L., also known as the oleaster or Russian olive, is a riparian bush native to Southern Europe and Western Asia and belongs to the Elaeagnaceae family, which comprises about 50 species. Used as an ornamental shrub as well as a soil stabilizer, but also regarded as an invasive species in Western North America, it is a highly drought- and cold-resistant plant, characterized by reddish sweet and sour berries [1
]. The edible fruits, consumed fresh or dried, are a rich source of vitamins such as tocopherol, vitamin C, B1, and α-carotene, as well as minerals (potassium, sodium, and phosphorous) [2
], and have traditionally been used in folk medicine for their analgesic, antipyretic, and diuretic activities, whereas the seeds are used for the extraction of an oil rich in polyphenols and other antioxidant molecules.
As reported in the literature [3
], many interesting compounds, such as flavonoids, vitamins, minerals, sugars, sterols, and alkaloids, detected in aqueous and organic extracts obtained from the berries, could justify their use as an ingredient in food supplements or drug formulations. In fact, due to this rich phytocomplex, antimicrobial, insecticidal, antiarthritic, anti-inflammatory, cardioprotective, hypolipidemic, antimutagenic, antitumor, antioxidant, and gastroprotective effects are well recognized [4
The proven relationship between oxidative stress and human diseases had led to increasing interest in polyphenol- and flavonoid-containing extracts, such as those derived from Elaeagnus angustifolia
leaves and fruits. Literature data reported the presence in oleaster fruits and pulp of phenolic acids, mainly represented by p
-hydroxybenzoic, caffeic and protocatechuic acid [5
], differently glycosylated isorhamnetin, quercetin and kaempferol derivatives, and catechins [3
]; the total phenolic and flavonoid contents of leaves and flowers were also evaluated, showing higher contents in the ethanol extracts from leaves [6
]. Moreover, anticancer properties and radical scavenging activity are attributed to flavonoids and pro anthocyanosides extracted from Elaeagnus angustifolia
Bioactive alkaloids were also detected in oleasters, such as eleagnin and calligonin, tetrahydroharmans that have a recognized inhibitory effect on monoamine oxidase [7
], as well as homeostatic effects on blood pressure and antimalarial activity. These compounds are mainly present in the roots, bark, and aerial parts of the plant [3
]. In reference to the essential oil, obtained by steam distillation of flowers or leaves, the chemical composition was recently investigated by GC-MS and GC-FID analyses and the radical scavenging by 2,2-diphenyl-1-picrylhydrazyl (DPPH) test; the toxicity against brine shrimp was also assessed. More than 50 and 25 compounds were extracted from flowers and leaves, respectively, among which cinnamates, farnesyl derivatives, and benzoates were detected [8
leaf and fruit extracts, traditionally employed for treating common illnesses, were recently re-evaluated by several scientific reports about their efficacy and their relationship with the content of bioactive compounds [2
]. The anti-ulcer activity of methanolic extracts obtained from the whole fresh fruit was tested with relevant results. A significant gastroprotective effect was also demonstrated for the separated carotenoid fraction from the fruit seed oil [9
]. Aqueous extracts obtained from the dry powdered fruit were tested for their antinociceptive and anti-inflammatory activity, showing promising effects on chronic pain and inflammation by inhibition of the cyclooxygenase type 2 enzyme [10
]. Conversely, the muscle-relaxant effect (in a dose-dependent manner) and antitumor activity exerted by Elaeagnus
fruits (as tested on HeLa cells) were attributed to the flavonoid content of seed aqueous and ethanolic extracts [11
] and of flesh ethyl acetate extracts, respectively.
The antioxidant and antiradical activities of seven genotypes of oleaster methanol extracts were recently evaluated [12
] by radical scavenging assays and by total phenolic and flavonoid content. The authors concluded that significant differences existed among the genotypes and that fruit seeds showed better antioxidant activity and higher phenolic contents with respect to the flesh and peels.
