Physico-Chemical and Phytochemical Characterization of Moroccan Wild Jujube “Zizyphus lotus (L.)” Fruit Crude Extract and Fractions

Wild jujube “Ziziphus lotus (L.) Desf.” belongs to the Rhamnaceae family and is a traditionally herbaceous medicinal plant. It is very common in arid and semi-arid regions and is currently used for its antidiabetic, sedative, analgesic, anti-inflammatory and hypoglycemic activities. The aim of the present work was to characterize the physico-chemical properties and the phytochemical profile of wild jujube sample collected from the Guercif region, in order to determine the polyphenolic compounds and the antioxidant ability Analyses were carried out directly after the harvest for the determination of pH, refractive index, total soluble solid (°Brix), dry matter, sugar/acidity, total sugars, reducing sugars, as well as lipid and protein content. Results showed that the investigated fruit is acidic (pH 4.9 ± 0.23) and rich in sugars (80.2 g/100 g ± 3.81). The GC-MS analysis of the fruit revealed a number of volatile compounds, as many as 97, belonging to different chemical classes. The HPLC-DAD-ESI/MS analysis showed the presence of a total of 20 polyphenolic compounds in both EtOAc and MeOH-water extracts. Among them, p-Hydroxybenzoic acid was the most abundant in the EtOAc extract (185.68 µg/100 mg ± 0.5) whereas Quercetin 3-O-rhamnoside-7-O-glucoside was found in higher amounts in the MeOH-water extract (25.40 µg/100 mg ± 0.5). These components have medical interest, notably for human nutrition, as well as health benefits and therapeutic effects. Therefore, Moroccan jujube “Zizyphus lotus (L.)” fruit may have potential industrial applications for food formulations.

Jujube fruits are spherical drupes with a size of a pupil, and are eaten at full maturity in October. Their taste evokes candied apple and their texture is similar to dates. They are marketed for human consumption as a fermented drink by mixing crushed fruits with water, and as flour after drying it [2].
This species is known worldwide for many different medical uses e.g. its antipyretic and antiviral properties [3,4]. In antiquity, Z. lotus was used for its emollient properties; the mixture of dried leaves and fruits was applied topically in the treatment of boils and throat and bronchopulmonary irritations [5]. In previous studies, it has been also reported that Z. lotus root bark has anti-inflammatory, analgesic, and antidiabetic activities [6][7][8].
These fruits are famous for their high biologically active material contents such as polyphenols, exhibiting antioxidant, antimicrobial, and immunomodulatory properties [9]. Some studies carried out on butanol extracts of Zizyphus spina-christi leaves showed that they are rich in saponins and improve the glucose-induced insulin release in type II diabetic rats [10]. Moreover, Z. lotus aqueous and organic extracts are characterized by the presence of flavonoids and tannins [11]. Particularly, cyclopeptide alkaloids, termed lotusines, and dammarane saponins have been isolated from this shrub, along with polyunsaturated fatty acids (oleic acid and linoleic acid), carbohydrates, and fibers [12][13][14][15][16][17][18][19].
A comparative study of two Zizyphus species, namely spina christi and lotus from Morocco, highlighted the presence of essential nutrients and phytochemical compounds in the fruits, pulps, seeds, and almonds. Flavonoid and anthocyanin contents were found to be highest in methanolic extract of the seeds and almonds of Z. lotus [16]. An earlier study reported the concentrations of different vitamins (vitamin A, C, and E) and fatty acids in root, stem, leaves, fruit pulp, and seed of Z. lotus L. The results achieved showed higher vitamin A and C contents in the fruit pulp and a richer source of linoleic acid (18:2n−6) than that in other parts of the plant [20].
To the best of our knowledge, there is a lack of information about the physico-chemical properties, as well as the phytochemical composition of such a shrub growing wild in Morocco; therefore, the present article will serve as an addition to the data that exists about Moroccan wild jujube. This work aims to evaluate the physico-chemical and bioactive properties of Z. lotus (L.) Desf fruit in order to potentially exploit them for industrial applications and incorporate them into food formulations to improve human health.

