Antioxidant, anti-inflammatory and cytotoxic activities of the unsaponifiable fraction of extra virgin olive oil ; Actividades antioxidantes, antiinflamatorias y citotóxicas de la fracción insaponificable de aceite de oliva virgen extra

The health benefits of olive oil are well-known. In this study, the unsaponifiable fraction of extra virgin olive oil (Unsap) was investigated for reducing power capacity, ferric reducing antioxidant power, ferrous chelating activity and nitric oxide inhibition. The present study was also designed to evaluate the in vitro cytotoxic effect of the Unsap against human carcinoma cells. The anti-inflammatory potential of Unsap has been determined via the inhibition of Human Cyclooxygenases. The results showed that Unsap is efficient for ferric reducing antioxidant power and nitric oxide inhibition. Unsap has a selective effect as anti-inflammatory agent. The results showed moderate to good in vitro antitumor activities of Unsap against human liver, lung and pancreas cancer cells with IC50 ranging from 19.6 to 30.4 μg/mL and good selectivity index (≥ 2). In conclusion, Unsap represents a promising and safe antitumor and antioxidant material that supports the need for further investigation.


INTRODUCTION
Extra virgin olive oil (EVOO) is the highest quality olive oil obtained from the olive fruit by mechanical processes. According to the Codex (2015) classification, EVOO category has free acidity, fatty acid ethyl esters, peroxide value and absorbance at 270 nm, and not higher than 0.8%, 35 mg/kg oil, 20 milliequivalents of active oxygen/kg oil and 0.22, respectively. In addition, it should be free of odor and taste defects. It consists of a major fraction of unsaturated fatty acids and the Unsap (1-2%) composed of aliphatic and triterpenic alcohols, sterols, hydrocarbons and α-tocopherol (Servili et al., 2014). Squalene represents the major hydrocarbon in the unsaponifiable fraction of olive oil (Cardeno et al., 2014).
The Unsap of EVOO as a natural product has anti-inflammatory and anticancer effects on human colon cancer in murine models (Cardeno et al., 2013). Escrich et al., (2011) attributed the beneficial effect of EVOO on the progression of breast cancer to its oleic acid content and its minor compounds. Dietary EVOO's Unsap prevented the damage in acute colitis and reduced pro-inflammatory protein expression to its basal levels in mice (Sánchez-Fidalgo et al., 2013). The Unsap of virgin olive oil prevented the development and progression of neuroinflammation-related diseases in obese mice (Toscano et al., 2019). A diet rich in EVOO exerts its effect on breast cancer by reducing the level of DNA damage (Solanas et al., 2010). Several studies have confirmed that the consumption of olive oil is effective in the prevention and treatment of cardiovascular diseases, and obesity and reduces the risk of cancer, especially breast, lung and stomach cancer (Covas et al., 2009). Squalene has chemopreventive activity against colon cancer (Rao et al., 1998). Smith et al., (1998) reported that a diet that contains olive oil or squalene could effectively inhibit lung tumorigenesis and decrease lung tumor multiplicity. Warleta et al., (2010) suggested that the squalene in virgin olive oils could be partially responsible for a lower incidence of breast cancer due to its protective activity against oxidative DNA damage in normal mammary cells. Liver cancer is one of the most widespread malignant diseases (Bray et al., 2018). A polyphenolic extract of EVOO reduced cell proliferation and increased cell death of liver cancer cell lines (HepG-2, Huh7 and Hep3B) (De Stefanis et al., 2019). Olive oil phenols such as hydroxytyrosol and oleuropein are not Unsap materials (Boskou, 2015). Although, squalene and γ-Tocopherol proved to have antioxidant and anticancer activities when applied as pure substances individually (Newmark, 1997;Abraham et al., 2019), Cardeno et al., (2014) reported that there is a great lack of knowledge on the biological activity of the Unsap of EVOO. The combination of these active compounds may be useful for enhancing the effectiveness, eliminating the free radicals' destructive effects or minimizing the doses needed for inhibiting the growth of cancer cells. Therefore, the novelty of this study is the evaluation of the combined effect of the constituents present in the Unsap of EVOO on scavenging radicals, reduction in COX-2 activity and selectivity against cancer cells compared to normal cells.

