Date Palm Seed Oil (Phoenix dactylifera L.) Green Extraction: Physicochemical Properties, Antioxidant Activities, and Phenolic and Fatty Acid Profiles

Date palm seed oil is among the precious vegetable oils with low yield, whose extraction is commonly done with organic solvents which cause serious problems. *is study aims to assess the effectiveness of orange peel essential oil as biosolvent for date seed oil extraction. Green extraction was conducted by Soxhlet apparatus as well as by soaking and compared with the Soxhlet method using petroleum ether. *e GC-MS analysis of orange peel essential oil confirmed its richness with limonene (94.31%), which justifies its usefulness as green solvent. *e latter gave higher yields, the extracted bio-oil was light brown with pleasant odor, and the characteristics were consistent with international standards. Based on the GC profiles, obtained oils were similar using both solvents, and the major compounds were oleic and lauric acids. *e bio-oil phenolic content and the antioxidant activity were high, and the major compounds were the protocatechuic, chlorogenic, and 4-O-caffeoylquinic acids. Gallic and p-coumaric acids were the major compounds for oil extracted by petroleum ether.


Introduction
In recent years, the use of date palm seeds oil (DPSO) has grown around the world especially in cosmetics manufacturing and pharmaceutical industries [1]. is oil is classified among the precious vegetable oils owing to its richness in fatty acids and phenolic and antioxidant compounds. Furthermore, DPSO has multiple benefits on human health [2]. In fact, Besbes et al. [3] have noted that, compared with olive oil, DPSO has higher oxidative stability. In addition, these authors have reported a good capacity of DPSO in the protection against UV light and therefore against cellular damage. Other studies revealed that date seed oils are good sources of α-tocotrienol [4], which is reported as an effective compound to reduce the breast cancer risk [5,6], cholesterol, and low-density lipoprotein cholesterol in humans [7]. Two processes were commonly used for oil extraction from the date seeds. e first was by pressing method allowing obtaining very pure oil containing no foreign chemical substances. Despite the very low yield (≈5.5%) of this method [8], interesting minor bioactive compounds were highlighted which have beneficial effects on human health and also play an important role in prolonging the shelf life of the cold-pressed oils by the increase of oxidative stability of the oil [9]. e second process was by using an organic solvent which is recovered by various methods. Jemni et al. [10] have discussed the usefulness of several solvents in the extraction of the date palm seed oil. ese authors concluded that nonpolar solvents such as toluene, chloroform, and hexane gave the best yields. After the extraction process, the oil must be completely purified to avoid the toxicity of organic solvents that had adverse effects on human health [11]. Despite its purification, there may be traces of the solvent that can affect the organoleptic quality of the oil. In addition, organic solvents are volatile compounds, and with high concentrations, ozone and photochemical oxidants are produced [12]. Moreover, solvents may be inhaled into the body, swallowed, or infiltrated through the skin. According to Costa and Aschner [13], most organic solvents may not only cause depression in the central nervous system, but also cause encephalopathy with intellect and memory deterioration.
Recently, the green chemistry focused on safer solvent named "biosolvent" such as terpenes [14]. ese latter have interesting chemical properties and exist in many plants especially in citrus fruits. In many industrial applications, terpenes are considered as new alternative to petroleum solvents. Recent attempts have been undertaken to use limonene to extract oil from vegetal materials, whose oil yield and quality are almost like those obtained using hexane [15,16].
is work was the first on the DPSO green extraction by using the essential oil of orange peel as a green solvent. e aim was to improve the DPSO quality by eliminating the risks resulting from organic solvent.

Vegetal Material Preparation.
e studied date palms in the present study were Phoenix dactylifera L. cv. Deglet Nour from Nefzaoua oases, which is a major part of the Tunisian continental oases located in the South West of Tunisia. Trees were irrigated at the rate of 17,000 m 3 /ha/year and winter fertilization was applied by an average amount per date palm tree of 20 kg for manure. e fresh date fruits were harvested from four date palm trees at full repining stage and during the season of 2020. e isolated seeds were washed, dried, and then ground to powder form in a grinder. Correspondingly, oranges from the Maltaise demi-sanguine variety (Citrus sinensis) were picked from mature trees. ey had spheroid shape, were concave at the base, and had a rounded apex shape and pecked surface texture. e mature orange trees were planted in an orchard located at the Cap-Bon region (North-East of Tunisia), spaced 7 m × 6 m, and were irrigated with drip line with four drippers per tree (4 l/h). e soil is lithic Leptosols and the average annual precipitation and reference evapotranspiration (ETo-PM) were about 651.3 and 1.080 mm/year, respectively.

