# Physical Fruit Traits, Proximate Composition, Antioxidant Activity, and Profiling of Fatty Acids and Minerals of Wild Jujube (Ziziphus lotus L. (Desf.)) Fruits from Eleven Moroccan Origins

Authors: Hasna Ait Bouzid; El Hassan Sakar; Laila Bijla; Mohamed Ibourki; Ahmed Zeroual; Jamila Gagour; Jamal Koubachi; Khalid Majourhat; Saïd Gharby
Journal: Journal of Food Quality
 (2022)

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## Abstract

This study aimed at investigating the effects of geographical origin on physical fruit traits, proximate composition, fatty acid, and elemental profiling of Moroccan wild jujube (Ziziphus lotus) fruits. Likewise, solvent effects on total phenolic content (TPC), total flavonoid content (TFC), tannin content, and antioxidant activity were also studied. Fruits were sampled from eleven sites where the species grows widely across Morocco (Tafraoute, Taroudant, Zagora, Rhamna, Beni Mellal, Zaouit Cheikh, Khenifra, B-Jaad, Lkhmissat, Sidi Hrazm, and Taounat). Physical fruit traits (length, width, and weight), proximate composition, and minerals were investigated. Fatty acid profiling of extracted oil was also evaluated. TPC and TFC as well as antioxidant activity (ABTS, DPPH, and FRAP) were determined on four different extracts, namely, ethanol extract (EE), methanol extract (ME), acetone extract (AE), and water extract (WE). Our outcomes revealed significant differences (p<0.05) among different origins for the measured fruit traits including ash (1.69–2.31%), moisture (2.56–5.69%), proteins (2.63–4.64%), oil (1.59–2.91%) and carbohydrates (86.82–89.20%). The most abundant minerals were K (548.93–828.44 mg/100 g) and Ca (137.50–211.78 mg/100 g). Major fatty acids were oleic acid (50.65 –60.25%), palmitic acid (12.03–18.67%), and linoleic acid (12.63–17.21%). Acetone performed better in terms of TPC (12.77–21.67 mg GAE/g DM), TFC (11.00–18.92 mg QE/g DM), and antioxidant activity using ABTS (22.96–29.32 mg TE/g DM), DPPH (27.96–96.64%), and FRAP (8.37–37.59 mg AAE/g DM). In conclusion, Z. lotus fruit could be considered as a source of carbohydrates and minerals and also natural antioxidants.


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## Body

## 1. Introduction

Plants are considered a source of medicines, especially for poor populations [1]. Recently, more attention has been devoted to the study of extracts from these plants in terms of their biological activities [2] and the search for natural additives or the development of new functional food products [3]. Thanks to its geographical diversity and well differentiated soils, Morocco has a very important ecological and floristic diversity [4]. Among more than 4,500 taxa of vascular plants, native or naturalized, 800 to 951 taxa are endemic [5]. Ziziphus lotus (Z. lotus) is a wild plant species from the Rhamnaceae family, which includes 135 to 179 species of Ziziphus [6]. In Arabic, it is commonly known as “Sedra,” and the fruit is a drupe called “Nbeg” [7]. This plant grows generally in arid and semi-arid regions around the world [8] in Asia, Africa, North America, South America, Oceania, and Europe [9]. Also known as jujube, it is a medicinal plant used in folk medicine as an anti-inflammatory, antidiabetic, antimicrobial, antipyretic, and antiviral drug [7]. Z. lotus contains many biologically active molecules, such as polyphenols (flavonoids and tannins), triterpenes, anthraquinones, alkaloids (cyclopeptides and isoquinolines), and saponosides [10, 11]. Many studies have shown its medicinal effects against several health issues such as digestive and urinary disorders, liver diseases, obesity, diabetes, skin infections, fever, diarrhea, and insomnia [12]. Besides these pharmacological properties, Z. lotus is also used in nutrition and cosmetics in various forms (honey, juice, tea, jam, loaf, vegetable oil, and cake). Its fruit (jujube); red and delicious is eaten fresh [13]. Z. lotus biological activity depend on plant part (leaves, fruits, pulps, roots, etc.) [14]. Jujube oil is composed of oleic (88.12%) and elaidic acid (7.88%) as major compounds [15], while seeds’ oil is rich in oleic and linoleic acids [14, 16]. Z. lotus leaves are rich in vitamin E with 155.71 mg/100 g, while their seeds are known for their high level of β-tocopherols with 130.47 mg/100 g [6]. Polyphenol family members such as flavonoids, phenolic acids, and other natural compounds are present in all parts of Z. lotus [17]. To the best of our knowledge, no detailed information is available about origin effect on Moroccan Z. lotus whole fruit composition. Hence, the originality of this paper, which had as objectives, (i) investigate physical fruit traits, proximate composition, antioxidant activity, fatty acid composition, and elemental profiling of wild jujube Ziziphus lotus L. (Desf.) fruits, (ii) to compare fruit nutritional value across the main Moroccan areas where Z. lotus grows, and (iii) to study the effect of solvent on phenolic, flavonoid content and antioxidant activity of Z. lotus fruit extracts.


## 2. Materials and Methods

### 2.1. Sample Collection and Preparation

This study was performed on fruit samples from main areas whereZ. lotus grows across Morocco. They consist of eleven different areas (Figure 1). In 2020, sampling was done at the jujube full ripening stage. Three samples of 1 kg were sampled from each location. After collection, the samples were air dried, and each of them was ground to a fine powder in an electric grinder and stored in bottles at room temperature.Figure 1
Map of Morocco showing sampling sites.


### 2.2. Physical Fruit Traits

Physical fruit traits consist of measuring dimensional traits (fruit length and fruit width) as well as fruit weight with aim of investigating differences amongZ. lotus from the different origins. To this end, from each above-described sample, sub-samples consisting each of thirty (30) dried fruits were considered from fruit physical trait measurement. Fruit axial dimensions (length and width) were determined with a digital caliper reading to an accuracy of 0.01 mm. Likewise, individual fruit weight was measured using an electronic balance with an accuracy of ± 0.001 g.


### 2.3. Proximate Composition

Oil content (OC) was determined using a Soxhlet extractor in which 15 g of fruit powder from each sample was placed and connected to a flask containing 150 mL ofn-hexane. The extraction was carried out for 6 hours at the boiling temperature of n-hexane. To determine the extract weight, the solvent (n-hexane) was evaporated using a rotary evaporator (R-200, Büchi, Zurich, Switzerland) at 40°C until complete removal. Subsequently, OC was determined gravimetrically according to the following formula and expressed as percent per dry matter (% DM):(1)OC%=weightofextractedoilweightoftotalsample×100.Protein content (PC) was determined using a LECO model elemental analyzer (LECO FP628, USA) by measuring total nitrogen, and PC was calculated by multiplying total nitrogen by 6.25. PC was expressed in percent per dry matter (% DM).Ash content (AC) was determined by incinerating fruit powders in a muffle furnace at 525°C for 4 hours. The moisture content (MC) was determined by drying the samples in an oven at 103°C until reaching a constant weight and MC was calculated using the following equation:(2)MC%=W2–W3W2–W1×100,where W1 represents the weight of container with lid, W2 is the weight of container with lid and sample before drying, and W3 is the weight of container with lid and sample after drying.Total carbohydrate content (CC) was calculated by subtracting the sum of moisture, ash, oil, and proteins from 100% according to Chouaibi et al. [18]:(3)carbohydratecontentCC,%=100%–%PC+%MC+%AC+%OC.


### 2.4. Mineral Profiling

Mineral content was determined accordingly as described in Ibourki et al. [19]. In brief, 1 g of the material was incinerated in a muffle furnace at 500°C for 2 hours and the obtained ash was treated with nitric acid (65%) and hydrochloric acid and then analyzed using a Perkin Elmer Model Optima 8000 DV spectrometer.


### 2.5. Fatty Acid Determination

To determine the fatty acid composition, 1 g of oil was mixed with 10 mL of methanol and 0.4 mL of 2 M potassium hydroxide prepared in methanol and allowed to reflux for 10 minutes. After cooling at room temperature, 2 mL of hexane was added to the mixture and washed with distilled water. The hexane layer containing fatty acid methyl esters (FAMEs) was collected and analyzed. The fatty acid composition was then determined as their corresponding methyl esters using gas chromatography (Agilent-6890) coupled to a flame ionization detector (GC-FID). The capillary column CP-Wax 52CB (30 m × 250μm i.d., 0.25 μm film thickness) was used. The carrier gas used was helium with a total gas flow rate of 1 mL/min. The initial oven temperature was set at 170 °C, and temperature gradient was 4°C/min to reach 230°C as the final temperature. Injector and detector temperature was set at 220°C. The injection volume was 2 μL in a split mode (split ratio 1 : 50). Results were expressed as a relative percentage of the area of each fatty acid methyl ester [20].


### 2.6. Total Phenolic Content, Flavonoid Content, and Antioxidant Activity

#### 2.6.1. Sample Preparation

The extraction of bioactive compounds was evaluated according to the method described by Yahia et al. [21] with slight modifications. In brief, 1 g of dry matter (DM) was macerated separately in 10 mL of various solvents (ethanol, methanol, acetone, and distilled water) for 24 h with stirring. Then obtained mixture was filtered through a Whatman filter and the extract solution was stored in dark bottles at 4°C until use. Four different solvents were used for extraction (ethanol 70%, methanol 70%, acetone 70%, and distilled water) to obtain ethanol extract (EE), methanol extract (ME), acetone extract (AE), and water extract (WE).


#### 2.6.2. Total Phenolic Content (TPC)

TPC was analyzed using the Folin–Ciocalteu reagent as described in Ismaili et al. [22]. 250 μL of diluted extract was mixed with 1.25 mL of Folin–Ciocalteu reagent (10%). 2 mL of sodium carbonate (Na2CO3 (7.5%)) was added, and the obtained mixture was placed into a water bath at 45°C for 30 min. The absorbance was measured using a UV-Vis spectrophotometer at 765 nm. A calibration curve was performed using gallic acid as a standard, and results were expressed as mg gallic acid equivalents per gram of dry matter (mg GAE/g DM).


#### 2.6.3. Total Flavonoid Content (TFC)

TFC was determined using the aluminum chloride as described by Rais et al. [23]. In a 10 mL volumetric flask, 1 mL of diluted extract was added, and then 4 mL of distilled water and 0.3 mL of sodium nitrite NaNO2 (5%) were added. After 5 min, 0.3 mL of aluminum chloride AlCl3 solution (10%) was added and left for 6 min, then 1 mL of NaOH (2 M) was added, and volume was completed to 10 mL with distilled water. After incubation at room temperature for 30 min, the absorbance was measured using an UV-Vis spectrophotometer at 415 nm. Quercetin was used as a standard for the calibration curve, and the results were expressed as mg quercetin equivalents per gram of dry matter (mg QE/g DM).


#### 2.6.4. Antioxidant Activity

(1) Free Radical Scavenging Activity by DPPH. DPPH (2-2-diphenyl-1-picrylhydrazyl) scavenging activity was measured as described by Nounah et al. [24] and Ismaili et al. [22]. 0.5 mL of DPPH ethanolic solution (0.2 mM) was added to 2.5 mL of diluted plant extract. The mixture was allowed to stand in dark at room temperature for 30 min, and then the absorbance at 517 nm was measured. The percentage of inhibition or radical scavenging activity (RSA) corresponds to the discoloration of the mixture and was calculated according to the following formula:(4)%RSA=AC–ASAC×100,where AC: absorbance of the control (0.5 mL DPPH and 2.5 mL ethanol) and AS: absorbance of the sample. A lower absorbance indicates a high radical scavenging activity.(2) Reducing/Antioxidant Power (FRAP). The ferric reducing antioxidant power (FRAP) determines the ability of extracts to reduce iron (III). It was measured according to Ben Yakoub et al. [25]. 0.5 mL of diluted extracts was mixed with 1.25 mL of a potassium phosphate buffer (0.2 M, pH 6.6) and 1.25 mL of 1% potassium ferricyanide K3 [Fe(CN)6]. The obtained mixture was allowed to react at 50°C for 20 min, then 1.25 mL of trichloroacetic acid CCl3COOH (10%) was added, and the mixture was centrifuged at 3000 rpm for 10 min. Finally, 1.25 mL of the supernatant was mixed with 1.25 mL of distilled water and 0.25 mL of 0.1% ferric chloride (FeCl3). The absorbance of each sample was measured at 700 nm. A calibration curve was prepared using ascorbic acid as a standard, and the results were expressed as mg ascorbic acid equivalents per gram of dry matter (mg AAE/g DM).(3) Trolox Equivalent Antioxidant Capacity. Trolox equivalent antioxidant capacity is based on reduction of ABTS+ radical. ABTS+ was prepared by reacting 10 mL of 2 mM ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) in H2O with 100 μL of 70 mM potassium persulphate. The obtained mixture was stored in dark at room temperature for 16 h [22]. The solution was diluted in ethanol to reach an absorbance of 0.700 ± 0.003 at 734 nm. 200 μL of the diluted sample was added to 2 mL of the ABTS+ solution and allowed to stand, for 10 minutes, before measuring the absorbance at 734 nm. Antioxidant activity was expressed as mg Trolox equivalents per gram of dry matter (TE/g DM) determined based on calibration curve using Trolox as a standard under the same conditions.


### 2.7. Statistical Analysis

All measurements and determinations were done at least in triplicate and then averaged. Quantitative differences were assessed using least significant difference (LSD) test. Mean values were expressed as mean ± standard deviation (SD). Differences were considered significant at 5% as a probability level. Correlation matrix and cluster analysis (CA) were carried out on data mean values. Principal component analysis (PCA) was performed on mean values to differentiate among various origins ofZ. lotus fruits using STATGRAPHICS package version XVII (Statpoint Technologies, Inc., Virginia, USA).


## 2.1. Sample Collection and Preparation

This study was performed on fruit samples from main areas whereZ. lotus grows across Morocco. They consist of eleven different areas (Figure 1). In 2020, sampling was done at the jujube full ripening stage. Three samples of 1 kg were sampled from each location. After collection, the samples were air dried, and each of them was ground to a fine powder in an electric grinder and stored in bottles at room temperature.Figure 1
Map of Morocco showing sampling sites.