Results and Discussion
Nutritional quality of fruits is usually first characterized by physicochemical parameters, which can indicate a general estimate of the overall composition of their nutrient content. The most discriminating criteria are related to their sweet and sour taste but also their firmness. These are important factors in the sensory quality determination and the food products acceptability by consumers. In addition, the organoleptic quality is mainly determined by these chemical indicators. The phytochemical screening is required to detect the majority of compounds possessing essential biological activity.

Variation of Physicochemical Parameters
Jujube fruits are almost unknown to the majority of the Moroccan population, and there is lack of knowledge about their nutritional quality. Therefore, their physicochemical parameters have not been previously reported. The results achieved for refractive index, acidity, total soluble solid ( • Brix), sugar/acidity, dry matter, ash, total sugars, and reducing sugars are shown in Table 1. Considering the moisture content of the harvested fruits, a value of 12.9% was attained. This value does not fall within the range (58.34-76.5%) reported for other species of jujube (Zizyphus jujuba Mill.) present in China [21]. The moisture content is considered as a critical parameter to evaluate the quality of jujube fruits and can be considerably affected by genotype and culture conditions [22]. In general, the water content can be influenced by the age of the plant, the period of the vegetative cycle, and even genetic factors [20]. This variation may also be due to the different soil and climatic conditions and to the geographic distribution [23]. Other parameters studied by Chen et al. revealed the following values: ash content (0.8-1.1), total sugars (TS) (27.19-31.7), acidity (1.98-3.12), and sugar/ acidity (S/A) (8.8-14.75) [21]. Studies on the same genus have found the percentage of dry matter values between these intervals: 7.88-77.93, 18.99-74.08, and 2.26-3.01, respectively, for % reducing sugars (RS), water content, and ash content. The same collected fruits in India revealed the following values, namely 1.4-6.2, 81.6-83, and 5.83, respectively, for RS, water content, and ash content [24]. The jujube fruits have a high total soluble solid (TSS) value, which is due to their high sugar content. This agrees with the results found by Zia-Ul-Haq et al. [25]. However, our values were different from those found by Gao et al. [26], probably due to the different extraction conditions. High quantities of TS were recovered from freeze-dried berries of jujube (80.2% ± 3.81). Cultivars of the Chinese jujube (Zizyphus jujuba cv) have shown values varying between 69.2% and 85.3% [27]. The average value of TS in the fractions was 6.23% ± 0.75 and 76.5% ± 1.21, respectively in EtOAc and MeOH-H 2 O fractions. The difference noted can be explained by the different polarity (p < 0.05) and the number of extraction cycles. There was a large gap between the values found in this study and those reported in China and India. This indicates that Moroccan jujubes, particularly from the Guercif region, have the sweetest character among the other studied jujubes. The soluble sugars of the Chinese jujube in five cultivars were identified as fructose, glucose, rhamnose, sorbitol, and sucrose [27]. Fructose and glucose were identified as the main sugars while sorbitol was present in small amounts. Other studies have also shown that glucose and fructose are the main present sugars [28]. The content of sucrose was found to be lower than the content of fructose and glucose. In fact, sucrose is synthesized in the leaves and is hydrolyzed to glucose and fructose with the invertase enzyme once translocated to the flesh of the fruit, which is known to occur during the ripening of the fruits [27]. TS content variations can be attributed to several factors such as the age of the plant, the burn load, the stage of ripening, and the fruit physiological state during analysis. Other factors such as the length of time in the sun, the climate, and the availability of water can also affect sugar content [21,[29][30][31][32]. Indeed, a high concentration of sugars prevents bacterial proliferation in jams and jellies, and this contributes to the transformation of the studied fruit into several food products, in particular, jams, compotes, marmalades, and juice [24]. The average refractive index (RI) values were 2.8 ± 0.00 (EtOAc) and 2.7 ± 0.02 (MeOH-H 2 O). RI is influenced by the polarity of the solvents employed, as demonstrated by the ANOVA test (p < 0.05) [33]. Regarding protein content, a very low content was determined: 0.9 mg/g. The total concentrations of vitamin C were 34.5 mg/g ± 0.30, 12.7 mg/g ± 0.51, and 33.6 mg/g ± 0.45, respectively, for fresh fruits, EtOAc fraction, and MeOH-H 2 O fraction. The ANOVA test demonstrated a significant effect (p < 0.05) of solvent polarity on vitamin C content of jujubes.

