Chemical Composition and Physicochemical Analysis of Opuntia dillenii Extracts Grown in Morocco

(e chemical composition and physicochemical properties of hexane and ethyl acetate extracts of skin, juice, and seeds of Opuntia dillenii fruit collected from three Moroccan regions (Oujda, Nador, and Essaouira) were studied.(e study revealed that the seed oil extracts presented the highest yield of 13.12%, followed by the skin fraction (1.77%) and the juice extract (0.49%). (e evaluation of fatty acid compositions using GC-MS analysis revealed the presence of linoleic acid as a dominating unsaturated fatty acid with a value of 72.39%, followed by palmitic acid, oleic acid, and stearic acid in all localities. Otherwise, the juice extract of Oujda locality was richer in margaric acid (37.41%), followed by Essaouira skin extract (10.7%) and Oujda seed extract (6.18%). However, the campesterol was detected only in trace in the juice extract. (e physicochemical properties of O. dillenii seed oils such as acid value, peroxide value, ester value, pH value, saponification value, density, and refractive index were all found to be in good agreement with the quality criteria for pure and fresh oils. In addition, principal component analysis (PCA) and hierarchical cluster analysis (HCA) were implemented to compare the difference in the chemical composition of the different O. dillenii extracts.


Introduction
Opuntia dillenii, commonly known as prickly pear, is a perennial plant belonging to the Cactaceae, Caryophyllales, family and the Caryophyllidae subclass [1,2]. It is extensively distributed in the desert, semidesert, and dry regions in the tropics and subtropics [3]. is plant is native to tropical America and India. However, it can be grown in Australia, Asia, and the Mediterranean region [4]. It was introduced in Europe and North Africa in the 16 th century [5]. Cactus has remarkable benefits to human and animal health [6]. It can be a source of various chemical compounds of biological and pharmacological importance.
Several studies have revealed its high efficacy in the treatment of antihyperlipidemic, antiatherosclerotic effects [7]. Its ability to protect nerve cells from Alzheimer's, Parkinson's, and cerebral vascular diseases has also been reported [8]. Particularly, juice of Opuntia fruits is a rich source of dietary fiber, vitamins (B1, B2, and C), and natural colorants (betanin and indicaxanthin) [9][10][11][12][13][14], which are responsible for the intense red coloring of the fruit [11]. It is also known by its analgesic, antihyperglycemic [15], inflammatory [16], antifungal [17], anticancer [1,18], hepatoprotective [19], and antidiabetic activities [18,20]. Indeed, the seeds of O. dillenii may contribute to the higher antioxidant activity due to the high concentrations of polyphenols and flavonoids and the presence of a large amount of unsaturated fatty acids that provide valuable natural antioxidants for the pharmaceutical and food industry [21]. e O. dillenii oil is well-known for its richness in essential fatty acids and vitamin E [6] which have efficiency potential on the skin and can be used as antiaging and antiwrinkle agents. e oil of O. dillenii could also be a source of linoleic acid (omega-6) [22], which is involved in the regulation of cardiovascular disorders. is oil has also shown a hepatoprotective and antidiabetic effect [23,24]. All these properties have been able to give a positive impact on the exploration of O. dillenii fruits.
In this paper, continuing our previous work on O. dillenii plant, we report the evaluation of the chemical and physicochemical properties of hexane and ethyl acetate extracts of skin, juice, and fruit seeds of O. dillenii grown in three different regions of Morocco (Oujda, Nador, and Essaouira). is investigation was performed to assess the potential nutritional value of O. dillenii fruits for human and animal consumption. e extracts were analyzed, characterized, and identified by GC-MS. e variation of O. dillenii extracts parameters between the three regions was investigated using statistical analyses (hierarchical cluster analysis (HCA) and principal component analysis (PCA)).

Chemicals and Reagents.
Hexane, ethyl acetate, and methanol were purchased from Merck (Karlsruhe, Germany). All chemicals and solvents were of highly analytical grade and were used as received from Merck without further purification.