Materials
EVOO was obtained from the Agriculture Research Center, Giza, Egypt. Ascorbic acid, butylated hydroxytoluene (BHT), MTT and trypan blue dye were purchased from Sigma (St. Louis, MO, USA). Fetal Bovine serum, DMEM, L-glutamine, gentamycin and 0.25% Trypsin-EDTA were purchased from Lonza (Walkersville, USA). Cell lines were obtained from the VACSERA Tissue Culture Unit, the Holding Company for Biological Products and Vaccines, Egypt.

Extraction of the unsaponifiable matter
The saponification of EVOO (5g) and extraction of the unsaponifiable matter were carried out according to the IUPAC (1992) method.

Ferric reducing antioxidant power (FRAP)
The reducing power of Unsap was evaluated using the method of Oyaizu (1986). This method is based on the reduction in ferricyanide in relation to different concentrations of Unsap (from 3.9 to 2000 μg/mL, in a geometric sequence). The absorbance was measured at 700 nm against a blank using a spectrophotometer (Milton Roy, Spectronic 1201). BHT was used as reference standard. The reducing capacity percentage (%) was calculated according to Canabady-Rochelle et al., (2015) as follows: Where, A 0 : absorbance of the control solution. As: sample absorbance.

Ferrous metal chelating activity
Different concentrations of the Unsap and reference standard (from 3.9 to 2000 μg/mL, in a geometric sequence) were added to a solution of FeCl 2 (2 mM). The reaction was initiated by the addition of ferrozine (5 mM) and the mixture was shaken vigorously and left to stand at room temperature for 10 min. Then, the absorbance of the solution was measured at 562 nm using a Milton Roy (Spectronic 1201) spectrophotometer according to Gülҫin et al., (2003). Decreasing absorbance in the metal chelating activity showed that metal ions were chelated before ferrozine bonding. The percentage of inhibition of ferrozine-Fe 2+ complex formation was given by the formula (2): Where, A 0 is the absorbance value of the control (in the presence of ferrozine and Fe 2+ ions), and A 1 is the absorbance value in the presence of the tested sample or standard. BHT was used as a reference standard.

Nitric oxide (NO) radical scavenging activity
The NO radical scavenging activity of Unsap was determined according to the method of Marcocci et al., (1994) by using a sodium nitroprusside (SNP). NO radical generated from SNP reacted with oxygen to produce nitrite ions, which were measured by the Greiss reagent. The concentrations of the tested Unsap ranged from 3.9 to 2000 μg/mL in a geometric sequence. The absorbance was measured at 546 nm against the corresponding blank solution (without SNP). Ascorbic acid was used as a reference standard. All the tests were performed in triplicate. The percent inhibition activity was calculated using the formula (3): where, A control is the absorbance of the control reaction at 546 nm and A test represents the absorbance of a test reaction at the same wavelength. Tested material concentration providing 50% inhibition (IC 50 ) was calculated from the graph by plotting inhibition percentage against concentration.

In-vitro anti-inflammatory activity (Cyclooxygenase inhibition assay)
The cyclooxygenase inhibition assay was carried out according to Larsen et al., (1996). Different concentrations of the tested sample (from 7.8 to 1000 μg/mL, in a geometric sequence) were preincubated with the cyclooxygenase enzymes (COX-1 and COX-2) at room temperature (25 °C) for 5 min in the presence of hematin. Phenol (500 μM), 1-leuco-dichlorofluorescein (20 μM), arachidonic acid (50 μM) and hematin (1 μM) in 1 mL of 0.1 M Tris-buffer (pH 8) were pre-mixed and added to the enzyme mixture. The absorbance was recorded within 15 seconds of the addition using a Milton Roy (Spectronic 1201) spectrophotometer at 502 nm. A blank was made without the addition of enzymes. Celecoxib was used as a standard. The IC 50 value (μg/mL), which is the concentration of the Unsap or standard that inhibited 50% of each cyclooxygenase, was calculated from the curve.

