1 Introduction

Numerous fruit seeds have been recognized as rich sources for many edible oils that have been revealed to contain high amounts of essential fatty acids and other biologically active phytochemicals such as phenolic compounds, sterols, tocopherols and carotenoids [1, 2]. Most of these natural compounds have been proven to possess substantial health promoting properties and could be employed as natural preservatives for food applications. Although the most common use of synthetic antioxidants in the food industries as an inhibitor of lipid oxidation, it has been restricted because of their toxicity [3]. Hence, much attention has been drawn to the extraction of natural antioxidants with plant origins.

Zizyphus lotus L. (Z. lotus), commonly known as jujube, belongs to the botanic family of the “Rhamanaceae” which is native to China and became extensively cultivated in tropical and subtropical regions, especially in East Asia (China, India), North Africa, and Middle Eastern countries [4, 5]. In Tunisia, it is a well-adapted plant growing in arid and sub-arid climates mainly in the south of the country [6]. Different parts of jujube such as fruits and leaves are used as food and a source of components of some traditional medicines for curing several diseases [4]. Examples are digestive disorder, urinary troubles, fever and diarrhea to mention a few [7, 8]. The chemical analyses of Z. lotus fruits have demonstrated that they contain peptide and cyclopeptide alkaloids, flavonoids, sterols, tannins, betulinic acid and triterpenoidal saponin glycosides [7, 9,10,11].

Interestingly, seeds of Z. lotus are considered important due to the great amount of their lipid fraction, reaching about 33% of the dry weight and providing several health maintaining compounds [12]. Particularly, Z. lotus seeds oil is rich in unsaturated fatty acids, such as oleic and linoleic acids, offering thus many advantages for human consumption. It contains also high amounts of phytosterols and tocopherols [6]. Nonetheless, the aforementioned properties are dependent on abiotic and biotic factors. Many studies highlighted the effect of the plant species [13], the season of harvest and location [14] on the chemical composition of edible oils extracted from seeds. Some other factors were also investigated. Ghafoor et al. [15] studied the effect of roasting temperature on chia seed’s oil quality and the obtained data indicated that heating at a temperature below or equal to 90 °C led to better preservation of the oil nutrients. Furthermore, the extraction methods affected the composition of total carotenoid and chlorophylls, phenolic compounds and fatty acids [16].

Apart from the medicinal and nutritional characters, and to the best of our knowledge, no research work pertaining to the evaluation of the effect of geographical origin of Z. lotus seed oils has been conducted so far.

Thus, this study’s utmost objective is the evaluation of the fatty acids and tocopherol composition as well as the antioxidant activity of Z. lotus cold pressed seed oils from different geographic origins in Tunisia. Physical properties of oils, such as dynamic viscosity and interfacial tension were also investigated. Such information would be an important contribution to a further valorization of the seed oil extracted from this species in the food industry and a proof of the product authenticity.

2 Materials and Methods

2.1 Chemicals and Reagents

All the chemicals and solvents, used in this study, were of the highest commercial grade.

2.2 Plant Materials

The collection of Z. lotus fruits was performed at the same maturity stage on the basis of fruit brown color and mucilaginous aspect, during August 2019, from different Tunisian locations, namely: Nabeul, Zaghouan, Siliana, Sousse, Mehdia, Kairouan, Kasserine, Sidi Bouzid, Sfax, Gabes, Gafsa and Tozeur. The geographic positions and climatic data of these locations are shown in Fig. 1. After being botanically identified according to the protocol of Tunisian flora [17], each sample (500 g) was air-dried and blended in a Retsch AS 200 Basic blender (Retsch GmbH, Germany). Next, the seeds were manually isolated from the edible parts and stored in the dark at − 18 °C. Besides, voucher specimens of each sample were kept for future reference in our lab.

Fig. 1
figure 1

Geographic position and climatic data (temperature (°C) and rainfall (mm)) of different locations for Z. lotus collection

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2.3 Cold Pressed Z. lotus Seeds Oil Extraction

To extract oil, we made use of a cold-pressing apparatus (CA59G, German Monforts Group, Moenchengladbach, Germany) to press Z. lotus seeds at 20 °C. The obtained mixture was centrifuged (Avanti J-26 XP, Beckman Coulter Inc., Brea, CA, USA) at 2500 g for 5 min to separate the oil. The extracted oils were kept in vials under a nitrogen atmosphere and kept in the dark at − 18 °C, till assays performing [18].

