Antioxidant and antibacterial activities of Opuntia ficus indica seed oil fractions and their bioactive compounds identification Opuntia ficus indica tohum yağ fraksiyonlarının antioksidan ve antibakteriyel aktiviteleri ve biyoaktif bileşikleri tanımlama

Aim: The two Opuntia ficus indica seed oil fractions (glyc-eridic and unsaponifiable) were compared to their biological activities and then assessed with their bioactive compounds. Methods: Based on the inhibition concentration at 50 percent (IC 50 ) values, the antioxidant activity of the two fractions was compared with three different tests. The antibacterial activity was assessed by the agar disk diffusion assay against five human pathogenic bacteria. More-over, the main fatty acids composition and unsaponifiable compounds in Opuntia ficus indica seed oil were identified by gas chromatography/mass spectrometryand (GC/MS) and NP-HPLC. Results: Results showed that antioxidant and antibacterial activities considerably varied depending on the fraction. In fact, unsaponifiable fraction has higher biological activities than the glyceridic ones. The unsaponifiable fraction has stronger effects of scavenging 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical and it is more efficient against bleaching of β -carotene than the saponifiable extract, unlike the experimental results of 2,2 ′ -azino-bis3-ethylbenzthiazoline-6- sulfonic acid (ABTS) scavenging test. Furthermore, the unsaponifiable fraction was more efficient against all pathogenic strains specially Escherichia coli . The major fatty acids identified were: linoleic (61.42%), oleic (20.55%) and palmitic acids (11.88%). Such a difference in biological capacities between the two fractions can be explained by the high content of some bioactive compounds in the unsaponifiable fraction. Low level of squalene was present in the seed oil. But high levels of terpenic alcohols (3169 mg/ kg) and of coenzyme Q9 (75.3 mg/kg) were detected in the Opuntia seed oil. Conclusion: Informations provided through this work are important for the valorization and use of this cactus vegetable oil in cosmetic, pharmaceutical and nutraceutical industries.


Introduction
Opuntia ficus indica belongs to the Cactaceae family and the order Centrospermae, their fruits are the cactus pear. In Tunisia, this plant grows wildly in arid and semi-arid regions, where the production of more succulent food plants is very limited. The cactus pear seed oil composition and its chemical characteristics were investigated by El Mannoubi et al. [1].
Dietary fatty acids have significant effects on plasma cholesterol and the levels of lipoproteins, which are linked to the incidence of coronary heart disease (CHD) [2]. Recently, the use of functional food and/or nutraceuticals increased due to their beneficial effects on human health [3]. Among Mediterranean plants, prickly pear seed oil exhibit hypoglycemic and hypo-cholesterolemic effects probably due to the fatty acid composition of the prickly pear seed oil [4].
Therefore, this work aims to compare glyceridic and unsaponifiable O. ficus indica seed oil on their antioxidant and antimicrobial activities. Our study of the saponifiable fraction which is the glyceridic fraction of the Opuntia ficus indica vegetebale oil is based on fatty acids which are also called "saponifiable lipids" (lipids which, treated with NaOH or KOH, give soap).
However, no researches concerning the biological effects of the different fractions (specially the biomolecules components of unsaponifiable fraction); prickly pear seeds oil were extensively studied. In fact, unsaponifiable fraction is the non-glyceride residual fraction which is insoluble in water (but soluble in organic solvents) after saponification.

Plant sampling and analysis of oil extract
Prickly pear fruits (O. ficus indica) were collected from Bani Wael (Nabeul locality), (45 Km east Tunis, 36°46′N, 10°60′E, semi arid bioclimate) during the fructification stage. The oil is obtained by the first cold pressure of prickly peer seeds. Acid value of seed oil was determined according to international standard ISO 660. Percentage of free fatty acids (% FFA) was calculated using oleic acid as factor. Iodine value of seed oil was determined according to international standard ISO 3961. The saponification value was determined according to international standard ISO 3657. The peroxide value was determined according to AFNOR NF T60-220. Refractive index of seed oil was determined at 20°C with an Abbe refractometer. The density was measured with a densimeter PAARDMA60.
The two fractions (glyceridic and unsaponifiable) were separated after hexane and ethanol 80% addition. The two fractions were then stored at 4°C until analysis.

