Plants and their fruits contain a wide variety of biological active secondary metabolites and, consequently, they are used for the development of drugs, dietary supplements, and functional foods, because of their antioxidant, anti-inflammatory, and antimicrobial properties [1
]. Lycium barbarum
is a perennial deciduous shrub with ellipsoid orange-red berries and a sweet-tangy flavor, also known as goji berry, wolfberry, barbary wolfberry, and Chinese boxthorn (or gouqizi in Chinese). The Lycium
genus includes up to 70 species that vegetate in regions from the temperate to the subtropical regions of Eurasia, Australia, southern Africa, and North and South America [3
species have a long history in China as they were used as medicine and functional food, and they are also referred to in the Traditional Chinese Pharmacopeia (TCP). Among the functional natural components, Goji berry fruits contain L. barbarum
polysaccharides (LPB) that are water-soluble glycoconjugates, and they are the most well-researched components [4
]. In addition to LBP, Goji berry fruits also contain carotenoids, wherein zeaxanthin dipalmitate is the predominant component (55.44% of total carotenoids), as well as polyphenols, which include caffeic acid, chlorogenic acid, p
-coumaric acid, quercetin, and kaempferol [5
It should be noted that extensive research was carried out on the bioactivity of L. barbarum
, while only a few papers are published for the L. chinense
]. Moreover, there are no reported studies on fatty acids (FA) for fruits of Greek origin. Free FA and complexes of lipids have key roles in metabolism as essential components of all membranes, as gene modulators, and as important energy sources, since they produce large quantities of ATP, carrying chemical energy within cells for metabolism [13
]. Τhe geographical origin is a significant quality parameter for many plant foods, since variations in soil composition, climate, and cultivation techniques cause differences in a plant’s chemical composition. The protected designation of origin (PDO) of some foods is of great importance in order to add high value to the product [14
Currently, L. barbarum
fruit is consumed by people in many ways, including drinking juice, eating raw and/or smoothies, mixed with tea, and added to trail mix, cereals, muffins, energy bars, or soups, and not only in China, where more than 95,000 tons of goji fruits are produced and derived from 82,000 hectares [15
]. In recent years, the goji berry cultivation expanded to other countries, such as Greece, due to the rising demands of consumers for superfoods and goji berry-related food products.
Regarding the growing interest in introducing goji cultivation in different areas in Greece, the sustainability of its production compared with the imported fruit should be evaluated. This study emphasizes the chemical fingerprint composition of the two varieties of fruits; however, it is also necessary to consider nutraceutical features, defining effective extraction processes in order to produce highly bioactive extracts that can be used in the pharmaceutical and food industry. For this reason, the aim of current study was to determine the physicochemical properties, such as the total carbohydrate and phenol content, present in goji berry fruit varieties (L. barbarum and L. chinensis Mill.) collected from the Thessaly region in central Greece, compared to imported fruit, measured and evaluated based on attenuated total reflection Fourier-transform infrared (ATR-FT-IR) spectra. In addition, the fatty-acid composition was estimated by gas chromatography coupled with mass spectrometry (GC-MS) analysis, while two in vitro methods (2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2’-azino-bis(3-ethyl-benzthiazoline-sulfonic acid) (ABTS)) were used for the evaluation of antioxidant activity. To the best of our knowledge, this is the first work that presents the concentration of the non-polar lipids of Greek goji berries obtained from the Thessaly region, and their chemical properties.
2. Materials and Methods
2.1. Goji Berry Samples
Goji berry fruits were collected from plantations of L. barbarum and L. chinense varieties located in the region of Thessaly in Greece. In total, eight samples were analyzed, six from the Thessaly region of Greece, one L. barbarum sample imported from Mongolia and another (L. barbarum) from China. More specifically, the three Thessaly samples of L. barbarum variety were collected during the months of June (LbC1), August (LbC2), and October (LbC3), while the other three Thessaly samples (LcC1, LcC2, and LcC3) were collected on the same days from the L. chinense plantation. All Thessaly samples were dried in order to reach a final moisture value on the order of 14%, which was similar to that of the imported ones. All samples were prepared in powder form with the use of a hummer mill.
In this study, methanol (MeOH), anhydrous sodium sulfate (Na2SO4), sodium chloride (NaCl), potassium hydroxide (KOH), methyl-tert-butyl ether (MTBE), and deionized water (>18 MΩ∙cm resistivity) were used. The aforementioned products were purchased from Sigma-Aldrich (Life Science Chemilab S.A., Athens, Greece). Supelco™ (Munich, Germany) 37 component fatty-acid methyl ester (FAME) mix was also used. The FAME mix contains the methyl esters of 37 fatty acids (catalog no. 47885-U) in a concentration of about 2% and 4% of each fatty-acid methyl ester. Palmitic acid methyl ester and tridecanoid acid in FAME mix were also purchased from Sigma-Aldrich in 6% and ≥99% concentrations, respectively.
