Chemical composition and antioxidant/antibacterial depictions of Zahidi date palm (Phoenix dactylifera) kernel Oil

2.4.1 Broth

Luria–Bertani broth was used following the instructions of the producer. A twenty-five (25 g) of broth powder was well mixed with 1 L of distilled water in a Schott bottle, and then the was covered and sterilised using an autoclave at 121 °C for 15 min. Then the sterilised flasks were removed, left to cool down and then kept at 4 °C in a chiller while waiting for further use.

2.4.2 The agar

A 37 g of agar powder (Luria–Bertani Agar) was mixed with 1 L of distilled water in a Schott bottle, then covered with aluminium foil and sterilised at 121 °C in an autoclave for 15 min. Then the sterilised flask was removed and left to cool down for about 10 to 15 min, then the solution was transferred into clean sterilised Petri dishes up to about one-third (1/3) full and allowed cool down by handling the lids loosely on top of the dishes avoiding condensation (Danial and Alkhalf, 2016).

2.4.3 Preparation of inoculum

As usual, a loopful of inoculum was taken from the pure culture of each bacteria grown on slants and inoculated into 10 mL of the prepared broth (Luria–Bertani). Then produced broth was incubated overnight at 37 °C. Then the obtained growth was used as an inoculum for the antimicrobial activity tests/assays. The cultures were obtained from the Microbiology Laboratory at the Department of Chemical Engineering and Sustainability, Kulliyyah of Engineering at the International Islamic University Malaysia (IIUM) that were ordered from the American Type Culture Collection.

2.4.4 Antibacterial activity test kernel extract

The antibacterial test/assay was performed as designated by (Adebolu and Oladimeji (2005), Gautam et al. (2007) and Karuppusamy and Rajasekaran (2009). The broth suspension was diluted to give an optical density at 625 nm (OD₆₂₅) equal to (=) 0.1 to achieve a microbial suspension of about 10⁶ CFU/mL. Separate agar plates (Luria–Bertani) were coated with different bacterial strains. Using a pipette, 100 μL of the prepared bacterial solution was added to the agar plates and evenly spread using a sterilised bent glass rod. The solution was almost absorbed into the agar for 5 min. Ten microliters (10 µL) of each extract -[(300 μg/disc) at a concentration of 30 mg/mL]- was applied to blank discs and observed/tested on the bacterial plates by means of Kirby–Bauer antimicrobial disc analysis.

2.4.5 Disc-Diffusion method (Kirby–Bauer)

Although many antibiotics were discovered, most of them become medically useless because of unacceptable poisonousness or serious evil side effects. One of the most universally used methods for antimicrobial susceptibility testing is the Kirby–Bauer disc-diffusion method (Bauer et al., 1966). In this study, the discs were impregnated with ten (10 μL) of the extracts plated onto the prepared agar plates which were prepared by swabbing the bacterial strain and incubating. Then the plates were placed into the incubator for 24 h 37 °C waiting for growing and inhibition of the bacteria to happen. A clear area around the disc that contains the extract indicates no bacterial evolution. This clear area is termed a zone of inhibition, which was used to quantify the susceptibility. The diameter of the zone of inhibition determined the result of the extracts. A great inhibition zone indicates that the organism was susceptible, whereas a lesser diameter of clear zones of inhibition indicates that the bacterial strain resisted the extract and continue growing (Bauer et al., 1966). The discs of antibiotics used in this research were pre-ordered from Merck Company (Merck Malaysia, Bandar Sunway, Petaling Jaya, Selangor DE, Malaysia). However, the discs used for standards were having low doses of tetracycline (30 µg/disc), gentamycin (10 µg/disc), chloramphenicol (30 µg/disc), streptomycin (10 µg/disc), and vancomycin (30 µg/disc). Ten percent (10%) of Dimethyl sulfoxide (DMSO) was used as the negative control.