## 2.2. Physical Fruit Traits

Physical fruit traits consist of measuring dimensional traits (fruit length and fruit width) as well as fruit weight with aim of investigating differences amongZ. lotus from the different origins. To this end, from each above-described sample, sub-samples consisting each of thirty (30) dried fruits were considered from fruit physical trait measurement. Fruit axial dimensions (length and width) were determined with a digital caliper reading to an accuracy of 0.01 mm. Likewise, individual fruit weight was measured using an electronic balance with an accuracy of ± 0.001 g.


## 2.3. Proximate Composition

Oil content (OC) was determined using a Soxhlet extractor in which 15 g of fruit powder from each sample was placed and connected to a flask containing 150 mL ofn-hexane. The extraction was carried out for 6 hours at the boiling temperature of n-hexane. To determine the extract weight, the solvent (n-hexane) was evaporated using a rotary evaporator (R-200, Büchi, Zurich, Switzerland) at 40°C until complete removal. Subsequently, OC was determined gravimetrically according to the following formula and expressed as percent per dry matter (% DM):(1)OC%=weightofextractedoilweightoftotalsample×100.Protein content (PC) was determined using a LECO model elemental analyzer (LECO FP628, USA) by measuring total nitrogen, and PC was calculated by multiplying total nitrogen by 6.25. PC was expressed in percent per dry matter (% DM).Ash content (AC) was determined by incinerating fruit powders in a muffle furnace at 525°C for 4 hours. The moisture content (MC) was determined by drying the samples in an oven at 103°C until reaching a constant weight and MC was calculated using the following equation:(2)MC%=W2–W3W2–W1×100,where W1 represents the weight of container with lid, W2 is the weight of container with lid and sample before drying, and W3 is the weight of container with lid and sample after drying.Total carbohydrate content (CC) was calculated by subtracting the sum of moisture, ash, oil, and proteins from 100% according to Chouaibi et al. [18]:(3)carbohydratecontentCC,%=100%–%PC+%MC+%AC+%OC.


## 2.4. Mineral Profiling

Mineral content was determined accordingly as described in Ibourki et al. [19]. In brief, 1 g of the material was incinerated in a muffle furnace at 500°C for 2 hours and the obtained ash was treated with nitric acid (65%) and hydrochloric acid and then analyzed using a Perkin Elmer Model Optima 8000 DV spectrometer.


## 2.5. Fatty Acid Determination

To determine the fatty acid composition, 1 g of oil was mixed with 10 mL of methanol and 0.4 mL of 2 M potassium hydroxide prepared in methanol and allowed to reflux for 10 minutes. After cooling at room temperature, 2 mL of hexane was added to the mixture and washed with distilled water. The hexane layer containing fatty acid methyl esters (FAMEs) was collected and analyzed. The fatty acid composition was then determined as their corresponding methyl esters using gas chromatography (Agilent-6890) coupled to a flame ionization detector (GC-FID). The capillary column CP-Wax 52CB (30 m × 250μm i.d., 0.25 μm film thickness) was used. The carrier gas used was helium with a total gas flow rate of 1 mL/min. The initial oven temperature was set at 170 °C, and temperature gradient was 4°C/min to reach 230°C as the final temperature. Injector and detector temperature was set at 220°C. The injection volume was 2 μL in a split mode (split ratio 1 : 50). Results were expressed as a relative percentage of the area of each fatty acid methyl ester [20].


## 2.6. Total Phenolic Content, Flavonoid Content, and Antioxidant Activity

### 2.6.1. Sample Preparation

The extraction of bioactive compounds was evaluated according to the method described by Yahia et al. [21] with slight modifications. In brief, 1 g of dry matter (DM) was macerated separately in 10 mL of various solvents (ethanol, methanol, acetone, and distilled water) for 24 h with stirring. Then obtained mixture was filtered through a Whatman filter and the extract solution was stored in dark bottles at 4°C until use. Four different solvents were used for extraction (ethanol 70%, methanol 70%, acetone 70%, and distilled water) to obtain ethanol extract (EE), methanol extract (ME), acetone extract (AE), and water extract (WE).


### 2.6.2. Total Phenolic Content (TPC)

TPC was analyzed using the Folin–Ciocalteu reagent as described in Ismaili et al. [22]. 250 μL of diluted extract was mixed with 1.25 mL of Folin–Ciocalteu reagent (10%). 2 mL of sodium carbonate (Na2CO3 (7.5%)) was added, and the obtained mixture was placed into a water bath at 45°C for 30 min. The absorbance was measured using a UV-Vis spectrophotometer at 765 nm. A calibration curve was performed using gallic acid as a standard, and results were expressed as mg gallic acid equivalents per gram of dry matter (mg GAE/g DM).


### 2.6.3. Total Flavonoid Content (TFC)

TFC was determined using the aluminum chloride as described by Rais et al. [23]. In a 10 mL volumetric flask, 1 mL of diluted extract was added, and then 4 mL of distilled water and 0.3 mL of sodium nitrite NaNO2 (5%) were added. After 5 min, 0.3 mL of aluminum chloride AlCl3 solution (10%) was added and left for 6 min, then 1 mL of NaOH (2 M) was added, and volume was completed to 10 mL with distilled water. After incubation at room temperature for 30 min, the absorbance was measured using an UV-Vis spectrophotometer at 415 nm. Quercetin was used as a standard for the calibration curve, and the results were expressed as mg quercetin equivalents per gram of dry matter (mg QE/g DM).


### 2.6.4. Antioxidant Activity

(1) Free Radical Scavenging Activity by DPPH. DPPH (2-2-diphenyl-1-picrylhydrazyl) scavenging activity was measured as described by Nounah et al. [24] and Ismaili et al. [22]. 0.5 mL of DPPH ethanolic solution (0.2 mM) was added to 2.5 mL of diluted plant extract. The mixture was allowed to stand in dark at room temperature for 30 min, and then the absorbance at 517 nm was measured. The percentage of inhibition or radical scavenging activity (RSA) corresponds to the discoloration of the mixture and was calculated according to the following formula:(4)%RSA=AC–ASAC×100,where AC: absorbance of the control (0.5 mL DPPH and 2.5 mL ethanol) and AS: absorbance of the sample. A lower absorbance indicates a high radical scavenging activity.(2) Reducing/Antioxidant Power (FRAP). The ferric reducing antioxidant power (FRAP) determines the ability of extracts to reduce iron (III). It was measured according to Ben Yakoub et al. [25]. 0.5 mL of diluted extracts was mixed with 1.25 mL of a potassium phosphate buffer (0.2 M, pH 6.6) and 1.25 mL of 1% potassium ferricyanide K3 [Fe(CN)6]. The obtained mixture was allowed to react at 50°C for 20 min, then 1.25 mL of trichloroacetic acid CCl3COOH (10%) was added, and the mixture was centrifuged at 3000 rpm for 10 min. Finally, 1.25 mL of the supernatant was mixed with 1.25 mL of distilled water and 0.25 mL of 0.1% ferric chloride (FeCl3). The absorbance of each sample was measured at 700 nm. A calibration curve was prepared using ascorbic acid as a standard, and the results were expressed as mg ascorbic acid equivalents per gram of dry matter (mg AAE/g DM).(3) Trolox Equivalent Antioxidant Capacity. Trolox equivalent antioxidant capacity is based on reduction of ABTS+ radical. ABTS+ was prepared by reacting 10 mL of 2 mM ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) in H2O with 100 μL of 70 mM potassium persulphate. The obtained mixture was stored in dark at room temperature for 16 h [22]. The solution was diluted in ethanol to reach an absorbance of 0.700 ± 0.003 at 734 nm. 200 μL of the diluted sample was added to 2 mL of the ABTS+ solution and allowed to stand, for 10 minutes, before measuring the absorbance at 734 nm. Antioxidant activity was expressed as mg Trolox equivalents per gram of dry matter (TE/g DM) determined based on calibration curve using Trolox as a standard under the same conditions.


## 2.6.1. Sample Preparation

The extraction of bioactive compounds was evaluated according to the method described by Yahia et al. [21] with slight modifications. In brief, 1 g of dry matter (DM) was macerated separately in 10 mL of various solvents (ethanol, methanol, acetone, and distilled water) for 24 h with stirring. Then obtained mixture was filtered through a Whatman filter and the extract solution was stored in dark bottles at 4°C until use. Four different solvents were used for extraction (ethanol 70%, methanol 70%, acetone 70%, and distilled water) to obtain ethanol extract (EE), methanol extract (ME), acetone extract (AE), and water extract (WE).


## 2.6.2. Total Phenolic Content (TPC)

TPC was analyzed using the Folin–Ciocalteu reagent as described in Ismaili et al. [22]. 250 μL of diluted extract was mixed with 1.25 mL of Folin–Ciocalteu reagent (10%). 2 mL of sodium carbonate (Na2CO3 (7.5%)) was added, and the obtained mixture was placed into a water bath at 45°C for 30 min. The absorbance was measured using a UV-Vis spectrophotometer at 765 nm. A calibration curve was performed using gallic acid as a standard, and results were expressed as mg gallic acid equivalents per gram of dry matter (mg GAE/g DM).


## 2.6.3. Total Flavonoid Content (TFC)

TFC was determined using the aluminum chloride as described by Rais et al. [23]. In a 10 mL volumetric flask, 1 mL of diluted extract was added, and then 4 mL of distilled water and 0.3 mL of sodium nitrite NaNO2 (5%) were added. After 5 min, 0.3 mL of aluminum chloride AlCl3 solution (10%) was added and left for 6 min, then 1 mL of NaOH (2 M) was added, and volume was completed to 10 mL with distilled water. After incubation at room temperature for 30 min, the absorbance was measured using an UV-Vis spectrophotometer at 415 nm. Quercetin was used as a standard for the calibration curve, and the results were expressed as mg quercetin equivalents per gram of dry matter (mg QE/g DM).


## 2.6.4. Antioxidant Activity

(1) Free Radical Scavenging Activity by DPPH. DPPH (2-2-diphenyl-1-picrylhydrazyl) scavenging activity was measured as described by Nounah et al. [24] and Ismaili et al. [22]. 0.5 mL of DPPH ethanolic solution (0.2 mM) was added to 2.5 mL of diluted plant extract. The mixture was allowed to stand in dark at room temperature for 30 min, and then the absorbance at 517 nm was measured. The percentage of inhibition or radical scavenging activity (RSA) corresponds to the discoloration of the mixture and was calculated according to the following formula:(4)%RSA=AC–ASAC×100,where AC: absorbance of the control (0.5 mL DPPH and 2.5 mL ethanol) and AS: absorbance of the sample. A lower absorbance indicates a high radical scavenging activity.(2) Reducing/Antioxidant Power (FRAP). The ferric reducing antioxidant power (FRAP) determines the ability of extracts to reduce iron (III). It was measured according to Ben Yakoub et al. [25]. 0.5 mL of diluted extracts was mixed with 1.25 mL of a potassium phosphate buffer (0.2 M, pH 6.6) and 1.25 mL of 1% potassium ferricyanide K3 [Fe(CN)6]. The obtained mixture was allowed to react at 50°C for 20 min, then 1.25 mL of trichloroacetic acid CCl3COOH (10%) was added, and the mixture was centrifuged at 3000 rpm for 10 min. Finally, 1.25 mL of the supernatant was mixed with 1.25 mL of distilled water and 0.25 mL of 0.1% ferric chloride (FeCl3). The absorbance of each sample was measured at 700 nm. A calibration curve was prepared using ascorbic acid as a standard, and the results were expressed as mg ascorbic acid equivalents per gram of dry matter (mg AAE/g DM).(3) Trolox Equivalent Antioxidant Capacity. Trolox equivalent antioxidant capacity is based on reduction of ABTS+ radical. ABTS+ was prepared by reacting 10 mL of 2 mM ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) in H2O with 100 μL of 70 mM potassium persulphate. The obtained mixture was stored in dark at room temperature for 16 h [22]. The solution was diluted in ethanol to reach an absorbance of 0.700 ± 0.003 at 734 nm. 200 μL of the diluted sample was added to 2 mL of the ABTS+ solution and allowed to stand, for 10 minutes, before measuring the absorbance at 734 nm. Antioxidant activity was expressed as mg Trolox equivalents per gram of dry matter (TE/g DM) determined based on calibration curve using Trolox as a standard under the same conditions.


## 2.7. Statistical Analysis

All measurements and determinations were done at least in triplicate and then averaged. Quantitative differences were assessed using least significant difference (LSD) test. Mean values were expressed as mean ± standard deviation (SD). Differences were considered significant at 5% as a probability level. Correlation matrix and cluster analysis (CA) were carried out on data mean values. Principal component analysis (PCA) was performed on mean values to differentiate among various origins ofZ. lotus fruits using STATGRAPHICS package version XVII (Statpoint Technologies, Inc., Virginia, USA).


## 3. Results

### 3.1. Physical Fruit Traits

Physical fruit trait is of great importance as they influence the consumer acceptance and fruit value on the market. The measured physical fruit traits are summarized in Table1. Fruits from Tafraout presented the highest size (14.47 ± 2.03 mm and 12.38 ± 1.37 mm for length and width, respectively). Fruits from Sidi Hrazm were marked by the smallest width (9.96 ± 0.71 mm) and the lowest weight (0.36 ± 0.07 g) while fruits of B-Jaad had the lowest score of length (10.74 ± 0.97 mm) and fruits of Beni Mellal displayed the greatest weight (0.76 ± 0.79 g).Table 1
Mean values of physical fruit traits ofZ. lotus fruits studied.
Origin
Length (mm)
Width (mm)
Weight (g)
Rhamna
11.43 ± 1.34a
11.03 ± 1.43a
0.49 ± 0.16a
Taroudant
13.06 ± 3.01a
12.12 ± 2.53a
0.67 ± 0.20a
Tafraout
14.47 ± 2.03a
12.38 ± 1.37a
0.75 ± 0.22a
Taounat
11.90 ± 1.15a
11.51 ± 1.22a
0.51 ± 0.13a
Zagora
11.28 ± 0.79a
11.15 ± 0.99a
0.52 ± 0.14a
Sidi Hrazm
10.89 ± 0.79a
9.96 ± 0.71a
0.36 ± 0.07a
Lkhmissat
12.92 ± 1.93a
11.56 ± 1.33a
0.50 ± 0.16a
B-Jaad
10.74 ± 0.97a
10.43 ± 0.80a
0.39 ± 0.13a
Beni Mellal
11.82 ± 2.58a
12.23 ± 1.24a
0.76 ± 0.30a
Khnifra
11.76 ± 0.95a
11,81 ± 0,89a
0.56 ± 0,03a
Zaouit Cheikh
11.85 ± 1.03a
11.59 ± 1.12a
0.51 ± 0.17a
Average
12.01 ± 2.01
11.43 ± 1.54
0.55 ± 0.21
Same letters within the same column indicate no significant differences (p<0.05).