A recent review summarized the efficacy of E. angustifolia
whole fruit aqueous extract on an osteoarthritis model, relating the anti-inflammatory effects via the inhibition of TNF-α, COX-1, COX-2, and IL-1β [13
]. Due to its anti-inflammatory activity, the hydroalcoholic extract of this plant was also tested as a wound-healing agent in oral mucositis. Oral mucositis, which represents a frequent chemotherapy side effect affecting the quality of life of patients during anticancer therapy, could be significantly reduced by a daily application of the tested extract [14
]. Hydroalcoholic extracts were also successfully tested on an experimental model of ovariectomy-induced osteoporosis in rats [15
]. Previous studies have shown that estrogen deficiency provoked oxidative stress reducing bone antioxidant defenses and induced IL-6, TNF-α, and IL-1. The role of pro-inflammatory cytokines was also highlighted. Therefore, a synergic effect of the phytosterols detected in the Elaeagnu
s extracts, in particular β-sitosterol and stigmasterol, with the anti-inflammatory polyphenols, could be taken into consideration for osteoporosis prevention. A significant chemopreventive effect of oleaster fruit against primary liver cancer induced in rats by diethylnitrosamine was revealed by Amereh et al. [16
]. GSH levels were significantly increased and lipid peroxidation lowered, according to the hepatoprotective activity mediated by the antioxidant, anti-inflammatory, and antimutagenic mechanisms.
fruit and leaf extracts were both tested as antimicrobial agents on antibiotic-resistant microorganisms, showing good activity towards Escherichia coli
and Salmonella typhimurium
]. Finally, Elaeagnus
polysaccharides were investigated for the best extraction conditions and their physicochemical and functional properties, and have been shown to exert anti-diabetic, antitumor, and immunological effects [19
]. In another report, two polysaccharide components from the fruit pulp were isolated, characterized for their average molecular weight and monosaccharide composition, and evaluated in relation to their antioxidant and free radical scavenging activity [20
]. Three crude polysaccharides tested for their immunological effects induced an increase in NO release and enhanced the macrophages’ phagocytic activity [21
], implying a potential use of oleaster as a functional food.
On the basis of prior knowledge, the present work aimed to compare the effect of different extraction techniques, performed on dried whole fruits and leaves of Elaeagnus angustifolia, in relation to the chemical composition of the obtained products. First, the leaves’ and fruits’ dry powders were submitted in their solid form to HS-GC/MS and CIEL*a*b* colorimetric analysis. Then, all the different obtained extracts were analyzed by colorimetric analyses and further studied for their chemical composition by HPLC-DAD, GC/MS, and 1H and 13C NMR analysis. Total carotenoid, chlorophyll, polyphenolic, and flavonoid contents were evaluated by UV-VIS spectroscopy. The extracts were also tested for some interesting biological activities, such as total antioxidant capacity, free radical scavenging activity, and enzyme inhibition activity.
3. Materials and Methods
Elaeagnus angustifolia leaves and fruits were collected from wild plants from Turkey (Konya) and were botanically and morphologically identified by Dr. Evren Yıldıztugay from the science faculty of Selcuk University, Konya, Turkey. Fruits were in a ready-to-eat ripeness state.
Double-distilled water, ethanol, 98% formic acid, acetonitrile RS, n-hexane for HPLC, and dichloromethane were purchased from Merck Life Sciences s.r.l (Milan, Italy), methanol for HPLC and diethyl ether were purchased from Carlo Erba Reagents (Milan, Italy), DMSO-d6 (99.80% D) was obtained from Eurisotop (Saint-Aubin, France) and CO2 was purchased from Sapio s.r.l. (Monza, Italy).
3.2. Sample Preparation
Whole fruits or mature long elliptic leaves were washed and then straightaway submitted to a dehydration process. The samples were distributed over a steel grid and dried in a dedicated box, avoiding light exposure. Forced ventilation at room temperature (25 °C) was applied until a constant weight was reached. After 10 days, the weight loss on drying was determined to be <10%, as recommended by the European Pharmacopeia guidelines. A laboratory mill (Retsch Cutting Mill SM 200, Haan, Germany) was used to grind the samples (powder size: about 1 mm). The powders (fruit powder, Fp
; leaf powder, Lp
) were stored in sealed bags in the dark at 4 °C until they were directly analyzed or subjected to the different extraction procedures (Figure 1
3.3. Extraction Methods
3.3.1. Hydro-Alcoholic Extraction (HAE) by Maceration
About 230 mg of the samples were extracted with 10 mL ethanol:water in 70:30 (v:v) ratio by stirring for 1 h at room temperature in the dark (FH and LH). The extraction mixture was decanted, filtered on paper, and the hydroalcoholic solvent was evaporated at 40 °C in the dark under a vacuum and immediately analyzed or stored at 4 °C.