Cytotoxicity evaluation
The cytotoxicity assay of Mosmann (1983) was performed as follows: the cells were seeded in 96-well plates at a cell concentration of 1×10 4 cells per well in 100 µL of growth medium. After 24 h of seeding, fresh medium containing different concentrations (from 3.9 to 500 μg/mL, in a geometric sequence) of the tested sample was added. The Microtiter plates were incubated at 37 ºC in a humidified incubator with 5% CO 2 for a period of 24 h. Control cells were incubated without the tested sample. Then, the yield of viable cells was determined by a MTT colorimetric method. The absorbances of the plates were measured at a wavelength of 570 nm using a Microplate reader (SunRise, TECAN, Inc., USA). Treated samples were compared with the cell control in the absence of the tested compounds. Vinblastine sulfate was used as a reference anticancer drug. All experiments were carried out in triplicate. The percentage of viability was calculated as illustrated in the following formula (4) Where: OD t is the mean optical density of wells treated with the tested sample and OD c is the mean optical density of the untreated cells. The relation between surviving cells and drug concentration is plotted to get the survival curve of each tumor cell line after treatment with the specified compound. The 50% inhibitory concentration (IC 50 ) or the cell cytotoxic concentration (CC 50 ) is the concentration required to cause toxic effects in 50% of intact cells. IC 50 was estimated from the graphic plots of the dose response curve for each concentration using Graphpad Prism software (San Diego, CA. USA).

Statistical analysis
The results are expressed as the mean values ± standard deviation (SD) of three experiments. The statistical analysis was carried out using Statistica software (StatSoft Inc., Tulsa, OK, USA). The Tuckey's test was used at a significance level of 5%.

RESULTS AND DISCUSSION
Squalene represented the main component of the Unsap while β-sitosterol, and γ-tocopherol were found in minor quantities as reported previously (Cardeno et al., 2014;Galal et al., 2020).

Antioxidant activity
The antioxidant activity of the Unsap was analyzed by different methods (FRAP, iron chelating activity and nitric oxide radical scavenging activity) to cover various ways of antioxidant protection and the results are illustrated in Figure1.
Previous studies indicated that the antioxidant activity of some compounds is correlated with their reducing power. Figure 1a presents the reducing capacity of Fe 3+ →Fe 2+ transformation in the presence of Unsap compared to the standard. The reducing power of the investigated Unsap and the positive standard BHT was concentration dependent and reached a plateau at concentrations higher than 1000 μg/mL. The reducing power of 1000 μg/mL of the Unsap was not significantly different from that of BHT (p > 0.05) at 250 μg/mL. The IC 50 value for Unsap (192.05 μg/mL) was higher than that of BHT (54.34 μg/mL), indicating lower activity of the Unsap. Abdallah et al., (2018) found that the polyphenol methanolic extract of olive oil samples, obtained from three Tunisian cultivars, had IC 50 values of FRAP ranging from 250 to 480 μg/mL. They ascribed this activity to the hydroxytyrosol and decarboxymethyl ligstroside aglycone in the polyphenol extract.
The generation of reactive oxygen species can be avoided by the chelating of metal ions. Extracts with iron chelating activity are effective for the reduction of lipid peroxidation (Atere et al., 2018). Chelating agents disrupt the formation of the Ferrozine-Fe 2+ complex and decrease the red color of the complex. The chelating effect of the Unsap on ferrous ions compared to BHT is illustrated in Figure1b. The chelating efficiency of 2000 μg/mL of radical scavenging activity compared to ascorbic acid (c). The results are presented as average values of three replicates ± SD. Values with different letters indicate significant differences (p < 0.05) according to Tukey's test. the Unsap was not significantly different from that of BHT (p > 0.05) at 500 μg/mL. The IC 50 value of the Unsap for chelating activity was 306.73 µg/mL, which was higher than the positive standard BHT (IC 50 = 50.20 µg/mL).
Based on Figure 1c, the evaluation of nitric oxide radical (generated by sodium nitroprusside at physiological pH) scavenging activity of Unsap and ascorbic acid showed a similar trend. The nitric oxide radical scavenging activity of the Unsap was not significantly different from that of the ascorbic acid (p > 0.05) at all the investigated concentrations. The IC 50 value for Unsap (116.7 μg/mL) was lower than that of the ascorbic acid (176.5 μg/mL) indicating higher antioxidant activity. Franco et al., (2014) found that NO antiradical activity of the oils of seven Spanish olive varieties ranged from 29.8 to 40.7% at 5.6 mg/mL of olive oil methanol solution.
Based on the data obtained from this study, the tested olive oil Unsap exhibits free radical inhibitor or scavenger activity for NO radical thus acting as primary antioxidant which reacts with free radicals, and may limit free radical damage in the human body.