Phytochemical Screening
The phytochemical screening of the wild jujube investigated in this work revealed the presence of different families of molecules, and results are presented in Table 2. Table 2. Phytochemicals detected in Z. lotus extracts.

Sugars
Deoxysugars Results showed the presence of flavonoids, tannins, anthocyanins, coumarins, saponosides, sterols, deoxysugars, and mucilages. These results are in agreement with previous studies carried on the same species [34,35] and in a similar species, Ziziphus mauritiana [36]. Concerning polyphenols, the best solvent extraction was MeOH-H 2 O. For anthocyanins, only traces were detected in the MeOH-H 2 O fraction whereas they were totally absent in the EtOAc one. These results are in agreement with previous findings reported by Tiwari et al. [37]. Total tannins were the most abundant in the MeOH-H 2 O, as demonstrated by other studies [38][39][40]. Sterols and steroids were present in higher amounts in the MeOH-H 2 O fraction, in agreement with data obtained by Tiwari et al. [37]. Unsaturated sterols and terpenes were present in the EtOAc extract [39]. Saponosides were detected in the crude and MeOH-H 2 O extracts [37,39]; the same results were attained for mucilages [41].

Polyphenols:
For each fraction, the average value of polyphenols was 3.0 ± 0.10 mg GAE/g dry matter (DM) and 4.8 ± 1.05 mg GAE/g DM, respectively, for EtOAc and MeOH. The statistical analysis of variance test revealed that there was a significant difference between the levels of polyphenols in the fractions according to the extraction solvent used (p < 0.05) [48]. In many published reports, the most suitable solvent for polyphenols extraction was represented by ethyl acetate [49,50]; in the present work, the MeOH-H 2 O mixture yielded the highest content of polyphenols, in agreement with other works [51,52]. As a consequence, the recovery of polyphenols from plant materials was indeed influenced by the solubility of the polyphenolic compounds in the extraction solvent.
Flavonoids: The average value of TFv was 2.0 ± 0.10 mg QE/g DM and 5.7 ± 0.05 mg QE/g DM, respectively, for jujube fractions of EtOAc and MeOH-H 2 O. The difference between the two results was significant (p < 0.05). A study conducted on the phytochemical composition of sea buckthorn exposed a level of flavonoids ranging from 2.18 to 6.6 mg QE/g DM [53]. A similar result was found by Vinatoru et al. [54] who extracted flavonoids from carrot powder using ultrasound extraction.
In another work carried out in Brazil, a research team found that acetonitrile could recover an optimal amount of Macela's flavonoids "Achyrolcine satureioides" [55].
Anthocyanins: The mean value of TA for each fraction was equal to 0.1 ± 0.00 for both EtOAc and MeOH-H 2 O fractions. The statistical analysis of variance test (ANOVA) showed that there was a significant difference depending on the diversity between all the anthocyanin contents (p < 0.05). In Mexico, pure methanol showed the greatest capacity to extract TA from the skin of Renealmia alpinia fruit compared to acetonitrile [56]. Similar results were reported by Ju and Howard [57] where MeOH 60% showed a greater capacity than ethanol 60% and water for the extraction of TA and phenolic compounds from grape skin. In addition, Khonkarn et al. [58] pointed out that MeOH was the solvent with the highest yield of anthocyanins from coconut, rambutan, and mangosteen barks. The difference between the levels of anthocyanins in each solvent can be explained by the stability of the anthocyanins, which can react with the solvent present in the mother solution, as reported by Benabdeljalil et al. [59]. It is also important to point out that, on a theoretical level, anthocyanins increase with the ripening of the fruits [60].
Tannins: The average values obtained for tannin concentrations of EtOAc and MeOH-H 2 O fractions were, respectively, equal to 5.2 ± 0.01 µg catechin equivalents (CE)/g DM and 11.1 ± 0.50 µg EC/g DM. The MeOH-H 2 O solvent mixture turned out to be the best solvent for extracting the maximum level of tannins. Mokhtarpour et al. reported that using 50% aqueous MeOH, revealed high tannin levels compared to that of other treatments [61]. Ghasemi et al. [62] evaluated pistachio shells and attained maximum tannin content in the MeOH fraction. In addition, in a Moroccan study on Acacia mollissima, the best yield of tannins was observed for the MeOH-H 2 O fraction [63].