2.9.1 DPPH RSC assay

Following the methods labelled by Musa et al. (2011), the antioxidant capacity was evaluated by employing the DPPH RSC. DPPH reagent was newly prepared by dissolving 40 mg DPPH in one (1.00) Litre of methanol, which is sufficient to get an absorbance of 1.00 ± 0.01 unit at 517 nm by a spectrophotometer (Spectro Nanostar, Germany). About 100 μL of the extract -(sample)- was then mixed with 1 mL of the newly made DPPH solution and reserved in the dark for about 30 min. The DPPH scavenging activity was then calculated and measured according to Eq. (1):

2.9.2 Phenolic acids extraction and quantification

High-performance liquid chromatographic (HPLC) following the method designed by Alasalvar et al. (2005) with slight adjustment was used to determine the phenolic acids in the Zahidi date palm kernel extract. The sample of the extract was filtered through a 0.45 µm PTFE filter (National Scientific Company, Rockwood, TN, USA) and then an HPLC analysis was performed. A 20-μL aliquot of a sample or standard extract was inserted by injecting it into a Prodigy ODS-2 column (250 mm × 3.2 mm, Phenomenex, Cheshire, UK). The equipment contained a Quat pump, a diode array detector (G1315A) and a degasser from (HP “hp” Agilent 1100 Series, Waldbronn, Germany). The filtered gradient elution was used with a mobile phase that contained 50 mM solution (A), which is a phosphoric acid, pH 2.5 and solution (B), which is acetonitrile. 0–5 min, isocratic elution 95% A and 5% B; 5–55 min, linear gradient to 80% A and 20% B; 55–60 min, linear gradient to 95% A and 5% B. The flow rate of the mobile phase was 0.9 mL/min. The wavelengths of the diode array detector were set at 254, 270, 280 and 329 nm to monitor the phenolic acids. Phenolic acid standards (2-hydroxyl cinnamic acid, vanillic acid, 4-hydroxyl benzoic, coumaric acid, 4-hydroxyl cinnamic acid, gallic acid and caffeic acid) were used for the identification of phenolic acids. However, the retention time, peak spectra and internal standards were used cautiously to identify & quantify phenolic acids. The phenolic acids were measured by calibration curves for standard concentrations that was ranging between 1 & 100 mg/mL of alcohol (methanol) and conveyed in milligrams of phenolic acid per 100 g of the sample of the extract.

2.9.3 Statistical analyses

Each value is the mean of three replications. Values of different parameters were expressed as the mean ± standard deviation (±SD). Student’s t-test was performed on the statistical analysis using MS Excel at the level of P < 0.05, to evaluate the significance of differences between mean values.

3.3.1 Antibacterial activity test of date palm seed oil

Fig. 1 shows the ability of some common antibiotics, such as tetracycline, chloramphenicol, streptomycin, gentamycin and vancomycin, to inhibit the growth of B. subtilis and S. aureus, representing the G-positive strains and P. aeruginosa and E. coli, representing the Gram-negative strains. Fig. 1 shows that all selected antibiotics affected the growth of all selected bacteria; however, gentamycin and tetracycline did a better job than the others as indicated by the inhibition zone diameters.

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Fig. 1

Positive controls: (tetracycline) Upper left; (chloramphenicol) Upper right; (streptomycin) Middle; (gentamycin) Lower left; and (vancomycin) Lower right. Plates I: B. subtilis, II: S. aureus, III: P. aeruginosa, IV: E. coli.

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The observation and measurement of the diameter of inhibition zones formed (Table 2 and Fig. 2) showed that the extracts from the P. dactylifera seed oil (Zahidi) had significant potential as sources of antimicrobial agents. The results showed that Zahidi seed oil extracts had greater antimicrobial activity hindering G-positive bacterial strains, such as B. subtilis and S. aureus, than did other G-negative strains, which were represented by P. aeruginosa and E. coli in this study. However, the solvents used to extract the active ingredients to be used were ethanol, methanol, acetone and distilled water, which were evaporated and replaced by DMSO which was utilised as a negative standard. The results showed that ethanol, methanol and acetone were good at extracting the active ingredients against Gram-positive bacteria from oil. The water extract showed some activity, but the activity was not consistent with that of G-positive bacteria and showed weak activity against G-negative bacteria.