Green Solvent Extraction and GC-MS Analysis.
e essential oil extraction from the prepared orange peel was carried out by steam distillation. e steam damages the plant cell structure and releases the volatile molecules which were then dragged towards the refrigerant. Fifty kilograms of fresh orange peel was distilled in a distillation unit operating on a steam-cum-water distillation principle for 3 hours. After the collection of the aqueous phase, the essential oil would subsequently be recovered and then placed in a dark flask.
e gas chromatography-mass spectrometry (GC-MS) analysis of the extracts was performed using a GC-MS (Model, QP 2010, Shimadzu) equipped with an RTX-5MS capillary column of 30 m in length, 0.25 mm in diameter, and 0.25 mm film in thickness. e detection was done by an electron ionization system (70 eV). Helium gas (99.99%), the carrier gas, was used at a constant flow rate of 1.20 ml/min. Injector and mass transfer line temperature were set at 250 and 200°C, respectively. e oven temperature program was from 50 to 250°C at 7°C/min, held isothermally for 2 min, and raised to 250°C at 5°C/min. Samples manual injection in the split mode was done with 50.0 split ratio and with 50-600 AMU mass scan. e total GC-MS running time is 35.50 min. e relative intensity of each volatile compound has been calculated as the ratio between the area of the specific molecule and the sum of the areas of all identified peaks (peak area normalization method) in the chromatogram [17].

DPSO Extraction by Soxhlet.
e DPSO was extracted by using pure solvent at their boiling point and azeotrope solvent at their critical solution temperature using Soxhlet apparatus for six hours. Hence, two solvents were used: petroleum ether as pure solvent for classical extraction method and "essential oil + water" (60% + 40%) as azeotrope solvent for the green extraction. Solvents were firstly removed by a rotary evaporator and then placed at 40°C overnight to remove the excess of the solvent.

DPSO Extraction Method by Soaking in Essential Oil.
All the steps are shown in Figure 1. Date seed powder was placed in a dark flask and homogenized with essential oil. After mixing for six hours at 45°C, the mixture was centrifuged. e liquid phase was used to recover the essential oil and the DPSO was yielded.

Physicochemical Characterization. Yields
were expressed in % of oil in the basis material. Color and odor were described and the acidity and peroxide values were carried out by standard IUPAC methods [18]. Density meter was used for oil density assessment.

Fatty Acid Analysis.
Fatty acid compositions were determined by GC analysis as described by Nehdi et al. [19]. e fatty acid methyl esters composition was determined by converting the oil to fatty acid methyl esters by addition of 1 ml of n-hexane to 40 mg of oil followed by 200 μl of sodium methoxide (2M). e mixture is heated in the bath at 50°C for few seconds followed by adding 200 μl HCl (2N). e analysis was done using a GC (Agilent 6890N, CA, USA) equipped with a flame ionization detector (FID) and a capillary column (MEGA-10, 25 m × 0.32 mm × 0.25 μm). e column temperature program was from 150 to 200°C at 2°C/min and the injector and detector temperature were set at 250°C. Helium was the carrier gas. Identification and analysis of the peaks were done with the Agilent Technologies Chemstation A09.01 software.

Polyphenol Extraction and Analysis.
Phenolic compounds from DPSO were extracted according to the method of Farrés-Cebrián et al. [20]. Extracted oils were added to ethanol 70% (1 : 1). e mixture was shaken vigorously for 2 min and then centrifuged (3500 rpm/5 min) and followed by 24 h at −18°C. Next, 2 ml of hexane was added and shaken vigorously for 2 min. After centrifugation (3500 rpm/5 min), the aqueous ethanolic extracts were directly analyzed.
Phenolic compounds quantification was done using liquid chromatography system (Hewlett-Packard 1100) with C-18 column (Teknokroma Tracer Extrasil ODS-2, 250 mm × 4.0 mm, i.d. 5 μm). e mobile phase (0.01% trichloroacetic acid in water and acetonitrile) had the following gradient over a total run time of 55 min: 95% A initially, 75% A-30 min, 50% A-45 min, 0% A-47 min, 75% A-95 min, and 95% A-52 min until completion of the run. e quantification of the compounds was carried out by peaks integration, which was done at different wavelengths, with reference to calibrations made using external standards.