### 3.2. Proximate Composition

Table2 shows the proximate composition of the studied Z. lotus fruits. The moisture content of the fruits from the different origins ranged from 2.56 ± 0.10% (Beni Mellal) to 5.69 ± 0.96% (Sidi Hrazm).Table 2
Proximate composition of the studiedZ. lotus fruits across 11 Moroccan origins (results are expressed as mean values ± standard deviation of percentage of moisture content (MC), ash content (AC), protein content (PC), oil content (OC), and carbohydrate content (CC)).
Origin/parameter
MC (%)
Ash (%)
PC (%)
OC (%)
CC (%)
Rhamna
2.70 ± 0.19a
2.01 ± 0.10a
3.64 ± 0.01f
2.90 ± 0.24a
88.86 ± 0.54a
Taroudant
3.20 ± 0.28ab
1.95 ± 0.09a
4.64 ± 0.01j
2.51 ± 0.43a
87.90 ± 0.34a
Tafraout
3.24 ± 0.50ab
1.83 ± 0.09a
4.03 ± 0.01g
2.38 ± 0.55a
88.51 ± 0.52a
Taounat
3.26 ± 0.69ab
1.95 ± 0.09a
2.76 ± 0.01b
2.82 ± 0.14a
89.20 ± 0.65a
Zagora
2.65 ± 0.30a
2.25 ± 0.05a
3.14 ± 0.02c
2.91 ± 0.91a
89.05 ± 0.76a
Sidi Hrazm
5.69 ± 0.96b
2.31 ± 0.11a
4.33 ± 0.01h
1.59 ± 0.34a
86.06 ± 0.32a
Lkhmissat
3.17 ± 0.21ab
2.02 ± 0.10a
4.50 ± 0.01i
2.69 ± 0.38a
87.43 ± 0.54a
B-Jaad
4.78 ± 0.76ab
1.69 ± 0.04a
3.43 ± 0.01e
2.04 ± 0.56a
88.04 ± 0.64a
Khnifra
2.63 ± 0.43a
1.79 ± 0.08a
2.76 ± 0.01b
2.19 ± 0.23a
88.56 ± 0.66a
Beni Mellal
2.56 ± 0.10a
2.31 ± 0.11a
3.37 ± 0.01d
2.75 ± 0.33a
86.82 ± 0.54a
Zaouit Cheikh
3.05 ± 0.54ab
1.78 ± 0.08a
2.63 ± 0.01a
2.25 ± 0.57a
88.46 ± 0.65a
Average
3.35 ± 1.09
2.06 ± 0.74
3.56 ± 0.70
2.45 ± 0.62
88.08 ± 1.10
Different letters within the same row indicate significant differences (p<0.05).Ash content is important to assess foods’ quality as it is linked to individual minerals. In our results, there were no significant differences among the studied eleven origins. The values varied from 1.69 ± 0.04% for B-Jaad to 2.31 ± 1.11% for Beni Mellal.Vegetable oil (VO) and protein are the most important components of seeds, and the economic value is related to oil content, followed by protein content [26]. In Z. lotus fruits, oil content was found to be low and ranged from 1.59 ± 0.34% (Sidi Hrazm) to 2.91 ± 0.91% (Zagora) and proteins are present in Z. lotus fruits in low amounts ranging from 2.63 ± 0.01% (Zaouit Cheikh) to 4.64 ± 0.01% (Taroudant), which were close to those obtained by El Maaiden et al. [9] (3.69 ± 0.10%) but higher than those found by El Cadi et al. [10] (0.09%).Carbohydrates are considered as a source of energy; in our results, they account for more than 87% of the constituents ofZ. lotus fruits and ranged from 86.06 ± 0.32 to 89.20 ± 0.65%.


### 3.3. Mineral Composition

Minerals are essential inorganic nutrients that our body needs for proper functioning, but the quantities supplied in should be well known [27]. They are usually divided into major minerals (macro-minerals) such as potassium, calcium, magnesium, and phosphorus and trace minerals (micro-minerals) like iron and zinc. These two groups of minerals are both of vital importance, but trace minerals are needed in smaller amounts than major minerals [28]. Ten minerals, namely, K, Ca, P, Mg, Na, Fe, Mn, Cu, Zn, and B, were analyzed in Z. lotus fruits in this study and the results are presented in Table 3.Table 3
Mean values ± standard deviation of mineral contents inZ. lotus fruits sampled from eleven Moroccan sites.
Element (mg/100 g)
Rhamna
Taroudant
Tafraout
Taounat
Zagora
Sidi Hrazm
Lkhmissat
B-Jaad
Khnifra
Beni Mellal
Zaouit Cheikh
Average
K
727.30 ± 3.85d
812.78 ± 10.84f
784.72 ± 7.63ef
721.91 ± 2.26cd
743.53 ± 0.60de
805.00 ± 9.74f
832.27 ± 17.80f
548.93 ± 7.26a
675.22 ± 0.97bc
828.44 ± 5.60f
628.33 ± 14.63b
737.13 ± 8.85
Ca
138.19 ± 0.45a
186.13 ± 1.95d
137.50 ± 0.18a
150.66 ± 1.45b
201.36 ± 1.28f
177.38 ± 0.46c
176.10 ± 1.01c
192.34 ± 0.01e
137.66 ± 0.82a
211.78 ± 0.74g
154.42 ± 1.46b
192.41 ± 2.64
P
90.62 ± 0.15e
87.51 ± 0.63d
72.46 ± 0.09c
73.96 ± 0.07c
100.74 ± 0.64f
103.45 ± 0.52g
100.30 ± 0.19f
88.73 ± 0.33de
57.14 ± 0.08a
87.33 ± 0.08d
62.63 ± 0.75b
84.08 ± 1.52
Mg
68.43 ± 0.92c
63.92 ± 0.22bc
54.40 ± 0.04a
57.00 ± 0.09ab
54.30 ± 5.45a
72.65 ± 0.01c
66.11 ± 0.37bc
71.26 ± 0.62c
52.66 ± 0.35a
72.34 ± 0.06c
58.02 ± 0.02ab
62.82 ± 0.78
Na
5.97 ± 0.08ab
7.40 ± 0.03ab
2.95 ± 0.02a
5.71 ± 0.03ab
3.66 ± 3.09ab
8.42 ± 0.00b
6.95 ± 0.08ab
4.50 ± 0.11ab
4.89 ± 0.11ab
3.78 ± 0.01ab
3.76 ± 0.05ab
5.27 ± 0.19
Fe
1.37 ± 0.02ab
2.17 ± 0.00ab
1.08 ± 0.00a
1.57 ± 0.01ab
1.14 ± 1.01ab
1.26 ± 0.04ab
2.35 ± 0.03ab
0.99 ± 0.02a
1.41 ± 0.05ab
2.84 ± 0.02b
0.84 ± 0.03a
1.54 ± 0.07
Mn
3.32 ± 0.02i
3.57 ± 0.01j
1.00 ± 0.00b
1.75 ± 0.01e
0.90 ± 0.00a
2.59 ± 0.02g
3.81 ± 0.02k
1.25 ± 0.00c
1.52 ± 0.02d
2.73 ± 0.02h
2.31 ± 0.02f
2.25 ± 0.10
Cu
0.69 ± 0.00a
0.59 ± 0.01a
1.18 ± 0.00a
1.86 ± 0.12a
1.24 ± 1.00a
1.42 ± 0.00a
0.58 ± 0.00a
3.35 ± 0.02b
0.35 ± 0.00a
0.51 ± 0.00a
0.35 ± 0.00a
2.63 ± 0.52
Zn
0.79 ± 0.00a
1.84 ± 0.01a
1.20 ± 0.00a
4.32 ± 0.01a
1.15 ± 1.00a
0.96 ± 0.00a
1.01 ± 0.00a
1.45 ± 0.02a
0.51 ± 0.01a
1.07 ± 0.00a
0.69 ± 0.00a
1.36 ± 0.10
B
1.11 ± 1.00a
1.49 ± 0.01a
1.06 ± 0.00a
1.22 ± 0.01a
0.77 ± 0.00a
1.16 ± 0.01a
1.41 ± 0.01a
0.76 ± 0.00a
0.92 ± 0.00a
1.02 ± 0.00a
0.70 ± 0.00a
1.05 ± 0.04
Different letters within the same row indicate significant differences (p<0.05).Z. lotus fruits from the different origins were dominated by K and Ca, which were abundant in fruits originated from Beni Mellal (828.44 ± 5.60 and 211.78 ± 0.74 mg/100 g, respectively) and their smallest concentrations were found in fruits collected from Khnifra (675.22 ± 0.97) and Tafraout (137.50 ± 0.18 mg/100 g), respectively. The remaining main minerals were P and Mg found at high concentrations in fruits from Sidi Hrazm (103.45 ± 0.52 and 72.65 ± 0.01 mg/100 g, respectively) and in small concentrations in fruits of Khnifra (57.14 ± 0.08 and 52.66 ± 0.35 mg/100 g, respectively). Na was also an important element in Z. lotus fruits, and it is found in abundance in fruits of Sidi Hrazm (8.42 ± 0.00 mg/100 g) and less in fruits of Tafraout (2.95 ± 0.02 mg/100 g). For trace elements (Table 3), namely, Fe, Mn, Cu, B, and Zn, they were also found in Z. lotus fruits with concentrations not exceeding 5 mg/100 g DM. Their highest amounts were 2.35 ± 0.03, 3.81 ± 0.02, 2.63 ± 0.52, 1.49 ± 0.01, and 4.32 ± 0.01 mg/100 g, respectively.


### 3.4. Fatty Acid Composition

VOs are food resources consumed by humans to satisfy their nutritional needs thanks to their profile of fatty acids (FAs), which are an essential indicator of the nutritional value of the oil [29–33].To determine this nutritional value ofZ. lotus oil, the composition of fatty acids was analyzed and the obtained results are shown in Table 4. As it can be seen in these outcomes, Z. lotus oil was dominated by MUFA ranging from 50.65 (Zaouit Cheikh) to 62.45% (Zagora). MUFA were followed by SFA, which varied between 21.80 (Taroudant) to 30.71% (Zaouit Cheikh), then finally PUFA representing small amounts, their level varied between 14.04 (Zagora) and 18.62% (Zaouit Cheikh). MUFA consisted mainly in oleic and palmitoleic acids. Oleic acid was the major fatty acid, and its values ranged from 50.65 (Zaouit Cheikh) to 60.25% (Zagora). The main saturated fatty acid of Z. lotus oil was palmitic acid (from 12.03% in Taroudant to 18.67% in Zaouit Cheikh), followed by stearic acid and then heneicosylic (heneicosanoic) acid and arachidic acid. Other saturated fatty acids were C8: 0, C10: 0, C12: 0, and C14: 0 detected in small quantities less than 1% of the total acids. Concerning PUFA, only linoleic and linolenic acids were detected with values varying from 12.63 (Zagora) to 17.21% (Zaouit Cheikh) and from 0.97 (Taroudant) to 2.15% (Tafraout), respectively.Table 4
Mean values ± SD (n = 3) of fatty acids in Z. lotus fruit oil of eleven Moroccan origins.
Fatty acid/origin
Rhamna
Taroudant
Tafraout
Taounat
Zagora
Sidi Hrazm
Lkhmissat
B-Jaad
Beni Mellal
Khnifra
Zaouit Cheikh
Average
Caprylic acid C8: 0
ND
ND
0.26 ± 0.01b
ND
0.27 ± 0.01b
0.24 ± 0.01b
ND
ND
0.09 ± 0.01a
ND
ND
0.21 ± 0.07
Capric acid C10: 0
ND
ND
0.92 ± 0.01d
0.40 ± 0.01b
0.80 ± 0.01c
0.40 ± 0.01b
ND
ND
0.32 ± 0.01a
ND
0.97 ± 0.01d
0.66 ± 0.27
Lauric acid C 12: 0
0.44 ± 0.01d
0.55 ± 0.01e
0.56 ± 0.01e
0.37 ± 0.01bc
0.42 ± 0.01cd
0.35 ± 0.01ab
0.47 ± 0.01d
ND
0.30 ± 0.01a
0.60 ± 0.01e
0.86 ± 0.01f
0.49 ± 0.15
Myristic acid C 14: 0
0.85 ± 0.01f
0.40 ± 0.01a
0.65 ± 0.01de
0.69 ± 0.01e
0.48 ± 0.01bc
0.65 ± 0.01de
0.62 ± 0.01d
0.53 ± 0.01c
0.50 ± 0.01dc
0.46 ± 0.01b
1.45 ± 0.01g
0.66 ± 0.27
Palmitic acid C 16: 0
16.57 ± 0.01h
12.03 ± 0.01a
14.38 ± 0.01g
14.19 ± 0.01e
13.79 ± 0.01d
13.63 ± 0.01c
14.30 ± 0.01f
13.63 ± 0.01c
13.32 ± 0.01b
13.35 ± 0.01b
18.67 ± 0.01i
14.35 ± 1.71
Palmitoleic acid C 16: 1
0.74 ± 0.01a
0.72 ± 0.01a
2.23 ± 0.01d
1.92 ± 0.01c
2.20 ± 0.01d
3.10 ± 0.01e
ND
1.96 ± 0.01c
1.65 ± 0.01b
ND
ND
1.81 ± 0.74
Stearic acid C 18:0
6.37 ± 0.01h
4.94 ± 0.01e
5.11 ± 0.01f
5.11 ± 0.01f
4.72 ± 0.01bc
4.77 ± 0.01c
4.68 ± 0.01ab
5.48 ± 0.01g
4.64 ± 0.01a
4.95 ± 0.01e
4.86 ± 0.01d
5.05 ± 0.47
Oleic acid C 18:1
55.24 ± 0.01b
59.15 ± 0.01i
57.32 ± 0.01e
57.19 ± 0.01d
60.25 ± 0.01k
56.81 ± 0.01c
58.66 ± 0.01h
58.32 ± 0.01f
58.5 ± 0.01g
59.64 ± 0.01j
50.65 ± 0.01a
57.43 ± 2.53
Linoleic acid C 18: 2
14.51 ± 0.01e
16.41 ± 0.01i
13.18 ± 0.01b
15.14 ± 0.01f
12.63 ± 0.01a
14.51 ± 0.01e
13.76 ± 0.01c
15.32 ± 0.01h
15.2 ± 0.01d
14.29 ± 0.01g
17.21 ± 0.01j
14.74 ± 1.27
Linolenic acid C 18: 3
1.25 ± 0.01c
0.97 ± 0.01a
2.15 ± 0.01g
1.46 ± 0.01d
1.41 ± 0.01d
1.90 ± 0.01f
1.29 ± 0.01c
1.57 ± 0.01e
1.30 ± 0.01c
1.10 ± 0.01b
1.41 ± 0.01d
1.43 ± 0.32
Arachidic acid C 20: 0
0.59 ± 0.01a
1.13 ± 0.01de
1.27 ± 0.01g
1.03 ± 0.01c
0.87 ± 0.01b
1.17 ± 0.01ef
1.09 ± 0.01d
0.98 ± 0.01c
1.21 ± 0.01f
1.73 ± 0.01i
1.37 ± 0.01h
1.13 ± 0.27
Heneicosylic acid C 21: 0
2.75 ± 0.01e
2.75 ± 0.01e
1.97 ± 0.01a
2.47 ± 0.01c
2.19 ± 0.01b
2.45 ± 0.01c
2.50 ± 0.01cd
2.20 ± 0.01b
2.98 ± 0.01f
3.07 ± 0.01g
2.53 ± 0.01d
2.53 ± 0.32
∑SFA∗
27.57
21.80
25.12
24.26
23.54
23.66
0
22.82
23.36
24.16
30.71
25.07
∑MUFA∗∗
55.98
59.87
59.55
59.11
62.45
59.91
0
60.28
60.15
59.64
50.65
59.42
∑PUFA∗∗∗
15.76
17.38
15.33
16.6
14.04
16.41
0
16.89
16.5
15.39
18.62
16.17
∗SFA: saturated fatty acids, ∗∗monounsaturated fatty acids, and ∗∗∗polyunsaturated fatty acids. Different letters within the same row indicate significant differences (p<0.05). ND = not detected.