Plant Material.
e matured purple fruits of prickly pear (O. dillenii) were collected in February 2017 from three regions in Morocco ( Figure 1): Oujda (Sidi Maafa), Nador (Kariat Arkmane), and Essaouira (Smimou). e geographical and climatic data of the studied regions are presented in Table 1. e plant species was authenticated by Prof. M. Bnouham (Faculty of Sciences, University Mohammed Premier, Oujda, Morocco). e sampled fruits were transported to the laboratory, washed thoroughly with water to remove dust and spines, then peeled, and mixed for 5 min using a Moulinex blender (Faciclic Glass LM310E, Groupe Seb, Mayenne, France) to separate seeds from the juice by passing through a sieve with a 2 mm screen ( Figure 2). e juice was dehydrated by heating at 40°C in the oven for 15 days. Seeds and skins were washed with distilled water, dried at room temperature (25°C), for one week, weighed to calculate the percentage in the edible fraction, then reduced to a fine powder using a Moulinex coffee grinder (DPA241, Groupe Seb, Lourdes, France), and stored at −20°C, as well as the juice, for a maximum of eight weeks.  [27] and NF ISO 6320 (January 1996) [25] were adopted to measure the density and refractive index at 20°C. e density of the oils was determined by a mass over volume measurement. e pH values were measured using a CPC-501 pH-meter (Elmetron, Chorzow, Poland). e measures were performed in triplicate for each parameter, and the results were presented as mean ± standard deviation.

Fatty Acid GC-MS Analysis of O. dillenii Extracts.
e fatty acid methyl esters of hexane and ethyl acetate extracts of O. dillenii fruits were prepared following the standard protocol NF T60-233 [28]. eir separation and identification were performed on a Shimadzu GC system (Kyoto, Japan) equipped with a BPX25 capillary column with 5% diphenyl, 95% dimethylpolysiloxane phase (30 m × 0.25 mm inner diameter × 0.25 µm film thickness), coupled to a QP2010 MS. Pure helium gas (99.99%) was used as carrier gas with a constant flow rate of 3 mL/min. e injection, ion source, and interface temperatures were all set at 250°C. e temperature program used for the column oven was 50°C (held for 1 min), heated to 250°C at 10°C/min and held for 1 min. e ionization of the sample components was done in the EI mode (70 eV). e mass range scanned was 40-300 m/z. 1 μL of each prepared extract diluted with an appropriate solvent was injected in a splitless mode (split ratio 90 : 1). All samples were analyzed in triplicate. Finally, compounds were identified by comparison of their retention times with those of authentic standards and their mass spectrum fragmentation patterns with those found in databases or those stored on the National Institute of Standards and Technology (NIST) 147, 198 compounds. LabSolutions (version 2.5) was used for data collection and processing.
2.6. Statistical Analysis. All the results are expressed as mean ± standard deviation (SD). e obtained data of the oil components with a percentage greater than 5% were analyzed statistically using the principal component (PCA) and hierarchical cluster (HCA) analyses. All the statistics were performed employing the XLSTAT software.

Yield Percentage of Skin, Pulp, and Seeds in Fruit.
e length and width (diameter) as well as the skin, juice, and seed content of the O. dillenii fruits, according to their locality, are summarized in Table 2. e morphological study showed that the Essaouira fruits were bigger than those of Oujda and Nador, while no large differences were observed between the morphological characteristics (length and width) of the O. dillenii belonging to Oujda and Nador locality. On the other hand, the percentage yield analysis of each dry part (skin, juice, and seeds) obtained from fresh Essaouira fruits revealed that the seed content was found to be the highest (14.7%), followed by skin (11.6%) and juice (4.4%). e Essaouira fruit contained the highest content of seeds, skin, and juice. In the literature, Ali Alsaad reported lower fruit seed content, 9.5%, in Iraq [22].  Journal of Chemistry ese findings demonstrated clearly that the fresh O. dillenii fruits contain more than 75% of water, which was in good agreement with previous data reported in the literature [29,30]. Furthermore, the juice fruit of Essaouira was less acidic compared to that of the two other localities, pH � 4.8, 3.9, and 3.2 for Essaouira, Oujda, and Nador, respectively.