Antioxidant Capacity.
Antioxidant activity was determined by the DPPH• method [21]. 10 μl of the DPSO was added to 190 μL of DPPH• (3.8 mg/50 mL methanol), after 30 min in the dark the measurement of the absorbance was done at 517 nm. e antioxidant activity was measured by decreasing the absorbance at 517 nm (TecanInfininte M200, Männedorf, Switzerland). e antioxidant capacity was expressed as mg ascorbic acid equivalent (AEAC) per 100 g FW.
ABTS assay was determined according to Wang et al. [22]. e ABTS radical cation (ABTS •+ ) solution was prepared (7 mM ABTS + 2.45 mM potassium persulphate) and incubated at 23°C in the dark for twelve hours. e ABTS •+ was diluted in ethanol (80%) until an absorbance of 0.700 (±0.005) at 734 nm. 2 ml of ABTS •+ solution was added to 100 μl of the DPSO sample and mixed vigorously. e incubation was done at room temperature during 5 min and the absorbance was immediately recorded at 734 nm. e absorbance was expressed as the Trolox-Equivalent Antioxidant Capacity (TEAC).

Statistical Analysis.
e average values of all the experiments were calculated and expressed as the mean value (±standard deviation).
e ANOVA with post hoc SNK comparisons was performed using SPSS 16.0 for Windows. Figure 2 shows the compounds of the orange peel essential revealed by GC-MS. Ten substances were identified and the summary is given in Table 1. e composition results were close to those found for samples from other geographical origin [23][24][25] with some detected differences. Typically, limonene was the major compound with slight differences; in our case it accounts for 94.31%. e other constituents were less than 1.7% and their order was as follows: [26] have compared the hydrodistillation with steam explosion for extraction of essential oil from orange peels and concluded that extraction process influences the limonene yield. e orange peel essential oil was used as biosolvent for the DPSO extraction, and the efficiency was compared with petroleum ether. e physicochemical properties of the extracted oils are exposed in Table 2. Concerning the Soxhlet method, the extracted DPSO using petroleum ether had a maximum yield of 9.8% and achieved after four hours of extraction (Figure 3), and the green solvent gave a maximum oil rate of 9.25% since the second hour of extraction. e soaking method using essential oil gave the highest yield and the maximum value was 13.88% obtained after two hours of extraction ( Figure 3). e oil yield extracted by petroleum ether is comparable to that found by Hamada et al. [27] (8.7-12.3% for 11 varieties of date kernels from Saudi Arabia) but lower than Tunisian Alig variety (12.73%) [28]. Petroleum ether has usually shown a high ability to extract oil in comparison with other organic solvents, such as chloroform-methanol. e efficiency of the green solvent was better in the soaking extraction method, which might be due to the dissolving power of the essential oil for Journal of Food Quality triglycerides when compared with petroleum ether. A previous study [29] has revealed that limonene is slightly more polar than hexane and could have better ability to extract oils from rice bran. In addition, the used green solvent is relatively fire-and explosion-safe, nontoxic to humans, and less volatile than hexane, and it comes from a renewable source. Petroleum ether gave, as expected, yellow oil (Supplementary File 1) with unpleasant odor and the density was 0.87 g/cm 3 . In other studies, Abdalla et al. [30] have noted higher density value of oil (0.91 g/cm 3 ) extracted from Sudan date seed variety by organic solvent. However, the DPSO extracted by the essential oil has different physical appearance, whose color is light brown (Supplementary File 1) with pleasant odor and density of 0.86 g/cm 3 . e bioextracted oil being slightly darker compared with petroleum ether-extracted oil might emanate from the higher temperature used during solvent recovery when essential oil is used, leading to the formation of oxidative materials, including polymers and other oil-soluble products as a result of Maillard reactions [29].