2.4 Fatty Acids Analysis

The determination of the fatty acid composition was realized using Gas chromatography (PerkinElmer Wallac Auto System XL autoinjector, San Jose, CA, USA) coupled to flame ionization detection (GC-FID) after conversion to fatty acid methyl esters (FAME). The procedure used was the same as our previous study [19]. For the methylation purposes, 0.2 mL sodium methoxide solution (2 mol L−1 in anhydrous methanol) was mixed, for 1 min using a vortex, with 0.05 g of oil dissolved in 1 mL of hexane. After sodium glycerolate sedimentation, 1 μL of the clear supernatant was injected into a Supelco Sp™ 2340 fused-silica capillary column (60 m × 0.25 mm, 0.20 μm film thickness) under the following conditions: Injection and detection temperatures were set at 250 °C. The oven temperature program was as follows: heating from 130 to 170 °C at a rate of 20 °C.min−1, then raised to 230 °C at the rate of 10 °C min−1, hold at 230 °C for 10 min, and finally raised to 250 °C, at the rate of 30 °C min−1. Nitrogen was used as a carrier gas at a flow rate of 50 mL min−1. Peaks of FAME were identified according to the retention times of a standard mixture. The peak areas were computed and using the direct normalization method, the FAME contents were expressed as area percentages.

2.5 Tocopherols Analysis

The evaluation of the tocopherols analysis was realized by high-performance liquid chromatography according to AOCS Method [20], using a Hewlett-Packard apparatus, series 1100 system (Santa Clara, CA). This apparatus is equipped with a model 168 UV detector (Beckman Coulter, Inc., Fullerton, CA) at a wavelength of 292 nm and a Lichrosorb Si60 (Merck) column with a 7 mm silica particle size. An amount of 15 µL of a mixture made up of 2 g of oil sample dissolved in 10 mL of n-hexane was eluted with (1% 2-propanol/ hexane) as a mobile phase with a flow rate set at 0.65 mL min−1.

Tocopherols were identified by comparing their retention times with those of standards.

The calculation of the conversion factors for vitamin E activities for each compound was carried out following the formulae below: α-tocopherol × 1.00; β-tocopherol × 0.40; γ-tocopherol × 0.10; δ-tocopherol × 0.01; α-tocotrienol × 0.30; β-tocotrienol × 0.05 and γ-tocotrienol × 0.01.

2.6 Physical Analysis

The physical properties such as dynamic viscosity and interfacial tension of Z. lotus seed oils were assessed according to the methods described by our previous study [19].

2.7 Antioxidant Activity

2.7.1 DPPH. Radical Scavenging Assay

The DPPH. radical scavenging activity of Z. lotus seed oil was evaluated following Essaidi et al. [21] procedure with a minor adjustment. 0.1 mL of seed oil, at different concentrations, was mixed with 2.2 mL DPPH. in ethanol (0.004%). The obtained mixture was thoroughly shaken and incubated for 60 min in the dark and at ambient temperature (± 20 °C); the absorbance was read at 515 nm. The radical scavenging activity was expressed as IC50 (mg/mL) which is the concentration providing the inhibition of 50% DPPH. radicals.

2.7.2 ABTS.+ Radical Scavenging Activity

The ABTS.+ Antiradical activity of Z. lotus seed oils was evaluated following the description of Zuleata et al. [22] on the basis of the capability of a sample to impede this radical (ABTS·+) in comparison with a reference antioxidant standard. First, the preparation of ABTS·+ radical was performed by the reaction of 25 mL of ABTS solution (7 mM) with 440 μl of potassium per sulphate (K2S2O8; 140 mM). The obtained mixture was not only kept at ambient temperature in the dark for 12–16 h, allowing the monocation radical (ABTS·+) to form, but also diluted with ethanol to acquire an absorbance of 0.700 at 734 nm. Subsequently, 2 mL of obtained ABTS·+ solution was mixed with 100 μL of each Z. lotus seed oil, at different concentrations, separately and the absorbance was measured after 6 min of mixing. Hence, the radical scavenging activity was expressed as inhibition concentration (IC50, mg/mL) which reduces 50% of free radicals present in the ABTS solution.

2.8 Statistical Analysis

The obtained results for at least three analyses for each sample are expressed as mean ± standard deviation. As for the assessment of the statistical analyses, it was realized using SPSS (version 18). The determination of the variance analyses was achieved through an ANOVA procedure. Duncan’s multiple range test was adopted to highlight significant differences (p < 0.05) between the means. To join Z. lotus origins in homogeneous groups, a cluster analysis was conducted taking into consideration the fatty acid composition and vitamin E composition [tocopherols (α, γ, δ) and tocotrienols (α, β, γ, δ)]. After performing a Hierarchical Cluster Analysis, using the nearest neighbor method and the squared Euclidean distance, the subjects were grouped. The total of clusters to retain was set up by the R-squared criteria, as explained by Maroco [23].