Assessment of antioxidant activities 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging activity
DPPH quenching ability of organ extracts was measured according to Hanato et al. [5]. One milliliter of the extract at known concentrations was added to 0.25 mL of a DPPH methanolic solution. The mixture was shaken vigorously and left standing at room temperature for 30 min in the dark. The absorbance of the resulting solution was then measured at 517 nm and corresponded to the ability of extracts to reduce the stable radical DPPH to the yellowcolored diphenylpicrylhydrazine. The antiradical activity was expressed as inhibition concentration at 50 percent (IC 50 ) (mg mL −1 ), the extract dose required to induce a 50% inhibition. A lower IC 50 value corresponds to a higher antioxidant activity of plant extract. The ability to scavenge the DPPH radical was calculated using the following equation: where A 0 is the absorbance of the control at 30 min, and A 1 is the absorbance of the sample at 30 min. Each fraction was analyzed in triplicate.

2,2′-Azino-bis 3-ethylbenzthiazoline-6-sulfonic acid (ABTS) radical cation scavenging
The ABTS + scavenging test is used to determine the antioxidant activity (by estimating peroxide formation) of both hydrophilic and hydrophobic compounds. The quantity of antioxidant in the test sample is inversely proportional to the ABTS radical development. ABTS + is generated by mixing 5 mL of 7 mM ABTS with 5 mL of 2.45 mM potassium persulphate and stored in the dark at room temperature for 16 h. The solution is diluted with ethanol to achieve an absorbance of 0.7 ± 0.02 at 734 nm. The radical-scavenging activity is assessed by mixing 950 μL of this ABTS + solution with 50 μL of each sample at different concentrations. After 6 min, the percentage inhibition was calculated at 734 nm for each concentration relative to blank absorbance [6]. IC values denote the concentration of sample required to scavenge 50% of ABTS free radicals. Low IC values indicate high radical -scavenging activity. Each fraction was analyzed in triplicate.

β-Carotene bleaching test (BCBT)
A modification of the method described by Koleva et al. [7] was employed. β-Carotene (2 mg) was dissolved in 20 mL chloroform and to 4 mL of this solution, linoleic acid (40 mg) and Tween 40 (400 mg) were added. Chloroform was evaporated under vacuum at 40°C and 100 mL of oxygenated ultra-pure water was added, then the emulsion was vigorously shaken. Sample extract were prepared in methanol. An aliquot (150 μL) of the β-carotene: linoleic acid emulsion was distributed in each of the wells of 96-well microtitre plates and methanolic solutions of the test samples (10 μL) were added. Three replicates were prepared for each of the samples. The microtitre plates were incubated at 50°C for 120 min, and the absorbance was measured using a model EAR 400 microtitre reader (Labsystems Multiskan MS) at 470 nm. Readings of all samples were performed immediately (t = 0 min) and after 120 min of incubation. The antioxidant activity (AA) of the extracts was evaluated in term of BCBT using the following formula: where A 0 is the absorbance of the control at 0 min, and A 1 is the absorbance of the sample at 120 min. The results are expressed as IC 50 values (mg/mL).

Antibacterial activities measurement
The antibacterial activity of the two fractions was assessed by the agar disk diffusion assay [8] against five human pathogenic bacteria: gram-positive cocci including Staphylococcus aureus (ATCC 25923), Enterococcus faecalis (ATCC 29212), and Gram-negative bacteria including Escherichia coli (ATCC 35218), Salmonella typhimurium (ATCC 1408) and Pseudomonas aeruginosa (ATCC 27853). The bacterial strains were first grown on Muller Hinton medium at 37°C for 24 h prior to seeding into the nutrient agar. One or several colonies of the indicator bacteria were transferred into API suspension medium (BioMérieux) and adjusted to the 0.5 McFarland turbidity standard with a Densimat (BioMérieux). A sterile filter disc with 6 mm in diameter (Whatman paper no. 3) was placed on the infusion agar seeded with bacteria, and 10 μL of 100 mg per disc extract concentrations were dropped into each paper disc. The treated Petri dishes were kept at 4°C for 1 h, and incubated at 37°C for 24 h. The antibacterial activity was assessed by measuring the zone of growth inhibition surrounding the disks [9]. Standard disks of Gentamycin (10 UI) served as positive antibiotic controls according to CASFM 2005 guidelines. Discs with 10 μL of pure methanol were used as negative controls.

