global adult population. In turn, pancreatic lipase breaks down dietary triglycerides into bioavailable forms—fatty acids and glycerol. Its inhibition can reduce energy intake at a meal, which is part of the strategy of overweight and obesity therapy. The lipoxygenase pathway, including lipoxygenase 5-LOX, 12-LOX, and 15-LOX, is associated with the production of hydroperoxy fatty acids and leukotrienes. Increased concentrations of these products correlate with the progression of, inter alia, inflammatory bowel disease, asthmatic bronchitis, rheumatoid arthritis, cancers, and cardiovascular diseases [13
Therefore, it was assumed that the results will allow the identification of significant differences in the pro-health potential and composition of the studied sea buckthorn cultivars for further use in the design of innovative functional products, nutraceuticals, and cosmeceuticals. Additionally, this study should indicate cultivars with the highest biological potential for further use in planning and expanding cultivations. Furthermore, the results of the cultivar Józef, bred in Poland, are presented for the first time. This creates promising perspectives for commercial production of this sea buckthorn cultivar and use in the food industry as a source of health-promoting substances and antioxidant properties.
2. Materials and Methods
Standards of sugars, organic acids, phenolic compounds, and carotenoid compounds were purchased from Extrasynthese (Genay, France), and the rest of the reagents were bought from Merck KgaA (Darmstadt, Germany). The samples before chromatographic analysis were filtered through a Hydrophilic PTFE 0.20 μm membrane (Millex Samplicity Filters, Merck KgaA, Darmstadt, Germany).
2.2. Plant Materials
The fruits of six sea buckthorn (Hippophaë rhamnoides
L.) cultivars—Aromatnaja, Botaniczeskaja-Lubitelskaja, Józef, Luczistaja, Moskwiczka, and Podarok Sadu—were tested (Figure 1
). Ripe berries were collected in early July and August 2018 from the Research Institute of Horticulture in Skierniewice (Poland). Fresh fruits were used to analyze the basic chemical composition. The second portion of selected berries was frozen, freeze-dried for 24 h (Christ Alpha 1–4 LSC, Martin Christ GmbH, Osterode am Harz, Germany) and crushed by a laboratory mill (A11, IKA, Darmstadt, Germany). The homogeneous materials were stored in a freezer at −80 °C until undergoing the other analysis.
2.3. Basic Chemical Composition
The soluble solids content was expressed in °Bx using a digital refractometer (Atago RX-5000, Atago Co. Ltd., Saitama, Japan). The instrument was calibrated using distilled water. Liquid and homogenized raw material was applied to the dry prism surface. The measurement was taken at 20 °C. The dry matter was determined by mixing the sample with diatomaceous earth, pre-drying, and final drying under reduced pressure. Titratable acidity (TA) was analyzed by the titration of homogenous fresh fruits with 0.1N NaOH to pH 8.1 and the result were expressed as g malic acid/100 g FW (fresh weight). TA and pH were determined using an automatic pH titrator system (TitroLine 5000, Xylem Analytics GmbH, Weilheim in Oberbayern, Germany). The soluble solids content, dry matter, and titratable acidity were taken according to European Standards, PN-EN 12143:2000, PN-EN 12145:2001, and PN-EN 12145:2000, respectively. Pectin content (g/100 g FW) was measured according to the Morris method reported by Pijanowski et al. [14
]. Ash (%), l
-ascorbic acid (mg/100 g FW), sugars, and organic acids contents (g/100 g FW) were determined, as reported previously by Wojdyło et al. [15
]. Sugars and organic acids were analyzed using high pressure liquid chromatography including the evaporative light scattering detector (HPLC-ELSD) and ultra performance liquid chromatography-photodiode array detector (UPLC-PDA) methods. All measurements were taken three times.
2.4. Analysis of Phenolic Compounds
The extraction of the samples for phenolic compounds and their chromatographic analysis were performed exactly as described by Wojdyło et al. [15
]. The samples were analyzed by an Ultra-Performance Liquid Chromatography Photodiode Array Detector (UPLC-PDA; Acquity UPLC System, Waters Corp., Milford, MA, US). The study identified phenolic acids and flavonols, and their sums were calculated as p
-coumaric acid and isorhamnetin-3-O
-rutinoside, respectively, which is based on dominant compounds and compared with reference standards. All results were taken in triplicate and shown as mg/100 g DM of berries (dry mass).