Antioxidant Activity
The mean IC 50 of each solvent fraction studied showed that Z. lotus has the highest antioxidant power in the MeOH-H 2 O fraction (smallest IC 50 ). Analysis by the ANOVA test showed a very significant difference between the results of the two fractions (p < 0.01). These values were lower than the ones by Najjaa et al. [64] but were higher than the ones reported by Ghazghazi et al. for methanol extracts [65].
Methanol has a great reduction capability and powerful free radical scavenging activity [66]. In this regard, in some medicinal plants, it has been found that the DPPH radical scavenging effects of methanolic extracts are greater than that of aqueous extracts. The same authors reported that the exhibited antioxidant activity by methanol extracts is due to the presence of phenolic substances such as rosmarinic acid from Salvia officinalis and Origanum vulgare [67]. Numerous in vitro studies have confirmed the ability of Z. lotus to scavenge free radicals and prevent cell damage [65,68]. In addition, it has been shown that Z. lotus do present antioxidant compounds belonging to different classes such as phenolic acids, flavonoids, and saponins. These components prevent oxidative stress by reducing reactive oxygen species (ROS), and a regular intake of natural antioxidants can lower the risk of various diseases by reducing oxidative stress [64].

GC-MS Analyses
The attained results of the GC-MS analysis of the n-hexane fraction showed the presence of lipids, alkanes, alcohols, sterols, and terpenoids. The studied fruits revealed a high number (N = 97) of these compounds as reported in Figure 1 and Table 4 with a % of similarity ranging from 83 to 97%.
Molecules 2020, 25, x FOR PEER REVIEW 6 of 18 confirmed the ability of Z. lotus to scavenge free radicals and prevent cell damage [65,68]. In addition, it has been shown that Z. lotus do present antioxidant compounds belonging to different classes such as phenolic acids, flavonoids, and saponins. These components prevent oxidative stress by reducing reactive oxygen species (ROS), and a regular intake of natural antioxidants can lower the risk of various diseases by reducing oxidative stress [64].

GC-MS Analyses
The attained results of the GC-MS analysis of the n-hexane fraction showed the presence of lipids, alkanes, alcohols, sterols, and terpenoids. The studied fruits revealed a high number (N = 97) of these compounds as reported in Figure 1 and Table 4 with a % of similarity ranging from 83 to 97%.  Table 4.   Table 4.      Table 7). These results are consistent with those of other studies carried out on the same species [20,35]. The difference in compounds detection can be due to their solubility in the extraction solvent, the degree of polymerization of the phenols, and the interaction of the phenols with other constituents of the plant [51,69].  Table 7). These results are consistent with those of other studies carried out on the same species [20,35]. The difference in compounds detection can be due to their solubility in the extraction solvent, the degree of polymerization of the phenols, and the interaction of the phenols with other constituents of the plant [51,69].

Samples
Wild jujube fruits (Z. lotus) were collected for 4 months (May-June-July and August), all the harvest areas were between the longitude 3 • 38 13007 and the latitude 34 • 23 45746 of the Guercif region. The fruits were harvested at their physiological maturity in the early morning, transported in well-closed boxes, and stored at −10 • C in the Materials and Resources Valorization Laboratory, Faculty of Sciences and Technology of Tangier.

Extraction Method
Five grams of lyophilized powder was defatted three times in 50 mL of n-hexane, dried, and homogenized with 50 mL of two solvents with increased polarity viz. EtOAc/water or MeOH/water (80:20 v/v) [70]. Each fraction was extracted by sonication in an ultrasound bath (130 kHz) for 45 min. The temperature was controlled by using a thermometer and gel ice box. After centrifugation for 5 min, the supernatant was filtered through a paper filter, dried, reconstituted with MeOH/water 80:20, v/v, and then filtered through a 0.45 µm Acrodisc nylon membrane (Merck Life Science, Merck KGaA, Darmstadt, Germany) prior to LC-PDA-MS analysis.