3.3.2. Microwave-Assisted Extraction (MAE)
MAE was performed using an automatic Biotage InitiatorTM
2.0 (Uppsala, Sweden) characterized by 2.45-GHz high-frequency microwaves and a power range of 0–300 W. The internal vial temperature was strictly monitored by the infrared (IR) sensor probe. About 230 mg of the samples were transferred to a sealed 10 mL vessel suitable for an automatic single-mode microwave reactor and 10 mL of ethanol:water in a 70:30 (v
) ratio were added to the sample. MAE was carried out by microwave irradiation for 7.3 min at 55 °C (corresponding to 1 h maceration at room temperature, following the Arrhenius equation and with the same solid-to-liquid ratio used in Section 3.3.1
). The extraction mixture was decanted, filtered on paper, evaporated at 40 °C in the dark under vacuum, and immediately analyzed or stored at 4 °C (FM
3.3.3. Supercritical CO2 Assisted Extraction (SCE)
SCE was carried out in a laboratory-scale supercritical fluid extraction system (Jasco Europe Srl, Cremella, Italy). The system was operated in a semi-continuous mode by pumping scCO2 with a Jasco-PU1580 unit, through a stainless-steel column, 10 mm i.d. and 200 mm length, equipped with 10 µ porous stainless steel 316 L sintered filter disks. The column was filled with the dehydrated samples, heated using a Jasco CO-4061 oven to the chosen temperature, and scCO2 was pumped to the target pressure. After a pre-equilibrating time of 30 min, SCE was performed.
The dried leaves (7.0 g) were transferred in the CO2 extractor added with 5 mL ethanol and extracted for 1 h at 150 bar and 55 °C. The dried fruits (7.0 g) were transferred to the CO2 extractor and extracted for 1 h at 250 bar and 80 °C. The extraction mixtures (FS and LS) were immediately analyzed by 1H and 13C NMR and HPLC analysis or stored at 4 °C until the analyses were performed.
3.4. Colorimetric Analysis
The powders and resulting dried extracts were analyzed for their color with an X-Rite SP-62 colorimeter (X-Rite Europe GmbH, Regensdorf, Switzerland), set with a D65 illuminant and a 10° observer angle, as previously described [43
]. Each experiment was performed four times and the results are expressed as the mean value ± standard deviation (SD).
3.5. Carotenoid and Chlorophyll Analysis
The total carotenoid and chlorophyll (a and b) contents in E. angustifolia
leaf and fruit samples (FP
) were detected as reported in [44
In brief, 50 mg of each sample were separately homogenized with a mortar and pestle in 6 mL of chloroform–methanol (2:1, v/v) in the presence of MgO (20 mg). The obtained homogenates were filtered on paper and distilled water was added to the amount of 20% of the extract volume. Finally, the mixture was centrifuged and the phases separated. The absorption spectrum of the chloroform phase was recorded with a Beckman Coulter (Pasadena, CA, USA) DU 800 spectrophotometer, in the range 350–800 nm with a spectral resolution of 0.5 nm, at a temperature of 20 °C. Both chlorophylls and total carotenoids were determined using their absorption coefficient. Data are reported as the mean of three replicates and expressed as μg/g dw (dry weight) ± SD.
3.6. HS-GC/MS Analysis
To investigate the volatile fraction of dried samples, a PerkinElmer Headspace (HS) Turbomatrix 40 autosampler (Waltham, MA, USA) connected to a Clarus 500 GC-MS was used for headspace analysis [45
]. To optimize the headspace procedure for the determination of volatile organic compounds (VOCs), parameters such as equilibration time and temperature were adjusted. The sampling procedure was performed as follows: fruit and leaf samples (14 mg and 120 mg, respectively) were put into a 20mL vial with 2 mL of diethyl ether and immediately tightly sealed with crimp aluminum caps and 20-mm white rubber septa (Merck KGaA, Darmstadt, Germany) using a vial crimper. The samples were incubated at 90 °C for 20 min, then the volume of headspace gas was transferred into the capillary column (Rtx-1) by a transfer line. The same temperature program, described in the next paragraph, was used for the GC/MS apparatus.