2.5. Analysis of Carotenoids, Tocopherols, and Tocotrienols
The extraction of the samples for carotenoid compounds was made as previously described by Wojdyło et al. [15
] and Nowicka et al. [16
]. The determination of carotenoids was made using the equipment as in Section 2.4
, according to the protocol given by Wojdyło et al. [15
]. The powder samples of fruits (0.20 g) containing 10% MgCO3
and 1% butylhydroxytoluene (BHT) to prevent oxidation were continuously shaken with 5 mL of a ternary mixture of methanol/acetone/hexane (1:1:2, v:v:v
) at 300 rpm (DOS-10L Digital Orbital Shaker, Elmi Ltd., Riga, Latvia) for 30 min in the dark. Recovered supernatants were obtained after 4–5 times being re-extracted of solid residue. All combined fractions collected after centrifugation (4 °C, 7 min at 19,000× g
, MPW-350, Warsaw, Poland) were evaporated to dryness. The pellet was diluted using 2 mL of 100% methanol, filtered through a hydrophilic polytetrafluoroethylene (PTFE) 0.20-μm membrane (Millex Samplicity Filter, Merck, Darmstadt, Germany) and used for analysis.
Carotenoids were carried out on an ACQUITY UPLC BEH RP C18 column being protected by the guard column of the same materials (1.7 mm, 2.1 mm 100 mm, Waters Corp., Milford, MA, USA) operated at 30 °C. The elution solvents were linear gradient of acetonitrile:methanol (70:30, v:v) (A) and 0.1% formic acid (B). The runs were monitored at 450 nm. The photodiode array detector PDA spectra were measured over the wavelength range of 200–700 nm in steps of 2 nm. The retention times and spectra were compared to those of the authentic standards. All incubations were done in triplicate. The results were expressed as mg per kg of dm. Samples for the analysis of tocopherols and tocotrienols were prepared as follows. The fresh sea buckthorn berries (∼3g) were homogenized with two times as much of the ethanol portion mixed with 0.05% butylated hydroxytoluene (BHT). Saponification was carried out using 60% CaOH, at a temperature of 50 °C for 2 h. Then, the samples were mixed with hexane:ethyl acetate with 0.05% BHT. After that, NaOH (saturated solution) was added. The upper layer was collected, evaporated, and dissolved in methanol with 0.05% BHT. The solutions were filtered through a Hydrophilic PTFE 0.20 μm membrane and used for UPLC analysis. The analysis of tocopherols and tocotrienols was carried out by using Ultra-Performance Liquid Chromatography with a fluorescence detector (UPLC-FL). The column ACQUITY UPLC BEH RP C18 (1.7 mm, 2.1 mm × 100 mm, Waters Corp., Milford, MA, US) being protected by a guard column of the same materials was operated at 30 °C. Identification and quantification was performed based on reference standards and calibration curves. The samples (5 μL) were injected, and the elution was completed in 12 min with an isocratic method of methanol with water (88:12, v:v) flow rates of 0.45 mL/min. All incubations were done in triplicate. The results were expressed as mg per kg of dm.
2.6. Analysis of Fatty Acids
Fatty acids were extracted and tested with the technique of gas chromatography with mass spectrometry (GC-MS), in the same way as described by Nowacki et al. [17
]. The samples were analyzed using a GC 6890 gas chromatograph coupled with a 5983 MS mass spectrometer (Agilent Technologies Inc., Santa Clara, CA, US) equipped in a quadrupole mass detector. Measurements were taken in triplicate. The results of fatty acid studies were expressed as the percentage of total fatty acids of sea buckthorn berries.
2.7. Determintation of Biological Activity: Anti-Oxidant, Anti-α-Amylase, Anti- α-Glucosidase, Anti-Lipase, and Anti-Lipoxygenase
The extraction procedure was the same for all determinations and was carried out identically, as described by Nowicka et al. [16
]. The ABTS, FRAP, and ORAC assays were conducted as previously reported by Re et al. [18
], Benzie and Strain [19
], and Ou et al. [20
], respectively. The ABTS•+
(2,2′-azine-bis-(3-ethylene-benzothiazoline-6-sulfonic acid) scavenging test is based on measuring the decrease in the color intensity inversely proportional to the antioxidant content. An ABTS•+
solution was prepared with an absorbance of 0.700 ± 0.02 at a wavelength of 734 nm. Sea buckthorn extracts and the ABTS•+
solution were mixed and, after 6 min, the absorption at the wavelength above was measured. Distilled water was blank. The results were calculated based on the calibration curve (R2
= 0.9950) for Trolox concentrations 0.100 to 0.900 mM.