Physical and Chemical Parameters of O. dillenii Seed Oils.
e study of the physicochemical properties of any product is a very useful way to measure its quality before its use or consumption. ese different parameters can be influenced by climatic conditions as well as cultural practices. In this context, some physicochemical parameters of O. dillenii seed oils (for the three sites) such as acid value, ester value, peroxide value, saponification value, pH value, density, and refractive index were measured following the appropriate protocols.
e obtained results were collected and are presented in Table 3.
According to the obtained data, the water content in seed oil for the three localities of O. dillenii was found to be below the max value proposed by Codex Stan 210-1999 and COI standards (max 0.2%). e higher content was shown by Nador sample with 0.189%, followed by Essaouira and Oujda samples with values of 0.154% and 0.148%, respectively. e peroxide value, which is the most used indicator to describe the relative stability to lipid oxidation of oils, was calculated [31]. Values greater than 10 meq/kg are highly susceptible to autooxidation as a consequence of moisture or presence of trace elements [32]. In our case, the peroxide values of the three localities ranged from 5.60 to 6.35, less than 10 meq O 2 /kg of oil. ese outcomes suggested that our oils possess a high shelf life. However, these values are higher than those obtained by El Mannoubi et al. [33] for O. ficus-indica seed oil and by Alsaad et al. [22] for O. dillenii.
e saponification values of the studied samples ranging from 210.37 to 252.45 were higher than those of O. stricta and O. ficus-indica reported by Ennouri et al. [29], but they always stayed in the range of Codex Stan 210-1999 and COI standards. is parameter indicates the presence of saturated fatty acids in the studied oil, and a low value points to the predominance of long-chain fatty acids, as suggested by Akintayo and Bayer [34]. e refractive index is another parameter that can be used to measure the quality of the oil. Low indices characterize oils of high quality. e refractive indices of our oils were found to be higher compared to the results of El Mannoubi [33] and Ennouri [29], but in accordance with the norm (Codex Stan 210-1999 and COI standards). e density of the O. dillenii seed oils at 20°C compared favorably with that of O. stricta seed oil but was higher than that of O. ficus-indica seed oil [18]. Essaouira oil was found to be less acidic with a pH value of 6.96, higher than that of Oujda (pH � 6.54) and Nador (pH � 6.50) samples.
e evaluation of the acid value is another possible way to determine the quality of fatty acids present in the oil, which could in turn explain the shelf life and stability of such oil. Its value gives an idea of the free acid level. A value below 2 presents proof of good conservation (low amount of free acids) [32]. In the present work, the acid values found in O. dillenii seed oils for the three samples were higher than 2.
ese high acidity levels could be attributed to the advanced maturity of the fruit and/or the harvesting period, as previously reported by Terouzi et al. [35]. is high acidity value could indicate strong enzymatic hydrolysis of the seeds during oil harvesting, handling, or processing [36]. It could also be explained by bad preservation. According to [37], when oil was subjected to poor storage conditions, its quality could deteriorate in various ways, but most often by hydrolysis or oxidation. e ester index test is an indirect method for determining the ester content of the oil. In fact, oils of excellent qualities have a large quantity of esters.
Generally, the studied oils extracted from O. dillenii seeds have been found to meet all quality criteria that confirm an excellent application. However, the observed differences between the samples could be due to geographical location, soil, climatic conditions, and maturation [38].