3.7. GC/MS Analyses
Gas chromatographic/mass spectrometric (GC/MS) analysis was carried out on FH
extracts, with both GC-MS and GC-FID, using a Turbomass Clarus 500 GC-MS/GC-FID from PerkinElmer Instruments [47
]. A Stabilwax fused-silica capillary column (Restek, Bellefonte, PA, USA) (60 m × 0.25 mm, 0.25 μm film thickness) was used with helium as the carrier gas (1.0 mL/min). GC oven temperature was kept at 60 °C and programmed to 220 °C at a rate of 6 °C/min, and kept constant at 220 °C for 20 min. All mass spectra were recorded in the electron impact ionization (EI) at 70 eV. The mass range was 30–400 m
. The FH
extracts (about 20 mg) were diluted in 1 mL of methanol and 2 μL of the obtained solutions were injected into the GC injector at a temperature of 280 °C. The identification of the main components was performed by comparison of their linear retention indices (LRIs) and spectral mass with those reported in the library data (Wiley 02 and Nist) of the GC/MS system. The LRI of each compound was calculated using a mixture of aliphatic hydrocarbons (C8
, Ultrasci, Bologna, Italy) injected directly into the GC injector with the same temperature program reported above. Relative abundances of the separated components were derived using the same instrumentation with the FID detector configuration, without the use of an internal standard or correction factors. Analyses were repeated twice.
3.8. HPLC-DAD Analyses
Dried extracts (FH, FM, FS and LH, LM, LS) were weighed, dissolved in methanol, and filtered before injection into an HPLC PerkinElmer apparatus consisting of a Series 200 LC pump, a Series 200 DAD, and a Series 200 autosampler, including a Totalchrom PerkinElmer software for the data acquisition. Chromatography was performed on a Luna RP18 column (250 × 4.6 mm i.d., 5 μm) using a mobile phase made by acetonitrile and water acidified by 5% formic acid, in a gradient with a flow rate of 1 mL/min, at 280 and 360 nm. Analyses were performed with a linear gradient from 98% acidified water to 50% acidified water over 33 min. Calibration curves were built and used for the quantitation of polyphenols, using at 280 nm epicatechin (R2 = 0.9878), chlorogenic acid (R2 = 0.9987), caffeic acid (R2 = 0.9984), p-coumaric acid (R2 = 0.9879), and ferulic acid (R2 = 0.9974) and at 360 nm quercetin-3-d-galactoside (R2 = 0.9999) as reference standards.
3.9. Semipreparative HPLC-Refractive Index Detector
Dried extract (FS) was weighed, dissolved in dichloromethane, and filtered before injection into an HPLC semipreparative apparatus, consisting of a Waters (Milford, MA, United States) Millipore 150 pump, a Gilson (Middleton, WI, USA) 132 refractive index detector, and Jasco (Easton, MD, United States) Borwin software for the data acquisition. Chromatography was performed on a Macherey-Nagel, (Bethlehem, PA, USA) 100-5 column. The eluent mixture used was 60/40 (v/v) n-hexane/ethyl acetate, and the flow rate was fixed at 5 mL/min. The analysis was performed at 25 °C.
3.10. H- and 13C-MR Analysis
1H and 13C NMR (400.13 and 100.03 MHz) analyses were recorded with a Bruker (Billerica, MA, United States)Avance 400 (Milano, Italy) spectrometer, equipped with a Nanobay console and Cryoprobe Prodigy Probe. Ten milligrams of FS sample were dissolved in 0.6 mL of DMSO-d6 (I.E% = 99.80%), transferred to a NMR tube, and analyzed. The resulting 1H NMR and 13C NMR spectra were processed using Bruker TOPSPIN software.
