Characterization of Defatted Cake Prepared from Egyptian Olive's Fruit (Wateeken Cultivar) and Its Biological Activity

INTRODUCTION

Functional foods, medicinal plants, fruits, or flowers are good sources of biologically active compounds that have different biological activities and are used as alternative and complementary medicine (Novais et al., 2021; Yu et al., 2021). Oxygen or nitrogen peroxy radical species produced inside cells from metabolism, drugs, and pollution can have dangerous effects on cells (Knekt et al., 1996; Moreno-González et al., 2020). These radicals can induce oxidative damage to subcellular components and subsequently lead to degenerative diseases (Hertog et al., 1995; Guinda et al., 2010). Squalene (2, 6, 10, 15, 19, 23-hexamethyl tetracosaheaxaene) is a precursor of cholesterol synthesis in animal tissue. It is one of the major components of the epidermal lipids. It protects skin from lipid peroxidation, acting as a peroxide scavenger and antiaging. It was found that a diet supplemented with squalene decreased cholesterol and triglyceride levels and protected against CVD in animals (Duffy et al., 2001). The tree Olea europaea L. belongs to the family Oleaceae. Fruits are the main source of olive oil which is considered to have high nutritional value worldwide. In the Mediterranean region, olive oil is the main dietary component that contributes to the improvement of health status by reducing the incidence of many disorders as metabolic syndrome, cardiovascular disease (CVD), and some tumors (Nijveldt et al., 2001). The quality of olive fruit and its composition are affected by species, genetics, acidity, and industrial production conditions (Nijveldt et al., 2001). Traditionally, the olive fruit and its leaves have been used in the management of different conditions such as diabetes, hypertension, respiratory infection, antioxidants, skin moister, hair growth, and hypercholesterolemia (Schreyer et al., 1998). The monounsaturated fatty acids are the main content of olive oil related to biological and nutritional properties. A previous study reported that olive fruit and oil showed antimicrobial activity against Salmonella enteritidis and Listeria monocytogenes. It is used for edible purposes in dietary components (Servili et al., 2013). The fruit has a powerful anti-oxidant activity and anticancer properties as breast and colon cancer has reported that the amounts of the phenolic and squalene compounds are relatively high in olive fruit and oil. This study aimed to characterize the quality of the Egyptian olive's fruit Olea europaea (Wateeken cultivar) by identifying phytochemical components using GC/MS (Gas chromatography/mass spectrum), quantifying the phenolic and squalene content, and assessing its biological activity as antioxidant, anti-inflammatory, antitumor activity against human hepatocellular carcinoma cell line and mechanism of action via apoptosis.

MATERIALS AND METHODS

Preparation of defatted cake prepared from Egyptian olive fruit Olea europaea (DCEOF)

The olive fruit Olea europaea (Wateeken cultivar) used in this study was collected from the local Egyptian markets. The fruits were washed and dried to remove moisture.  One kilogram of olive fruit was soaked, chopped, and extracted in one liter of pure methanol at room temperature for 24 h. The extract was filtered using Whatman #1 filter paper. After that, the solvent was removed using a rotatory vacuum till dry. The extract was defatted with pure petroleum ether olive fruit extract. The defatted extract was stored at 20 °C for analysis.

Identification of the composition of DCEOF by GC/MS

A hundred milligrams of extract were dissolved in one ml of high-grade acetonitrile and subjected to analysis. The components of DCEOF were identified by GC/MS  (Agilent, USA), Inj = 170 °C, Volume = 1 µL, Split = 1, Carrier Gas is helium, Solvent Delay = 6.00 min, Transfer Temp = 150 °C, Source Temp = 200 °C, Scan: 50 to 620 Da, Column (Elite-5MS, 30m 0.25mmID 0.25um).