Fatty acid methyl esters analysis (FAMEs)
Extract (0.2 mL) was saponified with 3 mL of 0.5 N NaOH in methanol, and the mixture was incubated for 15 min in a boiling water bath at 60°C. The fatty acids were later converted to methyl esters with 3 mL of a Boron trifluoride-methanol complex (14%) reagent, and the whole incubated at 60°C for 15 min. The FAME was washed with 2 mL of water and extracted with 10 mL of petroleum ether. The identification of FAMEs was performed using an HP-5980 Series II instrument, equipped with HP-5MS capillary column (30 m × 0.25 mm; 0.25 μm film thickness), split/splitless injector (220°C). The oven temperature was held at 150°C, then programmed at 15°C/min up to 220°C, and held isothermally at 220°C for 5 min. Helium was the carrier gas at an initial flow rate of 1 mL/min. Split ratio was 20:1. Injection volume was 2 μL. Quantification of FAMEs, expressed as percentage, was obtained directly from GC-MS peak area (pA) integration. The components were identified by comparing their relative retention times and mass spectra with the data from the library of oil constituents, Wiley, Mass-Finder and Adams gas chromatography/mass spectrometryand (GC/MS) library.

Chemical composition of unsaponific fraction Unsaponific content
The unsaponific content was determined with the extraction method by diethylic oxide ISO 3596.

Total phenolic contents
Total phenolic content were assayed by the Folin-Ciocalteu reagent, following Singleton's method slightly modified by Dewanto et al. [10]. An aliquot (0.125 mL) of suitable diluted samples was added to 0.5 mL of distilled water and 0.125 mL of the Folin-Ciocalteu reagent. The mixture was shaken and allowed to stand for 6 min, before adding 1.25 mL of 7% Na 2 CO 3 solution. The solution was then diluted with demonized water to a final volume of 3 mL and mixed thoroughly. After incubation for 90 min at 23°C, the absorbance versus prepared blank was read at 760 nm. Total phenolic content (three replicates for each fraction) was expressed as mg gallic acid equivalents (GAE) g −1 DW through the calibration curve with gallic.

Carotene content
AOAC (1998) official method, used to evaluate the oil carotenoid content, expressed as micrograms of β-carotene per gram of oil, was applied by calibration curve constructed by preparing solutions of increasing concentrations, from 0.5 to 2.5 μg of β-carotene/mL of hexane. The absorbance was recorded at 440 nm (JASCO V-530, WITEG Labotechnik). The oil was diluted with hexane before being analysed.

Quantification of coenzyme Q9 and coenzyme Q10 levels
Lipid components of the oils were extracted by mixing 990 μL of 1-propanol with 10 μL of the oil (note that a proportion of 900 μL of 1-propanol and 100 μL of the oil was tried in Opuntia oil samples to try to detect CoQ9). After 2 min of vortexing at 1400 rpm at room temperature, the mixed solution was centrifuged at 11,300 g for 5 min at room temperature. The subsequent supernatant was diluted 1/5 or 1/10 in 1-propanol prior to normal phase-high performance liquid chromatography (NP-HPLC) injection. CoQ9 and CoQ10 present in the oil extract were separated by reversed-phase high-performance liquid chromatography (HPLC; Gilson, WI, USA) with a C18 symmetry column (3.5 μm, 4.6 × 150 mm) using a mobile phase consisting of methanol, ethanol, 2-propanol, acetic acid glacial (500:500:15:15), and 50 mM sodium acetate at a flow rate of 0.9 mL/min. The electrochemical detector consisted of an ESA Coulochem III with the following setting:guard cell (upstream of the injector) at +900 mV and conditioning cell at _600 mV (downstream of the column) followed by the analytical cell at +350 mV.22 CoQ9 and CoQ10 concentrations were estimated by comparison of the pAs with those of standard solutions of known concentrations (0, 25, 100, 300, and 600 ng/mL). The results were expressed in milligrams of CoQ per kilogram of oil.