### 3.5. Total Phenolic Content, Flavonoid Content, and Antioxidant Activity

Table5 shows the mean values ± standard deviation of total phenolic contents, flavonoid contents, and antioxidant activities of different extracts of Z. lotus.Table 5
Mean values ± standard deviation of total phenolic contents, flavonoid contents, and antioxidant activities of different extracts ofZ. lotus (AE: acetone extract, EE: ethanolic extract, ME: methanolic extract, and WE: water extract).
Solvent
Origin
TPC (mg GAE/g DM)
TFC (mg QE/g DM)
DPPH (%)
FRAP (mg AA/g DM)
ABTS (mg TE/g DM)
EE
Rhamna
8.54 ± 0.08a
7.19 ± 0.12d
38.48 ± 0.02k
4.27 ± 0.33a
9.03 ± 0.57de
Taroudant
7.01 ± 0.02a
5.48 ± 0.04a
18.76 ± 0.01a
3.96 ± 0.10a
2.2 ± 0.68a
Tafraout
8.48 ± 0.11a
6.07 ± 0.04b
32.07 ± 0.04h
8.77 ± 0.66b
11.73 ± 0.01e
Taounat
7.24 ± 6.11a
7.9 ± 0.05e
31.35 ± 0.02f
3.79 ± 0.02a
6.55 ± 0.01bcd
Zagora
8.22 ± 0.12a
6.66 ± 0.04c
38.00 ± 0.00j
4.76 ± 0.08a
8.18 ± 0.12ce
Sidi Hrazm
7.63 ± 0.14a
5.4 ± 0.08a
23.04 ± 0.00c
3.26 ± 0.39a
2.98 ± 1.53ab
Lkhmissat
3.56 ± 0.01a
5.60 ± 0.12ab
19.58 ± 0.01b
3.43 ± 0.46a
4.73 ± 0.53ac
B-Jaad
7.83 ± 0.24a
5.51 ± 0.08a
30.88 ± 0.01e
4.06 ± 0.39a
8.46 ± 0.30ce
Khnifra
8.17 ± 0.22a
7.48 ± 0.04de
23.99 ± 0.00d
3.97 ± 0.61a
8.25 ± 1.28ce
Beni Mellal
10.35 ± 0.26a
7.45 ± 0.08de
34.20 ± 0.01i
5.17 ± 0.01a
10.55 ± 0.42e
Zaouit Cheikh
9.64 ± 0.34a
7.01 ± 0.20cd
31.59 ± 0.04g
4.66 ± 0.23a
10.51 ± 0.34e
Average
7.88 ± 0.25
6.52 ± 0.09
29.26 ± 0.67
4.55 ± 0.15
7.56 ± 0.31
ME
Rhamna
9.88 ± 0.10d
7.54 ± 0.29e
47.83 ± 0.02k
9.99 ± 0.32e
22.80 ± 0.96d
Taroudant
8.03 ± 0.16ac
6.13 ± 0.12ac
14.63 ± 0.01c
3.96 ± 0.00a
7.33 ± 1.14ab
Tafraout
10.13 ± 0.02d
6.98 ± 0.24ce
37.13 ± 0.01i
9.73 ± 0.49e
20.19 ± 1.86cd
Taounat
8.97 ± 0.26ac
6.51 ± 0.16bcd
19.24 ± 0.01d
5.02 ± 0.01ab
9.18 ± 1.96ab
Zagora
9.04 ± 0.36bcd
7.19 ± 0.04de
20.19 ± 0.01e
5.71 ± 0.17bc
11.64 ± 0.57ac
Sidi Hrazm
7.54 ± 0.15ab
6.01 ± 0.20ab
11.65 ± 0.01a
4.30 ± 0.13a
2.70 ± 0.53a
Lkhmissat
6.64 ± 0.12a
5.37 ± 0.04a
12.87 ± 0.01b
4.53 ± 0.17a
5.91 ± 1.50ab
B-Jaad
7.61 ± 0.69ab
7.28 ± 0.08de
24.39 ± 0.01h
6.20 ± 0.07c
11.89 ± 3.00ac
Khnifra
9.35 ± 0.01cd
7.22 ± 0.01de
44.85 ± 0.01j
8.25 ± 0.08d
20.72 ± 1.75cd
Beni Mellal
8.69 ± 0.12bcd
7.22 ± 0.08de
20.73 ± 0.01f
5.78 ± 0.19bc
11.43 ± 1.43ac
Zaouit Cheikh
9.37 ± 0.33cd
7.72 ± 0.20e
23.98 ± 0.01g
6.68 ± 0.10c
13.13 ± 2.39bc
Average
8.65 ± 0.11
6.83 ± 0.07
25.22 ± 0.12
6.37 ± 0.21
12.44 ± 0.65
AE
Rhamna
14.09 ± 0.63a
12.10 ± 0.08a
65.19 ± 0.01h
10.0 ± 0.11ab
25.87 ± 0.12ab
Taroudant
12.77 ± 0.22a
11.00 ± 0.37a
27.96 ± 0.01a
8.37 ± 0.56a
22.96 ± 2.23a
Tafraout
18.04 ± 0.52b
15.60 ± 0.24c
49.87 ± 0.01e
11.3 ± 1.10bc
29.36 ± 0.10b
Taounat
20.27 ± 0.47bc
17.90 ± 0.04b
52.28 ± 0.01f
11.5 ± 0.19bc
29.36 ± 0.10b
Zagora
21.67 ± 0.85c
18.90 ± 0.16c
96.64 ± 0.04j
37.5 ± 0.39e
29.32 ± 0.04b
Sidi Hrazm
13.81 ± 0.32a
11.00 ± 0 0.29a
86.16 ± 0.04i
16.6 ± 0.20d
29.32 ± 0.13b
Lkhmissat
14.85 ± 0.32a
12.00 ± 0.29a
29.44 ± 0.01b
8.24 ± 0.60a
29.13 ± 0.40b
B-Jaad
20.95 ± 0.29c
17.40 ± 0.09c
65.19 ± 0.01h
12.5 ± 0.28bc
26.51 ± 0.80ab
Khnifra
20.57 ± 0.06bc
18.20 ± 0.29c
58.60 ± 0.01g
12.4 ± 0.32bc
29.36 ± 0.10b
Beni Mellal
21.50 ± 0.50c
18.30 ± 0.62c
48.92 ± 0.01d
13.0 ± 0.37c
29.29 ± 0.17b
Zaouit Cheikh
21.28 ± 0.30c
18.00 ± 0.04c
38.71 ± 0.01c
13.2 ± 0.60c
29.45 ± 0.04b
Average
18.16 ± 0.35
15.49 ± 0.31
56.26 ± 0.21
14.05 ± 0.79
28.17 ± 0.22
WE
Rhamna
13.63 ± 0.13ab
12.6 ± 0.33a
11.51 ± 0.01g
9.12 ± 0.22a
13.63 ± 0.58ac
Taroudant
11.43 ± 0.61ab
11.8 ± 0.33a
6.05 ± 0.01a
8.12 ± 0.10a
9.80 ± 1.07a
Tafraout
15.29 ± 0.17b
17.56 ± 1.24b
10.23 ± 0.01e
9.99 ± 0.61a
14.83 ± 2.64ac
Taounat
13.97 ± 2.05ab
8.68 ± 1.33a
7.91 ± 0.01d
8.75 ± 0.24a
15.84 ± 0.31ac
Zagora
14.42 ± 0.25ab
11.8 ± 0.83a
12.79 ± 0.01h
9.59 ± 0.18a
17.58 ± 1.96bc
Sidi Hrazm
11.20 ± 0.35a
9.74 ± 0.49a
6.40 ± 0.01b
9.31 ± 0.54a
11.19 ± 0.17ab
Lkhmissat
11.08 ± 0.15a
12.9 ± 0.16a
12.91 ± 0.01i
7.29 ± 2.12a
10.75 ± 0.98ab
B-Jaad
12.77 ± 0.51ab
12.6 ± 0.66a
17.79 ± 0.01k
9.46 ± 0.15a
18.31 ± 1.92c
Khnifra
11.81 ± 0.31ab
13.0 ± 1.41a
11.28 ± 0.01f
9.77 ± 0.10a
19.13 ± 0.22c
Beni Mellal
13.39 ± 0.55ab
9.15 ± 0.01a
7.67 ± 0.01c
8.63 ± 0.69a
13.47 ± 0.08ac
Zaouit Cheikh
10.90 ± 0.25a
9.68 ± 0.41a
14.07 ± 0.01j
9.07 ± 0.52a
16.22 ± 0.31ac
Average
12.71 ± 0.16
11.77 ± 0.25
10.78 ± 0.35
9.01 ± 0.11
14.61 ± 0.33
Different letters within the same column, for each solvent, indicate significant differences (p<0.05).


#### 3.5.1. Total Phenolic Content

Phenols are gaining increasing importance, especially thanks to their beneficial effects on health [34]. Their role as natural antioxidants is attracting more and more interest in the prevention and treatment of cancer and inflammatory, cardiovascular, and neurodegenerative diseases [35].TPC was determined, and the obtained results are summarized in Table5. TPC levels varied considerably from a solvent to another and also between the different origins of Z. lotus. Acetone extracts exhibit the highest values of TPC regardless of origins. Fruits collected from Zagora presented the highest TPC in acetone extract (21.67 ± 0.85 mg GAE/g DM), while extracts of fruits from Taroudant displayed the lowest TPC value (12.77 ± 0.22 mg GAE/g DM). However, the fruits of Tafraout had the greatest content of TPC in both water and methanol extracts (15.29 ± 0.17 and 10.13 ± 0.02 mg GAE/g DM), while fruits of Zaouit Cheikh and Lkhmissat contained the lowest TPC content (10.90 ± 0.25 and 6.64 ± 0.12 mg GAE/g DM, respectively). For ethanol extracts, the highest TPC was observed in Khnifra (10.35 ± 0.26 mg GAE/g DM) and the lowest value was observed for Lkhmissat (3.56 ± 0.01 mg GAE/g DM).


#### 3.5.2. Total Flavonoid Content

Flavonoids, a group of natural substances with variable phenolic structures, are found in fruits, vegetables, and various plant parts including grains, bark, roots, stems, flowers, and so on. They are known for their nutraceutical, pharmaceutical, medicinal, and cosmetic properties [36].Table5 presents TFC in the studied eleven fruits of Z. lotus with different solvents. As for TPC, acetone extracts were the richest in TFC, followed by water extracts, and then methanol and ethanol extracts, respectively. Fruits from Zagora contained the highest TFC in acetone extracts (18.92 ± 0.16 mg QE/g DM), while fruits of Taroudant had the lowest TFC (11.07 ± 0.37 mg QE/g DM). Water extracted more flavonoids in fruits of Tafraout (17.56 ± 1.24 mg QE/g DM) but less in fruits of Taounat (8.68 ± 1.33 mg QE/g DM). In ethanol extracts, fruits collected from Beni Mellal had the highest TFC (7.48 ± 0.04 mg QE/g DM) and Sidi Hrazm fruits recorded the lowest value (5.40 ± 0.08 mg QE/g DM).