3 Results and Discussion

3.1 Oils Yield and Fatty Acids Composition

The harvest location significantly influenced the yield of the lipid fraction of Z. lotus seed. The obtained results showed that the percentage of oil extracted varied from 20.13 to 24.57% based on dry matter weight (Fig. 2).

Fig. 2
figure 2

Oil contents (%. v/m dry basis) of cold pressed Z. lotus seed oils from different origins. Nab, Zag, Sil, Sou, Kai, Meh, Kas, SiB, Sfa, Gaf, Gab and Toz: Oils extracted from Nabeul, Zaghouan, Siliana, Sousse, Mehdia, Kairouan, Kasserine, Sidi Bouzid, Sfax, Gabes, Gafsa and Tozeur, respectively. Each value is the mean of 3 independent trials ± SE

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Nonetheless, it is worthy to state that the highest and lowest oil yield values were found in samples collected from Sidi Bouzid and Siliana, respectively (p < 0.05). Nevertheless, no significant differences (p > 0.05) between samples collected from Sidi Bouzid and Kasserine were detected in terms of lipid amount.

The study conducted by El Aloui et al. [24] focused on the lipid fraction of Zizyphus jujube Mill. leaves from different locations in Tunisia. Their results showed differences in oil yields depending on the growing location. The highest yield was obtained for leaves collected in the region of Sfax and it did not exceed 10.31%. In the present study, the lowest oil seed yield was observed for the sample collected from Siliana and it was about twice higher than oil leaves content (20.13%). This difference is mainly attributed to the studied part of the plant.

The obtained oils were analyzed using GC-FID for the determination of their composition and results for fatty acids contents, total saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), and polyunsaturated fatty acids (PUFA) are summarized in Table 1.

Table 1 Fatty acid composition (%) of cold pressed Z. lotus seed oils from different origins
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Regardless of the location, MUFA (64.10–73.46%) predominated over SFA (6.14–17.96%) and PUFA (17.90–20.40%) due to the significant contribution of oleic acid. In all the samples, it was found that oleic acid was the predominant (60.45–70.36%), followed by linoleic acid (16.40–19.40%), two dominant acids accounting for 76.90–79.76% of the total fatty acids. The highest level in oleic acid was found in the sample harvested from Siliana followed by those from Zaghouan, Nabeul and Mehdia. Variations in oleic and linoleic acid contents observed in Z. lotus seed oil samples obtained from different Tunisian locations (Table 1) are probably related to region environmental interaction during the development and the maturity of the fruits. Furthermore, minor amounts of odd-chain fatty acids such as stearic, linolenic, and gadoleic acids were also observed in our study.

These results are in excellent agreement with those demonstrating that oleic acid is the most abundant fatty acid in Z. lotus seed oils [25]. Oleic acid was also found to be the most abundant acid in the lipid fraction of other parts of Z. Lotus. For instance, oils extracted from whole fruit [26] peel and pulp [27] were rich in C18:1. Oleic acid is characterized by its health benefits and its possible capacity to decrease blood pressure [28]. As a member of the family of main mono-unsaturated fatty acids of olive oil, oleic acid destroys the gene expression of breast cancer cells [29]. Hence, the edible oil industry’s main concern is the high quality of oleic vegetable oils. Indeed, since oil rich in unsaturated fatty acids are assumed to be advantageous agents, they are included into infant formula and several food products. Besides, they are available as nutraceutical supplements in many countries [30, 31]. Considering the elevated amount of unsaturated fatty acids, the seed oils of the twelve Z. lotus seeds under investigation are exceedingly advocated for human consumption, indicating a more beneficial fatty acid profile than other vegetable oils. Moreover, it is proven that trans fatty acids are likely to elevate LDL cholesterol and lower HDL cholesterol concentrations. It was demonstrated, however, that the analyzed oils are characterized by extremely low amounts in these fatty acids. Therefore, Z. lotus seed oil may be judged as a precious source of essential fatty acids.