Also, Folin–Ciocalteu, sodium carbonate in anhydrous crystal form, gallic acid, ethanol, 2,2-diphenyl-1-picrylhydrazyl (DPPH), methanol, and 2,2’-azino-bis(3-ethyl-benzthiazoline-sulfonic acid) (ABTS) were used as compounds and for antioxidant activity determinations.
2.3. Determination of Total Carbohydrate Content (TCC) of the Extracts
The phenol/sulfuric method according to Dubois et al. [16
] was used for the determination of TCC. Briefly, 1 mL of each sample solution was mixed with 0.5 mL of phenol (4%) and 2.5 mL of sulfuric acid (95%); after an incubation of 10 min, the optical density was measured at 490 nm using a Thermo Scientific (Evolution 201) spectrophotometer (Thermo Scientific, Madison, WI, USA). The TCC was calculated on the basis of the calibration curve of d
-glucose and expressed as g of carbohydrates/L of the extract. The linearity range of standard d
-glucose was determined as 0.01 to 0.1 mg/L (R2
= 0.9918). The equation was obtained by linear fitting of y
+ 0.0111 (data not shown), where y
is the absorbance at 490 nm and x
is the concentration of d
-glucose. The TCC was expressed as mg of d
-glucose equivalent per g of dried extract (mg/g dry weight).
2.4. Determination of Total Phenol Content (TPC) of the Extracts
Samples were aqueous ultrasound extracted according to the methodology of Skenderidis et al. [17
]. The TPC of the extracts was determined using the Folin–Ciocalteu microscale protocol [18
]. Briefly, 20 μL of goji berry extract was added to 1.58 mL of deionized water and 100 μL of Folin–Ciocalteu reagent. After the immediate addition of 300 µL of Na2
solution (20% w
), the mixture was left for 120 min of incubation in the dark. Finally, the optical density was measured at 765 nm using the abovementioned spectrophotometer. TPC was expressed as mg of Gallic Acid Equivalent (GAE) per g of dried fruit weight (mg GAΕ/g dry weight) after determination using a standard curve of absorbance values derived from standard concentration solutions of GA.
2.5. Determination of Total Antioxidant Capacity of Goji Berry Fruit
2.5.1. DPPH (2,2-diphenyl-1-picrylhydrazyl) Radical-Scavenging Activity
The DPPH radical-scavenging activity of the samples was evaluated according to the method described by Skenderidis et al. [17
]. Dissolved in distilled water at different concentrations, goji berry samples were mixed with 1 mL of a freshly made methanol solution of DPPH• radical (100 μM). The contents were vigorously mixed and incubated at room temperature in the dark for 20 min and the absorbance was read at 517 nm. Methanol solutions of tested extracts and DPPH• were used as blank and control measurements, respectively. All experiments were carried out three times on two separate occasions. The percentage of radical-scavenging capacity (RSC) of the tested extracts was calculated according to the following equation:
% DPPH• radical scavenging activity = ((Abscontrol − Abssample)/Abscontrol) × 100
are the absorbance values of the control and the tested sample, respectively. The half maximal inhibitory concentration (IC50
) value was calculated using the graph-plotted RSC percentage against extract concentration in order to compare radical-scavenging efficiency of the extracts. All experiments were repeated three times on two separate occasions.
2.5.2. ABTS•+ (2,2’-Azino-bis-(3-ethyl-benzthiazoline-sulfonic Acid) Radical-Scavenging Activity
radical-scavenging activity was measured according to the method described by Kerasioti et al. [19
]. The reaction was carried out in 1 mL of final volume, containing 400 μL of H2
O, 500 μL of ABTS (1 mM), 50 μL of H2
(30 μM), and 50 μL of 6 μΜ solution of the enzyme horseradish peroxidase (HRP). Contents immediately after enzyme addition were thoroughly mixed and incubated for 45 min in the dark at room temperature. After 45 min of incubation in the dark, different concentrations of the tested sample solution were added and mixed thoroughly; then, the absorbance was measured at 730 nm. In each experiment, blank (samples without HRP) and control samples (without goji berry extract) were used. All experiments were repeated three times and on at least two separate occasions. Furthermore, the RSC percentage of the ABTS•+
radical-scavenging activity and IC50
values were determined as described above.