#### 3.5.3. Antioxidant Activity

Variability of antioxidants and their properties requires the use of more than one method to assess the antioxidant activity of extracts. In order to study antioxidant activity of different extracts, three different techniques were used (DPPH, FRAP, and ABTS assay). DPPH assay measures the total antioxidant capacity (TAC) of compounds that are able to transfer hydrogen atoms, similar to ABTS, but it includes the action of polar and non-polar antioxidants, while FRAP assay measures the capacity to reduce iron ion Fe3+ to Fe2+ [8].The results of antioxidant activity by DPPH, FRAP, and ABTS of different fruits in different extracts are shown in Table5. The acetone extracts were more reductive than methanol, ethanol, and water extracts, respectively. Contrary to phenolic compounds, water extracts presented the lowest antioxidant activity. The percentages of reduction for acetone extracts reached more than 90% of DPPH radicals (Zagora) and more than 35% for fruits from the remaining origins. Methanol extracts scavenged between 11 and 47% of radicals. Ethanol extract percentages ranged from 19 to 38%, while water extract percentages of scavenging did not exceed 20% for fruits from all origins. El Maaiden et al. [8] reported that water extracts of Z. lotus whole fruits scavenged 61.91 ± 0.85% of DDPH radicals.Regarding antioxidant activity measured via FRAP, as observed for TPC and TFC, acetone extracts were more reductive than water, methanol, and ethanol extracts. With respect to origins, Zagora fruits had the highest power of reducing iron (37.59 ± 0.39 mg AAE/g DM) and fruits of Lkhmissat presented the lowest activity (8.24 ± 0.60 mg AAE/g DM). Water extracts presented similar activities for all origins (8.12 ± 0.10–9.99 ± 0.61 mg AAE/g DM). Methanol extract activities ranged from 3.96 ± 0.00 to 9.99 ± 0.32 mg AAE/g DM, and ethanol extracts had low activities (3.26 ± 0.39–8.77 ± 0.66 mg AAE/g DM).Antioxidant activity ofZ. lotus fruits using ABTS+ radical revealed that the highest antioxidant activity was found in acetone extracts (more than 22.00 mg TE/g DM) with no significant differences between different origins. The lowest activity was found in ethanol extracts (2.21 ± 0.08–11.86 ± 0.10 mg TE/g DM). Important variations were found for ABTS+ antioxidant activities for both water (9.80 ± 1.07–19.13 ± 0.22 mg TE/g DM) and methanol extracts (7.33 ± 1.14–22.80 ± 0.96 mg TE/g DM). Fruits from Taroudant were found to be the less active among the eleven origins, since they presented the lowest activities with different techniques (ABTS, DPPH, and FRAP) and for the four extraction solvents.


### 3.6. Principal Component Analysis

In this work, PCA was used as a multivariate statistical analysis with the aim to discriminate among various origins (sites). The first three components were retained since they allowed explaining over 69% of the total data variance. As it can be seen in Figure2(a), the eleven origins were separated via the first two components accounting for more than 54%. Different studied origins were separated according to PC1 (34.07%), PC2 (20.11%), and PC3 (15.01%) as evidenced in Figures 2(b)–2(d). These three components seem to be related to environmental variations among sampling sites including edaphic differences as well as climatic conditions (temperature and rainfall). Taroudant, Lkhmissat, Beni Mellal, andSidi Hrazm interacted with high records of minerals, which may be ascribed to soil fertility of these sites. Zaouit Cheikh was marked by high levels of PUFA and SFA. Likewise, Beni Mellal was associated to greater contents of OC, MUFA, and AC. Tafraout, Taroudant, and Beni Mellal were linked to greater values of physical fruit traits including fruit length (FL), fruit width (FW), and fruit weight (Fwe). The highest values of antioxidant capacity (FRAP and ABTS), TPC, and TFC were found in samples from Zagora and Tafraout. These outcomes seemed to be linked to water stress (low rainfall in these sites, Table 6).Figure 2
Principal component (PC) projections for the first three PCs that most impact influenced fruit traits. Blue segments represent dependent variables, while points plotted are mean values. FWe = weight, FL = fruit length, KW = fruit width, OC = oil content, CC = carbohydrates content, PC = protein content, AC = ash content, and MC = moisture content.
(a)
(b)
(c)
(d)Table 6
Geographical and climatic data for the sampling agro-ecological areas.
Site
Geographical coordinates
Altitude (m.a.s.l.)
Temperature (°C)
Rainfall (mm)
T
Min
T
max
Rhamna
32°46′48″N, 85°57′00″W
486
6
38
600.2
Taroudant
30°28′13″N, 8°52′37″W
237
3
43
272.0
Tafraout
29°43′28″N, 8°58′28″W
1 022
3
30
223.0
Taounat
34°32′11″N, 4°38′24″W
600
10
37
472.0
Zagora
30°19′56″ N, 5°50′18″W
733
4
42
56.0
Sidi Hrazm
31°1′43″N, 4°52′48″W
268
6
36
468.2
Lkhmissat
33°49′26″N, 6°3′58″W
409
5
33
594.4
B-Jaad
32°46′10″N, 6°23′49″W
642
4
35
357.5
Beni Mellal
32°20′14″N, 6°20′59″W
472
6
39
493.0
Khnifra
32°56′20″N, 5°40′6″W
860
4
35
564.0
Zaouit Cheikh
32°39′15″N, 5°55′17″W
667
6
39
477.0


### 3.7. Cluster Analysis

Cluster analysis (CA) was carried out to evaluate the connection among geographical origins. As it can be seen in Figure3, several clusters were extracted from our results confirming important variations among the 11 geographical origins in terms of fruit properties including physical fruit traits, proximate composition, profiling of minerals and fatty acids, and antioxidant capacity. A total of 11 clusters were highlighted. Both B-Jaad and Sidi Hrazm formed the first cluster with the highest similarity (higher Euclidean distance) as compared to the remaining origins. The smallest Euclidean distance (less than 10) was found for the cluster formed by Taroudant and Lkhmissat.Figure 3
Cluster analysis performed on mean values in theZ. lotus fruit samples collected from eleven Moroccan origins. Distance (Y axis) is the Euclidean distance among fruit samples based on the investigated fruit traits.


### 3.8. Correlation Study

Pearson correlations were performed on mean values to analyze different associations among studied dependent variables (Figure4). Based on our outcomes, important correlations were highlighted. Axial dimensions together with fruit weight (FL, FW, and FWe) were positively linked to each other. Also, FRAP, ABTS, DPPH, TPC, and TFC were positively and strongly associated to each other. The most relevant positive remaining correlations were K to (AC and PC), P to (AC and PC), Na to (PC, Fe to K, and Mn), Mn to (Fe and Na), Cu to MC, and B to (Mn, Fe, Na, K, and PC). On the contrary, there were important negative correlations. Among them, MUFA to (SFA and PUFA), MC to (OC, FW, and FWe), Mg to CC, both FW and FWe to (Mg and P), TPC to (B, Mn, Na, and PC), TFC to (B, Mn, Na, Mg, P, and PC), B to (DPPH and FRAP), and ABTS to (B, Mn, Na, P, and PC). The remaining correlations were low or insignificant.Figure 4
Coefficients of correlations (Pearson correlation) among studied dependent variables. FWe = weight, FL = fruit length, KW = fruit width, OC = oil content, CC = carbohydrates content, PC = protein content, AC = ash content, and MC = moisture content. SFA = saturated fatty acids, MUFA = monounsaturated fatty acids, and PUFA = polyunsaturated fatty acids.


## 3.1. Physical Fruit Traits

Physical fruit trait is of great importance as they influence the consumer acceptance and fruit value on the market. The measured physical fruit traits are summarized in Table1. Fruits from Tafraout presented the highest size (14.47 ± 2.03 mm and 12.38 ± 1.37 mm for length and width, respectively). Fruits from Sidi Hrazm were marked by the smallest width (9.96 ± 0.71 mm) and the lowest weight (0.36 ± 0.07 g) while fruits of B-Jaad had the lowest score of length (10.74 ± 0.97 mm) and fruits of Beni Mellal displayed the greatest weight (0.76 ± 0.79 g).Table 1
Mean values of physical fruit traits ofZ. lotus fruits studied.
Origin
Length (mm)
Width (mm)
Weight (g)
Rhamna
11.43 ± 1.34a
11.03 ± 1.43a
0.49 ± 0.16a
Taroudant
13.06 ± 3.01a
12.12 ± 2.53a
0.67 ± 0.20a
Tafraout
14.47 ± 2.03a
12.38 ± 1.37a
0.75 ± 0.22a
Taounat
11.90 ± 1.15a
11.51 ± 1.22a
0.51 ± 0.13a
Zagora
11.28 ± 0.79a
11.15 ± 0.99a
0.52 ± 0.14a
Sidi Hrazm
10.89 ± 0.79a
9.96 ± 0.71a
0.36 ± 0.07a
Lkhmissat
12.92 ± 1.93a
11.56 ± 1.33a
0.50 ± 0.16a
B-Jaad
10.74 ± 0.97a
10.43 ± 0.80a
0.39 ± 0.13a
Beni Mellal
11.82 ± 2.58a
12.23 ± 1.24a
0.76 ± 0.30a
Khnifra
11.76 ± 0.95a
11,81 ± 0,89a
0.56 ± 0,03a
Zaouit Cheikh
11.85 ± 1.03a
11.59 ± 1.12a
0.51 ± 0.17a
Average
12.01 ± 2.01
11.43 ± 1.54
0.55 ± 0.21
Same letters within the same column indicate no significant differences (p<0.05).


## 3.2. Proximate Composition

Table2 shows the proximate composition of the studied Z. lotus fruits. The moisture content of the fruits from the different origins ranged from 2.56 ± 0.10% (Beni Mellal) to 5.69 ± 0.96% (Sidi Hrazm).Table 2
Proximate composition of the studiedZ. lotus fruits across 11 Moroccan origins (results are expressed as mean values ± standard deviation of percentage of moisture content (MC), ash content (AC), protein content (PC), oil content (OC), and carbohydrate content (CC)).
Origin/parameter
MC (%)
Ash (%)
PC (%)
OC (%)
CC (%)
Rhamna
2.70 ± 0.19a
2.01 ± 0.10a
3.64 ± 0.01f
2.90 ± 0.24a
88.86 ± 0.54a
Taroudant
3.20 ± 0.28ab
1.95 ± 0.09a
4.64 ± 0.01j
2.51 ± 0.43a
87.90 ± 0.34a
Tafraout
3.24 ± 0.50ab
1.83 ± 0.09a
4.03 ± 0.01g
2.38 ± 0.55a
88.51 ± 0.52a
Taounat
3.26 ± 0.69ab
1.95 ± 0.09a
2.76 ± 0.01b
2.82 ± 0.14a
89.20 ± 0.65a
Zagora
2.65 ± 0.30a
2.25 ± 0.05a
3.14 ± 0.02c
2.91 ± 0.91a
89.05 ± 0.76a
Sidi Hrazm
5.69 ± 0.96b
2.31 ± 0.11a
4.33 ± 0.01h
1.59 ± 0.34a
86.06 ± 0.32a
Lkhmissat
3.17 ± 0.21ab
2.02 ± 0.10a
4.50 ± 0.01i
2.69 ± 0.38a
87.43 ± 0.54a
B-Jaad
4.78 ± 0.76ab
1.69 ± 0.04a
3.43 ± 0.01e
2.04 ± 0.56a
88.04 ± 0.64a
Khnifra
2.63 ± 0.43a
1.79 ± 0.08a
2.76 ± 0.01b
2.19 ± 0.23a
88.56 ± 0.66a
Beni Mellal
2.56 ± 0.10a
2.31 ± 0.11a
3.37 ± 0.01d
2.75 ± 0.33a
86.82 ± 0.54a
Zaouit Cheikh
3.05 ± 0.54ab
1.78 ± 0.08a
2.63 ± 0.01a
2.25 ± 0.57a
88.46 ± 0.65a
Average
3.35 ± 1.09
2.06 ± 0.74
3.56 ± 0.70
2.45 ± 0.62
88.08 ± 1.10
Different letters within the same row indicate significant differences (p<0.05).Ash content is important to assess foods’ quality as it is linked to individual minerals. In our results, there were no significant differences among the studied eleven origins. The values varied from 1.69 ± 0.04% for B-Jaad to 2.31 ± 1.11% for Beni Mellal.Vegetable oil (VO) and protein are the most important components of seeds, and the economic value is related to oil content, followed by protein content [26]. In Z. lotus fruits, oil content was found to be low and ranged from 1.59 ± 0.34% (Sidi Hrazm) to 2.91 ± 0.91% (Zagora) and proteins are present in Z. lotus fruits in low amounts ranging from 2.63 ± 0.01% (Zaouit Cheikh) to 4.64 ± 0.01% (Taroudant), which were close to those obtained by El Maaiden et al. [9] (3.69 ± 0.10%) but higher than those found by El Cadi et al. [10] (0.09%).Carbohydrates are considered as a source of energy; in our results, they account for more than 87% of the constituents ofZ. lotus fruits and ranged from 86.06 ± 0.32 to 89.20 ± 0.65%.