Determination of aliphatic and terpenic alcohols
The fatty substance, with 1-eicosanol added as internal standard, is saponified with ethanolic potassium hydroxide and then the unsaponifiable matter extracted with ethyl ether. The alcoholic fraction is separated from the unsaponifiable matter by chromatography on a basic silica gel plate; the alcohols recovered from the silica gel are transformed into trimethylsilyl ethers and analysed by capillary gas chromatography (GC, principle method of COI/T.20/Doc n 26 of 5th December 2003 and annex XIX of reglementation CEE2568/91). The guideline operating conditions for a chromatographic system with a split injection unit are as follows: column temperature: the initial isotherm is set at 180°C for 8 min and then programmed at 5°C/min to 260°C and a further 15 min at 260°C, temperature of injector: 280°C, temperature of detector: 290°C.
The contents of the individual aliphatic alcohols in mg/1000 g of fatty substance and the sum of the total aliphatic alcohols are reported.

Separation, identification and characterization of sterols
Separation of phytosterols was performed according to the ISO 12228 method. Lipids (250 mg) were refluxed for 15 min with 5 mL ethanolic KOH solution (3%, w/v) after addition of betulin (1 mg; Fluka) as an internal standard and a few antibumping granules. The mixture was immediately diluted with 5 mL of ethanol. The unsaponifiable part was eluted over a glass column packed with a slurry of aluminum oxide (Scharlau) in ethanol (1:2, w/v) with 5 mL of ethanol and 30 mL of diethyl ether at a flow rate of 2 mL/min. The extract was evaporated in a rotary evaporator at 40°C under reduced pressure, and the ether was completely evaporated under a steam of nitrogen.
For the characterization, a silica gel F254 plate (Fluka) was developed in the solvent system n-hexanediethyl ether (1:1, v/v). For the detection of sterols, the thin-layer plate was sprayed with methanol; the sterol bands were scraped from the plate and recovered by extraction with diethyl ether. The extract was then evaporated in a rotary evaporator and in nitrogen. Finally, the sterols were derivatized using a sylilant reagent (1 mL of pure 1-methyl-imidazole and 50 μL of N-methyl-N-(trimethylsilyl-heptafluorobutyramide purchased from Fluka). Preparation of standard solutions: a mixture of standard solutions of sterols was prepared by derivatization (cholesterol, sitosterol, stigmasterol, betulin, ergosterol, and campesterol).
Gas chromatographic analysis of sterols was carried out using a Hewlett Packard 6890 (Agilent) gas chromatograph equipped with an FID detector and a capillary column HP5 (5% phenylmethylsiloxane, 30 m × 0.25 mm id, film thickness 0.25 μm). The operational conditions were: injector temperature 320°C, column temperature: a gradient of 4°/min from 240°C to 255°C (65 min); carrier gas helium at a flow rate of 1 mL/min. Sterol peak identification was carried out according to the ISO 12228 reference method.

Separation, identification and quantification of tocopherols
Tocopherols were analyzed by NP-HPLC with fluorescent detection. A silica column (150 × 3.2 mm, Pinnacle II Silica 3 μm) was used. The mobile phase was the solvent system hexane-isopropanol (99.5:0.5, v/v). The flow rate was 0.5 mL/min, and the column was held at 30°C. The fluorescent detector was set at 290 and 330 nm, respectively, for excitation and emission. pAs are used for quantification. Tocopherol isomers were identified by comparing their retention times with those of pure standards. Standard solutions were prepared by serial dilution to concentrations of approximately 5 mg/mL of each tocopherol. Standard solutions were prepared from a stock solution stored in the dark at −20°C.

Extraction, identification and quantification of squalene
The content of squalene was conducted by simply diluting 1 g of an oil in 5.0 mL diethyl ether containing 5b-androstan-3,17-dione (100 mg/mL) as internal standard.
Analytical GC for the quantitation of squalene was conducted on a HP 5890 gas chromatograph fitted with a 30 m × 0.25 mm ID, glass column filled with 3% OV-1. Helium was used as carrier gas with a linear velociy of 30 mL/min. Sample aliquots of 2 mL was injected. The oven temperature was as follows: initial temperature, 160°C (held 1 min), 160 ± 260°C at 4°C min, 20 min at 260°C. The GC injector temperature was 250°C and the detector temperature was set at 270°C.

