Antioxidant and antibacterial activities of Opuntia ficus indica seed oil fractions and their bioactive compounds identification

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 yellow-colored diphenylpicrylhydrazine. The antiradical activity was expressed as inhibition concentration at 50 percent (IC₅₀) (mg mL⁻¹), the extract dose required to induce a 50% inhibition. A lower IC₅₀ value corresponds to a higher antioxidant activity of plant extract. The ability to scavenge the DPPH radical was calculated using the following equation:

DPPH Scavenging effect=[(A0−A1)/A0]×100

where A₀ is the absorbance of the control at 30 min, and A₁ 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:

AA (%)=[(A0−A1)/A0]×100

where A₀ is the absorbance of the control at 0 min, and A₁ is the absorbance of the sample at 120 min. The results are expressed as IC₅₀ values (mg/mL).

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% Na₂CO₃ 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-hexane–diethyl 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.

DPPH radical-scavenging activity

The scavenging effect of glyceridic and unsaponifiable fractions of O. ficus indica seed oil on the DPPH radical expressed as IC₅₀ 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 IC₅₀ 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 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 (IC₅₀) showed a higher ability in unsaponifiable fraction (IC₅₀=5 mg/mL) than in the glyceridic fraction (IC₅₀=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.

Fatty acid composition

The results of fatty acid compositions, total saturated fatty acids (SFA), monounsaturated and polyunsaturated fatty acids (MUFA and PUFA, respectively) of O. ficus indica seeds oil are shown in Table 5. The major fatty acids were linoleic acid (ω 6; C18:2) followed by oleic acid (C18:1) and palmitic acid (C16:0), representing, respectively 61.42; 20.55 and 11.88%. Besides these three main fatty acids 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].

Identification of unsaponific fraction components by HPLC and GC assay

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.

Figure 1:

Chromatographic profile of aliphatic and terpenic alcohols in Opuntia ficus indica seed oil.

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 pro-inflammatory cytokines [29] and induce cell cycle arrest and release of prostaglandin in response to the increased level of ROS [30].

Figure 2:

Chromatographic profile of sterols in Opuntia ficus indica seed oil.

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]. 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.

Figure 3:

Chromatographic profile of squalene determination inOpuntia ficus indica seed oil.