## 3.3. Mineral Composition

Minerals are essential inorganic nutrients that our body needs for proper functioning, but the quantities supplied in should be well known [27]. They are usually divided into major minerals (macro-minerals) such as potassium, calcium, magnesium, and phosphorus and trace minerals (micro-minerals) like iron and zinc. These two groups of minerals are both of vital importance, but trace minerals are needed in smaller amounts than major minerals [28]. Ten minerals, namely, K, Ca, P, Mg, Na, Fe, Mn, Cu, Zn, and B, were analyzed in Z. lotus fruits in this study and the results are presented in Table 3.Table 3
Mean values ± standard deviation of mineral contents inZ. lotus fruits sampled from eleven Moroccan sites.
Element (mg/100 g)
Rhamna
Taroudant
Tafraout
Taounat
Zagora
Sidi Hrazm
Lkhmissat
B-Jaad
Khnifra
Beni Mellal
Zaouit Cheikh
Average
K
727.30 ± 3.85d
812.78 ± 10.84f
784.72 ± 7.63ef
721.91 ± 2.26cd
743.53 ± 0.60de
805.00 ± 9.74f
832.27 ± 17.80f
548.93 ± 7.26a
675.22 ± 0.97bc
828.44 ± 5.60f
628.33 ± 14.63b
737.13 ± 8.85
Ca
138.19 ± 0.45a
186.13 ± 1.95d
137.50 ± 0.18a
150.66 ± 1.45b
201.36 ± 1.28f
177.38 ± 0.46c
176.10 ± 1.01c
192.34 ± 0.01e
137.66 ± 0.82a
211.78 ± 0.74g
154.42 ± 1.46b
192.41 ± 2.64
P
90.62 ± 0.15e
87.51 ± 0.63d
72.46 ± 0.09c
73.96 ± 0.07c
100.74 ± 0.64f
103.45 ± 0.52g
100.30 ± 0.19f
88.73 ± 0.33de
57.14 ± 0.08a
87.33 ± 0.08d
62.63 ± 0.75b
84.08 ± 1.52
Mg
68.43 ± 0.92c
63.92 ± 0.22bc
54.40 ± 0.04a
57.00 ± 0.09ab
54.30 ± 5.45a
72.65 ± 0.01c
66.11 ± 0.37bc
71.26 ± 0.62c
52.66 ± 0.35a
72.34 ± 0.06c
58.02 ± 0.02ab
62.82 ± 0.78
Na
5.97 ± 0.08ab
7.40 ± 0.03ab
2.95 ± 0.02a
5.71 ± 0.03ab
3.66 ± 3.09ab
8.42 ± 0.00b
6.95 ± 0.08ab
4.50 ± 0.11ab
4.89 ± 0.11ab
3.78 ± 0.01ab
3.76 ± 0.05ab
5.27 ± 0.19
Fe
1.37 ± 0.02ab
2.17 ± 0.00ab
1.08 ± 0.00a
1.57 ± 0.01ab
1.14 ± 1.01ab
1.26 ± 0.04ab
2.35 ± 0.03ab
0.99 ± 0.02a
1.41 ± 0.05ab
2.84 ± 0.02b
0.84 ± 0.03a
1.54 ± 0.07
Mn
3.32 ± 0.02i
3.57 ± 0.01j
1.00 ± 0.00b
1.75 ± 0.01e
0.90 ± 0.00a
2.59 ± 0.02g
3.81 ± 0.02k
1.25 ± 0.00c
1.52 ± 0.02d
2.73 ± 0.02h
2.31 ± 0.02f
2.25 ± 0.10
Cu
0.69 ± 0.00a
0.59 ± 0.01a
1.18 ± 0.00a
1.86 ± 0.12a
1.24 ± 1.00a
1.42 ± 0.00a
0.58 ± 0.00a
3.35 ± 0.02b
0.35 ± 0.00a
0.51 ± 0.00a
0.35 ± 0.00a
2.63 ± 0.52
Zn
0.79 ± 0.00a
1.84 ± 0.01a
1.20 ± 0.00a
4.32 ± 0.01a
1.15 ± 1.00a
0.96 ± 0.00a
1.01 ± 0.00a
1.45 ± 0.02a
0.51 ± 0.01a
1.07 ± 0.00a
0.69 ± 0.00a
1.36 ± 0.10
B
1.11 ± 1.00a
1.49 ± 0.01a
1.06 ± 0.00a
1.22 ± 0.01a
0.77 ± 0.00a
1.16 ± 0.01a
1.41 ± 0.01a
0.76 ± 0.00a
0.92 ± 0.00a
1.02 ± 0.00a
0.70 ± 0.00a
1.05 ± 0.04
Different letters within the same row indicate significant differences (p<0.05).Z. lotus fruits from the different origins were dominated by K and Ca, which were abundant in fruits originated from Beni Mellal (828.44 ± 5.60 and 211.78 ± 0.74 mg/100 g, respectively) and their smallest concentrations were found in fruits collected from Khnifra (675.22 ± 0.97) and Tafraout (137.50 ± 0.18 mg/100 g), respectively. The remaining main minerals were P and Mg found at high concentrations in fruits from Sidi Hrazm (103.45 ± 0.52 and 72.65 ± 0.01 mg/100 g, respectively) and in small concentrations in fruits of Khnifra (57.14 ± 0.08 and 52.66 ± 0.35 mg/100 g, respectively). Na was also an important element in Z. lotus fruits, and it is found in abundance in fruits of Sidi Hrazm (8.42 ± 0.00 mg/100 g) and less in fruits of Tafraout (2.95 ± 0.02 mg/100 g). For trace elements (Table 3), namely, Fe, Mn, Cu, B, and Zn, they were also found in Z. lotus fruits with concentrations not exceeding 5 mg/100 g DM. Their highest amounts were 2.35 ± 0.03, 3.81 ± 0.02, 2.63 ± 0.52, 1.49 ± 0.01, and 4.32 ± 0.01 mg/100 g, respectively.


## 3.4. Fatty Acid Composition

VOs are food resources consumed by humans to satisfy their nutritional needs thanks to their profile of fatty acids (FAs), which are an essential indicator of the nutritional value of the oil [29–33].To determine this nutritional value ofZ. lotus oil, the composition of fatty acids was analyzed and the obtained results are shown in Table 4. As it can be seen in these outcomes, Z. lotus oil was dominated by MUFA ranging from 50.65 (Zaouit Cheikh) to 62.45% (Zagora). MUFA were followed by SFA, which varied between 21.80 (Taroudant) to 30.71% (Zaouit Cheikh), then finally PUFA representing small amounts, their level varied between 14.04 (Zagora) and 18.62% (Zaouit Cheikh). MUFA consisted mainly in oleic and palmitoleic acids. Oleic acid was the major fatty acid, and its values ranged from 50.65 (Zaouit Cheikh) to 60.25% (Zagora). The main saturated fatty acid of Z. lotus oil was palmitic acid (from 12.03% in Taroudant to 18.67% in Zaouit Cheikh), followed by stearic acid and then heneicosylic (heneicosanoic) acid and arachidic acid. Other saturated fatty acids were C8: 0, C10: 0, C12: 0, and C14: 0 detected in small quantities less than 1% of the total acids. Concerning PUFA, only linoleic and linolenic acids were detected with values varying from 12.63 (Zagora) to 17.21% (Zaouit Cheikh) and from 0.97 (Taroudant) to 2.15% (Tafraout), respectively.Table 4
Mean values ± SD (n = 3) of fatty acids in Z. lotus fruit oil of eleven Moroccan origins.
Fatty acid/origin
Rhamna
Taroudant
Tafraout
Taounat
Zagora
Sidi Hrazm
Lkhmissat
B-Jaad
Beni Mellal
Khnifra
Zaouit Cheikh
Average
Caprylic acid C8: 0
ND
ND
0.26 ± 0.01b
ND
0.27 ± 0.01b
0.24 ± 0.01b
ND
ND
0.09 ± 0.01a
ND
ND
0.21 ± 0.07
Capric acid C10: 0
ND
ND
0.92 ± 0.01d
0.40 ± 0.01b
0.80 ± 0.01c
0.40 ± 0.01b
ND
ND
0.32 ± 0.01a
ND
0.97 ± 0.01d
0.66 ± 0.27
Lauric acid C 12: 0
0.44 ± 0.01d
0.55 ± 0.01e
0.56 ± 0.01e
0.37 ± 0.01bc
0.42 ± 0.01cd
0.35 ± 0.01ab
0.47 ± 0.01d
ND
0.30 ± 0.01a
0.60 ± 0.01e
0.86 ± 0.01f
0.49 ± 0.15
Myristic acid C 14: 0
0.85 ± 0.01f
0.40 ± 0.01a
0.65 ± 0.01de
0.69 ± 0.01e
0.48 ± 0.01bc
0.65 ± 0.01de
0.62 ± 0.01d
0.53 ± 0.01c
0.50 ± 0.01dc
0.46 ± 0.01b
1.45 ± 0.01g
0.66 ± 0.27
Palmitic acid C 16: 0
16.57 ± 0.01h
12.03 ± 0.01a
14.38 ± 0.01g
14.19 ± 0.01e
13.79 ± 0.01d
13.63 ± 0.01c
14.30 ± 0.01f
13.63 ± 0.01c
13.32 ± 0.01b
13.35 ± 0.01b
18.67 ± 0.01i
14.35 ± 1.71
Palmitoleic acid C 16: 1
0.74 ± 0.01a
0.72 ± 0.01a
2.23 ± 0.01d
1.92 ± 0.01c
2.20 ± 0.01d
3.10 ± 0.01e
ND
1.96 ± 0.01c
1.65 ± 0.01b
ND
ND
1.81 ± 0.74
Stearic acid C 18:0
6.37 ± 0.01h
4.94 ± 0.01e
5.11 ± 0.01f
5.11 ± 0.01f
4.72 ± 0.01bc
4.77 ± 0.01c
4.68 ± 0.01ab
5.48 ± 0.01g
4.64 ± 0.01a
4.95 ± 0.01e
4.86 ± 0.01d
5.05 ± 0.47
Oleic acid C 18:1
55.24 ± 0.01b
59.15 ± 0.01i
57.32 ± 0.01e
57.19 ± 0.01d
60.25 ± 0.01k
56.81 ± 0.01c
58.66 ± 0.01h
58.32 ± 0.01f
58.5 ± 0.01g
59.64 ± 0.01j
50.65 ± 0.01a
57.43 ± 2.53
Linoleic acid C 18: 2
14.51 ± 0.01e
16.41 ± 0.01i
13.18 ± 0.01b
15.14 ± 0.01f
12.63 ± 0.01a
14.51 ± 0.01e
13.76 ± 0.01c
15.32 ± 0.01h
15.2 ± 0.01d
14.29 ± 0.01g
17.21 ± 0.01j
14.74 ± 1.27
Linolenic acid C 18: 3
1.25 ± 0.01c
0.97 ± 0.01a
2.15 ± 0.01g
1.46 ± 0.01d
1.41 ± 0.01d
1.90 ± 0.01f
1.29 ± 0.01c
1.57 ± 0.01e
1.30 ± 0.01c
1.10 ± 0.01b
1.41 ± 0.01d
1.43 ± 0.32
Arachidic acid C 20: 0
0.59 ± 0.01a
1.13 ± 0.01de
1.27 ± 0.01g
1.03 ± 0.01c
0.87 ± 0.01b
1.17 ± 0.01ef
1.09 ± 0.01d
0.98 ± 0.01c
1.21 ± 0.01f
1.73 ± 0.01i
1.37 ± 0.01h
1.13 ± 0.27
Heneicosylic acid C 21: 0
2.75 ± 0.01e
2.75 ± 0.01e
1.97 ± 0.01a
2.47 ± 0.01c
2.19 ± 0.01b
2.45 ± 0.01c
2.50 ± 0.01cd
2.20 ± 0.01b
2.98 ± 0.01f
3.07 ± 0.01g
2.53 ± 0.01d
2.53 ± 0.32
∑SFA∗
27.57
21.80
25.12
24.26
23.54
23.66
0
22.82
23.36
24.16
30.71
25.07
∑MUFA∗∗
55.98
59.87
59.55
59.11
62.45
59.91
0
60.28
60.15
59.64
50.65
59.42
∑PUFA∗∗∗
15.76
17.38
15.33
16.6
14.04
16.41
0
16.89
16.5
15.39
18.62
16.17
∗SFA: saturated fatty acids, ∗∗monounsaturated fatty acids, and ∗∗∗polyunsaturated fatty acids. Different letters within the same row indicate significant differences (p<0.05). ND = not detected.