Statistical analysis
In all cases, three replicates were used for all assays. Results were processed by using the one-way analysis of variance (ANOVA) using the STATI-CF statistical program. Differences at p < 0.05 were considered to be significant.

Physicochemical characteristics of Opuntia seeds oil
Opuntia ficus indica seed oil shows a high iodine value (105.35) ( Table 1) due to its high content of unsaturated fatty acids (Table 2). This value is comparable with that of other seed oils such as corn oil (103-128), cotton seed oil (99-119), and mustard seed oil (92-125) [11]. The peroxide value of Opuntia ficus indica seed oil was evaluated as 1.41 meq/O2 (Table 1), showing the relative stability to oxidation of this oil. The high iodine value and oxidative stability shows that the seed oil upholds good qualities of edible oil [11]. The saponification value, determined as 175.2 mg of KOH/g of oil (Table 1), is lower than that of the safflower, sunflower, and corn oil [12] for which the average ranges of saponification value is 191-250. The density and refractive index are comparable to values reported by Ennouri et al. [13].

Assessment of antioxidant activities
There are several methods to determine the antioxidant activities. The chemical complexity of extracts which is often a mixture of compounds from different functional groups, polarity and chemical behavior, could lead to scattered results depending on the employed test [6]. Therefore, an approach with multiple assays for the evaluation of the potential antioxidant extracts would be more informative and even necessary. In this study, mainly three methods were used: DPPH radical scavenging activity, BCBT ability and ABTS.
According to the results of the present study, a significant difference of antioxidant capacity (DPPH tests, ABTS and bleaching of β-carotene) between saponific and unsaponific fractions of Opuntia seed oil.

DPPH radical-scavenging activity
The scavenging effect of glyceridic and unsaponifiable fractions of O. ficus indica seed oil on the DPPH radical expressed as IC 50 values was significantly different (13.5 and 11.5 mg/mL, respectively) ( Table 3). There are few researches on the antioxidant activity of the different fractions Opuntia seed oil. Rabhi et al. [14] have shown that the different ecotypes of Opuntia ficus indica in Tunisia have stronger effects of scavenging free radical than positive control butylated hydroxytoluene. However, our experimental data of this extremophile species revealed that the unsaponific extract have a stronger effect of scavenging free radical than the glyceridic one.

ABTS scavenging test
The scavenging effect of glyceridic and unsaponifiable fractions of O. ficus indica seed oil on the ABTS radical are expressed as IC 50 values and they were also significantly different (15 and 25.4 mg/mL, respectively) ( Table 3). Unlike the experimental results of DPPH activity, this test revealed that the glyceridic extract have a stronger effect of scavenging ABTS radical than the unsaponifiable one. In fact, the polarity and hydrophobicity antioxidants The bold values in the three major fatty acids of Opuntia seed oil. Means (three replicates) followed by at least one same letter are not significantly different at p < 0.05. 175.20 ± 0.03 Physical state of room temperature Liquid are two important factors in the biomembranes systems. Additionally, Lopez [15] argues that the implementation of phenolic compounds, most of them are polar; the lipid matrix is delicate and can be accompanied with a decrease in their effectiveness in protection against lipid oxidation. This is the reason why studies concerning antioxidant activity oils preferentially use the inhibition test of laundering the β-carotene as a mimetic model of lipid peroxidation in biological membranes [16].

BCBT antioxidant activity
Results displayed that the addition of O. ficus indica fraction oil extracts to this system prevents the bleaching of β-carotene at different degrees. Contrary to the ABTS test results, the unsaponifiable fraction is more efficient against the bleaching of β-carotene. Therefore, in term of BCBT effect, the extract concentration providing 50% inhibition (IC 50 ) showed a higher ability in unsaponifiable fraction (IC 50 = 5 mg/mL) than in the glyceridic fraction (IC 50 = 17.5 mg/mL) ( Table 3).
On the light of our results, the unsaponifiable fraction of Opuntia oil, rich in phenols which can be related with a remarkable antioxidant activities and also in other natural compounds, have proven inhibitory effect against lipid peroxidation by cleavage of lipid peroxidation chain reactions and trapping peroxyl radical [17]; therefore they are promising potential therapeutic sources. Table 4 shows the antibacterial activities of glyceridic and unsaponifiable seed oil fractions measured by the agar diffusion method against the selected pathogenic bacteria.