3.11. Determination of Total Bioactive Components: Total Phenolic Content (TPC) and Total Flavonoid Content (TFC)
The total phenolic content (TPC) was determined using the Folin–Ciocâlteu method according to Zengin et al. [49
], calculated and expressed as gallic acid equivalent (GAE) in mg/g extract of plant material. In brief, 50 µL of extract (1 mg/mL) and 100 µL of Folin–Ciocâlteu reagent, diluted 1:9 in distilled water, were mixed. After 3 min, 75 µL of sodium carbonate (Na2
, 2% w/v
) were added and then incubated at 25 °C in the dark for 120 min. The absorbances were measured at 765 nm.
The total flavonoid content (TFC) was calculated using a method previously described by Zengin et al. [28
]. Rutin was used as the reference compound and the results were expressed as equivalents of rutin (mg RE/g). In brief, 200 µL sample solution was mixed with AlCl3
(2% in methanol). After 15 min of incubation, the absorbance of each sample was recorded at 420 nm.
3.12. Antioxidant and Metal Chelating Spectrophotometric Assays
The scavenging capacity of the free radical DPPH was monitored according to Zengin et al. [28
]. In brief, 50 µL of the sample solution (1 mg/mL) were mixed with 0.004% methanolic solution (150 µL) of DPPH, a quite stable radical. The mixture was incubated for 30 min in the dark and the DPPH radical reduction was determined by measuring the absorption difference at 517 nm. Trolox was used as a standard reference and the DPPH results were expressed as Trolox equivalents per gram of dry extract (mg TE/g extract).
For the metal chelating activity [28
], the test solution (100 µL, 1 mg/mL) was added to a FeCl2
solution (50 µL, 2 mM). The reaction was initiated by adding 5 mM ferrozine (100 µL) solution. Similarly, a blank was prepared for each sample without ferrozine. Then, the absorbance of the sample and blank was noted at 562 nm after 10 min incubation at room temperature. The results were expressed as milligrams of ethylenediamine tetracetic acid (EDTA) equivalents (E) per sample amount (mg EDTAE/g extract).
An ABTS (2,2′-azino-bis(3-Ethylbenzothiazoline-6-sulfonic acid) radical cation scavenging assay was carried out by generating the ABTS+•
radical cation obtained by the reaction of 7 mM ABTS solution with 2.45 mM potassium persulfate in darkness for 12–16 h. Prior to starting the assay, methanol was used to dilute the ABTS+•
solution to an absorbance of 0.700 ± 0.02 at 734 nm. The resulting ABTS+•
solution (25 µL) was mixed with the extract solution (200 µL) and the mixture was incubated for 30 min at room temperature. The absorbance was then measured at 734 nm. Trolox was used as a standard reference and the results were expressed as Trolox equivalents per gram of dry extract (mg TE/g extract) [28
In FRAP (ferric ion reducing antioxidant power) assay, the reduction of Fe3+
-TPTZ (2,4,6-tris(2-pyridinyl)-1,3,5-triazine) to blue-colored Fe2+
-TPTZ complex was monitored by the method described by Zengin et al. [28
]. It is a simple and straightforward test. Ten volumes of acetate buffer (300 mM, pH 3.6), one volume of TPTZ solution (10 mM TPTZ in 40 mM HCl), and one volume of FeCl3
solution (20 mM FeCl3
O in 40 mM HCl) were mixed to prepare the FRAP reagent. The reaction mixture (25 µL of sample and 200 µL of FRAP reagent) was incubated for 30 min in the dark at 25 °C. The absorbance was then measured at 734 nm. Trolox was used as a standard reference and the results were expressed as Trolox equivalents per g of dry extract (mg TE/g extract).
The cupric ion reducing activity (CUPRAC) was determined according to the method of Zengin et al. [28
]. The sample solution (25 µL, 1 mg/mL) was added to a premixed reaction mixture (200 µL) containing CuCl2
(10 mM), neocuproine (7.5 mM) and NH4
Ac buffer (1 M, pH 7.0). The sample absorbances were read at 450 nm after a 30-min incubation at room temperature. CUPRAC activity was expressed as milligrams of Trolox equivalents (mg TE/g extract).
The total antioxidant activity of the samples was evaluated by the phosphomolybdenum method according to Zengin et al. [28
]. The sample solution (0.3 mL, 1 mg/mL) was added to 3 mL of reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate, and 4 mM ammonium molybdate). The sample absorbance was read at 695 nm after a 90-min incubation at 95 °C. The total antioxidant capacity was expressed as millimoles of Trolox equivalents (mmol TE/g extract).