## 3.5. Total Phenolic Content, Flavonoid Content, and Antioxidant Activity

Table5 shows the mean values ± standard deviation of total phenolic contents, flavonoid contents, and antioxidant activities of different extracts of Z. lotus.Table 5
Mean values ± standard deviation of total phenolic contents, flavonoid contents, and antioxidant activities of different extracts ofZ. lotus (AE: acetone extract, EE: ethanolic extract, ME: methanolic extract, and WE: water extract).
Solvent
Origin
TPC (mg GAE/g DM)
TFC (mg QE/g DM)
DPPH (%)
FRAP (mg AA/g DM)
ABTS (mg TE/g DM)
EE
Rhamna
8.54 ± 0.08a
7.19 ± 0.12d
38.48 ± 0.02k
4.27 ± 0.33a
9.03 ± 0.57de
Taroudant
7.01 ± 0.02a
5.48 ± 0.04a
18.76 ± 0.01a
3.96 ± 0.10a
2.2 ± 0.68a
Tafraout
8.48 ± 0.11a
6.07 ± 0.04b
32.07 ± 0.04h
8.77 ± 0.66b
11.73 ± 0.01e
Taounat
7.24 ± 6.11a
7.9 ± 0.05e
31.35 ± 0.02f
3.79 ± 0.02a
6.55 ± 0.01bcd
Zagora
8.22 ± 0.12a
6.66 ± 0.04c
38.00 ± 0.00j
4.76 ± 0.08a
8.18 ± 0.12ce
Sidi Hrazm
7.63 ± 0.14a
5.4 ± 0.08a
23.04 ± 0.00c
3.26 ± 0.39a
2.98 ± 1.53ab
Lkhmissat
3.56 ± 0.01a
5.60 ± 0.12ab
19.58 ± 0.01b
3.43 ± 0.46a
4.73 ± 0.53ac
B-Jaad
7.83 ± 0.24a
5.51 ± 0.08a
30.88 ± 0.01e
4.06 ± 0.39a
8.46 ± 0.30ce
Khnifra
8.17 ± 0.22a
7.48 ± 0.04de
23.99 ± 0.00d
3.97 ± 0.61a
8.25 ± 1.28ce
Beni Mellal
10.35 ± 0.26a
7.45 ± 0.08de
34.20 ± 0.01i
5.17 ± 0.01a
10.55 ± 0.42e
Zaouit Cheikh
9.64 ± 0.34a
7.01 ± 0.20cd
31.59 ± 0.04g
4.66 ± 0.23a
10.51 ± 0.34e
Average
7.88 ± 0.25
6.52 ± 0.09
29.26 ± 0.67
4.55 ± 0.15
7.56 ± 0.31
ME
Rhamna
9.88 ± 0.10d
7.54 ± 0.29e
47.83 ± 0.02k
9.99 ± 0.32e
22.80 ± 0.96d
Taroudant
8.03 ± 0.16ac
6.13 ± 0.12ac
14.63 ± 0.01c
3.96 ± 0.00a
7.33 ± 1.14ab
Tafraout
10.13 ± 0.02d
6.98 ± 0.24ce
37.13 ± 0.01i
9.73 ± 0.49e
20.19 ± 1.86cd
Taounat
8.97 ± 0.26ac
6.51 ± 0.16bcd
19.24 ± 0.01d
5.02 ± 0.01ab
9.18 ± 1.96ab
Zagora
9.04 ± 0.36bcd
7.19 ± 0.04de
20.19 ± 0.01e
5.71 ± 0.17bc
11.64 ± 0.57ac
Sidi Hrazm
7.54 ± 0.15ab
6.01 ± 0.20ab
11.65 ± 0.01a
4.30 ± 0.13a
2.70 ± 0.53a
Lkhmissat
6.64 ± 0.12a
5.37 ± 0.04a
12.87 ± 0.01b
4.53 ± 0.17a
5.91 ± 1.50ab
B-Jaad
7.61 ± 0.69ab
7.28 ± 0.08de
24.39 ± 0.01h
6.20 ± 0.07c
11.89 ± 3.00ac
Khnifra
9.35 ± 0.01cd
7.22 ± 0.01de
44.85 ± 0.01j
8.25 ± 0.08d
20.72 ± 1.75cd
Beni Mellal
8.69 ± 0.12bcd
7.22 ± 0.08de
20.73 ± 0.01f
5.78 ± 0.19bc
11.43 ± 1.43ac
Zaouit Cheikh
9.37 ± 0.33cd
7.72 ± 0.20e
23.98 ± 0.01g
6.68 ± 0.10c
13.13 ± 2.39bc
Average
8.65 ± 0.11
6.83 ± 0.07
25.22 ± 0.12
6.37 ± 0.21
12.44 ± 0.65
AE
Rhamna
14.09 ± 0.63a
12.10 ± 0.08a
65.19 ± 0.01h
10.0 ± 0.11ab
25.87 ± 0.12ab
Taroudant
12.77 ± 0.22a
11.00 ± 0.37a
27.96 ± 0.01a
8.37 ± 0.56a
22.96 ± 2.23a
Tafraout
18.04 ± 0.52b
15.60 ± 0.24c
49.87 ± 0.01e
11.3 ± 1.10bc
29.36 ± 0.10b
Taounat
20.27 ± 0.47bc
17.90 ± 0.04b
52.28 ± 0.01f
11.5 ± 0.19bc
29.36 ± 0.10b
Zagora
21.67 ± 0.85c
18.90 ± 0.16c
96.64 ± 0.04j
37.5 ± 0.39e
29.32 ± 0.04b
Sidi Hrazm
13.81 ± 0.32a
11.00 ± 0 0.29a
86.16 ± 0.04i
16.6 ± 0.20d
29.32 ± 0.13b
Lkhmissat
14.85 ± 0.32a
12.00 ± 0.29a
29.44 ± 0.01b
8.24 ± 0.60a
29.13 ± 0.40b
B-Jaad
20.95 ± 0.29c
17.40 ± 0.09c
65.19 ± 0.01h
12.5 ± 0.28bc
26.51 ± 0.80ab
Khnifra
20.57 ± 0.06bc
18.20 ± 0.29c
58.60 ± 0.01g
12.4 ± 0.32bc
29.36 ± 0.10b
Beni Mellal
21.50 ± 0.50c
18.30 ± 0.62c
48.92 ± 0.01d
13.0 ± 0.37c
29.29 ± 0.17b
Zaouit Cheikh
21.28 ± 0.30c
18.00 ± 0.04c
38.71 ± 0.01c
13.2 ± 0.60c
29.45 ± 0.04b
Average
18.16 ± 0.35
15.49 ± 0.31
56.26 ± 0.21
14.05 ± 0.79
28.17 ± 0.22
WE
Rhamna
13.63 ± 0.13ab
12.6 ± 0.33a
11.51 ± 0.01g
9.12 ± 0.22a
13.63 ± 0.58ac
Taroudant
11.43 ± 0.61ab
11.8 ± 0.33a
6.05 ± 0.01a
8.12 ± 0.10a
9.80 ± 1.07a
Tafraout
15.29 ± 0.17b
17.56 ± 1.24b
10.23 ± 0.01e
9.99 ± 0.61a
14.83 ± 2.64ac
Taounat
13.97 ± 2.05ab
8.68 ± 1.33a
7.91 ± 0.01d
8.75 ± 0.24a
15.84 ± 0.31ac
Zagora
14.42 ± 0.25ab
11.8 ± 0.83a
12.79 ± 0.01h
9.59 ± 0.18a
17.58 ± 1.96bc
Sidi Hrazm
11.20 ± 0.35a
9.74 ± 0.49a
6.40 ± 0.01b
9.31 ± 0.54a
11.19 ± 0.17ab
Lkhmissat
11.08 ± 0.15a
12.9 ± 0.16a
12.91 ± 0.01i
7.29 ± 2.12a
10.75 ± 0.98ab
B-Jaad
12.77 ± 0.51ab
12.6 ± 0.66a
17.79 ± 0.01k
9.46 ± 0.15a
18.31 ± 1.92c
Khnifra
11.81 ± 0.31ab
13.0 ± 1.41a
11.28 ± 0.01f
9.77 ± 0.10a
19.13 ± 0.22c
Beni Mellal
13.39 ± 0.55ab
9.15 ± 0.01a
7.67 ± 0.01c
8.63 ± 0.69a
13.47 ± 0.08ac
Zaouit Cheikh
10.90 ± 0.25a
9.68 ± 0.41a
14.07 ± 0.01j
9.07 ± 0.52a
16.22 ± 0.31ac
Average
12.71 ± 0.16
11.77 ± 0.25
10.78 ± 0.35
9.01 ± 0.11
14.61 ± 0.33
Different letters within the same column, for each solvent, indicate significant differences (p<0.05).


### 3.5.1. Total Phenolic Content

Phenols are gaining increasing importance, especially thanks to their beneficial effects on health [34]. Their role as natural antioxidants is attracting more and more interest in the prevention and treatment of cancer and inflammatory, cardiovascular, and neurodegenerative diseases [35].TPC was determined, and the obtained results are summarized in Table5. TPC levels varied considerably from a solvent to another and also between the different origins of Z. lotus. Acetone extracts exhibit the highest values of TPC regardless of origins. Fruits collected from Zagora presented the highest TPC in acetone extract (21.67 ± 0.85 mg GAE/g DM), while extracts of fruits from Taroudant displayed the lowest TPC value (12.77 ± 0.22 mg GAE/g DM). However, the fruits of Tafraout had the greatest content of TPC in both water and methanol extracts (15.29 ± 0.17 and 10.13 ± 0.02 mg GAE/g DM), while fruits of Zaouit Cheikh and Lkhmissat contained the lowest TPC content (10.90 ± 0.25 and 6.64 ± 0.12 mg GAE/g DM, respectively). For ethanol extracts, the highest TPC was observed in Khnifra (10.35 ± 0.26 mg GAE/g DM) and the lowest value was observed for Lkhmissat (3.56 ± 0.01 mg GAE/g DM).


### 3.5.2. Total Flavonoid Content

Flavonoids, a group of natural substances with variable phenolic structures, are found in fruits, vegetables, and various plant parts including grains, bark, roots, stems, flowers, and so on. They are known for their nutraceutical, pharmaceutical, medicinal, and cosmetic properties [36].Table5 presents TFC in the studied eleven fruits of Z. lotus with different solvents. As for TPC, acetone extracts were the richest in TFC, followed by water extracts, and then methanol and ethanol extracts, respectively. Fruits from Zagora contained the highest TFC in acetone extracts (18.92 ± 0.16 mg QE/g DM), while fruits of Taroudant had the lowest TFC (11.07 ± 0.37 mg QE/g DM). Water extracted more flavonoids in fruits of Tafraout (17.56 ± 1.24 mg QE/g DM) but less in fruits of Taounat (8.68 ± 1.33 mg QE/g DM). In ethanol extracts, fruits collected from Beni Mellal had the highest TFC (7.48 ± 0.04 mg QE/g DM) and Sidi Hrazm fruits recorded the lowest value (5.40 ± 0.08 mg QE/g DM).


### 3.5.3. Antioxidant Activity

Variability of antioxidants and their properties requires the use of more than one method to assess the antioxidant activity of extracts. In order to study antioxidant activity of different extracts, three different techniques were used (DPPH, FRAP, and ABTS assay). DPPH assay measures the total antioxidant capacity (TAC) of compounds that are able to transfer hydrogen atoms, similar to ABTS, but it includes the action of polar and non-polar antioxidants, while FRAP assay measures the capacity to reduce iron ion Fe3+ to Fe2+ [8].The results of antioxidant activity by DPPH, FRAP, and ABTS of different fruits in different extracts are shown in Table5. The acetone extracts were more reductive than methanol, ethanol, and water extracts, respectively. Contrary to phenolic compounds, water extracts presented the lowest antioxidant activity. The percentages of reduction for acetone extracts reached more than 90% of DPPH radicals (Zagora) and more than 35% for fruits from the remaining origins. Methanol extracts scavenged between 11 and 47% of radicals. Ethanol extract percentages ranged from 19 to 38%, while water extract percentages of scavenging did not exceed 20% for fruits from all origins. El Maaiden et al. [8] reported that water extracts of Z. lotus whole fruits scavenged 61.91 ± 0.85% of DDPH radicals.Regarding antioxidant activity measured via FRAP, as observed for TPC and TFC, acetone extracts were more reductive than water, methanol, and ethanol extracts. With respect to origins, Zagora fruits had the highest power of reducing iron (37.59 ± 0.39 mg AAE/g DM) and fruits of Lkhmissat presented the lowest activity (8.24 ± 0.60 mg AAE/g DM). Water extracts presented similar activities for all origins (8.12 ± 0.10–9.99 ± 0.61 mg AAE/g DM). Methanol extract activities ranged from 3.96 ± 0.00 to 9.99 ± 0.32 mg AAE/g DM, and ethanol extracts had low activities (3.26 ± 0.39–8.77 ± 0.66 mg AAE/g DM).Antioxidant activity ofZ. lotus fruits using ABTS+ radical revealed that the highest antioxidant activity was found in acetone extracts (more than 22.00 mg TE/g DM) with no significant differences between different origins. The lowest activity was found in ethanol extracts (2.21 ± 0.08–11.86 ± 0.10 mg TE/g DM). Important variations were found for ABTS+ antioxidant activities for both water (9.80 ± 1.07–19.13 ± 0.22 mg TE/g DM) and methanol extracts (7.33 ± 1.14–22.80 ± 0.96 mg TE/g DM). Fruits from Taroudant were found to be the less active among the eleven origins, since they presented the lowest activities with different techniques (ABTS, DPPH, and FRAP) and for the four extraction solvents.


## 3.5.1. Total Phenolic Content

Phenols are gaining increasing importance, especially thanks to their beneficial effects on health [34]. Their role as natural antioxidants is attracting more and more interest in the prevention and treatment of cancer and inflammatory, cardiovascular, and neurodegenerative diseases [35].TPC was determined, and the obtained results are summarized in Table5. TPC levels varied considerably from a solvent to another and also between the different origins of Z. lotus. Acetone extracts exhibit the highest values of TPC regardless of origins. Fruits collected from Zagora presented the highest TPC in acetone extract (21.67 ± 0.85 mg GAE/g DM), while extracts of fruits from Taroudant displayed the lowest TPC value (12.77 ± 0.22 mg GAE/g DM). However, the fruits of Tafraout had the greatest content of TPC in both water and methanol extracts (15.29 ± 0.17 and 10.13 ± 0.02 mg GAE/g DM), while fruits of Zaouit Cheikh and Lkhmissat contained the lowest TPC content (10.90 ± 0.25 and 6.64 ± 0.12 mg GAE/g DM, respectively). For ethanol extracts, the highest TPC was observed in Khnifra (10.35 ± 0.26 mg GAE/g DM) and the lowest value was observed for Lkhmissat (3.56 ± 0.01 mg GAE/g DM).


## 3.5.2. Total Flavonoid Content

Flavonoids, a group of natural substances with variable phenolic structures, are found in fruits, vegetables, and various plant parts including grains, bark, roots, stems, flowers, and so on. They are known for their nutraceutical, pharmaceutical, medicinal, and cosmetic properties [36].Table5 presents TFC in the studied eleven fruits of Z. lotus with different solvents. As for TPC, acetone extracts were the richest in TFC, followed by water extracts, and then methanol and ethanol extracts, respectively. Fruits from Zagora contained the highest TFC in acetone extracts (18.92 ± 0.16 mg QE/g DM), while fruits of Taroudant had the lowest TFC (11.07 ± 0.37 mg QE/g DM). Water extracted more flavonoids in fruits of Tafraout (17.56 ± 1.24 mg QE/g DM) but less in fruits of Taounat (8.68 ± 1.33 mg QE/g DM). In ethanol extracts, fruits collected from Beni Mellal had the highest TFC (7.48 ± 0.04 mg QE/g DM) and Sidi Hrazm fruits recorded the lowest value (5.40 ± 0.08 mg QE/g DM).


## 3.5.3. Antioxidant Activity

Variability of antioxidants and their properties requires the use of more than one method to assess the antioxidant activity of extracts. In order to study antioxidant activity of different extracts, three different techniques were used (DPPH, FRAP, and ABTS assay). DPPH assay measures the total antioxidant capacity (TAC) of compounds that are able to transfer hydrogen atoms, similar to ABTS, but it includes the action of polar and non-polar antioxidants, while FRAP assay measures the capacity to reduce iron ion Fe3+ to Fe2+ [8].The results of antioxidant activity by DPPH, FRAP, and ABTS of different fruits in different extracts are shown in Table5. The acetone extracts were more reductive than methanol, ethanol, and water extracts, respectively. Contrary to phenolic compounds, water extracts presented the lowest antioxidant activity. The percentages of reduction for acetone extracts reached more than 90% of DPPH radicals (Zagora) and more than 35% for fruits from the remaining origins. Methanol extracts scavenged between 11 and 47% of radicals. Ethanol extract percentages ranged from 19 to 38%, while water extract percentages of scavenging did not exceed 20% for fruits from all origins. El Maaiden et al. [8] reported that water extracts of Z. lotus whole fruits scavenged 61.91 ± 0.85% of DDPH radicals.Regarding antioxidant activity measured via FRAP, as observed for TPC and TFC, acetone extracts were more reductive than water, methanol, and ethanol extracts. With respect to origins, Zagora fruits had the highest power of reducing iron (37.59 ± 0.39 mg AAE/g DM) and fruits of Lkhmissat presented the lowest activity (8.24 ± 0.60 mg AAE/g DM). Water extracts presented similar activities for all origins (8.12 ± 0.10–9.99 ± 0.61 mg AAE/g DM). Methanol extract activities ranged from 3.96 ± 0.00 to 9.99 ± 0.32 mg AAE/g DM, and ethanol extracts had low activities (3.26 ± 0.39–8.77 ± 0.66 mg AAE/g DM).Antioxidant activity ofZ. lotus fruits using ABTS+ radical revealed that the highest antioxidant activity was found in acetone extracts (more than 22.00 mg TE/g DM) with no significant differences between different origins. The lowest activity was found in ethanol extracts (2.21 ± 0.08–11.86 ± 0.10 mg TE/g DM). Important variations were found for ABTS+ antioxidant activities for both water (9.80 ± 1.07–19.13 ± 0.22 mg TE/g DM) and methanol extracts (7.33 ± 1.14–22.80 ± 0.96 mg TE/g DM). Fruits from Taroudant were found to be the less active among the eleven origins, since they presented the lowest activities with different techniques (ABTS, DPPH, and FRAP) and for the four extraction solvents.