Chemical Composition of Hexane and Ethyl Acetate
O. dillenii Extracts. In this study, we compared the yields and the chemical composition of the hexane and ethyl acetate extracts of skin, juice, and seeds of O. dillenii fruits harvested from three geographical locations in Morocco (Tables 4-6).
In the case of Nador variety, the hexane seed extract showed the highest yield with a value of 13.12%, followed by Oujda extract (9.25%) and then Essaouira extract (5.85%). However, in the literature, Liu et al. [39] obtained a lower yield of 6.65% by using a supercritical carbon dioxide extraction of seed oil. Recently, Alsaad et al. [22] reported relatively the same yield of 6.5% by hydrodistillation extraction. Labuschagne and Hugo [40] reported that the oil content in cactus Opuntia dillenii seeds from South Africa was 5.69%, while Chang [21] reported that the oil content in cactus pear seeds from China was 6.01%. e preliminary report of gas-chromatography coupled with mass spectrometry analysis of the hexane extract revealed the presence of numerous fatty acids. eir amount varied according to the locality and the part of cactus fruit. e major constituents of the seed oils were linoleic acid with amounts varying from 66.57 to 72.39%, followed by palmitic acid (19.81 to 24.27%), stearic acid (2.37 to 9.61%), and myristic acid (0.97 to 2.91%). e Nador locality oil presented the highest percentage of linoleic acid (72.39%) followed by Essaouira and Oujda localities with 69.60 and 66.57%, respectively. A high unsaturated ratio of 2.75 was detected for the Nador locality oil, followed by Essaouira and Oujda localities, 2.29 and 2.04, respectively. ese values showed that the seed oils were rich in unsaturated fatty acids and confirmed their importance as nutritional value for human consumption.
In contrast, a very low amount of oil was recovered in the case of ethyl acetate seed extracts. e yield was about 1.35% for Essaouira, followed by 0.96 and 0.92% for Nador and Oujda localities, respectively. is oil was also dominated by linoleic acid whose values ranged from 36.21 to 72.39%, followed by butanoic acid, 0 to 21.68%, stearic acid, 5.49 to 23.25%, and palmitic acid, 9.40 to 20.17%. Oujda and Essaouira extracts were richer in linoleic acid, with a value of 72.39%, than Nador extract with 36.21%. However, a moderate quantity of butanoic acid as saturated fatty acid was found only in Nador extract with approximately 21.68%. e ethyl acetate seed extract showed the presence of less than 5.04% of oleic unsaturated fatty acid in only Oujda locality and a low amount of margaric acid (6.18%) in the same locality. On the other hand, the extract from Nador showed a higher value of unsaturated ratio (2.75), followed by Essaouira extract (2.29) and Oujda extract (2.04). Our results are in concordance with those reported in the literature by Alsaad et al. [22]. e major constituents of the seed oil were linoleic acid (72.9%), palmitic acid (15.12%), and stearic acid (7.51%). However, Liu et al. [39] reported a different profile of the Chinese seed oil. e main components obtained were linolenic acid (66.56%), palmitic acid (19.78%), stearic acid (9.01%), and linoleic acid (2.65%). is variation could be due to geographical location, soil, climatic conditions, and maturation. e yields of hexane and ethyl acetate extracts of O. dillenii juice from the three localities presented low values less than 0.86%. e chemical composition analysis of hexane extract (Table 5) showed that palmitic acid was the dominant fatty acid with a value of 55.89% for Oujda sample followed by linoleic (39.09%), oleic (24.82%), and stearic acid with less than 4.47%.  Interesting, the study showed the presence of small quantities of campesterol, which is a well-known beneficial agent with cholesterol-lowering and anticarcinogenic properties [41], concentrated especially in ethyl acetate extracts of Oujda and Nador varieties with contents of 2.48% and 2.15%. It is also  important to note the presence of margaric acid in ethyl acetate extract with a moderate amount of 37.41% and 34.15% in Oujda and Essaouira extracts, respectively. is compound was detected in a low value of 13.18% in Nador variety and was only present in trace amount, 0.15%, in the hexane extract of Oujda locality.
Concerning the skin extract, the highest yield of 1.77% was detected in the hexane extract of Oujda variety. However, the yield obtained in the ethyl acetate was about 1% in all the localities. Similar to previous results of seeds and juice extracts, the skin extracts were also found to be rich in linoleic and palmitic acids (Table 6). e highest percentage of linoleic acid, 68.09%, was found in Nador ethyl acetate extract. is compound was also abundant in the hexane extract with a quantity of 52.98% in the Essaouira locality. is study also showed that the Nador variety contained a higher amount of palmitic acid, 45.28% and 28.34% in hexane and ethyl acetate extracts, respectively. In contrast, oleic acid (16.67%) and 11hexadecenoic acid (15.39%) were present, as unsaturated fatty acids, only in the hexane extracts of both samples of Oujda and Essaouira.
In addition, the 4-fluorobenzoic acid was found in the ethyl acetate extract, with a percentage of 16.48%, as well as in the hexane extract, with a low content of 2.14%, of Oujda sample.
is compound was also detected in ethyl acetate extract of Nador and Essaouira samples but only in trace amounts.
Furthermore, a low quantity of margaric acid was identified only in the ethyl acetate extract of Essaouira. Pentadecanoic acid and margaric acid have a positive effect on health in several disease etiologies [42]. Holman [43] showed that both pentadecanoic and margaric acids have a role in reducing the development of multiple sclerosis, suggesting that fatty acids could increase membrane fluidity [44] to a similar degree to polyunsaturated fatty acids. Besides and based on previous studies, some of the constituents identified in O. dillenii fruits were biologically active compounds. ey were proven to possess pharmacologic activities which may contribute to the healing potential of the plant. Linoleic acid has beneficial properties for the skin, and, for this purpose, it is widely used in the cosmetics and pharmaceutical industry. Stearic acid has neutral effects on the concentration of LDL cholesterol in blood serum and no cholesterol-lowering impact on human health [45]. Based on UFA/SFA ratio, it is essential to mention that there is a higher amount of unsaturated fatty acids (UFA) in the skin and seed parts of the studied O. dillenii fruits than the juice part, which was found to be richer in saturated fatty acids (SFA). e ethyl acetate extract of Oujda skin extract presented the highest unsaturated ratio of 5.15. However, the hexane extract was less rich in unsaturated acids. Particularly, the study reported that the amount of linoleic acid is higher in comparison with other vegetable oils such as potato oil (52.69%), pomegranate oil (3.84%), and sesame oil (44.5%). ese findings were in accordance with recently published studies by Ghazi, where linoleic acid was the dominant fatty acid with an exceptional level up to 79.83% [46][47][48][49]. Finally, the evaluation of the chemical composition as well as the physicochemical properties of O. dillenii fruit extracts demonstrated that this plant could be an interesting natural source of edible oil containing high amounts of unsaturated fatty acids. In addition, it is interesting to note that the difference between the chemical compositions of the different extracts from the three varieties of O. dillenii fruits could be a consequence of many factors such as the degree of maturation, the growing conditions, and the nature of the solvent used during the extraction [35,36]. On the other hand, further detailed analyses and studies, along with isolation of active constituents, are needed to investigate this plant further.