The obtained results were similar to those obtained by Ghazghazi et al. (2014) [32] who studied the fatty acid composition of Ziziphus lotus L. fruits and they showed that oleic acid which was the dominant fatty acid presenting more than 88% of total fatty acids. Other studies focused on the identification and quantification of fatty acids from seed oils. Most of them indicated that seeds oil exhibit a common composition that includes both saturated and unsaturated fatty acids. Matthaus and Ozcan [33] studied the effect of habitat on the fatty acid composition of prickly pear seed oils. They reported that palmitic, oleic, and linoleic acids were found the major fatty acids. Their amounts ranged between 10.60 and 12.80; 13.00 and 23.50% and 49.30–62.10%, respectively.

A cluster method was used to classify the various Z. lotus seed oils according to their fatty acid compositions (Fig. 3).

Fig. 3
figure 3

Dendrogram of cold pressed Z. lotus seed oils from different origins according to their fatty acid compositions. Nab, Zag, Sil, Sou, Meh, Kai, Kas, SiB, Sfa, Gaf, Gab, and Toz: Oils extracted from Nabeul, Zaghouan, Siliana, Sousse, Mehdia, Kairouan, Kasserine, Sidi Bouzid, Sfax, Gabes, Gafsa and Tozeur, respectively

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Four clusters were found with an R-squared of 0.92. As far as the first cluster is concerned, it includes six regions, namely, Sidi Bouzid, Kasserine, Sfax, Gabes, Sousse, and Gafsa, on the basis of similarity in fatty acid composition. Concerning the second and third clusters, they included Tozeur and Kairouan, and Mehdia, Nabeul and Zaghouan, respectively. While the second cluster (Tozeur and Kairouan) demonstrated the highest levels of SFA and MUFA, the third cluster encompassing Mehdia, Nabeul and Zaghouan has the lowest levels of SFA and MUFA. Finally, the fourth cluster consists of Siliana region presenting the highest PUFA and MUFA levels.

3.2 Tocopherols Composition

As displayed in Table 2, the tocopherols composition of the twelve Tunisian Z. lotus seed oils was analyzed. Eight compounds were detected in almost all seed oils, comprising four tocopherols (α-, β-, δ- and γ) and four tocotrienols (α-, β-, δ-and γ). It is trusty to mention that all seed oils were richer in tocopherols than tocotrienols, with β-tocopherol as the most abundant, followed by δ -tocopherol and δ-tocotrienol.

Table 2 Tocopherol, tocotrienol and vitamin E amounts (mg/kg of oil) of cold pressed Z. lotus seed oils from different origins
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Generally, our findings are in good agreement with those reported by other studies found in the literature which have proven β-tocopherol as the most abundant tocopherol in Z. lotus seed oils [34]. However, α- and γ-tocopherols were only detected in lower amounts in all Z. lotus seed oils. Yet, Z. lotus seeds collected from Siliana and Mehdia, particularly comprised a considerable amount of total tocols and vitamin E, which were present in low amounts in all other samples collected from Gabes and Tozeur. However, there were no significant differences observed between all Z. lotus seed oils regarding δ-tocopherol amounts (p > 0.05). Z. lotus seed oils of the twelve Tunisian locations are likely to be utilized to postpone the body's aging process and to avoid the existence of many chronic diseases. Indeed, vitamin E is known for its numerous health benefits, given its power as a lipid-soluble antioxidant, and its ability to protect the human body's cells against the damage caused by free radicals.

After completing a dendrogram to the vitamin E amounts of the twelve seed oils samples used in the present investigation (Fig. 4), we detected four clusters with R-squared equal to 0.89, through which a substantial proportion of the total variation was maintained.

Fig. 4
figure 4

Dendrogram of cold pressed Z. lotus seed oils from different origins according to their vitamin E amounts. Nab, Zag, Sil, Sou, Meh, Kai, Kas, SiB, Sfa, Gaf, Gab and Toz: Oils extracted from Nabeul, Zaghouan, Siliana, Sousse, Mehdia, Kairouan, Kasserine, Sidi Bouzid, Sfax, Gabes, Gafsa and Tozeur, respectively

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Concerning the first identified cluster, it constituted seven Z. lotus seed oils, collected from Sidi Bouzid, Kasserine, Sfax, Kairouan, Sousse, Nabeul and Zaghouan regions, because all of them had similar tocopherols amounts. Oils extracted from seeds collected from Mehdia and Siliana were included in cluster 2 since both of them had relatively higher α- and β-tocopherols amounts. The Z. lotus seed oils of Gabes and Tozeur regions were included in cluster 3 due to their low α- and β-tocopherol amounts. The last cluster was represented by Gafsa region. This seed oil presented the lowest amount of δ-tocopherol.

3.3 Physical Properties

The physical properties of the twelve Z. lotus seed oils are presented in Table 3.