Physicochemical Characterization of the Extracted Oils.
Furthermore, the physical state of DPSO at 4°C was different as only the oil obtained by petroleum ether became solid, while the others remained liquid. In fact, oils have different physical proprieties and some of them freeze at low temperature and cause trouble in the industry process [31]. In the case of oils extracted from date palm seeds, the use of essential oil could be a good practice to avoid freeze problem. For olive oil, freezing temperature variability depends on variety and olive ripeness at processing. It can be concluded that extracted essential oil used as green solvent has slightly lightened the DPSO and affected its physical appearance.
A significant effect of the extraction method on the acidity was observed. Petroleum ether gave lower acidity value (0.87 g/g) which reflects its oxidative stability during extraction [3], which is in accordance with the results obtained previously [4,32]. e use of essential oil from orange peel was found to enhance the acid value to 1.03 and 1.01 mg/g, which is close to value of oil date seeds obtained from Libyan varieties [33]. Peroxide values were 2.96 meqO 2 /kg for petroleum ether solvent and 4.26 and 5.05 meqO 2 /kg for green solvent. ese indices have met the quality standards and show good stability of the extracted oils. In fact, Codex Alimentarius (2009) (CODEX-STAN, 210-1999) recommends that the acid and peroxide values of cold-pressed oils should not exceed 4.0 mg/g and 15 meqO 2 / kg, respectively. e low peroxide value indicates that the seed oil is fresh and is less prone to auto-oxidation. ese variations may come from various factors like the unsaturation degree of the fatty acids present in the oil, storage, the light, and metals or other compounds content that can catalyze the processes of oxidation [10,34]. Figure 4 and the percentages of fatty acids in the threeextracted DPSO are presented in Table 3. e same fatty acid profile was observed, and no significant difference was noted. Hence, the extracted DPSO are identical, and the new "green" extraction method did not change the fatty acid composition. e analysis has proven that oleic acids (C18 : 1) and lauric acids (C12 : 0) are the most abundant, followed by myristic (C14 : 0), palmitic (C16 : 0), linoleic (C18 : 2), and stearic acids (C18 : 0). However, caprylic (C10 : 0), palmitoleic (C16 : 1), and linolenic (C18 : 3) acids were found in small amounts. With respect to Sawaya et al. [35], they have indicated that DPSO is not a linoleic acid oil, but rather considered as an oleic-lauric oil. ese results agree well with other findings previously reported by Al-Hooti et al. [36], Al showiman [37], and Devshony et al. [38]. e proportion of USAFA (unsaturated fatty acids) is important to estimate the oil oxidation [39]. Indeed, the higher the USAFA is, the more the oil is prone to oxidation. Besbes et al. [32] have reported that PUFA (polyunsaturated fatty acids), essentially C18 : 2, are usually used to assess the oil deterioration level.     In our study and for all the extracted oils, PUFA values were around 8% which are lower than the previous findings by Besbes et al. [32] and by Tafti et al. [40], who found 14.1% and 14.08% of PUFA, respectively.

Phenolic Compounds and Antioxidant
Capacity. e current investigation demonstrated that petroleum ether solvent provided DPSO with a phenol content of 91.32 mg/ 100 g (Table 4). is quantity was higher than that reported by Besbes et al. [32] who found a quantity of 52.6 mg/100 g in the same variety. Green solvent was found to enhance the DPSO phenol content that became 345.23 mg/100 g by Soxhlet method and 402.67 mg/100 g by the soaking method (Table 4). ese variations between the two types of solvents could be attributed to the polarity, and thus the extractability of the bioactive substances [41]. Kumar et al. [14] have noted that terpenes have higher polarity than organic solvent as hexane. ese authors have recommended the use of terpenes to ensure a cleaner environment, safer handling, and nontoxicity. Furthermore, phenolic profiles were not the same. More phenolic compounds were detected in the case of extraction with essential oil. Although petroleum ether gave DPSO rich in gallic and p-coumaric acids, it also provided other components with a smaller amount (ocoumaric, trans-ferulic, and syringic acid and flavonoids components) ( Table 4). Essential oil solvent offered more phenolic compounds such as protocatechuic, chlorogenic, and 4-O-caffeoylquinic acids, which are detected with higher amounts than the gallic acid and p-coumaric acid (Table 4). e antioxidant activity had the same tendency as the phenolic content.
e DPPH test showed that DPSO extracted by essential oil exhibited the highest activity (39.22 and 47.06 mg AEAC/100 g FW) ( Figure 5). Similarly, ABTS assays showed that the antioxidant capacity of DPSO extracted by the soaking method had the highest value  ( Figure 5). Herch et al. [42] have studied the antioxidant capacity of flesh oil from the Kintichi variety and attributed the high antioxidant activity to the high phenolic content. DPSO extracted by green solvent was subjected to screening for its higher antioxidant activity and higher phenolic content. Polyphenols are natural antioxidants that offer this product the ability to repair the human skin [43]. Many studies proposed the DPSO as an ingredient of UV protector products [4,44]. Many biological effects were attributed to phenolic compounds, including antibacterial and anticarcinogenic [45,46]. Chlorogenic acids, which are detected only in the green extraction, prevent degenerative pathologies such as cardiovascular diseases and cancer [47]. (E)-Cinnamic acid that is also extracted only by the green method has a large spectrum of biological activities such as anti-inflammatory and anticancer properties. Furthermore, it has been detected as gut microbe-derived metabolites exerting various biological effects in the colon [48].

Conclusion
e use of essential oil extracted from orange peel as green solvent demonstrated its ability to extract oil from date palm seeds. is extraction method represents a promising approach for bio-oil extraction from date seeds. Besides, the comparison studies proved that this green solvent extracted higher oil amount and significantly improved its quality. e results of this study would also have broader implications for the extraction of other liposoluble compounds from date seeds. anks to its polar nature, it is possible that the used green solvent would be applied to extract more bioactive compounds. However, further research investigations should be carried out to improve the yield and its physicochemical properties. Future work should focus on the analysis of semivolatile compounds that could remain in the oil after essential oil evaporation.

Data Availability
No data were used to support this study.