Physico-Chemical Analyses
The AOAC international standard methods 16 were used to determine the physico-chemical characteristics: ashes and DM content. The pH measurement was performed using a digital pH meter. Titratable acidity (TAc) was measured by the titrimetric method. RI and %TSS were determined by a digital refractometer.

Phytochemical Screening
Phytochemical screening was performed according to the method of Trease, E. and Evans, W.C. (1987). The tests were based on visual observation of the color change or the formation of a precipitate after addition of specific reagents [71].

Determination of the Polyphenolic Content
3.6.1. Quantification of TPP, TFv, and TT in Z. lotus extracts TPP content was estimated using the Folin-Ciocalteu method [72] with a few modifications. Gallic acid was used as the standard (10/25/50/100/200 ppm). TPP content was recorded at 755 nm and was expressed as mg of GAE/g of DM. Flavonoids were quantified according to the method of Zhishen et al. [29], using AlCl 3 10%, NaOH 4%, and NaNO 2 5%. The absorbance was determined at 510 nm. A curve of catechin was also carried out. TFv content was expressed as mg QE/g DM. TT content was determined by the vanillin method of Julkunen-Tiitto and expressed as mg CE/g DW [30].

Quantification of Total Anthocyanin Content in Z. Lotus Extracts
TA content was estimated based on the differential pH (pH = 1 and pH = 4.5) by the method of Giusti and Wrolstad [31] with some modifications. Measurement was conducted at 510-700 nm in the UV-Vis spectrophotometer. The absorbance was calculated by the following formula: The total anthocyanin content was calculated by the molecular weight of pelargonidine-3-glucoside using the following equation: With M: molar mass of the pelargonidine-3-glucoside [g/mol], F: dilution factor, V: Volume of the extract (l), d: width of the Bowl (cm) and Q: quantity of homogenized fruit (g).

Determination of Antioxidant Activity
Free radical scavenging method DPPH(α, α-diphenyl-β-picrylhydrazyl) was carried out following the method described by Braca et al. [32] with minor modifications. Briefly, the fruit extracts were prepared from 25 µL of a methanolic solution and each of the pure compounds were added to 2 mL of DPPH (6.25 × 10 −5 M). After gentle mixing and incubation for 30 min at room temperature in the dark, allowing for reactions to take place, the absorbance values of the resulting solutions were measured at 517 nm using a blank containing the same concentration of DPPH radicals. Inhibitions of DPPH radical in percent (I%) were calculated as follow: where A blank is the absorbance of the control reaction (containing all reagents except the test compound) and A sample is the absorbance of the test compounds. The sample concentration provided 50% inhibition (half-maximal inhibitory concentration, IC 50 ) was calculated by plotting inhibition percentages against concentrations of the sample.

GC-MS
GC-MS analyses of volatile compounds was performed on an SLB-5ms column (30 m in length × 0.25 mm in diameter × 0.25 µm in thickness of film, Merck Life Science, Merck KGaA, Darmstadt, Germany). GC-MS detection involved an electron ionization system that utilized high energy electrons (70 eV). Pure helium gas (99.9%) was used as the carrier gas with a flow rate of 1 mL/min, and an injection volume of 0.7 µL was employed (a split ratio of 5:1). The initial temperature was set at 50 • C and increased up to 350 • C with an increase rate of 3 • C/min and holding time of about 5 min. Relative quantity of the chemical compounds present in each sample was expressed as percentage based on peak area produced in the chromatogram.
Compound identification was carried out by using complementary information coming from DAD, ESI-MS, and literature data.

Statistical Analysis
All analyses were performed in triplicate and data were reported as mean values and standard deviation (SD). The differences among treatments were detected by analysis of variance ANOVA (p < 0.05).

Conclusions
The present study shows that Z. lotus (L.) fruits possess interesting bioactive compounds as highlighted from the phytochemical profile. Results showed that the investigated fruits are acidic, rich in sugars, with a large array of volatile compounds belonging to different chemical classes. In addition, a total of 20 polyphenolic compounds were detected in both EtOAc and MeOH-water extracts. Such results demonstrate that Z. lotus (L.) is a potential source of bioactive compounds and can be potentially used for industrial applications in food formulations.