Statistical Analyses.
e statistical study was based on the hierarchical cluster analysis (HCA) and principal component analysis (PCA). e intermediate correlation matrices, correlation coefficients between the variables and the two axes F1 and F2, and the projection of the variables in the space of the axes F1 and F2 were obtained with XLSTAT software.
From the dendrogram produced by HCA (Figure 3), based on the Euclidean distances between collected samples, O. dillenii populations can be classified into five main clusters, which shows that there is a significant difference in the composition of O. dillenii extract harvested from three Moroccan regions (Oujda, Nador, and Essaouira). e samples linked by short distances are more similar than those connected by long ones. In the closer distances (18 units), the populations examined were divided into five groups (Gr 1 to 5). e first group (Cluster I), represented by five samples from the three regions of Morocco (Oujda, Nador, and Essaouira), SO1, SN1, SKN2, SKO2, and SE1, was rich in linoleic acid (32.27-72.39%). ese populations in the vicinity have a relatively different climate and soil, resulting in different oil contents. e second group (Cluster II), containing the following chemotypes: SN2, JN2, SKE1, SO2, and SE2, has been characterized by a significant percentage of linoleic acid (58.32-58.50%). e ethyl acetate juice extracts from Oujda and Essaouira samples (JE2 and JO2) were found to construct the third group (Cluster III). is latter group was found to be rich in margaric acid (34.15-37.41%), citric acid (10.18-17.01%), and stearic acid (5.29-13.11%). In the fourth group (Cluster IV), which was represented by SKE2, SKO1, JN1, and JE1 samples, palmitic acid (14.17-28.46%), oleic acid (5.15-22.57%), and linoleic acid (58.32-58.50%) were found to be the most abundant components. e group also contains significant to moderate levels of oleic acid (5.04-16.67%). Finally, the fifth group (Cluster V), represented by the two samples JO1 and SKN1, was found to be rich in linoleic acid (30.31-50.39%) and palmitic acid (45.28-55.89%). e analysis of the results showed that most of the information is explained by the first two factorial axes. In the F1 × F2 factorial design, the eigenvalues of the two components F1 and F2 and their contribution to the total inertia are represented in Table 7. For the PCA, it was found that the first two main components accounted for 56.21% of the phytochemical variance. As shown in Figure 4, by considering the PCA1 axis, it is possible to note that the extracts of O. dillenii, JO2, JE2, and JN2, are distinguished by a greater quantity of margaric acid (0.854), citric acid (0.977), and stearic acid (0.147). On the other hand, the extracts SN1, SO1, SE1, SE2, SN2, SO2, SE2, SO2, SKN2, SKE1, and JN2 are characterized by a low content of these constituents. Meanwhile, SN1, SO1, SE1, SE2, SN2, SO2, SE2, SO2, SKN2, SKE1, and JN2 are aligned on the PCA2 axis, where the most correlated variable is linoleic acid, which means that these extracts are rich in linoleic acid, and the reverse is true for JO2 and JE2. Table 8 shows that the higher the correlation is, the more the variable is related to the component. With the inertia of 21.02%, the second component (F2) was negatively correlated with palmitic acid (0.754) which existed in moderate to large quantities in the different O. dillenii extracts collected from three different regions (SKN1 and JO1). ese PCA data lead to the hierarchical upward classification of the AHA into four main groups that represent the following chemotypes: citric acid (0.977) (JO2 and JS2), myristic acid (0.738) (JN1, JS1, SKS2, and PO1), palmitic acid (0.754) (SKN1 and JO1), and linoleic acid (0.610) (SN1, SO1, SE1, SN2, SO2, SE2, SKO2, SKN2, SKS1, and JN2); their correlation matrices are represented in Table 9. is classification confirmed the results of HCA.