3.13. Enzyme Inhibition Assays
α-Amylase inhibition was measured using a reaction mixture of 25 µL of extract solution (1 mg/mL) and 50 µL of α
-amylase solution (10 U/mL) prepared in phosphate buffer containing 6 mM NaCl (pH 6.9). Following 10 min of incubation at 37 °C for 10 min, 50 µL of starch solution (0.05%) were added. To stop the reaction, 25 µL of HCl (1 M) were added. Subsequently, 100 µL of iodine-potassium iodide solution was included and, after another 10 min of incubation at 37 °C, absorbances were read at 630 nm. Acarbose was used as the standard inhibitor and the results were expressed as equivalents of acarbose (mmol ACAE/g extract) [28
α-Glucosidase inhibitory activity was performed as per the previous method [28
]. The sample solution (50 µL, 1 mg/mL) was mixed with glutathione as an antioxidant stabilizing agent for the enzyme (50 µL, 1 mg/mL), α-glucosidase solution (0.2 U/mL, from Saccharomyces cerevisiae
, EC 188.8.131.52, Sigma, Milan, Italy) (50 µL) in phosphate buffer (pH 6.8, 0.1 mM) and 50 µL of PNPG (4-nitrophenyl-α-D-glucopyranoside, 10 mM) in a 96-well microplate for 15 min at 37 °C. Similarly, a blank was prepared by adding the sample solution to all reaction reagents without the enzyme solution. The reaction was then stopped with the addition of 50 µL of sodium carbonate solution (0.2 M). The sample and blank absorbances were read at 405 nm. The absorbance of the blank was subtracted from that of the sample and the α-glucosidase inhibitory activity was expressed as millimoles of acarbose equivalents (mmol ACAE/g extract).
Cholinesterase (ChE) inhibitory activity was measured using Ellman’s method, as previously reported [28
]. The sample solution (100 µL, 1 mg/mL) was mixed with 100 µL of DTNB (5,5-dithio-bis(2-nitrobenzoic) acid, 0.3 mM) and 25 µL of AChE (acetylcholinesterase (Electric eel acetylcholinesterase, Type-VI-S, EC 184.108.40.206, Sigma, Milan, Italy), 0.026 U/mL), or BChE (butyrylcholinesterase (horse serum butyrylcholinesterase, EC 220.127.116.11, Sigma, Milan, Italy), 0.026 U/mL) solution in Tris-HCl buffer (pH 8.0, 50 mM) incubating in a 96-well microplate for 15 min at 25 °C. The reaction was then started with the addition of 25 µL of acetylthiocholine iodide (1.5 mM) or butyrylthiocholine chloride (1.5 mM). Similarly, a blank was prepared by adding the sample solution to all reaction reagents without the proper enzyme (AChE or BChE) solution. The sample and blank absorbances were read at 405 nm after a 10 min incubation at 25 °C. The absorbance of the blank was subtracted from that of the sample and the cholinesterase inhibitory activity was expressed as milligrams of galantamine equivalents (mg GALAE/g extract).
Evaluation of anti-tyrosinase potential was carried out by adding 25 µL of sample (1 mg/mL) to 40 µL of tyrosinase solution (200 U/mL) and 100 µL of phosphate buffer (40 mM, pH 6.8) in a 96-well microplate and was allowed to incubate at 25 °C for 15 min. The substrate L-DOPA (10 mM, 40 µL) was used to start the reaction. Following 10 min of incubation at room temperature, all absorbances were read at 492 nm. For all enzyme inhibition assays, a blank solution was prepared using the same respective procedures but without sample. Kojic acid was used as the positive control and results were expressed as kojic acid equivalents (mg KAE/g extract) [28
3.14. Statistical Analysis
Total bioactive compounds, antioxidant, and enzyme inhibition results were expressed as means ± SD of three replications. Then, one-way ANOVA (Tukey’s assay) was performed with Xlstat 2017 software Addinsoft (Paris, France), (p < 0.05 was considered statistically significant) for determining differences in the extracts.