Antimicrobial activity
The disk diffusion method was used to determine the antimicrobial activity in comparison with some selected food borne pathogens namely: E.coli, P. aeruginosa, S. aureus, E. faecalis and S. typhimurium. Results show that oil extracts inhibited microorganism growth, depending on the strains sensibility and fraction nature. In fact, the glyceridicic fraction does not inhibit the growth of bacteria and so it showed no antimicrobial activity, whereas unsaponifiable fraction was more active against all the studied pathogens. Therefore, the non-glyceride extract contains some interesting and bioactive compounds since they are able to inhibit some resistant stumps specially the negative gram as Echerchia coli. Several studies attributed the inhibitory effect of plant extracts against bacterial pathogens to their phenolic composition [18]. Therefore, there are other active compounds in the unsaponifiable fraction. These antibacterial actions of non-glyceride fraction can be attributed to the presence of aliphatic components (aliphatic alcohols, hydrocarbons) and terpenic components (carotenoids, sterols, tocopherols, terpenic alcohols, squalene and saponins). Thus, it is noted that the saponins of argan oil present a high antifungal and antibacterial activities when expressing low toxicity [19].

Glyceridic Unsaponifiable
Escherichia coli ATCC35218 6 ± 0.01 13 ± 2.2 27 Pseudomonas aeruginosa ATCC27853 6 ± 0 8.5 ± 0.8 16 Staphylococcus aureus ATCC25923 6 ± 0 12.7 ± 1.4 22 Enterococcus faecalis ATCC29212 6 ± 0 10 ± 0.5 16 Salmonella typhimurium ATCC1408 6 ± 0.01 11 ± 0.01 26 SD, standard deviation; IZ, inhibition zone. The diameter of disc was 6 mm. No antimicrobial activity (-), inhibition zone <1 mm. Weak inhibition zone, inhibition zone 1 mm. Slight antimicrobial activity, inhibition zone 2-3 mm. Moderate antimicrobial activity, inhibition zone 4-5 mm. High antimicrobial activity, inhibition zone 6-9 mm. Strong antimicrobial activity, inhibition zone >9 mm [18]. Inhibition zone calculated in diameter around the disc (mm ± SD). and other fatty acids were identified and quantified. PUFA was the main group of fatty acids, representing 61.65, followed by MUFA 21.37 and SFA 15.85%. The ratio of saturated/unsaturated acid O. ficus indica seed oil were 0.2, which is low because of the high level of unsaturated fatty acid such as C18:1n9 and C18:2n9. Linoleic and oleic acids are powerful antioxidant and antibacterial components [20]. These interesting unsaturated fatty acids are the main compounds of Helichrysum pedunculatum, Schotia brachypetala and flax seed oil [21] which is used to dress wounds during male circumcision rituals in South Africa [22,23]. Besides, studies led by Hu [24] indicates that the higher unsaturated fatty acids in oil, is more beneficial in the dietary food. In fact, these lipids have beneficial effects on health (diminishing inflammation and the risk of atherosclerosis and cardiovascular disease [13], contrary to the saturated ones which are responsible for heart problems and the increase of cholesterol in blood [20]. Table 5 shows that the unsaponifiable fraction presents a notable content of bioactive compounds. However, the high content in antioxidant molecules (such as polyphenols, carotene and Coenzyme Q9) can explain the fact that the unsaponifiable fraction presents higher biological activities than the glyceridic one. Opuntia ficus indica may play a potential role as a source of health promoting phenolics associated with antioxidant activity [25]. The detected phenolic compounds may be responsible for the important in vitro and ex vivo antioxidant activities of the plant crude extracts. We also note that Opuntia seed oil presents a higher content of CoQ9 and lower content of CoQ10 than the seed oil samples of virgin argan oil and other edible vegetebale oils (such as extra virgin olive oil and the virgin soybean oil) that presents higher CoQ10 levels determined by Venegas et al. [26]. The results may be relevant for the contribution of CoQ9 into the biological activities of O. ficus indica seed oil. The identification of aliphatic and terpenic alcohol of Opuntia seeds oil ( Figure 1) shows that the major aliphatic alcohol is the eicosanol and the major terpenic alcohol is the cycloartenol.