Analysis of squalene and phenolic in olive fruit by GC/MS

Briefly, one gm of the fruit was extracted with two ml of pure methanol for 4 hours at room temperature. The solvent was evaporated at 35 °C. The residue was dissolved in 5 ml hexane 5 ml for defatting. The residue was dissolved in 1 ml acetonitrile for subjection to GC/MS. The separation capillary column was 30 mm in length, 0.25 mm Id., and 0.25 mm in thickness. Helium was used as carrier gas. The oven's initial temperature was 100 °C to 270 °C for 20 min (Brahmi et al., 2013).

Assay of scavenger capacity of DCEOF using 1,1-Diphenyl-2-picrylhydrazyl free radical (DPPH)

The total antioxidant activity of DCEOF was determined according to (Ahmad et al., 2021). Serial dilution of extract (10-100 mg/ml) was prepared. Each sample was supplemented with 150 µL of a DPPH solution with a concentration of 400 µM. The sample was placed in an incubator at a temperature of 37 °C for 30 minutes. After incubation, the absorbance of the sample was measured at a wavelength of 517 nm. The obtained value was compared to the value provided by the DPPH solution, and each extract was used as a blank without DPPH. The data was determined by calculating the concentration at which a 50% decrease in the DPPH occurs.

Determination of the anti-inflammatory activity of DCEOF

One mg of extract was dissolved in 1ml DMSO and serial dilutions were prepared (20 to 300 μg/ml). Aspirin (100 μg/ml) was used as a positive control (Allouche et al., 2011). The in vitro anti-inflammatory activity by the assessment of inhibition of albumin denaturation and membrane stabilization.

Inhibition of albumin denaturation of DCEOF

To one milliliter of extract (1 mg/ml), one milliliter of 1% bovine albumin was added. Adjusting the pH to 6.3, the mixture was heated to 50 °C for 30 minutes and then incubated for 20 minutes at 37 °C. Cooling results in an absorbance of 660 nm. The equation below was used to calculate the percentage inhibition of albumin denaturation: (A_control – A_sample / A_control) X100.

Erythrocyte membrane stabilization by DCEOF

A volunteer participant who had not taken any analgesics for two weeks provided the fasting blood sample, which was centrifuged for ten minutes at 4000 rpm. One milliliter of extract was combined with one milliliter of RBC suspension and left to incubate for 30 minutes at 56 °C. After cooling, the mixture was centrifuged at 3000 rpm for 10 minutes to separate the supernatant. At 560 nm, the supernatant's absorbance was measured and contrasted with that of the control sample.

Effect of different concentrations of DCEOF on the viability of HepG2 cell line

The MTT assay is used to measure cellular viability and proliferation. Different concentrations of extract (0-200 µg/ml) were used and incubated for 24, 48, and 72 hours. Then, to each well, 10 µl of MTT reagent was added, and incubated for 3 hours at 37 °C. The absorbance was measured at 500 nm. The 0.3% H₂O₂ (v/v) was used as positive control.

Determination of caspase 3 and 9 levels in response to different concentrations of DCEOF

HepG2 was treated with 200 ug/ml extract and incubated for 24 hours with a CO2 incubator. The cells were collected and subjected to assay of caspase 3 and 9 by ELISA (enzyme-linked immunosorbent assay) technique kit obtained from BIORAD.

Statistical analysis

The data obtained was analyzed using SPSS, software V 20.0 using principal component analysis. Data was expressed as Mean ± SD. P value < 0.05 was considered as significant.

RESULTS AND DISCUSSION

The composition of DCEOF by GC/MS showed different active molecules as shown in Table 1. Including Branched alkene squalene [Dodecane, 2,6,11-trimethyl-, Tridecane, 2-methyl-], polyunsaturated fatty acids; Eicosane, Metabolites analogue [Decane, 2,3,5,8-tetramethyl-], Dodecanoic acid, Hexadecane, Sex prehormone [9(11)-Dehydrotestosterone], Sesquterpenoides [Dodecane, 2,6,11-trimethyl-, Undecane, 3,7-dimethyl-, 2,6-Dimethyldecane, Undecane, 3,8-dimethyl]. The squalene exerts an antibacterial effect. Vitamin A derivatives; Retinol, 4,14-Retro-retinol.

Table 1. The chemical composition of DCEOF with relative retention times.

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