Anti-inflammatory activity
The Unsap was investigated for its efficiency to inhibit human cyclooxygenases (COX-1 and COX-2) compared with celecoxib as a standard drug. The results are shown in Figure 2. The COX enzymes were inhibited by the tested Unsap in a dose dependent manner. The investigated Unsap demonstrated a weak potency towards COX-1 (inhibition did not exceed 37.25% at 1000 μg/mL), and moderate potency towards COX-2 (IC 50 = 412.20 μg/mL). The inhibitory effect of the Unsap at 1000 μg/mL on the activity of COX-2 was not significantly different from that of celecoxib (p > 0.05) at 250 μg/mL. On the other hand, the IC 50 value of the standard celecoxib on COX-1 and COX-2 was 400 μg/mL and 84.05 μg/mL, respectively.
The inhibition of nitric oxide (NO) and cyclooxygenase-2 (COX-2) production is considered a promising approach for the treatment of various diseases, including inflammation and cancer as reported by Park et al., (2003). Therefore, the tested olive oil Unsap may be a useful anti-inflammatory and anticancer agent.

Cytotoxic activity of Unsap against cancer cell lines
The cytotoxicity of the Unsap was investigated on different cancer cell lines including MCF-7, A-549, Panc-1 and HepG-2 carcinoma cells, as shown in Figure 3. The inhibitory activity of Unsap against human lung fibroblast normal cell lines was also assayed.
With regard to MCF-7, the Unsap at 125 µg/mL showed significantly (p < 0.05) the same inhibition activity of 62.5 µg/mL of the vinblastine sulfate drug. Treating Panc-1 cancer cells with Unsap at 62.5 µg/mL significantly induced (p < 0.05) the same inhibition effect as the standard drug at 15.6 µg/mL. The inhibition effect of the Unsap at 62.5 µg/mL on HepG-2 cancer cells was significantly (p < 0.05) similar to that of the standard drug at 31.25 µg/mL. These results indicated that those carcinoma cells were less susceptible to the cytotoxic effect of the Unsap compared to vinblastine sulfate. It is interesting to note that Unsap and vinblastine sulfate at the concentration of 62.5 µg/mL have significantly (p < 0.05) the same inhibition effect on A-549 lung cancer cells. The results clearly showed that Unsap at concentrations higher than 62.5 µg/mL was significantly (p < 0.05) more efficient in the inhibition of A-549 cancer cells than vinblastine sulfate at the equivalent concentration. Unsap exerts a low toxicity (CC 50 = 59 ± 3.9 µg/mL) on WI-38 human lung fibroblast normal cells (Figure 3e). However, the selective index (the ratio between IC 50 value for normal fibroblast cells and IC 50 value for cancer cells) towards HepG-2, A-549 and Panc-1 was 2.63, 1.94 and 3.01, respectively, indicating high selective toxicity (≥ 2) against those cancer cells as reported by Valderrama et al., (2016). Squalene has a chemopreventive effect against colon carcinogenesis (Rao et al., 1998). They attributed this effect to its inhibitory activity of HMG-CoA reductase and to suppressing the activation of oncogene proteins.

CONCLUSIONS
Unsap proved to have antioxidant activity and a selective cytotoxic effect against the A-549 cancer cell line. The data reveal that Unsap has anticancer activity on lung cancer cells (A-549) which is comparable to that of the drug vinblastine sulfate. These properties make Unsap a natural antioxidant and a promising anti-tumor agent for new pharmaceutical products to provide protection and treatment against the deleterious effects of free radicals.