Table 3 Physical properties of cold pressed Z. lotus seed oils from different origins
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The dynamic viscosity varied from 67.23 to 82.45 Pa.s. The highest values were observed in the samples collected from Nabeul and Siliana. While the lowest was found in the sample of Gabes. Also, the findings revealed that there are no significant variations observed in the samples collected from Sidi Bouzid and Sfax (p > 0.05). In this context, all Z. lotus seed oils demonstrated Newtonian behavior characterized by constant dynamic viscosity, which was estimated from the plotting shear stress as function of shear rate [19]. These obtained data were similar to those of Pistacia lentiscus seed oils [35].

Regarding the interfacial tension, the values ranged from 13.79 to 17.02 mN/m and the highest value was observed in the sample collected in Mehdia region. However, there was no significant differences between samples collected from Sidi Bouzid, Kasserine, and Sfax (p > 0.05). Some authors declared that the interfacial tension depends on the chemical composition of studied oil like free fatty acids, tocopherols, phospholipids as well as the polyphenol contents [36]. Z. lotus seed oil is known to possess high levels of carotenoids, polyphenols and tocopherols [34].

Besides, other compounds, generated by several chemical reactions during the extraction, storage and processing of vegetable oils could affect the interfacial properties [19, 34]. For instance, it has been reported that the oxidation of frying oils led to the formation of aldehydes, ketones and alcohols responsible of a decrease of the interfacial properties [37]. The interfacial film is progressively reinforced by fractions of higher molecular weight compounds that will diffuse more slowly, and contribute to a further decrease in the interfacial tension [36].These properties are highly used in certain food applications such as the formulation of emulsions [38].

3.4 Antioxidant Activities

Table 4 shows the DPPH. and ABTS+ radical scavenging activities of the Z. lotus seed oil samples which are expressed by IC50 referring to the lowest concentration of antioxidants necessary for 50% of radicals inhibition. Thus, the lower the IC50 value the more reactive the studied oil.

Table 4 Inhibitory concentrations (IC50 (mg/mL)) of DPPH. and ABTS+ radical scavenging activities of cold pressed Z. lotus seed oils from different origins
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Hence, the present study reveals that the DPPH. and ABTS.+ radical scavenging activities of Z. lotus seed oils obtained from Siliana are significantly higher (2.87 and 4.08 mg/mL) than all other seed oil samples (p < 0.05). However, Z. lotus seed oils obtained from Gafsa, Gabes and Tozeur had a significantly higher (p < 0.05) value of IC50 than those of studied seed oil samples, indicating thus a lower antioxidant potential for oils from these locations (Gafsa, Gabes and Tozeur). Nevertheless, high negative correlations were noticed between IC50 of ABTS+ radical scavenging activity (r = − 0.903, p < 0.01), between IC50 of DPPH. radical scavenging activity (r = − 0.922, p < 0.01) and the total tocols amounts in all studied oils. Negative correlations between IC50 of ABTS.+ radical scavenging activity (r = − 0.918, p < 0.01), and between IC50 of DPPH radical scavenging activity (− 0.922, p < 0.01) and vitamin E contents were also observed. Indeed, vitamin E is a well-known lipid-soluble antioxidant conferring increased oxidative stability to vegetable oils, particularly those rich in unsaturated fatty acids [39]. Thus, higher contents of Vitamin E in oils entail higher antioxidant activity and consequently lower IC50. Negative correlations between vitamin E content and methods determining the antioxidant activity were observed in currant and gooseberry fruits [40]. However, it is worth mentioning that studies focusing on correlations between vitamin E and methods for the evaluation of the antioxidant activity from plants, fruits or extracts have been very rare.

The antioxidant potential is attributed to different chemical compounds whose the amounts could be further affected by many factors such as the cultivar type, plant part, maturity and harvest period and environmental conditions of the locality. Though, synergistic or antagonistic effects of these compounds play a crucial role in the resulting antioxidant properties [41].

4 Conclusions

The present study undertakes for the first time the investigation of Z. lotus seeds oil, collected from different locations, for their fatty acids and tocopherols profiles, as well as their antioxidant activities. The obtained results revealed that Z. lotus seed oils were characterized by oleic acid as the predominant fatty acid. Such data represent great economic benefits due to the numerous potential applications of this component in the food, cosmetic and pharmaceutical industries. Beta-tocopherol was the main component. However, different compositional ratios between locations were observed. Moreover, Z. lotus seed oil collected from different locations are endowed with a promising antioxidant activities against DPPH. and ABTS+ radicals. The use of this oil may also serve in improving human health and the prevention of chronic diseases.