Conclusion
In this work, the GC-MS analysis showed that the studied extracts of O. dillenii fruit from the three localities contained a high percentage of unsaturated fatty acids and a low percentage of saturated fatty acids. Linoleic acid was the major fatty acid detected with a maximum content of 72.39%, followed by palmitic acid (maximum value of 55.89%). Otherwise, the juice extract of Oujda locality was richer in margaric acid (37.41%), followed by the Essaouira skin extract (10.7%) and Oujda seed extract (6.18%). However, the campesterol was detected only in trace in the juice extract. e physicochemical properties of O. dillenii seed oils such as acid value, peroxide value, ester value, pH value, saponification value, density, and refractive index were all found to be in good accordance with the quality criteria for pure and fresh oils, which opens the way for their use in various cosmetic applications and as sources of dietary lipids. Moreover, the presence of campesterol in the juice extract demonstrated that the O. dillenii fruits contained nutrients that are nutritionally beneficial to human health. Besides, the study also indicated that the chemical variation between the different extracts of O. dillenii depended especially on the environmental factors, climatic and geographical conditions, and the domestication of the species. Furthermore, statistical methods such as principal component analysis (PCA) and hierarchical cluster analysis (HCA) were employed based on the obtained results to highlight the existing correlation between the different parameters and the distribution of the variables in groups.

Data Availability
All data generated or analyzed during this study are included within this article.

Conflicts of Interest
e authors declare that they have no conflicts of interest.