Identification of unsaponific fraction components by HPLC and GC assay
Aliphatic and terpenic alcohols are widely used as active ingredients in pharmaceutical industry. The eicosanol and hexacosanol content is quite important in Opuntia seed oil and this allows the improvement of alterations induced by diabetes on the distal tubules of the kidney. It could also prevent diabetes-inducing nephropathy [21]. The docosanol, present also in the O. ficus indica seed oil, provides resistance to several infections among which the herpes virus [27]. Figure 2 and Table 6 present the sterol composition of Opuntia seed oil. The sterol amount represents 975 mg/100 g of seed oil. Fourteen sterol compounds were identified. The β-sitosterol is the major compound identified in the seed oil. Furthermore, the β-sitosterol is 70.8% of the total sterols. In fact, sterols are complex lipid substances synthesized exclusively by plants and possessing a broad spectrum of biological activities. The results of the sterol composition in the Opuntia seed oil  are in agreement with El Mannoubi et al. [1] who report that the β-sitosterol is the sterol marker as it accounts for 71.6% of the total sterol content. The wealth of sterols in Opuntia seed oil allows for it many applications. In fact, sterols comprise the bulk of the unsaponifiables in many oils and they are of interest due to their impact on health [17] phytosterols are widely applied in food and cosmetic industry and recently used as nutraceutical additive [28]. The next major sterol component of Opuntia oil was campesterol which is effective for the inhibition of proinflammatory cytokines [29] and induce cell cycle arrest and release of prostaglandin in response to the increased level of ROS [30]. In our study, all tocopherols and tocotrienols derivatives in O. ficus indica seed oil were identified (Table 7). Tocopherols also called Vitamin E is an important family of lipophilic compounds endowed with important antioxidant activity where the interest is determining the composition of tocopherols in O. ficus indica seed oil. The results allow us to conclude that the Opuntia seed oil is rich in tocopherols and tocotrienols (524 mg/kg). Thus, high levels of vitamin E, detected in oils, may contribute to great stability toward oxidation [27]. Moreover, this oil contains four isomers of tocopherol, which are α-tocopherol, β-tocopherol, γ-tocopherol and δ-tocopherol. The γ-tocopherol is the major tocopherol of Opuntia seed oil, confirming the results presented by El Manoubi et al. [1]. 12    The wealth of Opuntia seed oil in tocopherols can also add to it an important biological activity. Indeed, tocopherols are biological antioxidants that destroy reactive oxygen species (ROS) and protect the unsaturated fatty acids and membranes against oxidative damage [31].
Because of the antioxidant potential of squalene, we are concerned by the determination of its amount in the O. ficus indica seed oil. In fact, this triterpenic hydrocarbon is to some extent transferred to the skin (sebum is reported to contain 12%), its major protective effect is preventing skin cancer. The mechanism is probably by scavenging singlet oxygen generated by UV light. Our results (Table 5 and Figure 3) show that Opuntia seed oil does not contain a significant high amount of squalene compared to other oils, such as found by Owen et al. [28] in the olive oil.

Conclusion
Our results showed that unsaponifiable fraction of Opuntia seed oil has higher biological activities than the glyceridic one: the unsaponific fraction has a stronger effect of scavenging DPPH radical, is more efficient against bleaching of β-carotene and is more efficient against all pathogen strains specially Escherichia coli.
Such variability in antioxidant and antibacterial capacities between two fractions (glyceridic and unsaponifiable) can be explained by different active compounds (identified by HPLC and GC). However, a high level of terpenic alcohols and coenzyme Q9 were detected in the Opuntia seed oil.
These results might be of a great importance in terms of valorizing this cactacea as a source of natural bioactive molecules with high biological activities that can be used in nutraceutical and/or pharmaceutical industry.