## 3.6. Principal Component Analysis

In this work, PCA was used as a multivariate statistical analysis with the aim to discriminate among various origins (sites). The first three components were retained since they allowed explaining over 69% of the total data variance. As it can be seen in Figure2(a), the eleven origins were separated via the first two components accounting for more than 54%. Different studied origins were separated according to PC1 (34.07%), PC2 (20.11%), and PC3 (15.01%) as evidenced in Figures 2(b)–2(d). These three components seem to be related to environmental variations among sampling sites including edaphic differences as well as climatic conditions (temperature and rainfall). Taroudant, Lkhmissat, Beni Mellal, andSidi Hrazm interacted with high records of minerals, which may be ascribed to soil fertility of these sites. Zaouit Cheikh was marked by high levels of PUFA and SFA. Likewise, Beni Mellal was associated to greater contents of OC, MUFA, and AC. Tafraout, Taroudant, and Beni Mellal were linked to greater values of physical fruit traits including fruit length (FL), fruit width (FW), and fruit weight (Fwe). The highest values of antioxidant capacity (FRAP and ABTS), TPC, and TFC were found in samples from Zagora and Tafraout. These outcomes seemed to be linked to water stress (low rainfall in these sites, Table 6).Figure 2
Principal component (PC) projections for the first three PCs that most impact influenced fruit traits. Blue segments represent dependent variables, while points plotted are mean values. FWe = weight, FL = fruit length, KW = fruit width, OC = oil content, CC = carbohydrates content, PC = protein content, AC = ash content, and MC = moisture content.
(a)
(b)
(c)
(d)Table 6
Geographical and climatic data for the sampling agro-ecological areas.
Site
Geographical coordinates
Altitude (m.a.s.l.)
Temperature (°C)
Rainfall (mm)
T
Min
T
max
Rhamna
32°46′48″N, 85°57′00″W
486
6
38
600.2
Taroudant
30°28′13″N, 8°52′37″W
237
3
43
272.0
Tafraout
29°43′28″N, 8°58′28″W
1 022
3
30
223.0
Taounat
34°32′11″N, 4°38′24″W
600
10
37
472.0
Zagora
30°19′56″ N, 5°50′18″W
733
4
42
56.0
Sidi Hrazm
31°1′43″N, 4°52′48″W
268
6
36
468.2
Lkhmissat
33°49′26″N, 6°3′58″W
409
5
33
594.4
B-Jaad
32°46′10″N, 6°23′49″W
642
4
35
357.5
Beni Mellal
32°20′14″N, 6°20′59″W
472
6
39
493.0
Khnifra
32°56′20″N, 5°40′6″W
860
4
35
564.0
Zaouit Cheikh
32°39′15″N, 5°55′17″W
667
6
39
477.0


## 3.7. Cluster Analysis

Cluster analysis (CA) was carried out to evaluate the connection among geographical origins. As it can be seen in Figure3, several clusters were extracted from our results confirming important variations among the 11 geographical origins in terms of fruit properties including physical fruit traits, proximate composition, profiling of minerals and fatty acids, and antioxidant capacity. A total of 11 clusters were highlighted. Both B-Jaad and Sidi Hrazm formed the first cluster with the highest similarity (higher Euclidean distance) as compared to the remaining origins. The smallest Euclidean distance (less than 10) was found for the cluster formed by Taroudant and Lkhmissat.Figure 3
Cluster analysis performed on mean values in theZ. lotus fruit samples collected from eleven Moroccan origins. Distance (Y axis) is the Euclidean distance among fruit samples based on the investigated fruit traits.


## 3.8. Correlation Study

Pearson correlations were performed on mean values to analyze different associations among studied dependent variables (Figure4). Based on our outcomes, important correlations were highlighted. Axial dimensions together with fruit weight (FL, FW, and FWe) were positively linked to each other. Also, FRAP, ABTS, DPPH, TPC, and TFC were positively and strongly associated to each other. The most relevant positive remaining correlations were K to (AC and PC), P to (AC and PC), Na to (PC, Fe to K, and Mn), Mn to (Fe and Na), Cu to MC, and B to (Mn, Fe, Na, K, and PC). On the contrary, there were important negative correlations. Among them, MUFA to (SFA and PUFA), MC to (OC, FW, and FWe), Mg to CC, both FW and FWe to (Mg and P), TPC to (B, Mn, Na, and PC), TFC to (B, Mn, Na, Mg, P, and PC), B to (DPPH and FRAP), and ABTS to (B, Mn, Na, P, and PC). The remaining correlations were low or insignificant.Figure 4
Coefficients of correlations (Pearson correlation) among studied dependent variables. FWe = weight, FL = fruit length, KW = fruit width, OC = oil content, CC = carbohydrates content, PC = protein content, AC = ash content, and MC = moisture content. SFA = saturated fatty acids, MUFA = monounsaturated fatty acids, and PUFA = polyunsaturated fatty acids.


## 4. Discussion

Given its large use in different fields such as nutrition, health, and cosmetics and also for its edible fruit,Z. lotus has attracted the attention of several research works. Many studies were carried out on Z. lotus fruit chemical composition from different countries. In this study, nutritional value of Moroccan jujube was investigated.Our findings show that there were no much variations among different origins regarding the measured physical fruit traits. These traits in Moroccan jujube were similar to those reported by Yahia et al. [21] for Tunisian Z. lotus (12.19 ± 0.57 mm for length, 11.21 ± 0.02 mm for width, and 0.52 ± 0.02 mm for weight) and Boudraa et al. [37] for Algerian Z. lotus (14.65 ± 0.20 mm for length and 0.56 ± 0.01 mm for weight). In contrast, they were slightly higher than Algerian Z. lotus reported by Abdeddaim et al. (10.19 ± 0.04 mm for length and 10.26 ± 0.18 mm for width) [38]. As demonstrated by ANOVA, there are no significant differences (p<0.05) among different origins for the three parameters (length, width, and weight). The investigated origins showed a relative homogeneity of these physical fruit traits over areas where Z. lotus grows.Moisture content displayed an average of 3.35 ± 1.09% which was lower than the values reported in previous study on MoroccanZ. lotus from Khouribga (8.93 ± 0.25%) [9] and Algerian Z. lotus (12.32 ± 0.55%) [37]. An important difference was also observed between our results and values of five cultivars from Chinese jujube which accumulate high content of water (from 17.38 ± 1.21–22.52 ± 1.43%) [39]. Such differences could be attributed to the climatic conditions. For ash content, our values were smaller than those reported in the literature (3.26 ± 0.22% and 3.20 ± 0.54%) for Moroccan Z. lotus [9, 10]. Protein content found in our samples was close to that found in Moroccan jujube (3.69 ± 0.10%) [9] but higher than that found by El Cadi et al. [10] (0.09%) in Moroccan jujube and Li et al. for Chinese jujube (from 2.26 ± 0.03 to 3.01 ± 0.06%) [39]. The average oil content (2.45 ± 0.62%) was found to be higher than those obtained by other authors (0.23%, 1.11 ± 0.17%, and 0.37 ± 0.01–1.02 ± 0.05%) [9, 10, 38]. Carbohydrate content found was higher than those found by El Maaiden et al. [9] (83.01 ± 0.25%) [9] and El Cadi et al. [10] (80.2 ± 3.81%) [10]. This content of carbohydrate in Z. lotus whole fruit is close to that of fruit pulp (83 ± 0.41%) [9]. In terms of richness in carbohydrates, Z. lotus whole fruit was similar to dates (from 76.69 ± 1.10–90.18 ± 0.82%) [19] and Ziziphus spina-christi pulp (85.69 ± 0.06%) [40]. These results showed that Z. lotus fruits can be considered as a good source of carbohydrates.Regarding minerals, the obtained values were higher than those reviewed by other authors except for Na, which had smaller content [6, 8], and lower than those reported by Ibourki et al. for the pulp of Moroccan jujube [27]. Significant differences were observed among the different origins of Z. lotus fruits in terms of macro-elements including K, Ca, Mg, P, and Na and some macro-elements including Fe and Mn. In contrast, no significant differences were observed for Cu, Zn, and B. In comparison with individual parts of Z. lotus fruit, pulps were studied and they also contain important levels of K, Ca, Mg, and P. Z. lotus seeds are very rich in those minerals than pulps [38]. Z. lotus fruits are rich in essential minerals for human body and their amounts vary according to geographical sources.Z.
lotus vegetable oil is composed mainly of oleic, linoleic, and palmitic acids. Oleic acid is an omega-9 fatty acid that can be synthesized by the human body, but it is also found in foods [41]. Its content was smaller than the value found by Ghazghazi et al. [15]. High levels are found in olive oil (70–80%) [30, 42]. Oleic acid content in Z. lotus oil was smaller than that in olive and higher than that in palm, sunflower, soybean, but it was similar to peanut, canola, and argan oil [43–45]. Palmitic acid is the most common saturated fatty acid accounting for 20–30% of total fatty acids in the human body and can be provided by the diet or synthesized endogenously via de novo lipogenesis (DNL) [46]. Z. lotus fruits contain small amount of palmitic acid in comparison with palm oil but higher than palm kernel oil[47]. Linoleic acid content was smaller than the percentage found in argan oil (32%) [44], but it was higher than its percentage in olive oil (7%) [45]. Z. lotus vegetable oil profiling was in disagreement with results of Ghazghazi et al. who found mainly oleic acid (88.12% of cis and 7.88 of trans) [15]. It is also different from oil of the other parts of Z. lotus (pulp, stem, root, and leaves), but it is similar to that of seeds [14] in terms of major acids (oleic, palmitic, linoleic, and stearic acids) as illustrated in Table 5.Z.
lotus fruits contained important amounts of TPC. The recorded values were in the range reviewed by Abdoul-Azize et al. [6] (2.97–40.78 mg GAE/g DM) and smaller than values reported by El Maaiden et al. for water extract of Moroccan Z. lotus fruits from Khouribga (22.00 ± 0.15 mg GAE/g DM) [8]. Generally, important variations were observed in terms of TPC, TFC, and antioxidant capacity among extracts from different solvents and origins (Table 6). TPC was found to be higher than values reported by Ghazghazi et al. [15] for methanolic extract from Tunisian Z. lotus. Another study of phenolic compounds in Z. lotus fruits aqueous extract was conducted by Dahlia et al. [48], who found 436.25 ± 97.033–1349.46 ± 351.78 mg GAE/100 g DM with wide variations among sampling locations. In addition, solvent of extraction influenced TPC content in agreement with previously published literature [49, 50]. Aerial parts of Z. lotus were rich in phenolic compounds in both ethanol and acetone extracts (43.02 ± 0.04 mg/g and 31.52 ± 0.04, respectively) [2]. In view of these different results, acetone appears to be the most effective to extract phenolic compounds.Our values of TFC were higher than those reported by El Maaiden et al. [8] for Z. lotus fruit water extracts (5.49 ± 0.10 mg CE/g DM). They were also higher than those reviewed by Abdoul-Azize [6] (1.22 mg/g DM). TFC values from water extracts were higher than those obtained by Dahlia et al. (0.83 ± 0.09–0.98 ± 0.01 mg QE/g DM) [48] and Bencheikh et al. (2242.89 ± 25 μg QE/mg DM) [51]. According to outcomes, TFC of Z. lotus fruits was significantly influenced by the geographical origin as well as the solvent used. TFC contributes to the nutritional and medicinal importance of Z. lotus fruits.As revealed by the statistical test used, significant differences (p<0.05) were detected among the studied fruits for the different solvents except for water extracts (with FRAP) and acetone extracts (with ABTS). A comparative study for two extracts (acetone 70% and methanol 70%) of aerial parts of Z. lotus published by Tlili et al. showed that acetone extract contains more bioactive compounds and presents high antioxidant activity [2]. This is consistent with our findings, which show that acetone extracts had more phenolic content and flavonoids and exhibited high antioxidant activities than the remaining solvents. Antioxidant activity of Z. lotus fruits was also studied by Marmouzi et al. who demonstrated higher efficacy of Z. lotus extract compared to the reference compound used in the assays (quercetin) [17]. This proves the importance of Z. lotus fruits as source of natural antioxidants. The findings reported in this work provide scientific data about chemical composition of Moroccan jujube for different uses and also contribute to the enrichment of the database of medicinal and aromatic plants.Multivariate analysis includes various statistical approaches (PCA, CA, etc.), which are widely in use in the field of food science, chemometrics, etc. PCA was used previously in other works on different MAPs, fruit trees, and other crops to reduce data dimensionality and highlight correlation patterns in experimental data [51–57]. Likewise, CA was used to reveal similarities and variations in obtained data. Based on CA, important intern-origin variations were observed in our outcomes. Similar results were reported by other authors [58–60] confirming the effectiveness of CA in highlighting similarities and differences among geographic origins and fruit processing technologies among others.Regarding correlations, as in our results, similar trends of correlations were highlighted among fruit physical traits as well as other physicochemical attributes by other authors [56]. Correlation knowledge is very important for breeders and consumers. For instance, breeding for a desirable trait could foster the appearance or disappearance of another trait because of their correlation.


## 5. Conclusions

The present study was focused mainly on determining the nutritional value and the antioxidant activity of MoroccanZ. lotus of the whole fruits and highlighting the effect of geographical origin. The effect of solvent on the secondary metabolites was also investigated. The obtained results showed a good nutritional value with a high phenolic and flavonoid content and important antioxidant activities for Z. lotus fruits. Z. lotus oil consisted mainly of oleic, palmitic, and linoleic acids as major components, and it can be considered as a rich source of fatty acids. Proximate composition and fatty acid and mineral profiling were influenced by geographical origin. Regarding effect of solvent acetone was the most effective solvent for extraction of bioactive compounds from Z. lotus fruits. As a promising source of polyphenols, flavonoids, fatty acids, minerals, Z. lotus can be a potential candidate for human nutrition, health promoting and disease preventing.



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*Source: 9362366-2022-11-30.xml*