The FRAP method involves determining the ability to reduce Fe3+ ions by antioxidant substances contained in sea buckthorn extracts to the blue Fe2+ ions complex. Sea buckthorn extracts were mixed with distilled water. The absorbance of the samples was measured 10 min after the addition of the FRAP reagent (acetate buffer, 2,4,6-Tris(2-pyridyl)-s-triazine (TPTZ) in HCl and FeCl3 × 6H2O in a volume ratio of 10:1:1, v:v:v), at a wavelength of 593 nm. The results were calculated based on the calibration curve (R2 = 0.9899) for Trolox concentrations 0.050 to 0.900 mM.
The analysis of oxygen radical absorbance capacity (ORAC) consists of a spectrofluorometric measurement of the decrease in fluorescence caused by oxidation of a fluorescent substance under the influence of free radicals, but in the presence of antioxidant substances. Samples containing sea buckthorn extract, phosphate buffer, and fluorescein were incubated at 37 °C throughout the analysis period. 2,2’-Azobis(2-amidinopropane)dihydrochloride was added and the spectrofluorometric measurement was performed every 5 min at an excitation wavelength 493 nm and an emission wavelength of 515 nm. The blank was a phosphate buffer. The antioxidant activity of the tested samples was obtained by comparing the surface under the fluorescence decrease curves over time with the surface for pure Trolox solutions (12.5, 25.0, 50.0, and 75.0 µM).
The ABTS, FRAP, and ORAC results were expressed in mmol TE (Trolox)/100 g sample.
-glucosidase, and anti-lipase activity were studied, according to methods reported by Nowicka et al. [16
] and Podsędek et al. [21
]. Briefly, analysis of the anti-α
-amylase inhibitory activity is based on a spectrophotometric measurement of the color change as a result of a reaction of iodine in potassium iodide with the remaining starch after enzymatic hydrolysis. Basic samples contained sea buckthorn extracts, starch solution, and α
-amylase. After incubation at 37 °C, the reaction was stopped using 0.4 M HCl. A solution of potassium iodide with iodine was added. Reference samples contained phosphate buffer instead of an enzyme. The acarbose was included as a positive control and absorbance was measured at 600 nm.
The analysis of α-glucosidase inhibitory activity consists of the reaction of the enzyme with a β-d-glucosidase substrate producing a yellow solution upon cleavage. Basic samples containing sea buckthorn extracts and enzymes were incubated as above. After the addition of the substrate, the mixture was incubated again and measurement was made at 405 nm. As in the above analysis, the reference samples contained buffer instead of enzymes and the acarbose was included as a positive control.
The analysis of lipase inhibitory activity is based on a spectrophotometric measurement of the amount of p-nitrophenol formed from p-nitrophenyl acetate. Basic samples contained sea buckthorn extracts, Tris-HCl buffer, and the enzyme. After 5 min of incubation at 37 °C, the substrate was added. Then incubation continued for 15 min. Reference samples contained buffer instead of the enzyme and the orlistat was used as a positive control. Absorbance was measured at 400 nm. The results of anti-α-amylase, anti-α-glucosidase, and anti-lipase activity are presented as IC50 in mg/mL, i.e., the amount of the sample that is able to reduce enzyme activity by 50%.
Inhibitory activity toward 15-lipoxygenase was measured in accordance with Chung et al. [13
]. Basic samples containing sea buckthorn extract and enzymes were incubated at 37 °C. Then, linoleic acid was added and incubation continued for 20 min. The mixture was measured at 210 nm. Reference samples contained Tris-HCl buffer instead of the enzyme. The results were expressed as a percentage inhibition (at the concentration of 30 mg/mL).
All tests: anti-oxidant (ABTS, ORAC, FRAP), anti-α-amylase, anti- α-glucosidase, anti-lipase, and anti-lipoxygenase were performed in triplicate using a microplate reader SynergyTM H1 (BioTek, Winooski, VT, US).
2.8. Statistical Analysis
One-way analysis of variance (ANOVA, p < 0.05), Tukey’s test, Pearson’s correlation coefficients, and Principal Component Analysis (PCA) were carried out using XLSTAT for Microsoft Excel 2010 (Microsoft Corp., Redmond, WA, US) and Statistica 13.1 (StatSoft, Cracow, Poland). The results were presented as the mean value (n = 3) ± standard deviation (SD).