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Fig. 2

Inhibition zones; 1 (B. subtilis), 2 (S. aureus), 3 (P. aeruginosa), 4 (E. coli); Upper left = Zahidi + ethanol extract, upper right = Zahidi + methanol extract, lower left = Zahidi + acetone extract, lower right = Zahidi + distilled water extract “middle for all” (Negative controls = DMSO).

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We concluded that the oil extracted from Zahidi seed has a strong antibacterial act of inhibiting G-positive and G-negative bacterial strains. Such activity may be because of the presence of lauric (C_12:0) and other short-chain fatty acids, as previously reported (Batovska et al., 2009; Abbas et al., 2017).

3.3.2 MIC of ethanolic extract of Zahidi seed oil

The MIC was determined using the agar disc diffusion assay showed that S. aureus has the greatest sensitivity to the Zahidi palm kernel extract. S. aureus need only 10 mg/mL of extract to inhibit its growth at the MIC, the next was B. subtilis and P. aeruginosa, which had a MIC of 20 mg/mL, the most less sensitive was E. coli, which had a MIC of 44 mg/mL. These results are following the findings by Mirghani et al. (2011). The determination of the MIC of an ethanolic extract of Zahidi seed oil may be useful information for such industrial application of the date kernel oil in the food, pharmaceutical and cosmetics industries.

3.3.3 Determination of MBC

The MBCs of the selected ethanolic extract of Zahidi seed oil against S. aureus, B. subtilis, P. aeruginosa and E. coli were 20, 42, 81 and 165 mg/mL, respectively. These results are within the same range of results from the previous studies conducted by Mirghani et al. (2011), Othman et al. (2011) and Mahmud et al. (2019). The MBC of the selected ethanolic extract of Zahidi seed oil is important for applications of date seed oil to kill and/or inhibit the growth of unwanted microbial strains, such as in the food industry, or the treatment of infective diseases caused by resistant bacterial strains.

3.4.1 TPC

The TPCs in the oil extracted from Zahidi seeds was 32.96 ± 1.66 mg GAE/100 g oil, which is similar to the outcome obtained by Besbes et al. (2004b) who determined the TPC values in other varieties of date seeds oil from Tunisia, which were 520.8 and 220.3 mg/kg oil for the varieties Deglet Nour and Allig seed oils, respectively. Although phenolic compounds are part of the minor components of oils, they are factors for important oil characteristics, such as resistance against oxidation that may increase the shelf life, and they may have some effect on the oil’s flavour as well. Table 3 shows the major phenolic compounds in Zahidi seed oil determined by GC–MS.

3.5.1 Phenolic acids extraction and quantification

For the DPPH assay, the disappearance of DPPH is a well-known index used for the estimation of the RSC, which is usually spectroscopically measured as a decrease in the absorbance due to the change in the purple colour to yellow because of the radical scavenging by the antioxidants in the sample or the standard used via the hydrogen donation that stabilises the DPPH-H, as described in the following Eq. (2)

In this study, the occurrence of an antioxidant, lessening the absorbance at 517 nm as was measured until the antioxidant was exhausted under the assay conditions for oils.

The DPPH percent inhibition values were 42.5%, 9.1% and 68.3% used for MF, LF and TF, respectively. The total oil fraction showed the highest anti-scavenging activity for DPPH free radicals, which may be because of a synergistic effect of the whole oil constituents. Therefore, fractionation of Zahidi seed oil is not advisable if the maximum antioxidant activity of the oil is to be retained. The capacity for high scavenging activity may be predictable to the high amounts of TPC in Zahidi seed oil extract, which agrees with the conclusions of Besbes et al. (2004a) on oils extracted from the kernels of Deglet Nour and Allig cultivars of P. dactylifera L. obtained from Tunisia.