2.6. Chemical Components and Structure Analysis by Infrared Spectroscopy
The FT-IR measurements were obtained at room temperature using a Nicolet 6700 (Thermo Scientific) spectrophotometer in order to estimate the chemical composition of the samples. The OMNIC 9 software (Thermo Scientific) was used for the configuration of the spectrophotometer equipped with an attenuated total reflectance (ATR) accessory, a deuterated triglycine sulfate (DTGS) detector (Thermo Scientific), and a KBr beam splitter (Thermo Scientific). A diamond crystal sampling plate Smart iTX, (Thermo Scientific) clamped with a pointed tip was used in order to place and analyze the samples. Background (empty sample plate) scans were acquired and rationed against the sample spectrum. Furthermore, the ATR crystal and pointed tip were cleaned in order to remove any interference from the preceding sample. Spectra were collected at 4 cm−1 spectral resolution in the mid-infrared range of 4000–650 cm−1 with 32 successive scans. Furthermore, all spectra were ATR- and baseline-corrected, normalized, and transformed to absorbance spectra.
2.7. Fatty-Acid Methyl Ester (FAME) Synthesis
The method of FAME synthesis was applied as described by Gerasopoulos et al. [13
]. Firstly, 0.5 mL of sample was placed in heat-resistant pyrex glass (16 cm length, 1.6 cm diameter) sealed with a teflon screw cap. As an internal standard, 1 mL of methanolic solution of tridecanoidacid (C13:0) was added at a concentration of 600 μg/mL. Subsequently, 0.4 mL of 10 N KOH (potassium hydroxide) and 2.7 mL of pure methanol were added. The examined solution was contained in tubes placed in a water bath at 55 °C for 90 min, while vigorous stirring every 20 min followed in order to achieve proper hydrolysis of the samples. Tap water was used in order to cool down the tubes for about 15 min. Fatty-acid methyl esters, 0.3 mL of 24 N H2
were added in order to correct the composition. Thereafter, tubes were placed again in a water bath at 55 °C for 90 min followed by vigorous stirring every 20 min. A cooling bath with tap water followed, as previously. Finally, 3 mL of hexane was added as a solvent, and the samples were stirred with a vortex for 3 min. The treated samples were then placed in the centrifuge at 6000× g
for 15 min at room temperature, and the supernatant solution (hexane layer containing the FAME) was placed in GC vials of 2 mL. Finally, the supernatant solution was stored at −20 °C until GS/mass spectrometry (MS) analysis was carried out.
The fatty-acid profile assessment in goji berry samples was carried out in duplicate. A standard solution containing 37 FAMEs was used to identify the individual FA supplied by SupelcoTM
(Sigma-Aldrich, Munich, Germany) known as the 37 Component FAME Mix Standard. The peak area of the samples that was corrected with the respective correction factors was used for the calculation of the percentage of each FA [20
]. The GC-MS Agilent 7890A GC chromatography apparatus (Agilent, Frankfurt, Germany) was used. The GC silica column (J&W 112-88A7: 804.11246 HP-88 250 °C: 100 m × 250 µm × 0.25 µm) (Agilent, Frankfurt, Germany) was located inside a temperature-controlled oven (Agilent, Frankfurt, Germany). The following MS parameters were used: interface temperature, 250 °C; MS ionization mode; helium gas with 45.2 mL/min flow; electron ionization; detector voltage acquisition mass range, 20–850 amu. Data were collected and processed using the GCMS Agilent integrated software (Agilent).
2.8. Statistical Analysis
The standard deviation was calculated and the averaged values along with the standard deviations (SD) are documented in the respective tables or figures. Statistical differences among the means, as well as interactions between the variables used in chemical analyses, were detected by one-way Analysis Of Variance (ANOVA) followed by Tukey’s test, and the statistical significance was set at p ≤ 0.05. MiniTab®17.1.0 software (Minitab LCC, State College, PA, USA) was used as the tool to perform the abovementioned tests.
In the present study the composition of the bioactive substances of two Lycium species cultivated in Greece in different fruit periods of production were evaluated and compared with two samples which were imported from China and Mongolia. The results show that the Greek L. barbarum fruits from the Thessaly region contain higher amounts TCC and TPC than L. chinense. Among all samples, the Mongolian fruits showed better results against DPPH radical scavenging. Moreover, a significant variation in the fruit composition between the different cultivated periods emerged from the analysis results. The cultivated fruits in August showed higher amounts of the examined bioactive substances, which leads to the conclusion that this is probably due to the heat stress response of the plants because of the high temperatures during this particular month in the Thessaly area. Furthermore, the GC-MS analysis results show high levels of linoleic, palmitic, and oleic acids for the fruits of both species, which is in agreement with the results of other studies that appear in the literature. The analysis of the ATR-FT-IR spectra of the two varieties shows no differences between the two Lycium species. However, the intensity of the infrared peaks, which are related to the content of functional groups in the fruits, verifies the results obtained for the TP and TC contents. Nevertheless, for a better understanding of their phytochemicals and biological composition, as well as their effects, more investigations are needed.